bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phenotypic Screening of
Escherichia coli
K-12 Tn
5
Insertion Libraries, Using Whole-Genome Oligonucleotide Microarrays
Authors: Winterberg Kelly M. , Luecke John , Bruegl Amanda S. , Reznikoff William S. 
Published: January 2005

ABSTRACT

Complete genome sequences in combination with global screening methods allow parallel analysis of multiple mutant loci to determine the requirement for specific genes in different environments. In this paper we describe a high-definition microarray approach for investigating the growth effects of Tn
5
insertions in
Escherichia coli
K-12. Libraries of insertion mutants generated by a unique Tn
5
mutagenesis system were grown competitively in defined media. Biotin-labeled runoff RNA transcripts were generated in vitro from transposon insertions in each population of mutants. These transcripts were then hybridized to custom-designed oligonucleotide microarrays to detect the presence of each mutant in the population. By using this approach, the signal associated with 25 auxotrophic insertions in a 50-mutant pool was not detectable following nine generations of growth in glucose M9 minimal medium. It was found that individual insertion sites could be mapped to within 50 bp of their genomic locations, and 340 dispensable regions in the
E. coli
chromosome were identified. Tn
5
insertions were detected in 15 genes for which no previous insertions have been reported. Other applications of this method are discussed.
Methods To Increase Fidelity of Repetitive Extragenic Palindromic PCR Fingerprint-Based Bacterial Source Tracking Efforts
Authors: Hassan Wail M. , Wang Shiao Y. , Ellender Rudolph D. 
Published: January 2005

ABSTRACT

The goal of the study was to determine which similarity coefficient and statistical method to use to produce the highest rate of correct assignment (RCA) in repetitive extragenic palindromic PCR-based bacterial source tracking. In addition, the use of standards for deciding whether to accept or reject source assignments was investigated. The use of curve-based coefficients Cosine Coefficient and Pearson's Product Moment Correlation yielded higher RCAs than the use of band-based coefficients Jaccard, Dice, Jeffrey's x, and Ochiai. When enterococcal and
Escherichia coli
isolates from known sources were used in a blind test, the use of maximum similarity produced consistently higher RCAs than the use of average similarity. We also found that the use of a similarity value threshold and/or a quality factor threshold (the ratio of the average fingerprint similarity within a source to the average similarity of this source's isolates to an unknown) to decide whether to accept source assignments of unknowns increases the reliability of source assignments. Applying a similarity value threshold improved the overall RCA (ORCA) by 15 to 27% when enterococcal fingerprints were used and 8 to 29% when
E. coli
fingerprints were used. Applying the quality factor threshold resulted in a 22 to 32% improvement in the ORCA, depending on the fingerprinting technique used. This increase in reliability was, however, achieved at the expense of decreased numbers of isolates that were assigned a source.
Isolation of
Vibrio alginolyticus
and
Vibrio splendidus
from Aquacultured Carpet Shell Clam (
Ruditapes decussatus
) Larvae Associated with Mass Mortalities
Authors: Gómez-León J. , Villamil L. , Lemos M. L. , Novoa B. , Figueras A. 
Published: January 2005

ABSTRACT

Two episodes of mortality of cultured carpet shell clams (
Ruditapes decussatus
) associated with bacterial infections were recorded during 2001 and 2002 in a commercial hatchery located in Spain.
Vibrio alginolyticus
was isolated as the primary organism from moribund clam larvae that were obtained during the two separate events.
Vibrio splendidus
biovar II, in addition to
V. alginolyticus
, was isolated as a result of a mixed
Vibrio
infection from moribund clam larvae obtained from the second mortality event. The larval mortality rates for these events were 62 and 73%, respectively. Mortality was also detected in spat. To our knowledge, this is the fist time that these bacterial species have been associated with larval and juvenile carpet shell clam mortality. The bacterial strains were identified by morphological and biochemical techniques and also by PCR and sequencing of a conserved region of the 16S rRNA gene. In both cases bacteria isolated in pure culture were inoculated into spat of carpet shell clams by intravalvar injection and by immersion. The mortality was attributed to the inoculated strains, since the bacteria were obtained in pure culture from the soft tissues of experimentally infected clams.
V. alginolyticus
TA15 and
V. splendidus
biovar II strain TA2 caused similar histological lesions that affected mainly the mantle, the velum, and the connective tissue of infected organisms. The general enzymatic activity of both live cells and extracellular products (ECPs), as evaluated by the API ZYM system, revealed that whole bacterial cells showed greater enzymatic activity than ECPs and that the activity of most enzymes ceased after heat treatment (100°C for 10 min). Both strain TA15 and strain TA2 produced hydroxamate siderophores, although the activity was greater in strain TA15. ECPs from both bacterial species at high concentrations, as well as viable bacteria, caused significant reductions in hemocyte survival after 4 h of incubation, whereas no significant differences in viability were observed during incubation with heat-killed bacteria.
Synergy Tests by E Test and Checkerboard Methods of Antimicrobial Combinations against
Brucella melitensis
Authors: Orhan Gani , Bayram Aysen , Zer Yasemin , Balci Iclal 
Published: January 2005

ABSTRACT

Two different synergy testing methods, the checkerboard and the E test methods, were used to compare the in vitro efficacies of various antimicrobial combinations against 16
Brucella melitensis
strains isolated from blood cultures. The rate of agreement of the E test and checkerboard methods was found to be 55%. The most concordant results were found for the streptomycin-doxycycline combination in 12 (75%) tests, in which four strains showed synergistic activity by E test and antagonistic activity by the checkerboard method and in which one strain showed antagonistic activity by both methods. Even though each of these methods uses different conditions and endpoints, the results of both methods frequently agreed.
Rapid Multiplex Assay for Serotyping Pneumococci with Monoclonal and Polyclonal Antibodies
Authors: Yu Jigui , Lin Jisheng , Benjamin William H. , Waites Ken B. , Lee Che-hung , Nahm Moon H. 
Published: January 2005

ABSTRACTWe have developed and characterized a rapid semiautomated pneumococcal serotyping system incorporating a pneumococcal lysate preparation protocol and a multiplex serotyping assay. The lysate preparation incorporates a bile solubility test to confirm pneumococcal identification that also enhances assay specificity. The multiplex serotyping assay consists of 24 assays specific for 36 serotypes: serotypes 1, 2, 3, 4, 5, 6A, 6B, 7A/7F, 8, 9L/9N, 9V, 10A/10B/39/(33C), 11A/11D/11F, 12A/12B/12F, 14, 15B/(15C), 17F, 18C, 19A, 19F, 20, 22A/22F, 23F, and 33A/33F. The multiplex assay requires a flow cytometer, two sets of latex particles coated with pneumococcal polysaccharides, and serotype-specific antibodies. Fourteen newly developed monoclonal antibodies specific for common serotypes and a pool of polyclonal rabbit sera for some of the less-common serotypes are used. The two monoclonal antibodies specific for serotypes 18C and 23F recognize serotype-specific epitopes that have not been previously described. These monoclonal antibodies make the identification of the 14 common serotypes invariant. The specificity of the serotyping assay is fully characterized with pneumococci of all known (i.e., 90) serotypes. The assay is sensitive enough to use bacterial lysates diluted 20 fold. Our serotyping system can identify not only all the serotypes in pneumococcal vaccines but also most (>90%) of clinical isolates. This system should be very useful in serotyping clinical isolates for evaluating pneumococcal vaccine efficacy.
PCR Detection of Bacteria on Cardiac Valves of Patients with Treated Bacterial Endocarditis
Authors: Rovery Clarisse , Greub Gilbert , Lepidi Hubert , Casalta Jean-Paul , Habib Gilbert , Collart Frédéric , Raoult Didier 
Published: January 2005

ABSTRACTWe used broad-range PCR amplification and sequencing to detect and identify bacterial DNA in 156 valves of patients treated for infective endocarditis (IE). Bacterial DNA was found more frequently in patients who underwent valve replacement while on antibiotic treatment for IE (60%) than in patients who had completed antibiotic treatment for IE (37%;P= 0.02). We found specific bacterial DNA in valves removed from 11 of 30 patients who had completed antibiotic treatment for IE. Six had no histological evidence of IE. The presence of DNA was significantly correlated with the presence of histologic lesions (P= 0.001) and with the presence of bacteria detected by Gram staining (P< 0.001).Bartonellaand streptococci were detected for much longer after antibiotic treatment by PCR than other species (P= 0.047 and 0.04, respectively), and coagulase-negative staphylococci were detected for much shorter periods (P= 0.02). The finding that bacterial DNA was more likely to be detected in valves of patients with active IE than in patients who had completed antibiotic treatment for IE shows that bacterial DNA is cleared slowly. There was no significant correlation between the duration of antibiotic therapy and the presence of bacterial DNA in valves. Since the persistence of bacterial DNA in valves does not necessarily indicate the persistence of viable bacteria, the detection of bacterial DNA in valves from IE patients should be interpreted with caution, in particular in those patients with a past history of treated IE.
The Pathogen of Frogs
Amphibiocystidium ranae
Is a Member of the Order Dermocystida in the Class Mesomycetozoea
Authors: Pereira Cristiane N. , Di Rosa Ines , Fagotti Anna , Simoncelli Francesca , Pascolini Rita , Mendoza Leonel 
Published: January 2005

ABSTRACT

The pathogen of frogs
Amphibiocystidium ranae
was recently described as a new genus. Due to its spherical shape, containing hundred of endospores, it was thought to be closely related to the pathogens of fish, mammals, and birds known as
Dermocystidium
spp.,
Rhinosporidium seeberi
, and
Sphaerothecum destruens
in the Mesomycetozoea, but further studies were not conducted to confirm this relationship. To investigate its phylogenetic affinities, total genomic DNA was extracted from samples collected from infected frogs containing multiple cysts (sporangia) and endospores. The universal primers NS1 and NS8, used to amplify the 18S small-subunit rRNA by PCR, yielded ≈1,770-bp amplicons. Sequencing and basic local alignment search tool analyses indicated that the 18S small-subunit rRNA of
A. ranae
from both
Rana esculenta
and
Rana lessonae
was closely related to all of the above organisms. Our phylogenetic analysis placed this pathogen of frogs as the sister group to the genus
Dermocystidium
and closely related to
Rhinosporidium
. These data strongly supported the placement of the genus
Amphibiocystidium
within the mesomycetozoeans, which is in agreement with the phenotypic features that
A. ranae
shares with the other members of this class. Interestingly, during this study
Dermocystidium percae
did not group within the
Dermocystidium
spp. from fish; rather, it was found to be the sister group to
Sphaerothecum destruens
. This finding suggests that
D. percae
could well be a member of the genus
Sphaerothecum
or perhaps represents a new genus.
How Evolution of Mutations Conferring Drug Resistance Affects Viral Dynamics and Clinical Outcomes of Cytomegalovirus-Infected Hematopoietic Cell Transplant Recipients
Authors: Springer Kathryn L. , Chou Sunwen , Li Shaobing , Giller Roger H. , Quinones Ralph , Shira James E. , Weinberg Adriana 
Published: January 2005

ABSTRACT

Infection with cytomegalovirus (CMV) remains a significant cause of morbidity and mortality among hematopoietic cell transplant (HCT) recipients. We describe two pediatric HCT recipients who developed persistent and severe drug-resistant CMV infections. CMV resistance to foscarnet and ganciclovir was detected after only 6 and 11 weeks of therapy, respectively. Viral
pol
mutations associated with drug resistance in these patients included T838A (a novel mutation) and D588N, which were shown by marker transfer to confer foscarnet and multidrug resistance, respectively. Each of these mutations significantly reduced in vitro replication of CMV, suggesting that they may decrease viral fitness. This finding was further supported by the disappearance of mutations upon withdrawal of antiviral pressure in one patient. Novel antivirals or combination therapy may be required for the treatment of drug-resistant CMV in HCT recipients and perhaps in other severely immunocompromised patients.
Dry-Reagent-Based PCR as a Novel Tool for Laboratory Confirmation of Clinically Diagnosed
Mycobacterium ulcerans
-Associated Disease in Areas in the Tropics Where
M. ulcerans
Is Endemic
Authors: Siegmund V. , Adjei O. , Racz P. , Berberich C. , Klutse E. , van Vloten F. , Kruppa T. , Fleischer B. , Bretzel G. 
Published: January 2005

ABSTRACT

After tuberculosis and leprosy, Buruli ulcer (BU), caused by
Mycobacterium ulcerans
, is the third most common mycobacterial disease in immunocompetent humans. The disease occurs in tropical countries, with foci in West Africa, Central Africa, and the western Pacific. BU is defined as an infectious disease involving the skin and the subcutaneous adipose tissue characterized by a painless nodule, papule, plaque, or edema, evolving into a painless ulcer with undermined edges and often leading to invalidating sequelae. Due to the fundamental lack of understanding of modes of transmission, disease control in endemic countries is limited to early case detection through improved active surveillance and surgical treatment. The laboratory confirmation of BU is complicated by the absence of a diagnostic “gold standard.” Therefore, misclassification and delayed diagnosis of BU may occur frequently, causing a considerable socioeconomic impact in terms of treatment costs due to prolonged hospitalization. In order to respond to the urgent need to develop reliable tools for early case detection and to overcome technical difficulties accompanying the implementation of diagnostic PCR procedures in tropical countries, a dry-reagent-based PCR formulation for the detection of
M. ulcerans
in diagnostic specimens has been developed at the Bernhard Nocht Institute for Tropical Medicine. Following technical and clinical validation, the assay has been successfully installed and field tested at the Kumasi Centre for Collaborative Research in Tropical Medicine, Kumasi, Ghana. Preliminary results show an excellent diagnostic sensitivity of >95%.
Evaluation of the COBAS TaqMan HCV Test with Automated Sample Processing Using the MagNA Pure LC Instrument
Authors: Germer Jeffrey J. , Harmsen W. Scott , Mandrekar Jayawant N. , Mitchell P. Shawn , Yao Joseph D. C. 
Published: January 2005

ABSTRACT

The COBAS TaqMan HCV Test (TaqMan HCV; Roche Molecular Systems Inc., Branchburg, N.J.) for hepatitis C virus (HCV) performed on the COBAS TaqMan 48 Analyzer (Roche Molecular Systems) currently relies on a manual sample processing method. Implementation of an automated sample processing method would facilitate the clinical use of this test. In this study, we evaluated the performance characteristics of TaqMan HCV following automated sample processing by the MagNA Pure LC instrument (MP; Roche Applied Science, Indianapolis, Ind.). The analytical sensitivity of TaqMan HCV following sample processing by MP was 8.1 IU/ml (95% confidence interval, 6.1 to 15.2). The assay showed good linearity (
R
2
= 0.99) across a wide range of HCV RNA levels (25 to 5 × 10
6
IU/ml), with coefficients of variation ranging from 10% to 46%. Among 83 clinical specimens, the sensitivity and specificity of TaqMan HCV were 100% and 95%, respectively, when compared to the COBAS AMPLICOR hepatitis C virus test, version 2.0 (COBAS AMPLICOR; Roche Molecular Systems), with TaqMan HCV detecting two more HCV RNA-positive specimens than COBAS AMPLICOR. Both specimens were confirmed to be HCV RNA positive by the VERSANT HCV RNA qualitative test (Bayer HealthCare LLC, Tarrytown, N.Y.). There was also strong correlation (
R
2
= 0.95) and good agreement between the results from TaqMan HCV and the VERSANT HCV RNA 3.0 assay (bDNA) (Bayer HealthCare LLC) among a group of 93 clinical specimens. The MP is a versatile, labor-saving sample processing platform suitable for reliable performance of TaqMan HCV.
Epidemiology ofCampylobacter jejuniIsolated from Patients with Guillain-Barré and Fisher Syndromes in Japan
Authors: Takahashi Masaki , Koga Michiaki , Yokoyama Keiko , Yuki Nobuhiro 
Published: January 2005

ABSTRACTCampylobacter jejuniisolation is the standard for the diagnosis of this type of bacterial infection, but there have been no epidemiological studies of a large number ofC. jejuniisolates from patients with Guillain-Barré syndrome (GBS) and Fisher syndrome (FS). For 13 years, stool specimens from GBS/FS patients have been sent from 378 hospitals throughout Japan to the Tokyo Metropolitan Institute of Public Health. A total of 113 strains (11%) were isolated from the stool specimens from 1,049 patients. The isolation rate did not differ by region. The rates were 22% for 449 patients with a history of diarrhea and 2% for the others. An additional 18 isolates were provided by various hospitals. There was no noticeable seasonal distribution in the onset ofC. jejuniisolated from patients with GBS/FS. The male/female ratios were 1.7:1 for GBS and 2.2:1 for FS. The patient age range showed a peak in 10- to 30-year-old subjects who had GBS and in 10- to 20-year-old subjects who had FS. The predominance of young adults and male patients who hadC. jejuni-associated GBS/FS may be related to the preponderance of young adults and male patients who hadC. jejunienteritis. The median interval from diarrhea onset to neurologic symptom onset was 10 days for GBS/FS. Penner'sC. jejuniserotype HS:19 was more frequently present in GBS (67%) than in enteritis (6%) patients. HS:2 was more frequent in FS (41%) than in enteritis (14%) patients. These findings suggest that certainC. jejunistrains specifically trigger GBS and that others specifically trigger FS.
Molecular Epidemiology of Respiratory Syncytial Virus Infections among Children with Acute Respiratory Symptoms in a Community over Three Seasons
Authors: Sato Mizuho , Saito Reiko , Sakai Takatsugu , Sano Yasuko , Nishikawa Makoto , Sasaki Asami , Shobugawa Yugo , Gejyo Fumitake , Suzuki Hiroshi 
Published: January 2005

ABSTRACT
To study the molecular epidemiology of respiratory syncytial virus (RSV) in a community, children with acute respiratory symptoms at a pediatric outpatient clinic in Niigata, Japan, were analyzed over three seasons from November 2001 to July 2004. Of 499 nasopharyngeal aspirate specimens, 185 (37.1%) were RSV positive, and only 8 (4.5%) of 177 patients were shown by the reverse transcription (RT)-PCR method to be reinfected. RSV infection occurred beginning in the early winter, and the rates declined in the spring. The predominant subgroup changed from A to B and returned to A over the three seasons. Phylogenetic analysis also revealed that multiple genotypes cocirculated each year, with genotype GA5 of subgroup A predominating in the 2001-2002 and the 2003-2004 seasons. A new genotype of subgroup B (named BA, according to the nomenclature for viruses) with a 60-nucleotide insertion in the second variable region of the attachment glycoportein protein was predominant as an emerging strain in the 2002-2003 season, but this was not associated with new epidemiological or clinical features, unlike the cases of disease caused by other genotypes in the other seasons. In conclusion, our molecular analysis of RSV confirms that multiple genotypes cocirculate each year and that the genotype predominating may shift with the season. Support for determination of the genotype by RT-PCR as an effective tool for characterization of RSV circulation patterns in the community is provided.
Aspergillus Hyphae in Infected Tissue: Evidence of Physiologic Adaptation and Effect on Culture Recovery
Authors: Tarrand Jeffrey J. , Han Xiang Y. , Kontoyiannis Dimitrios P. , May Gregory S. 
Published: January 2005

ABSTRACT

Microbiologic cultures of fungi are routinely incubated at ambient temperatures in room air, and the rate of recovery of
Aspergillus
species from clinical specimens is poor. Failure of current culture methods to mimic the physiologic temperature and low-oxygen environment found in hypha-laden infected tissue may underlie this poor recovery. Experiments were performed to compare the recovery of
Aspergillus
spp. incubated at 35°C in 6% O
2
-10% CO
2
with that at 25°C in room air. The samples tested included
Aspergillus
-infected tissue specimens from a dog model and human autopsies, experimental anaerobically stressed
Aspergillus
inocula, and 10,062 consecutive clinical specimens. Culture at 35°C in 6% O
2
-10% CO
2
significantly enhanced the recovery of
Aspergillus
spp. from the infected autopsy tissue samples. Incubation at 35°C alone resulted in approximately 10-fold-improved culture recovery from the experimentally stressed hyphae, and the 6% O
2
-10% CO
2
atmosphere independently favored growth under temperature-matched conditions. Finally, incubation at 35°C (in room air) improved the overall recovery of
Aspergillus
spp. from clinical specimens by 31%. Culture at 35°C in a microaerobic atmosphere significantly enhances the recovery of
Aspergillus
spp. from various sources.
Aspergillus
hyphae growing in infected tissue appear to be adapted to the physiologic temperature and hypoxic milieu.
Evaluation of Nested PCR in Diagnosis of Typhoid Fever
Authors: Prakash Pradyot , Mishra Om Prakash , Singh Alok Kumar , Gulati Anil Kumar , Nath Gopal 
Published: January 2005

ABSTRACT

In this study, nested PCR using
H1-d
primers, which is specific for
Salmonella enterica
serovar Typhi, was compared to blood culture and the single-tube Widal test. Results indicate that nested PCR can be used as a gold standard to determine the cutoff titer of the Widal test for diagnosis of typhoid fever.
Evaluation of an Accelerated Protocol for Detection of Extended-Spectrum β-Lactamase-Producing Gram-Negative Bacilli from Positive Blood Cultures
Authors: Navon-Venezia Shiri , Leavitt Azita , Ben-Ami Ronen , Aharoni Yuval , Schwaber Mitchell J. , Schwartz David , Carmeli Yehuda 
Published: January 2005

ABSTRACT
We evaluated a protocol for the accelerated detection of extended-spectrum β-lactamases (ESBLs) in gram-negative bloodstream pathogens. Two hundred eighty-three blood culture bottles were subjected to direct ESBL testing by inoculating samples directly from blood culture bottles onto agar plates containing cefotaxime and ceftazidime disks, with and without clavulanate. Standard ESBL testing in accordance with the NCCLS guidelines after subculturing on agar plates was performed in parallel. Results of the direct ESBL testing were reported 2.3 days sooner and were comparable to those of the standard NCCLS method with sensitivity, specificity, and positive and negative predictive values of 100, 98, 94, and 100%, respectively.
PCR Amplification with Primers Based on IS
2404
and GC-Rich Repeated Sequence Reveals Polymorphism in
Mycobacterium ulcerans
Authors: Ablordey Anthony , Kotlowski Roman , Swings Jean , Portaels Françoise 
Published: January 2005

ABSTRACT

We describe a simple genotyping method for
Mycobacterium ulcerans
based on PCR amplification of genomic regions between IS
2404
and a frequently repeated GC-rich sequence. Application of this method to a global collection produced 10
M. ulcerans
genotypes corresponding to their geographic origin.