bluuebunny/crossref_metadata_2025 huggingface dataset:
Construction of an Integration-Proficient Vector Based on the Site-Specific Recombination Mechanism of Enterococcal Temperate Phage φFC1
Authors: Yang Hee-Youn , Kim Young-Woo , Chang Hyo-Ihl
Published: July 2002

The genome of temperate phage φFC1 integrates into the chromosome ofEnterococcus faecalisKBL 703 via site-specific recombination. In this study, an integration vector containing theattPsite and putative integrase genemj1of phage φFC1 was constructed. A 2,744-bp fragment which included theattPsite andmj1was inserted into a pUC19 derivative containing thecatgene to construct pEMJ1-1.E. faecalisKBL 707, which does not contain the bacteriophage but which has a putativeattBsite within its genome, could be transformed by pEMJ1-1. Southern hybridization, PCR amplification, and DNA sequencing revealed that pEMJ1-1 was integrated specifically at the putativeattBsite within theE. faecalisKBL 707 chromosome. This observation suggested that the 2,744-bp fragment carryingmj1and theattPsite of phage φFC1 was sufficient for site-specific recombination and that pEMJ1-1 could be used as a site-specific integration vector. The transformation efficiency of pEMJ1-1 was as high as 6 × 103transformants/μg of DNA. In addition, a vector (pATTB1) containing the 290-bpattBregion was constructed. pATTB1 was transformed intoEscherichia colicontaining a derivative of the pET14b vector carryingattPandmj1.This resulted in the formation of chimeric plasmids by site-specific recombination between the clonedattBandattPsequences. The results indicate that the integration vector system based on the site-specific recombination mechanism of phage φFC1 can be used for genetic engineering inE. faecalisand in other hosts.
Molecular Epidemiology of the Integron-Located VEB-1 Extended-Spectrum β-Lactamase in Nosocomial Enterobacterial Isolates in Bangkok, Thailand
Authors: Girlich Delphine , Poirel Laurent , Leelaporn Amornrut , Karim Amal , Tribuddharat Chanwitt , Fennewald Michael , Nordmann Patrice
Published: July 2002

Over a 2½-month period in 1999, 37 ceftazidime-resistant nonrepetitive enterobacterial isolates were collected from 37 patients in a Bangkok hospital, Thailand. Eighty-one percent of these strains expressed a clavulanic acid-inhibited extended-cephalosporin resistance profile. An identical extended-spectrum β-lactamase (ESBL), VEB-1, was found in 16 unrelated enterobacterial isolates (Escherichia coli,n= 10;Enterobacter cloacae,n= 2;Enterobacter sakazakii,n= 1; andKlebsiella pneumoniae,n= 3) and in two clonally relatedE. cloacaeisolates. TheblaVEB-1gene was located on mostly self-conjugative plasmids (ca. 24 to 200 kb) that conferred additional non-β-lactam antibiotic resistance patterns. Additionally, theblaVEB-1gene cassette was part of class 1 integrons varying in size and structure. TheblaVEB-1-containing integrons were mostly associated withblaOXA-10-like andarr-2-like gene cassettes, the latter conferring resistance to rifampin. These data indicated the spread ofblaVEB-1in Bangkok due to frequent transfer of different plasmids and class 1 integrons and rarely to clonally related strains. Plasmid- and integron-mediated resistance to rifampin was also found in enterobacterial isolates.
Detection and Initial Characterization of Novel Capsular Polysaccharide among Diverse
Campylobacter jejuni
Strains Using Alcian Blue Dye
Authors: Karlyshev Andrey V. , Wren Brendan W.
Published: July 2002

We have recently demonstrated that most strains of
Campylobacter jejuni
produce capsular polysaccharide (CPS), which can be detected by immunoblotting with homologous Penner antisera on polyvinylidene difluoride membranes (A. V. Karlyshev, D. Linton, N. A. Gregson, A. J. Lastovica, and B. W. Wren, Mol. Microbiol. 35:529–541, 2000). In this report, we describe a universal and rapid staining procedure using Alcian blue for
C. jejuni
CPS, which does not rely on the availability of antisera and identifies CPS in untypeable strains. Furthermore, Alcian blue staining identified CPS in its lipid-free form directly on Tricine gels, and we demonstrate that CPS is thermostable and is accumulated in the culture supernatant in a lipid-free form. The identification of a newly described CPS and its lipid-free form in
C. jejuni
should prove invaluable in studying the pathogenesis and epidemiology of this important pathogen.
Outbreak of Necrotizing Enterocolitis Associated with
Enterobacter sakazakii
in Powdered Milk Formula
Authors: van Acker Jos , de Smet Francis , Muyldermans Gaëtan , Bougatef Adel , Naessens Anne , Lauwers Sabine
Published: July 2002

We describe an outbreak of necrotizing enterocolitis (NEC) that occurred in the neonatal intensive care unit of our hospital. A total of 12 neonates developed NEC in June-July 1998. For two of them, twin brothers, the NEC turned out to be fatal.
Enterobacter sakazakii
, a known contaminant of powdered milk formula, was isolated from a stomach aspirate, anal swab, and/or blood sample for 6 of the 12 neonates. A review of feeding procedures revealed that 10 of the 12 patients were fed orally with the same brand of powdered milk formula.
E. sakazakii
was isolated from the implicated prepared formula milk as well as from several unopened cans of a single batch. Molecular typing by arbitrarily primed PCR (AP-PCR) confirmed, although partially, strain similarity between milk and patient isolates. No further cases of NEC were observed after the use of the contaminated milk formula was stopped. With this outbreak we show that intrinsic microbiological contamination of powdered milk formula can be a possible contributive factor in the development of NEC, a condition encountered almost exclusively in formula-fed premature infants. The use of sterilized liquid milk formula in neonatal care could prevent problems with intrinsic and extrinsic contamination of powdered milk formula.
Contamination of Transplantable Human Tumor-Bearing Lines by
Helicobacter hepaticus
and Its Elimination
Authors: Goto Kazuo , Ishihara Ken-Ichi , Kuzuoka Ayako , Ohnishi Yasuyuki , Itoh Toshio
Published: July 2002

Helicobacter hepaticus
contaminating nonfrozen tumors was transmissible to severe combined immunodeficient (SCID) mice, but the organism in cryopreserved samples was not. This suggests that
H. hepaticus
has the ability to spread via biomaterials and that freezing-thawing is able to reduce the numbers of organisms to levels insufficient for subcutaneous infection of SCID mice.
Analysis of Microsatellite Markers of
Candida albicans
Used for Rapid Typing
Authors: Botterel Françoise , Desterke Christophe , Costa Catherine , Bretagne Stéphane
Published: July 2002

To obtain a rapid genotyping method of
Candida albicans
, three polymorphic microsatellite markers were investigated by multiplex PCR. The three loci, called
CDC3
,
EF3
, and
HIS3
, were chosen because they are on different chromosomes so as to improve the chances of finding polymorphisms. One set of primers was designed for each locus, and one primer of each set was dye-labeled to read PCR signals by using an automatic sequencer. Amplifications were performed directly from the colonies harvested on the agar plate without a sophisticated DNA extraction step. At total of 27 reference strains and 73 clinical independent isolates were tested. The numbers of allelic associations were 10, 22, and 25 for the loci
CDC3
,
EF3
, and
HIS3
, respectively. The combined discriminatory power of the three microsatellites markers was 0.97. The markers were stable after 25 subcultures, and the amplifications were specific for
C. albicans
. An initial study of 17 clinical isolate pairs, including blood culture and peripheral sites, showed a similar genotype for 15 of them, confirming that candidemia usually originates from the colonizing isolate. Therefore, microsatellite marker analysis with multiplex PCR and automated procedures has a high throughput and should be suitable for large epidemiologic studies of
C. albicans
.
Risk Factors for Antibiotic-Resistant
Escherichia coli
Isolated from Hospitalized Patients with Urinary Tract Infections: a Prospective Study
Authors: Sotto Albert , De Boever Corinne Merle , Fabbro-Peray Pascale , Gouby Anne , Sirot Danielle , Jourdan Jacques
Published: July 2002

From November 1998 to February 1999 we prospectively evaluated the prevalence of resistance to penicillins, cephalosporins, carbapenem, quinolones, aminoglycosides, and trimethoprim-sulfamethoxazole (SXT) in 320
Escherichia coli
isolates isolated from hospitalized patients with acute urinary tract infections (UTIs). We also studied for these strains risk factors for resistance to amoxicillin-clavulanic acid (AMC), fluoroquinolones (FQs), and SXT. Resistance rates were consistent with those from major recent studies reported in the literature. Multivariate analyses selected the following factors as being significantly associated with
E. coli
resistance: (i) for resistance to AMC, prior (1 year) UTI (odds ratio [OR] = 2.71,
P
= 0.006), prior (1 year) urinary catheter (OR = 2.98,
P
= 0.0025), and prior (6 months) antibiotic exposure (OR = 2.68,
P
= 0.005); (ii) for resistance to FQs male sex (OR = 3.87,
P
= 0.03), with a trend toward significance for age >65 years (OR = 7.67,
P
= 0.06) and prior (1 year) UTI (OR = 2.98,
P
= 0.07); and (iii) for resistance to SXT, male sex (OR = 1.91,
P
= 0.046), hospitalization in an intermediate-term-care unit (OR = 2.18,
P
= 0.008), and prior (1 year) UTI (OR = 2.03,
P
= 0.03). Ours results suggest that prior UTI is a common risk factor for resistance to the different antibiotics tested. Although few studies on risk factors for
E. coli
resistance to antibiotics have been published, careful interpretation of their findings, taking into consideration the population, infection site, and period studied, should contribute to the formulation of a better strategy that can be used to overcome antibiotic resistance.
Kaposi's Sarcoma Associated with Previous Human Herpesvirus 8 Infection in Kidney Transplant Recipients
Authors: Cattani Paola , Capuano Maria , Graffeo Rosalia , Ricci Rosalba , Cerimele Francesca , Cerimele Decio , Nanni Giuseppe , Fadda Giovanni
Published: July 2002

This study investigates the prevalence of human herpesvirus 8 (HHV-8) infection in kidney transplant patients, evaluating the risk of HHV-8 transmission via transplantation and the association between pre- and posttransplantation HHV-8 infection and the subsequent development of Kaposi's sarcoma (KS). Immunofluorescence and an enzyme immunoassay were used to determine HHV-8 seroprevalence in 175 patients awaiting kidney transplantation and 215 controls who were attending our clinic for other reasons. All patients in the study came from central or southern Italy. Seroprevalence was similar in both groups (14.8 versus 14.9%), with no significant difference between the rates for male and female patients. Of the 175 patients, 100 were tested for anti-HHV-8 antibodies at various times during follow-up. During follow-up, seroprevalence increased from 12% on the date of transplantation to 26%. This increase was paralleled by an age-related increase in seroprevalence in the control group. During follow-up from 3 months to 10 years after transplantation, KS was diagnosed in seven patients (4.0%). Six of these patients were positive for HHV-8 prior to transplantation. Overall, 23.0% of patients who were HHV-8 positive before transplantation developed KS, whereas only 0.7% of seronegative patients developed the disease (relative risk, 34.4; 95% confidence interval, 4.31 to 274.0). This finding suggests that the key risk factor for KS is infection prior to transplantation and that antibody detection in patients awaiting transplantation could be useful in identifying patients at high risk for KS. In patients from geographic areas with a high prevalence of HHV-8, serological tests on donors may be less important.
Breast Milk Transmission of a Panton-Valentine Leukocidin-Producing
Staphylococcus aureus
Strain Causing Infantile Pneumonia
Authors: Le Thomas Isabelle , Mariani-Kurkdjian Patricia , Collignon Anne , Gravet Alain , Clermont Olivier , Brahimi Naı̈ma , Gaudelus Joël , Aujard Yannick , Navarro Jean , Beaufils François , Bingen Edouard
Published: July 2002

We report on a 38-day-old infant who developed pleuropneumonia due to a
Staphylococcus aureus
strain responsible for familial furunculosis, which was acquired by maternal breast-feeding. All isolates from the infant and parents were genetically related by randomly amplified polymorphic DNA analysis and produced Panton-Valentine leukocidin.
Fatal Pulmonary Infection Due to Multidrug-Resistant
Mycobacterium abscessus
in a Patient with Cystic Fibrosis
Authors: Sanguinetti Maurizio , Ardito Fausta , Fiscarelli Ersilia , La Sorda Marilena , D'Argenio Patrizia , Ricciotti Gabriella , Fadda Giovanni
Published: July 2002

We report a case of fatal pulmonary infection caused by
Mycobacterium abscessus
in a young patient with cystic fibrosis, who underwent bipulmonary transplantation after a 1-year history of severe lung disease. Fifteen days after surgery he developed septic fever with progressive deterioration in lung function.
M. abscessus
, initially isolated from a pleural fluid specimen, was then recovered from repeated blood samples, suggesting a disseminated nature of the mycobacterial disease. Drug susceptibility testing assay, performed on two sequential isolates of the microorganism, showed a pattern of multidrug resistance. Despite aggressive therapy with several antimycobacterial drugs, including clarithromycin, the infection persisted, and the patient died.
Diagnostics of
Neisseriaceae
and
Moraxellaceae
by Ribosomal DNA Sequencing: Ribosomal Differentiation of Medical Microorganisms
Authors: Harmsen Dag , Singer Christian , Rothgänger Jörg , Tønjum Tone , Sybren de Hoog Gerrit , Shah Haroun , Albert Jürgen , Frosch Matthias
Published: July 2002

Fast and reliable identification of microbial isolates is a fundamental goal of clinical microbiology. However, in the case of some fastidious gram-negative bacterial species, classical phenotype identification based on either metabolic, enzymatic, or serological methods is difficult, time-consuming, and/or inadequate. 16S or 23S ribosomal DNA (rDNA) bacterial sequencing will most often result in accurate speciation of isolates. Therefore, the objective of this study was to find a hypervariable rDNA stretch, flanked by strongly conserved regions, which is suitable for molecular species identification of members of the
Neisseriaceae
and
Moraxellaceae
. The inter- and intrageneric relationships were investigated using comparative sequence analysis of PCR-amplified partial 16S and 23S rDNAs from a total of 94 strains. When compared to the type species of the genera
Acinetobacter, Moraxella
, and
Neisseria
, an average of 30 polymorphic positions was observed within the partial 16S rDNA investigated (corresponding to
Escherichia coli
positions 54 to 510) for each species and an average of 11 polymorphic positions was observed within the 202 nucleotides of the 23S rDNA gene (positions 1400 to 1600).
Neisseria macacae
and
Neisseria mucosa
subsp.
mucosa
(ATCC 19696) had identical 16S and 23S rDNA sequences. Species clusters were heterogeneous in both genes in the case of
Acinetobacter lwoffii, Moraxella lacunata
, and
N. mucosa. Neisseria meningitidis
isolates failed to cluster only in the 23S rDNA subset. Our data showed that the 16S rDNA region is more suitable than the partial 23S rDNA for the molecular diagnosis of
Neisseriaceae
and
Moraxellaceae
and that a reference database should include more than one strain of each species. All sequence chromatograms and taxonomic and disease-related information are available as part of our ribosomal differentiation of medical microorganisms (RIDOM) web-based service (
http://www.ridom.hygiene.uni-wuerzburg.de/
). Users can submit a sequence and conduct a similarity search against the RIDOM reference database for microbial identification purposes.
Classification and Identification of Enterococci: a Comparative Phenotypic, Genotypic, and Vibrational Spectroscopic Study
Authors: Kirschner C. , Maquelin K. , Pina P. , Ngo Thi N. A. , Choo-Smith L.-P. , Sockalingum G. D. , Sandt C. , Ami D. , Orsini F. , Doglia S. M. , Allouch P. , Mainfait M. , Puppels G. J. , Naumann D.
Published: July 2002

Rapid and accurate identification of enterococci at the species level is an essential task in clinical microbiology since these organisms have emerged as one of the leading causes of nosocomial infections worldwide. Vibrational spectroscopic techniques (infrared [IR] and Raman) could provide potential alternatives to conventional typing methods, because they are fast, easy to perform, and economical. We present a comparative study using phenotypic, genotypic, and vibrational spectroscopic techniques for typing a collection of 18
Enterococcus
strains comprising six different species. Classification of the bacteria by Fourier transform (FT)-IR spectroscopy in combination with hierarchical cluster analysis revealed discrepancies for certain strains when compared with results obtained from automated phenotypic systems, such as API and MicroScan. Further diagnostic evaluation using genotypic methods—i.e., PCR of the species-specific ligase and glycopeptide resistance genes, which is limited to the identification of only four
Enterococcus
species and 16S RNA sequencing, the “gold standard” for identification of enterococci—confirmed the results obtained by the FT-IR classification. These results were later reproduced by three different laboratories, using confocal Raman microspectroscopy, FT-IR attenuated total reflectance spectroscopy, and FT-IR microspectroscopy, demonstrating the discriminative capacity and the reproducibility of the technique. It is concluded that vibrational spectroscopic techniques have great potential as routine methods in clinical microbiology.
Development of Reverse Transcriptase PCR Assays for Detection of Active Human Herpesvirus 6 Infection
Authors: Van den Bosch Glenn , Locatelli Giuseppe , Geerts Lieve , Fagà Giovanni , Ieven Margareta , Goossens Herman , Bottiger Disa , Öberg Bo , Lusso Paolo , Berneman Zwi N.
Published: July 2002

We developed reverse transcriptase (RT) PCR assays for the detection of mRNA from three spliced genes of human herpesvirus 6 (HHV-6), the immediate-early genes U16/U17 and U89/U90 and the late gene U60/U66. Sequence analysis determined the splicing sites of these genes. The new assays may be instrumental in investigating the association between HHV-6 and disease.
Effects of Two Different Growth Media on the Postantifungal Effect Induced by Polyenes on
Candida
Species
Authors: Shu M. , Ellepola A. N. B. , Samaranayake L. P.
Published: July 2002

There are no data on the effects of different growth media on polyene-induced postantifungal effect (PAFE) in
Candida
species. Hence, the nystatin- and amphotericin B-induced PAFEs in six
Candida
species (26 isolates) grown in Sabouraud's dextrose broth (SAB) and RPMI broth were evaluated, following limited exposure to the MICs of the two polyenes, using an automated turbidometric method. For nystatin, PAFE varied between 1.88 and 4.87 h in SAB and 0.66 and 6.89 h in RPMI, and for amphotericin B, the equivalent values were 3.13 to 10.98 h in SAB and 0.97 to 7.01 h in RPMI. These highly significant (
P
< 0.001) variations in the PAFE with both drugs, noted with most
Candida
strains grown in different media, call for standardization of intralaboratory methodology in measuring this parameter in order to obtain universally comparable data.
Genetic Variation among Hospital Isolates of Methicillin-Sensitive
Staphylococcus aureus
: Evidence for Horizontal Transfer of Virulence Genes
Authors: Moore P. C. L. , Lindsay J. A.
Published: July 2002

Staphylococcus aureus
strains often carry in their genomes virulence genes that are not found in all strains and that may be carried on discrete genetic elements. Strains also differ in that they carry one of four classes of an accessory gene regulator (
agr
) locus, an operon that regulates virulence factor expression and that has been proposed to be a therapeutic target. To look at their distribution among hospital strains, we investigated 38 methicillin-sensitive
S. aureus
isolates, classifying the isolates by
agr
class and screening them for the presence and restriction fragment length polymorphisms (RFLPs) of 12 core and 14 accessory virulence genes. Twenty-three (61%) were
agr
class I, 10 (26%) were
agr
class II, and 5 (13%) were
agr
class III. None were
agr
class IV. The
S. aureus
strains had distinguishable RFLP profiles, although clusters of isolates with clearly related core gene profiles were found among our strains, including all five
agr
class III strains, two sets of six strains within
agr
class I, and six strains within
agr
class II. Within these clusters there was evidence of horizontal acquisition and/or loss of multiple accessory virulence genes. Furthermore, two isolates from the same patient were identical except for the presence of the
sea
gene, indicating that movement of mobile elements may occur in vivo. Several strong correlations with the carriage of virulence genes between strains were seen, including a positive correlation between
tst
and
agr
class III and negative correlations between
tst
and
lukE-splB
and between
lukE-splB
and
seg-sei
. This suggests that the core genome or the presence of accessory genetic elements within a strain may influence acquisition and loss of other elements encoding virulence genes.
Evaluation of Laboratory Testing Methods for
Chlamydia trachomatis
Infection in the Era of Nucleic Acid Amplification
Authors: Battle Tamara J. , Golden Matthew R. , Suchland Kathleen L. , Counts Jon M. , Hughes James P. , Stamm Walter E. , Holmes King K.
Published: July 2002

Diagnostic tests presently available for
Chlamydia trachomatis
have widely varying performance characteristics. To assess evolving laboratory testing practices since the introduction of nucleic acid amplification tests (NAAT), we surveyed laboratories in Washington State about their testing practices in 1998 and compared our findings to a similar survey conducted in 1995. Laboratory directors of 61 (87%) of 70 laboratories performing chlamydial tests in 1998 returned a survey. Between 1995 and 1998, 36 laboratories discontinued chlamydial testing, and the total number of laboratories performing tests in the state decreased from 92 to 70, a 24% decline. Of the 36 laboratories that discontinued testing, 25 (69%) had previously used rapid tests. While no laboratory routinely used NAAT in 1995, ligase chain reaction (LCR) was used in 23% of laboratories in 1998 and accounted for 113,624 (36%) of the 318,133 tests performed that year. Among the remaining 204,509 tests performed in 1998, other tests employed included DNA probe (29%), enzyme immunoassay (20%), culture (12%), direct fluorescent antibody assays (3%), and rapid tests (<1%). The majority (65%) of tests performed in 1998 using technologies other than LCR or culture were done in laboratories that did more than 10,000 tests. Cost and loss of revenue to laboratories were the most frequently cited reasons for not adopting NAAT. We conclude that in Washington State, NAAT have been rapidly adopted in larger laboratories, but most patients are still tested with much less sensitive technologies. Financial constraints represent the major barrier to more widespread use of DNA amplification tests.
Distribution of Clinically RelevantBorreliaGenospecies in Ticks Assessed by a Novel, Single-Run, Real-Time PCR
Authors: Rauter Carolin , Oehme Rainer , Diterich Isabel , Engele Matthias , Hartung Thomas
Published: July 2002

A LightCycler-based PCR protocol was developed which targets theospAgene for the identification and quantification of the differentBorrelia burgdorferisensu lato species in culture and in ticks, based on the use of a fluorescently labeled probe (HybProbe) and an internally labeled primer. The detection limit of the PCR was 1 to 10 spirochetes. A melting temperature determined from the melting curve of the amplified product immediately after thermal cycling allowed the differentiation of the three differentB. burgdorferisensu lato genospecies (B. burgdorferisensu stricto,Borrelia garinii, andBorrelia afzelii) that are clinically relevant in Europe in a single PCR run. This method represents a simplified approach to study the association of differentBorreliaspecies in ticks, the risk of Lyme borreliosis, and the putatively species-specific clinical sequelae. To determine the reliability of the real-time PCR protocol, we studied the prevalence ofB. burgdorferisensu lato infection inIxodes ricinusticks. A total of 1,055 ticks were collected by flagging vegetation in five different sites in the region of Konstanz (south Germany) and were examined for the distribution ofB. burgdorferispecies by real-time PCR. The mean infection rate was 35%. Of 548 adult ticks, 40% were positive, and of 507 nymphs, 30% were positive. The predominant genospecies (with 18% mixed infections) in the examined areas wasB. afzelii(53%), followed byB. garinii(18%) andB. burgdorferisensu stricto (11%); 0.8% of the infectingBorreliacould not be identified.
Identification of the
Brucella melitensis
Vaccine Strain Rev.1 in Animals and Humans in Israel by PCR Analysis of the
Pst
I Site Polymorphism of Its
omp2
Gene
Authors: Bardenstein Svetlana , Mandelboim Michal , Ficht Thomas A. , Baum Miriam , Banai Menachem
Published: July 2002

Adverse effects of strain persistence and secretion in milk have been encountered with the
Brucella melitensis
vaccine strain Rev.1. Field isolates obtained from vaccinated animals and from a human resembled the vaccine strain Rev.1 by conventional bacteriological tests. The lack of a specific molecular marker that could specifically characterize the commercial vaccine strain prevented confirmation of the homology of the Rev.1-like field isolates to the vaccine strain. The composition of the
omp2
locus from two gene copies with differences in their
Pst
I restriction endonuclease sites was used to establish an epidemiologic fingerprint for the
omp2
gene in the Rev.1 vaccine strain. Primers designed to amplify DNA sequences that overlap the
Pst
I site revealed a single 282-bp DNA band common to all
Brucella
spp. Agarose gel electrophoresis of the
Pst
I digests of the PCR products from strains 16M and the vaccine strain Rev.1 revealed a distinctive profile that included three bands: one band for the intact 282-bp fragment amplified from
omp2a
and two bands resulting from the digestion of the amplified
omp2b
gene fragment, 238- and 44-bp DNA fragments, respectively. Amplified fragments of 37 Rev.1-like isolates, including 2 human isolates, also exhibited this pattern. In contrast, DNA digests of all other Israeli field isolates, including atypical
B. melitensis
biotype 1 and representatives of the biotype 2 and 3 isolates, produced two bands of 238 and 44 bp, respectively, corresponding with the digestion of both
omp2a
and
omp2b
genes. This method facilitates identification of the Rev.1 vaccine strain in both animals and humans in Israel.
Roleof Picornaviruses in Flu-Like Illnesses of Adults Enrolled in anOseltamivir Treatment Study Who Had No Evidence of Influenza VirusInfection
Authors: Boivin Guy , Osterhaus Albert D. , Gaudreau Annie , Jackson Helen C. , Groen Jan , Ward Penelope
Published: July 2002

The primary
objective of this study was to determine the role of picornavirus in
flu-like episodes (temperature of ≥38.0°C plus one respiratory and
one constitutional symptom) among otherwise healthy adults enrolled in a
placebo-controlled, double-blind, randomized oseltamivir treatment study.
Combined nasal and pharyngeal swabs were collected at baseline for
influenza cultures and picornavirus reverse transcription (RT)-PCR. In
addition, acute- and convalescent-serum samples were obtained for
serological studies of common respiratory pathogens. From a total of 719
subjects enrolled in the clinical trial within 36 h of the onset of
symptoms, 475 (66%) had evidence of recent influenza A or B virus
infections by means of culture and/or serological testing. Of the 244
remaining patients, 36 (15%) presented a seroconversion for at least one of
the common respiratory viruses or atypical pathogens. An RT-PCR assay for
the picornavirus 5" noncoding region (NCR) was positive in a subset of
15 (19%) of 78 patients with flu-like illnesses of undetermined etiology.
Sequence analysis of the picornavirus 5" NCR amplicons revealed that
14 (93%) of them had greater homology to rhinoviruses, whereas 1 (7%) was
related to enteroviruses. Interestingly, median total symptom scores and
oral temperatures of picornavirus-positive patients (
n
=
15) and placebo-treated influenza virus-positive patients (
n
=
161) were similar over a 3-week period. We conclude that, among
the influenza virus-negative preselected cases of this study, rhinoviruses
were relatively frequent pathogens associated with important respiratory
and systemic symptoms.
Rapid Detection of Pyrazinamide-Resistant
Mycobacterium tuberculosis
by a PCR-Based In Vitro System
Authors: Suzuki Yasuhiko , Suzuki Aya , Tamaru Aki , Katsukawa Chihiro , Oda Hajime
Published: July 2002

Pyrazinamide (PZA), an analog of nicotinamide, is a prodrug for tuberculosis which requires conversion to the bactericidal compound pyrazinoic acid by bacterial pyrazinamidase activity. Mutations leading to a loss of pyrazinamidase activity cause PZA resistance in
Mycobacterium tuberculosis
. Thus, the detection of pyrazinamidase activity makes the discrimination of PZA-resistant tuberculosis possible. However, the detection of the pyrazinamidase activity of
M. tuberculosis
isolates needs a large amount of bacilli and is therefore time consuming. In this paper, we describe a new method for the detection of pyrazinamidase activity with a PCR-based system. The genes encoding pyrazinamidase (
pncA
genes) in 30 resistant clinical isolates were amplified by PCR by using forward primers containing bacteriophage T7 promoter sequences at their 5" ends. Then the PCR products were directly subjected to an in vitro transcription-translation coupled system. All of the PZA-resistant isolates tested showed reduced pyrazinamidase activity compared to susceptible
M. tuberculosis
type strain H37Rv. In contrast, all of the 15 susceptible clinical isolates exhibited pyrazinamidase activities similar to that of H37Rv. This fact suggested the possibility of the usefulness of this system for the rapid detection of PZA-resistant
M. tuberculosis
.
Macrolide Efflux Genes
mef
(A) and
mef
(E) Are Carried by Different Genetic Elements in
Streptococcus pneumoniae
Authors: Del Grosso M. , Iannelli F. , Messina C. , Santagati M. , Petrosillo N. , Stefani S. , Pozzi G. , Pantosti A.
Published: July 2002

Susceptibilities to macrolides were evaluated in 267
Streptococcus pneumoniae
isolates, of which 182 were from patients with invasive diseases and 85 were from healthy carriers. Of the 98 resistant isolates, 20 strains showed an M phenotype and carried
mef
. Strains that carried both
mef
(A) and
mef
(E) were found: 17 strains carried
mef
(A) and 3 carried
mef
(E). The characteristics of the strains carrying the
mef
genes and the properties of the
mef-
containing elements were studied. Strains carrying
mef
(A) belonged to serotype 14, were susceptible to all the antibiotics tested except erythromycin, and appeared to be clonally related by pulsed-field gel electrophoresis (PFGE). The three
mef
(E) strains belonged to different serotypes, showed different susceptibility profiles, and did not appear to be related by PFGE. The sequences of a fragment of the
mef-
containing element, which encompassed
mef
and the
msr
(A) homolog, were identical among the three
mef
(E)-positive strains and among the three
mef
(A)-positive strains, although there were differences between the sequences for the two variants at 168 positions. In all
mef
(A)-positive strains, the
mef
element was inserted in
celB
, which led to impairment of the competence of the strains. In line with insertion of the
mef
(E) element at a different site, the competence of the
mef
(E)-positive strains was maintained. Transfer of erythromycin resistance by conjugation was obtained from two of three
mef
(A) strains but from none of three
mef
(E) strains. Due to the important different characteristics of the strains carrying
mef
(A) or
mef
(E), we suggest that the distinction between the two genes be maintained.
Extended-Spectrum β-Lactamase-ProducingKlebsiellapneumoniaein a Neonatal Intensive Care Unit in the High-Prevalence Area of Athens, Greece
Authors: Lebessi E. , Dellagrammaticas H. , Tassios P. T. , Tzouvelekis L. S. , Ioannidou S. , Foustoukou M. , Legakis N. J.
Published: July 2002

Extended-spectrum β-lactamase-producingKlebsiellapneumoniae(EPKP) strains are frequently implicated in outbreaks in neonatal intensive care units (NICUs). During the period from 1997 to 1998, 21 infections and 23 colonizations with EPKP were recorded in the NICU of a children's hospital in Athens, Greece. Seventeen of the infected and 12 of the colonized neonates had been referred from other hospitals. The remaining infections and colonizations occurred during the current hospitalization. Pulsed-field gel electrophoresis typing showed that the latter cases were due to an outbreak strain that persisted in the unit, while the repeated introduction of EPKP carriers was mostly due to clonal outbreaks in two maternity hospitals.
Quantitative Detection ofNeospora caninumin Bovine Aborted Fetuses and Experimentally Infected Mice by Real-Time PCR
Authors: Collantes-Fernández Esther , Zaballos Ángel , Álvarez-García Gema , Ortega-Mora Luis M.
Published: July 2002

We report the development of a real-time PCR assay for the quantitative detection ofNeospora caninumin infected host tissues. The assay uses the double-stranded DNA-binding dye SYBR Green I to continuously monitor product formation. Oligonucleotide primers were designed to amplify a 76-bp DNA fragment corresponding to the Nc5 sequence ofN. caninum. A similar method was developed to quantify the 28S rRNA host gene in order to compare the parasite load of different samples and to correct for the presence of potential PCR-inhibiting compounds in the DNA samples. A linear quantitative detection range of 6 logs with a calculated detection limit of 10−1tachyzoite per assay was observed with excellent linearity (R2= 0.998). Assay specificity was confirmed by using DNA from the closely related parasiteToxoplasma gondii. The applicability of the technique was successfully tested in a variety of host brain tissues: (i) aborted bovine fetuses classified into negative or positiveNeospora-infected animals according to the observation of compatible lesions by histopathological study and (ii) experimentally infected BALB/c mice, divided into three groups, inoculated animals with or without compatible lesions and negative controls. All samples were also tested by ITS1Neosporanested PCR and a high degree of agreement was shown between both PCR techniques (κ= 0.86). This technique represents a useful quantitative diagnostic tool to be used in the study of the pathogenicity, immunoprophylaxis, and treatment ofNeosporainfection.
Epidemiology of Extended-Spectrum β-Lactamase-Producing
Enterobacter
Isolates in a Spanish Hospital during a 12-Year Period
Authors: Cantón Rafael , Oliver Antonio , Coque Teresa M. , Varela María del Carmen , Pérez-Díaz José Claudio , Baquero Fernando
Published: July 2002

Fifteen
Enterobacter
clinical isolates (11
Enterobacter cloacae
isolates, 3
Enterobacter aerogenes
isolates, and 1
Enterobacter gergoviae
isolate), representing 0.4% of all
Enterobacter
isolates recovered in our hospital from 1989 to 2000, were suspected of harboring an extended-spectrum β-lactamase (ESBL). These isolates were recovered from 14 different patients. ESBLs were transferred by conjugation into an
Escherichia coli
recipient strain. Pulsed-field gel electrophoresis (PFGE) revealed a single clone of
E. aerogenes
and six different clones of
E. cloacae
. Four of these
E. cloacae
clonal types were represented by only one isolate each, but the other two were represented by three and four isolates, respectively. Isoelectric focusing, susceptibility phenotyping, PCR analysis, and sequencing demonstrated the presence of three different ESBLs. The most frequent was the recently characterized CTX-M-10 ESBL, which was found in the
E. gergoviae
isolate and in all but one of the
E. cloacae
isolates. The remaining
E. cloacae
isolate harbored a TEM-27 ESBL, and the three
E. aerogenes
isolates harbored a TEM-24 ESBL. PFGE revealed that our
E. aerogenes
strain was indistinguishable from the French TEM-24-producing
E. aerogenes
endemic clone. Although a low prevalence of ESBL-producing
Enterobacter
isolates was found in our institution over a 12-year period, a diversity of nonepidemic
E. cloacae
clones was detected, as was the persistence of the CTX-M-10 β-lactamase. The presence of the TEM-24-producing
E. aerogenes
French clone in our institution also demonstrates the intercountry dissemination of ESBL-producing isolates.
Emerging Extended-Spectrum β-Lactamases in
Proteus mirabilis
Authors: Pagani Laura , Migliavacca Roberta , Pallecchi Lucia , Matti Cecilia , Giacobone Ernesto , Amicosante Gianfranco , Romero Egidio , Rossolini Gian Maria
Published: July 2002

β-Lactamase production was detected in 147 (52%) of 282 consecutive nonduplicate
Proteus mirabilis
isolates obtained over a 1-year period from the S. Matteo Hospital of Pavia (northern Italy). Seventy isolates (48% of the β-lactamase producers) were found to produce extended-spectrum β-lactamases (ESBLs), identified as PER-1 (first report in this species) and TEM-52 in 52 and 18 isolates, respectively. Analysis of clonal diversity of the ESBL producers suggested different spreading patterns for the two ESBL determinants.
Molecular Epidemiology of Subgroup C Avian Pneumoviruses Isolated in the United States and Comparison with Subgroup A and B Viruses
Authors: Shin Hyun-Jin , Cameron Kjerstin T. , Jacobs Janet A. , Turpin Elizabeth A. , Halvorson David A. , Goyal Sagar M. , Nagaraja Kakambi V. , Kumar Mahesh C. , Lauer Dale C. , Seal Bruce S. , Njenga M. Kariuki
Published: July 2002

The avian pneumovirus (APV) outbreak in the United States is concentrated in the north-central region, particularly in Minnesota, where more outbreaks in commercial turkeys occur in the spring (April to May) and autumn (October to December). Comparison of the nucleotide and amino acid sequences of nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), and second matrix (M2) genes of 15 U.S. APV strains isolated between 1996 and 1999 revealed between 89 and 94% nucleotide sequence identity and 81 to 95% amino acid sequence identity. In contrast, genes from U.S. viruses had 41 to 77% nucleotide sequence identity and 52 to 78% predicted amino acid sequence identity with European subgroup A or B viruses, confirming that U.S. viruses belonged to a separate subgroup. Of the five proteins analyzed in U.S. viruses, P was the most variable (81% amino acid sequence identity) and N was the most conserved (95% amino acid sequence identity). Phylogenetic comparison of subgroups A, B, and C viruses indicated that A and B viruses were more closely related to each other than either A or B viruses were to C viruses.
L* Protein of Theiler's Murine Encephalomyelitis Virus Is Required for Virus Growth in a Murine Macrophage-Like Cell Line
Authors: Obuchi Masatsugu , Yamamoto Jiro , Odagiri Takato , Uddin Mohammed Nasir , Iizuka Hideaki , Ohara Yoshiro
Published: July 2002

We sought to confirm the importance of L* protein for growth ofTheiler's murine encephalomyelitis virus(TMEV) in a macrophage-like cell line, J774-1. The protein is out of frame with the polyprotein and synthesized in DA but not GDVII subgroup strains of TMEV. A recombinant virus, DANCL*/GD, which substitutes the DA 5′ noncoding and L* coding regions for the corresponding regions of GDVII and synthesizes L* protein, grew with little restriction in J774-1 cells. In contrast, another recombinant virus, DANCL*-1/GD, which has an ACG rather than an AUG as the starting codon of L* protein at nucleotide 1079, resulting in no synthesis of L* protein, did not grow well. No significant difference between the rates of adsorption to J774-1 cells of these viruses was observed. RNase protection assay demonstrated that DANCL*/GD viral RNA significantly increased, whereas only a minimal increase was observed for DANCL*-1/GD. The present study suggests that L* protein is required for virus growth in macrophages.
Characterization of Influenza Virus NS1 Protein by Using a Novel Helper-Virus-Free Reverse Genetic System
Authors: Enami Masayoshi , Enami Kazue
Published: July 2002

We have developed a novel helper-virus-free reverse genetic system to genetically manipulate influenza A viruses. The RNPs, which were purified from the influenza A/WSN/33 (WSN) virus, were treated with RNase H in the presence of NS (nonstructural) cDNA fragments. This specifically digested the NS RNP. The NS-digested RNPs thus obtained were transfected into cells together with the in vitro-reconstituted NS RNP. The NS-digested RNPs alone did not rescue viruses; however, cotransfection with the NS RNP did. This protocol was also used to rescue the NP transfectant. We obtained two NS1 mutants,
dl
12 and N110, using this protocol. The
dl
12 NS gene contains a deletion of 12 amino acids at positions 66 to 77 near the N terminus. This virus was temperature sensitive in Madin-Darby bovine kidney (MDBK) cells as well as in Vero cells. The translation of all viral proteins as well as cellular proteins was significantly disrupted during a later time of infection at the nonpermissive temperature of 39°C. The N110 mutant consists of 110 amino acids which are the N-terminal 48% of the WSN virus NS1 protein. Growth of this virus was significantly reduced at any temperature. In the virus-infected cells, translation of the M1 protein was reduced to 10 to 20% of that of the wild-type virus; however, the translation of neither the nucleoprotein nor NS1 was significantly interfered with, indicating the important role of NS1 in translational stimulation of the M1 protein.
The Core of the Respiratory Syncytial Virus Fusion Protein Is a Trimeric Coiled Coil
Authors: Matthews Jacqueline M. , Young Thomas F. , Tucker Simon P. , Mackay Joel P.
Published: July 2002

Entry into the host cell by enveloped viruses is mediated by fusion (F) or transmembrane glycoproteins. Many of these proteins share a fold comprising a trimer of antiparallel coiled-coil heterodimers, where the heterodimers are formed by two discontinuous heptad repeat motifs within the proteolytically processed chain. The F protein of human respiratory syncytial virus (RSV; the major cause of lower respiratory tract infections in infants) contains two corresponding regions that are predicted to form coiled coils (HR1 and HR2), together with a third predicted heptad repeat (HR3) located in a nonhomologous position. In order to probe the structures of these three domains and ascertain the nature of the interactions between them, we have studied the isolated HR1, HR2, and HR3 domains of RSV F by using a range of biophysical techniques, including circular dichroism, nuclear magnetic resonance spectroscopy, and sedimentation equilibrium. HR1 forms a symmetrical, trimeric coiled coil in solution (
K
3
≈ 2.2 × 10
11
M
−2
) which interacts with HR2 to form a 3:3 hexamer. The HR1-HR2 interaction domains have been mapped using limited proteolysis, reversed-phase high-performance liquid chromatography, and electrospray-mass spectrometry. HR2 in isolation exists as a largely unstructured monomer, although it exhibits a tendency to form aggregates with β-sheet-like characteristics. Only a small increase in α-helical content was observed upon the formation of the hexamer. This suggests that the RSV F glycoprotein contains a domain that closely resembles the core structure of the simian parainfluenza virus 5 fusion protein (K. A. Baker, R. E. Dutch, R. A. Lamb, and T. S. Jardetzky, Mol. Cell 3:309–319, 1999). Finally, HR3 forms weak α-helical homodimers that do not appear to interact with HR1, HR2, or the HR1-HR2 complex. The results of these studies support the idea that viral fusion proteins have a common core architecture.
An Enzymatic Footprinting Analysis of the Interaction of 40S Ribosomal Subunits with the Internal Ribosomal Entry Site of Hepatitis C Virus
Authors: Kolupaeva Victoria G. , Pestova Tatyana V. , Hellen Christopher U. T.
Published: July 2002

Hepatitis C virus translation is initiated on a ∼330-nucleotide (nt)-long internal ribosomal entry site (IRES) at the 5′ end of the genome. In this process, a 43S preinitiation complex (comprising a 40S ribosomal subunit, eukaryotic initiation factor 3 (eIF3), and a ternary [eIF2-GTP-initiator tRNA] complex) binds the IRES in a precise manner so that the initiation codon is placed at the ribosomal P site. This binding step involves specific interactions between the IRES and different components of the 43S complex. The 40S subunit and eIF3 can bind to the IRES independently; previous analyses revealed that eIF3 binds specifically to an apical half of IRES domain III. Nucleotides in the IRES that are involved in the interaction with the 40S subunit were identified by RNase footprinting and mapped to the basal half of domain III and in domain IV. Interaction sites were identified in locations that have been found to be essential for IRES function, including (i) the apical loop residues GGG
266-268
in subdomain IIId and (ii) the pseudoknot. Extensive protection from RNase cleavage also occurred downstream of the pseudoknot in domain IV, flanking both sides of the initiation codon and corresponding in length to that of the mRNA-binding cleft of the 40S subunit. These results indicate that the 40S subunit makes multiple interactions with the IRES and suggest that only nucleotides in domain IV are inserted into the mRNA-binding cleft of the 40S subunit.
Analysis of the Transmembrane Domain of Influenza Virus Neuraminidase, a Type II Transmembrane Glycoprotein, for Apical Sorting and Raft Association
Authors: Barman Subrata , Nayak Debi P.
Published: July 2002

Influenza virus neuraminidase (NA), a type II transmembrane protein, is directly transported to the apical plasma membrane in polarized MDCK cells. Previously, it was shown that the transmembrane domain (TMD) of NA provides a determinant(s) for apical sorting and raft association (A. Kundu, R. T. Avalos, C. M. Sanderson, and D. P. Nayak, J. Virol. 70:6508–6515, 1996). In this report, we have analyzed the sequences in the NA TMD involved in apical transport and raft association by making chimeric TMDs from NA and human transferring receptor (TR) TMDs and by mutating the NA TMD sequences. Our results show that the COOH-terminal half of the NA TMD (amino acids [aa] 19 to 35) was significantly involved in raft association, as determined by Triton X-100 (TX-100) resistance. However, in addition, the highly conserved residues at the extreme NH
2
terminus of the NA TMD were also critical for TX-100 resistance. On the other hand, 19 residues (aa 9 to 27) at the NH
2
terminus of the NA TMD were sufficient for apical sorting. Amino acid residues 14 to 18 and 27 to 31 had the least effect on apical transport, whereas mutations in the amino acid residues 11 to 13, 23 to 26, and 32 to 35 resulted in altered polarity for the mutant proteins. These results indicated that multiple regions in the NA TMD were involved in apical transport. Furthermore, these results support the idea that the signals for apical sorting and raft association, although residing in the NA TMD, are not identical and vary independently and that the NA TMD also possesses an apical determinant(s) which can interact with apical sorting machineries outside the lipid raft.