bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular Serotyping ofKlebsiellaSpecies Isolates by Restriction of the Amplified Capsular Antigen Gene Cluster
Authors: Brisse Sylvain , Issenhuth-Jeanjean Sylvie , Grimont Patrick A. D. 
Published: August 2004

ABSTRACTThe objective of the present work was to develop a molecular method that would enable determination of the capsular serotypes ofKlebsiellaisolates without the use of antiserum. PCR amplification of the capsular antigen gene cluster (cps) was followed by digestion with the restriction enzyme HincII (cpsPCR-restriction fragment length polymorphism [RFLP] analysis). The profiles (C patterns) obtained for 224 strains representing the 77 known K serotypes showed 3 to 13 fragments ranging in size from 0.2 to 4.4 kb. A total of 97 distinct C patterns were obtained; 100% of 61 pairs of samples tested twice showed reproducible C patterns. The C patterns were K-type specific; i.e., the C pattern(s) of any K serotype was distinct from the C patterns of all other K serotypes, with the only exceptions being serotypes K22 and K37, which are known to cross-react. For 12 of 17 K types for which at least two strains were included, C-pattern variations were found among strains with the same K serotype. Therefore,cpsPCR-RFLP analysis has a higher discriminatory power than classical K serotyping. C-pattern identity was observed among strains with a given K type that were collected many years apart and from distinct sources, indicating C-pattern stability. Only 4.5% of the strains were nontypeable, because of unsuccessful PCR amplification (whereas 8 to 23% are nontypeable by classical K serotyping). Three of four noncapsulated strains analyzed showed recognizable C patterns. The K serotypes of 18 (82%) of 22 recentKlebsiella pneumoniaeclinical isolates could be deduced from their C patterns. In conclusion,cpsPCR-RFLP analysis allows determination of the K serotype, while it is easier to perform and more discriminatory than classical serotyping.
Phenotypic and Genotypic Changes in
Salmonella enterica
subsp.
enterica
Serotype Typhimurium during Passage in Intestines of Broiler Chickens Fed on Diets That Included Ionophore Anticoccidial Supplements
Authors: Scalzo Salvatore , Corkill John E. , Shanks David J. , Rowan Timothy G. , Delaval José , Fleetwood Andrew , Murphy Martin , Hart C. Anthony 
Published: August 2004

ABSTRACT

The effect of continuous in-feed administration of anticoccidial agents on antimicrobial sensitivity and the level of bacterial shedding in poultry experimentally infected with
Salmonella enterica
subsp.
enterica
serotype Typhimurium definitive type 104 (DT104) were investigated. On day 0, 1,200 1-day-old
Salmonella
-free broiler chicks were placed into 50 pens, and the pens were randomly allocated to one of five treatments: nonsupplemented (negative control; T1), monensin at 120 mg/kg of diet (T2), salinomycin at 60 mg/kg of diet (T3), semduramicin at 20 mg/kg of diet (T4), or semduramicin at 25 mg/kg of diet (T5). Each bird was inoculated with a well-characterized strain of serotype Typhimurium DT104 on day 10. On day 49, the birds were euthanatized humanely. Bulk fecal samples were collected on days 13, 43, and 48 and were examined for organisms which had acquired resistance. The genetic basis of acquired resistance was determined from representative samples of isolates. Of 784
Salmonella
-selective plates supplemented with antimicrobial agents, only 33 showed growth. These isolates came from all treatment regimens, including the nonsupplemented control. A number of phenotypic changes were observed; these included changes in motility, phage type, and agglutination properties. Supplementation of the diet with an anticoccidial drug does not appear to affect antimicrobial resistance or the level of excretion of salmonellae. Most of the changes observed do not seem to be related to the presence of a supplement in feed. Salmonellae appear to be capable of acquiring antimicrobial resistance and phenotypic changes independently of specific antimicrobial selection pressures.
Evaluation of Immunoglobulin M (IgM) and IgG Rapid Cassette Test Kits for Diagnosis of Melioidosis in an Area of Endemicity
Authors: Wuthiekanun Vanaporn , Amornchai Premjit , Chierakul Wirongrong , Cheng Allen C. , White Nicholas J. , Peacock Sharon J. , Day Nicholas P. J. 
Published: August 2004

ABSTRACT
An enzyme-linked immunosorbent assay-based rapid cassette immunoglobulin G (IgG) and IgM immunochromogenic test kit was compared to the indirect hemagglutination test (IHA) for the diagnosis of acute melioidosis in northeastern Thailand. Admission sera from 70 culture-confirmed septicemic melioidosis patients and 30 patients with localized infections were tested. As a control group, 80 patients with other acute febrile illnesses (other bacterial infections, leptospirosis, or scrub typhus) and 119 healthy individuals were tested. The diagnostic sensitivity of the IgG and IgM tests and the IHA test were 79, 67, and 72%, respectively, with corresponding specificities of 90, 80, and 68%. This kit represents an improvement over IHA for the diagnosis of melioidosis an area of endemicity although, as with other serological tests, it has reduced diagnostic utility in a population with high background seropositivity.
Real-Time PCR Assay Using Molecular Beacon for Quantitation of Hepatitis B Virus DNA
Authors: Sum Simon Siu-Man , Wong Danny Ka-Ho , Yuen Man-Fung , Yuan He-Jun , Yu Jian , Lai Ching-Lung , Ho David , Zhang Linqi 
Published: August 2004

ABSTRACT

Levels of hepatitis B virus (HBV) DNA in the blood serve as an important marker in monitoring the disease progression and treatment efficacy of chronic HBV infection. Several commercial assays are available for accurate measurement of HBV genomic DNA, but many of them are hampered by relatively low sensitivity and limited dynamic range. The aim of this study was to develop a sensitive and accurate assay for measuring HBV genomic DNA using real-time PCR with a molecular beacon (HBV beacon assay). The performance of this assay was validated by testing serial dilutions of the two EUROHEP HBV DNA standards (
ad
and
ay
subtypes) of known concentrations. The assay showed low intra-assay (<7%) and interassay (<5%) variations for both subtypes. Its dynamic range was found to be 10
1
to 10
7
copies per reaction (1.0 × 10
2
to 1.0 × 10
9
copies ml
−1
). The assay was further evaluated clinically using serum samples from 175 individuals with chronic hepatitis B. The HBV DNA level measured by this assay showed good correlation with that measured by the commercially available COBAS AMPLICOR HBV Monitor test (
r
= 0.901;
P
< 0.001). The higher sensitivity and broader dynamic range of this assay compared to the existing commercial assays will provide an ideal tool for monitoring disease progression and treatment efficacy in HBV-infected patients, in particular for those with low levels of HBV viremia.
High-Resolution Genotyping ofCampylobacter upsaliensisStrains Originating from Three Continents
Authors: Lentzsch P. , Rieksneuwöhner B. , Wieler L. H. , Hotzel H. , Moser I. 
Published: August 2004

ABSTRACTNinety-sixCampylobacter upsaliensisstrains that originated from Australia, Canada, and Europe (Germany) and that were isolated from humans, dogs, and cats were serotyped for their heat-stable surface antigens. All of them were genotyped by enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) profiling, and 83 strains were genotyped by macrorestriction analysis with the endonuclease XhoI. Eighty-four percent of the strains belonged to five different serotypes (serotypes OI, OII, OIII, OIV, and OVI), with the proportions of strains in each serotype being comparable among the groups of strains from all three continents. Two serotypes, OIII and OIV, were prevalent at rates of 35 to 40%. Serotypes OI, OII, and OVI were detected at rates of 1.5 to 15%. Between 10 and 17.7% of the strains did not react with the available antisera. Analysis of the ERIC-PCR profiles revealed two distinct genotypic clusters, which represented the German and the non-European strains, respectively. XhoI macrorestriction yielded two genotypic clusters; one of them contained 80.2% of the German strains and 34.6% of the non-European strains, and the second cluster consisted of 65.4% of the non-European strains and 19.8% of the German strains. Fourteen strains from all three continents were analyzed for their 16S rRNA gene sequences. Only two minor variations were detected in four of the strains. In conclusion,C. upsaliensishas undergone diverging processes of genome arrangement on different continents during evolution without segregating into different subspecies.
Molecular Typing and Distribution of
Staphylococcus aureus
Isolates in Eastern Canadian Dairy Herds
Authors: Sabour P. M. , Gill J. J. , Lepp D. , Pacan J. C. , Ahmed R. , Dingwell R. , Leslie K. 
Published: August 2004

ABSTRACT

Macrorestriction analysis of SmaI-digested chromosomal DNA, using pulsed field gel electrophoresis (PFGE) was performed to type and estimate genetic relationships among 288
Staphylococcus aureus
isolates recovered from 58 Eastern Canadian dairy herds. In addition, a subset of the collection was phage typed and evaluated for sensitivity to 10 antimicrobial compounds. Of 288 isolates recovered, 29 distinct PFGE types were identified. Based on estimates of genetic relationships, the PFGE types were assigned to six lineage groups, designated A through F. Of all of the isolates, ca. 93% were assigned to lineage groups A, D, or F. In 58.6% of herds, only a single PFGE type was recovered, while the remainder had two to four types. Of the 212 isolates evaluated for antimicrobial resistance, 24.5% were resistant to one or more antimicrobials. Resistance to penicillin (9.9%) was most common, followed by resistance to sulfadimethoxine (7.5%). Isolates resistant to multiple antibiotics were rare. A total of 63% of isolates responded to phages from groups 1 and 3, and 32.8% could not be typed with any of the phage strains used. The other 4.1% belonged to a variety of phage types. Most of the PFGE lineage group A and F isolates corresponded to phage groups 3 and 1, respectively, and most group D isolates were not typeable. PFGE typing had better discriminatory power than phage typing in defining the relatedness of the
S. aureus
isolates. Distribution of PFGE types and phage types was independent across regions and within herds.
Interlaboratory Comparison of Results of Susceptibility Testing with Caspofungin against
Candida
and
Aspergillus
Species
Authors: Odds Frank C. , Motyl Mary , Andrade Roberto , Bille Jacques , Cantón Emilia , Cuenca-Estrella Manuel , Davidson Amanda , Durussel Christian , Ellis David , Foraker Elyse , Fothergill Annette W. , Ghannoum Mahmoud A. , Giacobbe Robert A. , Gobernado Miguel , Handke Rosemary , Laverdière Michel , Lee-Yang Wendy , Merz William G. , Ostrosky-Zeichner Luis , Pemán Javier , Perea Sophia , Perfect John R. , Pfaller Michael A. , Proia Laurie , Rex John H. , Rinaldi Michael G. , Rodriguez-Tudela Juan-Luis , Schell Wiley A. , Shields Christine , Sutton Deanna A. , Verweij Paul E. , Warnock David W. 
Published: August 2004

ABSTRACT

Seventeen laboratories participated in a study of interlaboratory reproducibility with caspofungin microdilution susceptibility testing against panels comprising 30 isolates of
Candida
spp. and 20 isolates of
Aspergillus
spp. The laboratories used materials supplied from a single source to determine the influence of growth medium (RPMI 1640 with or without glucose additions and antibiotic medium 3 [AM3]), the same incubation times (24 h and 48 h), and the same end point definition (partial or complete inhibition of growth) for the MIC of caspofungin. All tests were run in duplicate, and end points were determined both spectrophotometrically and visually. The results from almost all of the laboratories for quality control and reference
Candida
and
Aspergillus
isolates tested with fluconazole and itraconazole matched the NCCLS published values. However, considerable interlaboratory variability was seen in the results of the caspofungin tests. For
Candida
spp. the most consistent MIC data were generated with visual “prominent growth reduction” (MIC
2
) end points measured at 24 h in RPMI 1640, where 73.3% of results for the 30 isolates tested fell within a mode ± one dilution range across all 17 laboratories. MIC
2
at 24 h in RPMI 1640 or AM3 also gave the best interlaboratory separation of
Candida
isolates of known high and low susceptibility to caspofungin. Reproducibility of MIC data was problematic for caspofungin tests with
Aspergillus
spp. under all conditions, but the minimal effective concentration end point, defined as the lowest caspofungin concentration yielding conspicuously aberrant hyphal growth, gave excellent reproducibility for data from 14 of the 17 participating laboratories.
Rapid Differentiation of
Aspergillus
Species from Other Medically Important Opportunistic Molds and Yeasts by PCR-Enzyme Immunoassay
Authors: de Aguirre Liliana , Hurst Steven F. , Choi Jong Soo , Shin Jong Hee , Hinrikson Hans Peter , Morrison Christine J. 
Published: August 2004

ABSTRACT

We developed a PCR-based assay to differentiate medically important species of
Aspergillus
from one another and from other opportunistic molds and yeasts by employing universal, fungus-specific primers and DNA probes in an enzyme immunoassay format (PCR-EIA). Oligonucleotide probes, directed to the internal transcribed spacer 2 region of ribosomal DNA from
Aspergillus flavus
,
Aspergillus fumigatus
,
Aspergillus nidulans
,
Aspergillus niger
,
Aspergillus terreus
,
Aspergillus ustus
, and
Aspergillus versicolor
, differentiated 41 isolates (3 to 9 each of the respective species;
P
< 0.001) in a PCR-EIA detection matrix and gave no false-positive reactions with 33 species of
Acremonium
,
Exophiala
,
Candida
,
Fusarium
,
Mucor
,
Paecilomyces
,
Penicillium
,
Rhizopus
,
Scedosporium
,
Sporothrix
, or other aspergilli tested. A single DNA probe to detect all seven of the most medically important
Aspergillus
species (
A. flavus
,
A. fumigatus
,
A. nidulans
,
A. niger
,
A. terreus
,
A. ustus
, and
A. versicolor
) was also designed. Identification of
Aspergillus
species was accomplished within a single day by the PCR-EIA, and as little as 0.5 pg of fungal DNA could be detected by this system. In addition, fungal DNA extracted from tissues of experimentally infected rabbits was successfully amplified and identified using the PCR-EIA system. This method is simple, rapid, and sensitive for the identification of medically important
Aspergillus
species and for their differentiation from other opportunistic fungi.
Capillary Electrophoretic Restriction Fragment Length Polymorphism Patterns for the Mycobacterial
hsp65
Gene
Authors: Ho Hsin-Tsung , Chang Po-Ling , Hung Chia-Chien , Chang Huan-Tsung 
Published: August 2004

ABSTRACT

PCR-restriction fragment length polymorphism (RFLP) analysis is a nonprobe method for the rapid identification of
Mycobacterium
species. We demonstrate the separation of DNA or restriction fragments digested from the mycobacterial gene encoding the 65-kDa heat shock protein (
hsp65
) by capillary electrophoresis (CE). By using a pair of unlabeled primers, Tb11 and Tb12, and only one restriction enzyme, HaeIII, we investigated a total of 52 reference and clinical strains encompassing 12
Mycobacterium
species. The electrophoretic separation of high-resolution CE required <20 min and was capable of identifying fragments as small as 12 bp. A good agreement of measurement was observed between the sizes of restriction fragments resolved by CE, and the real sizes were deduced from the sequence analysis. Distinct differentiations were also well demonstrated between some species and subspecies by an extra HaeIII digestion site. With the advantage of the complete RFLP pattern available from CE, it appears to be more convenient to use an electropherogram rather than performing the cumbersome slab gel electrophoresis plus diagnostic algorithm to identify
Mycobacterium
species. Beyond the agarose and polyacrylamide gel electrophoresis, high-resolution CE provides an alternative for rapid identification of
Mycobacterium
species that is feasible for automation and routine use without the need for costly probes.
Use of 16S Ribosomal DNA PCR and Denaturing Gradient Gel Electrophoresis for Analysis of the Microfloras of Healing and Nonhealing Chronic Venous Leg Ulcers
Authors: Davies Charlotte E. , Hill Katja E. , Wilson Melanie J. , Stephens Phil , Hill C. Michael , Harding Keith G. , Thomas David W. 
Published: August 2004

ABSTRACT

The bacterial microfloras of 8 healing and 10 nonhealing chronic venous leg ulcers were compared by using a combination of cultural analysis and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene products. Cultural analysis of the microflora revealed that the majority of both wound types carried the aerobes
Staphylococcus
and
Pseudomonas
spp. (89 and 80%, respectively). Sequencing of 16S ribosomal DNAs selected on the basis of DGGE profiling allowed the identification of strains not detected by cultural means. Of considerable interest was the finding that more than 40% of the sequences represented organisms not cultured from the wound from which they were amplified. DGGE profiles also revealed that all of the wounds possessed one apparently common band, identified by sequencing as
Pseudomonas
sp. The intensity of this PCR signal suggested that the bacterial load of nonhealing wounds was much higher for pseudomonads compared to healing wounds and that it may have been significantly underestimated by cultural analysis. Hence, the present study shows that DGGE could give valuable additional information about chronic wound microflora that is not apparent from cultural analysis alone.
Use of a Genus- and Species-Specific Multiplex PCR for Identification of Enterococci
Authors: Jackson Charlene R. , Fedorka-Cray Paula J. , Barrett John B. 
Published: August 2004

ABSTRACT

The 16S rRNA gene has previously been used to develop genus-specific PCR primers for identification of enterococci. In addition, the superoxide dismutase gene (
sodA
) has been identified as a potential target for species differentiation of enterococci. In this study,
Enterococcus
genus-specific primers developed by Deasy et al. (E1/E2) were incorporated with species-specific primers based upon the superoxide dismutase (
sodA
) gene for development of a multiplex PCR. This assay provides simultaneous genus and species identification of 23 species of enterococci using seven different reaction mixtures. Accuracy of identification of the multiplex PCR was determined by comparisons to standard biochemical testing, the BBL Crystal kit, VITEK, and API Rapid ID 32 Strep. Isolates from swine feces, poultry carcasses, environmental sources, and retail food were evaluated and, overall, results for 90% of the isolates tested by PCR agreed with results obtained using standard biochemical testing and VITEK. Eighty-five percent and 82% of PCR results agreed with results from the API Rapid ID 32 Strep and BBL Crystal tests, respectively. With the exception of concurrence between identification using standard biochemical testing and VITEK (85%) and between BBL Crystal and VITEK (83%), the percent agreement for PCR was higher than or equal to any other pairwise comparison. Multiplex PCR for genus and species determination of enterococci provides an improved, rapid method for identification of this group of bacteria.
Use of Fatty Acid RPMI 1640 Media for Testing Susceptibilities of Eight
Malassezia
Species to the New Triazole Posaconazole and to Six Established Antifungal Agents by a Modified NCCLS M27-A2 Microdilution Method and Etest
Authors: Velegraki Aristea , Alexopoulos Evangelos C. , Kritikou Stavroula , Gaitanis George 
Published: August 2004

ABSTRACT

A novel formulation of RPMI 1640 medium for susceptibility testing of
Malassezia
yeasts by broth microdilution (BMD) and Etest is proposed. A modification of the NCCLS M27-A2 BMD method was used to test 53 isolates of
Malassezia furfur
(12 isolates),
M. sympodialis
(8 isolates),
M. slooffiae
(4 isolates),
M. globosa
(22 isolates),
M. obtusa
(2 isolates),
M. restricta
(2 isolates),
M. pachydermatis
(1 isolates), and
M. dermatis
(2 isolates) against amphotericin B, ketoconazole, itraconazole, fluconazole, voriconazole, terbinafine, and posaconazole by BMD and Etest. RPMI and antibiotic medium 3 (AM3) were supplemented with glucose, bile salts, a mixture of fatty acids, and
n
-octadecanoate fatty acids and Tween 20.
M. furfur
ATCC 14521 and
M. globosa
ATCC 96807 were used as quality control strains. Depending on the species, MICs were read after 48 or 72 h of incubation at 32°C. Low azole and terbinafine MICs were recorded for all
Malassezia
species, whereas amphotericin B displayed higher MICs (≥16 μg/ml) against
M. furfur
,
M. restricta
,
M. globosa
, and
M. slooffiae
strains, which were AM3 confirmed. Agreement of the two methods was 84 to 97%, and intraclass correlation coefficients were statistically significant (
P
< 0.001). Because of higher amphotericin B MICs provided by Etest for strains also displaying high BMD MICs (≥
1
μg/ml), agreement was poorer. The proposed media are used for the first time and can support optimum growth of eight
Malassezia
species for recording concordant BMD and Etest MICs.
Genomic Analysis Distinguishes
Mycobacterium africanum
Authors: Mostowy Serge , Onipede Anthony , Gagneux Sebastien , Niemann Stefan , Kremer Kristin , Desmond Edward P. , Kato-Maeda Midori , Behr Marcel 
Published: August 2004

ABSTRACT

Mycobacterium africanum
is thought to comprise a unique species within the
Mycobacterium tuberculosis
complex.
M. africanum
has traditionally been identified by phenotypic criteria, occupying an intermediate position between
M. tuberculosis
and
M. bovis
according to biochemical characteristics. Although
M. africanum
isolates present near-identical sequence homology to other species of the
M. tuberculosis
complex, several studies have uncovered large genomic regions variably deleted from certain
M. africanum
isolates. To further investigate the genomic characteristics of organisms characterized as
M. africanum
, the DNA content of 12 isolates was interrogated by using Affymetrix GeneChip. Analysis revealed genomic regions of
M. tuberculosis
deleted from all isolates of putative diagnostic and biological consequence. The distribution of deleted sequences suggests that
M. africanum
subtype II isolates are situated among strains of “modern”
M. tuberculosis
. In contrast, other
M. africanum
isolates (subtype I) constitute two distinct evolutionary branches within the
M. tuberculosis
complex. To test for an association between deleted sequences and biochemical attributes used for speciation, a phenotypically diverse panel of “
M. africanum-
like” isolates from Guinea-Bissau was tested for these deletions. These isolates clustered together within one of the
M. africanum
subtype I branches, irrespective of phenotype. These results indicate that convergent biochemical profiles can be independently obtained for
M. tuberculosis
complex members, challenging the traditional approach to
M. tuberculosis
complex speciation. Furthermore, the genomic results suggest a rational framework for defining
M. africanum
and provide tools to accurately assess its prevalence in clinical specimens.