bluuebunny/crossref_metadata_2025 huggingface dataset:
The Complex Flagellar Torque Generator of
Pseudomonas aeruginosa
Authors: Doyle Timothy B. , Hawkins Andrew C. , McCarter Linda L.
Published: September 2004

Flagella act as semirigid helical propellers that are powered by reversible rotary motors. Two membrane proteins, MotA and MotB, function as a complex that acts as the stator and generates the torque that drives rotation. The genome sequence of
Pseudomonas aeruginosa
PAO1 contains dual sets of
motA
and
motB
genes, PA1460-PA1461 (
motAB
) and PA4954-PA4953 (
motCD
), as well as another gene,
motY
(PA3526), which is known to be required for motor function in some bacteria. Here, we show that these five genes contribute to motility. Loss of function of either
motAB
-like locus was dispensable for translocation in aqueous environments. However, swimming could be entirely eliminated by introduction of combinations of mutations in the two
motAB
-encoding regions. Mutation of both genes encoding the MotA homologs or MotB homologs was sufficient to abolish motility. Mutants carrying double mutations in nonequivalent genes (i.e.,
motA motD
or
motB motC
) retained motility, indicating that noncognate components can function together.
motY
appears to be required for
motAB
function. The combination of
motY
and
motCD
mutations rendered the cells nonmotile. Loss of function of
motAB
,
motY
, or
motAB motY
produced similar phenotypes; although the swimming speed was only reduced to ∼85% of the wild-type speed, translocation in semisolid motility agar and swarming on the surface of solidified agar were severely impeded. Thus, the flagellar motor of
P. aeruginosa
represents a more complex configuration than the configuration that has been studied in other bacteria, and it enables efficient movement under different circumstances.
Modified Pathway To Synthesize Ribulose 1,5-Bisphosphate in Methanogenic Archaea
Authors: Finn Michael W. , Tabita F. Robert
Published: September 2004

Several sequencing projects unexpectedly uncovered the presence of genes that encode ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) in anaerobic archaea. RubisCO is the key enzyme of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway, a scheme that does not appear to contribute greatly, if at all, to net CO
2
assimilation in these organisms. Recombinant forms of the archaeal enzymes do, however, catalyze a bona fide RuBP-dependent CO
2
fixation reaction, and it was recently shown that
Methanocaldococcus
(
Methanococcus
)
jannaschii
and other anaerobic archaea synthesize catalytically active RubisCO in vivo. To complete the CBB pathway, there is a need for an enzyme, i.e., phosphoribulokinase (PRK), to catalyze the formation of RuBP, the substrate for the RubisCO reaction. Homology searches, as well as direct enzymatic assays with
M. jannaschii
, failed to reveal the presence of PRK. The apparent lack of PRK raised the possibility that either there is an alternative pathway to generate RuBP or RubisCO might use an alternative substrate in vivo. In the present study, direct enzymatic assays performed with alternative substrates and extracts of
M. jannsachii
provided evidence for a previously uncharacterized pathway for RuBP synthesis from 5-phospho-
d
-ribose-1-pyrophosphate (PRPP) in
M. jannaschii
and other methanogenic archaea. Proteins and genes involved in the catalytic conversion of PRPP to RuBP were identified in
M. jannaschii
(Mj0601) and
Methanosarcina acetivorans
(Ma2851), and recombinant Ma2851 was active in extracts of
Escherichia coli
. Thus, in this work we identified a novel means to synthesize the CO
2
acceptor and substrate for RubisCO in the absence of a detectable kinase, such as PRK. We suggest that the conversion of PRPP to RuBP might be an evolutional link between purine recycling pathways and the CBB scheme.
patS
Minigenes Inhibit Heterocyst Development of
Anabaena
sp. Strain PCC 7120
Authors: Wu Xiaoqiang , Liu Duan , Lee Martin H. , Golden James W.
Published: September 2004

The
patS
gene encodes a small peptide that is required for normal heterocyst pattern formation in the cyanobacterium
Anabaena
sp. strain PCC 7120. PatS is proposed to control the heterocyst pattern by lateral inhibition.
patS
minigenes were constructed and expressed by different developmentally regulated promoters to gain further insight into PatS signaling.
patS
minigenes
patS4
to
patS8
encode PatS C-terminal 4 (GSGR) to 8 (CDERGSGR) oligopeptides. When expressed by P

petE

, P

patS

, or P

rbcL

promoters,
patS5
to
patS8
inhibited heterocyst formation but
patS4
did not. In contrast to the full-length
patS
gene, P

hepA
-patS5

failed to restore a wild-type pattern in a
patS
null mutant, indicating that PatS-5 cannot function in cell-to-cell signaling if it is expressed in proheterocysts. To establish the location of the PatS receptor, PatS-5 was confined within the cytoplasm as a
gfp-patS5
fusion. The green fluorescent protein GFP-PatS-5 fusion protein inhibited heterocyst formation. Similarly, full-length PatS with a C-terminal hexahistidine tag inhibited heterocyst formation. These data indicate that the PatS receptor is located in the cytoplasm, which is consistent with recently published data indicating that HetR is a PatS target. We speculated that overexpression of other
Anabaena
strain PCC 7120 RGSGR-encoding genes might show heterocyst inhibition activity. In addition to
patS
and
hetN
, open reading frame (ORF)
all3290
and an unannotated ORF,
orf77
, encode an RGSGR motif. Overexpression of
all3290
and
orf77
under the control of the
petE
promoter inhibited heterocyst formation, indicating that the RGSGR motif can inhibit heterocyst development in a variety of contexts.
Insecticidal Pilin Subunit from the Insect Pathogen
Xenorhabdus nematophila
Authors: Khandelwal Puneet , Choudhury Devapriya , Birah Ajanta , Reddy M. K. , Gupta Gorakh Prasad , Banerjee Nirupama
Published: September 2004

Xenorhabdus nematophila
is an insect pathogen and produces protein toxins which kill the larval host. Previously, we characterized an orally toxic, large, outer membrane-associated protein complex from the culture medium of
X. nematophila
. Here, we describe the cloning, expression, and characterization of a 17-kDa pilin subunit of
X. nematophila
isolated from that protein complex. The gene was amplified by PCR, cloned, and expressed in
Escherichia coli
. The recombinant protein was refolded in vitro in the absence of its cognate chaperone by using a urea gradient. The protein oligomerized during in vitro refolding, forming multimers. Point mutations in the conserved N-terminal residues of the pilin protein greatly destabilized its oligomeric organization, demonstrating the importance of the N terminus in refolding and oligomerization of the pilin subunit by donor strand complementation. The recombinant protein was cytotoxic to cultured
Helicoverpa armigera
larval hemocytes, causing agglutination and subsequent release of the cytoplasmic enzyme lactate dehydrogenase. The agglutination of larval cells by the 17-kDa protein was inhibited by several sugar derivatives. The biological activity of the purified recombinant protein indicated that it has a conformation similar to that of the native protein. The 17-kDa pilin subunit was found to be orally toxic to fourth- or fifth-instar larvae of an important crop pest,
H. armigera
, causing extensive damage to the midgut epithelial membrane. To our knowledge, this is first report describing an insecticidal pilin subunit of a bacterium.
An Energy Taxis Transducer Promotes Root Colonization byAzospirillum brasilense
Authors: Greer-Phillips Suzanne E. , Stephens Bonnie B. , Alexandre Gladys
Published: September 2004

Motility responses triggered by changes in the electron transport system are collectively known as energy taxis. InAzospirillum brasilense, energy taxis was shown to be the principal form of locomotor control. In the present study, we have identified a novel chemoreceptor-like protein, named Tlp1, which serves as an energy taxis transducer. The Tlp1 protein is predicted to have an N-terminal periplasmic region and a cytoplasmic C-terminal signaling module homologous to those of other chemoreceptors. The predicted periplasmic region of Tlp1 comprises a conserved domain that is found in two types of microbial sensory receptors: chemotaxis transducers and histidine kinases. However, the function of this domain is currently unknown. We characterized the behavior of atlp1mutant by a series of spatial and temporal gradient assays. Thetlp1mutant is deficient in (i) chemotaxis to several rapidly oxidizable substrates, (ii) taxis to terminal electron acceptors (oxygen and nitrate), and (iii) redox taxis. Taken together, the data strongly suggest that Tlp1 mediates energy taxis inA. brasilense. Using qualitative and quantitative assays, we have also demonstrated that thetlp1mutant is impaired in colonization of plant roots. This finding supports the hypothesis that energy taxis and therefore bacterial metabolism might be key factors in determining host specificity inAzospirillum-grass associations.
Dynamic Cell Surface Hydrophobicity of
Lactobacillus
Strains with and without Surface Layer Proteins
Authors: Vadillo-Rodríguez Virginia , Busscher Henk J. , Norde Willem , de Vries Joop , van der Mei Henny C.
Published: September 2004

Variations in surface hydrophobicity of six
Lactobacillus
strains with and without an S-layer upon changes in ionic strength are derived from contact angle measurements with low- and high-ionic-strength aqueous solutions. Cell surface hydrophobicity changed in response to changes in ionic strength in three out of the six strains, offering these strains a versatile mechanism to adhere to different surfaces. The dynamic behavior of the cell surface hydrophobicity could be confirmed for two selected strains by measuring the interaction force between hydrophobic and hydrophilic tips with use of atomic force microscopy.
Detection of Intrahepatic Hepatitis B Virus DNA and Correlation with Hepatic Necroinflammation and Fibrosis
Authors: Wong Danny Ka-Ho , Yuen Man-Fung , Tse Eric , Yuan HeJun , Sum Simon Siu-Man , Hui Chee-Kin , Lai Ching-Lung
Published: September 2004

Assessment of intrahepatic hepatitis B virus (HBV) DNA levels in patients with chronic hepatitis B is important in understanding the natural history of the disease and designing antiviral therapy regimens. However, there is no standardized method for the measurement of intrahepatic HBV DNA levels. We describe a convenient novel method for the measurement of intrahepatic HBV DNA levels based on a modified COBAS Amplicor HBV Monitor test for HBV DNA measurement and real-time PCR β-actin gene detection for human genomic DNA (hgDNA) quantitation. Fifteen hepatitis B e antigen (HBeAg)-positive patients, 26 patients positive for antibody to HBeAg (anti-HBe), and 8 control patients were recruited. The mean between-run coefficient of variation for the β-actin real-time PCR assay was 15.4%. All eight control patients had undetectable intrahepatic and serum HBV DNA levels. All chronic hepatitis B patients had detectable intrahepatic HBV DNA levels, and all but one anti-HBe-positive patient had detectable serum HBV DNA levels. HBeAg-positive patients had higher median intrahepatic and serum HBV DNA levels than anti-HBe-positive patients (6,950 versus 676 HBV DNA copies/ng of hgDNA, respectively [
P
< 0.001] and 184 × 10
6
versus 6.65 × 10
6
copies/ml, respectively [
P
< 0.001]). The intrahepatic HBV DNA levels correlated strongly with the serum HBV DNA levels (
r
= 0.842;
P
< 0.001) and with the degree of fibrosis (
P
= 0.014). We conclude that the method that we describe is reliable and convenient for the measurement of intrahepatic HBV DNA levels and has potential clinical significance.
rpoB
Gene Sequencing for Identification of
Corynebacterium
Species
Authors: Khamis Atieh , Raoult Didier , La Scola Bernard
Published: September 2004

The genus
Corynebacterium
is a heterogeneous group of species comprising human and animal pathogens and environmental bacteria. It is defined on the basis of several phenotypic characters and the results of DNA-DNA relatedness and, more recently, 16S rRNA gene sequencing. However, the 16S rRNA gene is not polymorphic enough to ensure reliable phylogenetic studies and needs to be completely sequenced for accurate identification. The almost complete
rpoB
sequences of 56
Corynebacterium
species were determined by both PCR and genome walking methods. In all cases the percent similarities between different species were lower than those observed by 16S rRNA gene sequencing, even for those species with degrees of high similarity. Several clusters supported by high bootstrap values were identified. In order to propose a method for strain identification which does not require sequencing of the complete
rpoB
sequence (approximately 3,500 bp), we identified an area with a high degree of polymorphism, bordered by conserved sequences that can be used as universal primers for PCR amplification and sequencing. The sequence of this fragment (434 to 452 bp) allows accurate species identification and may be used in the future for routine sequence-based identification of
Corynebacterium
species.
Long-Term Follow-Up Study of Chinese Patients with YMDD Mutations: Significance of Hepatitis B Virus Genotypes and Characteristics of Biochemical Flares
Authors: Yuen Man-Fung , Yuan He-Jun , Sablon Erwin , Wong Danny Ka-Ho , Chan Annie On-On , Wong Benjamin Chun-Yu , Lai Ching-Lung
Published: September 2004

We sought to examine the role of hepatitis B virus (HBV) genotypes in virological breakthroughs and biochemical flares in patients with YMDD mutations during lamivudine therapy. Virologic breakthroughs (i.e., the reappearance of HBV DNA as determined by bDNA assay) and biochemical flares (mild flares = alanine aminotransferase [ALT] between 2 and 10 times the upper limit of normal [ULN]; severe flares = ALT >10 times ULN) were monitored in 154 hepatitis B e antigen-positive patients receiving long-term lamivudine. The HBV genotypes and YMDD mutations were determined. Forty-three patients had virological breakthroughs with YMDD mutations (median follow-up of 29.6 months [range, 22.3 to 61.4]). Twenty patients (47%) patients had mild biochemical flares; seven (16%) had severe flares. Two patients showed an elevation of bilirubin level that is >2 times the ULN. All patients recovered spontaneously. The cumulative risks for biochemical flares were 28, 47, and 58% for the first 3 years, respectively. Patients with biochemical flares compared to those without flares had a significantly higher median pretreatment ALT level (61 U/liter versus 34.5 U/liter [
P
= 0.012]). There were no differences in the cumulative risk of virological breakthroughs, risk, and severity of biochemical flares between patients with genotypes B (
n
= 11) and C (
n
= 32). There was an increase in the percentage of patients with single YMDD mutant at last follow-up compared to that at the time of virological breakthroughs (74% [
n
= 32] versus 47% [
n
= 20], respectively;
P
= 0.015). The chances of YMDD mutations with virological breakthroughs and biochemical flares were similar in patients with genotypes B and C. Biochemical flares were common, with 16% being severe in nature. High pretreatment ALT levels were associated with a higher chance of biochemical flares.
Characteristics of
Streptococcus pneumoniae
,
Haemophilus influenzae
, and
Moraxella catarrhalis
Isolated from the Nasopharynges of Asymptomatic Children and Molecular Analysis of
S. pneumoniae
and
H. influenzae
Strain Replacement in the Nasopharynx
Authors: Sulikowska Agnieszka , Grzesiowski Paweł , Sadowy Ewa , Fiett Janusz , Hryniewicz Waleria
Published: September 2004

Nasopharyngeal carriage of
Streptococcus pneumoniae
,
Haemophilus influenzae
, and
Moraxella catarrhalis
in 226 children in different settings (in a crèche [day care center], in an orphanage, and at home) during two seasons (winter and spring) was studied. The rates of carriage of
S. pneumoniae
and
H. influenzae
were markedly higher in the crèche and in the orphanage than in the home setting (e.g., 56.5, 63.3, and 25.9%, respectively, for
S. pneumoniae
in winter). Approximately 80% of the
S. pneumoniae
isolates identified in the crèche and in the orphanage belonged to the serotypes represented in the seven-valent pneumococcal vaccine, and 4.4% of the children were colonized by
H. influenzae
type b. Almost all
H. influenzae
isolates were fully susceptible to the antimicrobial agents tested, and only five (3.6%) produced β-lactamase; in contrast, 100% of the
M. catarrhalis
isolates were β-lactamase positive. Among
S. pneumoniae
isolates, 36.2% were nonsusceptible to penicillin (PNSP) and 11.8% were fully resistant to penicillin (PRP). All PNSP isolates were obtained from children at the crèche and at the orphanage but not among children brought up at home, and all PRP isolates showed a multiresistant phenotype. Colonization by PRP isolates correlated well with prior treatment with β-lactams. For the majority of children colonized at both sampling times, strain replacement of
S. pneumoniae
and
H. influenzae
was observed; long-term colonization by a single strain was rare.
Mutation in a Lordsdale Norovirus Epidemic Strain as a Potential Indicator of Transmission Routes
Authors: Dingle Kate E.
Published: September 2004

An increase in norovirus outbreaks was reported internationally during 2002 and 2003 and was also observed in Oxfordshire (United Kingdom) hospitals. To understand their epidemiological relationships, viruses from 22 outbreaks (15 from one hospital) were subjected to nucleotide sequencing. The 3′-terminal 3,255 nt or complete genomes were determined for 49 viruses. All outbreaks were caused by a genogroup II norovirus related to the Lordsdale virus (GII 4), common in healthcare settings. The norovirus mutation rate was sufficiently high that the 3,255-nucleotide sequences allowed separate and potentially connected outbreaks to be identified, since all outbreaks with identical sequences were temporally or geographically linked. The high mutation rate was further indicated by four mutations and three microheterogeneities in 3,255 nucleotides during 17 days of norovirus shedding by an immunocompromised patient. The data suggested that multiple virus introductions from the community, occasional transmission among wards, and one instance of ongoing environmental contamination had occurred. The accumulation, or lack, of mutations within an outbreak was also used to indicate the predominant transmission route. In an outbreak where person-to-person spread was thought to predominate, six mutations were detected throughout the genome, whereas one mutation was detected when point source infection was suspected. This norovirus epidemic strain differed from its closest previously described relative by 11.4 to 13.6% in the outer P2 domain of the capsid, which also had a single-amino-acid insertion. Alterations to the capsid structure compared to previous noroviruses may explain the increased number of outbreaks during 2002 and 2003.
The Species
Mycobacterium africanum
in the Light of New Molecular Markers
Authors: Niemann S. , Kubica T. , Bange F. C. , Adjei O. , Browne E. N. , Chinbuah M. A. , Diel R. , Gyapong J. , Horstmann R. D. , Joloba M. L. , Meyer C. G. , Mugerwa R. D. , Okwera A. , Osei I. , Owusu-Darbo E. , Schwander S. K. , Rüsch-Gerdes S.
Published: September 2004

The findings of recent studies addressing the molecular characteristics of
Mycobacterium tuberculosis
complex isolates have initiated a discussion on the classification of
M. africanum
, especially of those isolates originating from East Africa (cluster F, subtype II) and displaying phenotypic and biochemical characteristics more similar to those of
M. tuberculosis
. To further address this question, we analyzed a representative collection of 63
M. tuberculosis
complex strains comprising 30
M. africanum
subtype I strains, 20
M. africanum
subtype II strains, 10 randomly chosen
M. tuberculosis
isolates, and type strains of
M. tuberculosis
,
M. bovis
, and
M. africanum
for the following biochemical and molecular characteristics: single-nucleotide polymorphisms (SNPs) in
gyrB
and
narGHJI
and the presence or absence of RD1, RD9, and RD12. For all molecular markers analyzed, subtype II strains were identical to the
M. tuberculosis
strains tested. In contrast, the subtype I strains as well as the
M. africanum
type strain showed unique combinations of SNPs in
gyrB
and genomic deletions (the absence of RD9 and the presence of RD12), which proves their independence from
M. tuberculosis
and
M. bovis
. Accordingly, all subtype I strains displayed main biochemical characteristics included in the original species description of
M. africanum
. We conclude that the isolates from West Africa were proved to be
M. africanum
with respect to the phenotypic and genetic markers analyzed, while the isolates from East Africa must be regarded as phenotypic variants of
M. tuberculosis
(genotype Uganda). We propose the addition of the molecular characteristics defined here to the species description of
M. africanum
, which will allow clearer species differentiation in the future.
Comprehensive Detection and Serotyping of Human Adenoviruses by PCR and Sequencing
Authors: Sarantis Helen , Johnson Grant , Brown Martha , Petric Martin , Tellier Raymond
Published: September 2004

Human adenoviruses are common pathogens associated with many diseases, including respiratory, gastrointestinal, and ocular infections. Because they are now being increasingly recognized as agents of life-threatening disseminated infection in immunocompromised patients, robust and sensitive laboratory detection methods are needed for their rapid diagnosis. We describe here a PCR assay using a single primer pair, targeting a region of the hexon gene containing hypervariable region 7, that can detect all known human adenovirus serotypes and allows for serotype determination through the analysis of the nucleotide sequence. This comprehensive assay has proven effective for diagnosing adenoviruses at the serotype level in a broad range of patient specimens, including conjunctival, nasopharyngeal, stool, blood, and urine specimens.
Identification of Emerging Human-Pathogenic
Pythium insidiosum
by Serological and Molecular Assay-Based Methods
Authors: Vanittanakom Nongnuch , Supabandhu Jitwadee , Khamwan Chantana , Praparattanapan Jutarut , Thirach Sophit , Prasertwitayakij Narawudt , Louthrenoo Worawit , Chiewchanvit Siri , Tananuvat Napaporn
Published: September 2004

Pythium insidiosum
is a pathogen that causes disease in both animals and humans. Human infection is rare; however, when it does occur, most patients, especially those having underlying hemoglobinopathy syndromes, such as thalassemia, exhibit a severe form. We identified four isolates of
P. insidiosum
. Two were recovered from tissue biopsy specimens from thalassemic and leukemic patients, one was derived from brain tissue from a thalassemic patient, and another was isolated from a corneal ulcer from a fourth patient. Western blotting and an enzyme-linked immunosorbent assay (ELISA) were performed with a serum sample derived from one thalassemic patient. The methods used to identify the
P. insidiosum
isolates were based on morphology, nucleic acid sequencing, and a PCR assay. To confirm the identification, portions of the 18S rRNA genes of these four isolates were sequenced. The sequences were shown to be homologous to previously described
P. insidiosum
DNA sequences. In addition, PCR amplification of the internal transcribed spacer region specific for
P. insidiosum
was positive for all four isolates. The ELISA with the serum sample from the thalassemic patient gave a positive result from a serum dilution of 1:800. Finally, Western immunoblotting with this serum sample showed positive immunoglobulin G recognition for proteins of 110, 73, 56, 42 to 35, 30 to 28, 26, and 23 kDa. The results of this study show that both molecularly based diagnostic and serodiagnostic techniques are useful for the rapid identification of human pythiosis. The predominant antigens recognized by Western blotting may be useful in the development of a more sensitive and specific diagnostic tool for this disease.
Comparison of Two Culture Media and Three Sampling Techniques for Sensitive and Rapid Screening of Vaginal Colonization by Group B Streptococcus in Pregnant Women
Authors: Gupta Chakshu , Briski Laurence Edward
Published: September 2004

The Centers for Disease Control and Prevention (CDC) recommend universal screening of all pregnant women between 35 and 37 weeks of gestation for group B streptococci (GBS) by use of a selective broth medium. Recent reports suggest that Granada medium can be used for rapid and direct visual identification of GBS colonies. However, studies comparing the Granada medium method to the selective broth method are few, and while some report comparable sensitivities, others have found significant differences in detection rates between the two methods. This prospective study compared a method using Granada agar to a Todd-Hewitt broth method with subculture to blood agar in order to determine which GBS detection method is more sensitive and less labor-intensive and has a more rapid turnaround time. Detection rates for three sampling techniques (rectovaginal, vaginal only, and cervical only) were also compared. Consecutive specimens for GBS screening received over a 6-month period from 1,635 pregnant women were included. Overall, GBS was detected in 390 (23.8%) women. The Granada medium gave positive results for 348 of these women, and the selective broth gave positive results for 385, indicating sensitivities of 89.2% for the Granada medium and 98.7% for the selective broth. These findings show that the Granada medium method is less sensitive than the selective broth method and should not replace it as the only method for screening pregnant women for GBS. However, the Granada medium method reduced detection time to 1 day and also reduced the use of ancillary tests in approximately 90% of positive cases. Additionally, no significant differences were noted in the detection rates with rectovaginal, vaginal, and cervical specimens.
Nosocomial Transmission of CTX-M-2 β-Lactamase-Producing
Acinetobacter baumannii
in a Neurosurgery Ward
Authors: Nagano Noriyuki , Nagano Yukiko , Cordevant Christophe , Shibata Naohiro , Arakawa Yoshichika
Published: September 2004

Three strains of cefotaxime (CTX)-resistant
Acinetobacter baumannii
, FM0209680, FM0300106, and FM0301433, were isolated from transtracheal aspirate cultures of three patients with probable nosocomial infections in a neurosurgery ward in Japan. The CTX MICs for these isolates were greater than 128 μg/ml but were drastically reduced in the presence of 4 μg of clavulanic acid per ml. These strains were also resistant to ceftriaxone, cefpodoxime, and aztreonam but were susceptible to ceftazidime and imipenem. The profile of resistance to various broad-spectrum β-lactams was transferred by conjugation. Strain FM0209680 was not eradicated from case patient 1 by administration of imipenem, ceftazidime, and levofloxacin, even after a 6-month hospitalization period. Strains FM0300106 and FM0301433 were isolated from case patients 2 and 3 during the sixth week following admission, respectively, and then each patient was colonized for 3 weeks. Eradication of FM0300106 was successfully obtained from case patient 2 by imipenem treatment, while administration of imipenem was continued to prevent pneumonia. Prophylactic antimicrobial therapy was discontinued in case patient 3 because of the lack of pneumonic symptoms, and FM0301433 disappeared after the discontinuation of antimicrobial chemotherapy. All three strains carried the
bla
CTX-M-2
gene, and the appearance of colonies in the growth-inhibitory zones around disks of CTX and aztreonam in double-disk synergy tests suggested inducible β-lactamase production in these
A. baumannii
strains. The ribotyping investigation suggested that all these strains belong to the same clonal lineage. The plasmids harbored by
A. baumannii
had the same restriction profile as those harbored by
Proteus mirabilis
strains previously isolated in a urology ward of the Funabashi Medical Center.
Use of Culture, PCR Analysis, and DNA Microarrays for Detection of
Campylobacter jejuni
and
Campylobacter coli
from Chicken Feces
Authors: Keramas Georgios , Bang Dang Duong , Lund Marianne , Madsen Mogens , Bunkenborg Henrik , Telleman Pieter , Christensen Claus Bo Vöge
Published: September 2004

A DNA microarray for detection of
Campylobacter
spp. was recently developed and applied to detect
Campylobacter
spp. directly from chicken feces. Sixty-five pooled chicken cloacal swab samples from 650 individual broiler chickens were included in the study. The results of
Campylobacter
sp. detection obtained with DNA microarrays were compared to those obtained by conventional culture and gel electrophoresis. By conventional culture, 60% of the samples were positive for either
Campylobacter jejuni
or
Campylobacter coli
. By PCR and capillary electrophoresis, 95% of the samples were positive for
Campylobacter
spp., whereas with DNA microarrays all samples were positive for
Campylobacter
spp. By application of DNA microarray analysis, the isolates in 4 samples (6%) could not be identified to the species level, whereas by PCR-capillary electrophoresis, the isolates in 12 samples (19%) remained unidentified. Interestingly, PCR-capillary electrophoresis analysis revealed that two (3%) of the samples were positive for both
C. jejuni
and
C. coli
, while DNA microarray analysis revealed that nine (14%) of the samples were positive for both species. Of 65 samples, 2 samples were identified to contain
C. coli
by conventional culture but were positive for
C. jejuni
by both PCR-capillary electrophoresis and DNA microarray analysis. The discrepancy between the methods is discussed.
Enumeration and Isolation ofcpe-PositiveClostridium perfringensSpores from Feces
Authors: Heikinheimo Annamari , Lindström Miia , Korkeala Hannu
Published: September 2004

A hydrophobic grid membrane filter-colony hybridization (HGMF-CH) method for the enumeration and isolation ofcpegene-carrying (cpe-positive)Clostridium perfringensspores from feces was developed. A 425-bp DNA probe specific for thecpegene was sensitive and specific when tested with bacterial DNA and pure cultures. The enumeration ofcpe-positiveC. perfringensby the HGMF-CH method proved to be as sensitive as nested PCR combined with the most-probable number technique when tested with fecal samples from healthy individuals. With the aid of the HGMF-CH method, positive hybridization signals were detected from two out of seven fecal samples obtained from healthy individuals. Furthermore,cpe-positiveC. perfringenswas successfully isolated from both of these samples. The detection ofcpe-positiveC. perfringensby the HGMF-CH method is dependent on the ratio ofcpe-positiveC. perfringenscolonies to totalC. perfringenscolonies growing on the HGMF-tryptose-sulfite-cycloserine plate.cpe-positiveC. perfringenscould be isolated if the ratio ofcpe-positiveC. perfringensspores to totalC. perfringensspores was 6 × 10−5or higher. The HGMF-CH method provides an aid in the investigation of fecal samples of patients suffering from food poisoning or other diseases caused bycpe-positiveC. perfringens. The method also offers a new approach in the investigation of the epidemiology ofcpe-positiveC. perfringensstrains.
Epidemiology and Molecular Characterization ofStreptococcus pyogenesRecovered from Scarlet Fever Patients in Central Taiwan from 1996 to 1999
Authors: Chiou Chien-Shun , Liao Tsai-Ling , Wang Tzu-Hui , Chang Hsiu-Li , Liao Jui-Cheng , Li Chun-Chin
Published: September 2004

One hundred seventy-nineStreptococcus pyogenesisolates recovered from scarlet fever patients from 1996 to 1999 in central Taiwan were characterized byemm, Vir, and pulsed-field gel electrophoresis (PFGE) typing methods. The protocols for Vir and PFGE typing were standardized. A database of the DNA fingerprints for the isolates was established. Nineemmoremm-like genes, 19 Vir patterns, and 26 SmaI PFGE patterns were detected among the isolates. Among the three typing methods, PFGE was the most discriminatory. However, it could not completely replace Vir typing because some isolates with identical PFGE patterns could be further differentiated into several Vir patterns. The prevalentemmtypes wereemm4 (n= 81 isolates [45%]),emm12 (n= 64 [36%]),emm1 (n= 14 [8%]), andemm22 (n= 13 [7%]). Someemmtype isolates could be further differentiated into severalemm-Vir-PFGE genotypes; however, only one genotype in eachemmgroup was usually predominant. DNA from nine isolates was resistant to SmaI digestion. Further PFGE analysis with SgrAI showed that the SmaI digestion-resistant strains could be derived from indigenous strains by horizontal transfer of exogenous genetic material. The emergence of the new strains could have resulted in an increase in scarlet fever cases in central Taiwan since 2000. Theemmsequences, Vir, and PFGE pattern database will serve as a basis for information for the long-term evolutionary study of localS. pyogenesstrains.
Phage Types and Genotypes of Shiga Toxin-Producing
Escherichia coli
O157:H7 Isolates from Humans and Animals in Spain: Identification and Characterization of Two Predominating Phage Types (PT2 and PT8)
Authors: Mora Azucena , Blanco Miguel , Blanco Jesús E. , Alonso M. Pilar , Dhabi Ghizlane , Thomson-Carter Fiona , Usera Miguel A. , Bartolomé Rosa , Prats Guillermo , Blanco Jorge
Published: September 2004

Phage
typing and DNA macrorestriction fragment analysis by
pulsed-field electrophoresis (PFGE) were used for the epidemiological
subtyping of a collection of Shiga toxin-producing
Escherichia
coli
(STEC) O157:H7 strains isolated in Spain between 1980 and
1999. Phage typing distinguished a total of 18 phage types among 171
strains isolated from different sources (67 humans, 82 bovines, 12
ovines, and 10 beef products). However, five phage types, phage type 2
(PT2; 42 strains), PT8 (33 strains), PT14 (14 strains), PT21/28 (11
strains), and PT54 (16 strains), accounted for 68% of the study
isolates. PT2 and PT8 were the most frequently found among strains from
both humans (51%) and bovines (46%). Interestingly, we
detected a significant association between PT2 and PT14 and the
presence of acute pathologies. A group of 108 of the 171 strains were
analyzed by PFGE, and 53 distinct XbaI macrorestriction
patterns were identified, with 38 strains exhibiting unique PFGE
patterns. In contrast, phage typing identified 15 different phage
types. A total of 66 phage type-PFGE subtype combinations were
identified among the 108 strains. PFGE subtyping differentiated between
unrelated strains that exhibited the same phage type. The most common
phage type-PFGE pattern combinations were PT2-PFGE type 1 (1 human and
11 bovine strains), PT8-PFGE type 8 (2 human, 6 bovine, and 1 beef
product strains), PT2-PFGE subtype 4A (1 human, 3 bovine, and 1 beef
product strains). Nine (29%) of 31 human strains showed phage
type-PFGE pattern combinations that were detected among the bovine
strains included in this study, and 26 (38%) of 68 bovine
strains produced phage type-PFGE pattern combinations observed among
human strains included in this study, confirming that cattle are a
major reservoir of strains pathogenic for humans. PT2 and PT8 strains
formed two groups which differed from each other in their motilities,
stx
genotypes, PFGE patterns, and the severity of the
illnesses that they
caused.
Identification to the Species Level and Differentiation between Strains of
Aspergillus
Clinical Isolates by Automated Repetitive-Sequence-Based PCR
Authors: Healy M. , Reece K. , Walton D. , Huong J. , Shah K. , Kontoyiannis D. P.
Published: September 2004

A commercially available repetitive-sequence-based PCR (rep-PCR) DNA fingerprinting assay adapted to an automated format, the DiversiLab system, enables rapid microbial identification and strain typing. We explored the performance of the DiversiLab system as a molecular typing tool for 69
Aspergillus
isolates (38
A. fumigatus
, 15
A. flavus
, and 16
A. terreus
isolates) had been previously characterized by morphological analysis. Initially, 27
Aspergillus
isolates (10
A. fumigatus
, 9
A. flavus
, and 8
A. terreus
isolates) were used as controls to create a rep-PCR-based DNA fingerprint library with the DiversiLab software. Then, 42 blinded
Aspergillus
isolates were typed using the system. The rep-PCR-based profile revealed 98% concordance with morphology-based identification. rep-PCR-based DNA fingerprints were reproducible and were consistent for DNA from both hyphae and conidia. DiversiLab dendrogram reports correctly identified all
A. fumigatus
(
n
= 28),
A. terreus
(
n
= 8), and
A. flavus
(
n
= 6) isolates in the 42 blinded
Aspergillus
isolates. rep-PCR-based identification of all isolates was 100% in agreement with the contiguous internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) sequence-based identification of the respective isolates. Additionally, the DiversiLab system could demonstrate strain-level differentiation of
A. flavus
and
A. terreu
s. Automated rep-PCR may be a time-efficient, effective, easy-to-use, novel genotyping tool for identifying and determining the strain relatedness of fungi. This system may be useful for epidemiological studies, molecular typing, and surveillance of
Aspergillus
species.
Microevolution of
Candida albicans
Strains during Catheter-Related Candidemia
Authors: Shin Jong Hee , Park Mi-Ra , Song Jeong Won , Shin Dong Hyeon , Jung Sook-In , Cho Duck , Kee Seung Jung , Shin Myung Geun , Suh Soon Pal , Ryang Dong Wook
Published: September 2004

We examined microevolution in a series of
Candida albicans
strains isolated from patients with catheter-related candidemia. Sixty-one isolates (29 from blood, 18 from catheters, 10 from urine, and 4 from other sites) were obtained from 15 patients who were admitted to the same hospital over a 3-year period. Isolates were analyzed by using Southern hybridization with the C1 fragment of Ca3 as a probe (C1 fingerprinting) and pulsed-field gel electrophoresis (PFGE). PFGE typing consisted of electrophoretic karyotyping (EK) and restriction endonuclease analysis of genomic DNA (REAG) by using SfiI (REAG-S) and BssHII (REAG-B). When catheter isolates were compared with blood isolates from the same patient, catheter isolates from 5 of 14 patients (36%) exhibited minor band differences (microevolution) relative to blood isolates in either C1 fingerprinting (
n
= 4), REAG-S (
n
= 3), or REAG-B (
n
= 5) profiles, although they had identical EK patterns. However, the other sequential isolates from each patient, which had identical EK patterns, showed the same REAG and C1 fingerprinting patterns. Both fingerprinting methods revealed that two distinct genotypes were shared by isolates from seven patients in a neonatal intensive care unit, suggesting two nosocomial clusters. Except for two catheter isolates from the index patients of each cluster, no consecutive isolates collected from each of the two clusters showed any microevolution during the 2- or 7-month cluster periods. The findings suggest that in catheter-related candidemia, some
C. albicans
strains undergo microevolution during catheter colonization.
Characterizations of Adenovirus Type 41 Isolates from Children with Acute Gastroenteritis in Japan, Vietnam, and Korea
Authors: Li Lei , Shimizu Hideaki , Doan Lan Thi Phuong , Tung Phan Gia , Okitsu Shoko , Nishio Osamu , Suzuki Eiko , Seo Jeong Kee , Kim Kyo Sun , Müller Werner E. G. , Ushijima Hiroshi
Published: September 2004

Genetic and antigenic characterizations of 70 strains of adenovirus type 41 (Ad41), isolated between 1998 and 2001 from children in Japan, Vietnam, and Korea, were done by DNA restriction enzyme (RE) analysis, sequencing analysis, and monoclonal antibody (MAb)-based enzyme-linked immunosorbent assay (ELISA). Eight genome types were observed in the present study, among which D25, D26, D27, and D28 were novel genome types. These eight genome types were divided into two genome-type clusters (GTCs) based on phylogenetic analysis of the hypervariable regions (HVRs) of the hexon. GTC1 includes D1, D25, D26, D27, and D28, and the GTC2 contains D4, D12, and D22. The amino acid homologies among the members within a GTC were 97 to 100%, whereas between the members of different GTCs the homologies were 92 to 94%. The specificity of the GTC classification was confirmed by ELISA with MAb 1F, which was selected by the Ad41 prototype Tak strain. It was found that only the isolates of GTC1 but not of GTC2 reacted with MAb 1F. These results suggest that Ad41 isolates from the three countries should be classified into two subtypes. The accumulation of amino acid mutations located in HVRs of hexon are indicative for the classification of Ad41 subtype.
Definition of the Beijing/W Lineage of
Mycobacterium tuberculosis
on the Basis of Genetic Markers
Authors: Kremer Kristin , Glynn Judith R. , Lillebaek Troels , Niemann Stefan , Kurepina Natalia E. , Kreiswirth Barry N. , Bifani Pablo J. , van Soolingen Dick
Published: September 2004

Mycobacterium tuberculosis
Beijing genotype strains are highly prevalent in Asian countries and in the territory of the former Soviet Union. They are increasingly reported in other areas of the world and are frequently associated with tuberculosis outbreaks and drug resistance. Beijing genotype strains, including W strains, have been characterized by their highly similar multicopy IS
6110
restriction fragment length polymorphism (RFLP) patterns, deletion of spacers 1 to 34 in the direct repeat region (Beijing spoligotype), and insertion of IS
6110
in the genomic
dnaA-dnaN
locus. In this study the suitability and comparability of these three genetic markers to identify members of the Beijing lineage were evaluated. In a well-characterized collection of 1,020
M. tuberculosis
isolates representative of the IS
6110
RFLP genotypes found in The Netherlands, strains of two clades had spoligotypes characteristic of the Beijing lineage. A set of 19 Beijing reference RFLP patterns was selected to retrieve all Beijing strains from the Dutch database. These reference patterns gave a sensitivity of 98.1% and a specificity of 99.7% for identifying Beijing strains (defined by spoligotyping) in an international database of 1,084 strains. The usefulness of the reference patterns was also assessed with large DNA fingerprint databases in two other European countries and for identification strains from the W lineage found in the United States. A standardized definition for the identification of
M. tuberculosis
strains belonging to the Beijing/W lineage, as described in this work, will facilitate further studies on the spread and characterization of this widespread genotype family of
M. tuberculosis
strains.
Risk Factors for Community-Associated Methicillin-Resistant
Staphylococcus aureus
Infections in an Outbreak of Disease among Military Trainees in San Diego, California, in 2002
Authors: Campbell Katherine M. , Vaughn Andrew F. , Russell Kevin L. , Smith Besa , Jimenez Dinice L. , Barrozo Christopher P. , Minarcik John R. , Crum Nancy F. , Ryan Margaret A. K.
Published: September 2004

An outbreak of community-associated methicillin-resistant
Staphylococcus aureus
(CA-MRSA) skin infections was observed in a population of U.S. military trainees in the summer of 2002. A questionnaire was developed and administered to 206 trainees, 22 of whom had MRSA infections. Factors associated with infection were described by multivariable logistic regression modeling and included having a roommate in training with a prior skin infection (odds ratio [OR] = 3.44) or having a family member or friend who worked in a health care setting (OR = 2.79). Previous antibiotic use, hospitalization, or health problems were not associated with MRSA infection. This outbreak of MRSA skin infections in an otherwise-healthy, well-defined, military population provided an opportunity to describe risk factors for CA-MRSA which may help focus prevention efforts in this and other communities.
Evaluation of the Core Antigen Assay as a Second-Line Supplemental Test for Diagnosis of Active Hepatitis C Virus Infection
Authors: Krajden Mel , Shivji Rishma , Gunadasa Kingsley , Mak Annie , McNabb Gail , Friesenhahn Michel , Hendricks David , Comanor Lorraine
Published: September 2004

The British Columbia Center for Disease Control laboratory performs approximately 95% of all hepatitis C virus (HCV) antibody tests for the province's 4 million inhabitants. In 2002, the laboratory tested 96,000 specimens for anti-HCV antibodies, of which 4,800 (5%) were seroreactive and required confirmation of active infection. Although HCV RNA assays with a sensitivity of 50 IU/ml or less are recommended for the confirmation of active HCV infection, given the large number of seroreactive specimens tested annually, we evaluated the Ortho trak-C assay (OTCA) as a second-line confirmatory test and determined its limit of detection (LoD). Of 502 specimens from treatment-naïve anti-HCV-positive individuals, 478 had sufficient volumes for evaluation by the OTCA and HCV RNA tests. Core antigen was not detected in 147 of 478 (30.8%) of these specimens, of which 37 of 147 (25.2%) were shown to be viremic by the VERSANT HCV (version 3.0) (branched-DNA) assay and/or the VERSANT HCV qualitative assay. Testing of 144 replicates of a World Health Organization standard dilution series indicated that the LoD of OTCA was ∼27,000 IU/ml. This LoD is consistent with the inability of OTCA to detect core antigen in clinical specimens with low viral loads. We conclude that OTCA has limited value as a confirmatory test for the diagnosis of active HCV infection because 37 of 367 (10%) of viremic specimens had undetectable core antigen. Qualitative HCV RNA testing remains the present standard for the confirmation of active HCV infection in the diagnostic setting.
Evaluation of the New VITEK 2 Card for Identification of Clinically Relevant Gram-Negative Rods
Authors: Funke Guido , Funke-Kissling Pascale
Published: September 2004

The
VITEK 2 card for gram-negative bacteria (bioMérieux,Marcy-l′Etoile, France) has been redesigned to improve the
identification of fermenting and nonfermenting bacilli. Forty-seven
biochemical tests, including 19 enzymatic tests, are present in the new
card and interpreted in a kinetic mode. Final identification results
are available within 10 h. The database allows the
identification of 159 different taxa. Six hundred fifty-five
gram-negative rods (GNR; 511 fermenters and 144 nonfermenters),
representing 54 taxa, were tested. Strains were taken from fresh
routine primary isolation plates (
n
= 157), from
stored routine plates (
n
= 301), and from stock
cultures (
n
= 197). Six hundred thirty-seven strains
(97.3%) were correctly identified to the species level, 14
strains (2.1%) gave low discrimination results requiring
additional tests, and 4 strains (0.6%) gave discordant results;
not a single strain remained unidentified. Nearly 92% of all
isolates were correctly identified within 7 h of incubation.
The robustness of the system was demonstrated by the fact that strains
were grown on four different agar media before testing. The system may
also have the potential to be applied directly to primary isolation
plates, since in this instance 96.2% of 157 GNR were correctly
identified and 3.8% gave low discrimination results. The new
VITEK 2 card for gram-negative bacteria seems to be a promising new
tool for routine, rapid identification of
GNR.