bluuebunny/crossref_metadata_2025 huggingface dataset:
AphB Influences Acid Tolerance of
Vibrio vulnificus
by Activating Expression of the Positive Regulator CadC
Authors: Rhee Jee Eun , Jeong Hee Gon , Lee Jeong Hyun , Choi Sang Ho
Published: September 2006

A mutant of
Vibrio vulnificus
that was more sensitive to low pH was screened from a library of mutants constructed by random transposon mutagenesis. By use of a transposon-tagging method, an open reading frame encoding a LysR homologue, AphB, was identified and cloned from
V. vulnificus
. The deduced amino acid sequence of AphB from
V. vulnificus
was 80% identical to that reported from
V. cholerae
. A mutational analysis demonstrated that the gene product of
aphB
contributes to acid tolerance of
V. vulnificus
. The lysine decarboxylase activity and cellular level of the
cadA
transcript were decreased in the
aphB
mutant, indicating that AphB exerts its effect on the acid tolerance of
V. vulnificus
by enhancing the expression of
cadBA
. Western blot analyses demonstrated that the cellular level of CadC, a transcription activator of the
cadBA
operon, was significantly reduced by
aphB
mutation, and a primer extension analysis revealed that the
cadC
promoter (P

cadC

) activity was under the positive control of AphB. A direct interaction between AphB and the P

cadC

DNA was demonstrated by gel mobility shift assays. The AphB binding site mapped by deletion analyses of the P

cadC

regulatory region and confirmed by a DNase I protection assay was centered at the 61.5 bp upstream of the transcription start site. Accordingly, these results demonstrate that AphB and CadC function sequentially in a regulatory cascade to activate
cadBA
expression and that AphB activates the expression of
cadC
by directly binding to an upstream region of P

cadC

.
Inactivation of Rv2525c, a Substrate of the Twin Arginine Translocation (Tat) System of
Mycobacterium tuberculosis
, Increases β-Lactam Susceptibility and Virulence
Authors: Saint-Joanis Brigitte , Demangel Caroline , Jackson Mary , Brodin Priscille , Marsollier Laurent , Boshoff Helena , Cole Stewart T.
Published: September 2006

The twin arginine translocation (Tat) system is used by many bacteria to export fully folded proteins containing cofactors. Here, we show genetically that this system is essential for
Mycobacterium tuberculosis
, as the
tatAC
operon and
tatB
genes could be inactivated only in partially diploid strains. Using comparative genomics, the rv2525c gene of
M. tuberculosis
was identified as encoding a histidine-rich protein, with a twin arginine signal peptide, and orthologous genes were shown to be present in several but not all actinobacterial species. Conservation of this gene by
Mycobacterium leprae
, which has undergone reductive evolution, suggested an important role for rv2525c. An rv2525c knockout mutant was constructed, and biochemical analysis indicated that the mature Rv2525c protein is secreted. Upon exposure to antituberculous drugs, rv2525c expression is significantly up-regulated together with those of other genes involved in cell wall biogenesis. Phenotypic comparison of the mutant with the parental strain revealed an increase in susceptibility to some β-lactam antibiotics and, despite slower growth in vitro, enhanced virulence in both cellular and murine models of tuberculosis. The Tat system thus contributes in multiple ways to survival of the tubercle bacillus.
Expression and Assembly of a Functional Type IV Secretion System Elicit Extracytoplasmic and Cytoplasmic Stress Responses inEscherichia coli
Authors: Zahrl Doris , Wagner Maria , Bischof Karin , Koraimann Günther
Published: September 2006

Conditions perturbing protein homeostasis are known to induce cellular stress responses in prokaryotes and eukaryotes. Here we show for the first time that expression and assembly of a functional type IV secretion (T4S) machinery elicit extracytoplasmic and cytoplasmic stress responses inEscherichia coli. After induction of T4S genes by a nutritional upshift and assembly of functional DNA transporters encoded by plasmid R1-16, host cells activated the CpxAR envelope stress signaling system, as revealed by induction or repression of downstream targets of the CpxR response regulator. Furthermore, we observed elevated transcript levels of cytoplasmic stress genes, such asgroESL, with a concomitant increase of σ32protein levels in cells expressing T4S genes. AtraAnull mutant of plasmid R1-16, which lacks the functional gene encoding the major pilus protein pilin, showed distinctly reduced stress responses. These results corroborated our conclusion that the activation of bacterial stress networks was dependent on the presence of functional T4S machinery. Additionally, we detected increased transcription from therpoHp1promoter in the presence of an active T4S system. Stimulation ofrpoHp1was dependent on the presence of CpxR, suggesting a hitherto undocumented link between CpxAR and σ32-regulated stress networks.
Properties of Succinyl-Coenzyme A:d-Citramalate Coenzyme A Transferase and Its Role in the Autotrophic 3-Hydroxypropionate Cycle ofChloroflexus aurantiacus
Authors: Friedmann Silke , Alber Birgit E. , Fuchs Georg
Published: September 2006

The phototrophic bacteriumChloroflexus aurantiacususes the 3-hydroxypropionate cycle for autotrophic CO2fixation. This cycle starts with acetyl-coenzyme A (CoA) and produces glyoxylate. Glyoxylate is an unconventional cell carbon precursor that needs special enzymes for assimilation. Glyoxylate is combined with propionyl-CoA to β-methylmalyl-CoA, which is converted to citramalate. Cell extracts catalyzed the succinyl-CoA-dependent conversion of citramalate to acetyl-CoA and pyruvate, the central cell carbon precursor. This reaction is due to the combined action of enzymes that were upregulated during autotrophic growth, a coenzyme A transferase with the use of succinyl-CoA as the CoA donor and a lyase cleaving citramalyl-CoA to acetyl-CoA and pyruvate. Genomic analysis identified a gene coding for a putative coenzyme A transferase. The gene was heterologously expressed inEscherichia coliand shown to code for succinyl-CoA:d-citramalate coenzyme A transferase. This enzyme, which catalyzes the reactiond-citramalate + succinyl-CoA →d-citramalyl-CoA + succinate, was purified and studied. It belongs to class III of the coenzyme A transferase enzyme family, with an aspartate residue in the active site. The homodimeric enzyme composed of 44-kDa subunits was specific for succinyl-CoA as a CoA donor but also acceptedd-malate and itaconate instead ofd-citramalate. The CoA transferase gene is part of a cluster of genes which are cotranscribed, including the gene ford-citramalyl-CoA lyase. It is proposed that the CoA transferase and the lyase catalyze the last two steps in the glyoxylate assimilation route.
Control of Enterotoxin Gene Expression in
Bacillus cereus
F4430/73 Involves the Redox-Sensitive ResDE Signal Transduction System
Authors: Duport Catherine , Zigha Assia , Rosenfeld Eric , Schmitt Philippe
Published: September 2006

In contrast to
Bacillus subtilis
, the role of the two-component regulatory system ResDE has not yet been investigated in the facultative anaerobe
Bacillus cereus
. We examined the role of ResDE in the food-borne pathogen
B. cereus
F4430/73 by constructing
resDE
and
resE
mutants. Growth performances, glucose metabolism, and expression of hemolysin BL (Hbl) and nonhemolytic enterotoxin (Nhe) were analyzed in the three strains under distinct oxygenation and extracellular oxidoreduction potential (ORP) conditions. We show that growth and glucose metabolism were only moderately perturbed in both
resDE
and
resE
mutants under aerobiosis, microaerobiosis, and anaerobiosis generated under N
2
atmosphere (initial ORP = +45 mV). The major effects of
resDE
and
resE
mutations were observed under low-ORP anaerobic conditions generated under hydrogen atmosphere (iORP = −148 mV). These conditions normally favor enterotoxin production in the wild type. The
resE
mutation was more deleterious to the cells than the
resDE
mutation, causing growth limitation and strong deregulation of key catabolic genes. More importantly, the
resE
mutation abolished the production of enterotoxins under all of the conditions examined. The
resDE
mutation only decreased enterotoxin expression under anaerobiosis, with a more pronounced effect under low-ORP conditions. Thus, the ResDE system was found to exert major control on both fermentative growth and enterotoxin expression, and it is concluded that the ResDE system of
B. cereus
should be considered an anaerobic redox regulator. The data presented also provide evidence that the ResDE-dependent regulation of enterotoxins might function at least partially independently of the pleiotropic virulence gene regulator PlcR.
Intraspecific Variability of the Terminal Inverted Repeats of the Linear Chromosome of
Streptomyces ambofaciens
Authors: Choulet Frédéric , Gallois Alexandre , Aigle Bertrand , Mangenot Sophie , Gerbaud Claude , Truong Chantal , Francou François-Xavier , Borges Frédéric , Fourrier Céline , Guérineau Michel , Decaris Bernard , Barbe Valérie , Pernodet Jean-Luc , Leblond Pierre
Published: September 2006

The sequences of the terminal inverted repeats (TIRs) ending the linear chromosomal DNA of two
Streptomyces ambofaciens
strains, ATCC23877 and DSM40697 (198 kb and 213 kb, respectively), were determined from two sets of recombinant cosmids. Among the 215 coding DNA sequences (CDSs) predicted in the TIRs of strain DSM40697, 65 are absent in the TIRs of strain ATCC23877. Reciprocally, 45 of the 194 predicted CDSs are specific to the ATCC23877 strain. The strain-specific CDSs are located mainly at the terminal end of the TIRs. Indeed, although TIRs appear almost identical over 150 kb (99% nucleotide identity), large regions of DNA of 60 kb (DSM40697) and 48 kb (ATCC23877), mostly spanning the ends of the chromosome, are strain specific. These regions are rich in plasmid-associated genes, including genes encoding putative conjugal transfer functions. The strain-specific regions also share a G+C content (68%) lower than that of the rest of the genome (from 71% to 73%), a percentage that is more typical of
Streptomyces
plasmids and mobile elements. These data suggest that exchanges of replicon extremities have occurred, thereby contributing to the terminal variability observed at the intraspecific level. In addition, the terminal regions include many mobile genetic element-related genes, pseudogenes, and genes related to adaptation. The results give insight into the mechanisms of evolution of the TIRs: integration of new information and/or loss of DNA fragments and subsequent homogenization of the two chromosomal extremities.
FliT Acts as an Anti-FlhD
2
C
2
Factor in the Transcriptional Control of the Flagellar Regulon in
Salmonella enterica
Serovar Typhimurium
Authors: Yamamoto Shouji , Kutsukake Kazuhiro
Published: September 2006

Flagellar operons are divided into three classes with respect to their transcriptional hierarchy in
Salmonella enterica
serovar Typhimurium. The class 1 gene products FlhD and FlhC act together in an FlhD
2
C
2
heterotetramer, which binds upstream of the class 2 promoters to facilitate binding of RNA polymerase. Class 2 expression is known to be enhanced by a disruption mutation in a flagellar gene,
fliT
. In this study, we purified FliT protein in a His-tagged form and showed that the protein prevented binding of FlhD
2
C
2
to the class 2 promoter and inhibited FlhD
2
C
2
-dependent transcription. Pull-down and far-Western blotting analyses revealed that the FliT protein was capable of binding to FlhD
2
C
2
and FlhC and not to FlhD alone. We conclude that FliT acts as an anti-FlhD
2
C
2
factor, which binds to FlhD
2
C
2
through interaction with the FlhC subunit and inhibits its binding to the class 2 promoter.
Signal Transduction Genes Required for Heterocyst Maturation in
Anabaena
sp. Strain PCC 7120
Authors: Fan Qing , Lechno-Yossef Sigal , Ehira Shigeki , Kaneko Takakazu , Ohmori Masayuki , Sato Naoki , Tabata Satoshi , Wolk C. Peter
Published: September 2006

How heterocyst differentiation is regulated, once particular cells start to differentiate, remains largely unknown. Using near-saturation transposon mutagenesis and testing of transposon-tagged loci, we identified three presumptive regulatory genes not previously recognized as being required specifically for normal heterocyst maturation. One of these genes has a hitherto unreported mutant phenotype. Two previously identified regulatory genes were further characterized.
Genetic Variability in the G Protein Gene of Group A and B Respiratory Syncytial Viruses from India
Authors: Parveen Shama , Sullender Wayne M. , Fowler Karen , Lefkowitz Elliot J. , Kapoor Suresh Kumar , Broor Shobha
Published: September 2006

Respiratory syncytial virus (RSV) is the most commonly identified viral agent of acute respiratory tract infection (ARI) of young children and causes repeat infections throughout life. Limited data are available on the molecular epidemiology of RSV from developing countries, including India. This study reports on the genetic variability in the glycoprotein G gene among RSV isolates from India. Reverse transcription-PCR for a region of the RSV G protein gene was done with nasopharyngeal aspirates (NPAs) collected in a prospective longitudinal study in two rural villages near Delhi and from children with ARI seen in an urban hospital. Nucleotide sequence comparisons among 48 samples demonstrated a higher prevalence of group A (77%) than group B (23%) RSV isolates. The level of genetic variability was higher among the group A viruses (up to 14%) than among the group B viruses (up to 2%). Phylogenetic analysis revealed that both the GA2 and GA5 group A RSV genotypes were prevalent during the 2002-2003 season and that genotype GA5 was predominant in the 2003-2004 season, whereas during the 2004-2005 season both genotype GA5 and genotype BA, a newly identified group B genotype, cocirculated in almost equal proportions. Comparison of the nonsynonymous mutation-to-synonymous mutation ratios (
dN
/
dS
) revealed greater evidence for selective pressure between the GA2 and GA5 genotypes (
dN
/
dS
, 1.78) than within the genotypes (
dN
/
dS
, 0.69). These are among the first molecular analyses of RSV strains from the second most populous country in the world and will be useful for comparisons to candidate vaccine strains.
Differential In Vitro Expression of the
brkA
Gene in
Bordetella pertussis
and
Bordetella parapertussis
Clinical Isolates
Authors: Stefanelli Paola , Sanguinetti Maurizio , Fazio Cecilia , Posteraro Brunella , Fadda Giovanni , Mastrantonio Paola
Published: September 2006

In this study, we set up a real-time reverse transcriptase PCR assay to measure the relative amounts of
brkA
transcripts in 50
Bordetella
isolates. The results suggested that
brkA
expression is strain dependent and its level may play a role in determining the serum resistance or susceptibility phenotype. Pertussis immunocompetent sera were unable to kill
Bordetella parapertussis
via complement deposition.
Use of the Phoenix Automated System for Identification of
Streptococcus
and
Enterococcus
spp
Authors: Brigante Gioconda , Luzzaro Francesco , Bettaccini Alessia , Lombardi Gianluigi , Meacci Francesca , Pini Beatrice , Stefani Stefania , Toniolo Antonio
Published: September 2006

The Phoenix system (Becton Dickinson Diagnostic Systems, Sparks, MD) was evaluated for identification (ID) to the species level of streptococci and enterococci. Two hundred clinical isolates were investigated: beta-hemolytic streptococci (
n
= 50),
Streptococcus pneumoniae
organisms (
n
= 46), viridans group streptococci (
n
= 31),
Enterococcus faecium
(
n
= 36),
Enterococcus faecalis
(
n
= 25), and other catalase-negative cocci (
n
= 12). The API system (bioMérieux, Marcy l'Étoile, France) was used as a comparator. Molecular methods (sequencing of 16S rRNA and
zwf
and
gki
genes and
ddl
gene amplification) were used to investigate discordant results. Upon resolution of discrepancies, correct species ID was achieved by the Phoenix system for 121/129 (93.8%) streptococci and 63/70 (90.0%) enterococci. Excellent results were obtained for
S. pneumoniae
(45/45) and beta-hemolytic streptococci (49/50). With regard to viridans streptococci, the accuracy of the Phoenix system was 83.9%. Among the latter organisms, the best performance was obtained with isolates of the
Streptococcus sanguinis
group and
Streptococcus anginosus
group; problems were instead encountered with the
Streptococcus mitis
group. Four
E. faecium
and three
E. faecalis
isolates were misidentified as
Enterococcus casseliflavus/Enterococcus gallinarum
or
Enterococcus durans
. Thus, these isolates were identified only at the genus level. Compared with commercially available systems, the Phoenix system appears a reliable diagnostic tool for identifying clinically relevant streptococci and enterococci. The SMIC/ID-2 panel proved particularly effective for beta-hemolytic streptococci and pneumococci.
Relevance of Influenza A Virus Detection by PCR, Shell Vial Assay, and Tube Cell Culture to Rapid Reporting Procedures
Authors: Zitterkopf Nicole L. , Leekha Surbhi , Espy Mark J. , Wood Christina M. , Sampathkumar Priya , Smith Thomas F.
Published: September 2006

Influenza A virus was detected at higher rates and for more extended time periods with real-time PCR than with cell cultures. We show here that, using the theranostic approach, rapid viral detection and reporting can provide for early implementation and assessment of available antiviral therapy.
Molecular Epidemiology of Oral Treponemes in Patients with Periodontitis and in Periodontitis-Resistant Subjects
Authors: Moter Annette , Riep Birgit , Haban Vesna , Heuner Klaus , Siebert Gerda , Berning Moritz , Wyss Chris , Ehmke Benjamin , Flemmig Thomas F. , Göbel Ulf B.
Published: September 2006

The etiologic role of oral treponemes in human periodontitis is still under debate. Although seen by dark-field microscopy in large numbers, their possible role is still unclear since they comprise some 60 different phylotypes, most of which are still uncultured. To determine their status as mere commensals or opportunistic pathogens, molecular epidemiological studies are required that include both cultured and as-yet-uncultured organisms. Here we present such data, comparing treponemal populations from chronic periodontitis (CP) or generalized aggressive periodontitis (GAP) patients. As a periodontitis-resistant (PR) control group, we included elderly volunteers with more than 20 natural teeth and no history of periodontal treatment and no or minimal clinical signs of periodontitis. Almost every treponemal phylotype was present in all three groups. For most treponemes, the proportion of subjects positive for a certain species or phylotype was higher in both periodontitis groups than in the PR group. This difference was pronounced for treponemes of the phylogenetic groups II and IV and for
Treponema socranskii
and
Treponema lecithinolyticum
. Between the periodontitis groups the only significant differences were seen for
T. socranskii
and
T. lecithinolyticum
, which were found more often in periodontal pockets of GAP patients than of CP patients. In contrast, no difference was found for
Treponema denticola
. Our findings, however, strengthen the hypothesis of treponemes being opportunistic pathogens. It appears that
T. socranskii
,
T. lecithinolyticum
and group II and IV treponemes may represent good indicators for periodontitis and suggest the value of the respective probes for microbiological diagnosis in periodontitis subjects.
Rapid Identification of Ascomycetous Yeasts from Clinical Specimens by a Molecular Method Based on Flow Cytometry and Comparison with Identifications from Phenotypic Assays
Authors: Page Brent T. , Shields Christine E. , Merz William G. , Kurtzman Cletus P.
Published: September 2006

This study was designed to compare the identification of ascomycetous yeasts recovered from clinical specimens by using phenotypic assays (PA) and a molecular flow cytometric (FC) method. Large-subunit rRNA domains 1 and 2 (D1/D2) gene sequence analysis was also performed and served as the reference for correct strain identification. A panel of 88 clinical isolates was tested that included representatives of nine commonly encountered species and six infrequently encountered species. The PA included germ tube production, fermentation of seven carbohydrates, morphology on corn meal agar, urease and phenoloxidase activities, and carbohydrate assimilation tests when needed. The FC method (Luminex) employed species-specific oligonucleotides attached to polystyrene beads, which were hybridized with D1/D2 amplicons from the unidentified isolates. The PA identified 81 of 88 strains correctly but misidentified 4 of
Candida dubliniensis
, 1 of
C. bovina
, 1 of
C. palmioleophila
, and 1 of
C. bracarensis
. The FC method correctly identified 79 of 88 strains and did not misidentify any isolate but did not identify nine isolates because oligonucleotide probes were not available in the current library. The FC assay takes approximately 5 h, whereas the PA takes from 2 h to 5 days for identification. In conclusion, PA did well with the commonly encountered species, was not accurate for uncommon species, and takes significantly longer than the FC method. These data strongly support the potential of FC technology for rapid and accurate identification of medically important yeasts. With the introduction of new antifungals, rapid, accurate identification of pathogenic yeasts is more important than ever for guiding antifungal chemotherapy.
Abdominal Aortic Aneurysm Infected by
Yersinia pseudotuberculosis
Authors: Hadou Tahar , Elfarra Mazen , Alauzet Corentine , Guinet Françoise , Lozniewski Alain , Lion Christine
Published: September 2006

Infected aneurysms caused by
Yersinia
are very uncommon and are principally due to
Yersinia enterocolitica
. We describe the first case of an infected aneurysm caused by
Yersinia pseudotuberculosis
in an elderly patient with a history of atherosclerotic cardiovascular disease.
Diagnostic Value of Reverse Transcription-PCR for Detection of Cytomegalovirus pp67 in Samples from Solid-Organ Transplant Recipients
Authors: Meyer-Koenig Ursula , Romberg Ilona , Schneider Kerstin , Weidmann Manfred , Kern Winfried V. , Hufert Frank T.
Published: September 2006

We evaluated a highly sensitive quantitative real-time one-step reverse transcription-PCR (RT-PCR) for detection of human cytomegalovirus pp67 transcripts in monitoring of solid-organ transplant recipients. Results were compared with those of pp65 antigen testing and quantitative DNA-PCR. Due to a low clinical sensitivity, the pp67 RT-PCR was not able to replace these assays.
rpoB
Gene Mutations and Molecular Characterization of Rifampin-Resistant
Mycobacterium tuberculosis
Isolates from Shandong Province, China
Authors: Ma Xin , Wang Haiying , Deng Yunfeng , Liu Zhimin , Xu Yong , Pan Xi , Musser James M. , Graviss Edward A.
Published: September 2006

Sixty rifampin (RIF)-resistant and 75 RIF-susceptible
Mycobacterium tuberculosis
isolates from Shandong Province, China, were analyzed for
rpoB
gene mutations and genotyped. Mycobacterial interspersed repetitive unit (MIRU) genotype 223325173533 was overrepresented among RIF-resistant isolates. MIRU combined with IS
6110
restriction fragment length polymorphism analysis as the second-line genotyping method may reflect epidemiologic links more reliably than each method alone.
Evaluation of the SPF
10
-INNO LiPA Human Papillomavirus (HPV) Genotyping Test and the Roche Linear Array HPV Genotyping Test
Authors: van Hamont Dennis , van Ham Maaike A. P. C. , Bakkers Judith M. J. E. , Massuger Leon F. A. G. , Melchers Willem J. G.
Published: September 2006

The need for accurate genotyping of human papillomavirus (HPV) infections is becoming increasingly important, since (i) the oncogenic potential among the high-risk HPV genotypes varies in the pathogenesis of cervical cancer, (ii) monitoring multivalent HPV vaccines is essential to investigate the efficiency of the vaccines, and (iii) genotyping is crucial in epidemiologic studies evaluating HPV infections worldwide. Various genotyping assays have been developed to meet this demand. Comparison of different studies that use various HPV genotyping tests is possible only after a performance assessment of the different assays. In the present study, the SPF
10
LiPA version 1 and the recently launched Roche Linear Array HPV genotyping assays are compared. A total of 573 liquid-based cytology samples were tested for the presence of HPV by a DNA enzyme immunoassay; 210 were found to be positive for HPV DNA and were evaluated using both genotyping assays (163 with normal cytology, 22 with atypical squamous cells of undetermined significance, 20 with mild/moderate dysplasia, and 5 with severe dysplasia). Comparison analysis was limited to the HPV genotype probes common to both assays. Of the 160 samples used for comparison analysis, 129 (80.6%) showed absolute agreement between the assays (concordant), 18 (11.2%) showed correspondence for some but not all genotypes detected on both strips (compatible), and the remaining 13 (8.2%) samples did not show any similarity between the tests (discordant). The overall intertest comparison agreement for all individually detectable genotypes was considered very good (κ value, 0.79). The genotyping assays were therefore highly comparable and reproducible.
Molecular Characterization of Non-Penicillin-Susceptible
Streptococcus pneumoniae
in Norway
Authors: Sogstad Maren K. R. , Høiby E. Arne , Caugant Dominique A.
Published: September 2006

A total of 125 non-penicillin-susceptible
Streptococcus pneumoniae
isolates were received at the Norwegian Institute of Public Health in the period from 1995 to 2001. The strains were tested for antimicrobial susceptibility, serotyped, and genotyped by multilocus sequence typing (MLST); and their penicillin-binding proteins (PBPs) were typed by restriction fragment length polymorphism analysis of their
pbp
genes. Of the 125 strains, 48 (38%) were fully resistant to penicillin and 77 (62%) were intermediately resistant to penicillin. Most of the strains resistant to penicillin were also resistant to one or several additional antibiotics. The most frequent serotypes among the non-penicillin-susceptible strains were 14, 9V, 19F, 23F, and 6B. MLST analysis showed a high degree of genetic diversity among the 119 strains tested, with a total of 74 different sequence types. Six of the 26 internationally known resistant clones were present; the Spain
9V
-3 clone was the most frequent, with 19 isolates. A total of 74 (62%) of the isolates were related to 1 of the 26 international clones. Restriction enzyme analyses of the
pbp1a
,
pbp2b
, and
pbp2x
genes revealed 12, 12, and 19 different patterns, respectively; and a total of 43 different PBPs types were demonstrated. Our data indicate that the non-penicillin-susceptible strains in Norway are highly diverse genetically and that limited spread of the internationally known resistant strains occurred in the country in the period examined.
Highly Effective Detection of Human Papillomavirus 16 and 18 DNA by a Testing Algorithm Combining Broad-Spectrum and Type-Specific PCR
Authors: van Doorn Leen-Jan , Molijn Anco , Kleter Bernhard , Quint Wim , Colau Brigitte
Published: September 2006

The use of a single broad-spectrum human papillomavirus (HPV) DNA-based PCR test may fail to detect lower concentrations of HPV DNA due to competition between different genotypes in mixed infections. To improve HPV detection by PCR, broad-spectrum and type-specific (TS) PCRs were combined, with a focus on HPV-16 and HPV-18. Cervical and cervicovaginal cell samples were obtained from 1,113 healthy women (age range, 15 to 25 years) participating in an HPV-16/HPV-18 candidate vaccine efficacy trial. These samples were tested by a broad-spectrum SPF
10
PCR-DNA enzyme immunoassay, followed by a primer SPF
10
-based line probe assay (SPF
10
LiPA), and HPV-16- and HPV-18-TS PCRs. The results for the majority of the HPV-16/18 SPF
10
LiPA-positive samples were confirmed by TS-PCR (kappa values, 0.775 for HPV-16 and 0.785 for HPV-18). However, TS PCR revealed additional positive samples among those that contained other HPV genotypes due to competition. Conversely, SPF
10
LiPA identified HPV-16 or -18 in samples that remained negative by TS PCR as a result of sampling variation. Analysis of follow-up samples from more than 1,000 women confirmed that the combination of SPF
10
-LiPA with additional HPV-16- and HPV-18-TS PCR diminishes the rate of false-negative diagnosis. The combination of broad-spectrum and TS PCRs resulted in a novel testing algorithm. This combination of assays is more accurate than either method alone, and the novel algorithm offers a highly accurate and effective method for the analysis of HPV infections.
Characterization of
Bacillus cereus
Isolates Associated with Fatal Pneumonias: Strains Are Closely Related to
Bacillus anthracis
and Harbor
B. anthracis
Virulence Genes
Authors: Hoffmaster Alex R. , Hill Karen K. , Gee Jay E. , Marston Chung K. , De Barun K. , Popovic Tanja , Sue David , Wilkins Patricia P. , Avashia Swati B. , Drumgoole Rahsaan , Helma Charles H. , Ticknor Lawrence O. , Okinaka Richard T. , Jackson Paul J.
Published: September 2006

Bacillus cereus
is ubiquitous in nature, and while most isolates appear to be harmless, some are associated with food-borne illnesses, periodontal diseases, and other more serious infections. In one such infection,
B. cereus
G9241 was identified as the causative agent of a severe pneumonia in a Louisiana welder in 1994. This isolate was found to harbor most of the
B. anthracis
virulence plasmid pXO1 (13). Here we report the characterization of two clinical and one environmental
B. cereus
isolate collected during an investigation of two fatal pneumonia cases in Texas metal workers. Molecular subtyping revealed that the two cases were not caused by the same strain. However, one of the three isolates was indistinguishable from
B. cereus
G9241. PCR analysis demonstrated that both clinical isolates contained
B. anthracis
pXO1 toxin genes. One clinical isolate and the environmental isolate collected from that victim's worksite contained the
cap A
,
B
, and
C
genes required for capsule biosynthesis in
B. anthracis
. Both clinical isolates expressed a capsule; however, neither was composed of poly-
d
-glutamic acid. Although most
B. cereus
isolates are not opportunistic pathogens and only a limited number cause food-borne illnesses, these results demonstrate that some
B. cereus
strains can cause severe and even fatal infections in patients who appear to be otherwise healthy.
Clonal Relationship among
Vibrio cholerae
O1 El Tor Strains Causing the Largest Cholera Epidemic in Kenya in the Late 1990s
Authors: Scrascia M. , Maimone F. , Mohamud K. A. , Materu S. F. , Grimont F. , Grimont P. A. D. , Pazzani C.
Published: September 2006

Eighty
Vibrio cholerae
O1 strains selected to represent the 1998-to-1999 history of the largest cholera epidemic in Kenya were characterized by ribotyping, antimicrobial susceptibility, and random amplified polymorphic DNA patterns. Except for 19 strains from 4 local outbreaks in North Eastern Province along the Somalia border, the other 61 strains from 25 outbreaks occurring in districts scattered around the country were all ribotype B27 and resistant to chloramphenicol, spectinomycin, streptomycin, sulfamethoxazole, and trimethoprim. The 61 strains showed similar and specific amplified DNA patterns. These findings indicate that the predominant strains that caused the Kenyan epidemic had a clonal origin and suggest that ribotype B27 strains, which first appeared in West Africa in 1994, have had a rapid spread to eastern Africa.
Spread of Cholera with Newer Clones of
Vibrio cholerae
O1 El Tor, Serotype Inaba, in India
Authors: Dutta B. , Ghosh R. , Sharma N. C. , Pazhani G. P. , Taneja N. , Raychowdhuri A. , Sarkar B. L. , Mondal S. K. , Mukhopadhyay A. K. , Nandy R. K. , Bhattacharya M. K. , Bhattacharya S. K. , Ramamurthy T.
Published: September 2006

During 2004 and 2005, cholera was recorded in 15 states of India, with 7 outbreaks. The newly emerged
Vibrio cholerae
O1 Inaba had a different antibiogram and ribotype, different pulsotypes, and different mutations in the
wbeT
gene. Due to the absence of serogroup O139, the Inaba serotype may have acquired the potential to affect the population at large.
Development of an Internal Positive Control for Rapid Diagnosis of Avian Influenza Virus Infections by Real-Time Reverse Transcription-PCR with Lyophilized Reagents
Authors: Das Amaresh , Spackman Erica , Senne Dennis , Pedersen Jan , Suarez David L.
Published: September 2006

We developed an internal positive control (IPC) RNA to help ensure the accuracy of the detection of avian influenza virus (AIV) RNA by reverse transcription (RT)-PCR and real-time RT-PCR (RRT-PCR). The IPC was designed to have the same binding sites for the forward and reverse primers of the AIV matrix gene as the target amplicon, but it had a unique internal sequence used for the probe site. The amplification of the viral RNA and the IPC by RRT-PCR were monitored with two different fluorescent probes in a multiplex format, one specific for the AIV matrix gene and the other for the IPC. The RRT-PCR test was further simplified with the use of lyophilized bead reagents for the detection of AIV RNA. The RRT-PCR with the bead reagents was more sensitive than the conventional wet reagents for the detection of AIV RNA. The IPC-based RRT-PCR detected inhibitors in blood, kidney, lungs, spleen, intestine, and cloacal swabs, but not allantoic fluid, serum, or tracheal swabs The accuracy of RRT-PCR test results with the lyophilized beads was tested on cloacal and tracheal swabs from experimental birds inoculated with AIV and compared with virus isolation (VI) on embryonating chicken eggs. There was 97 to 100% agreement of the RRT-PCR test results with VI for tracheal swabs and 81% agreement with VI for cloacal swabs, indicating a high level of accuracy of the RRT-PCR assay. The same IPC in the form of armored RNA was also used to monitor the extraction of viral RNA and subsequent detection by RRT-PCR.
An Outbreak of Keratitis Caused by
Mycobacterium immunogenum
Authors: Sampaio Jorge Luiz Mello , Junior Doraldo Nassar , de Freitas Denise , Höfling-Lima Ana Luisa , Miyashiro Kozue , Alberto Fernando Lopes , Leão Sylvia Cardoso
Published: September 2006

From 8 October to 12 November 2003, 36 patients underwent surgical correction of myopia in a São Paulo, Brazil, clinic. Five patients had clinical signs of infectious keratitis, and a
Mycobacterium
species with previously unreported patterns determined by PCR restriction enzyme analysis of the
hsp65
gene and PCR restriction enzyme analysis of the 16S-23S rRNA internal transcribed spacer (ITS) was isolated from corneal scrapings from four of these patients. Subsequent evaluation by phenotypic tests and partial sequencing of the
hsp65
,
sodA
,
rpoB
, and 16S rRNA genes and the ITS supported the species identification as a variant of
Mycobacterium immunogenum
. The source of infection was not determined. The outbreak was caused by a single clone, as evidenced by identical pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus-PCR profiles. This is the first report of an outbreak where this species was isolated from infected tissues.
Multicenter Evaluation of the Etest and Disk Diffusion Methods for Differentiating Daptomycin-Susceptible from Non-Daptomycin-Susceptible
Staphylococcus aureus
Isolates
Authors: Jevitt Laura A. , Thorne Grace M. , Traczewski Maria M. , Jones Ronald N. , McGowan John E. , Tenover Fred C. , Brown Steven D.
Published: September 2006

Daptomycin is a novel cyclic lipopeptide that is approved by the U.S. Food and Drug Administration for the treatment of complicated skin and skin structure infections associated with
Staphylococcus aureus
and other gram-positive pathogens and also staphylococcal bacteremia, including right-sided endocarditis. The Clinical and Laboratory Standards Institute (CLSI) established “susceptible-only” interpretive criteria for broth microdilution (BMD) and disk diffusion (DD) testing of daptomycin in 2005. However, a series of
S. aureus
isolates have been recovered with daptomycin MICs in the nonsusceptible range (i.e., MICs of >1 μg/ml). The objective of this study was to determine the ability of the Etest and DD methods to differentiate daptomycin-susceptible from nonsusceptible isolates of
S. aureus
compared to the results of the CLSI BMD reference method. There was a good correlation between Etest MIC results and the results of BMD among laboratories (
r
= 0.86 to 0.88), with 95.3% of the Etest MICs within a ±1 log
2
dilution of the BMD MIC result. A total of 92 of 102 (90.2%) non-daptomycin-susceptible isolates of
S. aureus
identified by BMD in two participating laboratories were also classified as nonsusceptible by Etest. However, the very major and major error rates reported by one of the participating laboratories were 13.5 and 4.0%, respectively, primarily due to the absence of an intermediate category. The DD method, however, did not reliably differentiate daptomycin-susceptible from non-daptomycin-susceptible isolates. In 2005, daptomycin disks were voluntarily removed from the market by Cubist Pharmaceuticals. The disk diffusion breakpoints were subsequently removed from the CLSI M100 standard in 2006.
Performance of Human Immunodeficiency Virus Type 1 gp41 Assays for Detecting Enfuvirtide (T-20) Resistance Mutations
Authors: Marcial Michelle , Lu Jing , Deeks Steven G. , Ziermann Rainer , Kuritzkes Daniel R.
Published: September 2006

The performance of a gp41 assay, created using reagents designed for use with the OpenGene DNA Sequencing System, was evaluated using a panel of plasma samples obtained from enfuvirtide-naive and -experienced human immunodeficiency virus type 1-infected subjects. Resulting sequence data were highly accurate compared to a “home brew” assay and clonal sequence analysis.
Diagnosis of Gastric Syphilis by Direct Immunofluorescence Staining and Real-Time PCR Testing
Authors: Chen Cheng-Yen , Chi Kai-Hua , George Robert W. , Cox David L. , Srivastava Amitabh , Rui Silva Mário , Carneiro Fátima , Lauwers Gregory Y. , Ballard Ronald C.
Published: September 2006

We report on a case of gastric syphilis in a patient with chronic dyspepsia. The diagnosis was established by serology and the demonstration of spirochetes in diffusely inflammed gastric mucosa by staining with a fluorescent monoclonal antibody specific for pathogenic treponemes and by the detection of specific treponemal DNA sequences by a real-time PCR.
Clinical Significance of Coagulase-Negative Staphylococci Recovered from Nonsterile Sites
Authors: Tan Thean Yen , Ng Siew Yong , Ng Wan Xing
Published: September 2006

Laboratory criteria were used to select coagulase-negative staphylococci isolated from nonsterile body sites for further investigation. Fifty-seven percent of the study isolates were clinically significant, predominantly causing community-acquired soft tissue infections. There were species-related differences in virulence, with 91% of
Staphylococcus lugdunensis
isolates clinically significant compared with 11% of
S. haemolyticus
isolates.
Differential Detection of
Entamoeba histolytica
,
Entamoeba dispar
, and
Entamoeba moshkovskii
by a Single-Round PCR Assay
Authors: Hamzah Zulhainan , Petmitr Songsak , Mungthin Mathirut , Leelayoova Saovanee , Chavalitshewinkoon-Petmitr Porntip
Published: September 2006

A single-round PCR assay was developed for detection and differential diagnosis of the three
Entamoeba
species found in humans,
Entamoeba moshkovskii
,
Entamoeba histolytica
, and
Entamoeba dispar
, that are morphologically identical as both cysts and trophozoites. A conserved forward primer was derived from the middle of the small-subunit rRNA gene, and reverse primers were designed from signature sequences specific to each of these three
Entamoeba
species. PCR generates a 166-bp product with
E. histolytica
DNA, a 752-bp product with
E. dispar
DNA, and a 580-bp product with
E. moshkovskii
DNA. Thirty clinical specimens were examined, and the species present were successfully detected and differentiated using this assay. It was possible to detect as little as 10 pg of
E. moshkovskii
and
E. histolytica
DNA, while for
E. dispar
the sensitivity was about 20 pg of DNA. Testing with DNA from different pathogens, including bacteria and other protozoa, confirmed the high specificity of the assay. We propose the use of this PCR assay as an accurate, rapid, and effective diagnostic method for the detection and discrimination of these three morphologically indistinguishable
Entamoeba
species in both routine diagnosis of amoebiasis and epidemiological surveys.
Fourier-Transform Infrared Spectroscopic Analysis Is a Powerful Tool for Studying the Dynamic Changes in
Staphylococcus aureus
Small-Colony Variants
Authors: Becker Karsten , Laham Nahed Al , Fegeler Wolfgang , Proctor Richard A. , Peters Georg , von Eiff Christof
Published: September 2006

Infections due to small-colony variants (SCVs) of
Staphylococcus aureus
in patients with chronic and recurrent infections are an emerging problem; however, studies with this subpopulation are hampered by the fact that SCVs may exhibit unstable phenotypes, making them difficult to study, particularly in broth media. In this study, two
S. aureus
sets comprising the (i) normal and the (ii) SCV phenotype (clonal with normal phenotype) recovered from clinical specimens, as well as (iii) corresponding site-directed mutants displaying the SCV phenotype (knockout of
hemB
) and (iv) their complemented mutants were examined by Fourier-transform infrared (FTIR) spectroscopy. Phenotypes were defined on solid and in broth media. Using first-derivative infrared spectra to calculate spectral distances, hierarchical clustering based on spectral information resulted in a dendrogram with clear discrimination between SCV and normal phenotypes. The SCVs gave an FTIR fingerprint that was easily recognizable and that was much closer to other SCVs than to their parent strains. This technique offers for the first time a noninvasive approach to investigate dynamic processes of reversion of SCVs to the normal phenotype and vice versa. Thus, FTIR spectroscopy allowed a rapid and reproducible tool for the examination of different subpopulations of
S. aureus
on solid and in broth media for diagnostic and research purposes.
Detection of Hepatitis B Virus (HBV) Genotype E Carried—Even in the Presence of High Titers of Anti-HBs Antibodies—by an Argentinean Patient of African Descent Who Had Received Vaccination against HBV
Authors: Mathet Verónica L. , Cuestas María L. , Ruiz Vanesa , Minassian María L. , Rivero Cintia , Trinks Julieta , Daleoso Graciela , León Liliana M. , Sala Andrea , Libellara Beatriz , Corach Daniel , Oubiña José R.
Published: September 2006

Genotype E hepatitis B virus (HBV) was detected in two Argentine sisters exhibiting an African mitochondrial lineage. One of them (who had been vaccinated against HBV) exhibited anti-HBs cocirculating antibodies without HBsAg escape mutants, while her unvaccinated sister showed a D144A HBsAg escape mutant without anti-HBs antibodies. Both sisters carried an unusual L209V substitution within HBsAg.
Phenotypic Detection of Carbapenem-Susceptible Metallo-β-Lactamase-Producing Gram-Negative Bacilli in the Clinical Laboratory
Authors: Franklin Clare , Liolios Lisa , Peleg Anton Y.
Published: September 2006

Rapid detection of metallo-β-lactamase (MBL)-producing gram-negative pathogens is critical to prevent their widespread dissemination. Thus far, no standardized phenotypic method is available, and previously reported techniques have poor sensitivity for detecting carbapenem-susceptible MBL-carrying isolates, an increasingly described phenomenon. We developed a phenotypic detection method using both a double-disk synergy test and a combined-disk test with imipenem and 292 μg EDTA on one agar plate. Genotypic confirmation was used for validation. Of the 134 clinical isolates, 84 were confirmed to carry an MBL. Of these, 51 (61%) were susceptible to at least one carbapenem, and 22 (26%) were isolated from blood. The phenotypic method correctly differentiated all MBL-producing isolates (sensitivity, 100%). Fifty-one of the 52 MBL-negative isolates were correctly differentiated (specificity, 98%). This study reports the validation of a simple and accurate MBL detection method that can be easily incorporated into the daily routine of a clinical laboratory. Early detection of MBL-carrying organisms, including those with susceptibility to carbapenems, is of paramount clinical importance, as it allows rapid initiation of strict infection control practices as well as therapeutic guidance for confirmed infection.
Real-Time PCR Assays for Detection of Bocavirus in Human Specimens
Authors: Lu Xiaoyan , Chittaganpitch Malinee , Olsen Sonja J. , Mackay Ian M. , Sloots Theo P. , Fry Alicia M. , Erdman Dean D.
Published: September 2006

The recently discovered human bocavirus (HBoV) is the first member of the family
Parvoviridae
, genus
Bocavirus
, to be potentially associated with human disease. Several studies have identified HBoV in respiratory specimens from children with acute respiratory disease, but the full spectrum of clinical disease and the epidemiology of HBoV infection remain unclear. The availability of rapid and reliable molecular diagnostics would therefore aid future studies of this novel virus. To address this, we developed two sensitive and specific real-time TaqMan PCR assays that target the HBoV NS1 and NP-1 genes. Both assays could reproducibly detect 10 copies of a recombinant DNA plasmid containing a partial region of the HBoV genome, with a dynamic range of 8 log units (10
1
to 10
8
copies). Eight blinded clinical specimen extracts positive for HBoV by an independent PCR assay were positive by both real-time assays. Among 1,178 NP swabs collected from hospitalized pneumonia patients in Sa Kaeo Province, Thailand, 53 (4.5%) were reproducibly positive for HBoV by one or both targets. Our data confirm the possible association of HBoV infection with pneumonia and demonstrate the utility of these real-time PCR assays for HBoV detection.
Comparison of API 20E and
invA
PCR for Identification of
Salmonella enterica
Isolates from Swine Production Units
Authors: Nucera Daniele M. , Maddox Carol W. , Hoien-Dalen Patricia , Weigel Ronald M.
Published: September 2006

API 20E and
invA
PCR were evaluated for the identification of
Salmonella enterica
isolates from swine farms. API 20E had the highest agreement with other tests at the 99.9% likelihood level. Both tests had 100% sensitivity and 96% specificity compared to 16S rRNA sequencing. Compared to serotyping, both tests had 96% sensitivity; specificity was 86% for API 20E and 79% for
invA
PCR.