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Fiziologia - Physiology  2009.19.4(64)CONTENTS
1. Gene Expression of Programmed Death Ligand (Pd-L)-1, Pd-L2, Inducible Costimulator Ligand and B7-H3 on Human Alveolar Epithelial Cells and Regulation by Respiratory Syncytial Virus andType 1 And 2 Cytokines
Aurica G. Telcian, Adriana Muresan, Vasile Laza-Stanca, Sebastian L. Johnston and Luminita A. Stanciu . ........................................................................................................................................................................4
2. Assessment of Interleukin-1β and Interleukin-8 levels in the Gingival Fluid of Orthodontic Tractioned Teeth
Olteanu C, Muresan A, Craciun A, Serbanescu A, Olteanu I, Keularts MLW Irene.................................................................................................................................................................................................................8
3. Resistance to Beta-Lactam Antibiotics of Escherichia Coli Strains Isolated from Urocultures
Delia Berceanu Văduva, Monica Licker, Delia Muntean, Dana Velimirovici, Matilda Rădulescu, Dugăeşescu Dorina, Tatu Carmen, Marcela Adămuţ, Stângă Livia, Maria Rada, M. Berceanu Văduva, Roxana Moldovan...........12
4. Prevalence and Antimicrobial Resistance of Acinetobacter Baumannii Strains Isolated from the Cardiology Department
Dorina Dugăeşescu, Monica Licker, Delia Berceanu-Văduva, Mihaela Craciunescu, Liliana Dragomirescu, Elena Hogea, Carmen Tatu, Delia Muntean, Mihaela Popa, Ciprian Piluţ, Ramona Pîrvan, Denis Serban, Roxana
Moldovan ......................................................................................................................................................................................................................................................................................................................15
5. Antimicrobial Resistance of Escherichia Coli Strains Isolated from Urinary Tract Infections
Craciunescu Mihaela, Licker Monica, Muntean Delia, Hogea Elena, Berceanu-Vaduva Delia, Dugaesescu Dorina, Serban D., Popa Mihaela, Pilut C., Horhat F., Tatu Carmen, Moldovan Roxana . ...................................18
6. In vitro Characterization of Mechanisms Involved Adipogenic Differentiation of Mesenchymal Stem Cells and Tumor Associated Fibroblasts
Florina Bojin, Calin Tatu, Oana Gavriliuc, Valentin Ordodi, Alexandra Gruia, Adriana Rosca, Gabriela Tanasie, Carmen Tatu, Daciana Nistor, Mirabela Cristea, Simona Anghel, Alexandra Boleman, Carmen Bunu, Virgil
Paunescu ......................................................................................................................................................................................................................................................................................................................21
7. Normal and Abnormal in Medicine: Epidemiological Assessment of Cholesterol Levels in Medical Practice
Brumboiu Irina, Ursu Ligia, Simionca G, Chelaru E..........................................................................................................................................................................................................................................................27
8. Harvesting, Isolation and Characterization of Dog Periosteum - Derived Cells
Anghel Simona, Bojin Florina, Igna Cornel, Tanasie Gabriela, Seres Monica, Tatu Carmen, Luca Calin, Tatu Calin, Rosca Adriana, Gruia Alexandra, Ordodi Valentin, Georgescu Remus, Paunescu Virgil...........................31
9. Cavernous Hemangioma of the Parotid GlandAlexandra Faur, Elena Lazar, Marioara Cornianu, Alis Dema, Codruta Lazureanu, Anca Muresan, Camelia Gurban Vidita, Laura Smaranda Gotia...........................................................................................................36
10. Clinical Aspects of Aortic Coarctation in Small Children
Ana Lascu, Caius G. Streian, Ion Socoteanu, Corina Vernic...............................................................................................................................................................................................................................................38
11. Methicillin Resistant Staphylococcus Aureus Strains Isolated from Orthopaedics Ward and Ambulatory
M. Berceanu Văduva, H. Petrescu, D. Vermeşan, F. Tatu, A. Lupaşcu, V. Dumitraşcu, Elena Hogea, Delia Muntean, Matilda Rădulescu, Marcela Adămuţ, Carmen Tatu, Dana Velimirovici, Delia Berceanu Văduva...........41

CUPRINS
1. Expresia genelor pentru programmed death ligand (pd-l)-1, pd-l2, inducible costimulator ligand si b7-h3 in celulele epiteliale alveolare umane si modularea lor de catre virusul sincitial respirator
si tipul 1 sau 2 de citokine
Aurica G. Telcian, Adriana Muresan, Vasile Laza-Stanca, Sebastian L. Johnston and Luminita A. Stanciu..........................................................................................................................................................................4
2. Determinarea nivelului interleukinei-1β si a interleukinei-8 in lichidul gingival al dintilor tractionati ortodontic
Olteanu C, Muresan A, Craciun A, Serbanescu A, Olteanu I, Keularts MLW Irene.................................................................................................................................................................................................................8
3. Rezistenta la antibiotice betalactamice a unor tulpini de Escherichia Coli izolate din uroculturi
Delia Berceanu Văduva, Monica Licker, Delia Muntean, Dana Velimirovici, Matilda Rădulescu, Dugăeşescu Dorina, Tatu Carmen, Marcela Adămuţ, Stângă Livia, Maria Rada, M. Berceanu Văduva, Roxana Moldovan.......................12
4. Prevalenta şi rezistenta la antibiotice a tulpinilor de Acinetobacter Baumanii izolate intr-o sectie de cardiologie
Dorina Dugăeşescu, Monica Licker, Delia Berceanu-Văduva, Mihaela Craciunescu, Liliana Dragomirescu, Elena Hogea, Carmen Tatu, Delia Muntean, Mihaela Popa, Ciprian Piluţ, Ramona Pîrvan, Denis Serban, Roxana
Moldovan ......................................................................................................................................................................................................................................................................................................................15
5. Rezistenta la antibiotice a tulpinilor de Escherichia Coli izolate din infectii urinare
Craciunescu Mihaela, Licker Monica, Muntean Delia, Hogea Elena, Berceanu-Vaduva Delia, Dugaesescu Dorina, Serban D., Popa Mihaela, Pilut C., Horhat F., Tatu Carmen, Moldovan Roxana.....................................18
6. Caracterizarea mecanismelor implicate in diferentierea adipogenica a celulelor stem mezenchimale si a fibroblastelor tumorale
Florina Bojin, Calin Tatu, Oana Gavriliuc, Valentin Ordodi, Alexandra Gruia, Adriana Rosca, Gabriela Tanasie, Carmen Tatu, Daciana Nistor, Mirabela Cristea, Simona Anghel, Alexandra Boleman, Carmen Bunu, Virgil
Paunescu........................................................................................................................................................................................................................................................................................................................21
7. Normal si anormal in medicina: evaluarea epidemiologica a nivelelor de colesterol in activitatea practică medicala
Brumboiu Irina, Ursu Ligia, Simionca G, Chelaru E..........................................................................................................................................................................................................................................................27
8. Recoltarea, izolarea si caracterizarea celulelor derivate din periostul canin
Anghel Simona, Bojin Florina, Igna Cornel, Tanasie Gabriela, Seres Monica, Tatu Carmen, Luca Calin, Tatu Calin, Rosca Adriana, Gruia Alexandra, Ordodi Valentin, Georgescu Remus, Paunescu Virgil...........................31
9. Hemangiomul cavernos de glanda parotida
Alexandra Faur, Elena Lazar, Marioara Cornianu, Alis Dema, Codruta Lazureanu, Anca Muresan, Camelia Gurban Vidita, Laura Smaranda Gotia...........................................................................................................36
10. Aspecte clinice sugestive pentru coarctatia aortica la copilul mic
Ana Lascu, Caius G. Streian, Ion Socoteanu, Corina Vernic...............................................................................................................................................................................................................................................38
11. Rezistenţa la meticilină a tulpinilor de Staphylococcus Aureus izolate dintr-o secţie de ortopedie şi din ambulator
M. Berceanu Văduva, H. Petrescu, D. Vermeşan, F. Tatu, A. Lupaşcu, V. Dumitraşcu, Elena Hogea, Delia Muntean, Matilda Rădulescu, Marcela Adămuţ, Carmen Tatu, Dana Velimirovici, Delia Berceanu Văduva...........41

2009.19.4(64)  Fiziologia - Physiology

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Gene expression of Programmed Death Ligand(PD-L)-1, PD-L2, Inducible Costimulator Ligand andB7-H3 on human alveolar epithelial cells andregulation by respiratory syncytial virus andtype 1 and 2 cytokines
AURICA G. TELCIAN1, ADRIANA MURESAN2, VASILE LAZA-STANCA1, SEBASTIAN L.
JOHNSTON1 AND LUMINITA A. STANCIU1
1
Department of Respiratory Medicine, National Heart and Lung Institute, MRC & Asthma UK Centre in Allergic Mechanisms of Asthma and Centre for Respiratory Infection,
Imperial College London, Norfolk Place, London W2 1PG, UK
2
Department of Physiology, University of Medicine and Pharmacy “Iuliu Hatieganul”, Cluj-Napoca, Romania

ABSTRACTBronchiolitis caused by respiratory syncytial virus (RSV) during infancy is associated with the later development of asthma, and RSV is implicated in exacerbations of asthma. We had shown previously that RSV up-regulates the surface protein expression of co-stimulatory molecules (programmed death-1 ligand[PD-L1], PD-L2, inducible co-stimulator ligand [ICOS-L] and B7-H3) on alveolar epithelial cells. Here, we set up to extend these experiments and to investigate
the kinetic of gene expression of these molecules by RSV and the influence of type 1 and type 2 cytokines. Methods. We used quantitative RT-PCR to investigate
PD-L1, PD-L2, ICOS-L and B7-H3 gene expression on A549 cells and regulation of this expression by RSV, IFN-γ and IL-4. Results. The messenger RNA for allthese co-stimulatory molecules is present on A549 cells and is up-regulated by RSV infection in a replication-specific manner. IFN-γ and IL-4 is differentiallymodulating the gene expression of PD-L1 and PD-L2 on A549 cells. Conclusion. A549 cells are constitutively expressing a wide range of B7 family molecules,
even at messenger RNA level. Knowledge of the expression and function of these co-stimulatory molecules can be used to provide insights into mechanisms
of T cell activation and tolerance, with great therapeutic potential for controlling T cell responses.
Key words: B7 co-stimulatory molecules, respiratory syncytial virus, alveolar epithelial cells

INTRODUCTION
The immune system must rapidly respond to diverse microbial pathogens, yet
mount a highly specific response that engenders memory of the particular foreign
invader. As animal host and pathogenic microbes have coevolved, each in response
to other, families of immune system molecules with common structural features
have appeared. The co-stimulatory molecules deliver signals that serve to regulate
the magnitude and duration of adaptive immune responses. There are several
families of co-stimulatory molecules and of the best characterized co-stimulatory
pathway involves the B7:CD28 signalling molecules (20).
The programmed death ligand (PD-L)-PD-1 receptor pathway, which is part ofthe B7:CD28 family, consists of the two ligands PD-L1 and PD-L2 and their common
receptor PD-1. Investigators initially identified the PD-1 receptor by subtractive
hybridization studies using a T cell hybridoma undergoing programmed cell death,hence its name (8). Subsequent studies demonstrated that PD-1 was expressed on
activated T and B cells and was not required for apoptosis (1). PD-Ls expressed on
antigen presenting cells interact with their receptor on T cells and regulate primaryand memory T cell immune responses (5, 9). The PD-Ls–PD-1 pathway is also
important in tolerance and immunopathology (18).
ICOS-L was identified as the third member of the B7 family (2). It is binding
to ICOS and delivers a co-stimulatory signal to T cells (proliferative responses
and cytokine production). ICOS-L mRNA is expressed constitutively in a variety
of lymphoid and nonlymphoid tissues including kidney, liver and peritoneu. The

function of ICOS-L expression in nonlymphoid tissue is yet to be elucidated andfurther studies are required.
One of the latest B7 family members is B7-H3. It is closely related to the
ICOS-L but the identity of its counterligand remains to be determined. Althoughinitial studies showed a stimulator role of T cell responses (3), recent studies haveindicated that B7-H3 has an inhibitory function (21).
The role of co-stimulatory molecules (PD-L1/2, ICOS-L, B7-H3) in acute viral
infections is little studied. Respiratory syncytial virus (RSV) is the major cause of
acute bronchiolitis in infants. RSV infects nearly all infants in their first or second
winter (7), and though impaired Th1 immunity is implicated (12), it is largely
unknown why some infants become seriously ill while the majority have only
mild respiratory illness. Immunity to RSV is incomplete and re-infections occur
throughout life. The mechanisms for the deficient memory immune response to
RSV infection are poorly understood.
Respiratory epithelial cells are the primary site of RSV infection. They express
surface molecules associated with antigen presentation (15), including the PD-Ls,
ICOS-L and B7-H3 (6, 10, 19). We have previously reported co-stimulatory molecules
surface protein expression on respiratory epithelial cell lines and their induction by
RSV infection (19). Here we investigated PD-L1, PD-L2, ICOS-L and B7-H3 gene
expression in human alveolar epithelial cells (A549) and their regulation by RSV
infection and/or type 1 and 2 cytokines.

Received September 2009. Accepted October 2009. Address for correspondence: Dr. Aurica Telcian, Department of Respiratory Medicine, National Heart and Lung Institute, MRC & Asthma UK
Centre in Allergic Mechanisms of Asthma and Centre for Respiratory Infection, Imperial College London, Norfolk Place, London W2 1PG, UK

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Fiziologia - Physiology  2009.19.4(64)MATERIALS AND METHODS
Cell culture and virus infection
A549 cells, an alveolar adenocarcinoma epithelial cell line (European Collectionof Animal and Cell Cultures) were cultured and infected with RSV as previously
described (19). RSV A2 strain (gift from Prof. PJ Openshaw, Imperial College London,UK) was grown in Hep-2 cells and virus titre determined by plaque assay.
RNA extraction, reverse transcription, and quantitative PCR
Total RNA was extracted, and 1 µg was used for cDNA synthesis (Qiagenkits). Quantitative PCR was performed using specific primers and probes for PD-L1(forward primer 5’-GAA TTG GTC ATC CCA GAA CTA CCT-3’, reverse primer 5’-GCATAA TAA GAT GGC TCC CAG AAT-3’, probe FAM 5’-TGG CAC ATC CTC CAA ATG AAA
GGA CTC A-3’TAMRA), PD-L2 (forward primer 5’- GCT GTG GCA AGT CCT CAT ATC
A-3’, reverse primer 5’-GCT GCA ATT CCA GGC TCA AC-3’, probe FAM 5’-ATA CAG
AAC ATG ATC TTC CTC CTG CTA A-3’TAMRA), ICOS-L (forward primer 5’-CAT TGG CTG
CTG CAT AGA GAA C-3’, reverse primer 5’-AGA CAG GAA ATG ACA TCG GAG AG-3’,
probe FAM 5’-TCT GCA GCA GAA CCT GAC TGT CG-3’ TAMRA), B7-H3 (forward
primer 5’-AGC TGT GAG GAG GAG AAT GCA-3’, reverse primer 5’-CCA TCA TCT TCT
TTG CTG TCA GA-3’, probe FAM 5’-CAG CCC TGC AGC CTC TGA AAC A-3’TAMRA) and
18S rRNA (4). Data were analysed using version 1.4 ABI Prism 7500 SDS software,normalized to 18S rRNA and presented as 45-δCT.
Statistical analysis
The results were analysed using GraphPad Prism version 4.00 (GraphPad
Software, California). Results of at least three separate experiments were expressed
as means±standard errors of the means. When analysing multiple groups, a one way
ANOVA of all pairs and columns was used, followed if significant by paired t test for
paired comparisons. P values < 0.05 were considered statistically significant.

RESULTS AND DISCUSSION
PD-L1 mRNA

45-dCT

21

**

19

**

**

**

*

**

media
UV-RSV
RSV

17
15

PD-L2 mRNA

B 24
45-dCT

A 23

Up-regulation of B7 family molecule gene expression on RSV-infected A549
cells
We previously reported constitutive expression of PD-L1, PD-L2, ICOS-L andB7-H3 surface protein on A549 cells (19). To determine how their gene expression is regulated by RSV infection, time-course experiments were set up andco-stimulatory molecule expression was determined by quantitative RT-PCR(Taqman) (Fig. 1).
A549 cells were cultured up to 72 hours with RSV or UV-inactivated RSV (inorder to determine the role of virus replication), harvested at subsequent time-points
and Taqman was performed. PD-L1 gene expression was quickly up-regulated by
RSV infection, as early as 2 hours post virus infection then remained high, with nobig variations, up to 72 hours (Fig. 1A). PD-L2 gene expression was up-regulated by
RSV infection with a certain latency compared to PD-L1, but the expression showeda continuous increase up to 72 hours (Fig. 1B). ICOS-L gene expression was also
up-regulated by RSV infection at a later time-point and its expression continued
to increase up to 72 hours (Fig. 1C), however, B7-H3 mRNA was only slightly upregulated by RSV infection relative to media treated cells and its expression seemed
to be biphasic, with a peak at 8 hours and another one at 48 hours (Fig. 1D).
For all four molecules, the capacity of RSV to amplify their gene expression was
virus replication specific, as UV-inactivated RSV had no/small, transiently effect on
the gene expression of these molecules on A549 cells (Fig. 1A-D).
On A549 cells, Qian et al (16) also reported the presence of ICOS-L mRNAand the influence of different stimuli like TNF-α, LPS, IFN-γ or IL-4 on ICOS-L
gene expression but not in a quantitative manner; here, we report ICOS-L gene
expression by using quantitative RT-PCR, and its modulation in the context ofvirus infection (RSV).
Effects of IFN-γ and IL-4 on RSV induced PD-L1 and PD-L2 gene expression
on A549 cells
Differential regulation of PD-L1 / PD-L2 by type 1 (IFN-γ) and type 2 (IL-4)
cytokines has been previously demonstrated in different cell types (13). In our

18

*

**

**

8

24

media
UV-RSV
RSV

**

12

13
0

2

4

24

Time (hours)

48

*

20

*

*

**

18
16
14

6

72

ICOS-L mRNA

C 22
45-dCT

8

media
UV-RSV
RSV

0

2

4

Time (hours)

48

72

B7-H3 mRNA

D 25

media
UV-RSV
RSV*

24

45-dCT

11

23
22
21

0

2

4

8

24

Time (hours)

48

72

20

0

2

4

8

24

Time (hours)

48

72

Fig. 1. Respiratory syncytial virus (RSV)-induced PD-L1, PD-L2, ICOS-L and B7-H3 gene expression on A549 cells. Cells cultured in media only (white squares) or infected with RSV (MOI 1, black squares) or
UV-inactivated RSV (grey squares) were harvested at the subsequent time-points and PD-L1, PD-L2, ICOS-L and B7-H3 gene expression was measured by quantitative RT-PCR. Shown are the time courses of RSV inductionof PD-L1 [A], PD-L2 [B], ICOS-L [C] and B7-H3 [D]. Results are expressed as 45-δCT; data are means±SEs of 3-5 experiments; *p<0.05, **p<0.01 (RSV is compared to cells treated with media).

2009.19.4(64)  Fiziologia - Physiology

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5

study, un-infected or RSV-infected A549 cells were cultured with IFN-γ (50ng/mL) or IL-4 (10ng/mL) and mRNA expression for PD-L1 and PD-L2 molecules was*

45-dCT

22
18

*

*

**
*
*** **

**
***

**
**

*
**
*

*
***
**

media
IFN-g
RSV
RSV+IFN-g

20

245-dCT

4

824Time (hours)

48

11

72

PD-L2 mRNA

*

*

18

12

*
*
*** ** **
*
*

*
**
***

*
***

media
IFN-g
RSV
RSV+IFN- g

0

2

*

**

8

24

**

media
IL-4
RSV
RSV+IL-4

4Time (hours)

21

2

4

8

24

Time (hours)

48

72

6

*

*

16

*

11

0

48

72

PD-L2 mRNA

D 26
45-dCT

0

C 24

6

17

***

**

14

14
10

PD-L1 mRNA

B 23
45-dCT

PD-L1 mRNA

A 26

different settings is important since they can mediate diametric outcomes on T
cell mediated immune responses. Therefore reports about co-stimulatory molecule

0

2

4

*

*
*

*

***

*

*

8

24

48

Time (hours)

*
***

media
IL-4
RSV
RSV+IL-4

72

Fig. 2. Regulation by interferon (IFN)-γ and interleukin (IL)-4 of RSV-induced PD-L1 and PD-L2 gene expression on A549 cells. Cells treated with media (white squares) RSV (MOI 1, black squares) with orwithout either IFN-γ (50ng/mL) or IL-4 (10 ng/mL) (light grey squares correspond to cells treated with the cytokine alone and dark grey squares correspond to cells infected with RSV and treated with cytokines) were
harvested at the subsequent time-points and PD-L1 and PD-L2 gene expression was measured by quantitative RT-PCR. Results are expressed as 45-δCT; data are means±SEs of 3-5 experiments; *p<0.05, **p<0.01,***p<0.001 (RSV and IFN-γ or IL-4 alone are compared to cells treated with media, RSV plus IFN-γ or IL-4 is compared to RSV).

determined. IFN-γ treatment by itself up-regulated on A549 cells PD-L1 and PD-L2
mRNA expression (Fig. 2A and C). Also, IFN-γ treatment had a significant effect on
RSV induced PD-L1 and PD-L2 mRNA expression (Fig. 2A and C). IL-4 treatment
of A549 cells increased only the expression of PD-L2 mRNA, but not PD-L1 mRNA
(Fig. 2B and D). Furthermore, IL-4 treatment of A549 cells up-regulated also RSVinduced PD-L2 mRNA expression (Fig. 2D).
Comparing these results with PD-L1 and PD-L2 surface protein expression on
A549 cells and their modulation by IFN-γ and IL-4 (19), it looks like up-regulationof PD-L1 and PD-L2 mRNA by IFN-γ translates quickly into protein and by 24 hours
the surface protein is increased as well, in contrast to IL-4 up-regulation of PD-L2
gene expression which doesn’t lead to an increased surface protein expression, atleast up to 48 hours.
There is increasing evidence that allergic asthma, a chronic airway inflammatory
disease characterized by reversible airway obstruction, hyperreactivity, and lymphocyte/eosinophil recruitment, is driven and maintained by chronically activatedT cells with a Th2 phenotype. These T cells promote the activation and recruitmentof B cells and eosinophils and regulate the Ig class switch to the development of
antigen-specific IgE responses (14, 17). Although the significance of B7 molecules
expression on respiratory epithelial cells is not fully established, it has been hypothesized that PD-L1, PD-L2, ICOS-L and B7-H3 expression on airway epithelial
cells can productively interact with their ligands on T lymphocytes and may play
an important role in maintaining or regulating the activation of lymphocytes thathave migrated into the respiratory tract, expanding the mechanisms that underlie
T cell activation in airway inflammation.
Before developing immunotherapeutic reagents targeting these molecules,
dissecting the function of each of the B7 family members in different cells and6

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gene or protein expression from different groups on different respiratory epithelial
cells (6, 10, 11, 14, 16, 19) are more than welcome in the process of clarifying thefunction of these molecules.
In conclusion, we showed here that PD-L1, PD-L2, ICOS-L and B7-H3 gene
expression is up-regulated by RSV infection. RSV-induced gene-expression of
PD-L1 and PD-L2 is differentially regulated by IFN-γ and IL-4. Knowledge of theexpression and function of these co-stimulatory molecules can be used to provide
insights into mechanisms of T cell activation and tolerance, with great therapeuticpotential for controlling T cell responses.
REFERENCES
1. Agata Y, Kawasaki A, Nishimura H, et al: Expression of the PD-1
antigen on the surface of stimulated mouse T and B lymphocytes.
Int Immunol 1996; 8: 765-772
2. Aicher A, Hayden-Ledbetter M, Brady WA, et al: Characterizationof human inducible costimulator ligand expression and function.
J Immunol 2000; 164: 4689-4696
3. Chapoval AI, Ni J, Lau JS, et al: B7-H3: a costimulatory moleculefor T cell activation and IFN-gamma production. Nat Immunol
2001; 2: 269-274
4. Edwards MR, Johnson MW, and Johnston SL: Combination
therapy: Synergistic suppression of virus-induced chemokines inairway epithelial cells. Am J Respir Cell Mol Biol 2006; 34: 616-624
5. Greenwald RJ, Freeman GJ, and Sharpe AH: The B7 family revisited. Annu Rev Immunol 2005; 23: 515-548
6. Heinecke L, Proud D, Sanders S, et al: Induction of B7-H1 and
B7-DC expression on airway epithelial cells by the Toll-like receptor 3 agonist double-stranded RNA and human rhinovirus infection: In vivo and in vitro studies. J Allergy Clin Immunol 2008; 121:
1155-1160

Fiziologia - Physiology  2009.19.4(64)7. Hull J, Thomson A, and Kwiatkowski D: Association of respiratorysyncytial virus bronchiolitis with the interleukin 8 gene region in
UK families. Thorax 2000; 55: 1023-1027
8. Ishida Y, Agata Y, Shibahara K, et al: Induced expression of PD-1,
a novel member of the immunoglobulin gene superfamily, upon
programmed cell death. EMBO J 1992; 11: 3887-3895
9. Keir ME, Francisco LM, and Sharpe AH: PD-1 and its ligands inT-cell immunity. Curr Opin Immunol 2007; 19: 309-314
10. Kim J, Myers AC, Chen L, et al: Constitutive and inducible expression of b7 family of ligands by human airway epithelial cells.
Am J Respir Cell Mol Biol 2005; 33: 280-289
11. Kurosawa S, Myers AC, Chen L, et al: Expression of the costimulatory molecule B7-H2 (inducible costimulator ligand) by human
airway epithelial cells. Am J Respir Cell Mol Biol 2003; 28: 563-573
12. Legg JP, Hussain IR, Warner JA, et al: Type 1 and type 2 cytokine
imbalance in acute respiratory syncytial virus bronchiolitis. Am J
Respir Crit Care Med 2003; 168: 633-639
13. Loke P and Allison JP: PD-L1 and PD-L2 are differentiallyregulated by Th1 and Th2 cells. Proc Natl Acad Sci U S A 2003; 100:
5336-5341
14. Oei E, Kalb T, Beuria P, et al: Accessory cell function of airway
epithelial cells. Am J Physiol Lung Cell Mol Physiol 2004; 287: L318331
15. Papi A, Stanciu LA, Papadopoulos NG, et al: Rhinovirus infection

induces major histocompatibility complex class I and costimulatory molecule upregulation on respiratory epithelial cells. J Infect
Dis 2000; 181: 1780-1784
16. Qian X, Agematsu K, Freeman GJ, et al: The ICOS-ligand B7-H2,
expressed on human type II alveolar epithelial cells, plays a rolein the pulmonary host defense system. Eur J Immunol 2006; 36:906-918
17. Romagnani S: The role of lymphocytes in allergic disease. J
Allergy Clin Immunol 2000; 105: 399-408
18. Sharpe AH, Wherry EJ, Ahmed R, et al: The function of programmed cell death 1 and its ligands in regulating autoimmunity
and infection. Nat Immunol 2007; 8: 239-245
19. Stanciu LA, Bellettato CM, Laza-Stanca V, et al: Expression ofprogrammed death-1 ligand (PD-L) 1, PD-L2, B7-H3, and inducible
costimulator ligand on human respiratory tract epithelial cells andregulation by respiratory syncytial virus and type 1 and 2 cytokines.
J Infect Dis 2006; 193: 404-412
20. Subudhi SK, Alegre ML, and Fu YX: The balance of immune
responses: costimulation verse coinhibition. J Mol Med 2005; 83:
193-202
21. Suh WK, Gajewska BU, Okada H, et al: The B7 family member
B7-H3 preferentially down-regulates T helper type 1-mediatedimmune responses. Nat Immunol 2003; 4: 899-906

Expresia genelor pentru Programmed Death Ligand(PD-L)-1, PD-L2, Inducible Costimulator Ligand si B7-H3 incelulele epiteliale alveolare umane si modularea lor
de catre virusul sincitial respirator si tipul 1 sau 2 decitokine
REZUMAT

Virusul respirator sincitial (VRS) cauzeaza bronsiolite in copilarie iar acestea au fost asociate cu dezvoltarea ulterioara a bolii astmatice; in plus, VRS este
implicat in exacerbarile astmatice. Este publicat deja modul in care infectia cu VRS amplifica expresia moleculelor co-stimulatori (programmed death-1 ligand[PD-L1], PD-L2, inducible costimulator ligand [ICOSL] si B7-H3) ca proteine de suprafata pe celulele epiteliale alveolare. Obiectivul acestui articol a fost sa
investigam expresia genica a aceste molecule co-stimulatori si influenta VRS si tipul 1 sau 2 de citokine. Metode. qRT-PCR (quantitative reverse-transcription
polymerase chain reaction) a fost utilizat pentru a investiga expresia genelor pentru PD-L1, PD-L2, ICOS-L si B7-H3 in celulele alveolare A549 si modul in caresunt reglate de catre VRS, IFNγ sau IL-4. Rezultate. Pentru toate aceste molecule ARN-ul mesager este exprimat in celulele A549 in conditii bazale iar nivelul
lui este crescut in urma infectiei cu VRS intr-un mod dependent de replicarea virala. IFN-γ sau IL-4 moduleaza in mod diferit expresia genica pentru PD-L1 sau
PD-L2 in celulele A549. Concluzii. Celulele A549 exprima in mod constitutiv aceste molecule din familia B7 chiar si la nivelul ARN-ului mesager. Cunoasterea
expresiei si functiei acestor molecule co-stimulatori este utila pentru descifrarea mecanismelor in care limfocitele T sunt activate, avand potential terapeutic
pentru controlul functiilor limfocitelor T.
Cuvinte cheie: molecule co-stimulatoare B7, virusul respirator sincitial, celule epiteliale alveolare

2009.19.4(64)  Fiziologia - Physiology

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7

ASSESMENT OF INTERLEUKIN-1β AND INTERLEUKIN-8
LEVELS IN THE GINGIVAL FLUID OF ORTHODONTIC
TRACTIONED TEETH
OLTEANU C.1, MURESAN A.2, CRACIUN A.3, SERBANESCU A.1, OLTEANU I.3,
KEULARTS MLW IRENE4
1
Department of Orthodontics, „Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca
2
Department of Physiology, „Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca
3
Department of Biochemistry, „Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca
4
Department of Clinical Genetics, Maastricht University, Holland

ABSTRACT
Dental displacement, an obligatory state for a good result of an orthodontic treatment, is obtained by bone resorption and apposition, resulting from an
inflammatory process localized at this level. Lymphocytes produce a large number of peptides with regulatory functions upon the organism in autocrine,
paracrine or at high distance (endocrine) ways. These peptides were described as cytokines. Among these, interleukin 1β and interleukin 8 are the main proinflammatory cytokines, and they can be use as inflammation markers.
In these conditions, the objective of this paper is the assessment of the IL-1β and IL-8 levels in the gingival crevicular fluid of the orthodontic tractioned teeth
compared to the teeth upon which no orthodontic force is applied, before as well as after the device application.
The patients taken into this study were 12 girls and 6 boys, between 7 and 12 years, average age of 9.3 years, who exhibited an orthodontic tooth movementof a single tooth (experimental tooth), the homonym tooth from the antagonistic arcade being considered as control. The gingival fluid was sampled before
the beginning of the treatment, and after 1 hour, 24 hours and 7 days from the device application. The levels of IL-1β and IL-8 in the gingival crevicular fluid
was assessed by ELISA technique.
The level of the two pro-inflammatory interleukins in the gingival fluid of the tractioned teeth was significantly higher (p<0,01) at 24 hours from the device
application compared with the control teeth; at 7 days from the beginning of the treatment, only the IL-8 level in the experimental teeth being significantly
higher compared with the control teeth.
These results demonstrate that the levels of IL-1β and IL-8 in the gingival fluid increase with the dental movement taking place, due to a localized inflammatory process, as a response to mechanical stress.
Key words: interleukin 1β, interleukin 8, gingival fluid, inflammation, orthodontic tractioned teeth

Introduction
Lymphocytes produce a large number of peptides with regulatory functions
upon the organism in autocrine, paracrine or at high distance (endocrine) ways.
These peptides were described as cytokines.
Cytokines were first classified in lymphokines, interleukins and chemokines,
classification based on their functions, cells involved in their production, and thetarget for their action. Nowadays the general term of interleukins is used, without
other distinctions, because of the pronounced pleiotropism of cytokines.
Until now 35 interleukins (IL) are known, numbered from IL-1 to IL-35 (1).
Among these, the pro-inflammatory cytokines are represented by IL-1β, IL-2, IL-6,
IL-8, and IL-31.
Interleukin 1β has 153 amino acid residues, and in vivo is mainly produced bymonocytes/macrophages. It accomplishes a wide spectrum of stimulatory actions
upon the immune cells and upon some cells out of the immune system.
Interleukin 1β is considered as the major mediator of an inflammatory answer(4). It produces immediate, local, inflammatory reactions (edema), and systemic
answers as fever or the acute phase response of the liver.

Interleukin 8 has been discovered in 1987 and represents one of the mostimportant mediators of the defensive inflammatory answers (1). Is produced by avery large diversity of cells (monocytes, macrophages, epithelial cells, fibroblasts),
hereby any tissue in organism is able to produce this cytokine. IL-1β and TNF α
are the major stimuli for IL-8 releasing. IL-8 is released in infections, ischemia, ortraumatic injuries associated with high levels of IL-1β and TNF α. The target cells
for IL-8 are the neutrophile leucocytes.
Utilization of the orthodontic appliances in the treatment of various dentomaxillar anomalies most frequently suppose the application of some forces ofincreased intensity, non-physiological, which always will determine an inflammatory answer localized around the tooth or the teeth which are to be displaced.
A low-intensity inflammatory answer arisen around the orthodontic displaced
tooth is physiologic, simulating the activation of osteoclasts, the apparition of thephysiological process of bone remodeling and implicit the tooth displacement byresorption and bone apposition. On the other hand, a high-intensity inflammatory
answer could produce the apparition of some pathological phenomena, either localized around the displaced tooth, or generalized at the level of the all oral cavity.
The biologic active substances, such as cytokines and enzymes, are released

Received September 2009. Accepted October 2009. Address for correspondence: Dr. Cristi Olteanu, Department of Orthodontics, „Iuliu Hatieganu” University of Medicine and Pharmacy, ClujNapoca, e-mail: cristidolteanu@yahoo.com

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Fiziologia - Physiology  2009.19.4(64)by cells in periodontal tissue as an answer to the mechanical stress produced by
the orthodontic appliances (3, 9). The purposes of many researches were to better
understand the way by which the mechanical stress produced by orthodontic devices
determine an answer from the periodontal cells. A non-invasive modality to asses
the level of these active substances is the determination of their concentration in
the gingival fluid (6, 7).
A series of studies have proven changes in the interleukine-1β and IL-8
concentrations in the gingival fluid of the teeth upon which orthodontic forces
are exerted (7, 2, 12).
Thus, the objective of this paper is the assessment of the IL-1β and IL-8 levelsin the gingival crevicular fluid of the orthodontic tractioned teeth compared to theteeth upon which no orthodontic force is applied, before as well as after 1 hour, 24hours, and 7 days after the device application.

SUBJECTS AND METHODS
The written consent of the parents of the child patients taken into this study
was obtained after a previous description of the study’s protocol. In order to asses
the interleukin levels, 18 patients were taken into study, in which 288 measurements
were performed. The 18 patients, 12 girls and 6 boys (between 7 and 12 years, average age of 9.3 years), displayed the following criteria for including in study:•
Well general status, without known affections;•
Lack of antibiotic therapy within the last 6 months;•
Absence of anti-inflammatory drugs administration in the month
before the study;•
Healthy periodontal tissues and appropriate oral hygiene.
In each patient a brief instruction for teeth brushing and oral hygiene wasmade before the beginning of the orthodontic treatment. The tooth on which theorthodontic force has been applied was considered as experimental tooth, andthe homonym tooth on the antagonistic arcade was considered control tooth (i.e.
superior left canine/inferior left canine). The homonym teeth from the antagonistic
arcade were not exposed to any orthodontic force.
The orthodontic treatment in the case of all the 18 patients has been achieved
using biomechanical appliances. The level of the dental displacement during the
7 days of the study has been measured on the working models, after taking theimpressions.
Samples of gingival crevicular fluid (for the experimental tooth, and control
tooth respectively) have been collected after the following time intervals:•
Moment 0 – before the initiation of the orthodontic treatment;•
Moment 1 – at 1 hour from the device  application;•
Moment 2 – at 24 hours from the device application;•
Moment 3 – at 7 days from the device application.
The sampling of the gingival fluid was made as follows: the teeth were gently
washed with a water spray, then isolated with cotton bullets, and finally gently
dried with the air spray. Paper cones were used for sampling (Absorbent paper
points – Meta). A first cone was introduced in the gingival crevice, maintained herea minute, then a second cone was introduced in the same place. Cones contaminatedwith blood or saliva have been discarded.
The paper cones were introduced in Eppendorf tubes and kept at -80°C until
the measurements were performed. Before the experimental study was achieved,
over the cones 200 µl physiologic serum was added, then a centrifugation at 3600
rpm for 10 minutes was performed. The resulted supernatant was utilized for thedetermination of interleukin and of total protein concentrations.
The levels of IL-1β and IL-8 in the gingival crevicular fluid was assessed by
ELISA technique (Quantikine, R&D Systems Inc., Minneapolis, Minnesota, USA),2009.19.4(64)  Fiziologia - Physiology

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the results being exprimed as pg/µg total proteins in the gingival fluid. The level of
total proteins was assessed by Bradford method, using as a standard a BSA (Bovine
Serum Albumine) solution.
Concerning the statistical analysis, in order to compare the mean values
Student t-test was utilised, because the values have displaied a normal distribution.
The correlations between variables were determined with the Spearman test. The
statistical analysis was accomplished with the Statistical Package for Social Sciences(SPSS), and with the statistical software of Microsoft Office.

RESULTS
The average level of the dental displacements was of 1.5±0.3 mm, after the 7days of the study. No dental displacement was observed for the control teeth at any
subjects involved in this study. The mean concentration of interleukins 1β and 8 in
the gingival crevicular fluid of the experimental and control teeth for the patients
involved in study are emphasized in table I.
Table I. The mean concentration of interleukins 1β and 8 in the gingival crevicular fluidof the experimental and control teeth of the patients involved in the study (pg/µg total protein)

IL-1ȕ
IL-8

ZERO
Control
Experim.
0.216
0.223
114.9
114.3

MOMENT
1 HOUR
24 HOURS
Control
Experim.
Control
Experim.
0.222
0.267
0.219
0.571
112.6
118.4
116.7
350.7

7 DAYS
Control
Experim.
0.214
0.221
114.9
328.8

The variations of the average values of IL 1β and IL 8 at the time intervals considered are presented in the Figures 1 and 2.
0,6
0,5
0,4

Control

0,3Experimental

0,2
0,1
0

Zero

1 hour

24 hours

7 day sFig. 1. Modifications of IL-1β concentration in the gingival liquid during
the orthodontic treatment (pg/µg)
400
350
300
250
200

Control

150

Experimental

100
50
0

Zero

1 hour

24 hours

7 daysFig. 2. Modifications of IL-8 concentration in the gingival liquid during
the orthodontic treatment (pg/µg)

At moment 0 (before application of the device), the IL-1β level for the experimental tooth, and control respectively was having close values (0.216 pg/µg, and9

0.223 pg/µg respectively), the difference being statistically insignificant (p>0.5).
The same statistically insignificant difference (p>0.5) was also recorded at moment
0 for IL-8 (114.3 pg/µg in case teeth, 114.9 pg/µg in control teeth).
At 1 hour after the beginning of the force application, the values of the twointerleukins in the gingival fluid of the moved teeth (experimental) have slightly
increased levels, but statistically insignificant (1,2 times higher for IL-1β, and 1,04
times higher for IL-8). The concentrations of the two interleukins in control teeth
range in the same limits as in moment 0.
At 24 hours from the orthodontic device application, a pronounced increase,statistically significant (p<0.01), of the two interleukins levels has been observedin the experimental teeth (2.57 times for IL-1β, and 3.07 times for IL-8), while theselevels are keeping relative constant in the control teeth. In control teeth no modifications are noticed in the interleukins’ concentration as compared to moment 0.
At 7 days since the treatment has begun, the behavior of the two interleukins
diversifies:•
IL-1β levels returns to the initial values (0.221 pg/µg as comparedwith 0.223 pg/µg at moment 0)•
Il-8 level keeps increased (328.8 pg/µg as compared to 114.3 pg/µg at moment 0), but decreases as compared to the maximal level reached at 24hours – 350.7 pg/µg – the decreasing being statistically insignificant.
Another result of this study was the achievement of a statistically significant
correlation between the levels of the two interleukins before the orthodontic device
application and at 24 hours from the action of the orthodontic force.

inflammatory process.
At 24 hours from the orthodontic device application, in experimental
teeth a marked increase of the concentration of the two interleukins can be observed(2.57 times for IL-1β, and 3.07 times for IL-8), and which is statistically significant(p<0.01). A series of other studies have proved similar results, such as the reachingof a maximal concentration of the pro-inflammatory interleukins at 24 hours fromthe moment when the force was first applied (2,7,12,13).
Because the pro-inflammatory interleukins are markers of inflammation, thestatistically significant increase of their level in the gingival fluid of the orthodontic
tractioned tooth at 24 hours from the debut of the treatment denotes the initiationof a localized inflammatory process, inflammation strictly requested for dental
displacements. The pain sensation at 24-48 hours after the moment of orthodontic
device application is a clinical sign of this process.
At 7 days from the treatment beginning, the behavior of the two interleukins
is different:•
The level of IL-1β returns to the initial values (0.221 pg/µg as compared
to 0.223 pg/µg at moment 0)•
The level of IL-8 maintains increased (328.8 pg/µg as compared to114.3 pg/µg at moment 0), but decreases as compared to the maximal level reachedat 24 hours – 350.7 pg/µg – a decrease which is statistically insignificant.
The faster return to the initial values of IL-1β as compared to those of IL-8 could
be explained by the fact that IL-1β is essential for IL-8 induction, probable via an
autocrine, or paracrine mechanism (Maeda et al., 2007) (8).
Because in this study we used biomechanical orthodontic devices, and whichwere no activated in this period of time, the returns of IL-1β concentration to theinitial values after 7 days denotes the absence of the inflammatory process due tothe cessation of the bone remodeling process. These phenomena will restart after
a new activation of the device.

CONCLUSIONS

Fig. 3. Correlation between the IL-1β and IL-8 levels in the experimental teeth
before the beginning of the treatment

DISCUSSION
In this paper we proved the existence of an IL-8 level approximately 500
times higher as compared to those of the IL-1β in the gingival liquid at moment
0. These values reported in our study are in correspondence with the data inliterature (5,11).
The effect of application of a force upon the periodontal ligaments is rapid.
Thus, the cells localized at this level respond by releasing of modulators after several
minutes since the force was applied, as proved by Storey (10) on incisors guinea
pigs. In this study, the two interleukins have a slightly increased level at 1 hour fromthe beginning of the force application, as compared to moment 0, confirming theliterature data. Because of the application of the orthodontic force, an inflammatory
process of reduced intensity arises at 1 hour from the device application. This will
stimulate the resorption, and bone apposition processes necessary for the dental
displacements.
The fact that the IL-1β level has increased faster than the IL-8 level denotesthat IL-1β is the first of those taken into study to indicate the initiation of an
10

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1. The application of an orthodontic appliance is followed by an inflammatory
process localized strictly around the tooth or the teeth orthodontic tractioned.
2. The localized inflammatory process secondary arisen to the action of theorthodontic force is necessary for the tooth displacement, in order to ensure thebone remodeling by resorption and apposition.
3. The inflammatory process, localized around the tooth upon which theorthodontic force operates, is manifested at 1 hour since the force application, ismaxim at 24 hours, then at 7 days reduces near to disappearance.
4. These phenomena will repeat at a new activation of the orthodontic
device.
5. The studied markers, which demonstrate the existence of this inflammatory
process and to whose variation is statistically and clinically significant, are IL-1β, andIL-8, sampled from the gingival fluid of the orthodontic tractioned tooth.
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Dentofac Orthop. Jul 2006; 130(1): 71-6
3. Davidovitch Z, Nicolay OR, Ngan PW, Shanfeld JL. Neurotransmitters, cytokines, and the control of alveolar bone remodeling inorthodontics. Dent Clin North Am. 1988; 32: 411-35
4. Dinarello CA. Interleukin-1 and its biologically related cytokines.
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Fiziologia - Physiology  2009.19.4(64)5. Giannopoulou C, Mombelli A, Tsinidou K, Vasdekis V, Kamma
J. Detection of gingival crevicular fluid cytokines in children andadolescents with and without fixed orthodontic appliances. Acta
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6. Griffiths GS, Moulson AM, Petrie A, James IT. Evaluation of osteocalcin and pyridinium crosslinks of bone collagen as markers
of bone turnover in gingival crevicular fluid during different stagesof orthodontic treatment. J Clin Period. 1998; 25: 492-98
7. Iwasaki LR, Haack JE, Nickel JC, Reinhardt RA, Petro TM. Humaninterleukin-1 beta and interleukin-1 receptor antagonist secretionand velocity of tooth movement. Arch Oral Biol. 2001; 46: 185-89
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9. Saito M, Saito S, Ngan PW, Shanfeld JL, Davidovitch Z. Interleukin1β and prostaglandin E are involved in the response of periodontal

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28(3): 241-46

DETERMINAREA NIVELULUI INTERLEUKINEI-1β ŞI INTERLEUKINEI-8
ÎN LICHIDUL GINGIVAL AL DINŢILOR TRACŢIONAŢI ORTODONTICREZUMAT
Deplasarea dentară, condiţie obligatorie pentru reuşita unui tratament ortodontic, se realizează prin procese de resorbţie şi apoziţie osoasă,apărute secundar existenţei unui proces inflamator localizat la acest nivel. Limfocitele produc numeroase peptide care în mod autocrin, paracrin
sau la distanţă mare (endocrin) exercită funcţii reglatoare asupra organismului. Aceste peptide au fost descrise ca citokine. Dintre acestea,interleukina-1β şi interleukina-8 reprezintă principalele citokine cu rol proinflamator, putând fi astfel utilizate ca markeri ai inflamaţiei.
În aceste condiţii, obiectivul lucrării îl constituie determinarea nivelului IL-1β şi IL-8 în lichidul gingival crevicular al dinţilor tracţionaţi ortodontic,
comparativ cu dinţii asupra cărora nu se exercită nicio forţă ortodontică, înainte şi după aplicarea aparatului.
Pacienţii cuprinşi în studiu au fost 12 fete şi 6 băieţi, cu vârsta medie 9,3 ani, care au prezentat o deplasare în scop ortodontic a unui singur
dinte (dintele caz), dintele omonim de pe hemiarcada antagonistă fiind considerat dinte martor. Lichidul gingival a fost recoltat atât de ladintele caz, cât şi de la dintele martor, înainte de debutul tratamentului, la 1 oră, 24 ore şi 7 zile de la debutul deplasării dentare. Nivelul
interleukinelor în lichidul gingival a fost determinat utilizând un kit ELISA.
Concentraţia celor două interleukine în lichidul gingival al dinţilor tracţionaţi ortodontic a fost semnificativ crescută (p<0,01) la 24 ore dela aplicarea forţei comparativ cu dinţii martor, la 7 zile de la debutul tratamentului ortodontic, doar concentraţia interleukinei-8 la dinţii caz
fiind semnificativ crescută comparativ cu dinţii martor (p<0,01).
Aceste rezultate demonstrează că nivelul IL-1β şi IL-8 în lichidul gingival creşte odată cu desfăşurarea deplasărilor dentare, creştere datorată
apariţiei unui proces inflamator localizat, ca răspuns la existenţa unui stres mecanic.
Key words: interleukina 1β, interleukina 8, fluid gingival, inflamaţie, tracţionare ortodontică

2009.19.4(64)  Fiziologia - Physiology

cmyk

11

RESISTANCE TO BETA-LACTAM ANTIBIOTICS OFESCHERICHIA COLI STRAINS ISOLATED FROMUROCULTURESDELIA BERCEANU VADUVA, MONICA LICKER, DELIA MUNTEAN, DANA VELIMIROVICI,
MATILDA RADULESCU, DUGAESESCU DORINA, CARMEN TATU, MARCELA ADAMUT,
STANGA LIVIA, MARIA RADA, M. BERCEANU VADUVA, ROXANA MOLDOVAN
University of Medicine and Pharmacy”Victor Babeş”Timişoara

Abstract
Purpose: Considering the high incidence of urinary infections we made a study in order to determine the antibiotic susceptibility to beta-lactams of Escherichia
coli (E. coli) strains isolated from urocultures of patients admitted in the urology department, as well as from ambulatory patients.
Material and methods: We studied 526 strains of E. coli (350 from ambulatory patients and 176 from the urology department).
The germs were identified by the API method (BioMerieux) and the antibiotic susceptibility was performed by disk-diffusion Kirby-Bauer tests. Statistic analysisof the antibiograms and the determination of the resistance phenotypes were performed with automatic reading methods (Osiris – Bio Rad Laboratories).
For identifying ESBL (beta-lactamase with extended spectrum) producing strains, the sinergy tests were also performed.
Results: The wild phenotype was present in 49.15% of the strains isolated from ambulatory patients, and only in 17.04% in patients admitted in the urology
department. In ambulatory patients, as well as in patients admitted in the urology department, most of the strains were penicilinase-producing (46.85%,
respectively 57.95%).
Conclusions: We observed a constant lowering of the sensitivity of E. coli strains to beta-lactams, especially in the urology department, which draws attention
to the importance of monitoring antibiotics prescriptions.
Keywords: Escherichia coli, urocultures, resistance phenotype

Purpose350 were E. coli;•from 1871 urocultures obtained from urology sections, 462 were
Considering the high incidence of urinary infections, in order to prevent
positive
and
only
176
were E. coliselection of multiresistant bacterial strains and to choose the bestTable
drug therapy,
I and Figure 1 show the distribution of urocultures in the urology depart
Table
I
and
Figure1 show the distribution of urocultures in the urology
we made a study to determine the antibiotic susceptibilityambulatory
to beta-lactam patients.
of E. coli
department
andambulatory
patients.
strains isolated from urocultures of patients admitted in the urology department,
and from ambulatory patients, as well as to establish the resistance phenotype of Table nr. I: Distribution of urocultures in the urology department and ambulatory patients
Table nr. I: Distribution of urocultures in the urology department and ambulatory patientsthese strains.

Material and methods
We studied 526 strains of E. coli (350 from ambulatory patients and 176 fromthe urology department).
The germs were identified by the API method (BioMerieux) and the antibiotic
susceptibility was performed by disk-diffusion Kirby-Bauer tests. Statistic analysisof the antibiograms and and the determination of the resistance phenotypes were
performed with automatic reading methods (Osiris - Bio Rad Laboratories).
For identifying ESBL (beta-lactamase with extended spectrum) producing
strains, the sinergy tests were also performed.
In order to test the sensitivity to beta-lactam antibiotics the following drugs
were used: ampicillin (AMP), amoxicillin + clavulanic acid (AMC), ticarcillin (TIC),
cephalothin (CF), cefuroxime (CXM), ceftazidime (CAZ).

Results and discussions

Ambulatory
Urology
Total

Positive urocultures
431
462
893

Sterile urocultures
1458
1409
2867

1600
1400 1600
1409
1458
1400
1409
1458
1200
1200
1000
1000
800
431 462800
431 462600
600
400 400
200 200
00
Positive
SterilePositive
Sterileurocultures

Ambulatory
Ambulatory
Urology

Urologyuroculturesurocultures urocultures

We studied the 526 strains of E. coli isolated from 3760 urocultures:
Fig. 1. Distribution of urocultures in the urology department and ambulatory patients
Fig. 1. Distribution of urocultures in the urology department and ambulatory patients•from 1889 ambulatory urocultures, 431 were positive and only
Tables II and III show the distribution of species isolated from the positive u
Received September 2009. Accepted October 2009. Address for correspondence: Dr. Delia Berceanu, University of Medicine and Pharmacy”Victor Babeş”Timişoara, 2A Eftimie Murgu Square,(ambulatory and urology).
300041, Timisoara, Romania, e-mail: deliaberceanu@yahoo.com

Table II. Distribution of species isolated from the positive urocultures – ambulatory

12

cmyk

Fiziologia - Physiology  2009.19.4(64)Germs
Escherichia coli
Klebsiella pneumoniae
Proteus mirabilisEnterococcus spp.
Pseudomonas aeruginosa

Frequency
350
36
14
10
4

Percent
81.21%
8.35%
3.24%
2.32%
0.93%

600
400
200
0

Urology

Positiveurocultures

Sterileurocultures

Fig. 1. Distribution of urocultures in the urology department and ambulatory patientses II and III show the distribution of species isolated from the positive uroculturesand urology). Tables II and III show the distribution of species isolated from the positive
Ceftazidime
urocultures (ambulatory and urology).
Table II. Distribution of species isolated from the positive urocultures – ambulatory

Cefuroxime

Table II. Distribution of species isolated from the positive urocultures - ambulatory

Germs
Escherichia coli
Klebsiella pneumoniae
Proteus mirabilisEnterococcus spp.
Pseudomonas aeruginosaProteus vulgaris
Enterobacter cloacae
Staphylococcus aureus
coagulase-negative staphylococci
Citrobacter freundii
Streptococcus agalactiae
Morganella morganii
Acinetobacter baumannii
Total

Frequency
350
36
14
10
4
3
3
3
2
2
2
1
1
431

Percent
81.21%
8.35%
3.24%
2.32%
0.93%
0.69%
0.69%
0.69%
0.46%
0.46%
0.46%
0.23%
0.23%
100%

Cephalothin
Ticarcillin

345

05

308 349

Cefuroxime

345

185

0 18
1 24

0

Ampicillin

18 24

311

162

Amoxicillin+clavulanic acid

50
0

50

S

0

23

150

100

165 39 0
165
250

200

150

I

S

39 0

165

23
311

162
100

Ampicillin

05

165

308

185

Ticarcillin

0

0 1

Ceftazidime

Cephalothin

Amoxicillin+clavulanic acid

349

200

250

300
300

350

350

400

400

RR

I

Fig.2. Resistance to beta-lactams antibiotics of E. coli strains - ambulatory

Fig.2. Resistance to beta-lactams antibiotics of E. coli strains - ambulatory

V. Resistanceto beta-lactams
antibiotics
E. colistrains – urology
Table V.Table
Resistanceto beta-lactams
antibiotics
of E. coliofstrains- urology
ANTIBIOTICSENSITIVE
Nr.
%

RESISTANTNr.
%

AMPICILLIN

30
68

17.04
38.63

2

1.13

146
106

82.95
60.22

TICARCILLIN
CEPHALOTIN
CEFUROXIMECEFTAZIDIME

30
30
68
132

17.04
17.04
38.63
75

38
-

21.59
-

146
108
108
44

82.95
61.36
61.36
25

AMOXICILLIN +Table III. Distribution
speciesofisolated
frompositiveurocultures
Table III.ofDistribution
species isolated
from thethe positive
urocultures- urology – urology CLAVULANIC ACID

Resistance to beta-lactams antibiotics of isolated E. coli strains is shown in Tables IV and V, aswell as in Figure 2.

INTERMEDIATE
Nr.
%

Germs
Frequency
Percent
Escherichia coli
176
37.76%
Klebsiella pneumoniae
11925.53%
13244
0Enterococcus faecalis
23
4.93%
Germs
Frequency Percent
Ceftazidime
132 0
44
68
Cefuroxime
0 108
Pseudomonas
aeruginosa
23 37.76%
4.93%
Escherichia coli
176
0
Cefuroxime
68
108
Streptococcus
agalactiae
4.5%
Klebsiellapneumoniae
119 21 25.53%
30
38
108
Enterococcus
faecalis
23 19 4.93%
Staphylococcus
aureus
4.07%
Cephalothin
30
38
108
Pseudomonas aeruginosa
23 18 4.93%
30 0
146
Ticarcillin
Serratia marcescens
3.86%
Ticarcillin
30 0146
Streptococcus agalactiae
21
4.5%
Proteus mirabilis
12
2.57%
Staphylococcus aureus
19
4.07%
Amoxicillin+clavulanic
68
68
2 2
106 106
acid
Enterobactercloacae
2.57%
Serratia
marcescens
18 12 3.86%
Ampicillin 30
30 0
0146 146
Proteusmirabilis
12 12 2.57%
Enterobacter
gergoviae
2.57%
Ampicillin
Enterobacter cloacae
12
2.57%
0
20
40
60
80 100 120 140 160 180 200
Staphylococcus
saprophyticus
9 2.57%
1.93%Enterobacter gergoviae
12
0
50 S
100R
150
200
Acinetobacterbaumannii
5 1.93%
1.07%
I
Staphylococcus
saprophyticus
9
Acinetobacterbaumannii
5
Staphylococcus
haemolyticus
3 1.07%
0.64%
Fig. 3. Resistance to beta-lactams antibiotics
ofS
I E. coli strains
R - urology
Staphylococcus
haemolyticus
3
Stenotrophomnas
maltophilia
2 0.64%0.42%
Stenotrophomnas maltophilia
2
0.42%
CitrobacterCitrobacter
freundiifreundii
2 0.42%0.42%
2The interpretativeFig.
analysisof the antibiograms
the resistance phenotypes
3. Resistanceto beta-lactamsallowed
antibioticsestablishingof E. coli strainsof- urology
CitrobacterCitrobacter
koseri koseri
2 0.42%
0.42% of E. coli strains. Throughthe determination of the resistance phenotypes to antibiotics, information on2
theresistancemechanisms
couldbeobtained.
Based
on
these
mechanisms,
theantibiotics to which theSerratia liquefaciens
2Serratia liquefaciens
2 0.42%0.42%
tested bacteria was resistant could be detected.
Serratia odorifera
1
0.21%
The
interpretative
analysisoftheantibiograms
allowedestablishingof theSerratia odorifera
1 0.21%
0.21%
Klebsiella oxytoca
1
resistancephenotypes
of
E.
coli
strains.
ThroughthedeterminationoftheresistanceKlebsiella oxytoca
1 0.21%
0.21%
Providencia stuartii
1
phenotypes to antibiotics, information on the resistance mechanisms could be
1
ProvidenciaStaphylococcus
stuartii epidermidis
1 0.21%
0.21%
Pseudomonas
fluorescens
1
Staphylococcus
epidermidis
1 0.21%
0.21%
obtained. Based on these mechanisms, the antibiotics to which the tested bacteria
Aeromonas hydrophila
1
0.21%
Pseudomonas
fluorescens1
0.21%
was resistant could be detected.
Total
466
100%
Aeromonas hydrophila
1
0.21%
VI and Figure
showresistancethe resistancephenotypes
to beta-lactams
Table VI Table
and Figure
4 show4 thephenotypes
to beta-lactamsof E. ofcoliE. coli
strains isolated
Total
466 is shown in100%
Resistanceto beta-lactams antibiotics of isolated E. coli strains
Tables IV andV,
asstrains
isolatedfrompatientsfromtheurology
department
andtheambulatory.
from patients from the urology department and the ambulatory.

well as in Figure 2.

Table
VI.VI.
Resistantphenotypesof E.ofcoli
to beta-lactams
Table
Resistantphenotypes
E. coli
to beta-lactams

ance to beta-lactamsTable
antibiotics
isolated
E. colistrains
is shown in Tables IV and V, as
to beta-lactams
beta-lactams
antibiotics
coli
ambulatory
Table IV.
IV. Resistanceof
antibiotics
ofofE.E.coli
strains -- ambulatory
Phenotypeure 2.
ANTIBIOTICSENSITIVE
INTERMEDIATERESISTANT

Ambulatory
Nr.
%
172
49.15

Urology%
Wild phenotype (sensitive
17.04
phenotype)
AMPICILLIN
46.29% antibiotics
23
6.57%of E. 165
47.14% - ambulatory
Table
IV. Resistance to 162
beta-lactams
coli strains
Low PASE
128
36.57
38
21.59
AMOXICILLIN +311
88.86%
39
11.14%High PASE
36
10.28
64
36.36
ANTIBIOTICCLAVULANIC ACIDSENSITIVEINTERMEDIATERESISTANT
CASE
4
1.13
TICARCILLIN
52.86%- Nr. 165
Nr. 185%% 47.14% Nr.
ESBL %
1
0.29
40
22.72
CEPHALOTIN
308
88.00%
18
5.14%
24
6.86%
CHN
4
2.27
MPICILLIN
6.57%
47.14%
CEFUROXIME 162
345 46.29%
98.57%- 23 5
1.43% 165
Legend:PASE–
penicillinase,
CASE–
cephalosporinase,ESBL
beta-lactamasewithextended
99.71%- 39 1
0.69% Legend:
MOXICILLIN + CEFTAZIDIME 311 349 88.86%
11.14%- PASE – penicillinase,
CASE – cephalosporinase, ESBL - beta-lactamase with extended spectrum, CHN - quinolone

Nr.

%

Nr.

%

LAVULANIC ACIDCARCILLIN
185
52.86%EPHALOTIN 2009.19.4(64)308 Fiziologia
88.00%
18- Physiology
EFUROXIME
345
98.57%EFTAZIDIME
349
99.71%
-

cmyk

Nr.

%

Nr.
30

spectrum, CHN - quinolone

5.14%
-

165
24
5
1

47.14% 50
6.86% 45
1.43% 40
0.69% 35

49,15

13
36,57

36,36

Low PASE

30
25
20

Wild phenotype

21,59
17,04

22,72

High PASE
CASE

50

49,15

45
40

36,57

36,36

Wild phenotype

35

Low PASE

30
25

21,59

20

ESBL

10,28

10

CHN

1,13
0,29
0

5

Ambulatory

High PASE
CASE

17,04

15

0

22,72

0

2,27

irrational and long-term treatment with antibiotics led to the selection of multiresistant strains, as well as to the creation of an unbalance of saprophytic organisms
and dissemination of the pathogenic and facultative pathogenic ones.
5. An alarm signal is raised by the presence of multiresistant ESBL E. coli strains,most frequently isolated from the urology department (22.72%), compared to only
0.29% in ambulatory patients.
6. In establishing a correct anti-infectious therapy, the role of the microbiology laboratory is to perform the antibiogram, as well as to establish the resistancephenotypes, thus offering information on the development of the resistancemechanisms and the ways the phenotypes may spread.

UrologyFig. 4. Resistant phenotypes of E. coli to beta-lactams

The wild phenotype was present in 49.15% of the strains isolated from ambulatory patients, and only in 17.04% in patients admitted in the urology department.
In ambulatory patients, as well as in patients admitted in the urology department,
most of the strains were penicilinase-producing (46.85%, respectively 57.95%).
A warning sign is the presence of E. coli strains producing beta-lactamasewith extended spectrum (ESBL), multi-resistant strains that are usually isolated
in hospital environments. This can be explained by the empyrical, irrational andabusive use of antibiotics without an antibiogram.
ESBL strains were predominant in the urology department (22.27%), with
only 0.29% in the ambulatory.

Conclusions
1. We studied 526 strains of E. coli (350 from ambulatory patients and 176
from the urology department) isolated from 3760 urocultures.
2. The purpose of the study was to test the sensitivity to beta-lactams antibiotics of E. coli strains isolated from different ambulatory specimens, as well as
to establish the resistance phenotypes of these strains, in order to choose the best
chemotherapeutic agent and avoid multi-resistant bacterial strains.
3. We observed a constant lowering of the sensitivity of E. coli strains tobeta-lactams, especially in the urology department, which draws attention to theimportance of monitoring antibiotic prescriptions.
4. We consider that the development of resistance to antibiotics of these germs
is due to the antibiotic empyrical treatments, without an antibiogram. The abusive,

REFERENCES
1. Andronescu D, Olteanu D. Pielonephryta. Ed. Medicala, Bucharest, 1996.
2. Angelescu M. Antibiotics therapy. Ed. Medicala, Bucharest,1998.
3. Berceanu Vaduva D et al. Nosocomial infections. Ed. Mirton,
Timisoara, 1999.
4. Buiuc D, Negut M. Clinical microbiology book 2nd edition. Ed.
Medicala, Bucharest, 2008.
5. Gluhovschi G, Dragan I et al. Urinary tract infections in medical
practice. Ed. Helicon, Timisoara, 1992.
6. Greenwood D. Antimicrobial Chemotherapy, Oxford, University
Press, 1989.
7. Jacoby G, Medeiros AA. More extended-spectrum beta-lactamases. J. Antimocrob. Agents Chemother 1991; 35, 1697-1704.
8. Jehl F, Chomarat M, Weber M, Gerard A. From antibiogram toprescription. Ed. Stiintelor Medicale, Bucharest, 2004.
9. Licker M, Moldovan R et al. Special microbiology volume I - Bacteriology. Ed. Eurostampa, Timisoara, 2008.
10. Licker M, Moldovan R et al. Antibiotics resistence, history andactuality. Ed. Eurostampa, Timişoara, 2002.
11. Moldovan R et al. Microbiology – Practical laboratory support,
lito UMFT, 2002.
12. Moldovan R al. Medical microbiology volume II, lito UMFT, 2005.
13. Philippon A, Labia R, Jacoby G. Extended-spectrum betalactamases. J. Antimicrob. Agents Chemother1989; 33: 1131-36.
14. Sitor D et al. Resistance to cefotaxime and seven other beta-lactams in members of the family Enterobacteriaceae: a 3-year survey
in France. Antimicrob. Agents Chemother 1992; 36(8): 1677-81.
15. Sirot J. Resistance enzymatique des bacilles a Gram negatif
aux cephalosporines de 3-eme generation. Med. Mal. Infect1989;
24-30.

REZISTENŢA LA ANTIBIOTICE BETALACTAMICE A UNOR TULPINIDE ESCHERICHIA COLI IZOLATE DIN UROCULTURI
RezumatObiective: Compararea rezistenţei la betalactamine a tulpinilor de Escherichia coli (E. coli) izolate din uroculturi provenite de la pacienţi din ambulator
şi dintr-o secţie de urologie.
Material şi metodă: S-au luat în studiu 526 tulpini de E. coli (350 din ambulator şi 176 din secţia de urologie). Germenii au fost identificaţi cu ajutorul galeriilor
API (BioMerieux), iar testarea sensibilităţii la antibiotice s-a realizat prin metoda difuzimetrică Kirby-Bauer. Prelucrarea statistică a datelor, interpretarea
antibiogramelor şi încadrarea în fenotipuri de rezistenţă a tulpinilor de E. coli izolate s-a realizat cu ajutorul analizorului Osiris (Bio Rad Laboratories). Pentru
evidenţierea tulpinilor producătoare de betalactamaze cu spectru extins (BLSE) s-a utilizat şi testul de sinergie.
Rezultate: Fenotipul sălbatic a fost reprezentat de 49,15% din tulpinile izolate în ambulator şi numai de 17,04% din tulpinile provenite de la urologie.
Atât în ambulator, cât şi în secţia de urologie au predominat tulpinile secretoare de penicilinază, izolate într-un procent de 46,85% şi, respectiv, 57,95%.
Tulpinile BLSE au predominat în secţia de urologie 22,27%, în ambulator fiind izolate în procent de 0,29%.
Concluzii: S-a constatat o scădere continuă a sensibilităţii tulpinilor de E. coli la betalactamine, în special în secţia de urologie, ceea ce atrage atenţia
asupra necesităţii monitorizării prescrierii de antibiotice.
Cuvinte cheie: Escherichia coli, uroculturi, fenotipuri de rezistenţă

14

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Fiziologia - Physiology  2009.19.4(64)PREVALENCE AND ANTIMICROBIAL RESISTANCE OF
ACINETOBACTER BAUMANNII STRAINS ISOLATED FROMTHE CARDIOLOGY DEPARTMENTDORINA DUGAESESCU, MONICA LICKER, DELIA BERCEANU-VADUVA, MIHAELA CRACIUNESCU, LILIANA DRAGOMIRESCU, ELENA HOGEA, CARMEN TATU, DELIA MUNTEAN, MIHAELA POPA, CIPRIAN PILUT, RAMONA PIRVAN, DENIS SERBAN, ROXANA MOLDOVAN
University Of Medicine and Pharmacy “Victor Babes”Timisoara

ABSTRACT

Purpose: The study assessed the degree of microbial load of the air, surfaces, the carrier status of medical staff and patients hospitalized in the Cardiology
Department of Timisoara Municipal Hospital. Research has pursued the isolation and identification of Acinetobacter baumannii strains and their behavior
to antimicrobial agents.
Methods: Isolation was carried out on blood agar 5% and Mac Conkey agar, incubated at 37°C, 24h. Identification of Acinetobacter baumannii strains was
based on morphological, cultural and biochemical (API 20E galleries) characters. Antibiotic susceptibility testing was done by Kirby-Bauer method andautomated Vitek 2 compact system.
Results: Of the 362 samples were positive 51.65%. There were 35 strains of Acinetobacter baumannii from the total of 594 bacterial isolated strains. Thesecame from the different surfaces (65%), aeroflora (26%) and pharyngeal swabs (9%) of 4 hospitalized patients. Concerning their phenotypic patterns, moststrains of A. baumannii were included in penicilinase (PASE) producing phenotype (66%), followed by cephalosporinase producing (CASE) phenotype (20%)
and 5 strains (11%) belonged to the wild phenotype. Aminoglycosides wild type phenotype was 62.8%, followed by gentamycine netylmicin (GN) phenotype(14.3%) and gentamycine G phenotype (11.4%). Only 8.6% strains were resistant to fluoroquinolones.
Conclusions: In our study A. baumannii strains don’t represent a particular problem in terms of antibiotic resistance. Nosocomial potential can be represented
only by the 6 multirezistente strains (beta lactams + aminogliozydes + /-quinolones). It should therefore use appropriate and effective treatment against
the phenomenon of selection of microorganisms that acquire antimicrobial resistance.
Keywords: phenotype, strain, nosocomial potential

INTRODUCTION

Table I. Distribution
samplescollected
the Department
Cardiology Department
TableofI. Distributionof samples
collected fromfrom
the Cardiology

For over 30 years, nosocomial infections caused by gram-negative bacilli were foundto be a serious problem for developed, but especially for the developing countries.
Among the emerging pathogens involved in nosocomial infections, Acinetobactersp. draw attention on medical world, not as much through the wide spectrum ofclinical manifestations, but especially their gravity, directly linked to the lack ofeffective therapeutic means against multirezistente strains (2,3,6).

MATERIAL AND METHOD
362 samples were collected during december 2008 - june 2009, from Cardiology
Department in the Timisoara Municipal Hospital. Most samples were from patients(29%) and environment (aeroflora - 27% and surfaces - 22%). At a reduced rate
samples were collected from the sterile water, solutions and disinfectants (13%)
and medical nurses (9%).
Samples were sent to the Bacteriology Laboratory of Microbiology-Virology Department from the University of Medicine and Pharmacy "Victor Babes" Timisoara
where have been processed. All the samples were inoculated on Columbia 5%
sheep blood agar (bioMerieux) and selective media Mac Conkey (BioRad). Then
were incubated at 37°C for 24h. Identification of Acinetobacter baumannii strains
was generally based on morpho-tinctorial character (gram negative coccobacilli),
cultural and biochemical tests (API 20E galleries). Sensitivity tests were done

Patients
nasal swabs
pharyngeal swabs
lung aspirates
urinesMedical staff
nasal swabs
skin samples
Surface samples
Disinfectant
solutions / sterile
water
Aeroflora

Total

Samplescollected

Percent %

46
24
14
24

12.7
6.62
3.86
6.62

26
6
78
48

7.18
1.65
21.54
13.25

96

26.51

362

100

Received September 2009. Accepted October 2009. Address for correspondence: Dr. Dorina Dugaesescu, University of Medicine and Pharmacy”Victor Babeş”Timişoara, 2A Eftimie Murgu Square,
300041, Timisoara, Romania
Samples were sent to the Bacteriology Laboratory of Microbiology-Virology

De
University of Medicine and Pharmacy "Victor Babes" Timisoara where have been
15
2009.19.4(64)  Fiziologia - Physiology
samples were inoculated on Columbia 5% sheep blood agar (bioMerieux)
and seConkey (BioRad). Then were incubated at 37°C for 24h. Identification of Acine
cmykstrains was generally based on morpho-tinctorial character (gram negative coccob
biochemical tests (API 20E galleries). Sensitivity tests were done by disk diffusion - K

Medical staff
nasal swabs
skin samples
Surface samples
Disinfectant
solutions / sterile
water
Aeroflora

26
6
78
48

7.18
1.65
21.54
13.25

96

26.51

Total

362

100

30
20

11.42%

14.28%
5.71%

10
0

Susceptible
phenotyp

G

GN

GTN

5.71%
GT

Fig. 3. A. baumannii aminoglycosides resistance phenotypes
Samples were sent to the Bacteriology Laboratory of Microbiology-Virology Department from the
University of Medicine and Pharmacy "Victor Babes" Timisoara where have been processed. All thewereto fluoroquinolones(91.42%)andwereonly (8.57%) were
samples were
on -Columbia
sheep andblood
agar (bioMerieux)
and systemselectiveMost
media strains
Macstrains
by inoculated
disk diffusion
Kirby-Bauer5%method
automated
Vitek 2 compact
Most
weresusceptible
susceptible to fluoroquinolones(91.42%) andonly (8.57%)
Conkey (BioRad). Then were incubated at 37°C for 24h. Identification of Acinetobacterpercentage
baumannii was met in the case of sulphonamides (8.57%).
(whichallowedspecies
identification),
and
it
was
followedbyclassificationofresistant.
The
same
percentage
was
met
in
the
case
of
sulphonamides(8.57%).
strains was generally based on morpho-tinctorial character (gram negative coccobacilli), cultural andpatterns(4,9). Sensitivity tests were done by disk diffusion - Kirby-Bauer methodbiochemical phenotypic
tests (API 20E
galleries).
and automated Vitek 2 compact system (which allowed species identification), and it was followed by
91.42%%
classification of phenotypic patterns (4,9).

RESULTS AND DISCUSSION

100

90
RESULTS AND
Of theDISCUSSION
total 362 samples harvested 187 were positive (51.65%). We isolated 594
80
Of the total 362 samples harvested 187 were positive (51.65%). We isolated 594 bacterial strains withbacterial potential,
strains withfrom
possiblenosocomial
potential,
fromrepresented
which 35 strains(6%) were baumannii.
70
possible nosocomialwhich
35 strains(6%) wereby Acinetobacter
These strainsrepresented
came fromdifferent surfaces(65%),(26%)
4 hospitalized
by Acinetobacterbaumannii.
Theseaeroflorastrains camefromanddifferent
surfaces patients60ofpharyngeal swabs (9%).
50(65%), aeroflora (26%) and 4 hospitalized patients of pharyngeal swabs (9%).

40
30

aeroflora
26%

10
0

Susceptible phenotypes atquinolone

Resistance phenotypes atquinolone

Fig. 4. A. baumannii quinolones resistance phenotypes

pharyngeal
swabs
9%

surface samples
65%

8.57%

20

Entrance through a colonized patient is the most likely mode. However, introduction through contaminated materials (such as pillows) has also been documented.
Fig. 1. Distribution of A. baumannii strains isolated from Cardiology Department
Notably, introduction by healthy carriers is also conceivable, although it is not
Fig. 1. Distribution of A. baumannii strains isolated from Cardiology Department
known whether the rare strains that circulate in the community have epidemic
About beta-lactams resistance, most A. baumannii strains were included in PASE
potential. Once on a ward, A. baumannii can spread from the colonized patient to
About beta-lactams
resistance,
most
A. baumannii
strains were
includedin PASEphenotype (66.71%),
phenotype(66.71%),
followedby CASE phenotype(20%)
and 5 strains(11.42%)the environment and other susceptible patients (10). The direct environment offollowed by CASE phenotype (20%) and 5 strains (11.42%) belonged to wild type phenotype.
belonged to wild type phenotype.
the patient can become contaminated by excreta, air droplets and scales of skin.
Interestingly, A. baumannii can survive well in the dry environment, a feature it%
65.71%%
65.71%
shares with staphylococci. Hence, the contaminated environment can become
70
70
a reservoir from which the organism can spread. The acquisition of A. baumannii
60
60
by susceptible patients can occur through various routes, of which the hands of50
50
hospital staff are thought to be the most common, although the precise mode of40
40transmission is usually difficult to assess (5).
20%
20%
30
30
20

20

10

10

0

11.42% 11.42%

0Wild phenotype

PASE phenotype

CASE phenotype

Wild phenotype
PASE phenotype
CASE phenotype(penicillinase)(cephalosporinase)(penicillinase)(cephalosporinase)

Fig. 2. A. baumannii
resistancebeta-lactams
phenotypes resistance phenotypes
Fig. beta-lactams
2. A. baumannii
Fig. 2. A. baumannii beta-lactams resistance phenotypes

Most Most
strains
Acinetobacterbaumannii
isolates)
were resistant to: ticarcilin, piperacillin,
strains ofof Acinetobacterbaumannii(23 isolates) (23were resistant
to: ticarcilin,
Most strainsof butAcinetobacterbaumannii(23
isolates)
were cefixime,
resistant imipenem
to: ticarcilin,carbenicillin,
but susceptible
to tocefotaxime,
ceftazidime,cefoperazone,
and piperacillin,
piperacillin,carbenicillin,susceptible
cefotaxime,
ceftazidime,cefoperazone,
carbenicillin,susceptible
to resistancecefotaxime,
cefixime, imipenem andaztreonam.
The studyof aminoglycosides
showceftazidime,that,phenotype of Acinetobacter
cefixime,
imipenemandbutaztreonam.
The study of aminoglycosides
resistanceshow
that,sensitivecefoperazone,
baumannii
isolatedThestrains
hadof
theaminoglycosides
largest share (62.85%), followedby that,GN phenotype
aztreonam.
study
show
sensitive (14.28%)
phenotypeandofGAcinetobacter
sensitive
phenotype of Acinetobacterbaumannii isolated strains hadresistancethe largest sharephenotype(11.42%).
baumannii
isolated strains had the largest share (62.85%), followed by GN phenotype (14.28%) and G(62.85%), followed by GN phenotype (14.28%) and G phenotype (11.42%).
phenotype (11.42%).
%70

62.85%%

60

62.85%

70

50

60

40

50

30

40

20

11.42%

30

14.28%
5.71%

10
0

20

11.42%

Susceptible
10
phenotyp0

G

Susceptible

GN

5.71%

14.28%
GTN

GN

5.71%
GT
GTN

Fig. 3. A. baumannii G
aminoglycosides resistance phenotypes
phenotyp5.71%
GTFig. 5. The possible modes of Acinetobacter baumannii entry into a ward (5)As shown in many literature data, Acinetobacter sp. has a significant role in colo-Fig. 3. A. baumannii aminoglycosides resistance phenotypes

Most 16
strains were susceptible to fluoroquinolones (91.42%) and only (8.57%) were resistant.
The same
Fig. 3. A. baumannii
aminoglycosides resistance phenotypes Fiziologia - Physiology  2009.19.4(64)percentage was met in the case of sulphonamides(8.57%).

Most strains were susceptible to fluoroquinolones (91.42%) and only (8.57%) were resistant. The same
cmyk
91.42%%
percentage
was met in the case of sulphonamides (8.57%).
100

90

nization and infection of hospitalized patients. Estimation of the real frequency of
nosocomial infections produced by Acinetobacter sp. as etiologic agent is difficult,simple isolation of these microorganisms in clinical samples is not equivalent toinfection. In many cases it reflects only colonization of those sites. Furthermore,clear separation between the two situations is hampered by the fact that what
was originally isolated and labeled as "colonization" may, in a variable time then,clearly defined "infection". Depends on the anatomic site and time of collection,
but also a complex of factors, including biological and the patient's immune status,
pre-existing pathology and treatment, department’s profile and effectiveness ofestablished infection control are the most important unit. The use of molecularbiology techniques in the recent years made the necessary clarifications in manycases of this kind (5,7,10).

CONCLUSIONS
1. In our study of A. baumannii strains don’t represent a particular problem in termsof resistance to various classes of antibiotics. Only 6 strains were multirezistente(beta lactams + aminogliozydes + /-quinolones)
2. Good understanding of resistance phenotypes, inform us about precisely theresistance mechanisms and on the ways of spread.
3. With these data we can assess the development of resistance in the futureand establish a well thought f antibiotic policy, for preventing multiplication ofresistant strains.
4. The ability of these bacteria to develop antimicrobial resistance by various
mechanisms, their great capacity to survive long in the hospital environment
and multiple opportunities for transmission between patients, raise serious
problems in the treatment and management of nosocomial infections producedby Acinetobacter sp.

REFERENCES
1. Bassetti M, Righi E, Silvano E, Petrosillo N, Nicolini L. Drug Treatment for Multidrug-resistant Acinetobacter baumannii Infections.
Future Microbiology 2008; 3(6): 649-660.
2. Bergogne-Berezin E, Towner KJ. Acinetobacter spp. as nosocomial
pathogens: microbiological, clinical, and epidemiological features.
Clin Microbiol Rev. 1996; 9:148-65.
3. Bradford PA. Extended-spectrum β-lactamases in the 21st
century: characterization, epidemiology, and detection of thisimportant resistance threat. Clin Microbiol Rev, 2007, 48: 933-51.
4. Branea D. Comparative Study of resistance to chemotherapy
anti-microbial strains isolated from hospital and outpatient.
Doctoral Thesis, 2004.
5. Dijkshoorn L, Nemec A, Seifert H. An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii. Nature Rewiews
Microbiology 2007; 48: 933-51.
6. Falagas ME, Karveli EA The changing global epidemiology ofAcinetobacter baumannii infections: a development with major
public health implications. Journal Compilation European Societyof Clinical Microbiology and Infectious Deaseases, 2006; 13(2):117-119.
7. Gordon NC, Wareham W. A review of clinical and microbiologicaloutcomes following treatment of infections involving multidrugresistant Acinetobacter baumannii with tigecycline. Journal of
Antimicrobial Chemotherapy 2009; 63(4): 775-780.
8. Lortholary O, Fagon JY, Hoi AB, Slama MA, Pierre J, Giral P et al.
Nosocomial acquisition of multiresistant Acinetobacter baumannii:
risk factors and prognosis. Clin Infect Dis. 1995; 20:790-6.
9. Moldovan R, Licker M, Berceanu D, Craciunescu M, Dan L, Branea
D, Hogea E. Practical laboratory support, ed. Lito UMF Timisoara,
2002.
10. Mulin B, Talon D, Viel JF, Vincent C, Leprat R, Thouverez M et
al. Risk factors for nosocomial colonization with multiresistantAcinetobacter baumannii. Eur J Clin Microbiol Infect Dis. 1995;
14:569-76.

PREVALENŢA ŞI REZISTENŢA LA ANTIBIOTICE A TULPINILOR
DE ACINETOBACTER BAUMANII IZOLATE ÎNTR-O SECŢIE DECARDIOLOGIEREZUMAT

Scop: În urma acestui studiu am apreciat gradul de încărcătură microbiană a aerului, suprafeţelor şi starea de portaj a personalului medical şi pacienţilor spitalizaţi
într-o Secţie de Cardiologie din Timişoara. S-a urmărit izolarea şi identificarea tulpinilor de Acinetobacter baumannii din probe recoltate şi comportamentul lor laagenţii antimicrobieni.
Metodă: Probele biologice au fost însămânţate pe medii de cultură: geloză sânge 5% şi Mac Conkey, după care s-au incubat la 37°C 24h. Identificarea
tulpinilor de Acinetobacter baumanii s-a făcut pe baza caracterelor morfologice, culturale, biochimice (galerii API 20E). Testarea sensibilităţii la antibiotices-a făcut prin metoda difuzimetrică Kirby-Bauer şi sistemul automat Vitek 2.
Rezultate: Din cele 362 probe recolate au fost pozitive 51,65%. S-au izolat 35 tulpini de Acinetobacter baumannii din totalul celor 594 tulpini bacteriene
izolate. Aceste tulpini au provenit de la nivelul diferitelor suprafeţe (65%), aerofloră (26%) şi din exudatele faringiene (9%) a 4 pacienţi spitalizaţi. La betalactamine majoritatea tulpinilor de A. baumannii s-au încadrat în fenotipul PAZA (66%), urmat de fenotipul CAZA (20%) şi 5 tulpini (11%) au aparţinut fenotipului sălbatic.
La aminoglicozide fenotipul sălbatic a fost în proporţie de 62,8%, urmat de fenotipul GN (gentamicină, netilmicină) (14,3%) şi fenotipul G (gentamicină) (11,4%). La
fluoroquinolone doar 8,6% tulpini au fost rezistente.
Concluzii: În cadrul studiului nostru tulpinile de A. baumannii nu au pus o problemă deosebită în ceea ce priveşte rezistenţa la antibiotice. Potenţialul
nosocomial poate fi reprezentat doar prin cele 6 tulpini multirezistente (beta-lactamine+aminogliozide+/-quinolone). Se impune deci utilizarea unuitratament eficient şi adecvat împotriva fenomenului de selecţie a microorganismelor care dobândesc rezistenţă la antibiotice.
Cuvinte cheie: fenotip, tulpină, potenţial nosocomial2009.19.4(64)  Fiziologia - Physiology

cmyk

17

Antimicrobial Resistance of ESCHERICHIA COLISTRAINS isolated from urinary tract infections
CRACIUNESCU MIHAELA, LICKER MONICA, MUNTEAN DELIA, HOGEA ELENA, BERCEANUVADUVA DELIA, DUGAESESCU DORINA, SERBAN D, POPA MIHAELA, PILUT C, HORHAT F,TATU CARMEN, MOLDOVAN ROXANA
Victor Babes University of Medicine and Pharmacy, Timisoara

ABSTRACT
Purpose: For nosocomial urinary infections (NUI) monitoring, samples were taken between January 2008 and January 2009, from 629 patients hospitalsedin the Obstetrics and Gynecology Timisoara Hospital.
Methods: The Gram-negative Germs were identified at the specie level by biochemical testing using the API ID 32 E galleries (bioMerieux) and their antimicrobial
resistance was determined by agar mini API microdilution and the Kirby-Bauer disk diffusion (on standardized subculture) methods.
Results: From the totalnumber of 207 positive urocultures were isolated some strains with high nosocomial potential and the leader were E. coli, with 130
strains. Unfortunately, only 23.84% from the E. coli isolated strains kept unaffected their natural sensibility to the antimicrobial therapy. The penicillinase
producing strains represented 63.07 % and the CAZA and CHN strains represented 6.91 %. The ESBL producing strains (3.07%) associated aminoglycozides
and fluoroquinolones resistanceConclusions: The UTI, especially the NUI, become a health problem due to the increasing number of cases and the difficulty of treatment caused by the multiresistance to antimictobial treatment. The increased number of strains with acquired resistance to the antimicrobial therapy found by the present researchstudy is underlining the importance of permanent and systematic monitoring of the phenomenon.
Key words: nosocomial urinary infections, resistance phenotypes, E. coli

INTRODUCTION
Obstetrics and gynaecology departments have always had a leading role in
the history of nosocomial infections, starting with the research for the puerperal
fever of Pasteur and Semmelweis (3, 7). Although huge progresses have been
made regarding the microbiology, epidemiology, hygiene and the anti-infectious
chemotherapy, the urinary tract infections (UTI) are still a problem of the patients
hospitalized in these departments, due to the existing risk factors (1, 4).
The main risk factor, accountable for more than 80% of the nosocomial urinary
infections (NUI) is the instrumental approach of the urinary tract. NUI are favoured by
the probing of the urinary tract, depending on the length of time of probing, the typeof drainage (the open drainage being twice more frequent incriminated than theclosed drainage), the impaired integrity of the urinary tract by other gynaecologicalprocedures or by labour complications. The intrinsic factors are: the feminine gender,
the susceptibility of the host, the presence of gynaecological pathology (tumours,
infections, prior antibiotic therapy), and pregnancy (2).

MATERIAL AND METHODS
Urinary samples were taken between January 2008 and January 2009, from629 patients hospitalized in the Obstetrics and Gynaecology Timisoara Hospital.
Collection of specimens was performed, generally by the hospital’s medical
personnel, respecting the general rules of sampling for bacteriologic analysis. The
bacteriological culture media used for isolation were represented by Columbia typeagar (Bio Rad, Oxoid or bioMérieux).
The samples were cultivated by calibrated loops technique on nonselective solid
media agar type Columbia with 5% blood and on lactose media for enterobacteria,
most frequently used being Mac Conkey (Oxoid). The germs identification was

performed in concordance with their morpho-tinctorial characteristics in Gram
stain, the aspect of colonies on culture media, metabolic properties and somepathogenicity tests.
The Gram-negative germs were identified at specie level by biochemical
testing, the API ID 32 E galleries (bioMérieux) being used for the identificationof metabolic characteristics of these germs. The antimicrobial resistance of thegerms was performed by agar micro-dilutions method, using the mini API system(bioMerieux) and by the Kirby-Bauer disk diffusion method on a standardized
subculture.
The extended spectrum beta-lactamase (ESBL) production in enterobacteria and other gram-negative bacilli was suspected when a reduced inhibition
diameter zone surrounding the third generation of cephalosporin (cefotaxime 27
mm, ceftazidime 22 mm, ceftriaxone 25 mm), or at monobactams (aztreonam 27mm), was noticed.
For the correct interpretation of the ESBL producing phenotype, the presence of a positive synergy test (an image like a “champagne stopper” between
the cephalosporin or monobactam disks and the beta-lactamase inhibitor) is
conclusive (5).
The synergy test was performed using an amoxicillin/clavulanic acid disk of in
the middle, surrounded at a distance of 2 cm, by cefotaxim, ceftriaxon, ceftazidim
and aztreonam disks.

RESULTS
From the total number of 207 positive urocultures, we have isolated somestrains with a high nosocomial potential. These germs are represented in Figure 1.
One bacterial species was identified in 205 cultures and Candida spp. was isolated
in 2 samples.

Received September 2009. Accepted October 2009. Address for correspondence: Dr. Mihaela Craciunescu, University of Medicine and Pharmacy”Victor Babeş” Timişoara, 2A
Eftimie Murgu Square, 300041, Timisoara, Romania
18

cmyk

Fiziologia - Physiology  2009.19.4(64)Pefloxacin
Ciprofloxacin
Nitrofurantoin
Cotrimoxazol

107
107
104
112

82.3%
82.3%
80%
86.15%

-

-

23
23
26
18

1
1

1

Interpretation of these results, allowed framing of the germs in phenotypic patterns.
1
1
1
1
1
1
1
2
2
2
3
3
4
5
6

Streptococcus agalactiae
Pseudomonas aeruginosa
Stenotrophomonas maltophilia
Aeromonas sobria
Burkholderia cepacia
Morganella morganii
Citrobacter koseri
S. schleiferi
Serratia odorifera
K. ornithinolytica
S. saprophyticus
Enterobacter aerogenes
S. epidermidis
Proteus mirabilis
S. aureus
Enterococcus spp.

Table III. E. coli resistance phenotypes
Table III. E. coli resistance phenotypes

15

K. pneumoniae

28

130

E. coli

0

20

40

60

80

100

120

140

Fig. 1. Distribution of the isolated germs from the positive urocultures

As shown in theTable
previous
figure, theGram-negative
bacilli were better repreI. Beta-lactams
sensibilityof E.coli strainssented than the Gram-positive cocci, and the leader was E.coli with 130 strains.

Antimicrobial
chemotherapy

Antimicrobial
chemotherapy
Amoxicillin

Amoxiclav
Amoxicillin
Ticarcilin
Amoxiclav
Ticarcilin
Piperacilin
Piperacilin
Cefalotin
Cefalotin
Cefuroxim
Cefuroxim
Cefotaxim
Cefotaxim
Ceftazidim
Ceftazidim
Ceftriaxon
Ceftriaxon
CefepimCefepim
Cefoxitin
Imipenem
Imipenem

TableSensible
I. Beta-lactams sensibilityof E.coli strains
Intermediary

Table I. Beta-lactams sensibility of E.coli strains

Resistant

Associated resistancephenotypes
Wild strain
Low PASE
High PASE
CASE
CHN
Low PASE+IV Q
Low PASE+G
Low PASE+KTG+FT
Low PASE+G+IV Q
Low PASE+G+SXT+FT
Low PASE+SXT+FTHigh PASE+IV Q+FTHigh PASE+IV Q+SXT+FTHigh PASE+G+IV Q+FTHigh PASE+KTGANt+IV Q
CASE+SXT+FT
CASE+IV Q
KTG
KTGNt
KTGANt+IV Q
IV Q
ESBL+KTGANt+IV Q
Total

Number

Percent

31
28
22
6
1
3
1
3
2
10
2
3
5
2
1
1
1
1
1
1
1
4
130

23.84%
21.53%
16.92%
4.61%
0.76%
2.31%
0.76%
2.31%
1.53%
7.69%
1.53%
2.31%
3.84%
1.53%
0.76%
0.76%
0.76%
0.76%
0.76%
0.76%
0.76%
3.07%
100%

Intermediary
NumberSensible
Percent Number
Percent NumberResistant
Percent
35
26.92%
95
73.07%
Number Percent Number Percent Number Percent
84
64.61%
64.61%
40 73.07%
30.76%
35
26.92%
95
35
26.92%
95 30.76%
73.07%
84
64.61%
64.61%
40
35
26.92%
95
84
64.61%
1310%
33 73.07%
25.38%
84
64.61%
1310%
33
35
26.92%
49
37.69%
46 25.38%
35.38%
35
26.92%
4937.69%46
84
64.61%
46 35.38%
35.38%
84
64.61%
46
35.38%
Legend: low PASE- low level of penicillinase producing phenotype, high PASE- high level
125 96.15%
5
3.84%
penicillinase
producing phenotype,
CASE- induciblephenotype,
chromosomal cephalosporinase
producing phe125
96.15% Legend:-low PASE5
3.84%
low
level
of
penicillinase
producinghigh PASEhigh level pe
12596.15%
3.84%
notype, CHN- hyperproducing cephalosporinase phenotype, ESBL- extended spectrum beta-lactamase125
96.15% phenotype,--- CASE55 inducible
3.84%
chromosomal
cephalosporinase
producingphenotype,
CHNproducing phenotype, KTGANt – aminoglycozides (kanamycin, tobramycin, gentamycin, amikacin, hyperproduc12596.15%
3.84%
125
96.15% phenotype,--- ESBL55 extended
3.84%
spectrumbeta-lactamase
producingphenotype, KTGANt
netilmicin) resistance,
IVQ- quinolones crossresistance, SXT- trimethoprim-sulfamethoxazol
resistance, – aminogly
126
96.92%
3.07% amikacin,
FT- nitrofurantoin
resistance. resistance, IVQ- quinolones cross resistance,
126
96.92% tobramycin,--- gentamycin,
44
3.07%
netilmicin)
1resistance,
0.76% FT- nitrofurantoin resistance.
129 99.23% sulfamethoxazol
DISCUSSIONS
130
100%
-130
100%
-

The coli bacilli are natural sensible to several families of antimicrobial drugs:
DISCUSSIONS
beta-lactamines, aminoglycozides, fluoroquinolones, sulfamides and furans (6).
Table II. Aminoglycozides, quinolones, nitrofurantoin
and trimethoprim-sulfamethoxazol sensibility of E.coli strains
sensibleof antimicrobial
drugs
sensibility of E.coli strains The coli bacilli are natural
Unfortunately,
only 23.84%tofromseveral
E. coli isolatedfamilies
strains kept unaffected
their naturalaminoglycozides,
fluoroquinolones,
sulfamides
andfurans(6).
Unfortunately,
only
23.
sensibility to antimicrobial therapy.
Antimicrobial
SensibleIntermediary
Resistant
Antimicrobial
SensibleIntermediarystrains Resistantisolated
kept unaffected theirnaturalsensibility
to antimicrobial
therapy.
The penicillinase
producingstrains represented
63.07% while the CAZA
andchemotherapy
Table II. Aminoglycozides,
quinolones, nitrofurantoin
andnitrofurantoin
trimethoprim-sulfamethoxazol
sensibility of E.coli strains
Table II. Aminoglycozides,
quinolones,
and trimethoprim-sulfamethoxazol

chemotherapy

Number

Percent

Number

Percent

Number104
80% Number
26
Number
Percent
Percent
Number119
91.53%
11
104
80%
26
123
94.61%--11991.53%
117124
95.38%
6
123 94.61%
7
107
82.3%
23
124
95.38%
107
82.3%
236
107
82.3%
23
107
82.3%
23
107
82.3%
23
107
82.3%
--23
104
80%
26
107
82.3%
23
112
86.15%
--18
107
82.3%
23
Nitrofurantoin
104
80%
26
Interpretationof these results, allowed
Cotrimoxazol
112 framing
86.15%of the germs- in phenotypic- patterns.
18
Gentamycin
Tobramycin
Gentamycin
Netilmicin
Tobramycin
Amikacin
Netilmicin
Nalidixic acid
Amikacin
Norfloxacin
Nalidixic
Pefloxacinacid
Norfloxacin
Ciprofloxacin
Nitrofurantoin
Pefloxacin
Cotrimoxazol
Ciprofloxacin

Percent
20%
Percent
8.46%
20%
5.38%
8.46%
4.61%
5.38%
17.7%
4.61%
17.7%
17.7%
17.7%
17.7%
17.7%
20%
17.7%
13.84%
17.7%

20%
13.84%

Interpretation of these results, allowed framing of the germs in phenotypic

CHN strains represented only 6.91%. The ELSB producing strains (3.07%) associatedaminoglycozides and fluoroquinolones resistance.
We have found an increased number of strains resistant to penicillin’s: aminopenicillin (73.07%) and ureidopenicillin (25.38%). 30.76% from E. coli strains
were resistant to amoxicillin/clavulanic acid. We recorded a 3.07% fourth generationof cephalosporin resistant strains, 3.84% third generation cephalosporin resistanceand 35.38% first and second generation of cephalosporin resistance. Resistance to
cefamicin was recorded in only one strain and all the E. coli strains were sensible
to carbapenem.
E.coli strains were resistant to quinolones in a 17.7% and in different percentages to aminoglycozides (between 20% for gentamycin and 4.61% for amikacin).
13.84% of the E.coli strains were resistant to trimethoprim-sulfamethoxazol, but
the usage of these drugs is limited due to the contraindication during pregnancy.
20% of all the E.coli strains were nitrofurantoin resistant.

Interpretationof these results, allowed framing of the germs in phenotypic patterns.
patterns.
2009.19.4(64)  Fiziologia - Physiology

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19

CONCLUSIONS
1. The UTI, especially the NUI, become a health problem due to the increasing
number of cases and the difficulty of treatment caused by the multiresistance toantimicrobial treatment.
2. The increased number of strains with acquired antimicrobial resistance,found in the present research study is underlining the permanent and systematicnecessity of monitoring of this phenomenon.
3. These prevalence data are imposing a more active participation in international surveillance studies in order to compare the results of antimicrobial
resistance and to be able to implement correct methods of the infection control
in our hospitals.

REFERENCES
1. Berceanu-Vaduva D. Infecţiile nosocomiale. Ed. Mirton,Timişoara, 1999
2. Bocsan IS. Practical Epidemiology for family practitioners. Ed.
Medicala Universitara "Iuliu Hatieganu" Cluj Napoca, 1999
3. Holmes OW. The contagiousness of puerperal fever. N Engl QJ
Med Surg. 1843; 1: 503-530.
4. Licker M, Moldovan R et al. Antibiotics resistence, history andactuality. Ed. Eurostampa, Timişoara, 2002.
5. Lorian V. Antibiotics in laboratory medicine. Lippincott Williams&Wilkins, 2005
6. Mandell GL, Bennett JE, Dolin R. Principles and Practice of Infectious Diseases. 5th ed. New York: Churchill Livingstone, 2000
7. Pasteur L. Puerperal sepsis. Bull Acad Med (Paris). 1879; 8: 256260

Rezistenta la antibiotice a tulpinilor de ESCHERICHIACOLI izolate din infectii urinare
Rezumat
Scop: Pentru monitorizarea infecţiilor urinare nosocomiale, în perioada ianuarie 2008-ianuarie 2009, au fost prelevate probe de urină de la 629 paciente
din secţiile de Obstetrică şi Ginecologie ale Spitalului Clinic „Dr. Dumitru Popescu“ Timişoara.
Material si metode: Germenii au fost identificaţi utilizând galerii API (ID 32 E), iar pentru testarea sensibilităţii am folosit atât metoda microdiluţiilor în agar
pe sistemul mini API (bioMerieux) cât şi metoda difuzimetrică Kirby-Bauer pe subcultură standardizată.
Rezultate: Din cele 207 uroculturi pozitive s-au izolat o serie de tulpini bacteriene aparţinând unor specii cu pronunţat potenţial nosocomial, lider
fiind E.coli cu un număr de 130 tulpini. Numai 23,84% din tulpinile de E. coli pe care le-am izolat şi-au păstrat nealterată sensibilitatea naturală la chimioterapicele antiinfecţioase. Tulpinile producătoare de penicilinază au fost izolate într-un procent de 63,07%, iar fenotipurile CAZA şi respectiv CHN, într-un
procent de doar 6,91%. Tulpinile secretoare de BLSE (3,07%) au asociat rezistenţă la aminoglicozide şi fluoroquinolone.
Concluzii: Infecţiile tractului urinar, în special cele cu caracter nosocomial, au devenit o adevărată problemă de sănătate prin creşterea constantă
a numărului de cazuri precum şi prin dificultăţile de tratament cauzate de tulpinile multirezistente la antibiotice. Numărul ridicat de tulpini rezistente lachimioterapicele antiinfecţioase, cuprinse în prezentul studiu, subliniază încă o dată necesitatea monitorizării permanente şi sistematice a evoluţiei acestui
fenomen.
Cuvinte cheie: infecţii urinare nosocomiale, fenotipuri de rezistenţă, E. coli

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Fiziologia - Physiology  2009.19.4(64)IN VITRO CHARACTERIZATION OF MECHANISMS
INVOLVED IN ADIPOGENIC DIFFERENTIATION OF
MESENCHYMAL STEM CELLS AND TUMOR ASSOCIATED
FIBROBLASTSFLORINA BOJIN1, CALIN TATU2,3, OANA GAVRILIUC1, VALENTIN ORDODI2,3,
ALEXANDRA GRUIA2, ADRIANA ROSCA2, GABRIELA TANASIE1,2, CARMEN TATU1,2,
DANIELA CRISNIC1,2, DACIANA NISTOR1,2, MIRABELA CRISTEA2, SIMONA ANGHEL1,2,
ALEXANDRA BOLEMAN4, CARMEN BUNU1, VIRGIL PAUNESCU1,2
1
Department of Physiology and Immunology, “Victor Babes” University of Medicine and Pharmacy Timisoara
2
Department of Biology, “Victor Babes” University of Medicine and Pharmacy Timisoara
3
Transplant Immunology Center, County Clinical Emergency Hospital Timisoara
4
University of Agricultural Sciences and Veterinary Medicine of Banat Timisoara

ABSTRACTThe purpose of our study was to investigate the molecular mechanisms of adipogenesis regulation in different differentiation stages, both in mesenchymal
stem cells (MSCs), as well in tumor associated fibroblasts (TAFs), considering their potential to transform into cells exhibiting intra-cytoplasmic lipid vacuoles.
Starting with passage 2 for MSCs and passage 4 for TAFs, in appropriate culture conditions, cells were induced to differentiate towards the adipogenic lineage
within 21 days. Total RNA extraction followed by RT-PCR and qRT-PCR analysis and histochemical staining were performed in certain key points of differentiation time in order to confirm adipocytic induction. Immunocytochemical staining using anti-FABP4 antibody, specific for mature adipocytes revealed in bothcell types complete differentiation after 3 weeks of culture. Up- and down-regulation of different molecular markers, such as PPARγ, LPL and C/EBPα, can bejuxtaposed on adipocytic development in a similar manner in both cell types studied, being a promising pathway to study accumulation of mature adipocytes
and development of obesity, as well as role of adipose tissue in tumor progression.
Key words: mesenchymal stem cells, adipocytes, tumor-associated fibroblasts, adipogenesis, molecular marker

INTRODUCTION
Adipogenesis is a complex process involving proliferation of precursor cells,
commitment towards the adipogenic lineage and terminal differentiation into mature adipocytes (1). Although the terminal differentiation of adipocytes is intensively
studied and described using murine cell lines, key information about commitmentof human precursor cells to the adipogenic lineage is still lacking.
Human pre-adipocytes derived from adipose tissue through collagenase
digestion are widely used to study human adipogenesis; however, these cells are
already committed to the adipogenic lineage (2). Moreover, human pre-adipocytes
have reduced proliferative ability, un unpredictable variability based on different
donors and anatomical sites, and limited availability (1,3). Human mesenchymal
stem cells (hMSCs) (4) represent an alternative to this model, because they are
multipotent precursors of several cell types, including adipocytes, osteoblasts andchondrocytes (5,6), they can be isolated from donor bone marrow and separated
from other cell types by centrifugation on a density gradient. The variability between
hMSCs from different donors was reported to be low, while MSCs proliferate under invitro conditions and retain their adipogenic, chondrogenic and osteogenic potential(6). Theoretical concepts of MSCs differentiation pathway towards the adipocytic
lineage can be seen in Figure 1.
Large amount of data emerged in the last years regarding the supportiverole of tumor stroma, which can play a crucial role in tumorigenesis and invasion.

Mechanisms used in these processes remain unknown, but different chemokines,
cytokines and growth factors secreted by tumor stroma seem to induce selectionand expansion of neoplastic cells (7-9). Tumor associated fibroblasts (TAFs) areconsidered to form the majority of tumor stroma and posses similar characteristics
with MSCs in the respect of proliferation and differentiation potential (10), as wellFig. 1. Theoretical concepts of MSCs differentiation pathway towards mature adipocytes,
considering the pre-adipocytes stage, medium composition and genes involved in eachof these stages

Received September 2009. Accepted December 2009. Address for correspondence: Dr. Florina Bojin, Physiology and Immunology Department, “Victor Babes” University of Medicine andPharmacy Timisoara, 2A Eftimie Murgu Square, 300041, Timisoara, Romania, e-mail: florinabojin@umft.ro2009.19.4(64)  Fiziologia - Physiology

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21

in expression of different molecular and surface markers (11,12,13). Large amountsof adipose tissue surrounds TAFs and neoplastic cells within solid tumors, so thatthe question rising at this point is whether TAFs are the precursors of adipocytes,
and which is the role of this adipose tissue.
The main purpose of this study was to investigate the differentiation patternsof both MSCs and TAFs (isolated from breast cancer pieces) and to characterize thefactors involved in adipogenic differentiation of these cell types.

MATERIAL AND METHODS
Cell isolation and culture
Normal human mesenchymal stem cells (MSCs) were obtained from bonemarrow of 6 healthy Orthopedics patients undergoing hip replacement surgery.
Approximately 10 ml of bone marrow were placed in culture plates, and thefibroblastic-like, plastic adherent fraction, was isolated following multiple passages and used in our experiments. The MSCs were further cultured and expanded
in alpha-minimum essential medium (MEM; Gibco BRL, Invitrogen, Carlsbad,
CA, USA), supplemented with 10% fetal calf serum (FCS; PromoCell, Heidelberg,
Germany) and 2% Penicillin/Streptomycin mixture (Pen/Strep, 10,000 IU/ml;
PromoCell), by incubation at 37oC in 5% CO2 atmosphere. Medium was replaced
every three days and when the confluence was 80-90% the cells were passed using 0.25% Trypsin-EDTA solution (Sigma) followed by centrifugation (10 minutes,300g) and replated in T75 culture flasks at a density of 10,000 cells/cm2 to ensure
optimal proliferation. Starting with passage two, part of the cells were used forfurther phenotypical analyses and differentiation assays, while MSCs expanded topassages 2-5 were used in the subsequent experiments.
Human tumor-associated fibroblasts (TAFs) were isolated using both the
explant and collagenase type IV-S from Clostridium histolyticum (Sigma, St.
Louis, MO, USA) methods. Breast cancer surgical pieces of approximately 5 cm2
were obtained form 8 female patients, with the histopathological diagnosis of
infiltrative ductal mammary carcinoma. Tissue-isolated cells were washed several
times with phosphate buffered saline (PBS) solution, and passed through 0.70/0.40
μm strainer filters and were replated as single-cell suspension in adherent plastic
culture plates.
All tissue samples were obtained after signing the informed consent elaborated
under an approved protocol, according to the World Medical Association Declaration of Helsinki.
Differentiation experiments were conducted at different passages for both
MSCs and TAFs, starting with passage 2 for MSCs and passage 4 for TAFs (this latepassage was used in order to obtain a pure cell culture). Cells were seeded in appropriate culture flasks at a cellular density of 10,000 cells/cm2 being stimulated todifferentiate in Nonhematopoietic stem cell medium for generation of adipocytes(Miltenyi Biotec, Bergisch Gladbach, Germany) supplemented with 1% Penicillin/
Streptomycin.
Histochemical staining
Cells submitted to differentiation culture conditions were stained with Oil Red
O following the manufacturer protocol in the 21st day after induction of adipocytic
differentiation. Briefly, cells were washed twice with PBS, fixed in 10% solution offormaldehyde in aqueous phosphate buffer for 1 hour at room temperature, washedwith 60% isopropanol, and stained with Oil Red O (Sigma) solution. Red Oil O working solution was prepared by mixing 15 ml of a stoc (0.5% in 60% isopropanol) and
10 ml of distilled water and filtering through PDVF membrane (0.22 μm) filter. Red
Oil O solution stained the cells for 10 minutes, followed by repeated washing withwater. Counterstaining of the nucleus used hematoxilin solution.

22

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Immunocytochemical/immunofluorescence analysis
MSCs and TAFs differentiation experiments towards adipocytes were assessedusing anti-mFABP4 as primary antibody, respectively antibody from the Human
Mesenchymal Stem Cell Functional Identification Kit (R&D Systems), while thevisualization system was LSAB2 System-HRP (Dako); for immunofluorescence
we used AlexaFluor488 and AlexaFluor546 conjugated secondary antibodies(Molecular Probes, Invitrogen) and nuclear counterstaining with DAPI (Sigma).
Microscopy analysis was performed on a Nikon Eclipse E800 microscope equipped
Cells submitted to differentiation culture conditions were stained with Oil Red O following thewith adequate
fluorescencefilters.
manufacturer
protocolin the 21st day
after induction of adipocytic differentiation. Briefly, cells were

washed twice with PBS, fixed in 10% solution of formaldehyde in aqueous phosphate buffer for 1 hour atroom temperature, washed with 60% isopropanol, and stained with Oil Red O (Sigma) solution. Red Oil
O workingwas preparedby mixing
ml of a stoc (0.5% in 60% isopropanol) and 10 ml of
RNAsolution
extraction,
RT-PCR
and15 qRT-PCR
distilled water and filtering through PDVF membrane (0.22 ȝm) filter. Red Oil O solution stained the
Total
RNA
extraction
was
performed
different
passages andof incells for 10 minutes, followed by repeated washing atwithwater. Counterstaining
the crucial
nucleus day
usedhematoxilin solution.

points of differentiation (day 0, 3, 5, 7, and 21) for both MSCs and TAFs, from

Immunocytochemical/immunofluorescenceanalysis Total RNA Miniprep Kit (Sigma) andcultured cells using GenElute™ Mammalian
MSCs and TAFs differentiation experiments towards adipocytes were assessed using antiRNAconcentrationwasmeasuredon
Nanodrop(Wilmington,
mFABP4 as primary antibody, respectively aantibody
fromND-1000
the Human
Mesenchymal DE,
StemUSA)
Cell
Functional Identification Kit (R&D Systems), while the visualization system was LSAB2 System-HRP
spectrophotometer.
For
RT-PCR
we
used
100
ng
RNA/reaction
andweamplified(Dako); for immunofluorescence we used AlexaFluor488 and AlexaFluor546 conjugated secondary
antibodies(Molecular
Probes,Invitrogen)and nuclear
counterstaining with DAPI
(Sigma). Gamma),
Microscopy
fragmentsof 554 bp(PPARγ- Peroxisome
Proliferator-Activated
Receptor
analysis was performed on a Nikon Eclipse E800 microscope equipped with adequate fluorescence filters.

505 bp (LPL - LipoProtein Lipase), and 656 bp (C/EBPα – CCAAT/Enhancer Binding

RNA extraction, RT-PCR and qRT-PCR
ProteinTotalAlpha).
Total RNA (0.5 µg/reaction) from each sample was reverse tranRNA extraction was performed at different passages and in crucial day points ofdifferentiation(day 0,(SuperScript
3, 5, 7, and 21)IIIforFirst-Strand
both MSCs andTAFs, fromSystem;
cultured Invitrogen).
cells using GenElute™
scribed to cDNA
Synthesis
cDNA
Mammalian Total RNA Miniprep Kit (Sigma) and RNA concentration was measured on a Nanodrop NDsamples(10ng
template/reaction)were
analyzedbyquantitative
real-time
PCR,
1000 (Wilmington, DE, USA) spectrophotometer. For RT-PCR we used 100 ng RNA/reaction andweamplifiedfragments of 554 bp(PPARȖ
- Peroxisome
Proliferator-Activated
ReceptorSC,
Gamma),
505
usingaLightCycler480
SYBR
Green
I
Mastersystem(Roche,Florence,
USA))
andbp
(LPL - LipoProtein Lipase), and 656 bp (C/EBPĮ – CCAAT/Enhancer Binding Protein Alpha). Total
RNA(0.5LPLµg/reaction)from eachsampleAbsolute
was reverse
transcribed to cDNA
(SuperScript using
III First-Strand
PPARγ,and C/EBPαprimers.
quantification
was performed
the Fit
Synthesis System; Invitrogen). cDNA samples (10 ng template/ reaction) were analyzed by quantitative
Pointsmethod,withanexternal
standard
curve
andmolecular
markers
expression
real-time PCR, using a LightCycler480 SYBR Green I Master system (Roche, Florence, SC, USA)) andPPARȖ,
LPL and as
C/EBPĮ
Absolute quantification was performed using the Fit Points method,
was reported
copyprimers.
number/sample.
with an external standard curve and molecular markers expression was reported as copy number/sample.
Gene
PPARȖ

Sequence
Forward: 3’-AAGACCACTCCCACTCCTTTG-5’
Reverse: 3’-GTCAGCGGACTCTGGATTCA-5’

LPL

Forward: 3’-CTGAAGACACAGCTGAGGAC-5’
Reverse: 3’-CTGGTGAATGTGTGTAAGAC-5’

C/EBPĮ

Forward: 3’-TCGACATCAGCGCCTACATC-5’
Reverse: 3’-CTTGTCCACCGACTTCTTGG-5’

Role
Nuclear hormone receptor, transcriptional activator,
initially described as molecular target
Crucial role in adipogenesis, involved in cell proliferationand differentiation in C/EBPĮ deficient cells
Regulates development of adipocytes in response toendogenous lipid activators
Low levels are present in pre-adipocytes
Associated with differentiation process (Į and ȕ)
Increases fat uptake by cells
Indicator of pre-adipocytes – adipocytes conversion
Marker of final stages of adipogenesis
Decreases during adipogenesis process
Positively regulates expression with PPARȖ

Statistic analysis
Statistic
analysis was
performed using Excell Microsoft Office 2003 (Microsoft Corporation) software.
Statistic
analysis
The central tendencies of the variables were expressed as a mean (M), and the dispersion ones as standard
Statistic
was performed
Excell„t”-Student
MicrosofttestOffice2003(Microsoft
deviation(sd). Inanalysis
order to performthe statistic using
comparisons,and thevariance
analysis(ANOVA) were used for continuous variables. Differences were considered significant for p < 0.05.

Corporation) software. The central tendencies of the variables were expressed as a

RESULTSmean (M), and the dispersion ones as standard deviation (sd). In order to performthe statisticcomparisons,test and the variance analysis (ANOVA)
Microscopyof MSCs
and TAFs –„t”-Student
derived adipocytes
MSCs are known to be able to differentiate into adipocytes in 21 days of culture in appropriate
were
used
for
continuous
variables.
Differenceswere considered significant for
medium. The differentiation process is not involving 100% of the MSCs, but different proportions ofthese
p < cells
0.05.posses the characteristic of adipocytes. Spontaneous differentiation or early differentiated cells

RESULTS
Microscopy of MSCs and TAFs – derived adipocytes
MSCs are known to be able to differentiate into adipocytes in 21 days of culture
in appropriate medium. The differentiation process is not involving 100% of theMSCs, but different proportions of these cells posses the characteristic of adipocytes.
Spontaneous differentiation or early differentiated cells were not present in our
experiments involving MSCs-derived adipocytes. After 2 weeks of culture, MSCs
gained a more rounded shape, and by the end of 21 days, 50-60% of total MSCs
plated were containing lipid vacuoles inside the cytoplasm, the nuclei were pushed
towards the periphery and the cells began to detach from the culture flasks.
Compared to MSCs differentiation pathway, TAFs were able to differentiate evenin absence of specific medium, so that in day 3 there were approximately 1-2 cells/
well presenting lipid vacuoles when the cells were examined in light microscopy
Fiziologia - Physiology  2009.19.4(64)ded shape, and by the end of 21 days, 50-60% of total MSCs plated were containing
e the cytoplasm, the nuclei were pushed towards the periphery and the cells began toure flasks.
o MSCs differentiation pathway, TAFs were able to differentiate even in absence ofthat in day 3 there were approximately 1-2 cells/well presenting lipid vacuoles whenamined in light microscopy with high magnification (600x). Most of the cells
ocytes by the end of 3 weeks of culture in appropriate differentiation medium, but thewith high vacuoles
magnification (600x).
Mostlowerof the cells transformed
thepresenting lipid
wasthan inin adipocytes
MSCsby(40%).
TAFs-derived adipocytes
end
of
3
weeks
of
cultureinappropriate
differentiation
medium,but
theproportion
to round cells, but maintained their spindle-like shape, although the nuclei were side-of cells presenting lipid vacuoles was lower than in MSCs (40%). TAFs-derived
adipocytes did not transform into round cells, but maintained their spindle-likeal staining shape,using
OiltheRed
O showed
although
nuclei wereside-located. a better location of the lipid vacuoles within theuring the fat droplets
red,
the acounterstaining
Histochemicalin
staining
usingwhile
Oil Red O showed
better location of the lipid vacu-of the nuclei presented them inoles
withinthecytoplasm,bycolouringthefatdropletsin
red, while the counterstaining
al side of the cells (Figure 2).
of the nuclei presented them in blue at the peripheral side of the cells (Figure 2).

Fig. 3. Immunocytochemical staining of MSCs (up) and TAFs – derived adipocytes (FABP4)
Fig.
Immunocytochemical
stainingof MSCs
after
21 days3.
of culture.
Yellow droplets of lipids inside the cytoplasm(orange), counterstainedwithsolution (xadipocytes
100)(up) and TAFs –hematoxilin
derived(FABP4)afterdays of culture. Yellow
dropletsofFig. 3.21Immunocytochemical
stainingof MSCs
Immunocytchemical/immunofluorescencestaining
using
anti
mFABP4lipidsinsidethecytoplasm(orange),(up) and TAFs – derived adipocytes (FABP4)
antibody
bothwith
MSCs
TAFs were completely
differentiatedintoofadicounterstained
hematoxilin
solution(x 100)aftershowed
21 thatdaysof andculture.
Yellow
droplets
pocytes by the end of 21 days of culture, no intermediate phase presenting positive
lipidsinsidethecytoplasm (orange),
staining for this marker. We were able to count positive cells and to determine thecounterstained with hematoxilin solution (x 100)
ratio between differentiated/not-differentiated cells (Figures 3 and 4).

Fig. 2. Histochemical staining of MSCs (up) andTAFs– derived
adipocytesusingFig. 2.(down)
Histochemical stainingof MSCs (up) andTAFs (down) – derived
adipocytesOil
using
OilRedRed O, afterdays of culture.
Red droplets
lipids inside the Red
cytoplasm,
nuclei stained with
O,21after
21 daysofof culture.
dropletsofhematoxilin (x 200)
lipids inside the cytoplasm, nuclei stained withhematoxilin (x 200)

hemical/immunofluorescence staining using anti mFABP4 antibody showed that bothwere completely differentiated into adipocytes by the end of 21 days of culture, nopresenting positive staining for this marker. We were able to count positive cells ando between differentiated/not-differentiated cells (Figures 3 and 4).

2009.19.4(64)  Fiziologia - Physiology

cmyk

Fig. 4. Immunofluorescence MSCs (up) andTAFs (down) – derived adipocytes (FABP4)Fig.
4. Immunofluorescenceand TAFs (down)– derived adipocytes(FABP4) after
after
21 Immunofluorescencedays MSCsof (up)culture,secondaryantibody
4.
MSCs
and
21Fig.
days of culture,secondary antibody conjugated with AlexaFluor488(green (up)fluorescence)
andconjugatedwith
AlexaFluor488(greenTAFs (down)AlexaFluor546– derived
adipocytes(FABP4)(red fluorescence) (x 40)
after 21 days of culture, secondary antibody
conjugatedwith
AlexaFluor488(green23

fluorescence)andfluorescence) (x 40)

AlexaFluor546

C.

Fig. 5. RT-PCR for A. PPARȖ, B. LPL, C.
C/EBPĮ in random samples in day 3 ofdifferentiation of both MSCs and TAFs

(red

For quantitative analysis, we used qRT-PCR to determine relative value of genes conc
the beginning we wanted to test the presence of these genes in undifferentiated MSCs an
different passages. We concluded that all 3 genes analyzed were present in both MSCs a
increased amounts at early passages, but they tend to decrease in late passages. PPARȖ is dow
in MSCs and TAFs alongstarting
withwithpassagenumber,so14thatat werepassagevaluesGene expression patternpassage 6, whileat passage
the values
2.5 times14
lower,
in both measured we
thancellattypes
passageLPL
wasdown-regulatedwith passage 6, whil
RT-PCR was performed on random samples of lower
RNA for bothdif- 4 (Figure
cell types6).
havingalmostthe also
same value(Figure 7). While thestarting
rest of the molecularvalues whether
were 2.5 times
lower,
in both cellhavingalmostsame value (Figure 7
ferentiated towards the adipocytic lineage in day 3, in14
ordertheto determine
markers
are down-regulated
alongtypes
the passage
number,C/EBPαtheis up-regulated
ofgenes
the weremolecular
markers
are4 down-regulated
number,they express or not the specific markers of adipocytes.rest
All threetested:from passage
towards passage 14, whenalong
we havethe
doublepassage
amount comparedwith C/EBPĮ is u
passage
4 towards
passage14,
when
weis consistent
have double
amountcomparedwith the initial
PPARγ, LPL, and C/EBPα and we observed presence offrom
amplified
554 bp, 505
bp,
the initial one(Figure8). Thiswith the positive
regulationof expression
8). Thisthe positive
ofinexpression
and 656 bp fragments respectively in all samples analyzed,
even inisthisconsistent
early phase withbetween
PPARγ andregulation
C/EBPα described
the literature. between PPARȖ and C/EBPĮ
the literature.
of differentiation.

ed on random samples of RNA for both cell types differentiated towards thein order to determine whether they express or not the specific markers of
were tested: PPARȖ, LPL, and C/EBPĮ and we observed presence ofd 656 bp fragments respectively in all samples analyzed, even in this early
PPAR gamma
6

Reference value

5
4
TAF

3

MSC

2
1
0
P4

A.

P6

P9

P14

Fig.
6. PPARȖ
expressionin MSCs
anddown-regulation
TAFs at differentofpassages.
Noteexpression
thesignificant
times
Fig. 6. PPARγ
in MSCs and TAFs10
at different
passages. Note the significant 10
this genetimes
in latepassages
of
bot
cell
types
down-regulation of this gene in late passages of bot cell types
LPL
LPL
0,12
0,12

Reference
Referencevalue
value

0,1
0,1
0,08
0,08
TAF
TAF
MSC
MSC

0,06
0,06
0,04
0,04
0,02
0,02

00

B.

P4
P4

P6
P6

P9
P9

P14
P14

Fig. 7. LPL expression in MSCs and TAFs at different
passages.
Down-regulationof atLPL
present
in bothof LPL
cellFig. 7. LPL
expression in MSCs and TAFs
differentis
passages.
Down-regulationisin both cell types in a similar manner
types inin aa similar
similarpresent
mannertypes
mannerC/EBP alpha
alpha
C/EBP
1,4
1,4

Reference
Referencevalue
value

1,2
1,2

11
0,8
0,8

TAF
TAF
MSC
MSC

0,6
0,6
0,4
0,4
0,2
0,2
0
0

C.

P4
P4

P6
P6

P9
P9

P14
P14

Fig. 8.
8. C/EBPĮ
C/EBPĮ isis up-regulated
up-regulated progressively
progressively towards
towards ate
ate
Fig.
Fig. 8. C/EBPα
is up-regulated
progressively
towards
ate passages inin bothcell types,
morepassagesin bothbothcell types,
types,
more
significant
TAFs
when
passages
in
cell
more
significant
in TAFs
when
compared to MSCs (p < 0.001)
compared toto significant
MSCs in(p(pTAFs< when0.001)
comparedMSCs<
0.001)The differentiation program was submitted to qRT-PCR analysis in key time
The differentiationdifferentiation program
program was
was submitted toto qRT-PCR analysis
analysis inin keyThe
key timetime poin
poin
pointsfor both MSCs andsubmitted
TAFs. We analyzed qRT-PCR
the cells during the differentiation
Fig. 5. RT-PCRPPARȖ,B.
LPL,
C.
Fig. 5. RT-PCR forfor
A. PPARγ, B.A.
LPL, C. C/EBPα
inMSCsrandom samples
in
day
3
MSCs
and
TAFs.
We
analyzedthecells
duringthedifferentiation
process
in
days
3,
5,
77 aa
and TAFs. We analyzed
the
cells
duringthedifferentiation
process
in
days
3,
5,
process in days 3, 5, 7 and day 21, when the experiments were stopped and weof differentiation of both MSCs and
TAFs the experiments were
when
stoppedandweestimated
the
rate
of
differentiation
process
when inthe experiments
stoppedwe estimatedthe rate of differentiation
process
C/EBPĮ in random samples
daypresence
3 wereofestimated
the rate ofand
differentiation
process by staining
and presencetechniques andand presenceof molecularmolecular
markers.
PPARȖ,LPL techniquesand C/EBPĮC/EBPĮ
were themolecutechniquesofmarkers.
PPARȖ,
LPL
and
were
the
molecuof molecularmarkers. PPARγ, LPL Overall,and C/EBPα werethemolecular
markers
used toFor quantitativeof
analysis,
we usedMSCs
qRT-PCR to determine
relative
value
of
genes
differentiation
both
and
TAFs
used
to
assesstheevolutionofadipogenesis.
all
three
genes
presented
up-regulat
used to assess the evolutionofadipogenesis.
Overall,
all
three
genes
presented
up-regulatof adipogenesis.
all three genes presentedpathway
up-regulationin day 21. PP
concentration. In the beginning we wanted to test theexpression,presence of thesestarting
genes in withexpression,
startingwith assessdaythe00evolution
towards
the endendOverall,of differentiationdifferentiationday
towards
theofpathwayin day 21. PPof
theirexpression,
startingwithday
0
towards
theendof
differentiation
pathway
undifferentiated MSCs and TAFs, at different passages.
We concluded
that all 3in
regulated
mainlyin TAFs-derived
TAFs-derived adipocytes,
adipocytes, while
while thethe gene
gene maintains
maintainsa relative
constaregulated
mainlyarelativein day21. PPARγ
is up-regulated
mainlyin TAFs-derived
adipocytes, whileof
the mature consta
genes analyzed were present in both MSCs and TAFs inMSCs-derived
increased amounts at adipocytes,
early
MSCs-derived
adipocytes,
from
day
0
towards
thecompletedifferentiationfromdaya0relative
towards
differentiation
mature adipoc
adipocgene
maintainsconstantthe
valuecomplete
in pre-adipocytes-mature
MSCs-derived
adipocytes, from ofdaypassages, but they tend to decrease in late passages. 21
PPARγ
is down-regulatedin
21 (Figure(Figure
9).
LPL,
which
is
the
gene
involved
inadipocytes
con9). LPL, which
is
the
gene
involvedinpre-adipocytes-matureadipocytescon
0 towards
the complete
differentiationof mature adipocytes
in day 21 (Figure9). after 7 days
MSCs and TAFs along with passage number, so that at increasing
passage
14 valuesthemeasuredincreasing
the
lipid
uptakewithinthecells,
isincreasing
its
expression
mainlylipid uptake
withinthecells,
isincreasing
its
expression
mainlyafter
7
days
whichTAFs
is the gene(Figure
involved in pre-adipocytes-mature
conversion,by value in MS
were 10 times lower than at passage 4 (Figure 6). LPL
was also similar
down-regulated
almostsimilar forfor MSCs
MSCsLPL,and
and10). C/EBPĮC/EBPĮ adipocytes
maintainslowalmostTAFs (Figure
10).
maintains
aa low
value in MS
adipocytes, but
but its
its value
value increases
increases significantly
significantly (2x)(2x) inin TAFs-derived
TAFs-derived adipocytes
adipocytes inadipocytes,
in dayday 2121 ((Thisproves
that
in
this
cells
the
adipogenesis
processisnotsimilar
with
MSCs
and
that
Fiziologia- Physiology
2009.19.4(64)This proves that in this cells the adipogenesis
process
is not 
similarwith MSCs and that24
abnormal inter-relationinter-relation between
between thethe normal
normal markers
markers involved
involved inin generation
generation ofof adipocytes.
abnormaladipocytes.

sis, we used qRT-PCR to determine relative value of genes concentration. In
test the presence of these genes in undifferentiated MSCs and TAFs, atluded that all 3 genes analyzed were present in both MSCs and TAFs inssages, but they tend to decrease in late passages. PPARȖ is down-regulated
ith passage
cmyk number, so that at passage 14 values measured were 10 timesre 6). LPL was also down-regulated starting with passage 6, while at passage
PPAR gamma
PPAR gamma

2,5
2,5

e
lue

2
2

Reference value

differentiation program was submitted to qRT-PCR analysis in key time points for both
TAFs. We analyzed the cells during the differentiation process in days 3, 5, 7 and day 21,
xperiments were stopped and we estimated the rate of differentiation process by staining
and presence of molecular markers. PPARȖ, LPL and C/EBPĮ were the molecular markers
ess the evolution of adipogenesis. Overall, all three genes presented up-regulation of theirstarting with day 0 towards the end of differentiation pathway in day 21. PPARȖ is upmainly in TAFs-derived adipocytes, while the gene maintains a relative constant value inved adipocytes,increasingfrom theday
towards
thecells,
completeof mature adipocytes
in fibroblastsday (TAFs) are recently described (11) and characterlipid0uptake
within theis increasingdifferentiation
its expression mainly after
Tumor associated9). LPL, which
the geneinvolved
conversion,
byprogression and development is still unknown. They
7 daysisin culture,almost similar
for MSCsinandpre-adipocytes-mature
TAFs (Figure 10). C/EBPα maintainsadipocytes
ized, buttheir role in tumor
he lipid uptake
the cells, adipocytes,
is increasing
its increases
expression
mainly7 daysin characteristics
culture, with MSCs, including surface expression markers
a lowwithinvalue in MSCs-derived
but its value
significantly(2x) afterpossess
similarilar for MSCsin TAFs-derived
and TAFs(FigureC/EBPĮ
maintainsadipocytes
in day10).
21 (Figure11). This provesthat in this acellslow
the valueandintheMSCs-derived
ability to differentiate towards osteoblasts, chondrocytes and adipocytes.
but its value adipogenesis
increasesprocess
significantly(2x)MSCs
inand
TAFs-derived
adipocytes
21 (Figure
is not similar withthat we have an abnormalinter- in day
As demonstrated
by our11).
experiments, these cells can be easily differentiated ands that in this cells
adipogenesis
process
not similar
with MSCs andthattowehave
relation the
betweenthe normal markers
involved inisgenerationof adipocytes.
inducedtransform
intoanadipocytes, in almost the same proportion as MSCs, but
ter-relation between the normal markers involved in generation of adipocytes.
the molecular marker pattern is different, suggesting an abnormal regulation of
PPAR gamma
the differentiation and maturation processes. This can be a useful tool and TAFs
2,5
can become an in vitro model to study adipogenesis within tumors, as well as therelation with vasculature and tumor progression.
2
Considering the present findings we may conclude that some molecular
1,5
TAF
markers,
considered characteristic for adipogenic differentiation are phenotipicallyMSC
1
present in MSCs and TAFs, being up-regulated during the differentiation process
0,5
and down-regulated with passage number if no inductor is present within theirenvironment. TAFs also exhibiting the same characteristics as MSCs could be
0
D0
D3
D5
D7
D21
an indicator of their common origin and development, thus contributing to acFig. 9. Significant up-regulation of PPARȖ in TAFs comparedcumulation of tumor-associated adipcytes. This could be a promising pathway
9. Significant
up-regulationadipocytes.
of PPARγ in TAFs comparedto MSCs –thederivedgene
adipocytes.
to Fig.
MSCs– derived
In MSCsisfor targeting specific molecules involved in early adipocytic differentiation, thus
the gene isvalue
maintaining
maintainingInaMSCs
constant(p <a constant0.5) value (p < 0.5)
preventing accumulation of mature adipocytes and development of obesity, while
the tumor-associated adipocytes must be further investigated for determining their
LPL
LPLrole in tumor progression.
1,2
1,2

Reference
value
Reference
value

1
1
0,8
0,8

TAF
TAF
MSC
MSC

0,6
0,6
0,4
0,4
0,2
0,2
0
0

D0
D0

D3
D3

D5
D5

D7
D7

D21
D21

Fig.
10.
Significant
up-regulationof LPLinx both
both
cell
types
(5
Fig.
10.Significant
Significant up-regulationof LPL in bothofcellLPL
types (5increase)cell
proving
adipocytic
Fig.
10.
up-regulation
in
types
(5
increase) proving
proving adipocytic
adipocytic
differentiationdifferentiation
xx increase)differentiation

Acknowledgements
The work in this paper was supported by PNCD-II, Project 1748/2008(financing agency CNCSIS) and Project 8844/2009 (financed by UMF “Victor
Babes”Timisoara).
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while
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adipocytes there
there isis aa 33 xx increase
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rogenitor
cells
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are
unknown
(14).
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of
extracellularmatrix during tumor onset and progression.
rogenitor cells in vivo are unknown (14).
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used to study
adipogenic differentiationdifferentiaJof
Cellcells
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study
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rge
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differentiation 9.of
cells frombone
Wiseman
BS, Werb
Z. Stromal effects on mammary gland develtionuseof cellshas
frombeenbone marrow
or ofbone.murine
Extensivecell
use haslines
been madeof asmurinebone.
Extensive
made
such
BMS2
(15),
UAMS33(16),
bone. Extensive use has been made of murine cell lines such as BMS2 (15),
UAMS33(16),
opmentand breast
cancer. Science. 2002; 296: 1046-49.
cell lines
such as from
BMS2 (15),(16), and(18).
2T3 (17),
as wellhumanas line derived), as
as wellwell asas lineline
derivedp53UAMS33
null mice
mice
Also,
cell lines
lines have
have
beenJ. Mesenchymal
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one stem cells in cancer. Stem Cell Rev. 2008;
10. Stagg),
derivedfrom p53
null(18). Also,
human cell
been
used:
from p53(19)null mice(18).other
Also, humancell lines havefrom
been used:
one transformed
4:
119-24.used in thefromosteoblasts
andtransformed
MSC(20-22).
The
MSCs
from osteoblasts (19) and other transformed from MSC (20-22). The 11.
MSCs
usedDembinski
in theSpaeth EL,from osteoblasts
and otherfrom(20-22).
MSCsbeusedort have
have thethe advantage
advantage
that(19)they
they
are transformed
primary cells
cellsMSC
andtheyThecan
can
expanded rapidly
rapidlyin culture
culture JL, Sasser AK, et al. Mesenchymal stem cell
ort
that
are
primary
andthey
be expandedintransition to tumor-associated fibroblasts contributes to fibrovasin theof
present
report havetheadvantagethat they are primarycells and they candequate amounts
amounts
material
to study
study
adipogenesis
in detail.
detail.
cular network expansion and tumor progression. PLoS One. 2009;dequateof material
toadipogenesis
inexpanded rapidly
in cultureareto provide
adequate
amounts of material
to studyor associatedassociated befibroblasts(TAFs)
recently
described(11) andandcharacterized,
but their
their rolerole
4: e4992.
orfibroblasts(TAFs)
are recently
described(11)
characterized,
but
in detail.is still unknown. They possess similar characteristics
12. Singhwith
S, RossMSCs,
SR, Acena M, et al. Stroma is critical for preventing
ogression and adipogenesis
development

ogression and development is still unknown. They possess similar characteristics with MSCs,
urface expression
expression markers
markers andand thethe ability
ability toto differentiate
differentiate towards
towards osteoblasts,
osteoblasts, chondrocyteschondrocytes
urfacetes.
Asdemonstratedbyourexperiments,
these
cells
can
be
easilydifferentiatedand induced
inducedtes. As demonstrated
by ourexperiments,
these cells can be easily differentiated and2009.19.4(64)Fiziologia - Physiologyminto
adipocytes,
inalmostthesameproportion
as
MSCs,
but
the
molecular
markerpatternism into adipocytes, in almost the same proportion as MSCs, but the molecular marker pattern isggestinganabnormalregulationofthedifferentiation
andmaturation
processes.
This
can
be
ggesting an abnormal regulation of the differentiation and maturation processes. This can be aa
and
TAFs cancancmyk
become anan inin vitro
vitro model
model toto study
study adipogenesis
adipogenesis within
within tumors,tumors, as
as wellwell asas thetheand TAFs
becomehvasculature
and
tumor
progression.
h vasculature and tumor progression.
sidering thethe present
present findings
findings we
we maymay conclude
conclude thatthat some
some molecularmolecular markers,
markers, considered
consideredsidering

25

or permitting immunological destruction of antigenic cancer cells.
J Exp Med. 1992; 175: 139-46.
13. Sullivan NJ, Hall BM. Mesenchymal stem cells in tumor stroma.
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Springer; 2009. p. 29-38.
14. Tang W, Zeve D, Suh JM et al. White fat progenitor cells residein the adipose vasculature. Science 2008 ; 322 : 583-86.
15. Kelly KA, Gimble JM. 1,25-dihydroxy vitamin D3 inhibits
adipocyte differentiation and gene expression in murine bonemarrow stromal cells clones and primary cultures. Endocrinology
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16. Lecka-Czernik B, Gubrij I, Moerman EJ et al. Inhibition of Osf2/
Cbfa1 expression and terminal osteoblast differentiation by PPARgamma2. J Cell Biochem 1999; 74: 357-71.
17. Ghosh-Choudhury N, Windle JJ, Koop BA et al. Immortalized
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transgenic mice. Endocrinology 1996; 137: 331-9.

18. Thompson DL, Lum KD, Nygaard SC et al. The derivation andcharacterization of stromal cell lines from bone marrow of p53-/mice : New insights into osteoblasts and adipocyte differentiation.
J Bone Miner Res 1998; 13: 195-204.
19. Bodine PV, trailsmith M, Komm BS. Development and characterization of a conditionally transformed adult human osteoblastic
cell line. J Bone Miner Res 1996; 11: 806-19.
20. Colter DC, Sekiya I, Prockop DJ. Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells
in colonies of human stromal cells. Proc Natl Acad Sci USA 2001;
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21. Gronthos S, Zannettino AC, Hay SJ et al. Molecular and cellularcharacterization of highly purified stromal stem cells derived fromhuman bone marrow. J Cell Sci 2003; 116: 1827-35.
22. Prabhakar U, James IE, Dodds RA et al. A novel human bonemarrow stroma-derived cell line TF274 is highly osteogenic in vitro
and in vivo. Calcif Tissue Int 1998; 63: 214-20.

CARACTERIZAREA IN VITRO A MECANISMELOR IMPLICATEIN DIFERENTIEREA ADIPOGENICA A CELULELOR STEMMEZENCHIMALE SI A FIBROBLASTELOR ASOCIATE TUMORALREZUMAT
Scopul acestui studiu l-a constituit investigarea mecanismelor moleculare care intervin in reglarea adipogenezei in diferite stadii de diferentiere, atat la celulele stem mezenchimale (MSC), cat si la fibroblastele associate tumoral (TAF), luand in considerare potentialul acestora
de a se transforma in celule care prezinta vacuole lipidice intracitoplasmatice. Celulele aflate la pasajul 2 pentru MSC si respectiv la pasajul 4pentru TAF au fost induse timp de 21 de zile spre linia dipocitara, in mediu de cultura specific. La anumite intervale de timp sugestive pentru
diferentiere, s-a efectuat extractia ARN total, urmata de analiza RT-PCR si qRT-PCR, precum si colorarea histochimica a celulelor, pentru a
confirma inucerea spre linia adipocitara. Tehnicile de imunocitochimie au utilizat anticorpul anti-FABP4, care este specific pentru adipocitele
mature, relevand diferentierea completa a ambelor tipuri de celule dupa 3 saptamani de cultura. Cresterea si scaderea expresiei diferitilor
markeri adipocitari, cum ar fi PPARγ, LPL si C/EBPα se poate suprapune pe modelul diferentierii adipocitare in mod asemanator in ambele tipuri
de celule investigate, fiind o directie promitatoare pentru studiul acumularii adipocitelor mature si dezvoltarii obezitatii, precum si pentru
determinarea rolului tesutului adipos in progresia tumorala.
Cuvinte cheie: celule stem mezenchimale, adipocite, fibroblaste associate tumoral, adipogeneza, marker molecular

26

cmyk

Fiziologia - Physiology  2009.19.4(64)Normal and abnormal in medicine: epidemiologicalassessment of cholesterol levels in medical
practice
BRUMBOIU IRINA1, URSU LIGIA2, SIMIONCA G3, CHELARU E4
1
Epidemiology Department of “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca
2
Hospital of Infectious Diseases, Cluj-Napopca, Romania
3 st
1 year Intern Anatomopathology, “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca
4 nd
2 year student at “Iuliu Hatieganu” University of Medicine and Pharmacy Cluj-Napoca, Romania

ABSTRACTThe objective of this paper was to create a model for evaluation of cholesterol levels in medical current activity. For finding the key factors for an assessment model we analyzed comparatively the levels of total cholesterol and high-density lipoprotein (HDL) in 244 patients with atherosclerotic cardiovascular diseases and in 2301 subjects without cardiovascular pathology, identified between 2003 and 2007.
In cardiovascular patient group the average value of cholesterol was 230.1 mg% and in non cardiovascular subjects, 225.2 mg%. The HDL for male patientswith cardiovascular disease was agglomerated around 35-39 mg% and around 50-54 mg% for male without cardiovascular diseases. For women, the pattern was much more complex with a multimodal distribution of the cardiovascular female patients, placed in the high level area of HDL values.
In medical practice the cut-off cholesterol values are more complex to be established and more evaluations are necessaries. Usual assessment of normal
and abnormal cholesterol and HDL cholesterol levels can be used for decision analysis of the necessity of a treatment or the risk of complications.
Key words: normal, abnormal values, cholesterol

INTRODUCTION
The cardiovascular diseases are the lead cause of death and disability in thedeveloped world and in the last years became to be in the majority of in developing country. This deadly pathology has in the greatest majority of cases, theatherosclerotic nature. Lipids implication in the pathological mechanism of theatherosclerotic forming plaques was demonstrated to be very important (4, 6, 9).
A better knowledge about the lipid profile in different population groups, guide to
identification of some particular aspects who facilitate or represent different risks
factors for cardiovascular diseases and can have implications in prophylactic and/or therapeutic recommendations (1, 3, 5, 7, 10, 12).
To understand health problems, like atherosclerotic cardiovascular diseases,
the new method of modeling based on epidemiological methods, already used in
medical activity for solving different problems identified in clinical activity, seems
to be very useful. In the atherosclerotic pathology, for construction models, there arenecessary adequate epidemiological studies for a valid selection of right parameters
to be used in modeling process.
The objective of this work was to elaborate a model for the assessment of thecholesterol and HDL cholesterol levers in practical activity for caring patients withand without atherosclerotic cardiovascular diseases.

MATERIAL AND METHODS
The samples were constituted by patients with atherosclerotic cardiovasculardiseases and subjects without this pathology, apparently healthy, who voluntarily
participates in the period of 2003 – 2007 in our study. The including criteria were age
over 18 years, both gender, persons provided from urban and rural area, voluntary
participation and having some documentations signed by a medical doctor, withthe registration of cholesterol and HDL cholesterol levels measured in mg% anda diagnosis. The selection intended to elevate the likelihood of the sample to be
representative for regional adult population. We didn’t include the patients withgrave form of diseases and patients with immunodeficiency.
The tests for cholesterol levels were made in different laboratory of the north vest region of the country, where patients included in the study were assisted for any
reasons of the prophylactic or curative medical acts. Booth assistance like inpatientsand outpatients were accepted. The labs of the region usually use for measurementof cholesterol levels, the enzymatic colorimetric method and for HDL cholesterol,the direct method with enzyme PEG modified and dextral sulfate.
Data registration was directly in an informatics data base, performed in Excel
program of Microsoft Office Windows 2002. The confidentiality was assured byanonym registration of all data. For statistic we used Excel and EpiInfo 2002. The
statistic significance was accepted at level of p<0.05.
Possible errors may have been induced by the method of collecting data forbooth inpatients and outpatients, the utilization of all results of cholesterol levels
made by different laboratories, the voluntary participation and the possibility ofthe duplicates because of anonym registration.

RESULTS
The sample included 2545 adult subjects, divided in two groups, first composed by 244 patients with atherosclerotic cardiovascular diseases, and the second
composed by 2301 subjects without this pathology. In cardiovascular group, 63.9%
were male (36.1% female) and in no cardiovascular group, male were 54.5%(45.5% female), difference statistically significant (p<0.05).
The cholesterol in cardiovascular patient group, had average value 230.1 mg%

Received April 2008. Accepted July 2008. Address for correspondence: Brumboiu Irina, “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca, Epidemiology Department,
13 Emil Isac Street, Cluj-Napoca, Romania, 400023, tel. +0264-430444, +0264-594655, fax. +0264-430444, email ibrumboiu@umfcluj.ro, irinabrumboiu@yahoo.com
2009.19.4(64)  Fiziologia - Physiology

cmyk

27

8.5

1.2

2.6
0

1.2

1.2

5

5.7

6.9

10

0
<79

80-119120-159

160-199200-239240-279

280-319

peste 320

cholesterol (mg%)

No cardiovascular diseases

Cardiovascular diseases

Fig.2. The distribution of female patients with atherosclerotic cardiovascular diseases and subjects without cardiovascu
diseases, after total cholesterol levels

22.3
25

27.2
27

30

19.4

25

11.2

15

13.2

12.7

15.8

20
% of subjec

95%:
222.9
– 237.3),
higher(p<0.05)then
225.2 mg%
(IC 95%:
medical(IC
acts.
Booth
assistancelikesignificantly
inpatients andoutpatientswere
accepted.
The labsof the region
usually 222.6
use for– measurementofwas
cholesterollevels, the enzymaticcolorimetricmethod values
and for HDL
227.9),
witchinnocardiovasculargroup(TableI).
Cholesterol
cholesterol, the direct method with enzyme PEG modified and dextral sulfate.
distribution
had wasa predomination
subjects without
cardiovascular
in of
Data
registration
directly in an ofinformatics
data base,performed in diseases
Excel program
Microsoft Office Windows 2002. The confidentiality was assured by anonym registration of all data. Foralowvaluesarea,
inferior
to
240
mg%
and
in
highervaluesarea,
patientswithstatistic we used Excel and EpiInfo 2002. The statistic significance was accepted at level of p<0.05.
Possibleerrors may
have been
inducedthe methodof collecting
for distribution
booth inpatients andcardiovasculardiseases,becamemostbyfrequently(TableI, Figuredata1). Theoutpatients, the utilization of all results of cholesterol levels made by different laboratories, the voluntaryof cholesterol
value in offemale
subjectsbecausewith cardiovasculardiseases was in plateau
participationand the possibility
the duplicatesof anonym registration.

6.6

2.5
1.3

0.2
0

6.5

9.1

between 160 and 279 mg%, a very different aspect from female without that
RESULTS
10
Thesampleandincluded2545
adultwere
subjects,
divided indifferent
two groups,
244 patients
diseases,
averages
valuessignificantly
withfirst
225,8composed
mg% (ICby95%:
with atherosclerotic cardiovascular diseases, and the second composed by 2301 subjects without this
5
215.6
– 236) in cardiovasculargroup,
respectively
219.3
mg%
(IC 95%: group,
pathology.
In cardiovasculargroup, 63.9%patientwere male(36.1%
female) andin nocardiovascularmale were 54.5% (45.5% female), difference statistically significant (p<0.05).
215.3–
223.4)
infemalegroup
without
cardiovasculardiseases(TableI,
Figure
The cholesterol in cardiovascular patient group, had average value 230.1 mg% (IC 95%: 222.9 –
0237.3), 2).
significantly
higher(p<0.05) ofthen
225.2 mg%witch
was in no
In male, thedistribution
cholesterollevels(ICwas95%:
very222.6
similar– to227.9),
generalaspect<79
80-119 120-159 160-199 200-239 240-279 280-319
peste
cardiovascular group (Table I). Cholesterol values distribution had a predomination of subjects without320
of all subjects
includedin thearea,sample,withthemg%
predomination
cardiovascularcardiovasculardiseases ina low values
inferior
to 240and in higherofvalues
area, patients withcholesterol (mg%)
cardiovascular diseases, became most frequently (Table I, Figure 1). The distribution of cholesterol value
patients
inarea
higher
then
240
mg%,
and
the
averagevalues
of
242.2
mg%
(IC
in female subjects with cardiovascular diseases was in plateau between 160 and 279 mg%, a very
No cardiovascular diseases Cardiovascular diseases
different95%
aspectfrom–female
that diseases, patientsand averages
were significantly
different with
232.7
251.7)without
in cardiovascular
was values
significantly(p<.05) differ225,8 mg% (IC 95%: 215.6 – 236) in cardiovascular patient group,
respectively
219.3 mg%
(IC 95%:
Fig.3.
Distributionof malepatients with atherosclerotic cardiovascular diseases and subjects without cardiovascular disea
fromin229.7
mg%(IC without
95% 226.2– 233.2) indiseases
subjects(Table
without
cardiovascular
215.3 –ent
223.4)
femalegroup
cardiovascular
I, Figure2). In male, theFig.3. Distributionafterof maletotal
patients
with atheroscleroticcholesterollevelscardiovascular diseases and subjects
distribution of cholesterol levels was very similar to general aspect of all subjects included in the sample,
without cardiovascular diseases, after total cholesterol levels
diseases(TableI,
Figure3).
with the predomination of cardiovascular patients in area higher then 240 mg%, and the average values of242.2 mg% (IC 95% 232.7 – 251.7) in cardiovascular patients was significantly (p<.05) different fromin females with cardiovascular diseases had the average value 49.9 mg%
229.7 mg% (IC 95% 226.2 – 233.2) in subjects without cardiovascular diseasesHDL(Table I,cholesterol
Figure 3).
Table I. Levels of cholesterol and HDL cholesterol in patients with atherosclerotic cardiovascular

HDL higher
cholesterolin femaleswith cardiovasculardiseases
had thein females wit
95%: 46.9 – 52.8) significantly
then
47.7 mg%(IC 95%: 46.5
– 48.8)average value(Table
49.9 mg%
46.9values– 52.8)distribution
significantly was
higherGaussian in fem
cardiovascular diseases (0.1<p>0.05)
I). (IC
The95%:
HDL
then 47.7 mg%diseases,(IC 95%: 46.5
48.8)symmetricin females without
cardiovascularwithout atherosclerotic cardiovascularand –withagglomerationof values arounddiseases(0.1<p>0.05)cardiovascular(Table I). The HDL diseases
values distribution
was Gauss-distribution of H
44 mg% (fig. 4). Women withatherosclerotic
had a complex
values, with two conglomerations,
one without
aroundatherosclerotic
40-44 mg%cardiovascularand the second
around55-59 mg%. Men
ian in female
diseases,
and withsymmetric agglomeration of values around 40-44 mg% (fig. 4). Women
with atherosclerotic cardiovascular diseases had a complex distributionof HDL values, with two conglomerations, one around 40-44 mg% andthe second around 55-59 mg%. Men with cardiovascular diseases had anormal distribution of HDL values, with dispersion around 35-39 mg%,and thus without cardiovascular diseases was centered in 50-54 mg%(fig. 5). The average was 53.5 (IC 95%: 51.1 – 56) for cardiovascularpatients and masculine gender, and 56.6 (IC 95%: 55.5 – 57.7) in men
30
without cardiovascular diseases, difference statistically significant (Table
30
I). The distribution of HDL for all subjects without cardiovascular diseases
25
25
was relatively symmetric centered around 50-54 mg%, and for patients
20
20
with atherosclerotic cardiovascular diseases, the distribution was asym15
15
metrical with two peak around 40-44 mg% and 50-54 mg% (fig. 6). The
10
HDL average for all cardiovascular patients was 52.2 mg% (IC95%: 50.3
105
– 54.1) and 52.55 mg% (IC95%: 51.8 – 53.4) for all subjects without
5
that pathologies, no significant difference (Table I).
00<79
80-119120-159 160-199 200-239 240-279 280-319 peste 320
The average levels of total and HDL cholesterol in both cardiovascular and no<79
80-119120-159 160-199 200-239 240-279 280-319 peste 320
cardiovascular groups for female were constantly and significantly lower then in
No cardiovascular diseases Cardiovascular diseases
No cardiovascular diseases Cardiovascular diseases
g.1. The distribution of patientswithatherosclerotic
cardiovasculardiseasesand subjects
without
cardiovascular diseases,
correspondent male groups (Table I).
Fig.1.with
The atherosclerotic
distributionof patientswith atherosclerotic
cardiovasculardiseasesand subjects
1. The distribution of patients
cardiovasculardiseasesand subjects
without
cardiovascular diseases,
after totalcholesterol levels
withoutafter
cardiovasculardiseases,
after
total
cholesterollevelsVariability of parameters, measured by coefficient of variation, was the highest
total cholesterol levels
30
30
in women without cardiovascular diseases, for total cholesterol (29.9%) and for25
HDL cholesterol (38.5%) (Table I). Lowers variability was in the group of women
25
20
with atherosclerotic cardiovascular diseases for both total cholesterol (21.1%) and20
HDL cholesterol (28%) (Table I). The cardiovascular diseases groups, comparatively
15
15
to no cardiovascular groups, had lowers variability for total and HDL cholesterol
10levels to. Constantly, HDL cholesterol had highest variability then total cholesterol
105
in all analyzed groups.
50Validity assessment of tests for total cholesterol for utilization in care
<79
80-119120-159
160-199200-239240-279
280-319 peste 3200assistanceof cardiovascular patients was poor for cut-off at 200 mg% total<79
80-119120-159
160-199200-239240-279
280-319 peste 320
No cardiovascular diseases
Cardiovascular diseases
cholesterol with 45.1% for sensibility and 61.7% for specificity (Table II). The
No cardiovascularcardiovasculardiseases
Cardiovasculardiseasesig.2. The distribution of female patients with atherosclerotic
diseasesand subjects without cardiovascularsensibility of HDL cholesterol grows and the specificity low down to the highest
diseases,
after total cholesterollevels
.2. The distribution of female patients withatherosclerotic
cardiovasculardiseases and subjects without cardiovascularFig.2. The30distributionof femaleafter
patients
atherosclerotic
cardiovascular diseases and subjects
cut-off values, and for 45 mg% cut-off, sensibility was 44.4% and specificity
diseases,
totalwithcholesterollevelswithout cardiovascular diseases, after total cholesterol levels
30
55.8% (Table III).
7

7

6.1
6.1

10.8
10.8

13.1
13.1

21.4
21.4
25
25

2.72.70.80.80.80.82.12.1

6.5
6.5

10.3
10.3

% of diseas% of diseas

20.7
20.7
19.3
19.3

27.2
27.2
26.2
26.2

Table I. Levels of cholesterol and HDLcholesterolin patientswith atherosclerotic
diseasesand subjects
without
this pathologycardiovascular diseases and subjects
without this pathology
Patients category
Parameter
Average valueStandard
Confidence
Variation(mg %)
deviationintervalcoefficient(IC95%)(%)
Cardiovascularcholesterol
242,2
58,8
232.7 – 251.7
24,3
diseases, male patients HDL
53.5
15.4
51.1 – 56
28.7
cholesterol
Cardiovascularcholesterol
225.8
47.7
215.6 – 236
21.1
diseases, female
HDL
49.9
13.9
46.9 – 52.8
28
patients
cholesterol
Cardiovascularcholesterol
230.1
55.5
222.9 – 237.3
23.5
diseases, all patients
HDL
52.2
14.9
50.3 – 54.1
28.6
cholesterol
No cardiovascularcholesterol
229.7
61.5
226.2 – 233.2
26.8
diseases, male subjects HDL
56.6
19
55.5 – 57.7
33.6
cholesterol
No cardiovascularcholesterol
219.3
65.6
215.3 – 223.4
29.9
diseases, female
HDL
47.7
18.4
46.5 – 48.8
38.5
subjects
cholesterol
No cardiovascularcholesterol
225.2
63.3
222.6 – 227.9
28.1
diseases, all subjects
HDL
52.5
19.3
51.8 – 53.4
36.7
cholesterol

cholesterol (mg%)

26.426.4

8.5

5.7
1.2

5.7

8.5

11.5
11.5

20.420.4

27.327.3
26.426.4

26.426.4

6.9 6.9

1.2

0

2.6 2.60

1.2 1.2
1.2 1.2

11.811.8% of subjec% of subjec

22.522.5cholesterol (mg%)

cholesterol (mg%)

13.2

12.7

11.2

11.2

6.6

6.6

9.1 9.1
6.5
2.5
1.3

.5

5
0

0.2
0

10
5

13.2

15
10

cmyk

Fiziologia - Physiology  2009.19.4(64)
12.7

20
15

6.5

% of subjec% of subjec

28

19.4 19.4
15.8 15.8

25

25
20

22.3 22.3
25 25

27.2 27.2
27 27cholesterol (mg%)

6

6.8

8.7

5.7

6

3.4

1.1

0

<19

5.7
3.4

7.2
6.8

5.7

8.5

5.7

6.5

1.1

1.1

1.1

1.1

3

3.8

5

1.1

3

3.8

10

8.7

6.

the
women withatherosclerotic
diseasesdiseases
for both groups,
total
andgroup
HDLofcholesterol(28%)(Table I). cardiovascular
The cardiovascular
5
)
and
HDL
cholesterol(28%)(TableI).
The
cardiovasculardiseasesgroups,cardiovascular groups, had lowers variability for total and HDL cholesterol levels to.
cardiovasculargroups,had lowers
for totalin
andcholesterol
lesterolhad highest
variability
thenvariability
total cholesterol
allHDL
analyzedgroups.levels to.
0olesterol
hadhighestvariability
then
total
cholesterol
inallanalyzedgroups.
<19
20-24
25-29cardiovascular
30-34 35-39 40-44 45-49 50-54 55-59 60-64 65-69 >70
sment of tests for total cholesterol for utilization in care assistance of
tests
formg%
total cholesterol
for utilizationin carefor
assistanceof cardiovascularrssment
cut-offof at200
total cholesterol
with 45.1%
sensibility
and
61.7%
No cardiovasculardiseases for
Cardiovascular diseasesor
cut-off
at
200
mg%
total
cholesterol
with
45.1%
for
sensibility
and
61.7%tocardiovascularforthe. The sensibility of HDL cholesterol grows
andthe ofspecificity
lowdown
Fig.4. The
distribution
female patients withatherosclerotic
diseases and subjects without cardI).
The
sensibilityofHDL
cholesterol
grows
and
the
specificity
low
down
to
the
diseases,
after HDL
cholesterollevelsFig.4.
The
distributionoffemalepatients
withatherosclerotic
cardiovasculardiseasesand subjects without cardiov, and for 45 mg% cut-off, sensibility was 44.4% and specificity 55.8% (Table III).
diseases,
after HDL
cholesterol levels
es, and for 45
mg%
cut-off,
sensibility
was
44.4%
and
specificity
55.8%(TableIII).
Table II. The sensibility and specificity for different cut-of levels of total cholesterol in atheroscle30
HDL colesterol (mg%)

20-24 25-29 30-34 35-39 40-44

45-49 50-54 55-59 60-64 65-69>70

HDL colesterol (mg%)

No cardiovascular diseases

Cardiovascular diseases

20.2

20.2

0

0

1.6

1.6

8.6

3

3

8.6

10.8

12.4

10.8

12.4

4.8

4.8

4.8

4.8

14.3

10.314.3
14.3
15.2
15.2

14.3

8.2

8.2

4.24.2

3.73.72.72.7

1.21.2
4.84.82.52.5
1.21.2

10.3

14.3
14.3

13.1
13.1

% of subjectse% of subjectse

23.823.8

rotic cardiovascular diseases
30
and specificity for different cut-oflevels of total cholesterol in atherosclerotic
cardiovascular diseases
25
and specificity forCholesterol
different cut-of levelsoftotalcholesterolinatherosclerotic
cardiovascular diseases
Sensibility
Specificity
25
Cholesterol
Sensibility
Specificity
20levels(mg %)(%)(%)
20levels (mg %)
(%)
(%)
< 79
0.8
98
15
< 79
0.8
98
15
80-1193.4
97.1
80-1193.4
97.1
10120-159
13.6
90.6
10
120-159
13.6
90.6
160-19934.5
571.3
160-19934.5
71.3
5200-23961.7
45.1
200-239
61.7
0
45.1
0
<19 20-24 25-29 30-34 35-39 40-44 45-49 50-54 55-59 60-64 65-69 >70
240-279
83.1
20.1<19 20-24 25-29 30-34 35-39 40-44 45-49 50-54 55-59 60-64 65-69 >70
240-279
83.1
20.1
nivele HDL colesterol (mg%)
280-31993.9
13.1
280-31993.9
13.1
No cardiovascular diseases
Cardiovascular diseases
>>319
100
No cardiovascular diseases
Cardiovascular diseases
0
319
100
0
nivele HDL colesterol (mg%)

Fig.5.
withatherosclerotic
cardiovasculardiseasesandsubjects
without
cardi
Fig.5.The
Thedistribution
distributionofofmale
male patientspatients
withofatherosclerotic
cardiovasculardiseasesandsubjects
without
cardiova
Fig.5.
The distribution
male patients with atherosclerotic
cardiovasculardiseasesandsubjects
diseases,after
HDL
cholesterollevelsdiseases,after
HDL
cholesterollevelswithout
cardiovasculardiseases,after
HDL
cholesterollevelsTable
III.differentThe
sensibility andcut-of
specificity forlevels
different cut-ofof HDLcholesterol
cholesterollity andforlevelsoflevels
HDL
cholesterol
atheroscleroticcardiovascularcardiovascularbilityandspecificity
specificity
fordifferent
cut-ofof
HDL
ininatherosclerotic
in atherosclerotic
cardiovascular diseases
diseases

10.9

15

7.1

9.2
8.4
9.2
8.4
6.3
6.37.1

10.9

15

13.9
13.9 16.3
16.3
11.5
11.513
13

11.3

10.7

10.7

6.3
6.3
6.7
6.7

5.8
5.8
5.4
5.4

4.5
4.5

1.7
1.7

22
0.8
0.8

3.1
3.1
0.8
0.8

11.7
11.7
11.3

17.6
17.6

diseases25
25
Sensibility
Specificity
Sensibility
Specificity
(%)
(%)
(%)
20
20
0.8
96.9
96.9
94.9
1.7
94.9
15
15
3.3
90.5
90.5
8.8
84.6
10
84.6
10
15.5
78.3
78.3
5
33.1
67.5
5
67.5
44.4
55.8
44.4
55.8
0
0
60.7
41.9
<19 20-24 25-29 30-34 35-39 40-44 45-49 50-54 55-59 60-64 65-69 >70
60.7
41.9
<19 20-24 25-29 30-34 35-39 40-44 45-49 50-54 55-59 60-64 65-69 >70
73.7
30.5
HDL colesterol (mg%)
73.7
30.5
No cardiovascular diseases Cardiovascular diseases
No cardiovascular diseases Cardiovascular diseases
82
21.3
82
Fig. 6.21.3
The distribution of patientswithatherosclerotic
diseases andsubjects
without cardiovascular dFig. 6.
The distributionof patientscardiovascularwith atherosclerotic cardiovascularand subjects
Fig. 6. The
cardiovasculardiseases diseases
and subjects
without cardiovascu
89.1
15
after
HDL
cholesterollevels89.1
15distribution of patients with atherosclerotic
without cardiovascularafter HDL cholesterol
after HDLdiseases,cholesterollevels levels
100
00
100
% of subjec% of subjec

HDL
HDLcholesterol
cholesterollevelslevels(mg(mg %)
%)
<19
<19
20-24
20-24
25-29
25-29
30-34
30-34
35-39
35-39
40-44
40-44
45-49
45-49
50-54
50-54
55-59
55-59
60-64
60-64
65-69
65-69
>70
>70

HDL colesterol (mg%)

DISCUSSIONS

25

19.3

Clinical manifestations of atherosclerotic cardiovascular diseases are more
frequent in male then females, who before menopause have a relatively protection
DISCUSSIONS
conferred by estrogenic hormones who evict hypercholesterolemia (4, 8, 11). SameDISCUSSIONS
studies explain the differences between genders regarding cardio – vascular diseases
15
and lipids levels, by the modification of the expression patterns and effects of genes
and genes products induced by sex steroid and not by the distribution of specific
10alleles and genotype (8). Adder gender’s differentiations are made by activities oflipoprotein lipase and hormone sensitive lipase that are sex steroid modulated. The
5roll of different expression induced by sex hormones became important for clinicalevaluation, evolution prognostic, treatment and prevention.
0<19 20-24 25-29 30-34 35-39 40-44 45-49 50-54 55-59 60-64 65-69 >70
Opposite to our expectations, females with atherosclerotic cardiovasculardiseases had a significantly higher level of HDL cholesterol, than females without
No cardiovascular diseases
Cardiovascular diseases
cardiovasculardiseases. The explanation in part may be in regional nutritional habits
The distribution of female patients with atherosclerotic cardiovascular diseases and subjects without
cardiovascularFig.4. The distributiondiseases,of female patientsatherosclerotic
cardiovascularwith constant utilization of animal fat, usually pork and probably the modification
after with
HDL
cholesterollevels diseases and subjects
without cardiovascular diseases, after HDL cholesterol levelsof nutritional comportment after the disease onset (7).
30High levels of serum cholesterol and low level of HDL cholesterol are known
25
to be risks factors for atherosclerosis and because of that we considered the necessity of inclusion in elaboration of any model useful for clinical decisions (1, 11). In
20
practice the model of cholesterols levels are insufficient for cardiovascular prediction
15
or for other recommendation but in addition to adder factors in complex models
may become useful (2).
10
6.8

3.4

1.1

3

1.1

1.1

5.7

7.2
6.8

8.7

11.3

12.5
13.7

10.3

12.4

8.7

3.8

5.7

6.5

8.5

11.4

% of subjec

14

17

20

0

1.6

3

4.8

4.8

8.6

10.8

12.4

15.2

14.3

10.3

14.3

4.2

3.7

2.7

1.2

2.5
1.2

5

4.8

8.2

14.3

13.1

% of subjectse

20.2

23.8

HDL colesterol (mg%)

02009.19.4(64) Fiziologia - Physiology<19 20-24 25-29 30-34 35-39 40-44 45-49 50-54 55-59 60-64 65-69 >70
nivele HDL colesterol (mg%)

cmyk

No cardiovascular diseases

Cardiovascular diseases

The distribution of male patients with atherosclerotic cardiovascular diseases and subjects without cardiovasculardiseases, after HDL cholesterol levels

29CONCLUSIONSIn our study we observed:- the predominance of male between the patients with atherosclerotic
cardiovascular diseases;- higher levels and a complex distribution of HDL cholesterol for women withatherosclerotic cardiovascular diseases than women without this pathology;- for male patients, lower levels of HDL in atherosclerotic cardiovasculargroup;- the levels and the variability of total cholesterol was higher for all patientswith atherosclerotic cardiovascular diseases than patients without that pathology;- constantly higher variability of HDL cholesterol than total cholesterol in all
patients
REFERENCES
1. Basu AK, Pal SK, Saha S et al. Risk factor analysis in ischaemic
stroke: a hospital-based study. J Indian Med Assoc 2005;11:586588.
2. Brehm A, Pfeiler G, Pacini G et al. Relationship between Serum
Lipoprotein Ratios and Insulin Resistance in Obesity. Clinical
Chemistry 2004;50(12):2316-22.
3. Choi BG, Vilahur G, Yadegar D et al. The Role of High-Density

Lipoprotein Cholesterol in the Prevention and Possible Treatment of Cardiovascular Diseases. Current Molecular Medicine
2006;6:571-587.
4. Cullen P, Rauterberg J, Lorkowski S. The pathogenesis of atherosclerosis. Handb Exp Pharm 2005;170:3-70.
5. Elkind MS, Sciacca R, Boden-Albala B et al. Moderate alcohol
consumption reduces risk of ischemic stroke: the Northern Manhattan Study. Stroke 2006;1:13-19.
6. Falk E. Pathogenesis of atherosclerosis. J Am Coll Cardiol
2006;18:47-50.
7. Goulet J, Lapointe A, Lemieux S et al. Mediterranean Diet
and Cardiovascular Disease. Current Nutrition & Food Science
2006;2:265-273.
8. Husslein P, Huber J, Tempfer C. Gender Specificity, Genetic
Variation of Single Nucleotide Polymorphisms, and Blood Lipid
Parameters. Current Women’s Health Reviews 2006;2:87-90.
9. Kerenyi L, Mihalka L, Csiba L et al. Role of hyperlipidemia in
atherosclerotic plaque formation in the internal carotid artery. J
Clin Ultrasound 2006;6:283-288.
10. Perez-Castilon JL, Duenas-Laita A. New Approaches to Atherosclerotic Cardiovascular Disease. The Potentialities of Torcetrapib.
Recent Patents on Cardiovascular Drug Discovery 2006;1:109-114.
11. Plesca-Manea L. Tulburarile metabolismului lipidic. In Editura
Medicala Universitara “Iuliu Hatieganu”: Patofiziologie, PlescaManea, Cluj Napoca, 1998, 164-188.
12. Rizzo M, Berneis K. The Role of Small, Dense Low-DensityLipoproteins in Non-Coronary Forms of Atherosclerosis. Vascular
Disease Prevention 2006;3:269-274.

Normal ŞI ANORMAL În medicinĂ: EVALUAREA
epidemiologicĂ A NIVELELOR DE colesterol În
ACTIVITATEA practicĂ medicalĂREZUMAT

Obiectivul acestei lucrări a fost elaborarea unui model de evaluare a nivelelor colesterolului în activitatea medicală curentă. În găsirea factorilor cheie ai
unui model de evaluare am analizat comparativ nivelele colesterolului şi a high-density lipoprotein (HDL) la 244 pacienţi cu boli cardiovasculare de natură
aterosclerotică şi la 2301 subiecţi fără patologie cardiovasculară, identificaţi în perioada 2003 - 2007.
Valoarea medie a colesterolului la pacienţii cu boli cardiovasculare a fost de 230,1 mg% şi de 225,2 mg% la cei fără patologie cardiovasculară. Nivelele HDL
la pacienţii de gen masculin cu boli cardiovasculare s-au aglomerat în jurul valorilor de 35-39 mg%, iar în jurul valorilor de 50-54 mg% cele ale pacienţilor
de gen masculin fără boli cardiovasculare. Pentru femei, modelul a fost complex, cu distribuţie multimodală a pacientelor cu boli cardiovasculare, care s-au
plasat în zona nivelelor HDL cu mare risc.
În practica medicală valoarea prag pentru colesterol este dificil să fie stabilită, făcând necesară aprofundarea evaluărilor. În mod uzual, evaluarea valorilor
normale şi patologice ale colesterolului şi a HDL colesterol poate fi folosită în analiza deciziei necesităţii unei atitudini terapeutice sau a riscului apariţiei unor
complicaţii la boala de bază.
Cuvinte cheie: normal, valori anormale, colesterol

30

cmyk

Fiziologia - Physiology  2009.19.4(64)HARVESTING, ISOLATION AND CHARACTERIZATION OF
DOG PERIOSTEUM - DERIVED CELLSANGHEL SIMONA1,4, BOJIN FLORINA1, IGNA CORNEL5, TANASIE GABRIELA1,4, SERESMONICA5, TATU CARMEN 1,4, LUCA CALIN5, TATU CALIN2,4, ROSCA ADRIANA4, GRUIAALEXANDRA4, ORDODI VALENTIN2, GEORGESCU REMUS3, PAUNESCU VIRGIL1,4
1
Physiology and Immunology Department, University of Medicine and Pharmacy “Victor Babes”Timisoara
2
Biology Department, University of Medicine and Pharmacy “Victor Babes”Timisoara
3
Biophysics Department, University of Medicine and Pharmacy “Victor Babes”Timisoara
4
Transplant Immunology Center, County Clinical Emergency Hospital Timisoara
5
University of Agricultural Sciences and Veterinary Medicine of Banat Timisoara
HARVESTING, ISOLATION AND CHARACTERIZATION OF DOG
PERIOSTEUM - DERIVED CELLSABSTRACTANGHEL SIMONA1,4, BOJIN FLORINA1, IGNA CORNEL5, TANASIE GABRIELA1,4, SERES
The aim of the present study is to find an easy method to harvest, isolate and characterize dog periosteum-derived cells and expand these cells in vitro. In
MONICA5, TATU CARMEN 1,4, LUCA CALIN5, TATU CALIN2,4, ROSCA ADRIANA4, GRUIA
1,4
this study,2, periosteum
from adultdogs3femur
was harvested
and expanded.
ALEXANDRA4, ORDODI VALENTIN
GEORGESCU
REMUS, PAUNESCUVIRGIL1
Physiology and Immunology Department,
UniversityofMedicineandPharmacy“VictorBabes”The study evaluated and standardized the procedure for periosteum-derived cells harvesting and the optimal method for isolation. The isolated periosteumTimisoara
derived cells were maintained in vitro culture condition for 60 days. In that time the cells were evaluated regarding the morphological characteristics, viability
2Biology Department, University of Medicine and Pharmacy “Victor Babes” Timisoara
3
andofquantificationof Pharmacy
proliferation“Victor
capacityBabes”
in appropriate
culture conditions.
Biophysics Department, University
Medicine andTimisoara
4
Transplant Immunology Center, County
Clinicalperiosteum,
Emergencyperiosteum-derived
Hospital Timisoaracells, isolation methods
Keywords:
5
University of Agricultural Sciences and Veterinary Medicine of Banat Timisoara

Anesthetic procedure used was narco-neuroleptanal
body weight Acepromazine and 8 mg/kg body weight Ketami
and narcosis was induced and maintained with Halothan.
Preparationof surgicalandDogs
positioning
on themg/kg body weight Acepromazine
and 8 mg/kg bodyfieldweight Ketamine.
were intubated
INTRODUCTION
Received September 2009.
Accepted October 2009. Address for correspondence: Simona Anghel, 2A Eftimie Murgu Square,
300041, Timisoara, Romania,
e-mail: simoanghel@yahoo.com
Surgicalfield
wasprepared
byHalothan.
clipping with a h(endotracheal tube)
and narcosis
was induced
and maintained with
Bone fracture
healing and bone defects involve restoration by proliferation,
Preparationofsurgicalfield
andpositioningonthesurgical tablewith surgic
migrationanddifferentiationofcells
derived
from
the
inner
layersoftheperiosABSTRACT
positioned in lateral recumbence and were draped
The aim of the present
teum. study is to find an easy method to harvest, isolate and characterize dog periosteum-derivedSurgical field was prepared by clipping with a hair clipper and by antisepsis.
cells and expand these cells in vitro. In this study, periosteum from adult dogs femur was harvested and expanded.
Surgicaltechnique – approach to the femoral shaft
Dogs were positioned in lateral recumbence and were draped with surgical towels
order
to study cell/biomaterialinteractions,cells obtained
calvariaand the optimal
The study evaluatedInand
standardized
the procedure for
periosteum-derived
cells fromharvesting
method for isolation. The isolated periosteum-derived cells were maintained in vitro culture condition forThe
60 fixed
days. skin
was made along the craniolateral bwith towelincision
forceps.
(1,2,3,4) and long bones (5,6) are frequently used. Disadvantages of this type ofIn that time the cells were evaluated regarding the morphological characteristics, viability and quantification of
Surgical technique– approach tothe femoral(Figure
shaft
proliferation capacity
in appropriate
cultureconditions.
cells arerelated to theiradvanceddifferentiation stage. Consequently, it isthedifficultgreat trochanter
towards
patella1). The subcu
Key words: periosteum, periosteum-derived cells, isolation methods
The skin incision was made along the craniolateral border of the femoral bone,
to expand them into large amounts or to keep them in culture for long periods(7,8).
incised directly
under the skin incision.
from the level of the great trochanter towards patella (Figure 1). The subcutaneous
The latter renders these cells less suitable for long-term in vitro test systems. The
INTRODUCTION
The
lata
was
incisedthe cranial bordertissuefascia
and superficial
fascia wereinciseddirectly underalong
the skin incision.
use of progenitor cells (periosteum or bone marrow derived cells) may circumvent
Bone fracture healing and bone defects involve restoration by proliferation, migration and
The fasciaincisionlata was incisedextends
along the cranialthe
borderentireof the bicepslength
femoris muscle(Figure 2). Thisof the skin iproblems
10, the11, 12,13).layers of the periosteum.
differentiation theseof cells
derived(9,
from
inner

In order to Periosteum-derived
study cell/biomaterial
cells obtainedfromcalvaria(1,2,3,4) and aponeurosis
long(Figure 2). This incision extends the entire length of the skin incision.
cellsinteractions,
are in a less differentiatedstage,
which
may favor
bones (5,6) are frequently used. Disadvantages of this type of cells are related to their advanced
easierexpansionandmaintenanceforlongerperiods.
In
theliterature,
severalatdifferentiation stage. Consequently, it is difficult to expand them into large amounts or to keep them inculture for long
periods(7,8).andThelatter renders
these derived
cells lessforosteogenic
long-term in vitro test
temptsto culture
differentiate
periosteumcellssuitabletowards thesystems. The use of progenitor cells (periosteum or bone marrow derived cells) may circumvent theselineagewereperformed
(14,
15,
16).
problems (9, 10, 11, 12, 13).
Periosteum-derived cells are in a less differentiated stage, which may favor easier expansion andmaintenance for longer periods. In the literature, several attempts to culture and differentiate periosteumMATERIALSANDMETHODS
derived cells towards
the osteogenic lineage
wereperformed (14, 15, 16).

Femoral periosteal flaps sampling
Biologic material was obtained from five common-breed dogs which wereselected
based
on specific criteria, while data about age, gender, and weight ofFemoral periosteal
flaps
sampling
animals and also about types of tissue samples and sampling regions were noted,
Biologic material was obtained from five common-breed dogs which were selected based on
being
presented
in Tableage,
I. gender, and weight of animals and also about types of tissue
specific criteria,
whiledata about
MATERIALS AND METHODS

samples and sampling regions were noted, being presented in Table I.

Table I. Experimental animals’ characteristics

No.

Gender

C1
C2
C3
C4
C5

ƃ
ƃ
ƃ
Ƃ
ƃ

Table I. Experimental animals’ characteristics
Age
WeightPeriosteal flaps for
Periosteal flaps for(years)(kg)
digestion
explants
2
21
Left and right femora
Left and right femora
322Left and right femora
Left and right femora
325
Left femur
Left femur
3
16
Left and right femora
Left femur
4
19
Left femur
Left femur

Fig. 1. Skin incision
Fig. 1. Skin incision

Caudalof femoristhe biceps
femorisand craCaudalretraction
retraction of the biceps
muscle and cranial
retractionmuscleof thevastus shaft
lateralis muscle
revealedfemur.
the shaft of thenecessary to incise
revealed
theof theIt femur.
wasIt wasnecessary
to incise theAnesthetic procedure used was narco-neuroleptanalgesy (NNL-A). Premedication– 0.2
muscles, on the lateral shaft of the bone, in order to retract ade
Received September 2009. Accepted October 2009. Address for correspondence: Simona Anghel, 2A Eftimie Murgu Square, 300041, Timisoara, Romania, e-mail:simoanghel@yahoo.com
2009.19.4(64)  Fiziologia - Physiology

cmyk

31

the
femoralshaftunder the skin incision.
inciseddirectly
he craniolateralborderthe femoral
the levelof of the biceps femoris muscle aponeurosis
The fascia
lataofwas
incised bone,
along fromthe cranial
borderure 1). The subcutaneous tissue and superficial fascia were
(Figure 2). This incision extends the entire length of the skin incision.
rco-neuroleptanalgesy(NNL-A).
Premedication
0.2 mg/kg
thecranial border of thebiceps femorismuscle –aponeurosisdyweightKetamine.
Dogs were intubated (endotracheal tube)
engthof theskin incision.
ith Halothan.
ositioning on the surgical tableipping with a hair clipper and by antisepsis. Dogs wereraped with surgical towels fixed with towel forceps.
he femoral shaft
he craniolateral border of the femoral bone, from the level ofre 1). The subcutaneous tissue and superficial fascia were

he cranial border of the biceps femoris muscle aponeurosis
ength of the skin incision.
Fig. 1. Skin incision

Fig. 2. Fascia lata incision

Caudal retraction of the biceps femoris muscle and cranial retraction of the vastus lateralis muscle
revealed the shaft ofFig.
the2. femur.
It
was necessary to incise the fascial intermuscular septum between these
Fascialata
incisionFig. 2. Fascia
lata incision
Fig.
Detachment
periosteal
flap
Fig. 5. Detachment
ofof periosteal
flap
fascialintermuscular
septumbetweenon theinlateralshaft of themuscles,onthelateralretraction
shaftof thesethemuscles,
bone,
order
to retract
the5. vastus
lateralis.
orismuscle theandcranialofthevastuslateralismuscleadequatelybone, in order to retract adequately the vastus lateralis.
ssary to incise the fascial intermuscular septum between these
The periosteal flaps were segmented and put into:
order to retract adequately the vastus lateralis.
- wells plate with osteogenic aspect superficial – Fig
ml of isotonic and sterile sodium chloride solution + 1%
culture;- test tubes with the same content – for periosteal cellFig. 5. Detachment of periosteal flap
Fig. 2. Fascia lata incision

Fig. 6. Aspect of perioste

ris muscle and cranial retractionTheof thevastus lateralis
muscleperiosteal
flaps weresegmented and put into:
ssary to incise the fascial
septumbetweenthese
Fig. 6. Aspectof periosteal flaps flaps
Fig. 5.intermuscular
Detachmentofperiosteal
flap
Fig. 6.
Aspectof periosteal- wells plate with osteogenic aspect
superficial– Figure 7 (each wells conta
order to retract adequately
thevastuslateralis.
ml of isotonic and sterile sodium chloride solution + 1% Penicillin and Streptom
The periosteal flaps were segmented and put into:The
flapsof adductor
weresegmented and put into:
Fig. periosteal
3. Detachment
adductor
Fig.- wells
4. Detachmentofaspect
vastusintermedius
muscleFig.
3. Detachmentof
muscle muscle
plate with osteogenic
superficial– Figure 7 (eachwells conculture;- wells plate with osteogenic aspect superficialtained– approximately
Figure 71 ml
(each
wells
contained
1
of isotonic
and sterilesodium chlorideapproximately
solution + 1%
test
tubes
with
the
same
content–
forperiosteal
cells
culture(afterexposur
Penicillin
andStreptomycin)–
forexplants
culture;mlofisotonic
andsterilesodiumchloride
solution+1%
Penicillin
andStreptomycin)–
forexplantscle
4. Detachmentof vastus
intermedius
TheFig.adductor
muscle,which
insertsmuscle
on the caudal
aspects of the femoral shaft, was detached- test tubes with the same content – for periosteal cells culture (after exposure
culture;(Figure 3). The vastus intermedius muscle on the cranialto surface
collagenase). of the shaft were detached by freeing the- testtubes withthefemoral
same content– wasfor periosteal
cells culture (after exposure to collagenase).
s fasciaon thebetween
caudalaspectsoftheshaft,
detached
the muscle and the bone (Figure 4).
on the cranialof theshaft
were
detachedusing
by freeingtheThe surface
periosteal
flaps
were
detacheda periosteal
elevator (Figure 5). Aspects of periostealgure4).
Fig. 7. Periosteal flaps in w
flaps can be seen in Figure 6.
using a periosteal elevator (Figure 5). Aspects of periosteal

cle

Closure of the surgical wound consisted in attachinmuscle by simple continuous suture with catgut, suturin
subcutaneous tissue in the same manner and attaching the sk
thread.

Fig. 4. Detachmentof vastus
intermedius
muscleFig. 4. Detachmentof vastus intermedius
muscleIsolation methods of periostem-derived cells
The adductoraspects
muscle, whichof
insertsthe
on the femoral
caudal aspects ofshaft,the femoral shaft,s on the caudal
waswasdetacheddetached (Figure 3). The vastus intermedius muscle on the cranial surface of the shaft
Fig. 7. Periostealflaps
flaps in wellinplatewell platen the cranial
surface of the shaft were detached by freeing
the Fig. 7. Periosteal
were detached by freeing the fascia between the muscle and the bone (Figure
4).
Digestionof theplate
surgical wound consisted in attaching fascia lata to the cranial
Fig. 7. a.
Periosteal
flapsClosure
inmethodwell
ure 4).
The periosteal flaps were detached using a periosteal elevator (Figure 5).
borderof
bicepsmusclesimple continuous
suture with
catgut,phosphate
suturing the
1. periosteal
flapsby were
washedusing
bufferof the surgicalwound
attachinglata
Aspects of periosteal
flaps can be (Figure
seen inClosure
Figure 6.5). Aspects
using a periosteal
elevatorof periosteal
margins of theconsisted
superficial facia andin
subcutaneous
tissue in thefascia
same mannerand to the cra
werecarefully
removedmuch
asClosure ofmuscle
the surgicalwound continuous
consisted intissue
attachinglata
tosuture
theborderofasbiceps
by simple
suturecatgut,suturing
marginsof theattaching remainings
the withskinfascia
by simple
interrupted
withcranial
surgical thethread.
muscle by simple
continuous suture
suturing
the margins
ofthe
theskinsuperficial
facia
and an
2. resulting
werepassedinbysterile
test
tubes
subcutaneous
tissue within thecatgut,
samemanner segments
andattachingsimpleinterrupte
subcutaneous
tissueinthesamemannerandattaching
the
skin
by
simpleinterrupted
suturewithsurgicalColagenase from
Clostridium
Fiziologia- Physiology histolyticum
 2009.19.4(64) (Figure 8)
32
thread.
thread.

cmyk
methods ofcells
periostem-derived cells
Isolation methodsIsolationof periostem-derived

of the surgical wound consisted in attaching fascia lata to the cranial border of biceps
influenced
tissuesamplesizetheandmargins
the amountfibrous tissue(Figure 9);le continuous
suturebywith
catgut,suturingof the ofsuperficial
facia andsue4.
in thesameenzimaticmanner and digestion
attaching theskinfinished,by simple theinterrupted
suturewith surgicalwhenwasresultingcellularsuspensions were filtered using

ȝm strainers, passed in plastic test tubes and 20 ml of phosphate buffer saline were adde
complete the firsts of periostem-derived
cells washing step;

hod
Isolation methods of periostem-derived cells
flaps were washedusing
phosphate buffer saline and were cleaned (muscle and adipose
a. Digestion
methodmainings were carefully
as phosphatemuch buffer
as possible)
1. periosteal flapsremoved
were washed using
saline and were cleaned(muscleand adiposein
tissueremainings
weretubes
carefully removed
as much as possible)
segments werepassedsteriletest
and covered
with approximately 1 ml of type I
resultingsegments
werepassedinsterile
test
tubes
and
coveredwithapproximately
se from Clostridium histolyticum (Figure 8).
1 ml of type I Colagenase from Clostridium histolyticum (Figure 8).

Fig. 10. Aspect of cellular suspension after the first centrifugation

5.
6.

7.
8.

b. Explant method
Fig. 9. Aspect of samples at the end of
Fig.1. 10.
Aspectcellularsuspension
periosteal
flaps wereofwashedusing phosphatebuffer saline,after
plated thewiththeosteogenic
surfacefacingup,andtheresidual
adiposeandmuscletissuewasdigestion process
first centrifugation
removed as possible;
2. resulting segments of approximately 1 cm2 were placed in Petri dishes with
Fig.
8.Periosteal
Periosteal
aspectaftersolution
addingFig. 8.
flaps aspectflaps
after adding
the colagenase
diluted cellularsuspensions
were
centrifugedat1
500partrpm(Figure10);
theosteogenic
in directfor
contact10
withminutes
the culture plate, andwere incubatedforthe colagenase
solution
2. test tubes containing
the tissue samples
were incubated at 37oC, 5%o o10minutes
at
37
C
in
order
to
providethe
initialadherence;3.
test
tubes containing thetissuesampleswereincubated
37 C, 5% CO
2 for approximately
after
supernatantwasatremoved(approximately
19 30-45ml). The p
CO2 centrifugation,
for approximately 30-45 minutes,mostin order to of
finalizethe
the digestion
process.
3. adherent
flapsinterval
were then coveredrequiredwith an appropriatefor
amountdigestionof nutritive mediumwas
minutes,
in
order
to
finalizethedigestion
process.
Time
Time intervaldue
required for
was influencedsedimentation
by tissue sample size and the was(HAM/F-12),
resulted
todigestion
cellularmechanicallywith the remai
so that to prevent floatingomogenizedof the periosteal segments (Figure11).
influenced
size
andwastheamountof fibrous tissue (Figure 9);amount of fibrous bytissuetissue(Figure 9);sample
when enzimatic
digestionfinished,thesupernatant,and digestion
phosphatesalinewas added
over this cellular suspension up to 20
resulting cellularsuspensions
werefiltered usingwas
70 μmbuffer
strainers,
passedinthe
plasticresulting
4. whenenzimaticfinished,
cellular suspensions were filtered using 70
test tubeswash);and 20 ml of phosphate buffer saline were added to complete the first(secondȝm
strainers,
passed in plastic test tubes and 20 ml of phosphate buffer saline were added towashing step;
cellularsuspensions
were centrifugedagain at 1 500 rpm for 10 minutes;
completethe suspensions
first washingstep;
3. diluted cellularwere centrifugedat 1 500 rpm for 10 minutes
after(Figure
centrifugation,
almost the entire supernatant was removed. The pellet resulted due to cell
10);Fig. 11. Periosteal flaps adherent on Petri dshes, covered with nutrient medium

sedimentation was mechanically omogenized with a small amount of cell culture medof periostem-derived cells(nutritive medium – HAM/F Culture
121. +Culture
10%mediumfetalwasbovine+ 1%
Penicillin
andStreptomy
replaced serum
every 3 days,
carefully
analyzing the
microscopic
morph
Fig.
11.
Periosteal
flaps
adherentonPetridshes,
coveredwithnutrient
mediumchanges
occuringastheculturedeveloped(Figures
12
and
13).
approximately 1-2 ml);
Culture of periostem-derived cells
1. Culture medium
was
replaced every 3 days,cells
carefully analyzing the microscopic morphological
Cultureof periostem-derived
9. these cellular suspension were used for viability
tests
TripanBluechanges occuring
as theusing
culture developed(Figures 12
and 13). vital stainning: 10 ȝ
Culturemediumwasreplacedevery
3days, carefully analyzing the microscopic
Culture of periostem-derived cells
cell suspension / 90 ȝl of Tripan Blue, andofoccuring
this
were
1. Culture10
mediumȝl
was replaced
every 3 days,
carefully analyzing
the microscopic
morphologicalchangesas themixture
culturedeveloped (Figures
12 andtransfered13).morphological intochanges occuring as the culture developed (Figures 12 and 13).
hemacytometer for cell count and viability assessment using light microscopy. Cellular viab
was above 95%.
Viable cells count was determinant for choosing the size and type of cul
Fig. 9. Aspect of samples at the end of digestion process
flasks;4. after centrifugation, most of the supernatant was removed (approximately 19
Fig.
9. dueAspectofsedimentation
samples wasat theend ofomogenized Fig. 10. Aspect of cellular suspension after theml). The pellet
resulted
to cellularmechanically
10. cellularsuspensions
wereplatedinto culture
flaskscontaining culture medium in varidigestion
process
first centrifugation
A
with the remaining supernatant, and phosphate buffer saline was added over thisBamounts,
dependingonwash);their size. Cells
were plated
at a cellular density of 5 x 104 cells/cm2 .
cellular suspension
up to 20 ml (secondAABo
B
5. cellularsuspensions
were centrifugedagainwereat 1 500 centrifuged
rpm forinto
10 minutes;
dilutedcellularsuspensions
at 1 500 Fig.
rpm
for
10 minutes(Figure10);
11. 5.
Cultureflaskswerethen incubatedconditions:
37explant
C,
in
CO
Fig.
12.appropriate
Periosteum-derived
cells
emergingfromsample,Ob 20x:A. A.424 atmosphere.
day
12.
Periosteum-derived
cellstheemergingfrom theexplant5%
sample,Ob
20x:
day ofof periosteal flaps
Fig. 11. Periosteal flaps adherent on Petri dshes, covered with nutrient medium

Fig. 11. Periosteal flaps adherent on Petri dshes, covered with nutrient medium

Fig. 12. Periosteum-derived cells emerging from the explant sample, Ob 20x: A. 4th day of periosteal flaps
culture; B. after explant detachment
thth flaps
Fig. 12. Periosteum-derived cells emerging from the explant sample, Ob 20x: A. 4th day of periosteal
culture; B. after explant detachment

B.
detachment
centrifugation, almost themost
entire supernatant
was removed.
The pellet was removedculture;
periosteal
flapsafter
culture;explant
B. after explantdetachment
6. after6. after
centrifugation,of thesupernatant(approximately
19 ml). The pellet
resulted due to cellular sedimentation was mechanically omogenized with a small
resulted
due to cellular sedimentation was mechanically omogenized with the remaining
amount of cell culture medium (nutritive medium – HAM/F 12 + 10% fetal bovine
supernatant,phosphatebufferserum + 1% Penicillinandand Streptomycin,
approximately
1-2 ml);saline was added over this cellular suspension up to 20 ml. Explant(second
methodwash);
7. these cellularsuspension were used for viability tests using Tripan Blue vital
1. 7.
periosteal
were
using
buffersaline,plated with the osteogenic sur
stainning: 10suspensions
μlflapsof cell suspension/ 90
μlwashedof Tripan
Blue, and10 μl ofagain
thisphosphatemixturecellularwerecentrifugedat 1 500 rpm
forFig.10
minutes;
13. Day
6 of periosteum-derived cell culture (digestion
method) (Ob. 6x)
weretransferedintothehemacytometer
for
cell
count
and
viability
assessment
Fig. 13. Day 6 of periosteum-derived cell culture (digestion
facingand the residual
adiposemusclewas
tissuewas removedaspossible;
8.
afterup,centrifugation,
almost theentire andsupernatantremoved.
The method)pelletresulted
due to cellular(Ob. 6x)
using light microscopy. Cellular viability was above 95%. Viable cells count was 22. when reaching the confluence of approximately 90%, cells were passed using enzymatic
sedimentationwasmechanicallyomogenized
with
a
small
amountofcellculture(Trysin/EDTA
followedby 90%,
neutralization
20%medium
Fetalenzymatic
Calf Serum
2. resulting
approximately
1 cm wereplacedin0.25%),Petridisheswiththeosteogenicpa
2.detachment
when reaching
the confluenceof approximately
cells
werewith
passedusing
determinantsegments
for choosing the sizeof
and typeof culture flasks;
detachment(Trysin/EDTA
0.25%), followed
20% Fetal
solutionand 10minutes centrifugation
at 1500 byrpmneutralization(2 successivewith
washings).
CellsCalf
wereSerum
re-platedo2(nutritive
medium
–
HAM/F
12
+
10%
fetal
bovineserum+
1%
Penicillin
andStreptomycin,
solutionand10
minutes
centrifugation
at
1500
rpm
(2
successive
washings).
Cells
were
re-plated
8.
cellularsuspensions
wereplatedinto
cultureflaskscontaining
culture;ataconcentrationof5000
cells/cm
direct contact with the culture plate, and wereincubatedfor10
minutes
at 37 C in order to pro
2;
at aofconcentrationof 5000atcells/cm
the cells obtained
each passage
were frozen in order to be cryopreserved and maintained
medium in variable amounts,
dependingon their size. Cells were plated at a cellular 3. 3.part13.
Day 6usedofeachperiosteum-derived
cell
culture(digestion
approximately
1-2
ml);
part
ofFig.
thecells
obtainedatpassagewerefrozeninorder
tobe cryopreserved
for
a
long
time.
Medium
in
this
case
was
supplementedwith
15%
dimethylsulfoxide.
Fig. 13. Day 6 of periosteum-derived
cell6x)culture (digestion
method)(Ob. 6x)and maintained
the initial2
for a long time. Medium used in thiscase was(Ob.
supplemented
with 15% dimethylsulfoxide.
method)density ofcellular
5 x adherence;
104 cells/cmsuspension.
9. thesewere used for viability
tests
using
TripanBlue
vitalstainning:ȝl of
1. when reaching the confluence of approximately 90%, cells were10
passed9. Culture
flasks werethen incubatedintocovered
appropriate conditions:
37oC,anin appropriate amount of nutritive medium (HAM/F3. adherent
flaps
were
then
with
using
enzymatic
detachment(Trysin/EDTA
0.25%),
followedby neutralizationcellsuspension / 90 ȝl of Tripan
Blue,reaching
and
10the
ȝlconfluenceof thismixtureweretransferedintopassed
the using en
2. whenof
approximately
90%,
cells
were
RESULTS
5% CO2 atmosphere.
RESULTSso thattoprevent
floatingoftheperiosteal
segments(Figure11).
detachment(Trysin/EDTA
0.25%),
followedbyneutralizationwith20%
hemacytometer for cell count and viability assessment using light microscopy. Cellular viabilityFetal Calfand 10 minutes centrifugation at 1500 rpm (2 successive washings).
33 Cells were r
2009.19.4(64) Fiziologia- Physiology
wasabove 95%.
Viablecells count solution
was determinant
for choosing the size and type of culture
at a concentration of 5000 cells/cm2;
flasks;
3. part of the cells obtained at each passage were frozen in order to be cryopreserved and ma
cmyk suspensions were plated into
for a longtime. Medium
in this case culture
was supplemented
within15%variable
dimethylsulfoxide.
10. cellular
cultureflasks used
containing
mediumamounts, depending on their size. Cells were plated at a cellular density of 5 x 104 cells/cm2 .
11. Culture flasks were then incubated into appropriate conditions: 37oC, in 5% CO atmosphere.

14

cell count x 10E6

12
10

Explant

8

Digestio

642with 20% Fetal Calf Serum solution and 10 minutes centrifugation at 1500 rpm (20Cell count after P2
C1
C2
C3
C4
C5
successive washings). Cells were re-plated at a concentration of 5000 cells/cm2;
samples
2. part of the cells obtained at each passage were frozen in order to be cryopreserved and maintained for a long time. Medium used in this case
supplemented
Fig.was16.
Comparison between total cell count at P2 using both isolation methods, explan
with 15% dimethylsulfoxide.
steum surrounding the femoral bone of adult dogs (n = 5) was used. Cells from the
Afterpassage fragments.
2 (P2), proliferation
ayers of the periost were obtained by explant culturesof periosteal
Cells migratedrate was decreasing proportionally with
RESULTSsue within 3-5 days after harvest (Figureand12). All
samplesof
gave
rise to primarycellrespectively.
culturesduration
cellularculture,Cells obtained using both metho
Periosteum surrounding the femoral bone of adult dogs (n = 5) was used.
blast-like morphology, spindle-shape, andculture
increasedupconfluence(Figures
14,
15).
In
the
same
60 cultures
days,
Cells from the osteogenic layers of the periost were obtained bytoexplantof repeatedly passed when confluence was reaching the upper
l of 3-5 days, the samples processed by digestion method Fig.
inducedthe between
occurrenceof at P2 using both isolation methods, explant and enzymatic digestion
16. Comparison
totalcell countperiosteal fragments. Cells migrated from(P5).
the tissueInwithin
3-5
days
after
harvestthe last passage,
cellularviability
wasFig. 16.
Comparison
between
totalassessed
cell count at P2 usingusing
both isolationAcridin
methods, explant Orange met
ke adherentPeriosteum
cells withinthecultureflasks.
and enzymatic digestion
the femoralof adult
dogs(n with= 5) awas
used. Cells from the(Figure surrounding
12). All samples
gave risebone
to primarycell
culturesfibroblast-liketheproportionofviablecells
andapoptotic
cells,
andallowedusthetonumber
determine
Afterreachedpassage2within(P2), proliferation
rate was decreasing proportionally withof passages thular confluenceandbyenzymaticwasdays
osteogenic layers(explantof the periostculture
were obtained
explant culturesdigestion),of periosteal fragments.
Cells
migrated
amountof 10-14
cells still proliferating,which was of approximately 95% (Figure
17).
morphology,
spindle-shape,and increased
confluence(Figures14,and15).duration
In the
same
from
the
tissuewithin
3-5
days
after
harvest(Figure12).
Allsamples
gave
rise
to
primarycellculturesofcellularculture,respectively.
Cells
obtainedusing
bothmethods
weremaintainedinproliferating,which
wastheofsameapproximately
95%
17).
and 15).
Workingpassed
protocol whenfor further
cell (Figure
freezingwas
andreaching
cryopreservation
provedlimit,
to up to passage 5
with a fibroblast-likemorphology,
spindle-shape,and increased
confluence(Figures14, to15).
time intervalof 3-5 days,
the samples processedby digestion
methodinducedthe60In days,
cultureup
repeatedly
confluence
the upper
time interval of 3-5 days, the samples processed by digestionWorking
method(P5).
inducedthe last
occurrenceof optimal,forfurthercellwasandofAcridin
cryopreservation
provedto bbeconsidering
thefreezing
increased
cells
afterOrange
thawingmethod,
procedures,which
Inprotocol
thepassage,cellularviability
assessedviability
using
establishedoccurrence of fibroblast-like adherent cells within the culture
flasks.
fibroblast-like adherent cells within the culture flasks.
theproportion
viablecells
apoptotic
cells,
and allowed us which
to determine
the maintained
amount of cells stillwhich
wasandmaintained
within60-70%.
Cellular confluence(explant (explant
culture andenzymatic
digestion),wasreachedwithin of
10-14
days
the
increased
viability
of
cells
after
thawing
procedures,
waswit
Cellular confluence
cultureand enzymatic
digestion),wasreachedproliferating, which was ofapproximately
95%Orange(Figure17).
(Figures 14 and 15).
Fig.
17. Viability test Acridinof periosteum-derived
cells: viable cells-green nuclei;
within 10-14 days (Figures 14 and 15).
Working protocol for further cell freezing and cryopreservation proved to be optimal, considering
16
14cell count x 10E6

12
10

Explant

8

Digestion

6
4
2
0

C1

C2

C3

C4

C5

samplesthe increased viability of cells after thawing procedures, which was maintained within 60-70%.

A

A

Fig.14. Explant culture confluence, ob 15x (A), ob 4x (B)Fig.14.
Explantconfluence,
culture confluence,obob15x
15x (A),ob 4xob(B) 4x (B)Fig.14. Explant
culture(A),

B

B
Fig. 17. Viability test Acridin Orange of
periosteum-derived cells: viable cells-green
nuclei; apoptotic cells-orange nuclei(fluorescence microscopy, Ob. 20x)

Fig. 15. Confluence of enzymatically released
cells , ob 10x

Fig. 17. Viability test Acridin Orange ofapoptotic cells-orange nucleicells:(fluorescencemicroscopy,
Ob. 20x)
periosteum-derived
viablecells-green
DISCUSSION AND CONCLUSIONS
nuclei; apoptotic cells-orange nuclei
In bone fracture healing, it is important that restoration of the damaged area results in the
DISCUSSIONANDCONCLUSIONS
microscopy,
Ob. 20x)
formation of new bone, having (fluorescencethe same
integrity
as the surrounding
bone tissue. Cells from the

In bonefracture
healing, itareis important
restoration oftothethedamaged
area of damagedbonelininglayersof theperiosteum
the firstthat
contributors
regeneration
Next passage cultures showed adherent cells uniformly distributedosteogenic
over the tissue
culturedishbonetissue.
Therefore,
it could
bevaluableto bone,use these
specific
progenitor
cells
in culture models towith cells merely having a fibroblast-like morphology. Proliferation rate had
a greatvariance
dependingresults
intheformationofnewhavingthesameintegrityasthesurrounding
on subjects’ age and health status, proven by high periosteum-derived cell concentration/ cm2 obtained in and osteoinductive capacities of biomaterials, developed for bone regenerative
study the osteoconductive
bone tissue. Cells from the osteogenic bone lining layers of the periosteum are the firsta shorter time interval in case of young and healthy dogs. Total cell count obtainedat the first passage fortherapy.
6
Fig.Fig.
15.15.
Confluenceof6,enzymatically-2.7x10depending
oncellssize,released
the initial sample. Compared
the explant samples varied
between
1.5x10contributors
to thebyregenerationof damaged
Therefore,fragments
it could be valuable
Confluenceof enzymaticallyreleased
obof10x
Cell cultures wereobtained
migrationof cells bone
fromtissue.
periosteal(explant culture andto explant method, total cell count at first cells
passageof 10xsamples processed by enzymatic digestion was
, ob
6
6
to
use
these
specific
progenitor
cells
in
culturemodelstostudy
theosteoconductive
and as well as
enzymatic
digestion)andmonolayer-expanded.
In
theliterature,explant
culture (14,17)
Next
passageculturesshowedadherent
cells
uniformly
distributedoverthetissignificantly increased, variation between 3.5x10 -7x10 being obtained, also depending on the size ofof capacities
periosteal
fragmentsdeveloped
are describedtoisolate periosteal
cells, from a
initial periosteum
sample.
By
in vitro
expandingof athecells, at the morphology.
second digestion
passage(P2)method
we were(16,18,19,20)
able osteoinductive
toofbiomaterials,
forboneregenerative
therapy.
sue
culture
dish
with
cells
merelyhavingfibroblast-like
Proliferation
total cells/subject
with thehighestproliferation
rate,obtainculturesfrom the explant
samplesadherent
up to 10x106 cells
diversity
ofoveranimals
andhuman.
However,in our opinion, explant cultures have a higher probability topassageshoweduniformly
distributed
thewhile
tissueculturedish
6 status, proven by
Cellcultureswere
obtainedbymigration
of
cells
from
periosteal
fragments
rate
had
a
great
variance
dependingonsubjects’ageandhealthcells/subject
with the highest
in samples processed by digestion method, total cell count was up to 15x10 resultin cultures
osteogenic
potential. Isolation of cells by enzymatic treatment of periosteal
erely proliferation
having arate
fibroblast-likemorphology. Proliferation
rate hada greatwith
variance
depending(Figure 16).
(explantcells
andtheenzymatic
digestion)and monolayer-expanded.
the litera- layer), but alsohigh periosteum-derived
cell concentration / cm2 obtained in a shortertime intervalfragments
not only liberates
fromosteogenic
lineage(deriving from theIncambium
2cultureDISCUSSION AND CONCLUSIONSIn bone fracture healing, it is important that restoration of the damag
formation of new bone, having the same integrity as the surrounding boneosteogenic bone lining layers of the periosteum are the first contributors to the reage and health status, proven by high periosteum-derived
cell concentration/ cm
obtainedinbone tissue. Therefore,
it could
bevaluable
to
use
these
specificperiosteal
progenitor
cell
ture,
explant
culture(14,17)
as digestion(derivingfromtheas well
fibrouslayer).method
The (16,18,19,20)
periosteum ofpresent
around the longin case of young and healthy dogs. Total cell count obtained at thenon-osteogenic
first passage for fibroblasts
e interval in case of young and healthy dogs.
Total
cell
count
obtained
at
the
first
passageforof a thin
fromtowhichitis impossible
separateosteogenic
layer from6
the osteoconductive
osteoinductive
capacitiesofbiomaterials,
fragments
arelayer,described
isolate periosteal
cells, fromto
a diversityof theanimals
and developed-2.7x106, depending
onbonessize ofof
thedogs
initial consistsand
the explant samples varied between 1.5x10study
the fibrous
prior sample.
to enzymatic
digestion. Moreover, by using periosteal tissue fragments instead ofsamples varied between 1.5x106-2.7x106, depending on sizeof thelayer
initialComparedhuman.
However,in our opinion, explant cultures have a higher probability to result
therapy.
sample. Compared to explant method, total
cell count at first passage
ofsamplescell suspensions
to start thedigestion
cultures, theoutgrowing cells are derived from an intact tissue (layer), a
method, total cell count at first passage of samples processedby enzymaticwasin cultures with osteogenic
potential. Isolation
cells by fromenzymaticperiosteal
treatment ofprocessed by enzymatic digestion wasincreased,variation
betweenwere obtained
6 significantly
6
Cell
culturesbymigrationof ofcells
fragmenincreased, variation
between 3.5x10 -7x10 being obtained, also depending
on fragments
the size
periosteal
notof
only liberates cells from the osteogenic lineage (deriving
3.5x106-7x106 being obtained, also depending on the size of initial periosteum
enzymaticand(P2)monolayer-expanded.
In theliterature,
cultu
teum sample. By in vitro expanding of thecells, at thedigestion)
second passage
wefrom
the werecambiumablelayer),tobut also non-osteogenic
fibroblasts(deriving from explantthesample. By in vitro expanding of the cells, at the second passage (P2) we were able
the explant samples up to 10x106 total cells/subject
withthehighestproliferation
rate,
while
digestion
method(16,18,19,20)
of Theperiosteal
fragments
6
fibrous layer).
periosteum present
around the long arebones ofdescribed
dogs consists of to isolate p
to obtain from the explant samples up to 10x10
total cells/subject
with the highest
rocessed by digestion method, total cell count was up to 15x106 cells/subject
witha thinlayer,thefromhighest
which it is impossible
to separate
the osteogenic
layer fromcultures
thediversityof animals
However,in ouropinion,explant
have
proliferation rate, while in samples processedby digestion method,
total cell countand human.
rate (Figure 16).
fibrous
layer
prior
to
enzymatic
digestion.
Moreover,by
using
periosteal
tissuewas up to 15x106 cells/subject with the highest
proliferation
rate(Figure16).
result in cultures with osteogenic potential. Isolation of cells by enzymatic t
fragments instead of cell suspensions to start the cultures, the outgrowing cells
After passage 2 (P2), proliferation ratefragments
was decreasing proportionallywithliberates
thenot only
cells from the osteogenic lineage (deriving from the ca
are derived from an intact tissue (layer), a situation more similar to that in vivo
number of passages and duration of cellular culture, respectively. Cells obtained usnon-osteogenic
fibroblasts(derivingfromfibrous
The periosteum pr
when damaged bone
has to bethe
repaired.
Difficulties tolayer).
harvest the mesenchymal
ing both methods were maintained in culture up to 60 days, repeatedly passed when
dogs
thin
fromlayerwhich
it is impossible
cells layer,from the cambium
are often described.
This cambium layerto
has aseparate theconfluence was reaching the upper limit, bones
up to passageof5 (P5).
In the consists
last passage, of astem
tendencytoremainattached
totheunderlying
bone
when
the
explant
is
taken
(15).
cellular viability was assessed using Acridin
Orangemethod,whichestablishedthe fibrous layer prior to enzymatic digestion. Moreover, by using periosteal
tissu
Nevertheless, even when the cambium layer together with the fibrous layer was
the proportion of viable cells and apoptotic
cells,
andallowedustodeterminethecell suspensions to start the cultures, the outgrowing cells are derived from an
34

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Fiziologia - Physiology  2009.19.4(64)harvested successfully, contamination with other cell types and fibroblasts-like cells
is feasible. These contaminant non-progenitor cells can inhibit the differentiationof the mesenchymal stem cells in the cultures (16,21).
Morphology was dominated by spindle-shaped, fibroblast-like cells, suggesting the contamination of the cultures with cells derived from the fibrous layer of theperiosteum. The osteogenic layer was directed towards the bottom of the culture
dish, allowing attachment and expansion of the cells from the cambium layer andminimizing contamination with other cell types from the upper fibrous layer. For
the above mentioned reasons, we preferred working in future experiments withperiosteal explants instead of enzymatically released cells.
Despite the difficulties to obtain the correct material (periosteum with anintact osteogenic layer) and despite the fact that a lot of cultures were not capableof being induced to become differentiated osteoblast cultures, periosteum-derived
cultures have several advantages compared to bone derived cultures. First, there is
the biological relevance of the periosteal cells being the first contributors to repair
damaged bone. Secondly, their osteogenic potential persists even after prolonged
cultivation in osteogenic medium. Longer culture periods (3–4 months) can be
performed before final differentiation is obtained, making these cultures suitable forlong-term in vitro evaluation of biomaterials (10,11). These less differentiated cells
can be cultured without losing the potential of osteogenesis. As a consequence, thecells can be expanded for up to 7 passages before terminal osteoblastic differentiation. Compared to osteoblast cultures derived from long bones and calvaria fromadult or fetal rat (7) which have a higher differentiation stage, periosteum-derived
cultures have the advantage of prolonged cultivation before terminal differentiationand the consequent dying off of the cultures.
In conclusion, we want to emphasize the importance of periosteum-derived
cell cultures to study biomaterial/cell interactions.
Acknowledgements
The work in this paper was supported by PNCDI-II Project 130/2008 (financing
agency CNCSIS-Idei) and Project 41-091/2007 (financing agency CNMP).
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scanning microscopy studies of osteoblast growth and phenotypic expression
in three-dimensional degradable matrices. J.Biomed. Mater.Res., 1995, 29:
843-48.
2. Burdick JA, Padera RF, Huang JV, Anseth KS. An investigation of the cytotoxicity
and histocompatibility of in situ forming lactic acid based orthopedic biomaterials.
J. Biomed. Mater. Res. (Appl. Biomater.), 2002, 63: 484-91.
3. Dalby MJ, Di Silvio L, Harper EJ, Bonfield W. Increasing hydroxyapatite incorporation into poly (methylmethacrylate) cement increases osteoblast adhesion
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4. Moursi AM, Winnard AV, Lannutti JJ, Seghi R. Enhanced osteoblast response
to a polymethylmethacrylate – hydroxyapatite composite. Biomaterials, 2002;
23: 133-144.
5. Calandrelli L, Immirzi B, Malinconico M, Orsello G, Volpe MG, Della Ragione F,

Zappia V. Biocompatibility studies on biodegradable polyester-based compositesof human osteoblasts: a preliminary screening. J. Biomed. Mater. Res., 2002,
59: 611-17.
6. Scotchford CA, Cascone MG, Downes S, Giusti P. Osteoblast responses tocollagen–PVA bioartificial polymersin vitro: the effects of cross-linking method andcollagen content. Biomaterials, 1998, 19: 1-11.
7. Declercq H, Van den Vreken N, De Maeyer E, Verbeeck R, Schacht E, De Ridder L , Cornelissen R. Isolation, proliferation and differentiation of osteoblastic cells
to study cell/biomaterial interactions: comparison of different isolation techniquesand source. Biomaterials, 2004, 25: 757-68.
8. Salgado AJ, Coutinho OP, Reis RL. Bone tissue engineering: state of the art
and future trends. Macromol. Biosci., 2004, 4: 743-65.
9. Ishaug SL, Crane GM, Miller MJ, Yasko AW, Yasemski MJ, Mikos AG. Bone
formation by threedimensional stromal osteoblast culture in biodegradable polymer
scaffolds. J. Biomed. Mater. Res., 1997, 37: 17-28.
10. Perka C, Schultz O, Spitzer R, Lindenhayn K, Burmester G, Sittinger M. Segmental bone repair by tissue engineered periosteal cell transplants with bioresorbable fleece and fibrin scaffolds in rabbits. Biomaterials, 2000, 21: 1145-53.
11. Arnold U, Lindenhayn K and Perka C. In vitro-cultivation of human periosteum
derived cells in bioresorbable polymer-TCP-composites. Biomaterials, 2002, 23:2303-10.
12. Spitzer RS, Perka C, Lindenhayn K, Zippel H. Matrix engineering for osteogenic
differentiation of rabbit periosteal cells using a-tricalcium phosphate particles in athree-dimensional fibrin culture. J. Biomed. Mater.Res. 2002, 59:690-96.
13. Lisignoli G, Zino N, Remiddi G, Piacentini A, Puggioli A, Trimarchi C, Fini M,
Maraldi NM, Facchini A. Basic fibroblast growth factor enhances in vitro mineralization of rat bone marrow stromal cells grown on non-woven hyaluronic acid based
polymer scaffold. Biomaterials, 2001, 22: 2095-105.
14. Koshihara Y, Kawamura M, Endo S, Tsutsumi C, Kodama H, Oda H, Higaki
S. Establishment of human osteoblastic cells derived from periosteum in culture.
In Vitro Cell Dev. Biol., 1989, 25(1): 37-43.
15. Breitbart AS, Grande DA, Kessler R, Ryaby JT, Fitzsimmons RJ, Grant RT.
Tissue engineered bone repair of calvarial defects using cultured periosteal cells.
Plast. Reconstr. Surg., 1998; 101: 567-76.
16. Solchaga LA, Cassie`de P, Caplan AI. Different response to osteo-inductive
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17. Bahrami S, Stratmann U, Wiesmann HP, Mokrys K, Bruckner P, Szuwart T.
Periosteally derived osteoblast-like cells differentiate into chondrocytes in suspension culture in agarose. Anat. Rec., 2000, 259(2): 124-130.
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RECOLTAREA, IZOLAREA SI CARACTERIZAREA CELULELORDERIVATE DIN PERIOSTUL CANINREZUMAT

Scopul acestui studiu a fost identificarea unei metode simple de recoltare, izolare, caracterizare si expandare in vitro a celulelor derivate din periostul canin.
In acest scop au fost folosite fragmente de periost obtinute din femurul cainilor adulti, care a fost recoltat, procesat si expandat in vitro. Studiul a evaluat si
standardizat procedura de recoltare a celulelor derivate din periost si a optimizat metodele de izolare si cultivare ale acestora. Celulele derivate din periost au
fost mentinute in cultura timp de 60 de zile. In acest timp celulele au fost evaluate din punct de vedere al caracteristicilor morfologice, al viabilitatii si a fostevaluata capacitatea de proliferare in conditii de cultura adecvate.
Cuvinte cheie: periost, celule derivate din periost, metode de izolare
2009.19.4(64)  Fiziologia - Physiology

cmyk

35

CAVERNOUS HEMANGIOMA OF THE PAROTID GLAND
ALEXANDRA FAUR1, ELENA LAZAR1, MARIOARA CORNIANU1, ALIS DEMA1, CODRUTA LAZUREANU1, ANCA MURESAN1, CAMELIA GURBAN VIDITA2, LAURA SMARANDA GOTIA3
1
Department of Pathology
2
Department of Biochemistry3
Department of Physiology“Victor Babes” University of Medicine and Pharmacy Timisoara

ABSTRACTWe evaluated the medical record of patients with salivary gland neoplasms diagnosed at Timisoara City Hospital from 2002 to 2009. A study has been carriedout for seven years on 220 cases of salivary gland tumors and only one case of salivary gland hemangioma was diagnosed. Although hemangioma is a common soft tissue tumor that frequently occurs in infancy, in our experience it is rarely biopsied and rather unfamiliar to the surgical pathologist. We present acase of a cavernous hemangioma involving the parotid salivary gland in a one year infant.
Key words: hemangioma, salivary gland, parotid

INTRODUCTION
Salivary glands can be divided into two distinct exocrine groups: the major
salivary glands-witch includes parotid, submandibular and sublingual glands andin the mucosa of the upper aerodigestive tract there are the minor salivary glands.
The major function of the salivary glands is to secrete saliva, witch plays a significant
role in lubrication, digestion, immunity and overall maintenance of homeostasis
within the human body (12).
Salivary gland tumors are rare disorders. The global annual incidence when
all salivary gland tumors were considered varied from 0.4-13.5 cases per 100,000
populations. Hemangiomas of the salivary glands account for approximately 0.4%
of salivary tumours and occur almost exclusively in the parotid gland (8). In thepediatric population hemangiomas represents about 50% of parotid neoplasms.
Most infantile hemangiomas appear in the first 6 months of life and increase in size
slowly and involutes by the age of 5-6 years. Hemangiomas are diagnosed usuallyclinically being easily noticeable, bright red, flat or raised patches on the skin, soft,
warm to the touch and can swell when the baby is crying. Deeper hemangiomas
may require imaging studies to confirm the diagnosis. Adult hemagioma is rare in
the salivary glands (1,3,4).
Hemangiomas are benign tumors characterized by a proliferation of endothelial
cells and pericytes and are seen mainly in the first two decades of life. They are classified as capillary and cavernous type. The congenital capillary type of hemangiomaor the juvenile type is the predominant subtype in the first year of life. The juvenile
hemangioma is common in females and there has a histologic appearance of acellular proliferation of capillary sized vessels and pericytes that extend diffusely
through the gland. The mitoses can be present. Cavernous hemangiomas are seenin older children (15).
Being a benign tumor witch eventually involutes, the treatment of hemangioma is commonly delayed. This benign vascular tumor seen in children and adults
is only rarely biopsied leaving the pathologist relatively inexperienced with thisclinically common lesion (1,4,6,7).
We present a case of a cavernous hemangioma involving the parotid glandof a one-year-male infant.

MATERIALS AND METHODS
We evaluated the medical record of patients with salivary gland neoplasms
diagnosed at Timisoara City Hospital from 2002 to 2009. We analyzed 220 cases ofsalivary gland tumors and only one case of parotid hemangioma was diagnosed.
Our patient was a male infant of 1 year old witch had a large mass in theright submndibular region. Clinical examination revealed a solitary soft swelling,mobile from side to side witch due to its large size caused asymmetry of the face.
The overlying skin was normal. The facial nerve was intact. This mass developed in
the last four months and had approximately 5/3 cm in size. The mass was excisedin toto and submitted for histopathological examination. On the cut surface it had
cystic and hemorrhagic areas.
Microscopic examination on hematoxilin-eosin stain (HE) revealed a single lineof endothelial cells with multiple thin-walled medium to large-sized blood vessels
present in between the glandular tissue with acinar preservation (Fig.1). Many red
blood cells were seen. There was no mitotic activity in the cells. The diagnosis ofcavernous hemangioma was made (Fig.2).

Fig. 1. Cavernous hemangioma of the parotid gland of the one-year-old male showing
thin-walled vessels (right) and parotid gland parenchyma (left). HE x 4.

Received July 2009. Accepted August 2009. Address for correspondence: Dr. Alexandra Corina Faur, Department of Pathology, Victor Babes University of Medicine and Pharmacy, P-ta Eftimie
Murgu 2A, 300041, Timisoara, Romania, e-mail: alexandra_pantu@yahoo.com

36

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Fiziologia - Physiology  2009.19.4(64)tumor growth, disfigurement, failure to involutes or intralesional hemorrhage thetrue incidence of this lesion might be underestimated.
In conclusion although hemangioma is considered one of the most common
soft tissue tumors, in our surgical pathology files is very rare and therefore is often
unfamiliar to the surgical pathologist. The rarity of salivary gland hemangioma in
our experience prompted us to report this case.
Acknowledgements: This work was supported partially by grant forscientific research from CNCSIS-PNII-Resurse Umane, type: TD, code CNCSIS: 314,
contract no. 138/15.09.08
REFERENCES
Fig.2. Higher magnification showing large diameter vessels with bland endothelial lining. HE x 20.

DISSCUSION
Neonatal and infantile lesions have a self-limiting clinical course so the treatment of infantile parotid hemangioma is usually conservative. Steroides reduce
growth and are the main treatment. Pressure therapy or embolisation may beconsidered also (1,14,15). Surgery is performed in only few cases with increase intumor size, disfigurement, failure to involutes or intralesional hemorrhage (9,16).
In our case the tumor had a large size causing disfigurement and was localized deepin the parotid region so the surgical treatment was performed.
The differential diagnosis for hemangioma includes lymphangioma, pleomorphic adenoma, branchial cleft cyst, lymphoepithelial lesions, reactive adenopathy,
abcess and cellulitis. The inflammatory lesions respond to antibiotics. The masses
without signs of inflammation can be cystic or solid. Cystic lesions are commonly
hemangiomas and lymphangiomas. For the lesions with a solid character magnetic
resonance imaging, computer tomography is useful. (1,2)
In a study over a period of 30 years, Childers E LB et al identified only 10 casesof hemangioma located in salivary glands: nine in the parotid gland and one in aminor salivary gland in the lower lip (4). Bentz BG et all reported 324 masses ofthe salivary gland region in children of witch 192 were hemangiomas, followed
by lymphangiomas and pleomorphic adenomas (2). The most common neoplasm
found in a large study of 420 pediatric salivary tumors made by AFIP was pleomorphic adenoma followed by hemangiomas (10). Mayo Clinic rewied a 52-years
experience of 74 pediatric salivary gland tumors and the most common lesion was
pleomorphic adenoma followed by hemangioma also.(5)CONCLUSION
The incidence of hemangioma cases is variable. Because the surgery is
performed only on hemangiomas demonstrating an increase in tumor size, rapid

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Washington, Mosby, St.Louis, 4th ed., 2001
16. Werner JA, Dünne AA, Folz BJ et al. Current concepts in theclassification, diagnosis and treatment of hemangiomas and vascular malformations of the head and neck. Eur Arch Otolaryngol
2001, 258:141-149.

HEMANGIOM CAVERNOS DE GLANDA PAROTIDAREZUMATIn studiul nostru, am evaluat cazurile existente in arhiva Spitalului Clinic Municipal de Urgenta Timisoara si raportate in perioada 2002-2009, selectand pacientii cu diagnostic de tumori ale glandelor salivare. Astfel ca, pe o perioada de 7 ani, din cele 220 de tumori ale glandelor salivare raportate doar un singur
caz a fost diagnosticat cu hemangiom salivar cu localizare parotidiana. Desi hemangioamele sunt tumori de tesuturi moi frecvent diagnosticate in perioada
copilariei in experienta nostra s-au dovedit a fi foarte rar biopsiate ceea ce le face sa fie putin cunoscute de catre patolog. In lucrarea de fata prezentam un
caz de hemangiom cavernos de glanda salivara parotida la un copil de 1 an.
Cuvinte cheie: hemangiom, glanda salivara, parotida
2009.19.4(64)  Fiziologia - Physiology

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37

CLINICAL ASPECTS OF AORTIC COARCTATION IN SMALL
CHILDREN
ANA LASCU1, CAIUS G. STREIAN1, ION SOCOTEANU1, CORINA VERNIC2
University of Medicine and Pharmacy ”Victor Babes”Timisoara, Department of Heart Surgery, Institute of Heart Disease Timisoara, Romania
2
Departament of Medical Informatics and Biostatistics, ”Victor Babes” University of Medicine and Pharmacy Timisoara, Romania

1

ABSTRACTThe aortic coarctation is a relatively frequent congenital cardiac malformation consisting in the narrowing of the aortic isthmus. We compared the demographic(age, sex, environment) and medical (blood pressure, heart rate, femoral pulse) data between a group of 13 patients with aortic coarctation (group B) and acontrol group of 22 children (group A) with similar background and age (1-3 year).
Using the paired t test there were no important differences between left and right arm systolic blood pressure in group A (p>0.05). For group B there were
important differences between left and right sistolic blood pressure in females (p=0.03840 S) but not in males. Also, the absence of the femural pulse wassignificantly more frequent in group B compared to group A (p<0.0001ES). Associated cardiac lesions as bicuspid aortic valve were found more frequently
in group B (38.46%) as in group A (9.09%).
In conclusions, clinical signs of disease like the absence of femoral pulse and hypertension are specific for the presence of aortic coarctation. Furthermore,other cardiac malformations, like bicuspid aortic valve are often associated with aortic coarcation.
Key words: aortic coarctation, blood pressure, heart rate, femoral pulse.

INTRODUCTION
Aortic coarctation is a congenital malformation consisting in the narrowing
in the aortic isthmus, near the origin of the left subclavian artery. It is caused byanomalies in the development of the 4th and 6th aortic arches. The aortic coarctation is relatively frequent (5-10% of all congenital cardiac malformations), withan occurrence of 5/10000 of live newborns and a gender ratio M:F of ~2:1 (1). The
age of diagnosis is related with the severity of the obstruction, the clinical severity
and the correct interpretation of the symptoms: a complicating factor is that aortic
coarctation may be associated with other cardiac lesions. The preferred treatment isinterventional or surgical, but there is the chance of relapse (3, 4). The life expectancy
is low, 20% live to reach 50 of age (2, 5).
The purpose of our study is to improve the diagnosis of the aortic coarctationin order to render the proper treatment.

coarctation diagnosed after birth at the Dept. of Heart Surgery of The Institute ofHeart Disease Timisoara in between 1998-2008. The children in both groups were in
between 1-3 years old. The appraisal of the data was necessary for the comparative
study and to underline the clinical and diagnostic particularities of each group.
The group A of the study was formed by 22 subjects (9 girls representing
40.91%, 13 boys representing 59.09%) with a 1.86 ± 1.05 year average (1-3
years), 14 patients for rural environment (representing 63.64%), respectively 8
patients urban environment (representing 36.36%). The B group was represented
by 13 patients of which 3 girls (23.08) and 10 boys (76.92%), with an average of
2.07 ± 1.24 years, 6 patients from urban environment (46.15%) and 7 patientsfrom rural environment (53.85%). Regarding gender distribution and origin, there
were no significant differences (chi square test, p>0.05) between the control grup
(group A ) and B (13 patients with aortic coarctation) (Figures 1 and 2).
15

MATERIAL AND METHOD
In a retrospective study, there were evaluated the demographic (age, sex,
environment) and medical (blood pressure, heart rate) data. The statistical analysis
consisted in a quantitative and qualitative study of the data. For the quantitative
analysis the data defined as an average of standard deviation; and for the qualitative
analysis the data is presented as frequency.
To compare the means, we used the paired and unpaired t test and to2compare the percentages we used the chi square test ( c ). The value p<0.05
was considered statistically significant. The statistical analysis was done on
specialized software OpenEpi 2.3 and Microsoft Excel.
The patients were divided in two groups that had similar background and age:group A (control) comprised 22 children; group B comprised 13 patients with aortic

13

13
11

10

9

9

Male
Female75331GrupA

GrupB

Fig. 1. Gender distribution of the tow groups

Fig. 1. Gender distribution of the tow groups

Received September 2009. Accepted October 2009. Address for correspondence: Dr. Ana Lascu, University of Medicine and Pharmacy”Victor Babeş”Timişoara, 2A Eftimie Murgu Square, 300041,
Timisoara, Romania

38

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16
11
6

14Fiziologia - Physiology  2009.19.4(64)
Rural
Urban
8

7

6

3

Maximum

1
GrupA

GrupBp (unpaired t test)

Fig. 1. Gender distribution of the tow groups

137.5

130

127.5

120

80

75

7

p<0.001p<0.001p<0.001p<0.001

0.003573

0.088

0

ES

ES

ES

ES

FS

4 FS

8

Table II. Comparative aspects of heart frequency values.
16

14

Measured

Rural
Urban

11

8

valuesGroups A

7

6

6

(22)

1
GrupA

GrupB

Fig. 2. Distribution according to the backgroundFig. 2. Distribution according to the backgroundWe included systolic blood pressure (SBP) and diastolic blood pressure (DBP)on the left arm respectively right arm, and heart frequency (HR) values as clinicaldata to evaluate the group. Also, the change in femoral pulse (weak/ absent) forthe children with CoAo was compared with that of the control group.
The blood pressure measurement was made with a sphygmomenometer, using
the ascultatory method (Korotkow) at the upper and lower level of the listening witha stethoscope placed at the level of humeral (brachial) artery. The noise appears at
the slow decompression of the cuff, due to turbulent blood pressure.
Measurements were made in clinostatism using different sized cuffs eccording
to the patients size. The width of the cuff must be at least 40% of the arm circumference (measured at the half distance between the olecron and the acronion).
The heart rate measurement was carried out by listening the sounds of the heartwith a stethoscope placed on the thorax. Examining the absence or presence of thefemoral pulse was performed by palpation of the femoral artery at the groin.

Groups B(13)

Table I. Comparative aspects of blood presure values
SBPleft(mmHg)

SBPright(mmHg)

M

F

M

F

M

F

M

F

Ratio

80.18

79.67

80.18

83.60

49.88

52.85

52.77

54.66

Groups A

Standard

20.35

29.25

14.04

15.87

17.99

26.28

10.57

16.44

(22)

deviation
45

51.5

30

36.5

42.5

40

30

30

Minimum

DBPleft(mmHg)

DBPright(mmHg)

Maximum

119

115

80.5

75

118.5

107.5

67.5

72.5

Ratio

116.56120

107.56

110

67

73.33

60.68

66.66

GroupsB

Standard

17.69

15.27

18.94

8.66

10.13

2.88

7.68

11.54

(13)

deviation
Minimum

90

100

80

105

65

70

52.5

60

Maximum

137.5

130

127.5

120

80

75

72.5

80

p<0.001p<0.001p<0.001p<0.001

0.003573

0.088

0.024

0.02725

ES

ES

ES

ES

FS

4 FS

8S

S

p (unpaired t test)

Table II. Comparative aspects of heart frequency values.

Also, the change inMeasured
femoral pulse(weak/ absent)
was highlighted for theParameterHR
F
children group with CoAo values(group B) compared withM the control
group (p<0.0001ES).
Ratio
110
125
Groups A
For the group A the femoral(22) pulse was present in 20 subjects (90.90%) and absent
Standard deviation
12.85
14.45
in only 2 subjects (9.09%), compared
Minimum with group
105 B where
110 the femoral pulse was
Maximum
155
present in only one patient (7.69%),
for the rest140of 12 (92.30%)
being absent.
Groups B
(13)

Ratio

114

137

Standard deviation

13.72

15.27

Minimum

100

120

Maximum

130

150

P>0.05N

P=0.02S

636 S

2009.19.4(64)  Fiziologia - Physiologyp (unpaired t test)

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Ratio

110

125

Standard deviation

12.85

14.45

Minimum

105

110

Maximum

140

155

Ratio

114

137

Standard deviation

13.72

15.27

Minimum

100

120

Maximum

130

150

P>0.05N

P=0.02S

636 S

Table II. Comparative aspects of heart frequency values.

20Group A

20

Group B

12

15

2

5

Table I. Comparative aspects of blood presure values
valuesF

10Using the paired t test to compare the left sistolic blood pressure with the rightsistolic blood pressure in females, respectively in males from the A group, there were
found no significant differences (p>0.05).
For the B group, there were no significant differences (p>0.05) in males,
but there were significant differences in females, comparing SBPleft-SBPright(p=0.03840 S) and DBPleft-DBPright (p=0.01378), according to Table I. Heartrate compared between males and females was highly significant p=0.003138 ingroup A and p<0.001 ES in group B (Table II).

ParametersHRMp (unpaired t test)

RESULTS AND DISCUsIONS

MeasuredParameter

1

0
Femural pulse absent

Femural pulse prezent

Fig.3. Modification of the femural pulse in Group B vs. Group A

The aortic coarctation diagnosis was the first to be taken into account for 8 of
the children with aortic coarctation (61.58%) compared with the 22 subjects ofgroup A (62.85%) for which the aortic coarctation was not primarly diagnosed;
they were examined within a routine consultation or for other pathology by thefamily doctor, p>0.05 NS. The weight of the associated diagnoses is also similar,
the most frequent being the bicuspid aortic valve (6 cases for group B (46.15%),
followed by aortic stenosis.
Prime aortic
coarctation

22

25

Other diag.

20
15
8

10
5

5

0

0
Group AGroup B

Fig.4. Primary diagnosis of aortic coarctation in Group B vs. Group A

39

CONCLUSIONSREFERENCES

Patients with aortic coarctation (aged 1-3 yrs old) represent a significant
percentage (37.14%) of the total number of children aged 1-3 year old integrated
in this study.
As associated diagnoses, bicuspid aortic valve is discovered most often in thegroup with aortic coarcation 5 patients (38.46%) as compared with the control groupwere bicuspid aortic valve was shown in only 2 cases (9.09%), p=0.9657 NS.
Statistical results indicate that clinical signs of disease (the absence of femoral
pulse) and hypertension were evident for the children with aortic coarctation (1-3
years) as compare to group A.

1. McBride KL, Marengo L, Canfield M, Langlois P, Fixler D, Belmont
JW. Epidemiology of Noncomplex Left Ventricular Outflow Tract
Obstruction Malformations (Aortic Valve Stenosis, Coarctation of
the Aorta, Hypoplastic Left Heart Syndrome) in Texas, 1999 –2001,
BirthDefects. Res A Clin Mol Teratol, 2005 August; 73(8): 555-561.
2. Campbell M. Natural history of coarctation of the aorta. Br. HeartJ. 1970 September; 32 (5): 633-640.
3. Socoteanu. Tratat de Patologie Chirurgicală Cardiovasculară,Editura Medicală Bucureşti, 2007, Vol I, p.462-496.
4. Socoteanu. Tratat de Cardiopatii Congenitale, Editura AcademieiBucureşti 2009, în curs de apariţie.
5. Verheugt CL, Uiterwaal CS, Grobbee DE, Mulder BJ. Long-term
prognosis of congenital heart defects: A systematic review. Int J
Cardiol, 2008 August; 131(1): 25-32.

ASPECTE CLINICE SUGESTIVE PENTRU COARCTAŢIA AORTICĂ
LA COPILUL MIC
REZUMAT

Coarctaţia de aortă (CoAo) este o malformaţie cardiacă congenitală relativ frecventă constând în îngustarea lumenului aortei istmice. Au fost comparate
date demografice (vârsta, sex, mediu) şi medicale (tensiunea arterială, frecvenţa cardiacă, pulsul femural) la un grup de 13 pacienţi cu coarctaţie de aortă
(grupul B) şi un grup de control de 22 de copii (grupul A) proveniţi dintr-un studiu populaţional asemănător din punct de vedere al mediului de provenienţă
şi al vârstei (1-3 ani).
Aplicând testul t pereche diferenţele sunt nesemnificative între tensiunea arterială sistolică măsurată la braţul drept şi stâng în grupul A (p>0,05). În grupul
B, diferenţele sunt semnificative între tensiunea arterială sistolică dreaptă şi stângă la femei (p=0,03840 S), dar nu la sexul masculin. De asemenea, absenţa
pulsului femural este semnificativ evidentă în grupul B comparativ cu grupul A (p<0,0001 ES).
În concluzie, semnele clinice de boală ca: absenţa pulsului femural şi hipertensiunea arterială sunt specifice pentru prezenţa coarctaţiei de aortă. Asocierea
leziunilor cardiace ca bicuspidia valvei aortice este mai frecventă în grupul B (38,46%) comparativ cu grupul A (9,09%).
Cuvinte cheie: coarctaţie aortică, tensiunea arterială, frecvenţa cardiacă, puls femural.

40

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Fiziologia - Physiology  2009.19.4(64)METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS
STRAINS ISOLATED FROM AN ORTHOPAEDICS WARD AND
AMBULATORY
M. BERCEANU VADUVA, H. PETRESCU, D. VERMESAN, F. TATU, A. LUPASCU, V.
DUMITRASCU, ELENA HOGEA, DELIA MUNTEAN, MATILDA RADULESCU, MARCELAADAMUT, CARMEN TATU, DANA VELIMIROVICI, DELIA BERCEANU VADUVAUniversity of Medicine and Pharmacy”Victor Babeş”Timişoara

Abstract

Purpose: Staphylococcus aureus is one of the species most frequently isolated in in ambulatory, as well as in hospital environments, and can cause infections
in both patients with immune deficiencies and healthy patients. It is one of the most pyogenic bacteria, capable of producing infections in any part of thehuman body. These are produced through the multiplication of the microorganisms (with direct invasion and tiss damage), or through release of toxins. The
aim of the present paper is a comparative study of the sensitivity to antiinfectious chemotherapy drugs of S. aureus strains isolated from various pathologicalproducts collected from an orthopaedics ward and from ambulatory, and also to establish the resistance phenotypes of these strains. Of special interest was
the study of the frequency of the Peni-R Meti-R (MRSA - Staphylococcus aureus methicillin-resistant) phenotype in the orthopaedics ward and ambulatory.
Material and methods: 951 S. aureus strains were studied: 60 isolated from 255 samples from the orthopaedics ward, and 891 isolated from 1620 samples
from ambulatory. Testing of the antibiotic resistance was performed through Kirby-Bauer disk-diffusion method. Interpretative analysis was used to establish
the resistance phenotypes of the germs.
Results: A high percentage of multiresistant strains was isolated, 21 strains (35.00%) isolated from the orthopaedics ward and 89 strains (9.99%) fromambulatory being identified as Peni-R Meti-R (MRSA) resistance phenotype.
Conclusions: The high frequency of Staphylococcus aureus Peni-R Meti-R (MRSA) resistance phenotype in the hospital environment explains the involvementof this germ in the ethiology of nosocomial infections. In order to limit the spread of these strains, especially within the hospital, enforcement of efficientnosocomial infections control techniques is necessary, as well as introducing policies for the rational antibiotic use.
Keywords: Staphylococcus aureus, MRSA, resistance phenotype.

INTRODUCTION

Material and methods

Staphylococcus aureus represents one of the most frequently isolated species
both in ambulatory and in hospital; it can cause infections to patients with low
infection resistance or to healthy, immune persons.
It is one of the most pyogenic bacteria, able to produce infections anywhere in
the organism, starting with simple skin staphylococcic infections to infections withsevere development. They are produced through the proliferation of microorganisms,with direct invasion and tissue destruction or through toxin release.
S. aureus provisionally colonizes mucous membranes and wet tegumentaryareas. Almost 15-30% of normal adults are naso-pharynx bearers of S. aureus. For
the medical staff and patients who were hospitalized for long time, the bearing
rate may reach 40-80%, which explains the various nosocomial infections withthis species.
The aim of the present paper is to test the drug sensitivity of S aureus strains
isolated from various pathological productes collected from patients admitted in
an orthopaedics ward and ambulatory, as well as to establish the resistance phenotypes of these strains. This way the best chemotherapeutic agent can be chosen
and multi-drug resistance can be avoided. Of special interest was the study of thefrequency of the Peni-R Meti-R (MRSA - Staphylococcus aureus methicillin-resistant)phenotype in the ambulatory and orthopaedics ward.

951 S. aureus strains were studied: 60 isolated from 255 samples from theorthopaedics ward, and 891 isolated from 1620 samples from ambulatory. Testingof the antibiotic resistance was performed through Kirby-Bauer disk-diffusion
method. The results were interpreted according to CLSI standard.
In order to test the sensitivity to antibiotics, the following drugs wereused: penicillin (P), oxacillin (OX), kanamycin (K), tobramycin (TM), gentamicin(GM), erythromycin (E), clindamycin (CLI), vancomycin (VA), ciprofloxacin (CIP),
trimethoprim-sulfamethoxazole (SXT). Although some of the antibiotics included inpanel for staphylococci susceptibility testing are not usually used in clinical practice,
the testing panel was designed to detect the main mechanisms of resistance toantibiotics, and to allow identifying of the resistance phenotypes.
Interpretative analysis was used to establish the resistance phenotypes of
the germs.

Results and discussions
Of the total 951 S. aureus strains studied, 891 were isolated from 1620 samples
from ambulatory, and 60 were isolated from 255 samples from the orthopaedics
ward.

Received September 2009. Accepted October 2009. Address for correspondence: Dr. Delia Berceanu, University of Medicine and Pharmacy”Victor Babeş”Timişoara, 2A Eftimie Murgu
Square, 300041, Timisoara, Romania, e-mail: deliaberceanu@yahoo.com
2009.19.4(64)  Fiziologia - Physiology

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41

uded in panel for staphylococci susceptibility testing are not usuallyin clinical
4. used
Blood
3
5.00
ting panel was designed to detect the main mechanisms of resistance to5.antibiotics,
andtoSputum1
1.67
SSIONSUSSIONSg of the resistance phenotypes.
6.
Bronchial aspirates
1
1/67. aureusaureusstrainsstudied,891891
wereisolatedfrom 1620
fromambulatory,strainsstudied,isolatedfrom1620
samplesfrom ambulatory,
ative
analysis
was used
to establish
the were
resistancephenotypes
ofsamplesthegerms.
Table III. Specimens from which the S. aureus strains were isolated – orthopaedics

255 samples
thetheorthopaedics
ward.ward.
mm 255
samplesfrom
fromorthopaedics

Total

60

Nr.

Specimens

100%

Nr.

%

D DISCUSSIONSInterpretative
analysis
wasfrom
usedtoWoundestablish
phenotypes of the germs
Table I.
Sample891
distribution
1. ambulatory,
secretion the resistance46
76.66
otal 951 S. aureus strainsstudied,isolatedfrom 1620
samples
Table
I. Samplewere
distribution
determinationoftheresistancephenotypes
toantibiotics,
information
2.
Pharingealswab
610.00on the resistance mec
lated from 255 samples from theorthopaedics
Table
I. Sample distributionward.
Interpretative
analysiswasusedantibiotics
to establish 3the toresistancephenotypesofbe obtained
and based on thesemechanisms
the
which
the tested
bacteria
OriginNr.
%
3.
Sinovial fluid
5.00
thegerms.
Throughthedeterminationoftheresistancephenotypes
toantibiotics,
4.
Blood
3
5.00
could
be
detected.
Origin Table I. SampleNr.
%
distribution
information on5.the resistancemechanisms could be1obtained andbased on theseAmbulatory
1620
86.40%
Sputum
1.67
The following phenotype
resistanceto beta-lactams
were estabished:
Peni-S Meti-S
mechanisms
theantibiotics
to
which
the
testedbacteria
wasresistantcould beOriginNr.S, Peni-R%86.40%
1
1/67
Ambulatory
Orthopaedics
255 1620
13.60%Meti-R (MRSA). detected. 6. Bronchial aspirates
Total
100%
Most of the isolated S. aureus
be 60Peni-R
Meti-SPeni-S(83.50% - ambu
Ambulatory
1620
86.40%
The followingstrains
phenotypeproved
resistance totobeta-lactams
were estabished:
Orthopaedics
255
13.60%
TOTAL
1875 orthopaedics).
100%
Only
6.51%
of
the
ambulatory
strais
and
5%
of
the
orthopaedics
strains we
Peni-R
Meti-S,
Meti-R (MRSA).
Interpretative Meti-S,
analysiswasusedPeni-R
to establish
the resistance phenotypes of the germs. ThroughOrthopaedics
255
13.60%
beta-lactamsdetermination(Peni-S Meti-S).
The
MRSA
presence
in
9.99%
of
the
ambulatory
cases
andc
of the resistancephenotypes
to antibiotics,
information
the (83.50%
resistance
Most of theisolated S. aureus
strains proved
to be Peni-Ron
Meti-S- mechanisms
TOTAL
1875
100%
TOTAL
1875
100%
obtainedand basedon alarm
these
thetoambulatory
whichstrains
thewas straresi
orthopaedicsbeward
represents
anbecausetheof theMRSA
hospital
ambulatory,
60%mechanisms-signal,orthopaedics).
Onlyantibiotics
6.51%
straistested
andare5%bacteria
could
detected. of(Table
multi-resistanttobeantibiotics
IV andFigure2). Theypresent
crossedresistance to al
the orthopaedics
strains
were sensitive
to beta-lactams(Peni-SMeti-S). The
The following phenotype resistance to beta-lactams were estabished: Peni-S Meti-S, Peni-R M
MRSAcarbapenemes,presence in 9.99% of the ambulatory
cases and 35.00%
in the orthopaedics(penicillins, S, cephalosporins,
betalactamase
inhibitors).
The resistanc
100,00%Peni-R Meti-R (MRSA).
ward
representsanalarm
signal,becausethe
MRSA
strainsarehospitalstrains,
erythromycin, clindamycin,
chloramphenicol,
tetracycline,
trimethoprim-sulfamethoxazol
Most of the isolated S. aureus strains proved to be Peni-R Meti-S (83.50% - ambulatory, 60
80,00%100,00%
which
areofmulti-resistantto antibiotics(Tableand2).sensitivity
They present
orthopaedics).
Onlythe6.51%
the ambulatory
strais and
5%IVoforthopaedics
strainssensitivaminoglycosides,
althoughdiffusimetrictest
may
indicate
atheFigure
false
towerethese
ant
100,00%beta-lactams (Peni-Scrossed
Meti-S).
The toMRSA
presencein 9.99%of the ambulatory
cases and 35.00% in
resistanceall beta-lactams(penicillins,
cephalosporins,
carbapenemes,
orthopaedics ward represents
an IV.
alarm
signal,becausethe MRSA
strains clindamycin,
are hospital strains, which
Table
Resistancephenotypes
beta-lactams
betalactamase
inhibitors).
The resistanceextends
totoerythromycin,
multi-resistant to antibiotics (Table IV and Figure 2). They present crossed resistance to all beta-lac
chloramphenicol, tetracycline, trimethoprim-sulfamethoxazole, quinolones,(penicillins, Nr.
cephalosporins,
carbapenemes, betalactamase
inhibitors).Orthopaedics
The resistance extend
Phenotype
Ambulatory
aminoglycosides,
although the diffusimetric
test mayindicate a false sensitivity
erythromycin, clindamycin,
chloramphenicol,
tetracycline,
trimethoprim-sulfamethoxazole,quinoloto thesethe
antibiotics.
Nr. indicate %Nr. to these% antibiotics.
aminoglycosides, althoughdiffusimetric test may
a false sensitivity

60,00% 80,00%
60,00%

80,00%
40,00%

40,00%

20,00%
60,00%
0,00%

20,00%

40,00%0,00%
Ambulatory

Ambulatory

20,00%

1.

Orthopaedics
Orthopaedics

type)
Nr.

Fig. 1. Sample distribution

Fig. 1.Fig.
Sampledistribution
1. Sample
distribution

Peni-S Meti-S
(wild
58
Table
IV. Resistancephenotypes
tobeta-lactams
beta-lactams 3
Table
IV. Resistancephenotypes to6.51

0,00%
The
specimens from which the S. aureus strains were isolated are presented
in Tables II and III.

Phenotype

Ambulatory

Peni-R Meti-S

744
Nr.

83.50
%

Nr.

36

%

60.00

3.

1. Meti-R
Peni-S Meti-S(wild
Peni-R(MRSA)

8958

6.51
9.99

3

21

5.00

35.00

2.

Peni-R Meti-S

3.

Specimens

Nr.

Nr.

60

83.50

36

Peni-R Meti-R (MRSA)

89

9.99

21

35.00

Total

891

100

60

100

Table II. Specimens from which the S. aureus strains were isolated - ambulatory

Specimens

100

744

Table II. Specimens from which the S. aureus strains were isolated - ambulatory
able II. Specimens fromwhichFig.
the 1.
S. Sample
aureus strains
were isolated - ambulatory
distribution

Nr.

Orthopaedics

2.

type)
cimensfromthewhich
the S. aureus
strains
isolated
arepresented
presented in
andIII.III.891
Ambulatory
Orthopaedics
TotalII II
om which
S. aureus
strains
werewere
isolatedareinTablesTables
and

Nr.

5.00

60.00

100

%

%

100,00%83,50%
m which the1. S. aureus
strains were
Pharingeal swab
635 isolated
71.26 are presented in Tables II and III.

1.

Pharingeal
swab
2.
Nasal
swab

635 188

80,00%100,00%

71.26
21.09

83,50%

60,00% 80,00%

ble II. Specimensfromaureusambulatory
3. swabSputumwhich the S. 188
14 strains
1.57were isolated
2.
Nasal
21.09
40,00%-60,00%
3.

4.
Bronchial aspirates
Sputum

14 3

0.33
1.57

4.

Bronchial aspirates

3

0.33

5.
1.

Ear
secretion swab
Pharingeal

15

6.

Pus
8.

Nr.

5.

SpecimensEarsecretion

6.

Pus

7.

Wound secretion

15 Nr.
16

1.68

%

1.80

1.68
7 635
0.78 71.26

20,00%

40,00%

6,51%

20,00%

5,00%

6,51%

5,00%

60,00%

60,00%

35,00%
9,99%35,00%

9,99%

0,00% 0,00%
Peni-S
Meti-S
Meti-S Peni-R
Peni-R
Meti-R
Peni-S
Meti-S Peni-R
Peni-R Meti-S
Meti-R
Ambulatory

Ambulatory

Orthopaedics

Orthopaedics

Fig. 2. Resistance phenotypes to beta-lactams
16 9
1.80
2. Resistance phenotypes
to beta-lactams
188 1.01 21.09
Fig. Fig.
2. Resistancephenotypes
to beta-lactams
7.
Wound
secretion
7 4
0.78
9.
Endocervical swab
0.45In order to determine the three resistance phenotypes to aminoglycosides (K, KT, K
tobramycin andgentamicin
used.
Thus,
susceptibility
to amikacinca3.
Sputum
14 kanamycin,
1.57
Inthe
order
tothreedeterminewere
the threeresistancephenotypes
to aminoglycosides(K,and netilmicin (Kto determine
resistancephenotypes
toaminoglycosides
8.
Conjunctival
9 884In order
1.01
Total swab
100%KTG) kanamycin,weretobramycin
and gentamicin
were used. Thus, susceptibility
tokanamycin,
and KT,
gentamicin
used.
Thus, susceptibility
to amikacin
and net4.
Bronchial aspirates
3 tobramycin
0.33
9.
Endocervical swab
4
0.45
amikacin and netilmicin can be readily deduced from the resistance or susceptibility
Table III. Specimens from which the S. aureus strains were isolated – orthopaedics
Table
III. Specimens from which the S. aureus strains were15
isolated – orthopaedics 1.68 profile to the three aminoglycosides tested (Table V and Figure 3).
5.
Earsecretion
Total
884
100%
The K phenotype (resistance to kanamycin) – encountered in 15.71% ofNr.
Specimens
Nr.
%
6.
Pus
16
1.80 the ambulatory strains and 11.66% of the orthopaedics strains, as well as the KT
1.
Wound secretion
46
76.66
phenotype (resistance to kanamycin and tobramycin) – encountered in 0.45% of
7.
Woundsecretion
7
0.78 the ambulatory strains and 3.33% of the orthopaedics ward, are also predictive for2.
Pharingeal swab
6
10.00
resistance to amikacin; netilmicin being active.
3.
Sinovial fluid
3 9
5.00
8.
Conjunctival
swab
1.01
Resistance to kanamycin, tobramycin and gentamicin (KTG phenotype) –
4.
Blood
3
5.00
encounteredin 11.87% of the ambulatory strains and 15.00% of the orthopaedics
9.
Endocervical
swab
4
0.45
5.
Sputum1
1.67
ward, is predictive to resistance to amikacin and netilmicin.

2.

swab
NasalConjunctivalswab

6.
Bronchial aspirates
Total
Total

1 884

1/67

60

100%

100%

ative analysis was used to establish the resistance phenotypes of the germs. Through the
Fiziologia - Physiology  2009.19.4(64)42f the resistance phenotypes to antibiotics, information on the resistance mechanisms couldd based on these mechanisms the antibiotics to which the tested bacteria was resistant
cmyk
d.
owing phenotype resistance to beta-lactams were estabished: Peni-S Meti-S, Peni-R MetiR (MRSA).

38,33%80,00%
ilyfrom thesusceptibility
profileto thethreeaminoglycosides
tested0,00%(TableV 71,38%) –deduced
encounteredin resistance
0.45% oforthe
ambulatory
strainsand3.33%
of the orthopaedics
ward,
are
40,00%
FigureWild type 16,50%
MLSB 56,66%MLSB
M
ive for3).resistance to amikacin; netilmicin being active.
60,00%
9,99% 3,33% 2,13% 1,66%
The Ktophenotype(resistanceto kanamycin)– encountered
15.71% of –theencounteredambulatory20,00%
strains
inducible constitutive
istancekanamycin,
tobramycin
and gentamicin(KTG inphenotype)in 11.87% 38,33%
ly deduced
the resistancestrains,or susceptibility
to thethree aminoglycosides
tested (Table
V
11.66%
of from
the andorthopaedics
as well asprofile
theward,KT
phenotype(resistanceto kanamycin
and40,00%
0,00%
ulatorystrains
15.00% of the orthopaedics
is predictive
to resistanceto amikacinand 16,50%
Figure 3).– encountered in 0.45% of the ambulatory strains and 3.33% of the orthopaedics ward, areWild type
MLSB Orthopaedics
MLSB
M
Ambulatory
amycin)20,00%
3,33% 2,13% 1,66%
inducible 9,99%
constitutive
The K phenotype(resistanceto kanamycin)– encounteredpredictive
for resistanceto amikacin;
netilmicin being
active. in 15.71% of the ambulatory strains
11.66%of theto orthopaedics
strains, as andwellgentamicin
as the KT(KTG
phenotype(resistanceto kanamycin
andResistancekanamycin,
tobramycin
phenotype)– encounteredin 11.87%
0,00%
Table V.
Resistance phenotypes
to aminoglycosides
Orthopaedics
Fig.type
4.Ambulatory
Resistanceto MLS M
MLSBphenotypes
MLSB
amycin)– encountered
0.45% of the orthopaedics
ambulatory strains
and3.33% ofto theorthopaedics
ward, andareWild
e ambulatory
strains andin15.00%
ward, ispredictive
resistanceto amikacin
inducible
constitutive
predictive for Nr.
resistance toamikacin; netilmicin
being active.
micin.
Phenotype
Ambulatory
Orthopaedics
In orderto detect theresistanceFig.
to 4.
fluoroquinolones,a single quinolone was used – ciprof
Resistance to kanamycin, tobramycin and gentamicin (KTG phenotype)– encounteredin 11.87%
Resistance phenotypes to MLS
Ambulatory
Orthopaedics
staphylococci
being
cross-resistanttoallfluoroquinolones.
Most
were sensitive – 86.98%
Table
V.
Resistancephenotypes
toaminoglycosides
Nr.
%
Nr.
%
Table
V.
Resistancephenotypes
toaminoglycosides
used–ciprofloxacin;
staphylococci
being
cross-resistant
tostrains
all fluoroquinolones.
e ambulatory strains and 15.00% of the orthopaedics ward, is predictive to resistance to amikacin and
In
order
to
detecttheresistancetofluoroquinolones,asinglequinolonewasused acquired– ciprofloxa
ambulatory
strains
and61.67%
oftheothopaedics
strains,
and
only
few
of
them
re
micin.
Most
strains
weresensitive–
86.98%
oftheambulatory
strains
and61.67%of the1. Nr.Wild typePhenotype
642Ambulatory
72.05
42
70.00
Orthopaedics
staphylococci
being38.33%
cross-resistant
toFig.
all4.fluoroquinolones.
Most
strains
were sensitive – 86.98% of(13.02%- ambulatory,
othopaedics)(TableVII
andFigure5).
Resistancephenotypes
toMLS
othopaedics
strains,
and
only
few
of
them
acquiredresistance(13.02%
ambula2.
K
140
15.71
11.66strains and 61.67% of the othopaedics strains, and only few of them acquired resista
Nr.
%
Nr.7ambulatory%
Table V. Resistance phenotypes
toaminoglycosides
tory,38.33%
38.33%(TableVII andVII
FigureIn3.33
to detecttheTable
resistanceto fluoroquinolones,
aFiguresingle 5).
quinolone was used – ciprofloxa
VII. Resistancephenotypes
to5).
fluoroquinolones- orderambulatory,-- othopaedics)
othopaedics)(Table
and
3.
4642
0.45
1.KT Wild type
72.05
422(13.02%
70.00
Nr.
Phenotype
Ambulatory
Orthopaedics
staphylococci
being
cross-resistanttoallfluoroquinolones.
Most strains were sensitive – 86.98% of
2.
K
140
15.71
7
11.66
4.
KTG
105
11.78
9
15.00
Table
phenotypes
to tofluoroquinolones
Nr. 61.67%
Phenotype
Ambulatory
Table
VII. Resistancephenotypes
fluoroquinolones
Nr.
%
Nr. ambulatory%
strains andof VII.
the Resistanceothopaedics
strains,
andonlyOrthopaedics
few of them acquired resist
3.
KT
4
0.45
2
3.33
Total
891
100
60
100
(13.02%
ambulatory,
38.33%
othopaedics)(TableVII
andFigure
5).
Nr.
Nr.
%
1.
Wild type
642
72.05
42
70.00
Nr.
Phenotype
Ambulatory%
Orthopaedics
4.
2.

KTG
K

3.

KT

4.

KTG

Total

Total 70,00%
80,00% 72,05%
80,00% 72,05%

105
140
891
4

11.78
15.71
100
0.45

9
7
60
2

15.00
11.66
100
3.33

105
891

11.78

9

15.00

100

60

100

60,00%

WildTable
type VII. Resistance phenotypes
775
86.98
37
Nr.
%fluoroquinolones
Nr.
to
2. Total
Resistant

1.

Total
Wild type2.

Resistant

100,00% Total

40,00%

80,00%
40,00% 72,05%

70,00% 11,66%
11,78% 15,00%
15,71%
0,45% 3,33%11,78% 15,00%
15,71% 11,66%
0,45% 3,33%

20,00%

60,00%
20,00%

0,00%

40,00%
0,00%
Wild type
Wild type
20,00%

K

KT

KTG

11,78%
K 11,66% KT
KTG 15,00%
15,71%
0,45% 3,33%
Ambulatory
Ambulatory Orthopaedics
Orthopaedics

100,00%80,00%

60,00%
100,00%40,00%
40,00%
80,00%
20,00%
20,00%
60,00%
0,00%
0,00%
40,00%

20,00%
0,00%

Orthopaedics%61.67

38.33
3723
61.67
Orthopaedics
2360
38.33
Nr.
%100
60
100
37
61.67

116
891
Nr.

891
775

13.02
100
%
100
86.98

116

13.02

23

38.33

86,98% 891

100

60

100

86,98%

61,67%

61,67%

80,00%

60,00%

WildFig.
type3. Resistance
K phenotypes toKT
aminoglycosidesKTG
3. Resistancephenotypes toto aminoglycosides
aminoglycosides
Fig.Fig.
3. Resistancephenotypes
Ambulatory

116
13.02
775
86.98
Ambulatory

2. Nr.
1. ResistantWild type
Phenotype70,00%

60,00%

0,00%

1.

38,33%

86,98%
61,67%

38,33%

13,02%
13,02%

38,33%
13,02%
Resistant
Resistant

Wildtype
type
Wild
Ambulatory
Ambulatory

Orthopaedics
Orthopaedics

Resistant
In orderto determine
resistancetotoMLS
MLS (macrolides,(macrolides, lincosamides,
streprogramines) the Fig.
order
to determine
the tothe
resistancelincosamides,
the 5. Resistance phenotypes to fluoroquinolones
In orderdetermine the resistanceto MLS (macrolides, lincosamides,
strepro- streprogramines)Fig.
5.
Resistancephenotypes
to fluoroquinolones
Fig.
5.
Resistancephenotypes
tofluoroquinolones
ciationoferythromycin
andclindamycin
wastestedusing
thediffusimetricmethod(TableVI
andof erythromycin
and theclindamycin
was tested
thewasdiffusimetric(Table VI and Ambulatory Orthopaedics
gramines)
association
erythromycin
and usingclindamycin
tested using
the method
Fig. 3. ofResistancephenotypes
to aminoglycosidesre 4).
71.83%of the ambulatory strains
and 56.66% of the orthopaedics strains were sensitiv
71.83%
oftheambulatory
strains and 56.66% of the orthopaedics strains were sens
diffusimetricmechanism
method(Tablewas
VI andFigure4).(macrolides,In
determine
the resistancetoMLS
streprogramines)
trimethoprim-sulfamethoxazole,therestofResistance
28.16%,
43.33%
respectively
being resistant.
Theordermaintoresistancethemodifyingof the lincosamides,
ribosomal
target,connected
to theFig.
5.
to fluoroquinolones
71.83%
oftheambulatory
strainsphenotypes
and 56.66%
of the orthopaedics
main resistancemechanism
was thewas
modifyingof thetheribosomal
target,connected
theof 28.16%,
thetorest
43.33%
respectively
beingstrains
resistant.
ciationandclindamycin
testedusing
diffusimetricmethod(TableVI Theandisition of erythromycin
the erm gene(whichis responsible
fortheresistancethroughribosomal
modification).
The mainresistancemechanism was
modifying
oftrimethoprim-sulfamethoxazole,theribosomal
target,weresensitivetotrimethoprim-sulfamethoxazole,
the
rest
of
28.16%,
43.33%
Table
VIII.
Resistancephenotypes
totrimethoprim-sulfamethoxazole
of
the
erm
gene
(whichis
responsibleforresistancethroughribosomal
modification).
The
71.83%
oftheambulatory
strains
and56.66%
oftheorthopaedics
strains
were sensitivre4). expression
otypicofresistancecanbeinducible
orconstitutive:
inducible
MLS
phenotype,B
connected to the acquisition of the erm gene (which is responsible for resistance
Table
VIII.being
Resistancephenotypes to trimethoprim-sulfamethoxazole
respectively
resistant.
expressionof resistancecan wasbe inducible
or constitutive:
inducible
MLS
B phenotype,
trimethoprim-sulfamethoxazole,
rest of 28.16%, Ambulatory
43.33% respectively
being resistant.
The mainmechanism
the modifyingof the ribosomal
target, connectedto thethephenotype.
titutive
MLSBresistanceNr.
Phenotype
Orthopaedics
through ribosomal modification). The phenotypic expression of resistance can be inMLS
isitionthe
erm gene28.62%(which isresistancemodification).
In theambulatoryof responsible
the strains forwereresistantthroughto theribosomal
MLS, and
61.65%
in ThetheBofphenotype.
Nr.
Phenotype
Ambulatory
Orthopaedics
Nr. to trimethoprim-sulfamethoxazole%
Nr.
%
Table VIII.
Resistancephenotypes
constitutive:
inducible
phenotype,resistantconstitutive
MLS
phenotype.
Table
VIII. Resistancephenotypes
to trimethoprim-sulfamethoxazole
B were orotypicexpressionof orresistancecan strains
be MLS
inducible
constitutive:
inducible
phenotype,
opaedics
ward. ducible
he ambulatory
28.62%of theto Bthe
MLS, MLS
and B 61.65%
in the
Nr.
%
Nr.
%
In
the
ambulatory
28.62%
ofthestrains
wereresistant
to
the
MLS,
and
61.65%
1.
Wild
type
640
71.83
34
56.66
Table
VI.
Resistancephenotypes
toMLS
titutive
MLS
phenotype.
Nr.
Phenotype
Ambulatory
Orthopaedics
cs ward. B
251
28.17
2634
43.33
in theorthopaedics
ward.
In the ambulatory
28.62%
ofstrains phenotypes
were resistant to the MLS, and 61.65%
in Resistantthe
1. 2. Wild
type
640
71.83
56.66
Table
VI.theResistanceNr.
Phenotype
Ambulatory to MLS
Orthopaedics

opaedics ward.

Nr. phenotypes% to%Table Table
VI. ResistancePhenotype
Ambulatory
Orthopaedics
VI. Resistancephenotypes
to MLSMLS Nr.

Nr.

1.
2.
3.
4.

1.
Nr.
2.

Wild type
Phenotype
MLSB inducilble

Wild type3.
MLSB constitutive
1.MLSBWild
type4.
Minducilble(active efflux)
2.
MLSB inducilble
constitutive
MLSBTotal
3.
MLSB constitutive

M (active efflux)

4.

636

71.38

147
Nr.
636
89
636
147
19
89147
891
89

16.50
%
71.38
9.99
71.38
16.50
2.13
16.50
9.99
100
9.99

23
38.33
Orthopaedics
Nr.
%
34
56.66
Nr.
%
23
38.33
2
3.33
23
38.33
56.66
134
1.66
34
56.66
2
3.33
60
100
2
3.33

19

2.13

1

1.66

100

60

100

Nr.Ambulatory%19

M (active efflux)

Total

891

Total

891

2.13

100

1

60

1.66
100

2.

Wild type

Total
Resistant

891
251

640

%
100
28.17
71.83

Nr.
6026
34

%
100
43.33
56.66

2. Total
Resistant

891
251

100
28.17

2660

100
43.33

891

100

60

100

1.

Nr.

Wild type

Total

80,00%

71,83%
56,66%

60,00%

28,17%

40,00%

43,33%

20,00%

80,00%
60,00%

71,38%
38,33%

40,00%

16,50%

20,00%
0,00%

0,00%

56,66%

Wild type

MLSB
inducible

Wild type
Ambulatory

9,99%

3,33% 2,13% 1,66%

Resistant
Orthopaedics

Fig. 6. Resistance phenotypes to trimethoprim-sulfamethoxazoleFig. 6. Resistance phenotypes to trimethoprim-sulfamethoxazole

CONCLUSIONS
MLSBMConclusions
A high frequency,
especially in the orthopaedics ward, was observed for the multire
constitutive

aureus strains, which drawsA attention
toespeciallythe needmonitoring
antibitic
We behigh frequency,
in thefor
orthopaedics
ward, wasobservedprescription.
for thedvelopmentof
antibiotic
resistance
in
these
germs
is
causedbyempirycal
treatments,
not
based
Ambulatory Orthopaedics
multiresistant S. aureus strains, which draws attention to the need for monitoring
antibiogram.
antibitic prescription. We believe that dvelopment of antibiotic resistance in theseFig. 4. Resistance phenotypes to MLS
Presence of MRSA
– is35.00
in the orthopadics
ward
andin the ambulatory – r
germs
caused %by empirycal
treatments, not based
upon
the 9.99%
antibiogram.
Fig. 4. Resistance phenotypes to MLS
an alarm signal, because thePresence
MRSA
strainsarehospitalstrains,
multiresistantto antibiotics, wof MRSA – 35.00 % in the orthopadics ward and 9.99% in theorder to detecttothefluoroquinolones,
resistance to fluoroquinolones,a singlequinolonewasresistanceto allwas
beta-lactams.
Resistance is frequently extended to other antibiotic families.
order to detect theInresistancea singlequinoloneused – ciprofloxacin;highfrequencyof Staphylococcus
aureus Peni-R Meti-R (MRSA) resistance phenot
occi being cross-resistant to all fluoroquinolones. Most strainsThe
were
sensitive– 86.98%of thehospital
environment
explainsthe
involvementof this germ in the ethiology of43
nosocomial infe
ry strains and 2009.19.4(64)61.67% of theothopaedics
strains,
and
only
few
of
them
acquiredresistance
Fiziologia - Physiology- ambulatory, 38.33% - othopaedics) (Table VII and Figure5). to limit the spread of these strains, especially within the hospital, enforcement ofordernosocomial infections control techniques is necessary, as well as introducing policies for the ra
cmykTable VII. Resistance phenotypes to fluoroquinolonesof antibiotics.
No strains resistant to vancomycin were described, but vancomycin must be used ca
Nr.
Phenotype
Ambulatory
Orthopaedics
preserve
utility. It is a back-up antibiotic, used as treatment of choise in infections d
Nr.
%
Nr. its clinical%ambulatory – represents an alarm signal, because the MRSA strains are hospitalstrains, multiresistant to antibiotics, with cross-resistance to all beta-lactams.
Resistance is frequently extended to other antibiotic families.
The high frequency of Staphylococcus aureus Peni-R Meti-R (MRSA) resistancephenotype in the hospital environment explains the involvement of this germ in
the ethiology of nosocomial infections. In order to limit the spread of these strains,
especially within the hospital, enforcement of efficient nosocomial infections
control techniques is necessary, as well as introducing policies for the rational useof antibiotics.
No strains resistant to vancomycin were described, but vancomycin must be
used carefully to preserve its clinical utility. It is a back-up antibiotic, used as treatment of choise in infections determined by methicillin-resistant strains (MRSA).
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REZISTENŢA LA METICILINĂ A TULPINILOR DE
STAPHYLOCOCCUS AUREUS IZOLATE DINTR-O SECŢIE
DE ORTOPEDIE ŞI DIN AMBULATOR
rezumatIntroducere: Staphylococcus aureus reprezintă una din speciile izolate cel mai frecvent atât în ambulator, cât şi în mediul spitalicesc, ce poate
determina infecţii atât la persoanele cu rezistenţă antiinfecţioasă scăzută, cât şi la persoanele sănătoase. Este una dintre cele mai piogene bacterii, capabilă
de a produce infecţii cu orice sediu în organism. Ele se produc prin proliferarea microorganismelor (cu invazie directă şi distrugerea ţesuturilor) sau prineliberarea de toxine.
Scopul prezentei lucrări este reprezentat de studiul comparativ sensibilităţii la chimioterapicele antiinfecţioase a tulpinilor de Staphylococcus aureus izolate
din diverse produse patologice provenite dintr-o clinică de ortopedie şi din ambulator, precum şi stabilirea fenotipurilor de rezistenţă în care se încadrează
aceste tulpini. S-a urmărit incidenţa tulpinilor de Staphylococcus aureus Peni-R Meti-R (MRSA - Staphylococcus aureus meticilino-rezistent) în secţia deortopedie şi în ambulator.
Material şi metodă: S-au luat în studiu 951 tulpini de Staphylococcus aureus: 60 izolate din 255 probe provenite din clinica de ortopedie şi 891 izolate
dintr-un număr de 1620 probe din ambulator. Testarea sensibilităţii la antibiotice s-a realizat prin metoda difuzimetrică Kirby-Bauer. Prin analiza interpretativă
a antibiogramelor s-a făcut încadrarea germenilor în fenotipuri de rezistenţă.
Rezultate: S-a izolat un procent crescut de tulpini de Staphylococcus aureus cu multirezistenţă la antibiotice, 21 tulpini (35,00%) izolate din clinica deortopedie şi 89 tulpini (9,99%) provenite din ambulator încadrându-se în fenotipul de rezistenţă Peni-R Meti-R.
Concluzii: Frecvenţa crescută a tulpinilor Staphylococcus aureus Peni-R Meti-R (MRSA) din spital explică implicarea acestui germen în etiologia infecţiilor
nosocomiale. Pentru limitarea răspândirii în special în mediul spitalicesc a acestor tulpini, se impune instituirea unor măsuri eficace de control al infecţiilor
nosocomiale şi introducerea unor politici de utilizare raţională a antibioticelor.
Cuvinte cheie: Staphylococcus aureus, MRSA, fenotip de rezistenţă.

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Fiziologia - Physiology  2009.19.4(64)