bluuebunny/crossref_metadata_2025_split huggingface dataset:
In Vivo Killing of
Porphyromonas gingivalis
by Toluidine Blue-Mediated Photosensitization in an Animal Model
Authors: Kömerik N. , Nakanishi H. , MacRobert A. J. , Henderson B. , Speight P. , Wilson M. 
Published: February 2003

ABSTRACT

Porphyromonas gingivalis
is one of the major causative organisms of periodontitis and has been shown to be susceptible to toluidine blue-mediated photosensitization in vitro. The aims of the present study were to determine whether this technique could be used to kill the organism in the oral cavities of rats and whether this would result in a reduction in the alveolar bone loss characteristic of periodontitis. The maxillary molars of rats were inoculated with
P. gingivalis
and exposed to up to 48 J of 630-nm laser light in the presence of toluidine blue. The number of surviving bacteria was then determined, and the periodontal structures were examined for evidence of any damage. When toluidine blue was used together with laser light there was a significant reduction in the number of viable
P. gingivalis
organisms. No viable bacteria could be detected when 1 mg of toluidine blue per ml was used in conjunction with all light doses used. On histological examination, no adverse effect of photosensitization on the adjacent tissues was observed. In a further group of animals, after time was allowed for the disease to develop in controls, the rats were killed and the level of maxillary molar alveolar bone was assessed. The bone loss in the animals treated with light and toluidine blue was found to be significantly less than that in the control groups. The results of this study show that toluidine blue-mediated lethal photosensitization of
P. gingivalis
is possible in vivo and that this results in decreased bone loss. These findings suggest that photodynamic therapy may be useful as an alternative approach for the antimicrobial treatment of periodontitis.
Alteration of
Escherichia coli
Topoisomerase IV to Novobiocin Resistance
Authors: Hardy Christine D. , Cozzarelli Nicholas R. 
Published: February 2003

ABSTRACT

DNA gyrase and topoisomerase IV (topo IV) are the two essential type II topoisomerases of
Escherichia coli
. Gyrase is responsible for maintaining negative supercoiling of the bacterial chromosome, whereas topo IV's primary role is in disentangling daughter chromosomes following DNA replication. Coumarins, such as novobiocin, are wide-spectrum antimicrobial agents that primarily interfere with DNA gyrase. In this work we designed an alteration in the ParE subunit of topo IV at a site homologous to that which confers coumarin resistance in gyrase. This
parE
mutation renders the encoded topo IV approximately 40-fold resistant to inhibition by novobiocin in vitro and imparts a similar resistance to inhibition of topo IV-mediated relaxation of supercoiled DNA in vivo. We conclude that topo IV is a secondary target of novobiocin and that it is very likely to be inhibited by the same mechanism as DNA gyrase.
Proteomic Approach to Understanding Antibiotic Action
Authors: Bandow Julia Elisabeth , Brötz Heike , Leichert Lars Ingo Ole , Labischinski Harald , Hecker Michael 
Published: February 2003

ABSTRACT

We have used proteomic technology to elucidate the complex cellular responses of
Bacillus subtilis
to antimicrobial compounds belonging to classical and emerging antibiotic classes. We established on two-dimensional gels a comprehensive database of cytoplasmic proteins with pIs covering a range of 4 to 7 that were synthesized during treatment with antibiotics or agents known to cause generalized cell damage. Although each antibiotic showed an individual protein expression profile, overlaps in the expression of marker proteins reflected similarities in molecular drug mechanisms, suggesting that novel compounds with unknown mechanisms of action may be classified. Indeed, one such substance, a structurally novel protein synthesis inhibitor (BAY 50-2369), could be classified as a peptidyltransferase inhibitor. These results suggest that this technique gives new insights into the bacterial response toward classical antibiotics and hints at modes of action of novel compounds. Such a method should prove useful in the process of antibiotic drug discovery.
Ergosterol Biosynthesis Inhibitors Become Fungicidal when Combined with Calcineurin Inhibitors against
Candida albicans
,
Candida glabrata
, and
Candida krusei
Authors: Onyewu Chiatogu , Blankenship Jill R. , Del Poeta Maurizio , Heitman Joseph 
Published: February 2003

ABSTRACT

Azoles target the ergosterol biosynthetic enzyme lanosterol 14α-demethylase and are a widely applied class of antifungal agents because of their broad therapeutic window, wide spectrum of activity, and low toxicity. Unfortunately, azoles are generally fungistatic and resistance to fluconazole is emerging in several fungal pathogens. We recently established that the protein phosphatase calcineurin allows survival of
Candida albicans
during the membrane stress exerted by azoles. The calcineurin inhibitors cyclosporine A (CsA) and tacrolimus (FK506) are dramatically synergistic with azoles, resulting in potent fungicidal activity, and mutant strains lacking calcineurin are markedly hypersensitive to azoles. Here we establish that drugs targeting other enzymes in the ergosterol biosynthetic pathway (terbinafine and fenpropimorph) also exhibit dramatic synergistic antifungal activity against wild-type
C. albicans
when used in conjunction with CsA and FK506. Similarly,
C. albicans
mutant strains lacking calcineurin B are markedly hypersensitive to terbinafine and fenpropimorph. The FK506 binding protein FKBP12 is required for FK506 synergism with ergosterol biosynthesis inhibitors, and a calcineurin mutation that confers FK506 resistance abolishes drug synergism. Additionally, we provide evidence of drug synergy between the nonimmunosuppressive FK506 analog L-685,818 and fenpropimorph or terbinafine against wild-type
C. albicans
. These drug combinations also exert synergistic effects against two other
Candida
species,
C. glabrata
and
C. krusei
, which are known for intrinsic or rapidly acquired resistance to azoles. These studies demonstrate that the activity of non-azole antifungal agents that target ergosterol biosynthesis can be enhanced by inhibition of the calcineurin signaling pathway, extending their spectrum of action and providing an alternative approach by which to overcome antifungal drug resistance.
Modulation of the Activity of Secretory Phospholipase A
2
by Antimicrobial Peptides
Authors: Zhao Hongxia , Kinnunen Paavo K. J. 
Published: February 2003

ABSTRACT

The antimicrobial peptides magainin 2, indolicidin, and temporins B and L were found to modulate the hydrolytic activity of secretory phospholipase A
2
(sPLA
2
) from bee venom and in human lacrimal fluid. More specifically, hydrolysis of phosphatidylcholine (PC) liposomes by bee venom sPLA
2
at 10 μM Ca
2+
was attenuated by these peptides while augmented product formation was observed in the presence of 5 mM Ca
2+
. The activity of sPLA
2
towards anionic liposomes was significantly enhanced by the antimicrobial peptides at low [Ca
2+
] and was further enhanced in the presence of 5 mM Ca
2+
. Similarly, with 5 mM Ca
2+
the hydrolysis of anionic liposomes was enhanced significantly by human lacrimal fluid sPLA
2
, while that of PC liposomes was attenuated. These results indicate that concerted action of antimicrobial peptides and sPLA
2
could improve the efficiency of the innate response to infections. Interestingly, inclusion of a cationic gemini surfactant in the vesicles showed an essentially similar pattern on sPLA
2
activity, suggesting that the modulation of the enzyme activity by the antimicrobial peptides may involve also charge properties of the substrate surface.
Efflux-Mediated Resistance to Tigecycline (GAR-936) in
Pseudomonas aeruginosa
PAO1
Authors: Dean Charles R. , Visalli Melissa A. , Projan Steven J. , Sum Phaik-Eng , Bradford Patricia A. 
Published: February 2003

ABSTRACT

Pseudomonas aeruginosa
strains are less susceptible to tigecycline (previously GAR-936; MIC, 8 μg/ml) than many other bacteria (P. J. Petersen, N. V. Jacobus, W. J. Weiss, P. E. Sum, and R. T. Testa, Antimicrob. Agents Chemother. 43:738-744, 1999). To elucidate the mechanism of resistance to tigecycline,
P. aeruginosa
PAO1 strains defective in the MexAB-OprM and/or MexXY (OprM) efflux pumps were tested for susceptibility to tigecycline. Increased susceptibility to tigecycline (MIC, 0.5 to 1 μg/ml) was specifically associated with loss of MexXY. Transcription of
mexX
and
mexY
was also responsive to exposure of cells to tigecycline. To test for the emergence of compensatory efflux pumps in the absence of MexXY-OprM, mutants lacking MexXY-OprM were plated on medium containing tigecycline at 4 or 6 μg/ml. Resistant mutants were readily recovered, and these also had decreased susceptibility to several other antibiotics, suggesting efflux pump recruitment. One representative carbenicillin-resistant strain overexpressed OprM, the outer membrane channel component of the MexAB-OprM efflux pump. The
mexAB-oprM
repressor gene,
mexR
, from this strain contained a 15-bp in-frame deletion. Two representative chloramphenicol-resistant strains showed expression of an outer membrane protein slightly larger than OprM. The
mexCD-OprJ
repressor gene,
nfxB
, from these mutants contained a 327-bp in-frame deletion and an IS element insertion, respectively. Together, these data indicated drug efflux mediated by MexCD-OprJ. The MICs of the narrower-spectrum semisynthetic tetracyclines doxycycline and minocycline increased more substantially than did those of tigecycline and other glycylcyclines against the MexAB-OprM- and MexCD-OprJ-overexpressing mutant strains. This suggests that glycylcyclines, although they are subject to efflux from
P. aeruginosa
, are generally inferior substrates for
P. aeruginosa
efflux pumps than are narrower-spectrum tetracyclines.
New Insertion Sequence Elements in the Upstream Region of
cfiA
in Imipenem-Resistant
Bacteroides fragilis
Strains
Authors: Kato Naoki , Yamazoe Kikuo , Han Chang-Gyun , Ohtsubo Eiichi 
Published: February 2003

ABSTRACT

The 747-bp
cfiA
gene, which encodes a metallo-β-lactamase, and the regions flanking
cfiA
in six imipenem-resistant and four imipenem-susceptible
Bacteroides fragilis
strains isolated in Japan were analyzed by PCR and DNA sequencing. The nucleotide sequences of the
cfiA
genes (designated

cfiA
1

to

cfiA
10

) of all 10 strains tested varied from that of the standard
cfiA
gene from
B. fragilis
TAL2480. However, putative proteins encoded by the
cfiA
variants contained conserved amino acid residues important for zinc binding and hairpin loop formation, suggesting that
cfiA
variants have the capability of producing metallo-β-lactamases with full catalytic activities. PCR assay indicated that six metallo-β-lactamase-producing, imipenem-resistant strains had an insertion mutation in the region immediately upstream of
cfiA
. Nucleotide sequencing of the PCR-amplified fragments along with the upstream region of
cfiA
revealed that there were five new kinds of insertion sequence (IS) elements (designated IS
612
, IS
613
, IS
614
, IS
615
, and IS
616
, with a size range of 1,594 to 1,691 bp), of which only IS
616
was found to be almost identical to IS
1188
, one of the IS elements previously identified in the upstream region of
cfiA
. These elements had target site duplications of 4 or 5 bp in length, terminal inverted repeats (14, 15, or 17 bp in size), and a large open reading frame encoding a putative transposase which is required for the transcription of IS elements. Each element was inserted such that the transcriptional direction of the transposase was opposite to that of
cfiA
. A computer-aided homology search revealed that, based on the homology of their putative transposases, the sizes of their terminal inverted repeat sequences, and their target site duplications, IS
612
, IS
613
, IS
614
, and IS
615
belong to the IS
4
family, which includes IS
942
, previously found in some drug-resistant
B. fragilis
strains, but that IS
616
belongs to the IS
1380
family. All the IS elements appear to have putative promoter motif sequences (the −7 region's TAnnTTTG motif and the −33 region's TTG or TG) in their end regions, suggesting that the IS elements provide a promoter for the transcription of
cfiA
upon insertion. These data provide additional proof that various IS elements may exist to provide a promoter to express the
cfiA
gene.
High Variability of Plasma Drug Concentrations in Dual Protease Inhibitor Regimens
Authors: Guiard-Schmid Jean-Baptiste , Poirier Jean-Marie , Meynard Jean-Luc , Bonnard Philippe , Gbadoe Ayi Hola , Amiel Corinne , Calligaris Frédérique , Abraham Bruno , Pialoux Gilles , Girard Pierre-Marie , Jaillon Patrice , Rozenbaum Willy 
Published: February 2003

ABSTRACT

Ritonavir (RTV) strongly increases the concentrations of protease inhibitors (PIs) in plasma in patients given a combination of RTV and another PI. This pharmacological interaction is complex and poorly characterized and shows marked inter- and intraindividual variations. In addition, RTV interacts differently with saquinavir (SQV), indinavir (IDV), amprenavir (APV), and lopinavir (LPV). In this retrospective study on 542 human immunodeficiency virus-infected patients, we compared inter- and intraindividual variability of plasma PI concentrations and correlations between the
C
min
(minimum concentration of drug in plasma) values for RTV and the coadministered PI
C
min
values. Mean RTV
C
min
s are significantly lower in patients receiving combinations containing APV or LPV than in combinations with SQV or IDV. With the most common PI dose regimens (600 mg of IDV twice a day [BID], 800 mg of SQV BID, and 400 mg of LPV BID), the interindividual
C
min
variability of patients treated with a PI and RTV seemed to be lower with APV and LPV than with IDV and SQV. As regards intraindividual variability, APV also differed from the other PIs, exhibiting lower
C
min
variability than with the other combinations. Significant positive correlations between RTV
C
min
and boosted PI
C
min
were observed with IDV, SQV, and LPV, but not with APV. Individual dose adjustments must take into account the specificity the pharmacological interaction of each RTV/PI combination and the large inter- and intraindividual variability of plasma PI levels to avoid suboptimal plasma drug concentrations which may lead to treatment failure and too high concentrations which may induce toxicity and therefore reduce patient compliance.
Pharmacokinetics of Intravenous Acyclovir, Zidovudine, and Acyclovir-Zidovudine in Pregnant Rats
Authors: Brown Stacy D. , Bartlett Michael G. , White Catherine A. 
Published: February 2003

ABSTRACTThe pharmacokinetics and placental transfer of acyclovir and zidovudine monotherapies and acyclovir-zidovudine combination therapy were compared in the pregnant rat. Timed-pregnancy Sprague-Dawley rats were used for the study. Doses of 60 mg of each drug/kg of body weight in monotherapy and in combination therapy were given by intravenous bolus, and samples of maternal plasma, amniotic fluid, fetal tissue, and placental tissue were collected over a period of 8 h postdose. Concentrations of each drug in the various matrices were measured by high-performance liquid chromatography. All data were analyzed by using WinNonlin. A one-compartment model with first-order elimination was used to fit the AZT plasma data from the combination therapy rats, but the plasma data from the other groups were fit to a two-compartment model. Tissue data were analyzed by noncompartmental analysis to generate area-under-the-concentration-time-curve values. Implementation of the combination therapy altered the pharmacokinetics of each drug compared to its monotherapy pharmacokinetics. The combination of these two drugs may potentiate fetal and amniotic fluid exposures to each drug.
Comparing Pharmacokinetics of Amoxicillin Given Twice or Three Times per Day to Children Older than 3 Months with Pneumonia
Authors: Fonseca Walter , Hoppu Kalle , Rey Luís C. , Amaral João , Qazi Shamim 
Published: February 2003

ABSTRACT
For children with ambulatory pneumonia, the World Health Organization (WHO) recommends oral amoxicillin (15 mg/kg of body weight/dose) thrice daily (t.i.d.) or oral cotrimoxazole (4 mg of trimethoprim/kg/dose) twice daily (b.i.d.). The more frequent amoxicillin dosing may lead to compliance problems. To compare the pharmacokinetics and levels of amoxicillin in plasma in the current WHO acute respiratory infection recommendations with the 25-mg/kg/dose b.i.d. regimen, we performed a two-group parallel study of 66 children ages 3 to 59 months with pneumonia. Amoxicillin was given orally at 25 mg/kg/dose b.i.d. or 15 mg/kg/dose t.i.d. Amoxicillin concentrations were determined by high-performance liquid chromatography after the first dose on days 1 and 3. After the first dose on day 1, the mean area under the concentration-time curve (AUC) for amoxicillin after the 25-mg/kg dose was 54.7 versus 24.9 μg · h/ml after the 15-mg/kg dose. After the first dose on day 3, the mean AUC was 44.1 versus 28.5 μg · h/ml. All but two children had plasma amoxicillin concentrations above 0.5 μg/ml for >50% of the dose interval on both days. Six children on day 1 and five children on day 3 had concentrations above 1.0 μg/ml for <50% of the dose interval. On day 1, 16 of 27 children in the b.i.d. group and 11 of 26 children in the t.i.d. group had concentrations that were above 2.0 μg/ml for <50% of the dose interval, and on day 3, 18 of 31 children in the b.i.d. group and 8 of 31 children in the t.i.d. group had concentrations that were above 2.0 μg/ml for <50% of the dose interval. Amoxicillin b.i.d. is a feasible alternative for t.i.d. dosing. To lengthen the time above the MIC at higher concentration levels, a 30- to 40-mg/kg/dose b.i.d. should be considered instead of the 25 mg/kg/dose used in this study.
Marine Bacteria Cause False-Positive Results in the Colilert-18 Rapid Identification Test for
Escherichia coli
in Florida Waters
Authors: Pisciotta John M. , Rath Damon F. , Stanek Paul A. , Flanery D. Michael , Harwood Valerie J. 
Published: February 2003

ABSTRACT

The Colilert-18 system for enumeration of total coliforms and
Escherichia coli
is approved by the U.S. Environmental Protection Agency for use in drinking water analysis and is also used by various agencies and research studies for enumeration of indicator organisms in fresh and saline waters. During monitoring of Pinellas County, Fla., marine waters, estimates of
E. coli
numbers (by Colilert-18) frequently exceeded fecal coliform counts (by membrane filtration) by 1 to 3 orders of magnitude. Samples from freshwater sites did not display similar discrepancies. Fecal coliforms, including
E. coli
, could be cultured from 100% of yellow fluorescent wells (denoting
E. coli
-positive results) inoculated with freshwater samples but could be cultured from only 17.1% of the “positive” wells inoculated with marine samples.
Ortho
-nitrophenyl-β-
d
-galactopyranoside (ONPG)-positive or 4-methylumbelliferyl-β-
d
-glucuronide (MUG)-positive noncoliform bacteria were readily cultured from Colilert-18 test wells inoculated with marine samples. Filtered cell-free seawater did not cause false positives. Coculture preparations of as few as 5 CFU of
Vibrio cholerae
(ONPG positive) and
Providencia
sp. (MUG positive) ml
−1
inoculated into Colilert-18 caused false-positive
E. coli
results. Salinity conditions influenced coculture results, as the concentration of coculture inoculum required to cause false positives in most wells increased from about 5 CFU ml
−1
in seawater diluted 1:10 with freshwater to ≈5,000 CFU ml
−1
in seawater diluted 1:20 with freshwater. Estimated
E. coli
numbers in various marine water samples processed at the 1:10 dilution ranged from 10 to 7,270 CFU·100 ml
−1
, while
E. coli
numbers in the same samples processed at the 1:20 dilution did not exceed 40 CFU·100 ml
−1
. The lower estimates of
E. coli
numbers corresponded well with fecal coliform counts by membrane filtration. This study indicates that assessment of
E. coli
in subtropical marine waters by Colilert-18 is not accurate when the recommended 1:10 sample dilution is used. The results suggest that greater dilution may diminish the false-positive problem, but further study of this possibility is recommended.
Regulation of Endo-Acting Glycosyl Hydrolases in the Hyperthermophilic Bacterium
Thermotoga maritima
Grown on Glucan- and Mannan-Based Polysaccharides
Authors: Chhabra Swapnil R. , Shockley Keith R. , Ward Donald E. , Kelly Robert M. 
Published: February 2003

ABSTRACT

The genome sequence of the hyperthermophilic bacterium
Thermotoga maritima
encodes a number of glycosyl hydrolases. Many of these enzymes have been shown in vitro to degrade specific glycosides that presumably serve as carbon and energy sources for the organism. However, because of the broad substrate specificity of many glycosyl hydrolases, it is difficult to determine the physiological substrate preferences for specific enzymes from biochemical information. In this study,
T. maritima
was grown on a range of polysaccharides, including barley β-glucan, carboxymethyl cellulose, carob galactomannan, konjac glucomannan, and potato starch. In all cases, significant growth was observed, and cell densities reached 10
9
cells/ml. Northern blot analyses revealed different substrate-dependent expression patterns for genes encoding the various endo-acting β-glycosidases; these patterns ranged from strong expression to no expression under the conditions tested. For example,
cel74
(TM0305), a gene encoding a putative β-specific endoglucananse, was strongly expressed on all substrates tested, including starch, while no evidence of expression was observed on any substrate for
lam16
(TM0024),
xyl10A
(TM0061),
xyl10B
(TM0070), and
cel12A
(TM1524), which are genes that encode a laminarinase, two xylanases, and an endoglucanase, respectively. The
cel12B
(TM1525) gene, which encodes an endoglucanase, was expressed only on carboxymethyl cellulose. An extracellular mannanase encoded by
man5
(TM1227) was expressed on carob galactomannan and konjac glucomannan and to a lesser extent on carboxymethyl cellulose. An unexpected result was the finding that the
cel5A
(TM1751) and
cel5B
(TM1752) genes, which encode putative intracellular, β-specific endoglucanases, were induced only when
T. maritima
was grown on konjac glucomannan. To investigate the biochemical basis of this finding, the recombinant forms of Man5 (
M
r
, 76,900) and Cel5A (
M
r
, 37,400) were expressed in
Escherichia coli
and characterized. Man5, a
T. maritima
extracellular enzyme, had a melting temperature of 99°C and an optimun temperature of 90°C, compared to 90 and 80°C, respectively, for the intracellular enzyme Cel5A. While Man5 hydrolyzed both galactomannan and glucomannan, no activity was detected on glucans or xylans. Cel5A, however, not only hydrolyzed barley β-glucan, carboxymethyl cellulose, xyloglucan, and lichenin but also had activity comparable to that of Man5 on galactomannan and higher activity than Man5 on glucomannan. The biochemical characteristics of Cel5A, the fact that Cel5A was induced only when
T. maritima
was grown on glucomannan, and the intracellular localization of Cel5A suggest that the physiological role of this enzyme includes hydrolysis of glucomannan oligosaccharides that are transported following initial hydrolysis by extracellular glycosidases, such as Man5.