bluuebunny/crossref_metadata_2025 huggingface dataset:
Several
Salmonella enterica
subsp.
enterica
Serotype 4,5,12:i:− Phage Types Isolated from Swine Samples Originate from Serotype Typhimurium DT U302
Authors: de la Torre E. , Zapata D. , Tello M. , Mejía W. , Frías N. , García Peña F. J. , Mateu E. M. , Torre E.
Published: June 2003

Pulsed-field gel electrophoresis, plasmid profiling, and phage typing were used to characterize and determine possible genetic relationships between 48
Salmonella enterica
subsp.
enterica
isolates of pig origin collected in Catalonia, Spain, from 1998 to 2000. The strains were grouped into 23 multidrug-resistant
fljB
-lacking
S. enterica
serovar 4,5,12:i:− isolates, 24
S. enterica
serovar Typhimurium isolates, and 1
S. enterica
serovar 4,5,12:−:− isolate. After combining the
Xba
I and
Bln
I macrorestriction profiles (
XB
profile), we observed 29 distinct subtypes which were grouped into seven main patterns. All 23 of the 4,5,12:i:− serovar strains and 10 serovar Typhimurium isolates were found to have pattern AR, and similarities of >78% were detected among the subtypes. Three of the serovar Typhimurium DT U302 strains (strains T3, T4, and T8) were included in the same 4,5,12:i:− serovar cluster and shared a plasmid profile (profile I) and a pattern of multidrug resistance (resistance to ampicillin, chloramphenicol, streptomycin, sulfonamide, tetracycline, gentamicin, and trimethoprim-sulfamethoxazole) commonly found in monophasic isolates. This led us to the conclusion that strains of the
S. enterica
4,5,12:i:− serovar might have originated from an
S. enterica
serovar Typhimurium DT U302 strain.
Transient and PersistentHelicobacter pyloriColonization in Native American Children
Authors: Pérez-Pérez Guillermo I. , Sack R. Bradley , Reid Raymond , Santosham Mathuram , Croll Janne , Blaser Martin J.
Published: June 2003

Helicobacter pyloriis chiefly acquired in childhood, but the exact timing of acquisition is not well understood. The main goal of this study was to assessH. pyloriacquisition in a pediatric population. We studied two cohorts of Native American children: a birth cohort of 50 children and 58 older children (mean age, 53 months). We measured serum immunoglobulin G (IgG), IgM, and IgA antibodies toH. pyloriwhole-cell antigen and IgG antibodies to CagA. Among 44 birth cohort children monitored for more than 12 months, 24 (54.5%) had seroconversions, 7 (15.9%) were transient, and 17 (38.6%) were persistent. Among the older children, 49 (84.5%) of the 58 children were monitored for 1 year; 34 (69.4%) hadH. pyloriantibodies at study entry. During the next year, 7 (20.6%) children seroreverted, and of 15 initially negative children, 5 (33.3%) seroconverted. In both groups, evaluation of CagA antibodies increased the sensitivity ofH. pyloridetection. Serum pepsinogen I (PGI) levels inH. pylori-negative children rose significantly until age 6 months and remained constant for the next 19 months. At the time ofH. pyloriseroconversion, PGI peaked to levels significantly higher than in the never-seroconverted (P= 0.02) and the pre-seroconverted (P= 0.03) children, but then declined to levels paralleling those ofH. pylori-negative children. Thus,H. pyloriacquisition, accompanied by a transient PGI increase, was frequent in this population, especially in the second and third years of life, but often was brief.
Development of Group- and Serotype-Specific One-Step SYBR Green I-Based Real-Time Reverse Transcription-PCR Assay for Dengue Virus
Authors: Shu Pei-Yun , Chang Shu-Fen , Kuo Yu-Chung , Yueh Yi-Yun , Chien Li-Jung , Sue Chien-Lin , Lin Ting-Hsiang , Huang Jyh-Hsiung
Published: June 2003

A quantitative one-step SYBR Green I-based reverse transcription (RT)-PCR system was developed for the detection and differentiation of four different dengue virus serotypes in acute-phase serum samples. A set of group- and serotype-specific primer pairs was designed against conserved sequences in the core region and evaluated for clinical diagnosis. A linear relationship was obtained between the amount of input RNA and cycle threshold (Ct) value over a range of 10 to 10
7
PFU per ml of cell culture-derived dengue viruses. The detection limit of the group-specific primer pair was between 4.1 and 43.5 PFU/ml for four dengue serotypes. The detection limit of each of the serotype-specific primer pairs was calculated to be 10 PFU/ml for dengue virus serotype 1 (DEN-1), 4.6 PFU/ml for DEN-2, 4.1 PFU/ml for DEN-3, and 5 PFU/ml for DEN-4. Comparisons between the one-step SYBR Green-based RT-PCR assay and the conventional cell culture method in the clinical diagnosis of dengue virus infection from acute-phase serum samples of confirmed dengue patients were performed. The results showed that 83 and 67% of 193 acute-phase serum samples tested were positive by the one-step SYBR Green-based RT-PCR method and cell culture method, respectively. Further analysis showed that the one-step SYBR Green-based RT-PCR method could detect twice as many acute-phase serum samples with positive dengue-specific immunoglobulin M (IgM) and/or IgG antibodies than cell culture method. Our results demonstrate the potential clinical application of the one-step SYBR Green I-based RT-PCR assay for the detection and differentiation of dengue virus RNA.
Limitations of TaqMan PCR for Detecting Divergent Viral Pathogens Illustrated by Hepatitis A, B, C, and E Viruses and Human Immunodeficiency Virus
Authors: Gardner Shea N. , Kuczmarski Thomas A. , Vitalis Elizabeth A. , Slezak Tom R.
Published: June 2003

Recent events illustrate the imperative to rapidly and accurately detect and identify pathogens during disease outbreaks, whether they are natural or engineered. Particularly for our primary goal of detecting bioterrorist releases, detection techniques must be both species-wide (capable of detecting all known strains of a given species) and species specific. Due to classification restrictions on the publication of data for species that may pose a bioterror threat, we illustrate the challenges of finding such assays using five nonthreat organisms that are nevertheless of public health concern: human immunodeficiency virus (HIV) and four species of hepatitis viruses. Fluorogenic probe-based PCR assays (TaqMan; Perkin-Elmer Corp., Applied Biosystems, Foster City, Calif.) may be sensitive, fast methods for the identification of species in which the genome is conserved among strains, such as hepatitis A virus. For species such as HIV, however, the strains are highly divergent. We use computational methods to show that nine TaqMan primer and probe sequences, or signatures, are needed to ensure that all strains will be detected, but this is an unfeasible number, considering the cost of TaqMan probes. Strains of hepatitis B, C, and E viruses show intermediate divergence, so that two to three TaqMan signatures are required to detect all strains of each virus. We conclude that for species such as hepatitis A virus with high levels of sequence conservation among strains, signatures can be found computationally for detection by the TaqMan assay, which is a sensitive, rapid, and cost-effective method. However, for species such as HIV with substantial genetic divergence among strains, the TaqMan assay becomes unfeasible and alternative detection methods may be required. We compare the TaqMan assay with some of the alternative nucleic acid-based detection techniques of microarray, chip, and bead technologies in terms of sensitivity, speed, and cost.
Detection of Marek's Disease Virus DNA in Chicken but Not in Human Plasma
Authors: Hennig Holger , Osterrieder Nikolaus , Müller-Steinhardt Michael , Teichert Hanns-Martin , Kirchner Holger , Wandinger Klaus-Peter
Published: June 2003

Marek's disease virus (MDV) causes a common lymphomatous and neuropathic disease in domestic chickens and, less commonly, turkeys and quail. It is a member of the α-herpesviruses and until now was considered to be strongly cell associated. In 1991, MDV was suggested to be the causative infectious agent of multiple sclerosis (MS) in humans. In a previous study, we investigated the leukocytes of 107 well-defined MS patients for the presence of MDV DNA but were unable to confirm a role for MDV in the pathogenesis of MS. A recent report (S. Laurent, E. Esnault, G. Dambrine, A. Goudeau, D. Choudat, and D. Rasschaert, J. Gen. Virol. 82:233-240, 2001) described the detection of MDV DNA in 20% of 202 human serum samples, regardless of whether the individuals were exposed to poultry. The detection of MDV DNA in chicken serum samples was reported as well. The aim of the present study was to investigate whether we can confirm the presence of MDV DNA in chickens and humans if we use plasma as the source for nucleic acid isolation. Leukocytes and plasma specimens from 16 chickens experimentally infected with MDV serotype 1 and plasma specimens from 300 volunteer blood donors were tested for MDV DNA by two different TaqMan PCR assays. MDV DNA was repeatedly found in the leukocytes as well as in the plasma specimens of all 16 animals. All human samples analyzed, however, tested negative by both assays. Accordingly, Marek's disease in chickens can be diagnosed by detection of MDV DNA in plasma as well as in leukocytes. Once again, we found no evidence for the spread of MDV to humans.
Development of a Heteroduplex Mobility Assay To Identify Field Isolates of Porcine Reproductive and Respiratory Syndrome Virus with Nucleotide Sequences Closely Related to Those of Modified Live-Attenuated Vaccines
Authors: Key K. F. , Guenette D. K. , Yoon K.-J. , Halbur P. G. , Toth T. E. , Meng X. J.
Published: June 2003

Porcine reproductive and respiratory syndrome has been devastating the swine industry since the late 1980s. The disease has been controlled, to some extent, through the use of modified live-attenuated (MLV) vaccines once available. However, such a practice periodically resulted in isolation or detection of vaccine-like viruses from pigs as determined by a partial genomic sequencing. In this study, we developed a heteroduplex mobility assay (HMA) for quickly identifying porcine reproductive and respiratory syndrome virus (PRRSV) isolates with significant nucleotide sequence identities (≥98%) with the modified live-attenuated vaccines. The major envelope gene (ORF5) of 51 PRRSV field isolates recovered before and after the introduction of the vaccines was amplified, denatured, and reannealed with the HMA reference vaccine strains Ingelvac PRRS MLV and Ingelvac PRRS ATP, respectively. Nine of the 51 field isolates and the VR2332 parent virus of Ingelvac PRRS MLV, which were all highly related to Ingelvac PRRS MLV with ≤2% nucleotide sequence divergence as determined by sequence analysis, were all identified by the HMA to form homoduplexes with the reference Ingelvac PRRS MLV. No homoduplex-forming field isolate was identified when Ingelvac PRRS ATP was used as the HMA reference except for its parent virus JA142. Other field isolates with more than 2% nucleotide sequence divergence with the respective reference vaccine strain resulted in the formation of heteroduplexes with reduced mobility in polyacrylamide gel electrophoresis. The HMA results also correlated well with the results of phylogenetic analyses. The data indicated that the HMA developed in the study may be a rapid and efficient method for large-scale screening of potential vaccine-like PRRSV field isolates for further genetic characterization.
Comparison of Commercial DNA Extraction Kits for Extraction of Bacterial Genomic DNA from Whole-Blood Samples
Authors: Smith K. , Diggle M. A. , Clarke S. C.
Published: June 2003

The demand for molecular diagnostic tests in medical microbiology has highlighted the need for efficient methods of DNA extraction. In addition, it is preferable for these methods to be automated. An example of such a requirement is for the confirmation of meningococcal disease where rapid, sensitive, and specific procedures are required for public health management purposes. Previous studies have shown that whole blood is the preferred method for the isolation of bacterial DNA in meningococcal disease, and in this study, we compare five commercially available kits for the extraction of bacterial genomic DNA from whole-blood samples. These include kits in a 96-well binding plate, 96-well filter plate, and metallic bead formats. The method for all five kits is described, and the sensitivity, specificity, ease of automation, and overall efficiency are determined.
Infection of a Ventriculoatrial Shunt with Phenotypically Variable
Staphylococcus epidermidis
Masquerading as Polymicrobial Bacteremia Due to Various Coagulase-Negative Staphylococci and
Kocuria varians
Authors: Ben-Ami Ronen , Navon-Venezia Shiri , Schwartz David , Carmeli Yehuda
Published: June 2003

The diagnosis of bloodstream infection with coagulase-negative staphylococci is frequently based on the isolation of the same organism from more than one blood culture. Phenotypic variation is a common characteristic of pathogenic strains of
Staphylococcus epidermidis
which may affect species identification by the microbiology laboratory. We describe a patient with a new onset of nephritis and gram-positive bacteremia. Gram-positive cocci grew in multiple blood cultures and were identified by the Vitek 2 system as
Kocuria varians
,
Staphylococcus hyicus
, and
S. epidermidis
. Bacterial isolates grew on blood agar and Congo red agar plates as two distinct morphotypes and exhibited phenotypic variation. Neither morphotype could be identified by the API-Staph assay. Cellular fatty acid analysis identified one of the morphotypes as
S. epidermidis
but could not identify the other morphotype. All isolates were found to be identical by pulsed-field gel electrophoresis, and both colonial morphotypes were identified as
S. epidermidis
by 16S ribosomal gene sequencing. Phenotypic variation of
S. epidermidis
may affect identification to the species level by phenotype-based identification systems. Caution should be exercised when differentiating between true infection and contamination based on strain identification.
Shiga Toxin 1c-Producing
Escherichia coli
Strains: Phenotypic and Genetic Characterization and Association with Human Disease
Authors: Friedrich Alexander W. , Borell Julia , Bielaszewska Martina , Fruth Angelika , Tschäpe Helmut , Karch Helge
Published: June 2003

The distribution of the
stx
1c
allele among Shiga toxin (Stx)-producing
Escherichia coli
(STEC) and the virulence characteristics of
stx
1c
-harboring STEC are unknown. In this study, we identified
stx
1c
in 76 (54.3%) of 140
eae
-negative, but in none of 155
eae
-positive, human STEC isolates (
P
< 0.000001). The 76
stx
1c
-harboring
E. coli
isolates belonged to 22 serotypes, and each produced Stx1c as demonstrated by latex agglutination. Characterization of putative virulence factors demonstrated the presence of the locus of proteolysis activity (LPA) and the high-pathogenicity island in 65.8 and 21.1%, respectively, of the 76 Stx1c-producing
E. coli
isolates. Moreover, all but three of these strains contained
saa
, the gene encoding an STEC autoagglutinating adhesin. The virulence profiles of Stx1c-producing
E. coli
isolates were mostly serotype independent and heterogeneous. This enabled us to subtype the isolates within the same serotype. The individuals infected with Stx1c-producing
E. coli
strains were between 3 months and 72 years old (median age, 23.5 years) and usually had uncomplicated diarrhea or were asymptomatic. We conclude that Stx1c-producing
E
.
coli
strains represent a significant subset of
eae
-negative human STEC isolates, which belong to various serotypes and frequently possess LPA and
saa
as their putative virulence factors. The phenotypic and molecular characteristics determined in this study allow the subtyping of Stx1c-producing STEC in epidemiological and clinical studies.
Evaluation of Repetitive Element Sequence-Based PCR as a Molecular Typing Method for
Clostridium difficile
Authors: Spigaglia Patrizia , Mastrantonio Paola
Published: June 2003

Repetitive element sequence-based PCR (rep-PCR) is a typing method that enables the generation of DNA fingerprinting that discriminates bacterial strains. In this study, we evaluated the applicability of rep-PCR in typing
Clostridium difficile
clinical isolates. The results obtained by rep-PCR were compared with those obtained by pulsed-field gel electrophoresis (PFGE) and PCR ribotyping. A high correspondence between pattern differentiations produced by rep-PCR and PFGE was observed, whereas PCR ribotyping showed a lower level of discriminatory power.
Genetic Relatedness of Levofloxacin-Nonsusceptible
Streptococcus pneumoniae
Isolates from North America
Authors: Johnson Crystal N. , Benjamin, William H. , Moser Stephen A. , Hollingshead Susan K. , Zheng Xiaotian , Crain Marilyn J. , Nahm Moon H. , Waites Ken B.
Published: June 2003

We characterized 32 levofloxacin-nonsusceptible
Streptococcus pneumoniae
(LNSP) isolates obtained from a broad geographic region of North America over a 5-year period by using capsular serotypes, antimicrobial susceptibility profiles, BOX-PCR, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Sixteen international clones identified by the Pneumococcal Molecular Epidemiology Network also were included for comparison. Fifteen serotypes were represented, with serogroups 6, 9, 14, 19, and 23 accounting for 63% of isolates. Among isolates whose quinolone resistance-determining regions were sequenced, all contained
gyrA
and
parC
point mutations. Sixty-three percent were penicillin susceptible, and 84% were erythromycin susceptible. BOX-PCR analysis identified 39 different band patterns among 32 LNSP and 16 international clones and grouped 16 isolates, including 2 international clones, into seven unrelated groups of 2 to 4 isolates each. PFGE analysis identified 35 different band patterns among 32 LNSP and 16 international clones and grouped 21 isolates, including 3 international clones, into eight unrelated groups of 2 to 6 isolates each. MLST performed on 10 isolates identified five allelic profiles and separated 9 isolates into four groups of 2 to 3 isolates each. Overall, each typing method indicated that the LNSP were heterogeneous and that resistance to fluoroquinolones was not closely associated with a particular serotype or with coresistance to other antimicrobial classes and suggests that LNSP have likely arisen through independent mutational events as a result of selective pressure. However, seven LNSP were found to be related to three international clones by PFGE.
Quantitation of Human Immunodeficiency Virus Type 1 in Breast Milk
Authors: Ghosh M. K. , Kuhn L. , West J. , Semrau K. , Decker D. , Thea D. M. , Aldrovandi G. M.
Published: June 2003

The distribution and stability of human immunodeficiency virus type 1 (HIV-1) in breast milk (BM) components remain largely unknown. Inhibitory effects, if any, of BM on HIV RNA and DNA PCR amplification are poorly understood. We have addressed these issues by using virus-spiked BM samples from HIV-negative women. BM samples from HIV-negative women were spiked with HIV-1 virions or cells containing a single integrated copy of HIV DNA (8E5/LAV). After incubation under different experimental conditions, viral RNA was detected by the Roche Amplicor UltraSensitive assay in whole-milk, skim milk, and lipid fractions. We found excellent correlation between HIV-1 input copy and recovery in whole milk (
r
= 0.965,
P
< 0.0001), skim milk (
r
= 0.972,
P
< 0.0001), and the lipid fraction (
r
= 0.905,
P
< 0.001). PCR inhibition was observed in less than 10% of the spiked samples. Similar levels of inhibition were noted in BM samples collected from HIV-infected women. HIV proviral DNA was detected in BM samples using real-time PCR (linear correlation between the threshold cycle versus log DNA copy number, >0.982). The effects of incubation duration and temperature and repeated freeze-thaw cycles on HIV RNA recovery were analyzed. HIV RNA levels were remarkably stable in whole milk after three freeze-thaw cycles and for up to 30 h at room temperature. Our findings improve the understanding of the dynamics of HIV detection in BM and the conditions for BM sample collection, storage, and processing.
Identification by Subtractive Hybridization of a Novel Insertion Element Specific for Two Widespread
Burkholderia cepacia
Genomovar III Strains
Authors: Liu Lixia , Spilker Theodore , Coenye Tom , LiPuma John J.
Published: June 2003

Species of the
Burkholderia cepacia
complex cause chronic and life-threatening infections in persons with cystic fibrosis. Epidemic strains infect multiple patients, reside primarily in genomovar III, and have an apparent enhanced capacity for human infection and/or interpatient transmission. By using subtractive hybridization, a novel insertion element, designated IS
1363
, was identified in epidemic strain PHDC, known to infect many cystic fibrosis patients in the mid-Atlantic region of the United States. IS
1363
was also found in most isolates of the ET12 lineage, responsible for infecting large numbers of patients in Ontario, Canada, and the United Kingdom. Southern blot analysis demonstrated that whereas multiple copies of IS
1363
were present in strain PHDC, only one copy was present in ET12 isolates. IS
1363
was used to probe a collection of 943
B. cepacia
complex isolates, representing all nine genomovars, recovered from 761 cystic fibrosis patients or the natural environment. IS
1363
was not found in other genomovar III strains and, with the exception of
B. ambifaria,
was absent from other
B. cepacia
complex species. Genotyping analyses of all IS
1363
-positive isolates demonstrated that strain PHDC was more widely distributed in the United States than previously appreciated; 212 cystic fibrosis patients in 24 states were identified as being infected with PHDC.
Dissemination of SHV-12 and Characterization of New AmpC-Type Beta-Lactamase Genes among Clinical Isolates of
Enterobacter
Species in Korea
Authors: Lee Sang Hee , Kim Jae Young , Shin Sang Heum , An Young Jun , Choi Young Wook , Jung Yeun Chang , Jung Ha Il , Sohn Eui Suk , Jeong Seok Hoon , Lee Kye Joon
Published: June 2003

To determine the prevalence and genotype of an extended-spectrum beta-lactamase and new chromosomal AmpC beta-lactamases among clinical isolates of
Enterobacter
species, we performed antibiotic susceptibility testing, pI determination, induction tests, transconjugation, enterobacterial repetitive consensus (ERIC) PCR, sequencing, and phylogenetic analysis. Among the 51 clinical isolates collected from a university hospital in Korea, 6 isolates have been shown to produce SHV-12 and inducible AmpC beta-lactamases. These also included three isolates producing TEM-1b and one strain carrying TEM-1b and CMY-type beta-lactamases with a pI of 8.0. The results from ERIC PCR revealed that six isolates were genetically unrelated, suggesting that dissemination of SHV-12 was responsible for the spread of resistance to extended-spectrum beta-lactams in Korea. Six genes of inducible AmpC beta-lactamases that are responsible for the resistance to cephamycins (cefoxitin and cefotetan), amoxicillin, cephalothin, and amoxicillin-clavulanic acid were cloned and characterized. A 1,165-bp DNA fragment containing the
ampC
genes was sequenced and found to have an open reading frame coding for a 381-amino-acid beta-lactamase. The nucleotide sequence of four
ampC
genes (
bla
EcloK992004.1
,
bla
EcloK995120.1
,
bla
EcloK99230
, and
bla
EareK9911729
) shared considerable homology with that of AmpC-type class C beta-lactamase genes of gram-negative bacteria, especially that of the chromosomal
ampC
gene (
bla
EcloMHN1
) of
Enterobacter cloacae
MHN1 (99.9, 99.7, 99.6, and 99.6% identity, respectively). The sequences of two
ampC
genes (
bla
EcloK9973
and
bla
EcloK9914325
) showed close similarity to the chromosomal
ampC
gene (
bla
EcloQ908R
) of
E. cloacae
Q908R (99.7% identity). The results from phylogenetic analysis suggested that six
ampC
genes could originate from
bla
EcloMHN1
or
bla
EcloQ908R
.
Rapid Detection of
vanA
and
vanB
Genes Directly from Clinical Specimens and Enrichment Broths by Real-Time Multiplex PCR Assay
Authors: Palladino Silvano , Kay Ian D. , Flexman James P. , Boehm Ingrid , Costa Anna Maria G. , Lambert Erica J. , Christiansen Keryn J.
Published: June 2003

A real-time PCR assay previously developed for use on the Roche LightCycler platform was investigated as an alternative to culture for the direct detection of vancomycin-resistant enterococci (VRE) in clinical specimens. PCR primers and fluorescence resonance energy transfer hybridization probes specific for the
vanA
and
vanB
genes were combined in a multiplex real-time PCR assay performed directly with fecal material obtained by rectal swabbing and with enrichment broth samples. DNA was prepared from the rectal swabs and enrichment broths with a commercially available DNA preparation column designed specifically for use with fecal specimens. One hundred eighty duplicate rectal swabs were obtained from 42 patients who were previously found to be positive for VRE and who were being monitored for carriage of VRE. Direct and enrichment broth cultures were performed with one swab, while PCR was performed with the other swab as well as any corresponding presumptive positive enrichment broth. In total, 100 specimens from 30 patients remained positive for VRE by at least one method. The multiplex real-time PCR was positive for 88 enrichment broths of rectal swabs from 27 patients but for only 45 rectal swabs from 15 patients. Direct culture was positive for VRE for only 43 specimens from 11 patients, while enrichment broth culture was positive for VRE for 75 specimens from 22 patients. Inhibition studies for the multiplex real-time PCR assay, performed by spiking the DNA extracts from 50 negative rectal swabs and the corresponding enrichment broths with between 1 and 10 CFU of a VanB
Enterococcus faecium
strain, detected inhibition rates of 55.1 and 10%, respectively. PCR performed directly with enrichment broths was found to be significantly more sensitive than enrichment broth culture (
P
< 0.025). Negative samples were identified significantly earlier by PCR than by culture alone.
High Percentage of Methicillin-ResistantStaphylococcus aureusIsolates with Reduced Susceptibility to Glycopeptides in The Netherlands
Authors: Van Griethuysen Arjanne , Van 't Veen Annemarie , Buiting Anton , Walsh Timothy , Kluytmans Jan
Published: June 2003

While testing the in vitro activities of 14 antimicrobial agents against 107 methicillin-susceptibleStaphylococcus aureus(MSSA) and 250 methicillin-resistantS. aureus(MRSA) isolates collected in The Netherlands, we found to our surprise that 19 (7.6%) MRSA isolates were suspected of having reduced susceptibilities to the glycopeptides when the Etest system (AB Biodisk, Solna, Sweden) was used with a large inoculum (no. 2 McFarland standard) and an extended incubation time (48 h) on brain heart infusion agar for MIC testing. Eventually, 15 of these isolates were classified as heterogeneously resistant to glycopeptides (heterogeneously glycopeptide-intermediateS. aureus[hGISA] isolates) according to the population analysis profile-area under the curve analysis. The MICs at which 50 and 90% of isolates are inhibited obtained with the Etest system with the large inoculum were as follows: for MSSA isolates, 3.0 and 4.0 μg/ml, respectively, for both teicoplanin and vancomycin; for MRSA isolates, 3.0 and 8.0 μg/ml, respectively, for teicoplanin, and 3.0 and 4.0 μg/ml, respectively, for vancomycin. This is the first report of hGISA isolates in The Netherlands.
Molecular Analysis of Human Herpesvirus 8 by Using Single Nucleotide Polymorphisms in Open Reading Frame 26
Authors: Endo Tokiomi , Miura Toshiyuki , Koibuchi Tomohiko , Nakamura Hitomi , Takahashi Takashi , Odawara Takashi , Goto Mieko , Ajisawa Atsushi , Iwamoto Aikichi , Nakamura Tetsuya
Published: June 2003

Human herpesvirus 8 (HHV-8) can be classified into distinct subtypes on the basis of sequence polymorphisms in several open reading frames (ORFs). We analyzed the subtypes of HHV-8 in 59 human immunodeficiency virus-infected Japanese patients by using polymorphisms in ORF26 and found that over two-thirds of the HHV-8 isolates fell into major subtype A. We also found that single nucleotide polymorphisms (SNPs) at nucleotide positions 1032 (C-to-A substitution) and 1055 (G-to-T substitution) in HHV-8 ORF26 were correlated with increased susceptibility to Kaposi's sarcoma, compared to the results obtained with HHV-8 with wild-type nucleotides at these positions (
P
= 0.0106). This observation suggests that molecular heterogeneity of the HHV-8 genome affects the biological properties of HHV-8, resulting in different clinical phenotypes of HHV-8 infection. Since sensitive PCR of ORF26 allowed us to analyze the SNPs by using peripheral blood from HHV-8-infected patients, the ORF26 SNPs will be a potent tool for investigating the pathogenesis of HHV-8 infection.
Analysis of Genetic Diversity of
Streptococcus suis
Clinical Isolates from Pigs in Spain by Pulsed-Field Gel Electrophoresis
Authors: Vela Ana I. , Goyache Joaquin , Tarradas Carmen , Luque Inmaculada , Mateos Ana , Moreno Miguel A. , Borge Carmen , Perea J. Anselmo , Domínguez Lucas , Fernández-Garayzábal José F.
Published: June 2003

Pulsed-field gel electrophoresis (PFGE) was used to investigate the diversity of
Streptococcus suis
isolates of various serotypes recovered from swine clinical samples in Spain. Capsular types 9 (64.9%) and 2 (14.8%) were the most frequently isolated serotypes followed by serotype 7 (5.9%) and serotype 8 (4.3%). The PFGE results of this study with 60 different pulsotypes indicate a great genetic diversity among the
S. suis
isolates, which is consistent with the broad distribution of
S. suis
in the swine population. Forty-five percent of the pulsotypes corresponded to single isolates, no pulsotype was common to all farms, and at least 3 different pulsotypes were isolated in 56% of herds in which more than 3 clinical isolates were analyzed. These results reveal a great diversity both between and within herds throughout the strains of
S. suis
studied, demonstrating that different strains of
S. suis
are associated with infection in pigs. Some pulsotypes were more frequently isolated and exhibited a wider distribution over herds than others, and were the unique or predominant strains in several herds, suggesting the existence of a prevalent or a few prevalent clones responsible for a large proportion of clinical cases. Overall, the great genetic heterogeneity of the clinical strains of
S. suis
, the isolation of different strains within the same herd, and the predominance of particular strains in some herds are evidence that infection by
S. suis
is a dynamic process and reinforce the idea that the epidemiology of
S. suis
infection is very complex.
A Hospital-Associated Outbreak of Legionnaires' Disease Caused by
Legionella pneumophila
Serogroup 1 Is Characterized by Stable Genetic Fingerprinting but Variable Monoclonal Antibody Patterns
Authors: Bernander Sverker , Jacobson Kerstin , Helbig Jürgen Herbert , Lück Paul Christian , Lundholm Monica
Published: June 2003

An outbreak of 18 pneumonia cases caused by
Legionella pneumophila
serogroup 1 occurred at a Swedish university hospital 1996 to 1999. Eight clinical isolates obtained by culture from the respiratory tract were compared to 20 environmental isolates from the hospital and to 21 epidemiologically unrelated isolates in Sweden, mostly from patients, by using pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism analysis (AFLP), and monoclonal antibody (MAb) typing. All patients and most environmental isolates from the outbreak hospital belonged to the same genotypic cluster in both PFGE and AFLP. This genotype was distinctly different from other strains, including a cluster from a second hospital in a different part of the country. The MAb subtype of the outbreak clone was Knoxville except for three isolates that were Oxford. A variation in the MAb reactivity pattern was also found in a second genotypic cluster. These changes in the MAb reactivity pattern were due to the absence or presence of the
lag-
1 gene coding for an
O
-acetyltransferase that is responsible for expression of the lipopolysaccharide epitope recognized by MAb 3/1 of the Dresden Panel. In all MAb 3/1-positive strains, the
lag-
1 gene was present on a genetic element that was bordered by a direct repeat that showed a high degree of sequence homology. Due to this homology, the
lag-
1 gene region seemed to be an unstable element in the chromosome. MAb patterns are thus a valuable adjunct to genotyping methods in defining subgroups inside a genotypic cluster of
L. pneumophila
sg 1.
General Acquisition of Human Papillomavirus Infections of Skin Occurs in Early Infancy
Authors: Antonsson Annika , Karanfilovska Silvana , Lindqvist Pelle G. , Hansson Bengt Göran
Published: June 2003

The human skin papillomaviruses (HPVs) represent a group of ubiquitous viruses detected at a high prevalence in the normal skin of healthy adults. In the present study, we analyzed skin swab samples from babies during their first days of life and from infants at various ages up to age 4 years. Specimens from their parents and, for the newborn babies, environmental samples were also investigated. HPV DNA was already detected on the day of birth in samples from 2 of the 16 babies, and 45% of the samples from the babies were positive for HPV in the days following birth. Seventy-seven percent of the skin samples collected from the mothers were HPV DNA positive. The prevalence of HPV DNA among children from the ages of 1 month to 4 years varied between 50 and 70%. The HPV DNA sequences detected revealed a great diversity of genotypes and putative genotypes. Among 115 samples from 38 infants and 31 parents and 7 environmental samples, a total of 73 HPV types or putative types were isolated. Of these, 26 putative HPV types have not been described before. Our data suggest that asymptomatic HPV infections of normal skin are acquired very early in infancy and are caused by a great multiplicity of HPV types.
Novel Reporter T-Cell Line Highly Susceptible to Both CCR5- and CXCR4-Using Human Immunodeficiency Virus Type 1 and Its Application to Drug Susceptibility Tests
Authors: Miyake Hiroshi , Iizawa Yuji , Baba Masanori
Published: June 2003

CCR5-using (R5) human immunodeficiency virus type 1 (HIV-1) is a major viral population that is transmitted by sexual intercourse and that replicates in infected individuals during the asymptomatic stage of HIV-1 infection, suggesting that agents effective against R5 HIV-1 can be expected to prevent viral transmission and delay disease progression. However, R5 HIV-1 is unable to replicate in human T-cell lines, which is an apparent obstacle to efficient and reliable susceptibility tests of compounds for their activities against R5 HIV-1. To establish a simple and rapid assay system for the monitoring of R5 HIV-1 replication and drug susceptibility, we have established a novel reporter T-cell line, MOCHA (which represents MOLT-4 cells stably expressing CCR5 and carrying the HIV-1 long terminal repeat-driven secretory alkaline phosphatase). Cells of this cell line express CD4, CXCR4, and CCR5 on their surfaces and secrete human placental alkaline phosphatase into the culture supernatants during HIV-1 infection. MOCHA cells proved to be highly permissive for the replication of R5 HIV-1 as well as CXCR4-using (X4) HIV-1, and the alkaline phosphatase activity increased in parallel with increasing HIV-1 p24 antigen levels in the culture supernatants. When HIV-1 reverse transcriptase inhibitors, protease inhibitors, and entry inhibitors, including the CCR5 antagonist TAK-779 and the CXCR4 antagonist AMD3100, were examined for their inhibitory effects on R5 and X4 HIV-1 replication in MOCHA cells, the antiviral activities of these compounds were found to be almost identical to those previously reported in peripheral blood mononuclear cells. Thus, MOCHA cells are an extremely useful tool for detection of R5 and X4 HIV-1 replication and drug susceptibility tests.
Characterization ofTreponema phagedenis-Like Spirochetes Isolated from Papillomatous Digital Dermatitis Lesions in Dairy Cattle
Authors: Trott Darren J. , Moeller Michelle R. , Zuerner Richard L. , Goff Jesse P. , Waters W. Ray , Alt David P. , Walker Richard L. , Wannemuehler Michael J.
Published: June 2003

Four spirochete strains were isolated from papillomatous digital dermatitis (PDD) lesions in Iowa dairy cattle and compared with two previously described spirochete strains isolated from dairy cattle in California. These six strains shared an identical 16S ribosomal DNA sequence that was 98% similar toTreponema phagedenisand 99% similar to the uncultivated PDD spirochete sequence DDLK-4. The whole-cell protein profiles resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these six strains were similar. However, these strains showed differences in the antigenic diversity of lipopolysaccharide (LPS). Genetic diversity was also detected by pulsed-field gel electrophoresis of genomic DNA digests, revealing differences among five of the six strains. Serum immunoglobulin G antibodies from dairy cattle with active PDD lesions reacted with the LPS of all but one PDD spirochete strain. Likewise, peripheral blood mononuclear cells from cattle with active PDD lesions produced blastogenic responses to one of the two California isolates. Both antibody and lymphocyte blastogenic responses were reduced in convalescent dairy cattle, suggesting the immune response to these spirochetes has short duration. These results demonstrate genetic and antigenic diversity amongT. phagedenis-like treponemes and provide further evidence for the involvement of these spirochetes in the pathogenesis of PDD.
Multilocus Sequence Typing System for Group B Streptococcus
Authors: Jones Nicola , Bohnsack John F. , Takahashi Shinji , Oliver Karen A. , Chan Man-Suen , Kunst Frank , Glaser Philippe , Rusniok Christophe , Crook Derrick W. M. , Harding Rosalind M. , Bisharat Naiel , Spratt Brian G.
Published: June 2003

A multilocus sequence typing (MLST) system was developed for group B streptococcus (GBS). The system was used to characterize a collection (
n
= 152) of globally and ecologically diverse human strains of GBS that included representatives of capsular serotypes Ia, Ib, II, III, V, VI, and VIII. Fragments (459 to 519 bp) of seven housekeeping genes were amplified by PCR for each strain and sequenced. The combination of alleles at the seven loci provided an allelic profile or sequence type (ST) for each strain. A subset of the strains were characterized by restriction digest patterning, and these results were highly congruent with those obtained with MLST. There were 29 STs, but 66% of isolates were assigned to four major STs. ST-1 and ST-19 were significantly associated with asymptomatic carriage, whereas ST-23 included both carried and invasive strains. All 44 isolates of ST-17 were serotype III clones, and this ST appeared to define a homogeneous clone that was strongly associated with neonatal invasive infections. The finding that isolates with different capsular serotypes had the same ST suggests that recombination occurs at the capsular locus. A web site for GBS MLST was set up and can be accessed at http://sagalactiae.mlst.net. The GBS MLST system offers investigators a valuable typing tool that will promote further investigation of the population biology of this organism.
Species-Specific Identification of Campylobacters by Partial 16S rRNA Gene Sequencing
Authors: Gorkiewicz Gregor , Feierl Gebhard , Schober Caroline , Dieber Franz , Köfer Josef , Zechner Rudolf , Zechner Ellen L.
Published: June 2003

Species-specific identification of campylobacters is problematic, primarily due to the absence of suitable biochemical assays and the existence of atypical strains. 16S rRNA gene (16S rDNA)-based identification of bacteria offers a possible alternative when phenotypic tests fail. Therefore, we evaluated the reliability of 16S rDNA sequencing for the species-specific identification of campylobacters. Sequence analyses were performed by using almost 94% of the complete 16S rRNA genes of 135 phenotypically characterized
Campylobacter
strains, including all known taxa of this genus. It was shown that 16S rDNA analysis enables specific identification of most
Campylobacter
species. The exception was a lack of discrimination among the taxa
Campylobacter jejuni
and
C. coli
and atypical
C. lari
strains, which shared identical or nearly identical 16S rDNA sequences. Subsequently, it was investigated whether partial 16S rDNA sequences are sufficient to determine species identity. Sequence alignments led to the identification of four 16S rDNA regions with high degrees of interspecies variation but with highly conserved sequence patterns within the respective species. A simple protocol based on the analysis of these sequence patterns was developed, which enabled the unambiguous identification of the majority of
Campylobacter
species. We recommend 16S rDNA sequence analysis as an effective, rapid procedure for the specific identification of campylobacters.
Genetic Biodiversity of
Mycobacterium tuberculosis
Complex Strains from Patients with Pulmonary Tuberculosis in Cameroon
Authors: Niobe-Eyangoh Sara Ngo , Kuaban Christopher , Sorlin Philippe , Cunin Patrick , Thonnon Jocelyn , Sola Christophe , Rastogi Nalin , Vincent Veronique , Gutierrez M. Cristina
Published: June 2003

We analyzed DNA polymorphisms in 455
Mycobacterium tuberculosis
complex isolates from 455 patients to evaluate the biodiversity of tubercle bacilli in Ouest province, Cameroon. The phenotypic and genotypic identification methods gave concordant results for 99.5% of
M. tuberculosis
isolates (413 strains) and for 90% of
Mycobacterium africanum
isolates (41 strains).
Mycobacterium bovis
was isolated from only one patient. Analysis of regions of difference (RD4, RD9, and RD10) proved to be an accurate and rapid method of distinguishing between unusual members of the
M. tuberculosis
complex. Whereas
M. africanum
strains were the etiologic agent of tuberculosis in 56% of cases 3 decades ago, our results showed that these strains now account for just 9% of cases of tuberculosis. We identified a group of closely genetically related
M. tuberculosis
strains that are currently responsible for >40% of smear-positive pulmonary tuberculosis cases in this region of Cameroon. These strains shared a spoligotype lacking spacers 23, 24, and 25 and had highly related IS
6110
ligation-mediated (LM) PCR patterns. They were designated the “Cameroon family.” We did not find any significant association between tuberculosis-causing species or strain families and patient characteristics (sex, age, and human immunodeficiency virus status). A comparison of the spoligotypes of the Cameroon strains with an international spoligotype database (SpolDB3) containing 11,708 patterns from >90 countries, showed that the predominant spoligotype in Cameroon was limited to West African countries (Benin, Senegal, and Ivory Coast) and to the Caribbean area.
Diagnosis of Pneumococcal Pneumonia by
psaA
PCR Analysis of Lung Aspirates from Adult Patients in Kenya
Authors: Scott J. Anthony G. , Marston Eric L. , Hall Andrew J. , Marsh Kevin
Published: June 2003

psaA
is the gene encoding pneumococcal surface adhesin A (PsaA), a 37-kDa protein expressed on the surface of
Streptococcus pneumoniae
. PCR primers for
psaA
have been shown to amplify the target DNA sequence in all 90 serotypes of
S. pneumoniae
and in none of 67 heterologous pathogens and colonizing bacteria of the upper respiratory tract. Pathogenic bacteria identified in lung aspirate specimens cannot normally be dismissed as contaminants or colonizers, which limit the assay specificity of other respiratory tract specimens.
psaA
PCR analysis was evaluated in 171 lung aspirates from Kenyan adults with acute pneumonia. The limit of detection was one genome equivalent. Sensitivity, estimated in 35 culture-positive lung aspirates, was 0.83 (95% confidence interval, 0.70 to 0.95).
psaA
PCR analysis extended the number of identifications of
S. pneumoniae
in lung aspirates from 35 on culture to 61 by both methods. Of 26 new pneumococcal diagnoses, 19 were corroborated by results of blood culture or urine antigen detection. Sequences of the PCR products from 12 positive samples were identical to the
psaA
target gene fragment. Using an internal control for the PCR, inhibition of
psaA
PCR was demonstrated in 17% (8 of 47) of false-negative specimens. The results of a control PCR for the human gene β-actin suggested that false-negative
psaA
PCR results are attributable to problems of specimen collection, processing, or DNA extraction in 30% of cases (14 of 47).
psaA
PCR analysis is a sensitive tool for diagnosis of pneumococcal pneumonia in adults.
Nocardia veterana
as a Pathogen in North American Patients
Authors: Conville Patricia S. , Brown June M. , Steigerwalt Arnold G. , Lee Judy W. , Byrer Dorothy E. , Anderson Victoria L. , Dorman Susan E. , Holland Steven M. , Cahill Barbara , Carroll Karen C. , Witebsky Frank G.
Published: June 2003

The molecular methodologies used in our laboratories have allowed us to define a group of
Nocardia
isolates from clinical samples which resemble the type strain of
Nocardia veterana
. Three patient isolates and the type strain of
N. veterana
gave identical and distinctive restriction fragment length polymorphisms (RFLPs) for an amplified portion of the 16S rRNA gene. These three isolates and the
N. veterana
type strain also gave identical RFLPs for an amplified portion of the 65-kDa heat shock protein gene, but this pattern was identical to that obtained for the
Nocardia nova
type strain. Sequence analysis of both a 1,359-bp region of the 16S rRNA gene and a 441-bp region of the heat shock protein gene of the patient isolates showed 100% identities with the same regions of the
N. veterana
type strain. DNA-DNA hybridization of the DNA of one of the patient isolates with the DNA of the
N. veterana
type strain showed a relative binding ratio of 82%, with 0% divergence, confirming that the isolate was
N. veterana
. Biochemical and susceptibility testing showed no significant differences among the patient isolates and the
N. veterana
type strain. Significantly, the results of antimicrobial susceptibility testing obtained for our isolates were similar to those obtained for
N. nova
, indicating that susceptibility testing alone cannot discriminate between these species. We present two case studies which show that
N. veterana
is a causative agent of pulmonary disease in immunocompromised patients residing in North America. We also describe difficulties encountered in using 16S rRNA gene sequences alone for discrimination of
N. veterana
from the related species
Nocardia africana
and
N. nova
because of the very high degree of 16S rRNA gene similarity among them.