bluuebunny/crossref_metadata_2025 huggingface dataset:
Statistical Approaches for Estimating Actinobacterial Diversity in Marine Sediments
Authors: Stach James E. M. , Maldonado Luis A. , Masson Douglas G. , Ward Alan C. , Goodfellow Michael , Bull Alan T.
Published: October 2003

Bacterial diversity in a deep-sea sediment was investigated by constructing actinobacterium-specific 16S ribosomal DNA (rDNA) clone libraries from sediment sections taken 5 to 12, 15 to 18, and 43 to 46 cm below the sea floor at a depth of 3,814 m. Clones were placed into operational taxonomic unit (OTU) groups with ≥99% 16S rDNA sequence similarity; the cutoff value for an OTU was derived by comparing 16S rRNA homology with DNA-DNA reassociation values for members of the class
Actinobacteria.
Diversity statistics were used to determine how the level of dominance, species richness, and genetic diversity varied with sediment depth. The reciprocal of Simpson's index (1/
D
) indicated that the pattern of diversity shifted toward dominance from uniformity with increasing sediment depth. Nonparametric estimation of the species richness in the 5- to 12-, 15- to 18-, and 43- to 46-cm sediment sections revealed a trend of decreasing species number with depth, 1,406, 308, and 212 OTUs, respectively. Application of the LIBSHUFF program indicated that the 5- to 12-cm clone library was composed of OTUs significantly (
P
= 0.001) different from those of the 15- to 18- and 43- to 46-cm libraries.
F
ST
and phylogenetic grouping of taxa (P tests) were both significant (
P
< 0.00001 and
P
< 0.001, respectively), indicating that genetic diversity decreased with sediment depth and that each sediment community harbored unique phylogenetic lineages. It was also shown that even nonconservative OTU definitions result in severe underestimation of species richness; unique phylogenetic clades detected in one OTU group suggest that OTUs do not correspond to real ecological groups sensu Palys (T. Palys, L. K. Nakamura, and F. M. Cohan, Int. J. Syst. Bacteriol. 47:1145-1156, 1997). Mechanisms responsible for diversity and their implications are discussed.
Assessment of the Effects of Holding Time and Temperature on
Escherichia coli
Densities in Surface Water Samples
Authors: Pope Misty L. , Bussen Michelle , Feige Mary Ann , Shadix Lois , Gonder Sharon , Rodgers Crystal , Chambers Yildiz , Pulz Jessica , Miller Ken , Connell Kevin , Standridge Jon
Published: October 2003

Escherichia coli
is a routinely used microbiological indicator of water quality. To determine whether holding time and storage conditions had an effect on
E. coli
densities in surface water, studies were conducted in three phases, encompassing 24 sites across the United States and four commonly used monitoring methods. During all three phases of the study,
E. coli
samples were analyzed at time 0 and at 8, 24, 30, and 48 h after sample collection. During phase 1, when 4°C samples were evaluated by Colilert or by placing a membrane onto mFC medium followed by transfer to nutrient agar containing 4-methylumbelliferyl-β-
d
-glucuronide (mFC/NA-MUG), three of four sites showed no significant differences throughout the 48-h study. During phase 2, five of seven sites showed no significant difference between time 0 and 24 h by membrane filtration (mFC/NA-MUG). When evaluated by the Colilert method, five of seven sites showed no significant difference in
E. coli
density between time 0 and 48 h. During phase 3, 8 of 13 sites showed no significant differences in
E. coli
densities between time 0 and the 48-h holding time, regardless of method. Based on the results of these studies, it appears that if samples are held below 10°C and are not allowed to freeze, most surface water
E. coli
samples analyzed by commonly used methods beyond 8 h after sample collection can generate
E. coli
data comparable to those generated within 8 h of sample collection. Notwithstanding this conclusion,
E. coli
samples collected from surface waters should always be analyzed as soon as possible.
Use of Bromodeoxyuridine Immunocapture To Identify Active Bacteria Associated with Arbuscular Mycorrhizal Hyphae
Authors: Artursson Veronica , Jansson Janet K.
Published: October 2003

Arbuscular mycorrhizae are beneficial for crops grown under low-till management systems. Increasingly, it is becoming apparent that bacteria associated with mycorrhizae can enhance the beneficial relationship between mycorrhizae and plants. However, it has been difficult to study these relationships by conventional techniques. In this study actively growing bacteria were identified in soil from an undisturbed fallow field known to contain arbuscular mycorrhizae by using molecular tools to eliminate the need for cultivation. A thymidine analog, bromodeoxyuridine (BrdU), was added to the soil and incubated for 2 days. DNA was extracted, and the newly synthesized DNA was isolated by immunocapture of the BrdU-containing DNA. The active bacteria in the community were identified by 16S rRNA gene PCR amplification and DNA sequence analysis. Based on 16S rRNA gene sequence information, a selective medium was chosen to isolate the corresponding active bacteria.
Bacillus cereus
strain VA1, one of the bacteria identified by the BrdU method, was isolated from the soil and tagged with green fluorescent protein. By using confocal microscopy, this bacterium was shown to clearly attach to arbuscular mycorrhizal hyphae. This study was the first to use this combination of molecular and traditional approaches to isolate, identify, and visualize a specific bacterium that is active in fallow soil and associates with arbuscular mycorrhizal hyphae.
Nuclease Activity of
Saccharomyces cerevisiae
Mre11 Functions in Targeted Nucleotide Alteration
Authors: Liu Li , Usher Michael , Hu Yiling , Kmiec Eric B.
Published: October 2003

Oligonucleotides can be used to direct site-specific changes in genomic DNA through a process in which mismatched base pairs in the oligonucleotide and the target DNA are created. The mechanism by which these complexes are developed and resolved is being studied by using
Saccharomyces cerevisiae
as a model system. Genetic analyses have revealed that in all likelihood the reaction occurs in two phases: DNA pairing and DNA repair. While the former phase involves strand assimilation, the latter phase likely involves an endonucleolytic processing step that leads to joint resolution. In this study, we established the importance of a functioning
MRE11
gene in the overall reaction, as yeast strains deficient in
MRE11
exhibited severely reduced activity. The activity could be rescued by complementation with wild-type
MRE11
genes but not with
MRE11
alleles lacking the nuclease function. Taken together, the data suggest that Mre11 provides nuclease activity for targeted nucleotide exchange, a process that could be used to reengineer yeast genes.
Prospecting for Novel Biocatalysts in a Soil Metagenome
Authors: Voget S. , Leggewie C. , Uesbeck A. , Raasch C. , Jaeger K.-E. , Streit W. R.
Published: October 2003

The metagenomes of complex microbial communities are rich sources of novel biocatalysts. We exploited the metagenome of a mixed microbial population for isolation of more than 15 different genes encoding novel biocatalysts by using a combined cultivation and direct cloning strategy. A 16S rRNA sequence analysis revealed the presence of hitherto uncultured microbes closely related to the genera
Pseudomonas
,
Agrobacterium
,
Xanthomonas
,
Microbulbifer
, and
Janthinobacterium.
Total genomic DNA from this bacterial community was used to construct cosmid DNA libraries, which were functionally searched for novel enzymes of biotechnological value. Our searches in combination with cosmid sequencing resulted in identification of four clones encoding 12 putative agarase genes, most of which were organized in clusters consisting of two or three genes. Interestingly, nine of these agarase genes probably originated from gene duplications. Furthermore, we identified by DNA sequencing several other biocatalyst-encoding genes, including genes encoding a putative stereoselective amidase (
amiA
), two cellulases (
gnuB
and
uvs080
), an α-amylase (
amyA
), a 1,4-α-glucan branching enzyme (
amyB
), and two pectate lyases (
pelA
and
uvs119
). Also, a conserved cluster of two lipase genes was identified, which was linked to genes encoding a type I secretion system. The novel gene
aguB
was overexpressed in
Escherichia coli
, and the enzyme activities were determined. Finally, we describe more than 162 kb of DNA sequence that provides a strong platform for further characterization of this microbial consortium.
A Novel Extracellular Multicopper Oxidase from
Phanerochaete chrysosporium
with Ferroxidase Activity
Authors: Larrondo Luis F. , Salas Loreto , Melo Francisco , Vicuña Rafael , Cullen Daniel
Published: October 2003

Lignin degradation by the white rot basidiomycete
Phanerochaete chrysosporium
involves various extracellular oxidative enzymes, including lignin peroxidase, manganese peroxidase, and a peroxide-generating enzyme, glyoxal oxidase. Recent studies have suggested that laccases also may be produced by this fungus, but these conclusions have been controversial. We identified four sequences related to laccases and ferroxidases (Fet3) in a search of the publicly available
P. chrysosporium
database. One gene, designated
mco1
, has a typical eukaryotic secretion signal and is transcribed in defined media and in colonized wood. Structural analysis and multiple alignments identified residues common to laccase and Fet3 sequences. A recombinant MCO1 (rMCO1) protein expressed in
Aspergillus nidulans
had a molecular mass of 78 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the copper I-type center was confirmed by the UV-visible spectrum. rMCO1 oxidized various compounds, including 2,2′-azino(bis-3-ethylbenzthiazoline-6-sulfonate) (ABTS) and aromatic amines, although phenolic compounds were poor substrates. The best substrate was Fe
2+
, with a

K
m

close to 2 μM. Collectively, these results suggest that the
P. chrysosporium
genome does not encode a typical laccase but rather encodes a unique extracellular multicopper oxidase with strong ferroxidase activity.
An Alternative Efficient Procedure for Purification of the Obligate Intracellular Fish Bacterial Pathogen
Piscirickettsia salmonis
Authors: Henríquez Vitalia , Rojas María Verónica , Marshall Sergio H.
Published: October 2003

Piscirickettsia salmonis
is an obligate intracellular bacterial pathogen of salmonid fish and the etiological agent of the aggressive disease salmonid rickettsial syndrome. Today, this disease, also known as piscirickettsiosis, is the cause of high mortality in net pen-reared salmonids in southern Chile. Although the bacteria can be grown in tissue culture cells, genetic analysis of the organism has been hindered because of the difficulty in obtaining
P. salmonis
DNA free from contaminating host cell DNA. In this report, we describe a novel procedure to purify in vitro-grown bacteria with iodixanol as the substrate to run differential centrifugation gradients which, combined with DNase I digestion, yield enough pure bacteria to do DNA analysis. The efficiency of the purification procedure relies on two main issues: semiquantitative synchrony of the
P. salmonis
-infected Chinook salmon embryo (CHSE-214) tissue culture cells and low osmolarity of iodixanol to better resolve bacteria from the membranous structures of the host cell. This method resulted in the isolation of intact piscirickettsia organisms and removed salmon and mitochondrial DNA effectively, with only 1.0% contamination with the latter.
Species-Specific Peptide Ligands for the Detection of
Bacillus anthracis
Spores
Authors: Williams David D. , Benedek Orsolya , Turnbough Charles L.
Published: October 2003

Currently available detectors for spores of
Bacillus anthracis
, the causative agent of anthrax, are inadequate for frontline use and general monitoring. There is a critical need for simple, rugged, and inexpensive detectors capable of accurate and direct identification of
B. anthracis
spores. Necessary components in such detectors are stable ligands that bind tightly and specifically to target spores. By screening a phage display peptide library, we identified a family of peptides, with the consensus sequence TYPXPXR, that bind selectively to
B. anthracis
spores. We extended this work by identifying a peptide variant, ATYPLPIR, with enhanced ability to bind to
B. anthracis
spores and an additional peptide, SLLPGLP, that preferentially binds to spores of species phylogenetically similar to, but distinct from,
B. anthracis
. These two peptides were used in tandem in simple assays to rapidly and unambiguously identify
B. anthracis
spores. We envision that these peptides can be used as sensors in economical and portable
B. anthracis
spore detectors that are essentially free of false-positive signals due to other environmental
Bacillus
spores.
Development of a
mariner
-Based Transposon for Use in
Sorangium cellulosum
Authors: Julien Bryan , Fehd Ruby
Published: October 2003

In order to generate marked insertions in the myxobacterium
Sorangium cellulosum
, a transposon based on the eukaryotic
mariner
transposon was developed. The transposition frequency was increased with the use of a mutated
tnp
gene. The transposon randomly inserts into the chromosome, as demonstrated by targeted mutagenesis of the
epoK
gene.
Effects of Minerals on Resistance of
Bacillus subtilis
Spores to Heat and Hydrostatic Pressure
Authors: Igura Noriyuki , Kamimura Yuya , Islam M. Shahidul , Shimoda Mitsuya , Hayakawa Isao
Published: October 2003

Among
Bacillus subtilis
IFO13722 spores sporulated at 30, 37, and 44°C, those sporulated at 30°C had the highest resistance to treatments with high hydrostatic pressure (100 to 300 MPa, 55°C, 30 min). Pressure resistance increased after demineralization of the spores and decreased after remineralization of the spores with Ca
2+
or Mg
2+
, whereas the resistance did not change when spores were remineralized with Mn
2+
or K
+
, suggesting that former two divalent ions were involved in the activation of cortex-lytic enzymes during germination.
Teschoviruses as Indicators of Porcine Fecal Contamination of Surface Water
Authors: Jiménez-Clavero Miguel Angel , Fernández Carlos , Ortiz José Antonio , Pro Javier , Carbonell Gregoria , Tarazona José Vicente , Roblas Neftalí , Ley Victoria
Published: October 2003

Teschoviruses specifically infect pigs and are shed in pig feces. Hence, their presence in water should indicate contamination with pig fecal residues. To assess this hypothesis, we have developed a real-time reverse transcriptase PCR (RT-PCR) method that allows the quantitative detection of pig teschovirus (PTV) RNA. The method is able to detect 92 fg of PTV RNA per ml of sample. Using this method, we have detected the presence of PTV RNA in water and fecal samples from all pig farms examined (
n
= 5). Feces from other animal species (cattle, sheep, and goats) were negative in this test. To compare the PTV RNA detection method with conventional chemical determinations currently in use for evaluation of water contamination, we analyzed water samples collected downstream from a pig slurry spillage site. We have found a positive correlation within both types of determinations. The sensitivity of the PTV detection assay was similar to that achieved by unspecific organic matter determination and superior to all other conventional chemical analyses performed. Furthermore, the new method is highly specific, revealing the porcine origin of the contamination, a feature that is lacking in currently available methods for the assessment of water contamination.
Comparison of
Campylobacter
Isolates from Poultry and Humans: Association between In Vitro Virulence Properties, Biotypes, and Pulsed-Field Gel Electrophoresis Clusters
Authors: Nadeau Éric , Messier Serge , Quessy Sylvain
Published: October 2003

The in vitro virulence properties of 197 temporally and geographically related
Campylobacter
isolates from chicken broilers and humans were compared. Comparisons of the virulence properties associated with genotypes and biotypes were made. All isolates adhered to, and 63% invaded, INT-407 cells, whereas 13% were cytotoxic for CHO cells. CHO cell-cytotoxic extracts were also cytotoxic for INT-407 cells, but the sensitivity for Vero cells was variable. The proportion of isolates demonstrating a high invasiveness potential (>1,000 CFU ml
−1
) or Vero cell cytotoxicity was significantly higher for human than for poultry isolates. Invasiveness was associated with
Campylobacter jejuni
isolates of biotypes 1 and 2, whereas CHO and INT-407 cell cytotoxicity was associated with
C. jejuni
isolates of biotypes 3 and 4. Cytotoxic isolates were also clustered according to pulsed-field gel electrophoresis profiles.
Formate-Dependent Growth and Homoacetogenic Fermentation by a Bacterium from Human Feces: Description of
Bryantella formatexigens
gen. nov., sp. nov
Authors: Wolin Meyer J. , Miller Terry L. , Collins Matthew D. , Lawson Paul A.
Published: October 2003

Formate stimulates growth of a new bacterium from human feces. With high formate, it ferments glucose to acetate via the Wood-Ljungdahl pathway. The original isolate fermented vegetable cellulose and carboxymethylcellulose, but it lost this ability after storage at −76°C. 16S rRNA gene sequencing identifies it as a distinct line within the
Clostridium coccoides
supra-generic rRNA grouping. We propose naming it
Bryantella formatexigens
gen. nov., sp. nov.
Multiplex Real-Time PCR Method To Identify Shiga Toxin Genes
stx1
and
stx2
and
Escherichia coli
O157:H7/H
−
Serotype
Authors: Jinneman Karen C. , Yoshitomi Ken J. , Weagant Stephen D.
Published: October 2003

A multiplex real-time PCR method to simultaneously detect the
stx1
and
stx2
genes of Shiga toxin-producing
Escherichia coli
and a unique conserved single-nucleotide polymorphism in the
E. coli
O157:H7/H
−
uidA
gene has been developed. There is more than 98.6% sensitivity and 100% specificity for all three gene targets based on a panel of 138 isolates. The PCR efficiencies were ≥1.89, and as few as 6 CFU/reaction could be detected.
The High Salt Requirement of the Moderate Halophile
Chromohalobacter salexigens
DSM3043 Can Be Met Not Only by NaCl but by Other Ions
Authors: O'Connor Kathleen , Csonka Laszlo N.
Published: October 2003

The growth rate of
Chromohalobacter salexigens
DSM 3043 can be stimulated in media containing 0.3 M NaCl by a 0.7 M concentration of other salts of Na
+
, K
+
, Rb
+
, or NH
4
+
, Cl
−
, Br
−
, NO
3
−
, or SO
4
2−
ions. To our knowledge, growth rate stimulation by a general high ion concentration has not been reported for any organism previously.
Distribution of Bovine Herpesvirus Type 5 DNA in the Central Nervous Systems of Latently, Experimentally Infected Calves
Authors: Vogel Fernanda Silveira Flôres , Caron Luizinho , Flores Eduardo Furtado , Weiblen Rudi , Winkelmann Evandro Reinoldo , Mayer Sandra Vanderli , Bastos Reginaldo Gaspar
Published: October 2003

Bovine herpesvirus type 5 (BHV-5) is an alphaherpesvirus associated with meningoencephalitis, a disease highly prevalent in South America. In this study, we investigated the distribution of BHV-5 DNA in the brains of latently, experimentally infected calves by using a PCR for the glycoprotein B gene. Twelve calves inoculated intranasally with a Brazilian BHV-5 isolate were divided into two groups: group A calves (n= 4) were euthanized 55 days postinoculation (p.i.) for tissue collection; group B calves (n= 8) were submitted to dexamethasone administration at day 60 p.i. for reactivation of latent infection and were euthanized 50 days later. Latent infection was reactivated in all group B calves, as demonstrated by virus isolation from nasal secretions and/or seroconversion. Three calves developed neurological disease and died or were euthanized in extremis. For group A calves, viral DNA was consistently detected in the trigeminal ganglia (4/4), midbrain (4/4), thalamus (4/4), and olfactory cortex (4/4) and less frequently in the pons (3/4), cerebellum (3/4), anterior cerebral cortex (2/4), and olfactory bulb (2/4). For calves previously submitted to reactivation (group B), viral DNA was detected with roughly the same frequency in the same areas as for the group A calves. In addition, viral DNA was detected in the posterior (5/5) and dorso-lateral cortex (3/5). All DNA-positive tissues were negative for infectivity and viral antigens. These results demonstrated that latent BHV-5 DNA is present in several areas of the brain during latent infection and that virus reactivation may result in the establishment of latent infection in additional sites of the brain.
Etiology of Acute Diarrhea in Adults in Southwestern Nigeria
Authors: Okeke Iruka N. , Ojo Oladipupo , Lamikanra Adebayo , Kaper James B.
Published: October 2003

Stool specimens from 113 adult outpatients with diarrhea in southwestern Nigeria and 63 controls were examined for bacterial and parasitic enteric pathogens. Enterohemorrhagic
Escherichia coli
(EHEC) (
P
< 0.02), enteroaggregative
E. coli
(EAEC) (
P
< 0.02), and
Entamoeba histolytica
(
P
< 0.0002) were significantly associated with diarrhea.
Salmonella
,
Shigella
, nontoxigenic
Vibrio cholerae
, other categories of diarrheagenic
E. coli
, as well as a variety of helminths were recovered more frequently from the stools of patients than from the stools of controls but did not show a significant association with disease. Multiple pathogens were recovered from 36.3% of specimens, and bloody diarrhea was commonly associated with
E. histolytica
and diarrheagenic
E. coli
infections. The majority of EHEC isolates were non-O157 strains that carried the
stx
2
gene. Of the 23 EHEC-infected patients, 12 (52.2%) presented during the 10th week of the study. EHEC strains isolated within this cluster were more likely to hybridize with the enterohemolysin gene probe, to be nonmotile and sorbitol positive, and to fail to agglutinate O157 antisera. Pulsed-field gel electrophoresis demonstrated that the only strains with
Xba
I profiles that occurred more than once were isolated during the 10th and 11th weeks of the study, suggesting an outbreak. The study has demonstrated that
E. histolytica
, EHEC, and EAEC are important diarrheal pathogens within the study area and that sporadic and epidemic EHEC infections occur in developing as well as developed countries. Routine surveillance for diarrheagenic
E. coli
, even only at the tertiary-care level, would be useful in identifying outbreaks and assist in identifying environmental reservoirs and transmission routes.
Molecular Detection and Identification of Influenza Viruses by Oligonucleotide Microarray Hybridization
Authors: Sengupta Srikumar , Onodera Kenji , Lai Alexander , Melcher Ulrich
Published: October 2003

Microarrays of virus-specific oligonucleotides may provide a method of screening samples for the presence or absence of a large variety of viruses simultaneously. Influenza viruses are ideal for evaluating such microarrays because of their genetic and host diversity, and the availability of an extensive sequence database. A collection of 476 influenza virus-specific oligonucleotides was spotted onto glass slides as probes. Viral RNAs were reverse transcribed and amplified by PCR, and the products were labeled with cyanine dyes. The presence of viruses and their identities were determined by hybridization. The fluorescence intensities of oligonucleotide spots were highly reproducible within each slide and satisfactorily proportional between experiments. However, the intensities of probe spots completely complementary to target sequences varied from background to saturation. The variations did not correlate with base composition, nucleotide sequence, or internal secondary structures. Therefore, thresholds for determining whether hybridization to a spot should be judged as positive were assigned individually. Considering only positive spots from probes predicted to be monospecific for influenza virus species, subtype, host source, or gene segment, this method made correct identifications at the species, hemagglutinin subtype, and gene segment levels. Monospecific neuraminidase (NA) subtype probes were insufficiently diverse to allow confident NA subtype assignment. Incorporating positive spots from polyspecific probes into the identification scheme gave similar results. Overall, the results demonstrate the potential of microarray-based oligonucleotide hybridization for multiple virus detection.
Contribution of the PlateliaCandida-Specific Antibody and Antigen Tests to Early Diagnosis of SystemicCandida tropicalisInfection in Neutropenic Adults
Authors: Sendid Boualem , Caillot Denis , Baccouch-Humbert Bénédicte , Klingspor Lena , Grandjean Monique , Bonnin Alain , Poulain Daniel
Published: October 2003

The PlateliaCandida-specific antigen and antibody assays (Bio-Rad Laboratories) were used to test serial serum samples from seven neutropenic adult patients with hematological malignancies who had developed systemicCandida tropicalisinfections. The diagnosis of candidiasis was based on a positive blood culture (all seven patients) and the isolation ofC. tropicalisfrom a normally sterile site (six patients). All patients received early antifungal therapy with amphotericin B and/or an azole derivative and had successful outcomes. When the combined assays were applied to sera collected at different time points before and after the first positive blood culture, all patients tested positive. In six patients, at least one positive test was obtained with sera collected, on average, 5 days (range, 2 to 10 days) prior to the first positive blood culture, while blood cultures were constantly negative. High and persistent mannanemias were detected in all patients during the neutropenic period. In five patients, an increased antibody response was detected when the patients recovered from aplasia. Controls consisted of 48 serum samples from 12 febrile neutropenic patients with aspergillosis (n= 4), bacteremia (n= 4), or no evidence of infection (n= 4). A low level of mannanemia was detected in only one serum sample, and none showed significantCandidaantibody titers. Our data thus confirm the value of the combined detection of mannanemia and antimannan antibodies in individuals at risk of candidemia and suggest that in neutropenic patients, an approach based on the regular monitoring of both markers could contribute to the earlier diagnosis ofC. tropicalissystemic infection.
Rapid Diagnosis of Mycobacterial Infections and Quantitation of
Mycobacterium tuberculosis
Load by Two Real-Time Calibrated PCR Assays
Authors: Broccolo Francesco , Scarpellini Paolo , Locatelli Giuseppe , Zingale Anna , Brambilla Anna M. , Cichero Paola , Sechi Leonardo A. , Lazzarin Adriano , Lusso Paolo , Malnati Mauro S.
Published: October 2003

Sensitive and specific techniques to detect and identify
Mycobacterium tuberculosis
directly in clinical specimens are important for the diagnosis and management of patients with tuberculosis (TB). We developed two real-time PCR assays, based on the IS
6110
multicopy element and on the
senX3
-
regX3
intergenic region, which provide a rapid method for the diagnosis of mycobacterial infections. The sensitivity and specificity of both assays were established by using purified DNA from 71 clinical isolates and 121 clinical samples collected from 83 patients, 20 of whom were affected by TB. Both assays are accurate, sensitive, and specific, showing a complementary pattern of
Mycobacterium
recognition: broader for the IS
6110
-based assay and restricted to the
M. tuberculosis
complex for the
senX3
-
regX3
-based assay. Moreover, the addition of a synthetic DNA calibrator prior to DNA extraction allowed us to measure the efficiency of DNA recovery and to control for the presence of PCR inhibitors. The mycobacterial burden of the clinical samples, as assessed by direct microscopy, correlates with the
M. tuberculosis
DNA load measured by the
senX3
-
regX3
-based assay. In addition, reduced levels of
M. tuberculosis
DNA load are present in those patients subjected to successful therapy, suggesting a potential use of this assay for monitoring treatment efficacy. Therefore, these assays represent a fully controlled high-throughput system for the evaluation of mycobacterial burden in clinical specimens.
Fast and Accurate Quantitative Detection of
Helicobacter pylori
and Identification of Clarithromycin Resistance Mutations in
H
.
pylori
Isolates from Gastric Biopsy Specimens by Real-Time PCR
Authors: Lascols Christine , Lamarque Dominique , Costa Jean-Marc , Copie-Bergman Christiane , Le Glaunec Jeanne-Marie , Deforges Lionel , Soussy Claude-James , Petit Jean-Claude , Delchier Jean-Charles , Tankovic Jacques
Published: October 2003

Rapid identification of patients infected with clarithromycin-resistant
Helicobacter pylori
without the need for culture can help to avoid useless prescriptions of clarithromycin. We developed and tested a routine real-time quantitative PCR assay dedicated to that purpose. One hundred ninety-six consecutive gastric biopsy specimens were examined by culture, histology performed by a trained physician, and rapid PCR with the LightCycler apparatus. Infection was defined as (i) positivity of culture, (ii) positivity of histology, or (iii) positivity of PCR if confirmed by positivity of a concomitant indirect test (serology or urea breath test). Susceptibility to clarithromycin was tested by E-test and PCR. The prevalence of infection was 33.7% (66 of 196 samples). The sensitivities of culture, histology, and PCR were 90.9% (60 of 66 samples), 87.9% (58 of 66 samples), and 97.0% (64 of 66 samples), respectively. The specificity of PCR was 94.6% (123 of 130 samples). The linearity of the PCR results was achieved over a 6-log range of input DNA, and we were able to accurately quantify as few as 300 bacteria and to qualitatively detect as few as 30 bacteria per DNA sample. For clarithromycin susceptibility testing, there was 98.2% (55 of 56 samples) concordance between E-test and PCR. Forty-eight strains were clarithromycin susceptible, and 9 strains were clarithromycin resistant. The single discrepancy concerned a culture which was a mixture of mutant and wild type, with a susceptible-to-resistant ratio of 11.5: the resistant population was detected by E-test but not by PCR. Our PCR assay is accurate for fast detection of
H. pylori
as well as of clarithromycin resistance and is also able to objectively determine bacterial density.
Association between Handling of Pet Treats and Infection with
Salmonella enterica
Serotype Newport Expressing the AmpC β-Lactamase, CMY-2
Authors: Pitout Johann D. D. , Reisbig Mark D. , Mulvey Mike , Chui Linda , Louie Marie , Crowe Larry , Church Deirdre L. , Elsayed Sameer , Gregson Dan , Ahmed Rafiq , Tilley Peter , Hanson Nancy D.
Published: October 2003

Resistance to the extended-spectrum cephalosporins can occur in
Salmonella
species via the production of extended-spectrum and AmpC β-lactamases. We describe human infections with
Salmonella enterica
serotype Newport phage type 14 strains resistant to ceftazidime (CAZ) and cefoxitin (FOX) related to the handling of pet treats containing dried beef. These strains were isolated from five patients in Calgary, Alberta, Canada, during 2002 and were compared to a strain cultured from a commercial pet treat present at the property of one of the patients. The strains were resistant to FOX, CAZ, cefpodoxime, ampicillin, and chloramphenicol; intermediate resistant to ceftriaxone and cefotaxime; and sensitive to the aminoglycosides, ciprofloxacin, cefepime, and imipenem. Isoelectric focusing, multiplex PCR, and sequencing of the amplicons showed that all strains produced the plasmid-encoded AmpC β-lactamase, CMY-2. Restriction analysis of plasmid DNA following transformation demonstrated that
bla
CMY-2
was encoded on an approximately 140-kb plasmid. Pulsed-field gel electrophoresis showed the human and pet treat
Salmonella
strains to be highly related. This study is the first to implicate the transfer of multidrug-resistant
Salmonella
species through the handling of commercial pet treats containing animal products. In addition to documenting the first cases of human infection caused by CMY-2-producing
S. enterica
serotype Newport strains in Canada, this study illustrates the necessity of rapid and accurate laboratory-based surveillance in the identification of novel types of antimicrobial resistance.
Restriction Fragment Length Polymorphism Analysis of Ribosomal DNA Intergenic Regions Is Useful for Differentiating Strains of
Trichophyton mentagrophytes
Authors: Mochizuki Takashi , Ishizaki Hiroshi , Barton Richard C. , Moore Mary K. , Jackson Colin J. , Kelly Steven L. , Evans E. Glyn V.
Published: October 2003

Twenty isolates of
Tricophyton mentagrophytes
var.
mentagrophytes
and 47 isolates of
T. mentagrophytes
var.
interdigitale
, identified by morphological characteristics, were screened by restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). Sixty isolates (14 of 20
T. mentagrophytes
var.
mentagrophytes
isolates and 46 of 47
T. mentagrophytes
var.
interdigitale
isolates) shared an identical ITS RFLP profile and were further investigated by using a probe targeted to the rDNA nontranscribed spacer (NTS) region. Polymorphisms were observed in the NTS regions of both
T. mentagrophytes
var.
mentagrophytes
and
T. mentagrophytes
var.
interdigitale
isolates. Twenty-three individual RFLP patterns (DNA types P-1 to P-12 and A-1 to A-11) were recognized and divided into two groups depending on the presence (P) or absence (A) of a 2.5-kb band, which correlated to a large extent with the morphological variety. Eleven of 14
T. metagrophytes
var.
mentagrophytes
isolates were A types, and all of the 46
T. mentagrophytes
var.
interdigitale
isolates were P types. A majority of strains (23 of 60 [38.3%]) were characterized by one RFLP pattern (pattern P-1), and eight types (P-1 to P-6, P-8, and P-9) accounted for 75% (45 of 60) of all strains, including all of the
T. mentagrophytes
var.
interdigitale
isolates. The remaining 15 types were represented by one only isolate and included all of the
T. mentagrophytes
var.
mentagrophytes
isolates. We conclude that RFLP analysis of the rDNA NTS region is a valuable technique for differentiation of
T. mentagrophytes
strains. Furthermore, by use of this method, there appears to be a greater degree of diversity among
T. mentagrophytes
var.
mentagrophytes
isolates than among
T. mentagrophytes
var.
interdigitale
isolates.
Effect of Duplicate Isolates of Methicillin-Susceptible and Methicillin-ResistantStaphylococcus aureuson Antibiogram Data
Authors: Horvat Rebecca T. , Klutman Neil E. , Lacy Melinda K. , Grauer Dennis , Wilson Marsha
Published: October 2003

DuplicateStaphylococcus aureusisolates were analyzed to determine the impact of multiple isolates from the same patient on annual antibiogram data. During a 6-year period (1996 to 2001), 3,227 patients with 4,844S. aureusisolates were evaluated. A total of 39% of patients with methicillin-resistantS. aureus(MRSA) (n= 860) and 23% of patients with methicillin-susceptibleS. aureus(MSSA) (n= 2,367) infections had duplicate isolates. Cumulative data show that 91% of the patients during this 6-year period with duplicate isolates (2 to 13 duplicates/year) did not switch between MSSA and MRSA but retained the originalS. aureusstrain whether it was MSSA or MRSA. Rates of MRSA were calculated for each year by using all isolates and then eliminating duplicates. The impact of duplicate MRSA and MSSA isolates was evaluated by using the ratio of isolates per patient such that ratios of >1.0 indicate >1 isolate per patient. The 6-year ratio for MRSA was 1.90 isolates/patient, and the ratio for MSSA was 1.35. A significant difference (P< 0.05) was noted in the MRSA rates in 4 of 6 years when duplicate isolates were removed. Common phenotypic antibiogram patterns were compared for all MRSA isolates during the 6-year period, and 64% were of a single antibiogram phenotype. Eighty-eight percent of patients with duplicate MRSA isolates had phenotypically identical multiple isolates. The rate of MRSA differs when duplicate isolates are removed from the antibiogram data.
emm
Typing of M Nontypeable Invasive Group A Streptococcal Isolates in Israel
Authors: Moses Allon E. , Hidalgo-Grass Carlos , Dan-Goor Mary , Jaffe Joseph , Shetzigovsky Ilanit , Ravins Miriam , Korenman Zinaida , Cohen-Poradosu Ronit , Nir-Paz Ran
Published: October 2003

We performed
emm
typing of M nontypeable invasive group A streptococcal (GAS) isolates collected in a prospective population-based study in Israel. One hundred twenty of 131 isolates (92%) had
emm
sequences compatible with GAS, consisting of 51 different
emm
types. Eleven isolates were found to be group G streptococcus. Of the 120 isolates, 55 (46%) belonged to 32 types for which there were no typing sera available in the Streptococcal Reference Laboratory in Israel. The other 65 (64%) isolates, consisting of 19 types, had sera available and therefore could have been serotyped. Forty-three isolates had T and
emm
types which were not correlated according to standard M-typing protocols and were therefore missed. The principal effect of
emm
typing was the addition of 32 types not previously identified in Israel and the discovery of new associations between
emm
and T types.
emm
typing did not significantly change the proportion of M types; the five most common types were 3, 28, 2, 62, and 41. Twenty different types comprised 80% of all isolates. No new
emm
sequences were discovered.
emm
typing emphasized the unusually low incidence of M1 strains causing severe disease in Israel. As serological typing of GAS becomes more problematic due to lack of sera and the appearance of new
emm
types, reference laboratories should replace M typing with
emm
sequence typing. Development of a GAS vaccine relies on the
emm
type distributions in different geographical locations. In our study, 7% of isolates (types 41 and 62) are not included in a 26-valent vaccine that is being studied.
Decreased Vancomycin Susceptibility of Coagulase-Negative Staphylococci in a Neonatal Intensive Care Unit: Evidence of Spread of
Staphylococcus warneri
Authors: Center Kimberly J. , Reboli Annette C. , Hubler Robin , Rodgers Gail L. , Long Sarah S.
Published: October 2003

Coagulase-negative staphylococci (CoNS) are important pathogens in premature neonates; decreasing glycopeptide susceptibility has been observed among these isolates. The epidemiology of colonization with CoNS, the organisms' vancomycin susceptibilities, and genetic relatedness were studied over 6 months in a tertiary-care neonatal unit. A total of 321 isolates of CoNS were isolated. Seventy-five percent of the infants were colonized at admission, and virtually all were colonized thereafter. Common species were
Staphylococcus epidermidis
(69%),
S. warneri
(12%),
S. haemolyticus
(9.7%), and
S. hominis
(5.6%). A total of 3.9% of CoNS isolates had decreased vancomycin susceptibility (DVS) (MICs > 2.0 μg/ml); isolate recovery was associated with a stay in a neonatal intensive care unit for >28 days (
P
= 0.039), vancomycin exposure (
P
= 0.021), and
S. warneri
colonization (
P
< 0.0001). Nine of 12 (75%) CoNS with DVS were
S. warneri
, had enhanceable high-level resistance in vitro, were indistinguishable or closely related by pulsed-field gel electrophoresis, and were different from 29 vancomycin-susceptible
S. warneri
isolates. Epidemiological analysis suggested unsuspected nosocomial spread. Species determination in certain settings may aid in the understanding of emerging nosocomial problems.
Prediction of Decreased Susceptibility to Penicillin of
Neisseria meningitidis
Strains by Real-Time PCR
Authors: Stefanelli Paola , Carattoli Alessandra , Neri Arianna , Fazio Cecilia , Mastrantonio Paola
Published: October 2003

Sequence analysis of the
penA
gene, encoding penicillin-binding protein 2 (PBP2), in 30 penicillin-intermediate (PenI)
Neisseria meningitidis
strains showed altered gene sequences due to the translocation of exogenous DNA blocks derived from commensal neisseriae, which are known to have PBP2 proteins with decreased affinity for the antibiotic. In order to obtain a rapid and reproducible method for predicting the PenI phenotype, a real-time PCR assay was set up with primers and probes designed on the basis of the
penA
gene. The A→G mutation at codon 566, in the transpeptidase domain of the
penA
gene (which is present in the whole sample of 30 PenI strains and in all the 41 sequences of PenI meningococci isolated worldwide and has been deposited in the sequence databank), was chosen as a marker of
penA
translocations. Two hybridization probes were designed to distinguish the wild-type
penA
gene in penicillin-susceptible (PenS) meningococci from the mutated
penA
gene at codon 566 in PenI strains. Thermal analysis of probe hybridization revealed a melting temperature difference of at least 6°C between PenI and PenS strains. This real-time PCR protocol characterizes the penicillin phenotype of
N. meningitidis
in a few hours without DNA sequencing and is useful for rapid screening of the penicillin-intermediate genotype among meningococcal isolates.
Comparison of a Shiga Toxin Enzyme-Linked Immunosorbent Assay and Two Types of PCR for Detection of Shiga Toxin-Producing
Escherichia coli
in Human Stool Specimens
Authors: Pulz Matthias , Matussek Andreas , Monazahian Masyar , Tittel Andreas , Nikolic Elisabet , Hartmann Maike , Bellin Tobias , Buer Jan , Gunzer Florian
Published: October 2003

Shiga toxin (Stx)-producing
Escherichia coli
(STEC) is a major cause of sporadic cases of disease as well as serious outbreaks worldwide. The spectrum of illnesses includes mild nonbloody diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome. STEC produces one or more Stxs, which are subdivided into two major classes, Stx1 and Stx2. The ingestion of contaminated food or water, person-to-person spread, and contact with animals are the major transmission modes. The infective dose of STEC may be less than 100 organisms. Effective prevention of infection is dependent on rapid detection of the causative bacterial pathogen. In the present study, we examined 295 stool specimens for the presence of Stx-producing
E
.
coli
by three different methods: an Stx enzyme-linked immunosorbent assay, a conventional PCR assay, and a LightCycler PCR (LC-PCR) assay protocol recently developed by our laboratory at the Institute of Medical Microbiology at Hannover Medical School. Our intent was to compare these three methods and to examine the utility of the STEC LC-PCR protocol in a clinical laboratory. The addition of a control DNA to each sample to clearly discriminate inhibited specimens from negative ones enhanced the accuracy of the LC-PCR protocol. From our results, it can be concluded that LC-PCR is a very useful tool for the rapid and safe detection of STEC in clinical samples.
Genotypic Analyses of
Vibrio parahaemolyticus
and Development of a Pandemic Group-Specific Multiplex PCR Assay
Authors: Okura Masatoshi , Osawa Ro , Iguchi Atsushi , Arakawa Eiji , Terajima Jun , Watanabe Haruo
Published: October 2003

A total of 54
Vibrio parahaemolyticus
strains including pandemic O3:K6 strains and newly emerged O4:K68, O1:K25, O1:K26, and O1:K untypeable strains (collectively referred to as the “pandemic group”) were examined for their pulsed-field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR) profiles and for the presence or absence of genetic marker DNA sequences,
toxRS
/
new
or
orf8
, that had been reported elsewhere to be specific for the pandemic group. Both PFGE and AP-PCR analyses indicated that all strains of the pandemic group formed a distinct genotypic cluster, suggesting that they originated from the same clone. In addition to the pandemic group, four O3:K6 strains that did not possess the thermostable direct hemolysin (
tdh
) gene also belonged to this cluster and possessed the
toxRS
/
new
sequence. However, three O3:K6 strains that clearly belonged to the pandemic group by PFGE and AP-PCR did not possess the
orf8
sequence. The evidence suggests that neither the
toxRS
/
new
nor the
orf8
sequence is a reliable gene marker for definite identification of the pandemic group. We therefore developed a novel multiplex PCR assay specific for the pandemic group. The assay successfully distinguished pandemic group strains from other
V. parahaemolyticus
strains by yielding two distinct PCR products for
tdh
(263 bp) and the
toxRS
/
new
sequence (651 bp).
Description of a New Yeast Species,
Malassezia japonica
, and Its Detection in Patients with Atopic Dermatitis and Healthy Subjects
Authors: Sugita Takashi , Takashima Masako , Kodama Minako , Tsuboi Ryoji , Nishikawa Akemi
Published: October 2003

Lipophilic yeasts of the genus
Malassezia
are part of the normal cutaneous microflora and are considered one of the factors that trigger atopic dermatitis (AD). We isolated two strains of
Malassezia
from a healthy Japanese female. Analysis of the D1/D2 26S ribosomal DNA and internal transcribed spacer region sequences of the isolates suggested that they are new members of the genus
Malassezia
. We propose the name
Malassezia japonica
sp. nov. for the isolates.
M
.
japonica
is easily distinguished from the seven known lipophilic species by its ability to assimilate Tween 40 and Tween 60 and its inability to assimilate Tween 20 and Tween 80 and to grow at 40°C. Furthermore, by applying transparent dressings to the skin lesions of 36 patients with AD and the skin of 22 healthy subjects,
M
.
japonica
DNA was detected by a non-culture-based method consisting of nested PCR with
M
.
japonica
species-specific primers.
M
.
japonica
DNA was detected from 12 of the 36 patients (33.3%) and 3 of the 22 healthy subjects (13.6%). Although it is not known whether
M
.
japonica
plays a role in AD, this species was part of the microflora in both patients with AD and healthy subjects.
Diagnosis of
Helicobacter pylori
Infection in Adults with Intellectual Disability
Authors: Wallace Robyn A. , Schluter Philip J. , Forgan-Smith Ross , Wood Robyn , Webb Penelope M.
Published: October 2003

Helicobacter pylori
infection is common among adults with intellectual disability. The acceptabilities and accuracies of different diagnostic tests in this population are unknown. We aimed to determine (i) patient acceptability and (ii) performance characteristics of serology, fecal-antigen, and urea breath tests among adults with intellectual disability. One hundred sixty-eight such adults underwent
H. pylori
testing with serology and fecal-antigen tests, and a portion underwent treatment. One year later, the participants were retested with fecal-antigen, serology, and urea breath tests. The numbers of specimens obtained and difficulties in collection reported by caregivers were noted. Test performance characteristics were assessed among participants and 65 of their caregivers, using serology as the reference. All participants provided at least one specimen, despite reported collection difficulties for 23% of fecal and 27% of blood specimens. Only 25% of the participants provided breath specimens; failure to perform this test was associated with lower intellectual ability and higher maladaptive behavior. The sensitivity, specificity, and positive and negative predictive values of the fecal test (baseline and 12 months versus caregivers) were 70 and 63 versus 81, 93 and 95 versus 98, 96 and 92 versus 93, and 53 and 74 versus 93%, respectively; those of the urea breath test (12 months versus caregivers) were 86 versus 100, 88 versus 95, 75 versus 89, and 94 versus 100%, respectively. With assistance, fecal or blood specimens for
H. pylori
assessment can be provided by most patients with intellectual disability regardless of their level of function or behavior. Only those with greater ability can perform the urea breath test. Using serology as the reference test, the limitations of performance characteristics of the fecal-antigen and urea breath tests are similar to those among a control group of caregivers.