bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the
cydAB
-Encoded Cytochrome
bd
Oxidase from
Mycobacterium smegmatis
Authors: Kana Bavesh D. , Weinstein Edward A. , Avarbock David , Dawes Stephanie S. , Rubin Harvey , Mizrahi Valerie
Published: July 2002

The
cydAB
genes from
Mycobacterium smegmatis
have been cloned and characterized. The
cydA
and
cydB
genes encode the two subunits of a cytochrome
bd
oxidase belonging to the widely distributed family of quinol oxidases found in prokaryotes. The
cydD
and
cydC
genes located immediately downstream of
cydB
encode a putative ATP-binding cassette-type transporter. At room temperature, reduced minus oxidized difference spectra of membranes purified from wild-type
M. smegmatis
displayed spectral features that are characteristic of the γ-proteobacterial type cytochrome
bd
oxidase. Inactivation of
cydA
or
cydB
by insertion of a kanamycin resistance marker resulted in loss of
d-
heme absorbance at 631 nm. The
d
-heme could be restored by transformation of the
M. smegmatis cyd
mutants with a replicating plasmid carrying the highly homologous
cydABDC
gene cluster from
Mycobacterium tuberculosis
. Inactivation of
cydA
had no effect on the ability of
M. smegmatis
to exit from stationary phase at 37 or 42°C. The growth rate of the
cydA
mutant was tested under oxystatic conditions. Although no discernible growth defect was observed under moderately aerobic conditions (9.2 to 37.5 × 10
2
Pa of pO
2
or 5 to 21% air saturation), the mutant displayed a significant growth disadvantage when cocultured with the wild type under extreme microaerophilia (0.8 to 1.7 × 10
2
Pa of pO
2
or 0.5 to 1% air saturation). These observations were in accordance with the two- to threefold increase in
cydAB
gene expression observed upon reduction of the pO
2
of the growth medium from 21 to 0.5% air saturation and with the concomitant increase in
d
-heme absorbance in spectra of membranes isolated from wild-type
M. smegmatis
cultured at 1% air saturation. Finally, the
cydA
mutant displayed a competitive growth disadvantage in the presence of the terminal oxidase inhibitor, cyanide, when cocultured with wild type at 21% air saturation in an oxystat. In conjunction with these findings, our results suggest that cytochrome
bd
is an important terminal oxidase in
M. smegmatis.
Novel Genes Affecting Urease Activity inActinobacillus pleuropneumoniae
Authors: Bossé Janine T. , Gilmour Hali D. , MacInnes Janet I.
Published: July 2002

Characterization of a series of urease-negative transposon mutations ofActinobacillus pleuropneumoniaerevealed that many of the mutants had insertions 2 to 4 kbp upstream of the urease gene cluster. A 5-kbp upstream region of DNA was sequenced and found to contain six open reading frames (ORFs) transcribed in the same orientation as the urease genes. As well, a partial ORF,apuR, 202 bp upstream of these six ORFs, is transcribed in the opposite orientation. The predicted product of this partial ORF shows homology with many members of the LysR family of transcriptional regulators. Five of the ORFs (cbiKLMQO) appear to form an operon encoding a putative nickel and cobalt periplasmic permease system. ThecbiMandcbiQgenes encode proteins that have sequence similarity with known cobalt transport membrane proteins, and thecbiOgene encodes a cobalt transport ATP-binding protein homologue. The product of thecbiKgene is predicted to be the periplasmic-binding-protein component of the system, though it does not show any sequence similarity with CbiN, the cobalt-binding periplasmic protein.Escherichia coliclones containing this putative transport operon together with the urease genes ofA. pleuropneumoniaewere urease positive when grown in unsupplemented Luria-Bertani broth, whereas a clone containing only the minimal urease gene cluster required the addition of high concentrations of NiCl2for full urease activity. This result supports the hypothesis that nickel is a substrate for this permease system. The sixth ORF,utp, appears to encode an integral membrane protein which has significant sequence identity with mammalian urea transport proteins, though its function inA. pleuropneumoniaeremains to be determined.
Recruitment of the
mecA
Gene Homologue of
Staphylococcus sciuri
into a Resistance Determinant and Expression of the Resistant Phenotype in
Staphylococcus aureus
Authors: Wu Shang Wei , de Lencastre Herminia , Tomasz Alexander
Published: July 2002

Strains of methicillin-resistant
Staphylococcus aureus
(MRSA) have become the most important causative agents of hospital-acquired diseases worldwide. The genetic determinant of resistance,
mecA
, is not a gene native to
S. aureus
but was acquired from an extraspecies source by an unknown mechanism. We recently identified a close homologue of this gene in isolates of
Staphylococcus sciuri
, a taxonomically primitive staphylococcal species recovered most frequently from rodents and primitive mammals. In spite of the close sequence similarity between the
mecA
homologue of
S. sciuri
and the antibiotic resistance determinant
mecA
of
S. aureus
,
S. sciuri
strains were found to be uniformly susceptible to β-lactam antibiotics. In an attempt to activate the apparently “silent”
mecA
gene of
S. sciuri
, a methicillin-resistant derivative, K1M200 (for which the MIC of methicillin is 200 μg/ml), was obtained through stepwise exposure of the parental strain
S. sciuri
K1 (methicillin MIC of 4 μg/ml) to increasing concentrations of methicillin. DNA sequencing of the
mecA
homologue from K1M200 revealed the introduction of a point mutation into the −10 consensus of the promoter: the replacement of a thymine residue at nucleotide 1577 in the susceptible strain K1 by adenine in the resistant strain K1M200, which was accompanied by a drastic increase in transcription rate and the appearance of a new protein that reacted with monoclonal antibody prepared against the penicillin-binding protein 2A (PBP2A), i.e., the gene product of
S. aureus mecA
. Transduction of
mecA
from K1M200 (cloned into a plasmid vector) into a methicillin-susceptible
S. aureus
mutant resulted in a significant increase of methicillin resistance (from a methicillin MIC of 4 μg/ml to 12 and up to 50 μg/ml), the appearance of a low-affinity PBP detectable by the fluorographic assay, and the production of a protein that reacted in a Western blot with monoclonal antibody to PBP2A. Antibiotic resistance and the protein products disappeared upon removal of the plasmid-borne
mecA
homologue. The observations support the proposition that the
mecA
homologue ubiquitous in the antibiotic-susceptible animal species
S. sciuri
may be an evolutionary precursor of the methicillin resistance gene
mecA
of the pathogenic strains of MRSA.
The Evolving Genome of
Salmonella enterica
Serovar Pullorum
Authors: Liu Gui-Rong , Rahn Andrea , Liu Wei-Qiao , Sanderson Kenneth E. , Johnston Randal N. , Liu Shu-Lin
Published: July 2002

Salmonella enterica
serovar Pullorum is a fowl-adapted bacterial pathogen that causes dysentery (pullorum disease). Host adaptation and special pathogenesis make
S. enterica
serovar Pullorum an exceptionally good system for studies of bacterial evolution and speciation, especially regarding pathogen-host interactions and the acquisition of pathogenicity. We constructed a genome map of
S. enterica
serovar Pullorum RKS5078, using I-
Ceu
I,
Xba
I,
Avr
II, and
Spe
I and Tn
10
insertions. Pulsed-field gel electrophoresis was employed to separate the large DNA fragments generated by the endonucleases. The genome is 4,930 kb, which is similar to most salmonellas
.
However, the genome of
S. enterica
serovar Pullorum RKS5078 is organized very differently from the majority of salmonellas, with three major inversions and one translocation. This extraordinary genome structure was seen in most
S. enterica
serovar Pullorum strains examined, with different structures in a minority of
S. enterica
serovar Pullorum strains. We describe the coexistence of different genome structures among the same bacteria as genomic plasticity. Through comparisons with
S. enterica
serovar Typhimurium, we resolved seven putative insertions and eight deletions ranging in size from 12 to 157 kb. The genomic plasticity seen among
S. enterica
serovar Pullorum strains supported our hypothesis about its association with bacterial evolution: a large genomic insertion (157 kb in this case) disrupted the genomic balance, and rebalancing by independent recombination events in individual lineages resulted in diverse genome structures. As far as the structural plasticity exists, the
S. enterica
serovar Pullorum genome will continue evolving to reach a further streamlined and balanced structure.
The
Escherichia coli
Cell Division Protein FtsW Is Required To Recruit Its Cognate Transpeptidase, FtsI (PBP3), to the Division Site
Authors: Mercer Keri L. N. , Weiss David S.
Published: July 2002

The bacterial cell division protein FtsW has been suggested to perform two functions: stabilize the FtsZ cytokinetic ring, and facilitate septal peptidoglycan synthesis by the transpeptidase FtsI (penicillin-binding protein 3). We show here that depleting
Escherichia coli
cells of FtsW had little effect on the abundance of FtsZ rings but abrogated recruitment of FtsI to potential division sites. Analysis of FtsW localization confirmed and extended these results; septal localization of FtsW required FtsZ, FtsA, FtsQ, and FtsL but not FtsI. Thus, FtsW is a late recruit to the division site and is essential for subsequent recruitment of its cognate transpeptidase FtsI but not for stabilization of FtsZ rings. We suggest that a primary function of FtsW homologues—which are found in almost all bacteria and appear to work in conjunction with dedicated transpeptidases involved in division, elongation, or sporulation—is to recruit their cognate transpeptidases to the correct subcellular location.
Analysis of the
Bacillus subtilis spoIIIJ
Gene and Its Paralogue Gene,
yqjG
Authors: Murakami Takako , Haga Koki , Takeuchi Michio , Sato Tsutomu
Published: July 2002

The
Bacillus subtilis spoIIIJ
gene, which has been proven to be vegetatively expressed, has also been implicated as a sporulation gene. Recent genome sequencing information in many organisms reveals that
spoIIIJ
and its paralogous gene,
yqjG
, are conserved from prokaryotes to humans. A homologue of SpoIIIJ/YqjG, the
Escherichia coli
YidC is involved in the insertion of membrane proteins into the lipid bilayer. On the basis of this similarity, it was proposed that the two homologues act as translocase for the membrane proteins. We studied the requirements for
spoIIIJ
and
yqjG
during vegetative growth and sporulation. In rich media, the growth of
spoIIIJ
and
yqjG
single mutants were the same as that of the wild type, whereas
spoIIIJ yqjG
double inactivation was lethal, indicating that together these
B. subtilis
translocase homologues play an important role in maintaining the viability of the cell. This result also suggests that SpoIIIJ and YqjG probably control significantly overlapping functions during vegetative growth.
spoIIIJ
mutations have already been established to block sporulation at stage III. In contrast, disruption of
yqjG
did not interfere with sporulation. We further show that high level expression of
spoIIIJ
during vegetative phase is dispensable for spore formation, but the sporulation-specific expression of
spoIIIJ
is necessary for efficient sporulation even at the basal level. Using green fluorescent protein reporter to monitor SpoIIIJ and YqjG localization, we found that the proteins localize at the cell membrane in vegetative cells and at the polar and engulfment septa in sporulating cells. This localization of SpoIIIJ at the sporulation-specific septa may be important for the role of
spoIIIJ
during sporulation.
Critical Regions of
secM
That Control Its Translation and Secretion and Promote Secretion-Specific
secA
Regulation
Authors: Sarker Shameema , Oliver Donald
Published: July 2002

SecA is an essential ATP-driven motor protein that binds to presecretory or membrane proteins and the translocon and promotes the translocation or membrane integration of these proteins.
secA
is subject to a protein secretion-specific form of regulation, whereby its translation is elevated during secretion-limiting conditions. A novel mechanism that promotes this regulation involves translational pausing within the gene upstream of
secA
,
secM
. The
secM
translational pause prevents formation of an RNA helix that normally blocks
secA
translational initiation. The duration of this pause is controlled by the rate of secretion of nascent SecM, which in turn depends on its signal peptide and a functional translocon. We characterized the atypical
secM
signal peptide and found that mutations within the amino-terminal region specifically affect the
secM
translational pause and
secA
regulation, while mutations in the hydrophobic core region affect SecM secretion as well as translational pausing and
secA
regulation. In addition, mutational analysis of the 3′ end of
secM
allowed us to identify a conserved region that is required to promote the translational pause that appears to be operative at the peptide level. Together, our results provide direct support for the
secM
translational pause model of
secA
regulation, and they pinpoint key sequences within
secM
that promote this important regulatory system.
Molecular Analysis of a Hospital Cafeteria-Associated Salmonellosis Outbreak Using Modified Repetitive Element PCR Fingerprinting
Authors: Johnson James R. , Clabots Connie , Azar Miguel , Boxrud David J. , Besser John M. , Thurn Joseph R.
Published: July 2002

A hospital cafeteria-associated outbreak of gastroenteritis due to
Salmonella enterica
serotype Infantis was retrospectively evaluated using modified repetitive element PCR (rep-PCR) fingerprinting with the ERIC2 and BOXA1R primers and computer-assisted gel analysis and dendrogram construction. Rep-PCR yielded objective between-cycler, same-strain similarity values of from 92% (composite fingerprints) to 96% (ERIC2 fingerprints). The 70
Salmonella
isolates (which included 19 serotype Infantis isolates from the hospital outbreak, 10 other serotype Infantis isolates, and 41 isolates representing 14 other serotypes) were resolved well to the serotype level with each of the three fingerprint types (ERIC2, BOXA1R, and composite). Rep-PCR typing uncovered several historical serotyping errors and provided presumptive serotype assignments for other isolates with incomplete or undetermined serotypes. Analysis of replicate fingerprints for each isolate, as generated on two different thermal cyclers, indicated that most of the seeming subserotype discrimination noted in single-cycler dendrograms actually represented assay variability, since it was not reproducible in combined-cycler dendrograms. Rep-PCR typing, which would have been able to identify the presence of the hospital-associated serotype Infantis outbreak after the second outbreak isolate, could be used as a simple surrogate for serotyping by clinical microbiology laboratories that are equipped for diagnostic PCR.
Quantitative, Fluorogenic Probe PCR Assay for Detection of Human Herpesvirus 8 DNA in Clinical Specimens
Authors: Stamey Felicia R. , Patel Mitesh M. , Holloway Brian P. , Pellett Philip E.
Published: July 2002

A quantitative, fluorescence-based PCR assay (TaqMan-based system) was developed for detection of human herpesvirus 8 (HHV-8) DNA in clinical specimens. Primers and probes chosen from each of five 10-kb segments from the unique region of the HHV-8 genome were evaluated for sensitivity with dilution series of DNA extracted from a cell line (BCBL-1) that harbors HHV-8 DNA. Although several of the primer-probe sets performed similarly with BCBL-1 DNA that had been diluted in water, their performance differed when target DNA was diluted in a constant background of uninfected cell DNA, an environment more relevant to their intended use. The two best primer-probe combinations were specific for HHV-8 relative to the other known human herpesviruses and herpesvirus saimiri, a closely related gammaherpesvirus of nonhuman primates. PCRs included an enzymatic digestion step to eliminate PCR carryover and an exogenous internal positive control that enabled discrimination of false-negative from true-negative reactions. The new assays were compared to conventional PCR assays for clinical specimens (saliva, rectal brushings, rectal swab specimens, peripheral blood lymphocytes, semen, and urine) from human immunodeficiency virus-positive patients with or without Kaposi's sarcoma. In all instances, the new assays agreed with each other and with the conventional PCR system. In addition, the quantitative results obtained with the new assays were in good agreement both for duplicate reactions in the same assay and between assays.
Ultrastructural Study ofChlamydia pneumoniaeIn a Continuous-Infection Model
Authors: Kutlin Andrei , Flegg Cameron , Stenzel Deb , Reznik Tamara , Roblin Patricia M. , Mathews Sarah , Timms Peter , Hammerschlag Margaret R.
Published: July 2002

We have established an in vitro model of long-term continuousChlamydia pneumoniaeinfection in HEp-2 cells. Using transmission electron microscopy, we demonstrated the presence of spontaneous abnormal chlamydial inclusions similar in appearance to the persistent chlamydial forms induced in vitro by treatment with cytokines or antibiotics or by nutrient deprivation.
Multiplex PCR for Simultaneous Identification of
Staphylococcus aureus
and Detection of Methicillin and Mupirocin Resistance
Authors: Pérez-Roth E. , Claverie-Martı́n F. , Villar J. , Méndez-Álvarez S.
Published: July 2002

In this work, we describe a multiplex PCR assay for the detection of clinically relevant antibiotic resistance genes harbored by some
Staphylococcus aureus
isolates and for the simultaneous identification of such isolates at the species level. Conditions were optimized for the simultaneous detection of the 310-, 456-, and 651-bp regions of the
mecA
(encoding high-level methicillin resistance),
ileS
-
2
(encoding high-level mupirocin resistance), and
femB
(encoding a factor essential for methicillin resistance) genes, respectively, from a single colony in a single reaction tube. The
femB
PCR fragment allows the specific identification of
S
.
aureus
. Validation of the method was performed using 50 human isolates of methicillin-resistant
S
.
aureus
(MRSA) and the appropriate control strains. This assay offers a rapid, simple, feasible, specific, sensitive, and accurate identification of mupirocin-resistant MRSA clinical isolates and could be systematically applied as a diagnostic test in clinical microbiology laboratories, facilitating the design and use of antibiotic therapy.
Typing and Subtyping Influenza Virus Using DNA Microarrays and Multiplex Reverse Transcriptase PCR
Authors: Li Jiping , Chen Shu , Evans David H.
Published: July 2002

A model DNA microarray has been prepared and shown to facilitate typing and subtyping of human influenza A and B viruses. Reverse transcriptase PCR was used to prepare cDNAs encoding ∼500-bp influenza virus gene fragments, which were then cloned, sequenced, reamplified, and spotted to form a glass-bound microarray. These target DNAs included multiple fragments of the hemagglutinin, neuraminidase, and matrix protein genes. Cy3- or Cy5-labeled fluorescent probes were then hybridized to these target DNAs, and the arrays were scanned to determine the probe binding site(s). The hybridization pattern agreed perfectly with the known grid location of each target, and the signal-to-background ratio varied from 5 to 30. No cross-hybridization could be detected beyond that expected from the limited degree of sequence overlap between different probes and targets. At least 100 to 150 bp of homology was required for hybridization under the conditions used in this study. Combinations of Cy3- and Cy5-labeled DNAs can also be hybridized to the same chip, permitting further differentiation of amplified molecules in complex mixtures. In a more realistic test of the technology, several sets of multiplex PCR primers that collectively target influenza A and B virus strains were identified and were used to type and subtype several previously unsequenced influenza virus isolates. The results show that DNA microarray technology provides a useful supplement to PCR-based diagnostic methods.
Molecular Detection of
Mycoplasma pneumoniae
in Adults with Community-Acquired Pneumonia Requiring Hospitalization
Authors: Dorigo-Zetsma J. Wendelien , Verkooyen Roel P. , van Helden H. Pieter , van der Nat Hans , van den Bosch Jules M.
Published: July 2002

Mycoplasma pneumoniae
infection was diagnosed in 18 (12.5%) of 144 adults hospitalized with community-acquired pneumonia. The infection was demonstrated by PCR in 15 patients and by serology, using two methods, in 10 patients. The mean age of the 8 patients with positive
M. pneumoniae
PCR and negative serology was significantly higher than that of the 10 patients with positive serology.
Evaluation of a Group-Specific 16S Ribosomal DNA-Based PCR for Detection of
Helicobacter bizzozeronii, Helicobacter felis
, and
Helicobacter salomonis
in Fresh and Paraffin-Embedded Gastric Biopsy Specimens
Authors: De Groote Dominic , Haesebrouck Freddy , van Doorn Leen-Jan , Vandamme Peter , Ducatelle Richard
Published: July 2002

A new specific and sensitive 16S ribosomal DNA-based PCR assay was developed. The assay targets a 78-bp DNA fragment unique to
Helicobacter bizzozeronii, Helicobacter felis
, and
Helicobacter salomonis
and can be used with freshly frozen and formalin-fixed paraffin-embedded gastric biopsy specimens.
Molecular Evidence ofBartonellaspp. in Questing AdultIxodes pacificusTicks in California
Authors: Chang C. C. , Chomel B. B. , Kasten R. W. , Romano V. , Tietze N.
Published: July 2002

Ticks are the vectors of many zoonotic diseases in the United States, including Lyme disease, human monocytic and granulocytic ehrlichioses, and Rocky Mountain spotted fever. Most knownBartonellaspecies are arthropod borne. Therefore, it is important to determine if someBartonellaspecies, which are emerging pathogens, could be carried or transmitted by ticks. In this study, adultIxodes pacificusticks were collected by flagging vegetation in three sites in Santa Clara County, Calif. PCR-restriction fragment length polymorphism and partial sequencing of 273 bp of thegltAgene were applied forBartonellaidentification. Twenty-nine (19.2%) of 151 individually tested ticks were PCR positive forBartonella. Male ticks were more likely to be infected withBartonellathan female ticks (26 versus 12%,P= 0.05). None of the nine ticks collected at Baird Ranch was PCR positive forBartonella. However, 7 (50%) of 14 ticks from Red Fern Ranch and 22 (17%) of 128 ticks from the Windy Hill Open Space Reserve were infected withBartonella. In these infected ticks, molecular analysis showed a variety ofBartonellastrains, which were closely related to a cattleBartonellastrain and to several known human-pathogenicBartonellaspecies and subspecies:Bartonella henselae, B. quintana, B. washoensis,andB. vinsoniisubsp.berkhoffii. These findings indicate thatI. pacificusticks may play an important role inBartonellatransmission among animals and humans.
Comparison of Nucleic Acid Amplification, Serology, and Microbiologic Culture for Diagnosis ofRhodococcus equiPneumonia in Foals
Authors: Sellon Debra C. , Besser Thomas E. , Vivrette Sally L. , McConnico Rebecca S.
Published: July 2002

Recently, a technique was described for amplification ofRhodococcus equi-specific chromosomal andvapADNA from blood and tracheal wash fluids. It was hypothesized that this technique would be more sensitive than standard culture techniques or serology for diagnosis ofR. equipneumonia in foals. Tracheal wash fluid, nasal swabs, whole blood samples, and serum samples from 56 foals with pneumonia were analyzed. Final clinical diagnosis was determined by the attending clinician on the basis of final interpretation of all available information about each foal, including clinical presentation, diagnostic test results, response to therapy, and outcome. Clinical diagnosis was used as a final reference standard for calculation of sensitivity, specificity, and predictive values for PCR, serology using an agar gel immunodiffusion test, and tracheal wash fluid culture. PCR of tracheal wash fluid using primers that recognized thevapAvirulence plasmid ofR. equihad a diagnostic sensitivity of 100% and specificity of 90.6%. Sensitivity and specificity were 57.1 and 93.8%, respectively, for standard microbiologic culture of tracheal wash fluid and 62.5 and 75.9%, respectively, for serology. PCR of tracheal wash fluid is more sensitive and specific for diagnosis ofR. equipneumonia than are other available diagnostic tests.
Type-Specific Detection of Echovirus 30 Isolates Using Degenerate Reverse Transcriptase PCR Primers
Authors: Kilpatrick David R. , Quay Jacqueline , Pallansch Mark A. , Oberste M. Steven
Published: July 2002

Following an approach used to specifically identify polioviruses and enterovirus 71, we have developed reverse transcriptase (RT) PCR primers containing mixed-base residues or deoxyinosine at positions of codon degeneracy. These primers permit specific RT-PCR amplification of echovirus 30 (E30) sequences by targeting sites that encode conserved amino acid motifs within the major capsid protein, VP1. All 221 E30 strains tested, isolated in 16 countries over a 44-year period, yielded the predicted 158-bp PCR product. No specific products were obtained by PCR assays containing templates from any of the other 63 EV serotypes. Inosine-containing degenerate primers may be widely applicable to the identification of echovirus serotypes by PCR.
Outer Membrane Proteins and DNA Profiles in Strains ofHaemophilus parasuisRecovered from Systemic and Respiratory Sites
Authors: Ruiz Alvaro , Oliveira Simone , Torremorell Montserrat , Pijoan Carlos
Published: July 2002

Polyserositis caused byHaemophilus parasuisis an important disease that affects mostly weaned pigs. Recent studies have shown that virulence can differ among strains recovered from distinct body sites and also that it may be related to the presence of certain outer membrane proteins (OMPs). The objective of this study was to compare the OMP and DNA profiles ofH. parasuisstrains isolated from systemic and respiratory sites from diseased and healthy pigs. Strains evaluated in this study were processed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and repetitive-PCR techniques. Two experiments were conducted in order to better define the relationship among genotype, phenotype, and site of isolation. Experiment 1 included 53H. parasuisisolates recovered from healthy and diseased pigs from unrelated herds. Experiment 2 included 31 isolates ofH. parasuisobtained from diseased pigs involved in an outbreak in a large, multifarm system. Results showed that strains recovered from systemic sites had more homogeneous OMP and DNA profiles than those isolated from respiratory sites. Evaluation of isolates involved in the multifarm outbreak showed that only twoH. parasuisstrains were causing disease. These strains had homogeneous OMP and DNA profiles. However, it was noted that these two parameters were unrelated, since strains classified in the same genotype group expressed different OMP profiles. The homogeneity of OMP and DNA profiles of strains isolated from systemic sites strongly suggests the existence of clonal relationships between virulent strains and also suggests that expression of certain OMP profiles may be related to virulence.
Use of Subgenic 18S Ribosomal DNA PCR and Sequencing for Genus and Genotype Identification of Acanthamoebae from Humans with Keratitis and from Sewage Sludge
Authors: Schroeder Jill M. , Booton Gregory C. , Hay John , Niszl Ingrid A. , Seal David V. , Markus Miles B. , Fuerst Paul A. , Byers Thomas J.
Published: July 2002

This study identified subgenic PCR amplimers from 18S rDNA that were (i) highly specific for the genus
Acanthamoeba
, (ii) obtainable from all known genotypes, and (iii) useful for identification of individual genotypes. A 423- to 551-bp
Acanthamoeba
-specific amplimer ASA.S1 obtained with primers JDP1 and JDP2 was the most reliable for purposes i and ii. A variable region within this amplimer also identified genotype clusters, but purpose iii was best achieved with sequencing of the genotype-specific amplimer GTSA.B1. Because this amplimer could be obtained from any eukaryote, axenic
Acanthamoeba
cultures were required for its study. GTSA.B1, produced with primers CRN5 and 1137, extended between reference bp 1 and 1475. Genotypic identification relied on three segments: bp 178 to 355, 705 to 926, and 1175 to 1379. ASA.S1 was obtained from single amoeba, from cultures of all known 18S rDNA genotypes, and from corneal scrapings of Scottish patients with suspected
Acanthamoeba
keratitis (AK). The AK PCR findings were consistent with culture results for 11 of 15 culture-positive specimens and detected
Acanthamoeba
in one of nine culture-negative specimens. ASA.S1 sequences were examined for 6 of the 11 culture-positive isolates and were most closely associated with genotypic cluster T3-T4-T11. A similar distance analysis using GTSA.B1 sequences identified nine South African AK-associated isolates as genotype T4 and three isolates from sewage sludge as genotype T5. Our results demonstrate the usefulness of 18S ribosomal DNA PCR amplimers ASA.S1 and GTSA.B1 for
Acanthamoeba
-specific detection and reliable genotyping, respectively, and provide further evidence that T4 is the predominant genotype in AK.
Evaluation of PCR Using TRC
4
and IS
6110
Primers in Detection of Tuberculous Meningitis
Authors: Narayanan Sujatha , Parandaman Vijayalakshmi , Narayanan P. R. , Venkatesan P. , Girish C. , Mahadevan S. , Rajajee Sarala
Published: July 2002

We have evaluated a new set of primers (TRC
4
) in comparison with the IS
6110
primers commonly used in PCR to detect tuberculous meningitis among children. The levels of concordance between the results of IS
6110
PCR and TRC
4
PCR with cerebrospinal fluid specimens from patients with clinically confirmed tuberculous meningitis were 80 and 86%, respectively. Results with the two primer sets were concordant for 55 positive and 22 negative specimens (
n
= 98). We conclude that the sensitivity of PCR can be increased by using both IS
6110
and TRC
4
primers.
Rapid Receptor-Clustering Assay To Detect Uropathogenic and Diarrheal
Escherichia coli
Isolates Bearing Adhesins of the Dr Family
Authors: Goluszko P. , Selvarangan R. , Nowicki B. J. , Nowicki S. , Hart A. , Pawelczyk E. , Nguyen K.
Published: July 2002

Infections caused by
Escherichia coli
isolates expressing adhesins of the Dr family are associated with diarrhea and urinary tract infections, and these
E. coli
strains recognize the complement regulatory protein decay-accelerating factor (DAF) as their receptor. Clustering of the DAF receptor at the sites of bacterial adherence to epithelial cells is proposed as an alternative to PCR assay for rapid detection of Dr-positive
E. coli
.
Standardization of Antifungal Susceptibility Variables for a Semiautomated Methodology
Authors: Rodrı́guez-Tudela Juan L. , Cuenca-Estrella Manuel , Dı́az-Guerra Teresa M. , Mellado Emilia
Published: July 2002

Recently, the methodology that will serve as a basis of the standard for antifungal susceptibility testing of fermentative yeasts of the European Committee on Antibiotic Susceptibility Testing has been described. This procedure employs a spectrophotometric method for both inoculum adjustment and endpoint determination. However, the utilization of a spectrophotometer requires studies for standardization. The present work analyzes the following parameters: (i) accuracy of inoculum preparation, (ii) correlation between optical density and CFU per milliliter, (iii) influence of the wavelength on the endpoint determination, and (iv) influence of the dimethyl sulfoxide concentration on the growth kinetics. The main results can be summarized as follows: (i) inoculum preparation following the methodology recommended by the National Committee for Clinical Laboratory Standards is an exact procedure; (ii) the relationship between optical density and CFU per milliliter is linear (coefficient of determination,
r
2
= 0.84); (iii) MICs obtained by means of spectrophotometric readings at different wavelengths are identical (for amphotericin B, an intraclass correlation coefficient of 0.98 was obtained; for fluconazole, the intraclass correlation coefficient was 1); and (iv) a 2% concentration of dimethyl sulfoxide produces a significantly slower and lower growth curve of
Candida
spp. than other concentrations.
Development of a PCR Assay for Identification of Staphylococci at Genus and Species Levels
Authors: Martineau Francis , Picard François J. , Ke Danbing , Paradis Sonia , Roy Paul H. , Ouellette Marc , Bergeron Michel G.
Published: July 2002

We have developed a PCR-based assay which allows the detection of staphylococci at the genus level by targeting the
tuf
gene, which encodes the elongation factor Tu. Degenerate PCR primers derived from consensus regions of several
tuf
genes were used to amplify a target region of 884 bp from 11 representative staphylococcal species. Subsequently, the entire nucleotide sequence of these amplicons was determined. The analysis of a multiple alignment of these sequences revealed regions conserved among staphylococci but distinct from those of other gram-positive bacteria genetically related to staphylococci. PCR primers complementary to these regions could amplify specifically and efficiently a DNA fragment of 370 bp for all of 27 different staphylococcal species tested. There was no amplification with genomic DNA prepared from 53 nonstaphylococcal species tested to verify the specificity of the assay (20 gram positive and 33 gram negative). Furthermore, this assay amplified efficiently all 27 American Type Culture Collection (ATCC) staphylococcal reference strains as well as 307 clinical isolates of staphylococci from the Québec City region. Analysis of the multiple sequence alignment for the 884-bp fragment for the 11 staphylococcal species as well as comparison of the sequences for the 370-bp amplicon from five unrelated ATCC and clinical strains for each of the species
S. aureus, S. epidermidis, S. haemolyticus, S. hominis
, and
S. saprophyticus
demonstrated sufficient interspecies polymorphism to generate genus- and species-specific capture probes. This sequence information allowed the development of
Staphylococcus
-specific and species-specific (targeting
S. aureus, S. epidermidis, S. haemolyticus, S. hominis
, or
S. saprophyticus
) capture probes hybridizing to the 370-bp amplicon. In conclusion, this PCR assay is suitable for detection of staphylococci at both genus and species levels.
Evaluation of a Novel Heminested PCR Assay Based on the Phosphoglucosamine Mutase Gene for Detection of
Helicobacter pylori
in Saliva and Dental Plaque
Authors: Goosen C. , Theron J. , Ntsala M. , Maree F. F. , Olckers A. , Botha S. J. , Lastovica A. J. , van der Merwe S. W.
Published: July 2002

A novel heminested PCR protocol was developed for the specific detection of
Helicobacter pylori
at low copy numbers. A set of primers specific for the phosphoglucosamine mutase gene (
glmM
) of
H. pylori
produced a 765-bp fragment that was used as template for the heminested primer pair delineating a 496-bp fragment. By using agarose gel electrophoresis for detection of the heminested PCR-amplified products, amplification of
H. pylori
genomic DNA was achieved at concentrations as low as 0.1 pg, equivalent to 5 × 10
2
bacteria. A study was subsequently undertaken to evaluate the heminested PCR for detection of
H. pylori
in dental plaque and saliva. Specimens collected from 58 individuals were cultured, and PCR was subsequently performed on the oral cultures. Identification of
H. pylori
in the same series of saliva and dental plaque specimens was carried out with PCR using a primer pair specific for the
H. pylori
urease B gene and by the heminested PCR assay. The identity of the amplified products was confirmed by DNA sequencing. Our results demonstrate that the heminested PCR assay was specific for detection of
H. pylori
, yielding no false-positive results, and that
H. pylori
had a low prevalence (approximately 3%) in specimens obtained from the oral cavity.
Sensitive Enzyme Immunoassay for Hepatitis B Virus Core-Related Antigens and Their Correlation to Virus Load
Authors: Kimura Tatsuji , Rokuhara Akinori , Sakamoto Yoko , Yagi Shintaro , Tanaka Eiji , Kiyosawa Kendo , Maki Noboru
Published: July 2002

A sensitive enzyme immunoassay (EIA) specific for hepatitis B virus core antigen (HBcAg) and hepatitis B e antigen (HBeAg) was developed. We designated the precore/core gene products as hepatitis B virus (HBV) core-related antigens (HBcrAg). In order to detect HBcrAg even in anti-HBc/e antibody-positive specimens, the specimens were pretreated in detergents. The antibodies are inactivated by this pretreatment and, simultaneously, the antigens are released and the epitopes are exposed. The assay demonstrated 71 to 112% recovery using HBcrAg-positive sera. We observed no interference from the tested anticoagulants or blood components. When the cutoff value was tentatively set at 103U/ml, all healthy control (HBsAg/HBV-DNA negative;n= 108) and anti-HCV antibody-positive (n= 59) sera were identified as negative. The assay showed a detection limit of 4 × 102U/ml using recombinant antigen. Detection limits were compared in four serially diluted HBV high-titer sera. The HBcrAg assay demonstrated higher sensitivity than HBV-DNA transcription-mediated amplification (TMA) or HBeAg radio immunoassay (RIA) in the dilution test. HBcrAg concentrations correlated well with HBV-DNA TMA (r= 0.91,n= 29) and in-house real-time detection-PCR (r= 0.93,n= 47) in hepatitis B patients. On HBeAg/anti-HBe antibody seroconversion panels, the HBcrAg concentration changed in accordance with HBV-DNA levels. HBcrAg concentration provides a reflection of HBV virus load equivalent to HBV-DNA level, and the assay therefore offers a simple method for monitoring hepatitis B patients.
Quantitative Microbiological Study of Human Carious Dentine by Culture and Real-Time PCR: Association of Anaerobes with Histopathological Changes in Chronic Pulpitis
Authors: Martin F. Elizabeth , Nadkarni Mangala A. , Jacques Nicholas A. , Hunter Neil
Published: July 2002

The bacteria found in carious dentine were correlated with the tissue response of the dental pulps of 65 teeth extracted from patients with advanced caries and pulpitis. Standardized homogenates of carious dentine were plated onto selective and nonselective media under anaerobic and microaerophilic conditions. In addition, real-time PCR was used to quantify the recovery of anaerobic bacteria. Primers and fluorogenic probes were designed to detect the total anaerobic microbial load, the genera
Prevotella
and
Fusobacterium
, and the species
Prevotella melaninogenica
,
Porphyromonas endodontalis
,
Porphyromonas gingivalis
, and
Micromonas
(formerly
Peptostreptococcus
)
micros
. The pulpal pathology was categorized according to the cellular response and degenerative changes. Analysis of cultured bacteria showed a predominance of gram-positive microorganisms, particularly lactobacilli. Gram-negative bacteria were also present in significant numbers with
Prevotella
spp., the most numerous anaerobic group cultured. Real-time PCR analysis indicated a greater microbial load than that determined by colony counting. The total number of anaerobes detected was 41-fold greater by real-time PCR than by colony counting, while the numbers of
Prevotella
and
Fusobacterium
spp. detected were 82- and 2.4-fold greater by real-time PCR than by colony counting, respectively. Real-time PCR also identified
M. micros
,
P. endodontalis
, and
P. gingivalis
in 71, 60, and 52% of carious samples, respectively. Correlation matrices of the real-time PCR data revealed significant positive associations between
M. micros
and
P. endodontalis
detection and inflammatory degeneration of pulpal tissues. These anaerobes have been strongly implicated in endodontic infections that occur as sequelae to carious pulpitis. Accordingly, the data suggest that the presence of high levels of these bacteria in carious lesions may be indicative of irreversible pulpal pathology.
Utility of a Multiplex PCR Assay for Detecting Herpesvirus DNA in Clinical Samples
Authors: Druce Julian , Catton Mike , Chibo Doris , Minerds Kirsty , Tyssen David , Kostecki Renata , Maskill Bill , Leong-Shaw Wendy , Gerrard Marie , Birch Chris
Published: July 2002

A multiplex PCR was designed to amplify herpes simplex virus types 1 and 2, cytomegalovirus, and varicella-zoster virus DNA present in a diverse range of clinical material. The susceptibility of these viruses to in vivo inhibition by at least one antiviral drug was an important consideration in their inclusion in the multiplex detection system. An aliquot of equine herpesvirus was introduced into each specimen prior to extraction and served as an indicator of potential inhibitors of the PCR and a detector of suboptimal PCR conditions. Compared to virus isolation and immunofluorescence-based antigen detection, the multiplex assay yielded higher detection rates for all viruses represented in the assay. The turnaround time for performance of the assay was markedly reduced compared to those for the other techniques used to identify these viruses. More than 21,000 tests have been performed using the assay. Overall, the multiplex PCR enabled the detection of substantially increased numbers of herpesviruses, in some cases in specimens or anatomical sites where previously they were rarely if ever identified using traditional detection methods.
Population Diversity ofStaphylococcus intermediusIsolates from Various Host Species: Typing by 16S-23S Intergenic Ribosomal DNA Spacer Polymorphism Analysis
Authors: Bes Michèle , Saidi Slim Leila , Becharnia Fatma , Meugnier Hélène , Vandenesch François , Etienne Jerome , Freney Jean
Published: July 2002

Twelve 16S-23S ribosomal DNA intergenic spacer (ITS-PCR) types were identified among 57Staphylococcus intermediusisolates from humans and other animals. Six ITS-PCR types were host specific, and most human and canine strains belonged to the same types (A and J). Pigeon, horse, and mink strains appeared more heterogeneous.
A Yeast-Based Genetic System for Functional Analysis of Viral mRNA Capping Enzymes
Authors: Ho C. Kiong , Martins Alexandra , Shuman Stewart
Published: July 2002

Virus-encoded mRNA capping enzymes are attractive targets for antiviral therapy, but functional studies have been limited by the lack of genetically tractable in vivo systems that focus exclusively on the RNA-processing activities of the viral proteins. Here we have developed such a system by engineering a viral capping enzyme—vaccinia virus D1(1-545)p, an RNA triphosphatase and RNA guanylyltransferase—to function in the budding yeast
Saccharomyces cerevisiae
in lieu of the endogenous fungal triphosphatase (Cet1p) and guanylyltransferase (Ceg1p). This was accomplished by fusion of D1(1-545)p to the C-terminal guanylyltransferase domain of mammalian capping enzyme, Mce1(211-597)p, which serves as a vehicle to target the viral capping enzyme to the RNA polymerase II elongation complex. An inactivating mutation (K294A) of the mammalian guanylyltransferase active site in the fusion protein had no impact on genetic complementation of
cet1Δceg1
Δ cells, thus proving that (i) the viral guanylyltransferase was active in vivo and (ii) the mammalian domain can serve purely as a chaperone to direct other proteins to the transcription complex. Alanine scanning had identified five amino acids of vaccinia virus capping enzyme—Glu37, Glu39, Arg77, Glu192, and Glu194—that are essential for γ phosphate cleavage in vitro. Here we show that the introduction of mutation E37A, R77A, or E192A into the fusion protein abrogates RNA triphosphatase function in vivo. The essential residues are located within three motifs that define a family of viral and fungal metal-dependent phosphohydrolases with a distinctive capacity to hydrolyze nucleoside triphosphates to nucleoside diphosphates in the presence of manganese or cobalt. The acidic residues Glu37, Glu39, and Glu192 likely comprise the metal-binding site of vaccinia virus triphosphatase, insofar as their replacement by glutamine abolishes the RNA triphosphatase and ATPase activities.
Influenza Virus NS1 Protein Counteracts PKR-Mediated Inhibition of Replication
Authors: Bergmann Michael , Garcia-Sastre Adolfo , Carnero Elena , Pehamberger Hubert , Wolff Klaus , Palese Peter , Muster Thomas
Published: July 2002

The availability of an influenza virus NS1 gene knockout virus (delNS1 virus) allowed us to establish the significance of the biological relationship between the influenza virus NS1 protein and double-stranded-RNA-activated protein kinase (PKR) in the life cycle and pathogenicity of influenza virus. Our results show that the lack of functional PKR permits the delNS1 virus to replicate in otherwise nonpermissive hosts, suggesting that the major function of the influenza virus NS1 protein is to counteract or prevent the PKR-mediated antiviral response.
Evidence that Herpes Simplex Virus VP16 Is Required for Viral Egress Downstream of the Initial Envelopment Event
Authors: Mossman Karen L. , Sherburne Richard , Lavery Carol , Duncan Joanne , Smiley James R.
Published: July 2002

During infection with herpes simplex virus type 1 (HSV-1), VP16 serves multiple functions, including transcriptional activation of viral immediate early genes and downregulation of the virion host shutoff protein vhs. Furthermore, VP16 has been shown to be involved in some aspect of virus assembly and/or maturation. Experiments with a VP16 null virus, 8MA, suggested that VP16 plays a direct role in virion assembly, since removal of VP16 from the HSV-1 genome results in reduced levels of encapsidated DNA and a failure to produce extracellular enveloped particles. However, VP16 null mutants display a severe translational arrest due to unrestrained vhs activity, thus complicating interpretation of these data. We examine here the role of VP16 in virion assembly and egress in the context of a vhs null background, using the virus 8MA/ΔSma (VP16
−
vhs
−
). Comparison of 8MA and 8MA/ΔSma with respect to viral DNA accumulation and encapsidation and accumulation of the major capsid protein, VP5, revealed that the 8MA lethal phenotype is only partially due to uncontrolled vhs activity, indicating that VP16 is required in HSV-1 virion formation. Electron microscopy confirmed these results and further showed that VP16 is required for HSV-1 egress beyond the perinuclear space. In addition, we describe the isolation and characterization of an 8MA derivative capable of propagation on Vero cells, due to second site mutations in the vhs and UL53 (gK) genes. Taken together, these results show that VP16 is required for viral egress downstream of the initial envelopment step and further underscore the importance of VP16 in controlling vhs activity within an infected cell.