bluuebunny/crossref_metadata_2025 huggingface dataset:
Production of Cyclic Lipopeptides byPseudomonas fluorescensStrains in Bulk Soil and in the Sugar Beet Rhizosphere
Authors: Nielsen Tommy Harder , Sørensen Jan
Published: February 2003

The production of cyclic lipopeptides (CLPs) with antifungal and biosurfactant properties byPseudomonas fluorescensstrains was investigated in bulk soil and in the sugar beet rhizosphere. Purified CLPs (viscosinamide, tensin, and amphisin) were first shown to remain highly stable and extractable (90%) when applied (ca. 5 μg g−1) to sterile soil, whereas all three compounds were degraded over 1 to 3 weeks in nonsterile soil. When a whole-cell inoculum ofP. fluorescensstrain DR54 containing a cell-bound pool of viscosinamide was added to the nonsterile soil, declining CLP concentrations were observed over a week. By comparison, addition of the strains 96.578 and DSS73 without cell-bound CLP pools did not result in detectable tensin or amphisin in the soil. In contrast, when sugar beet seeds were coated with the CLP-producing strains and subsequently germinated in nonsterile soil, strain DR54 maintained a high and constant viscosinamide level in the young rhizosphere for ∼2 days while strains 96.578 and DSS73 exhibited significant production (net accumulation) of tensin or amphisin, reaching a maximum level after 2 days. All three CLPs remained detectable for several days in the rhizosphere. Subsequent tests of five other CLP-producingP. fluorescensstrains also demonstrated significant production in the young rhizosphere. The results thus provide evidence that production of different CLPs is a common trait among manyP. fluorescensstrains in the soil environment, and further, that the production is taking place only in specific habitats like the rhizosphere of germinating sugar beet seeds rather than in the bulk soil.
Arthrobacter
Strain VAI-A Utilizes Acyl-Homoserine Lactone Inactivation Products and Stimulates Quorum Signal Biodegradation by
Variovorax paradoxus
Authors: Flagan Suvi , Ching Weng-Ki , Leadbetter Jared R.
Published: February 2003

Many
Proteobacteria
produce acyl-homoserine lactones (acyl-HSLs) and employ them as dedicated cell-to-cell signals in a process known as quorum sensing. Previously,
Variovorax paradoxus
VAI-C was shown to utilize diverse acyl-HSLs as sole sources of energy and nitrogen. We describe here the properties of a second isolate,
Arthrobacter
strain VAI-A, obtained from the same enrichment culture that yielded
V. paradoxus
VAI-C. Although strain VAI-A grew rapidly and exponentially on a number of substrates, it grew only slowly and aberrantly (i.e., linearly) in media amended with oxohexanoyl-HSL as the sole energy source. Increasing the culture pH markedly improved the growth rate in media containing this substrate but did not abolish the aberrant kinetics. The observed growth was remarkably similar to the known kinetics of the pH-influenced half-life of acyl-HSLs, which decay chemically to yield the corresponding acyl-homoserines. Strain VAI-A grew rapidly and exponentially when provided with an acyl-homoserine as the sole energy or nitrogen source. The isolate was also able to utilize HSL as a sole source of nitrogen but not as energy for growth.
V. paradoxus
, known to release HSL as a product of quorum signal degradation, was examined for the ability to support the growth of
Arthrobacter
strain VAI-A in defined cocultures. It did. Moreover, the acyl-HSL-dependent growth rate and yield of the coculture were dramatically superior to those of the monocultures. This suggested that the original coenrichment of these two organisms from the same soil sample was not coincidental and that consortia may play a role in quorum signal turnover and mineralization. The fact that
Arthrobacter
strain VAI-A utilizes the two known nitrogenous degradation products of acyl-HSLs, acyl-homoserine and HSL, begins to explain why none of the three compounds are known to accumulate in the environment.
Terminal Restriction Fragment Length Polymorphism Data Analysis for Quantitative Comparison of Microbial Communities
Authors: Blackwood Christopher B. , Marsh Terry , Kim Sang-Hoon , Paul Eldor A.
Published: February 2003

Terminal restriction fragment length polymorphism (T-RFLP) is a culture-independent method of obtaining a genetic fingerprint of the composition of a microbial community. Comparisons of the utility of different methods of (i) including peaks, (ii) computing the difference (or distance) between profiles, and (iii) performing statistical analysis were made by using replicated profiles of eubacterial communities. These samples included soil collected from three regions of the United States, soil fractions derived from three agronomic field treatments, soil samples taken from within one meter of each other in an alfalfa field, and replicate laboratory bioreactors. Cluster analysis by Ward's method and by the unweighted-pair group method using arithmetic averages (UPGMA) were compared. Ward's method was more effective at differentiating major groups within sets of profiles; UPGMA had a slightly reduced error rate in clustering of replicate profiles and was more sensitive to outliers. Most replicate profiles were clustered together when relative peak height or Hellinger-transformed peak height was used, in contrast to raw peak height. Redundancy analysis was more effective than cluster analysis at detecting differences between similar samples. Redundancy analysis using Hellinger distance was more sensitive than that using Euclidean distance between relative peak height profiles. Analysis of Jaccard distance between profiles, which considers only the presence or absence of a terminal restriction fragment, was the most sensitive in redundancy analysis, and was equally sensitive in cluster analysis, if all profiles had cumulative peak heights greater than 10,000 fluorescence units. It is concluded that T-RFLP is a sensitive method of differentiating between microbial communities when the optimal statistical method is used for the situation at hand. It is recommended that hypothesis testing be performed by redundancy analysis of Hellinger-transformed data and that exploratory data analysis be performed by cluster analysis using Ward's method to find natural groups or by UPGMA to identify potential outliers. Analyses can also be based on Jaccard distance if all profiles have cumulative peak heights greater than 10,000 fluorescence units.
In Situ Production of Exopolysaccharides during Sourdough Fermentation by Cereal and Intestinal Isolates of Lactic Acid Bacteria
Authors: Tieking Markus , Korakli Maher , Ehrmann Matthias A. , Gänzle Michael G. , Vogel Rudi F.
Published: February 2003

EPS formed by lactobacilli in situ during sourdough fermentation may replace hydrocolloids currently used as texturizing, antistaling, or prebiotic additives in bread production. In this study, a screening of >100 strains of cereal-associated and intestinal lactic acid bacteria was performed for the production of exopolysaccharides (EPS) from sucrose. Fifteen strains produced fructan, and four strains produced glucan. It was remarkable that formation of glucan and fructan was most frequently found in intestinal isolates and strains of the species
Lactobacillus reuteri
,
Lactobacillus pontis
, and
Lactobacillus frumenti
from type II sourdoughs. By the use of PCR primers derived from conserved amino acid sequences of bacterial levansucrase genes, it was shown that 6 of the 15 fructan-producing lactobacilli and none of 20 glucan producers or EPS-negative strains carried a levansucrase gene. In sourdough fermentations, it was determined whether those strains producing EPS in MRS medium modified as described by Stolz et al. (37) and containing 100 g of sucrose liter
−1
as the sole source of carbon also produce the same EPS from sucrose during sourdough fermentation in the presence of 12% sucrose. For all six EPS-producing strains evaluated in sourdough fermentations, in situ production of EPS at levels ranging from 0.5 to 2 g/kg of flour was demonstrated. Production of EPS from sucrose is a metabolic activity that is widespread among sourdough lactic acid bacteria. Thus, the use of these organisms in bread production may allow the replacement of additives.
Growth of a
Dehalococcoides
-Like Microorganism on Vinyl Chloride and
cis
-Dichloroethene as Electron Acceptors as Determined by Competitive PCR
Authors: Cupples Alison M. , Spormann Alfred M. , McCarty Perry L.
Published: February 2003

A competitive PCR (cPCR) assay targeting 16S ribosomal DNA was developed to enumerate growth of a
Dehalococcoides
-like microorganism, bacterium VS, from a mixed culture catalyzing the reductive dehalogenation of
cis
-1,2-dichloroethene (cDCE) and vinyl chloride (VC), with hydrogen being used as an electron donor. The growth of bacterium VS was found to be coupled to the dehalogenation of VC and cDCE, suggesting unique metabolic capabilities. The average growth yield was (5.2 ± 1.5) × 10
8
copies of the 16S rRNA gene/μmol of Cl
−
(number of samples, 10), with VC being used as the electron acceptor and hydrogen as the electron donor. The maximum VC utilization rate (
q̂
) was determined to be 7.8 × 10
−10
μmol of Cl
−
(copy
−1
day
−1
), indicating a maximum growth rate of 0.4 day
−1
. These average growth yield and

q̂

values agree well with values found previously for dechlorinating cultures. Decay coefficients were determined with growth (0.05 day
−1
) and no-growth (0.09 day
−1
) conditions. An important limitation of this cPCR assay was its inability to discriminate between active and inactive cells. This is an essential consideration for kinetic studies.
Functional Expression of a Fungal Laccase in
Saccharomyces cerevisiae
by Directed Evolution
Authors: Bulter Thomas , Alcalde Miguel , Sieber Volker , Meinhold Peter , Schlachtbauer Christian , Arnold Frances H.
Published: February 2003

Laccase from
Myceliophthora thermophila
(MtL) was expressed in functional form in
Saccharomyces cerevisiae
. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together with a 22-fold increase in
k
cat
, the total activity was enhanced 170-fold. Specific activities of MtL mutants toward 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations. The most effective mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host. The C terminus is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold. Mutations accumulated during nine generations of evolution for higher activity decreased enzyme stability. Screening for improved stability in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity. The molecular mass of MtL expressed in
S. cerevisiae
is 30% higher than that of the same enzyme expressed in
M. thermophila
(110 kDa versus 85 kDa). Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible for this increase. This
S. cerevisiae
expression system makes MtL available for functional tailoring by directed evolution.
New PCR Assay Using Glucose-6-Phosphate Dehydrogenase for Identification of
Leishmania
Species
Authors: Castilho Tiago M. , Shaw Jeffrey Jon , Floeter-Winter Lucile M.
Published: February 2003

Glucose-6-phosphate dehydrogenase (G6PD) is one of the multilocus enzymes used to identify
Leishmania
by zymodeme analysis. The polymorphic pattern revealed by partial characterization of the gene encoding G6PD generated molecular markers useful in the identification of different
Leishmania
species by PCR. Initially degenerate oligonucleotides were designed on the basis of data on the conserved active center described for other organisms. Primers for reverse transcription-PCR experiments, designed from the nucleotide sequence of the PCR product, enabled us to characterize the 5′ and 3′ untranslated regions and the
G6PD
open reading frame of reference strains of
Leishmania
(
Viannia
)
braziliensis
,
Leishmania
(
Viannia
)
guyanensis
,
Leishmania
(
Leishmania
)
mexicana
, and
Leishmania
(
Leishmania
)
amazonensis
. Sets of paired primers were designed and used in PCR assays to discriminate between the parasites responsible for tegumentar leishmaniasis of the subgenera
Leishmania
(
Leishmania
) and
Leishmania
(
Viannia
) and to distinguish
L.
(
Viannia
)
braziliensis
from others organisms of the subgenus
Leishmania
(
Viannia
). No amplification products were detected for the DNA of
Crithidia fasciculata
,
Trypanosoma cruzi
, or
Leishmania
(
Sauroleishmania
)
tarentolae
or DNA from a healthy human control. The tests proved to be specific and were sensitive enough to detect parasites in human biopsy specimens. The successful discrimination of
L.
(
Viannia
)
braziliensis
from other parasites of the subgenus
Leishmania
(
Viannia
) opens the way to epidemiological studies in areas where more than one species of the subgenus
Leishmania
(
Viannia
) exist, such as Amazonia, as well as follow-up studies after chemotherapy and assessment of clinical prognoses.
Detection and Identification of Fungi from Fungus Balls of the Maxillary Sinus by Molecular Techniques
Authors: Willinger Birgit , Obradovic Alexandra , Selitsch Brigitte , Beck-Mannagetta Johann , Buzina Walter , Braun Hannes , Apfalter Petra , Hirschl Alexander M. , Makristathis Athanasios , Rotter Manfred
Published: February 2003

The aim of this study was to find a reliable method for the detection and identification of fungi in fungus balls of the maxillary sinus and to evaluate the spectrum of fungi in these samples. One hundred twelve samples were obtained from patients with histologically proven fungal infections; 81 samples were paraffin-embedded tissue sections of the maxillary sinus. In 31 cases, sinus contents without paraffin embedding were sent for investigation. PCR amplification with universal fungal primers for 28S ribosomal DNA and amplicon identification by hybridization with species-specific probes for
Aspergillus fumigatus
,
Aspergillus flavus
,
Aspergillus niger
,
Aspergillus terreus
,
Aspergillus glaucus
,
Pseudallescheria boydii
,
Candida albicans
, and
Candida glabrata
were performed for all samples. Furthermore, PCR products were sequenced. Fresh samples were also cultivated. Fungal DNA was detected in all of the fresh samples but only in 71 paraffin-embedded tissue samples (87.7%). Sequence analysis was the most sensitive technique, as results could be obtained for 28 (90.3%) fresh samples by this method in comparison to 24 (77.4%) samples by hybridization and 16 (51.6%) samples by culture. However, sequence analysis delivered a result for only 36 (50.7%) of the paraffin-embedded specimens. Hybridization showed reliable results for
A. fumigatus
, which proved to be the most common agent in fungus balls of the maxillary sinus. Other
Aspergillus
species and other genera were rarely found.
Multiple Patterns of Resistance to Fluconazole in
Candida glabrata
Isolates from a Patient with Oropharyngeal Candidiasis Receiving Head and Neck Radiation
Authors: Redding Spencer W. , Kirkpatrick William R. , Saville Stephen , Coco Brent J. , White William , Fothergill Annette , Rinaldi Michael , Eng Tony , Patterson Thomas F. , Lopez-Ribot Jose
Published: February 2003

Candida glabrata
has emerged in recent years as a significant cause of systemic fungal infection. We have previously reported on the first three patients receiving radiation for head and neck cancer to develop oropharyngeal candidiasis due to
C. glabrata
. The goal of this study was to track the development of increased fluconazole resistance in
C. glabrata
isolates and to evaluate previously described genetic mechanisms associated with this resistance from one of these three patients. The patient was a 52-year-old man with squamous cell carcinoma treated with radiation. At week 7 of his radiation, he developed oropharyngeal candidiasis, which was treated with 200 mg of fluconazole daily for 2 weeks. Serial cultures from this and three subsequent time points yielded
C. glabrata
. Isolates from these cultures were subjected to antifungal susceptibility testing, DNA karyotyping, and evaluation of the expression of genes previously associated with
C. glabrata
resistance to fluconazole,
CgCDR1
,
CgCDR2
, and
CgERG11
. Two strains (A and B) of
C. glabrata
were identified and found to display different patterns of resistance development and gene expression. Strain A developed resistance over a 2-week period and showed no overexpression of these genes. In contrast, strain B first showed resistance 6 weeks after fluconazole therapy was discontinued but showed overexpression of all three genes. In conclusion, development of resistance to fluconazole by
C. glabrata
is a highly varied process involving multiple molecular mechanisms.
Multicenter Evaluation of the VERSANT HCV RNA Qualitative Assay for Detection of Hepatitis C Virus RNA
Authors: Hendricks David A. , Friesenhahn Michel , Tanimoto Lorine , Goergen Bernd , Dodge Deborah , Comanor Lorraine
Published: February 2003

We report the results of a multicenter evaluation of a new assay for the detection of hepatitis C virus (HCV) RNA in human serum or plasma based on transcription-mediated amplification (HCV TMA). Analysis of combined data obtained from 15 independent sites, including 4 sites in the United States and 11 in Europe, by using preproduction kits showed a limit of detection of 9.8 IU/ml and an overall mean specificity of 97.9%. In addition, assay runs and samples were valid consistently (97.8% of assay runs and 98.0% of specimen results). Of the 15 sites that participated in the multicenter evaluation, 2 subsequently carried out additional performance studies with production kits in support of the assay's registration in France. Comparison of the findings from these two sites during the multicenter evaluation and during the registration studies showed an overall improvement in assay performance. A statistically significant (
P
< 0.001) improvement was achieved for both specificity and specimen validity in the registration studies, which were 99.4 and 98.1%, respectively. Combined data from the two sites showed a lower limit of detection of approximately 2.4 IU/ml and an improved assay validity of 100%, although the sample size was too small to show statistical significance at the 0.05 level. In summary, the performance characteristics of HCV TMA indicate that this assay is a reliable tool for the detection of HCV RNA in serum or plasma. Improvement in assay performance has been demonstrated with refinement of assay reagents, instrumentation, and operator experience.
Identification of Major Streptococcal Species by
rrn
-Amplified Ribosomal DNA Restriction Analysis
Authors: Schlegel Laurent , Grimont Francine , Grimont Patrick A. D. , Bouvet Anne
Published: February 2003

Amplified ribosomal DNA restriction analysis (
rrn
-ARDRA) is based on PCR amplification and restriction of a fragment of rRNA genes including 16S and 23S genes and the intergenic spacer.
rrn
-ARDRA was evaluated for the identification of species within the genus
Streptococcus
. A total of 148 type and reference strains of pyogenic, oral, and group D streptococci were examined in order to construct a database for identification of streptococci. The amplified product was a single band approximately 4,500 bp long. This amplicon was digested separately with three (
Hha
I,
Mbo
II, and
Sau
3A) restriction endonucleases. Respectively, 27, 26, and 28 major patterns were observed after
Hha
I,
Mbo
II, and
Sau
3A restrictions. Streptococcal strains belonging to different species had different patterns or different combination of patterns. An identification system based upon a combination of the three restriction patterns in a single database was then proposed.
rrn
-ARDRA was successfully applied to 11 clinical isolates whose identification to the species level was difficult to obtain by phenotypic analysis. Using a database of well-characterized strains,
rrn
-ARDRA is a powerful method for the identification of streptococcal isolates.
Six Rapid Tests for Direct Detection of
Clostridium difficile
and Its Toxins in Fecal Samples Compared with the Fibroblast Cytotoxicity Assay
Authors: Turgeon David K. , Novicki Thomas J. , Quick John , Carlson LaDonna , Miller Pat , Ulness Bruce , Cent Anne , Ashley Rhoda , Larson Ann , Coyle Marie , Limaye Ajit P. , Cookson Brad T. , Fritsche Thomas R.
Published: February 2003

Clostridium difficile
is one of the most frequent causes of nosocomial gastrointestinal disease. Risk factors include prior antibiotic therapy, bowel surgery, and the immunocompromised state. Direct fecal analysis for
C. difficile
toxin B by tissue culture cytotoxin B assay (CBA), while only 60 to 85% sensitive overall, is a common laboratory method. We have used 1,003 consecutive, nonduplicate fecal samples to compare six commercially available immunoassays (IA) for
C. difficile
detection with CBA: Prima System
Clostridium difficile
Tox A and VIDAS
Clostridium difficile
Tox A II, which detect
C. difficile
toxin A; Premier Cytoclone A/B and Techlab
Clostridium difficile
Tox A/B, which detect toxins A and B; and ImmunoCard
Clostridium difficile
and Triage Micro
C. difficile
panels, which detect toxin A and a species-specific antigen. For all tests, Triage antigen was most sensitive (89.1%; negative predictive value [NPV] = 98.7%) while ImmunoCard was most specific (99.7%; positive predictive value [PPV] = 95.0%). For toxin tests only, Prima System had the highest sensitivity (82.2%; NPV = 98.0%) while ImmunoCard had the highest specificity (99.7%; PPV = 95.0%). Hematopoietic stem cell transplant (HSCT) patients contributed 44.7% of all samples tested, and no significant differences in sensitivity or specificity were noted between HSCT and non-HSCT patients. IAs, while not as sensitive as direct fecal CBA, produce reasonable predictive values, especially when both antigen and toxin are detected. They also offer significant advantages over CBA in terms of turnaround time and ease of use.
Longitudinal Reliability of Focus Glycoprotein G-Based Type-Specific Enzyme Immunoassays for Detection of Herpes Simplex Virus Types 1 and 2 in Women
Authors: Cherpes Thomas L. , Ashley Rhoda L. , Meyn Leslie A , Hillier Sharon L.
Published: February 2003

Serologic assays that utilize herpes simplex virus (HSV) type-specific glycoproteins G-1 (HSV-1) and G-2 (HSV-2) to discriminate between antibodies against HSV-1 and HSV-2 are sensitive and specific. However, the high rates of seroreversion, defined as the change in an individual's antibody status from positive to negative over time, previously reported in longitudinal evaluations of glycoprotein G type-specific tests suggests that their use in HSV acquisitional studies would be problematic. To further explore the reliability of the glycoprotein G-based serologic tests, we evaluated HSV-1 and HSV-2 enzyme immunoassays from Focus Technologies in a longitudinal cohort of 1,207 young women from Pittsburgh, Pa. On enrollment of the women in the study, HSV-1 and HSV-2 antibodies were detected in 46.6 and 24.9% of the women, respectively. Among the women with at least three visits, 3.4% (15 of 447) of those who were HSV-1 antibody positive had a subsequent negative result while fewer than 1% (2 of 227) of those who were HSV-2 antibody positive seroreverted. The median of mean positive index values for women who seroreverted to HSV-1 antibody was lower than that for women who remained seropositive (1.25 versus 7.06;
P
< 0.001). Similarly, the median of mean positive index values for women whose HSV-2 antibody status reverted from positive to negative was lower than that for those women who did not serorevert (1.83 versus 7.46;
P
= 0.02). Comparative Western blot analysis demonstrated that the lower positive index values, seen more often among the HSV seroreverters, often signified false-positive immunoassay results. Overall, the seroreversion rates were low; the use of glycoprotein G-based serologic tests for the measurement of HSV-1 and HSV-2 antibodies in incidence studies therefore appears warranted.
Multilocus Sequence Typing Reveals a Lack of Diversity among
Escherichia coli
O157:H7 Isolates That Are Distinct by Pulsed-Field Gel Electrophoresis
Authors: Noller Anna C. , McEllistrem M. Catherine , Stine O. Colin , Morris, J. Glenn , Boxrud David J. , Dixon Bruce , Harrison Lee H.
Published: February 2003

Escherichia coli
O157:H7 is a major cause of foodborne illness in the United States. Pulsed-field gel electrophoresis (PFGE) is the molecular epidemiologic method mostly commonly used to identify food-borne outbreaks. Although PFGE is a powerful epidemiologic tool, it has disadvantages that make a DNA sequence-based approach potentially attractive. Multilocus sequence typing (MLST) analyzes the internal fragments of housekeeping genes to establish genetic relatedness between isolates. We sequenced selected portions of seven housekeeping genes and two membrane protein genes (
ompA
and
espA
) of 77 isolates that were diverse by PFGE to determine whether there was sufficient sequence variation to be useful as an epidemiologic tool. There was no DNA sequence diversity in the sequenced portions of the seven housekeeping genes and
espA
. For
ompA
, all but five isolates had sequence identical to that of the reference strains.
E. coli
O157:H7 has a striking lack of genetic diversity in the genes we explored, even among isolates that are clearly distinct by PFGE. Other approaches to identify improved molecular subtyping methods for
E. coli
0157:H7 are needed.
Detection ofFrancisella tularensiswithin Infected Mouse Tissues by Using a Hand-Held PCR Thermocycler
Authors: Emanuel Peter A. , Bell Ryan , Dang Jessica L. , McClanahan Rebecca , David John C. , Burgess Robert J. , Thompson Joseph , Collins Lisa , Hadfield Ted
Published: February 2003

The diagnosis of human cases of tularemia often relies upon the demonstration of an antibody response toFrancisella tularensisor the direct culturing of the bacteria from the patient. Antibody response is not detectable until 2 weeks or more after infection, and culturing requires special media and suspicion of tularemia. In addition, handling liveFrancisellaposes a risk to laboratory personnel due to the highly infectious nature of this pathogen. In an effort to develop a rapid diagnostic assay for tularemia, we investigated the use of TaqMan 5′ hydrolysis fluorogenic PCR to detect the organism in tissues of infected mice. Mice were infected to produce respiratory tularemia. ThefopAandtul4genes ofF. tularensiswere amplified from infected spleen, lung, liver, and kidney tissues sampled over a 5-day period. The samples were analyzed using the laboratory-based Applied Biosystems International 7900 and the Smiths Detection-Edgewood BioSeeq, a hand-held portable fluorescence thermocycler designed for use in the field. A comparison of culturing and PCR for detection of bacteria in infected tissues shows that culturing was more sensitive than PCR. However, the results for culture take 72 h, whereas PCR results were available within 4 h. PCR was able to detect infection in all the tissues tested. Lung tissue showed the earliest response at 2 days when tested with the ABI 7900 and in 3 days when tested with the BioSeeq. The results were in agreement between the ABI 7900 and the BioSeeq when presented with the same sample. Template preparation may account for the loss of sensitivity compared to culturing techniques. The hand-held BioSeeq thermocycler shows promise as an expedient means of forward diagnosis of infection in the field.
Candida tropicalis
in a Neonatal Intensive Care Unit: Epidemiologic and Molecular Analysis of an Outbreak of Infection with an Uncommon Neonatal Pathogen
Authors: Roilides Emmanuel , Farmaki Evangelia , Evdoridou Joanna , Francesconi Andrea , Kasai Miki , Filioti Joanna , Tsivitanidou Maria , Sofianou Danai , Kremenopoulos George , Walsh Thomas J.
Published: February 2003

From June to July 1998, two episodes of
Candida tropicalis
fungemia occurred in the Aristotle University neonatal intensive care unit (ICU). To investigate this uncommon event, a prospective study of fungal colonization and infection was conducted. From December 1998 to December 1999, surveillance cultures of the oral cavities and perinea of the 593 of the 781 neonates admitted to the neonatal ICU who were expected to stay for >7 days were performed. Potential environmental reservoirs and possible risk factors for acquisition of
C. tropicalis
were searched for. Molecular epidemiologic studies by two methods of restriction fragment length polymorphism analysis and two methods of random amplified polymorphic DNA analysis were performed. Seventy-two neonates were colonized by yeasts (12.1%), of which 30 were colonized by
Candida albicans
, 17 were colonized by
C. tropicalis
, and 5 were colonized by
Candida parapsilosis
. From December 1998 to December 1999, 10 cases of fungemia occurred; 6 were due to
C. parapsilosis
, 2 were due to
C. tropicalis
, 1 was due to
Candida glabrata
, and 1 was due to
Trichosporon asahii
(12.8/1,000 admissions). Fungemia occurred more frequently in colonized than in noncolonized neonates (
P
< 0.0001). Genetic analysis of 11 colonization isolates and the two late blood isolates of
C. tropicalis
demonstrated two genotypes. One blood isolate and nine colonization isolates belonged to a single type. The fungemia/colonization ratio of
C. parapsilosis
(3/5) was greater than that of
C. tropicalis
(2/17,
P
= 0.05), other non-
C. albicans Candida
spp. (1/11,
P
= 0.02), or
C. albican
s (0/27,
P
= 0.05). Extensive environmental cultures revealed no common source of
C. tropicalis
or
C. parapsilosis
. There was neither prophylactic use of azoles nor other risk factors found for acquisition of
C. tropicalis
except for total parenteral nutrition. A substantial risk of colonization by non-
C. albicans Candida
spp. in the neonatal ICU may lead to a preponderance of
C. tropicalis
as a significant cause of neonatal fungemia.
Evaluation of Neuraminidase Enzyme Assays Using Different Substrates To Measure Susceptibility of Influenza Virus Clinical Isolates to Neuraminidase Inhibitors: Report of the Neuraminidase Inhibitor Susceptibility Network
Authors: Wetherall N. T. , Trivedi T. , Zeller J. , Hodges-Savola C. , McKimm-Breschkin J. L. , Zambon M. , Hayden F. G.
Published: February 2003

The increasing use of influenza virus neuraminidase (NA) inhibitors (NIs) necessitates the development of reliable methods for assessing the NI susceptibility of clinical isolates. We evaluated three NA inhibition assays against a panel of five clinical isolates each of influenza virus A/H1N1, A/H3N2, and B strains and four viruses with a defined resistance genotype (R292K, H274Y, R152K, and E119V). For fluorometric enzyme assay (FA) 1 (FA-1), 2′-(4-methylumbelliferyl)-α-
d
-
N
-acetylneuraminic acid (MUNANA) at 100 μM was used as the substrate, with pretitration of the virus input. For FA-2, MUNANA at 200 μM was used as the substrate, with a fixed 1:10 dilution of input virus. For the chemiluminescence (CL) assay, the 1,2-dioxetane derivative of sialic acid at 100 μM was used as the substrate, with pretitration of the virus. Four different operators repeated the assays several times in a blinded fashion with both zanamivir and oseltamivir carboxylate (GS4071) to determine intra- and interassay variations. Mean 50% inhibitory concentration (IC
50
) values were lower and generally less variable with the CL assay. FA-1 displayed greater variation than the CL assay or FA-2 and the highest IC
50
values with zanamivir; FA-2 showed the highest values with oseltamivir, particularly for influenza virus B, and was more variable with zanamivir than was the CL assay. All three assays detected 40-fold or greater changes in IC
50
values for the resistant viruses with at least one drug. Mixing experiments, whereby increasing fractions (0, 20, 40, 60, 80, and 100%) of NA from a known NI-resistant virus were mixed with the corresponding NI-sensitive parental NA, indicated that the resolution of IC
50
values was clearer with the CL assay than with FA-2 for two of the resistant variants (R152K and E119V). The FA and CL methods were reliable for the detection of NI resistance, but all assays have certain limitations. Based on reproducibility, ease of automation, time required for the assay, and greater sensitivity, the CL assay was selected for future susceptibility testing of influenza virus isolates circulating globally.
Molecular Fingerprinting of Fish-Pathogenic
Lactococcus garvieae
Strains by Random Amplified Polymorphic DNA Analysis
Authors: Ravelo Carmen , Magariños Beatriz , López-Romalde Sonia , Toranzo Alicia E. , Romalde Jesús L.
Published: February 2003

In this work, we used the random amplified polymorphic DNA (RAPD) technique to evaluate the genetic diversity in
Lactococcus garvieae
, an important pathogen for fish. Fifty-seven strains with different hosts and geographical origins, including Japan and several countries of the Mediterranean area such as Spain, Portugal, France, Italy, England, and Turkey, were analyzed. Two primers, oligonucleotides 5 and 6 (Pharmacia Biotech) were utilized; primer 5 was the most discriminative, since allowed us to differentiate 10 RAPD -types related to the origin of the strains. Regardless of the oligonucleotide primer employed, the 57 isolates of
L. garvieae
studied were separated into three genetic groups, composed of the Spanish, Portuguese, English, and Turkish strains (group A), the Italian and French strains (group B), and the Japanese strains (group C). The similarity of isolates within each group, estimated on the basis of the Dice coefficient, ranged from 75 to 100%. Our findings also indicate that RAPD profiling constitutes a useful tool for epidemiological studies of this fish pathogen.
Occurrence of Extended-Spectrum and AmpC Beta-Lactamases in Bloodstream Isolates of
Klebsiella pneumoniae:
Isolates Harbor Plasmid-Mediated FOX-5 and ACT-1 AmpC Beta-Lactamases
Authors: Coudron Philip E. , Hanson Nancy D. , Climo Michael W.
Published: February 2003

We tested 190
Klebsiella pneumoniae
bloodstream isolates recovered from 189 patients in 30 U.S. hospitals in 23 states to determine the occurrence of extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase producers. Based on growth inhibition by clavulanic acid by disk and MIC test methods, 18 (9.5%) of the isolates produced ESBLs. Although the disk diffusion method with standard breakpoints identified 28 cefoxitin-nonsusceptible isolates, only 5 (18%) of these were confirmed as AmpC producers. Of two AmpC confirmatory tests, the three-dimensional extract test was easier to perform than was the double-disk approximation test using a novel inhibitor, Syn2190. Three of the five AmpC producers carried the
bla
FOX-5
gene, while the other two isolates harbored the
bla
ACT-1
gene. All AmpC genes were transferable. In vitro susceptibility testing with standard inocula showed that all five AmpC-producing strains were susceptible to cefepime, imipenem, and ertapenem but that with a high inoculum, more of these strains were susceptible to the carbapenems than to cefepime. All but 1 of 14 screen-positive AmpC nonproducers (and ESBL nonproducers) were susceptible to ceftriaxone and cefepime at the standard inoculum as were 6 of 6 isolates that were randomly selected and tested with a high inoculum. These results indicate that (i) a significant number of
K. pneumoniae
bloodstream isolates harbor ESBL or AmpC β-lactamases, (ii) confirmatory tests are necessary to identify true AmpC producers, and (iii) in vitro, carbapenems are active against AmpC-producing strains of
K. pneumoniae
.
Apophysomyces elegans
: an Emerging Zygomycete in India
Authors: Chakrabarti A. , Ghosh A. , Prasad G. S. , David J. K. , Gupta S. , Das A. , Sakhuja V. , Panda N. K. , Singh S. K. , Das S. , Chakrabarti T.
Published: February 2003

Apophysomyces elegans
was considered a rare but medically important zygomycete. We analyzed the clinical records of eight patients from a single center in whom zygomycosis due to
A. elegans
was diagnosed over a span of 25 months. We also attempted a DNA-based method for rapid identification of the fungi and looked for interstrain polymorphism using microsattelite primers. Three patients had cutaneous and subcutaneous infections, three had isolated renal involvement, one had rhino-orbital tissue infection, and the final patient had a disseminated infection involving the spleen and kidney. Underlying illnesses were found in two patients, one with diabetes mellitus and the other with chronic alcoholism. A history of traumatic implantation was available for three patients. All except two of the patients responded to surgical and/or medical therapy; the diagnosis for the two exceptions was made at the terminal stage of infection. Restriction enzyme (
Mbo
I,
Msp
I,
Hin
fI) digestion of the PCR-amplified internal transcribed spacer region helped with the rapid and specific identification of
A. elegans
. The strains could be divided into two groups according to their patterns, with clustering into one pattern obtained by using microsatellite [(GTG)
5
and (GAC)
5
] PCR fingerprinting. The study highlights the epidemiology, clinical spectrum, and diagnosis of emerging
A. elegans
infections.
Use of Multienzyme Multiplex PCR Amplified Fragment Length Polymorphism Typing in Analysis of Outbreaks of Multiresistant
Klebsiella pneumoniae
in an Intensive Care Unit
Authors: van der Zee Anneke , Steer Niels , Thijssen Eveline , Nelson Jolande , van't Veen Annemarie , Buiting Anton
Published: February 2003

We developed and optimized a new modified amplified fragment length polymorphism (AFLP) typing method to obtain a multibanding fingerprint that can be separated by agarose gel electrophoresis. Both to maximize the discriminatory power and to facilitate the computer-assisted analysis, bacterial DNA was digested with four different restriction enzymes. After ligation of adaptors to the DNA fragments, PCR testing of various single primers was performed. Two single primers that gave optimal results with regard to band resolution and discriminatory power were selected and combined. The computer-assisted analysis of fingerprint patterns was performed with Pearson's product-moment correlation values of densitometric curves, without assigning bands to peaks. Thus, the analysis is not subject to human interpretation errors. With this method, we investigated two outbreaks of multiresistant
Klebsiella pneumoniae
in an intensive care unit and various sporadic isolates of
K. pneumoniae
and
Klebsiella oxytoca.
Cluster analysis of isolates analyzed in different experiments and on different gels showed that fingerprint patterns clustered correctly according to subspecies or to the outbreaks. Multienzyme multiplex PCR AFLP revealed that the first outbreak was caused by two different types of strains. Outbreak two was caused by yet another strain of
K. pneumoniae
. In conclusion, the typing method used here is easy to perform and highly reproducible, and due to generation of complex banding patterns, it has a higher discriminatory power. Furthermore, the multienzyme multiplex PCR fingerprints are easy to analyze, and a reliable database can be stored in the computer to facilitate comparison of future isolates of
Klebsiella
spp. The method can be performed in every clinical microbiology laboratory.
Rapid Detection of Adenovirus in Throat Swab Specimens by PCR during Respiratory Disease Outbreaks among Military Recruits
Authors: Echavarria Marcela , Sanchez Jose L. , Kolavic-Gray Shellie A. , Polyak Christina S. , Mitchell-Raymundo Felicia , Innis Bruce L. , Vaughn David , Reynolds Richard , Binn Leonard N.
Published: February 2003

We evaluated the performance of a generic PCR test to detect adenoviruses (AdV) in throat swab specimens collected from asymptomatic and ill military recruits with acute respiratory disease. Samples (
n
= 210) were collected at entry to basic training and at the time of large outbreaks of AdV-associated acute respiratory disease among military recruits at Fort Jackson, South Carolina, from 1997 to 1998. Compared to cell culture, a sensitivity of 99% and a specificity of 98% were noted for the PCR method to detect AdV in throat swabs. Similar results were obtained with or without DNA extraction, suggesting the absence of significant inhibitors for the PCR method in throat swab samples. No AdV was detected by culture or PCR in throat swabs from healthy recruits, suggesting the absence of latency or asymptomatic shedding. Throat swab specimens proved to be adequate, noninvasive samples to rapidly diagnose respiratory disease in young adults. This generic direct PCR proved to be a useful test for the rapid diagnosis of AdV-associated respiratory disease, detecting all serotypes tested to date and furnishing results within 6 h of specimen arrival. The use of this direct, rapid, sensitive, and specific assay would assist health care providers and public health practitioners in the early diagnosis, management, and control of AdV-associated respiratory disease.
Detection of
Helicobacter pylori
in Paraffin-Embedded and in Shock-Frozen Gastric Biopsy Samples by Fluorescent In Situ Hybridization
Authors: Rüssmann Holger , Feydt-Schmidt Anne , Adler Kristin , Aust Daniela , Fischer Almuth , Koletzko Sibylle
Published: February 2003

We report on the successful application of fluorescent in situ hybridization for detection of
Helicobacter pylori
and determination of its clarithromycin susceptibility in formalin-fixed and paraffin-embedded gastric biopsy specimens that had been prepared for pathological examination. This method is useful when results from conventional culturing with antibiotic susceptibility testing are not available.
Detection of
Candida albicans
mRNA from Formalin-Fixed, Paraffin-Embedded Mouse Tissues by Nested Reverse Transcription-PCR
Authors: Schofield David A. , Westwater Caroline , Paulling Emily E. , Nicholas Peter J. , Balish Edward
Published: February 2003

Histopathology archives represent a vast source of infectious disease specimens that can be used to elucidate important disease processes. In this report, we describe a method to detect
Candida albicans
gene expression from infected, formalin-fixed, paraffin-embedded mouse tissue. By use of glass beads to break open the fungal cells and proteinase K treatment, RNA was extracted routinely from tissue sections that had been fixed for up to 72 h. Upon reverse transcription of the RNA and nested PCR, the procedure detected
C. albicans
“housekeeping” and putative virulence genes.
Clinical Utility of Urinary Antigen Detection for Diagnosis of Community-Acquired, Travel-Associated, and Nosocomial Legionnaires' Disease
Authors: Helbig Jürgen H. , Uldum Søren A. , Bernander Sverker , Lück Paul Christian , Wewalka Günther , Abraham Bill , Gaia Valeria , Harrison Timothy G.
Published: February 2003

The clinical utility of
Legionella
urinary antigen assays for the diagnosis of Legionnaires' disease was assessed by using samples from 317 culture-proven cases. The sensitivities of the Binax enzyme immunoassay (EIA) and Biotest EIA were found to be 93.7 and 94.4% for travel-associated infection and 86.5 and 76.0% for community-acquired infection but only 44.2 and 45.7% for nosocomially acquired infection, respectively.
Phylogeny of the Genus
Nocardia
Based on Reassessed 16S rRNA Gene Sequences Reveals Underspeciation and Division of Strains Classified as
Nocardia asteroides
into Three Established Species and Two Unnamed Taxons
Authors: Roth Andreas , Andrees Sebastian , Kroppenstedt Reiner M. , Harmsen Dag , Mauch Harald
Published: February 2003

Conventional identification of
Nocardia
in the routine laboratory remains problematic due to a paucity of reliable phenotypic tests and due to the yet-unresolved taxonomy of strains classified as belonging to the species
Nocardia asteroides
, which comprises the type strain and isolates with drug pattern types II and VI. The 16S rRNA gene of 74 representative strains of the genus
Nocardia
, encompassing 25 established species, was sequenced in order to provide a molecular basis for accurate species identification and with the aim of reassessing the phylogeny of taxons assigned to the species
N. asteroides
. The result of this phylogenetic analysis confirms that the interspecies heterogeneity of closely related nocardial species can be considerably low (a sequence divergence of only 0.5% was found between
N. paucivorans
and
N. brevicatena
). We observed a sequence microheterogeneity (sequence divergence of fewer than five bases) in 8 of 11 species of which more than one strain in the species was studied. At least 10 taxons were found that merit description as new species. Strains previously classified as
N. asteroides
fell into five distinct phylogenetic groups: the type strain cluster (
N. asteroides
sensu strictu),
N. abscessus
,
N. cyriacigeorgica
, and two clusters closely related to
N. carnea
or
N. flavorosea
. The strains within the latter two groups probably represent new species, pending further genetic and phenotypic evaluation. Restricted phenotypic data revealed that
N. abscessus
,
N. cyriacigeorgica
, and the two
Nocardia
species taxons are equivalent to drug patterns I, VI, and II, respectively. In the future, these data will help in finding species-specific markers after adoption of a more precise nomenclature for isolates closely related to
N. asteroides
and unravel confusing phenotypic data obtained in the past for unresolved groups of strains that definitely belong to separate taxons from a phylogenetic point of view.
Novel Observation of Hot-Cold-Hot Hemolysis Exhibited by Group B Streptococci
Authors: Okazaki Norio , Osawa Ro , Suzuki Rieko , Nikkawa Takayasu , Whiley Robert A.
Published: February 2003

Six human isolates of group B streptococci (GBS) were cultured on blood agar anaerobically at 37°C for 18 h and then at 4°C for 6 h and reincubated anaerobically at 37°C for 6 h. Three of the strains showed a marked enlargement of the hemolysis zone compared with that obtained after hot-only (37°C for 18 h) or hot-cold (37°C for 18 h and then 4°C for 6 h) treatment. Subsequent broth culture experiments revealed that enhanced hemolytic activity due to hot-cold-hot treatment was observed in all 6 GBS strains when cultured in the presence of starch.
Value of Indirect Hemagglutination and Coagglutination Tests for SerotypingHaemophilus parasuis
Authors: Del Río M. L. , Gutiérrez C.B. , Rodríguez Ferri E. F.
Published: February 2003

An indirect hemagglutination test (IHA) and a coagglutination test (CA) were evaluated using saline, boiled, and autoclaved extracts for serotypingHaemophilus parasuis. CA showed several cross-reactions, whereas IHA gave rise to specific reactions, with minor exceptions. IHA was further compared with the immunodiffusion test (the “gold standard”) for the serotyping of 67 field isolates. As a conclusion, IHA is recommended as a useful method for sensitive and specific serotyping ofH. parasuis.