bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of Promoters Recognized by PvdS, an Extracytoplasmic-Function Sigma Factor Protein from
Pseudomonas aeruginosa
Authors: Wilson Megan J. , McMorran Brendan J. , Lamont Iain L.
Published: July 2002

The alternative sigma factor PvdS is required by
Pseudomonas aeruginosa
for initiation of transcription from pyoverdine (
pvd
) promoters. Two divergent PvdS-dependent promoters (
pvdE
and
pvdF
) were characterized by deletion analysis, and the minimal promoter region for each included a sequence element, the iron starvation (IS) box, that is present in other
pvd
promoters. Site-directed mutagenesis showed that the IS box elements were essential for promoter activity in vivo. Band shift assays and in vitro transcription experiments showed that a complex of PvdS and core RNA polymerase required the presence of an IS box in order to bind to and initiate transcription from
pvd
promoters. These results indicate that IS box elements participate in sequence-specific recognition by PvdS to enable initiation of transcription from
pvd
promoters and are likely to represent a −35 sequence element for this sigma factor.
Regulation of Ornibactin Biosynthesis and
N
-Acyl-
l
-Homoserine Lactone Production by CepR in
Burkholderia cepacia
Authors: Lewenza Shawn , Sokol Pamela A.
Published: July 2002

The CepR-CepI quorum-sensing system has been shown to regulate production of the siderophore ornibactin, extracellular proteases, and
N
-octanoyl-homoserine-
l
-lactone (OHL) in
Burkholderia cepacia
strain K56-2. To examine the effect of
cepIR
on production of other siderophores,
cepR
mutants were constructed in strains that produce pyochelin in addition to salicylic acid and ornibactins. Pc715j-R1 (
cepR
::tp) hyperproduced ornibactin but produced parental levels of pyochelin and salicylic acid, suggesting that CepR is a negative regulator of ornibactin synthesis but not pyochelin or salicylic acid. Pc715j-R1 was also protease deficient and OHL negative. The effects of
cepR
on ornibactin biosynthetic genes were examined by constructing
cepR pvdA-lacZ
and
cepR pvdD-lacZ
mutants and monitoring β-galactosidase activity. There was an increase in expression of
pvdA
in the
cepR
mutant compared to the level in its parent strain in both low- and high-iron media during stationary phase. When the outer membrane protein profiles of a
cepR
mutant and the wild-type strain were compared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, there did not appear to be any difference in levels of expression of the ornibactin receptor. Experiments with
cepI-lacZ
and
cepR-lacZ
transcriptional fusions indicated that
cepI
was not expressed in the
cepR
mutant and that
cepR
acts as a negative regulator of its own expression. By a thin-layer chromatography assay for
N
-acyl homoserine lactones, OHL and
N
-hexanoyl-
l
-homoserine lactone (HHL) were detectable in K56-2 and Pc715j, both wild-type strains. OHL was not detectable and HHL was only weakly detectable in the
cepI
and
cepR
mutants. These results suggest that CepR is both a positive and negative transcriptional regulator and that CepR may influence the expression of ornibactin biosynthetic genes in addition to the expression of the
cepIR
quorum-sensing system.
SirA Orthologs Affect both Motility and Virulence
Authors: Goodier Robert I. , Ahmer Brian M. M.
Published: July 2002

The
sirA
gene of
Salmonella enterica
serovar Typhimurium encodes a two-component response regulator of the FixJ family that has a positive regulatory influence on the expression of type III secretion genes involved with epithelial cell invasion and the elicitation of bovine gastroenteritis. SirA orthologs in
Pseudomonas, Vibrio
, and
Erwinia
control the expression of distinct virulence genes in these genera, but an evolutionarily conserved target of SirA regulation has never been identified. In this study we tested the hypothesis that
sirA
may be an ancient member of the flagellar regulon. We examined the effect of a
sirA
mutation on transcriptional fusions to flagellar promoters (
flhD, fliE, fliF, flgA, flgB, fliC, fliD, motA
, and
fliA
) while using fusions to the virulence gene
sopB
as a positive control. SirA had only small regulatory effects on all fusions in liquid medium (less than fivefold). However, in various types of motility agar plates,
sirA
was able to activate a
sopB
fusion by up to 63-fold while repressing flagellar fusions by values exceeding 100-fold. Mutations in the
sirA
orthologs of
Escherichia coli, Vibrio cholerae, Pseudomonas fluorescens
, and
Pseudomonas aeruginosa
result in defects in either motility or motility gene regulation, suggesting that control of flagellar regulons may be an evolutionarily conserved function of
sirA
orthologs. The implications for our understanding of virulence gene regulation in the gamma
Proteobacteria
are discussed.
Roles for Sigma Factors in Global Circadian Regulation of the Cyanobacterial Genome
Authors: Nair Usha , Ditty Jayna L. , Min Hongtao , Golden Susan S.
Published: July 2002

The circadian clock of the unicellular cyanobacterium
Synechococcus elongatus
PCC 7942 imposes a global rhythm of transcription on promoters throughout the genome. Inactivation of any of the four known group 2 sigma factor genes (
rpoD2
,
rpoD3
,
rpoD4
, and
sigC
), singly or pairwise, altered circadian expression from the
psbAI
promoter, changing amplitude, phase angle, waveform, or period. However, only the
rpoD2
mutation and the
rpoD3 rpoD4
and
rpoD2 rpoD3
double mutations affected expression from the
kaiB
promoter. A striking differential effect was a 2-h lengthening of the circadian period of expression from the promoter of
psbAI
, but not of those of
kaiB
or
purF
, when
sigC
was inactivated. The data show that separate timing circuits with different periods can coexist in a cell. Overexpression of
rpoD2
,
rpoD3
,
rpoD4
, or
sigC
also changed the period or abolished the rhythmicity of P
psbAI
expression, consistent with a model in which sigma factors work as a consortium to convey circadian information to downstream genes.
α-Galactosidase Aga27A, an Enzymatic Component of theClostridium josuiCellulosome
Authors: Jindou Sadanari , Karita Shuichi , Fujino Emi , Fujino Tsuchiyoshi , Hayashi Hidenori , Kimura Tetsuya , Sakka Kazuo , Ohmiya Kunio
Published: July 2002

TheClostridium josui aga27Agene encodes the cellulosomal α-galactosidase Aga27A, which comprises a catalytic domain of family 27 of glycoside hydrolases and a dockerin domain responsible for cellulosome assembly. The catalytic domain is highly homologous to those of various α-galactosidases of family 27 of glycoside hydrolases from eukaryotic organisms, especially plants. The recombinant Aga27A α-galactosidase devoid of the dockerin domain preferred highly polymeric galactomannan as a substrate to small saccharides such as melibiose and raffinose.
Genetic Analysis of the RcsC Sensor Kinase from
Escherichia coli
K-12
Authors: Clarke D. J. , Joyce S. A. , Toutain C. M. , Jacq A. , Holland I. B.
Published: July 2002

The Rcs two-component pathway is involved in the regulation of capsule production in
Escherichia coli
. RcsC is predicted to be the sensor component of this two-component pathway, and in this study we present the first genetic data that support the role of RcsC as a hybrid sensor kinase.
Tetracycline Induces Stabilization of mRNA in
Bacillus subtilis
Authors: Wei Yi , Bechhofer David H.
Published: July 2002

The
tet
(L) gene of
Bacillus subtilis
confers low-level tetracycline (Tc) resistance. Previous work examining the >20-fold-inducible expression of
tet
(L) by Tc demonstrated a 12-fold translational induction. Here we show that the other component of
tet
(L) induction is at the level of mRNA stabilization. Addition of a subinhibitory concentration of Tc results in a two- to threefold increase in
tet
(L) mRNA stability. Using a plasmid-borne derivative of
tet
(L) with a large in-frame deletion of the coding sequence, the mechanism of Tc-induced stability was explored by measuring the decay of
tet
(L) mRNAs carrying specific mutations in the leader region. The results of these experiments, as well as experiments with a
B. subtilis
strain that is resistant to Tc due to a mutation in the ribosomal S10 protein, suggest different mechanisms for the effects of Tc on translation and on mRNA stability. The key role of the 5" end in determining mRNA stability was confirmed in these experiments. Surprisingly, the stability of several other
B. subtilis
mRNAs was also induced by Tc, which indicates that addition of Tc may result in a general stabilization of mRNA.
Two Different Lantibiotic-Like Peptides Originate from the Ericin Gene Cluster of
Bacillus subtilis
A1/3
Authors: Stein Torsten , Borchert Stefan , Conrad Birgit , Feesche Jörg , Hofemeister Brigitte , Hofemeister Jürgen , Entian Karl-Dieter
Published: July 2002

A lantibiotic gene cluster was identified in
Bacillus subtilis
A1/3 showing a high degree of homology to the subtilin gene cluster and occupying the same genetic locus as the
spa
genes in
B. subtilis
ATCC 6633. The gene cluster exhibits diversity with respect to duplication of two subtilin-like genes which are separated by a sequence similar to a portion of a
lanC
gene. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analyses of
B. subtilis
A1/3 culture extracts confirmed the presence of two lantibiotic-like peptides, ericin S (3,442 Da) and ericin A (2,986 Da). Disruption of the
lanB
-homologous gene
eriB
resulted in loss of production of both peptides, demonstrating that they are processed in an
eriB
-dependent manner. Although precursors of ericins S and A show only 75% of identity, the matured lantibiotic-like peptides reveal highly similar physical properties; separation was only achieved after multistep, reversed-phase high-performance liquid chromatography. Based on Edman and peptidase degradation in combination with MALDI-TOF MS, for ericin S a subtilin-like, lanthionine-bridging pattern is supposed. For ericin A two C-terminal rings are different from the lanthionine pattern of subtilin. Due to only four amino acid exchanges, ericin S and subtilin revealed similar antibiotic activities as well as similar properties in response to heat and protease treatment. For ericin A only minor antibiotic activity was found.
Malonyl-Coenzyme A Reductase from
Chloroflexus aurantiacus
, a Key Enzyme of the 3-Hydroxypropionate Cycle for Autotrophic CO
2
Fixation
Authors: Hügler Michael , Menendez Castor , Schägger Hermann , Fuchs Georg
Published: July 2002

The 3-hydroxypropionate cycle is a new autotrophic CO
2
fixation pathway in
Chloroflexus aurantiacus
and some archaebacteria. The initial step is acetyl-coenzyme A (CoA) carboxylation to malonyl-CoA by acetyl-CoA carboxylase, followed by NADPH-dependent reduction of malonyl-CoA to 3-hydroxypropionate. This reduction step was studied in
Chloroflexus aurantiacus
. A new enzyme was purified, malonyl-CoA reductase, which catalyzed the two-step reduction malonyl-CoA + NADPH + H
+
→ malonate semialdehyde + NADP
+
+ CoA and malonate semialdehyde + NADPH + H
+
→ 3-hydroxypropionate + NADP
+
. The bifunctional enzyme (aldehyde dehydrogenase and alcohol dehydrogenase) had a native molecular mass of 300 kDa and consisted of a single large subunit of 145 kDa, suggesting an α
2
composition. The N-terminal amino acid sequence was determined, and the incomplete gene was identified in the genome database. Obviously, the enzyme consists of an N-terminal short-chain alcohol dehydrogenase domain and a C-terminal aldehyde dehydrogenase domain. No indication of the presence of a prosthetic group was obtained; Mg
2+
and Fe
2+
stimulated and EDTA inhibited activity. The enzyme was highly specific for its substrates, with apparent

K
m

values of 30 μM malonyl-CoA and 25 μM NADPH and a turnover number of 25 s
−1
subunit
−1
. The specific activity in autotrophically grown cells was 0.08 μmol of malonyl-CoA reduced min
−1
(mg of protein)
−1
, compared to 0.03 μmol min
−1
(mg of protein)
−1
in heterotrophically grown cells, indicating downregulation under heterotrophic conditions. Malonyl-CoA reductase is not required in any other known pathway and therefore can be taken as a characteristic enzyme of the 3-hydroxypropionate cycle. Furthermore, the enzyme may be useful for production of 3-hydroxypropionate and for a coupled spectrophotometric assay for activity screening of acetyl-CoA carboxylase, a target enzyme of potent herbicides.
The Superoxide Dismutase Gene
sodM
Is Unique to
Staphylococcus aureus
: Absence of
sodM
in Coagulase-Negative Staphylococci
Authors: Valderas Michelle Wright , Gatson Joshua W. , Wreyford Natalie , Hart Mark E.
Published: July 2002

Superoxide dismutase (SOD) profiles of clinical isolates of
Staphylococcus aureus
and coagulase-negative staphylococci (CoNS) were determined by using whole-cell lysates and activity gels. All
S. aureus
clinical isolates exhibited three closely migrating bands of activity as previously determined for laboratory strains of
S. aureus
: SodM, SodA, and a hybrid composed of SodM and SodA (M. W. Valderas and M. E. Hart, J. Bacteriol. 183:3399-3407, 2001). In contrast, the CoNS produced only one SOD activity, which migrated similarly to SodA of
S. aureus
. Southern analysis of eight CoNS species identified only a single
sod
gene in each case. A full-length
sod
gene was cloned from
Staphylococcus epidermidis
and determined to be more similar to
sodA
than to
sodM
of
S. aureus
. Therefore, this gene was designated
sodA
. The deduced amino acid sequence of the
S. epidermidis sodA
was 92 and 76% identical to that of the SodA and SodM proteins of
S. aureus
, respectively. The
S. epidermidis sodA
gene expressed from a plasmid complemented a
sodA
mutation in
S. aureus
, and the protein formed a hybrid with SodM of
S. aureus
. Both hybrid SOD forms as well as the SodM and SodA proteins of
S. aureus
and the
S. epidermidis
SodA protein exist as dimers. These data indicate that
sodM
is found only in
S. aureus
and not in the CoNS, suggesting an important divergence in the evolution of this genus and a unique role for SodM in
S. aureus
.
Identification of Epidemic Strains of
Acinetobacter baumannii
by Integrase Gene PCR
Authors: Koeleman Johannes G. M. , Stoof Jeroen , Van Der Bijl Madelon W. , Vandenbroucke-Grauls Christina M. J. E. , Savelkoul Paul H. M.
Published: July 2002

Forty-eight clinical
Acinetobacter
isolates with different epidemic behavior were investigated for the presence of integrons and plasmids and for antibiotic susceptibility. Integrons were demonstrated in 50% of the strains by an integrase gene PCR. Epidemic strains of
Acinetobacter baumannii
were found to contain significantly more integrons than nonepidemic strains. Also, the presence of integrons was significantly correlated with simultaneous resistance to several antibiotics. Plasmids were detected in 42% of the strains. However, there was no significant correlation between the numbers of plasmids and integrons in
Acinetobacter
species strains, no significant difference in the number of plasmids between epidemic and nonepidemic
A. baumannii
strains, and no significant correlation between the presence of plasmids and antibiotic resistance. Hence, it is likely that integrons play an important role in antibiotic resistance and thereby in the epidemic behavior of
A. baumannii
. Because the integrase gene PCR identified almost three-quarters of the epidemic
A. baumannii
isolates (17 of 23), this seems to be a rapid and simple technique for the routine screening and identification of clinical
A. baumannii
isolates with epidemic potential.
Relative Accuracy of Nucleic Acid Amplification Tests and Culture in Detecting
Chlamydia
in Asymptomatic Men
Authors: Cheng Hong , Macaluso Maurizio , Vermund Sten H. , Hook Edward W.
Published: July 2002

Published estimates of the sensitivity and specificity of PCR and ligase chain reaction (LCR) for detecting
Chlamydia trachomatis
are potentially biased because of study design limitations (confirmation of test results was limited to subjects who were PCR or LCR positive but culture negative). Relative measures of test accuracy are less prone to bias in incomplete study designs. We estimated the relative sensitivity (
RSN
) and relative false-positive rate (
RFP
) for PCR and LCR versus cell culture among 1,138 asymptomatic men and evaluated the potential bias of
RSN
and
RFP
estimates. PCR and LCR testing in urine were compared to culture of urethral specimens. Discordant results (PCR or LCR positive, but culture negative) were confirmed by using a sequence including the other DNA amplification test, direct fluorescent antibody testing, and a DNA amplification test to detect chlamydial major outer membrane protein. The RSN estimates for PCR and LCR were 1.45 (95% confidence interval [CI] = 1.3 to 1.7) and 1.49 (95% CI = 1.3 to 1.7), respectively, indicating that both methods are more sensitive than culture. Very few false-positive results were found, indicating that the specificity levels of PCR, LCR, and culture are high. The potential bias in RSN and RFP estimates were <5 and <20%, respectively. The estimation of bias is based on the most likely and probably conservative parameter settings. If the sensitivity of culture is between 60 and 65%, then the true sensitivity of PCR and LCR is between 90 and 97%. Our findings indicate that PCR and LCR are significantly more sensitive than culture, while the three tests have similar specificities.
DNA Relatedness, Phenotypic Characteristics, and Antimicrobial Susceptibilities of
Globicatella sanguinis
Strains
Authors: Shewmaker P. Lynn , Steigerwalt Arnold G. , Shealey LaShondra , Weyant Robbin , Facklam Richard R.
Published: July 2002

DNA-DNA reassociation was performed on 15 strains of
Globicatella sanguinis
to compare their taxonomic status with phenotypic characterization. All 15 strains selected for DNA-DNA reassociation readily met the criteria for species relatedness. The relative binding ratio was 81% or greater at the optimal temperature and 76% or greater at the stringent temperature, and the divergence was less than 3% for all strains hybridized with the type strain. These strains included nine strains from the Centers for Disease Control Streptococcus Laboratory culture collection that were previously included in comparative 16S rRNA gene sequencing studies as well as six additional phenotypically variant isolates. DNA-DNA relatedness was less than 18% at the optimal reassociation temperature to
Aerococcus viridans, Enterococcus avium
, and
Streptococcus uberis
, which are phenotypically similar to
G. sanguinis
. This study confirms these
Globicatella
strains were previously misidentified as
S. uberis
or
S. uberis
-like strains based on biochemical characteristics. The biochemical data from 28 strains was compiled to further define the phenotypic criteria for identification of this species. A revised description of the species should be variable reaction for pyrrolidonylarylamidase production (75% positive), positive reaction for the bile esculin test (100%), growth at 45°C (96%), variable reaction for acid production from arabinose (45% positive), and negative starch hydrolysis (0% positive). We also evaluated four rapid identification systems, the Biomerieux rapid ID32 STREP (ID32), the Crystal rapid gram-positive identification (Cry4), the BBL Crystal gram-positive identification (Cry24), and the Remel IDS RapID STR (IDS) systems for their ability to identify these strains.
Nucleic Acid Sequence-Based Amplification Assays for Rapid Detection of West Nile and St. Louis Encephalitis Viruses
Authors: Lanciotti Robert S. , Kerst Amy J.
Published: July 2002

The development and application of nucleic acid sequence-based amplification (NASBA) assays for the detection of West Nile (WN) and St. Louis encephalitis (SLE) viruses are reported. Two unique detection formats were developed for the NASBA assays: a postamplification detection step with a virus-specific internal capture probe and electrochemiluminescence (NASBA-ECL assay) and a real-time assay with 6-carboxyfluorescein-labeled virus-specific molecular beacon probes (NASBA-beacon assay). The sensitivities and specificities of these NASBA assays were compared to those of a newly described standard reverse transcription (RT)-PCR and TaqMan assays for SLE virus and to a previously published TaqMan assay for WN virus. The NASBA assays demonstrated exceptional sensitivities and specificities compared to those of virus isolation, the TaqMan assays, and standard RT-PCR, with the NASBA-beacon assay yielding results in less than 1 h. These assays should be of utility in the diagnostic laboratory to complement existing diagnostic testing methodologies and as a tool in conducting flavivirus surveillance in the United States.
Evaluation of Two Enzyme Immunoassays for Detection of Human Rotaviruses in Fecal Specimens
Authors: Eing Bodo R. , May Guenter , Baumeister Horst G. , Kühn Joachim E.
Published: July 2002

The two assays evaluated in this study (the Ridascreen rotavirus and the Pathfinder rotavirus) exhibited comparable sensitivities (100%) but highly divergent positive predictive values (93.74 and 57.7%, respectively) when compared on 393 specimens. This difference should be considered when using these tests on collectives with an unknown or low prevalence.
Association of the Urease Gene with Enterohemorrhagic
Escherichia coli
Strains Irrespective of Their Serogroups
Authors: Nakano Masayuki , Iida Tetsuya , Ohnishi Makoto , Kurokawa Ken , Takahashi Akira , Tsukamoto Teizo , Yasunaga Teruo , Hayashi Tetsuya , Honda Takeshi
Published: July 2002

Among various diarrheagenic
Escherichia coli
strains from clinical sources, we found that the urease gene was specifically associated with enterohemorrhagic
E. coli
(EHEC) strains irrespective of their serogroups. The results suggest that the urease gene can be a useful genetic marker for the detection of EHEC strains and for the diagnosis of infections caused by EHEC strains in the clinical situation.
A Shared Noncapsular Antigen Is Responsible for False-Positive Reactions by
Staphylococcus epidermidis
in Commercial Agglutination Tests for
Staphylococcus aureus
Authors: Blake Janet E. , Metcalfe Mark A.
Published: July 2002

Many of the commercial slide agglutination tests for
Staphylococcus aureus
incorporate antibodies against cell surface antigens associated with methicillin resistance, including capsular polysaccharides and an uncharacterized antigen, serotype 18. These tests are more sensitive than the first-generation agglutination procedures that detected only bound coagulase and protein A, but they suffer from false-positive reactions with some coagulase-negative staphylococci. The aim of this study was to elucidate the mechanism for false-positive agglutination by
S. epidermidis
in these tests. A group of methicillin-resistant
S. aureus
(MRSA) isolates, including a serotype 18 strain, that were not detectable in the first-generation tests were found to be of capsular polysaccharide type 8. All of these isolates were deficient in bound coagulase and/or protein A, and they possessed a heat-stable, proteinaceous antigen that was absent from a prototype capsule type 8 strain. Enzyme-linked immunosorbent assay and agarose gel immunodiffusion experiments demonstrated that this proteinaceous antigen was also present on both methicillin-sensitive and methicillin-resistant
S. epidermidis
clinical isolates.
S. epidermidis
strains that gave false-positive agglutination test results had a considerably higher level of this antigen than strains that gave the correct negative result. These findings reveal the importance of the careful selection of MRSA strains for raising anti-capsular type 8 antibodies for use in agglutination tests. Strains devoid of the antigen shared with
S. epidermidis
should be used to eliminate potential cross-reactions with this coagulase-negative coccus.
Direct Identification of
Mycobacterium avium
Complex and
Mycobacterium gordonae
from MB/BacT Bottles Using AccuProbe
Authors: Louro Ana Paula S. , Waites Ken B. , Georgescu Ecaterina , Benjamin William H.
Published: July 2002

We evaluated the ability of the AccuProbe (Gen-Probe, San Diego, Calif.) to detect
Mycobacterium gordonae
and
Mycobacterium avium
complex directly in liquid medium flagged positive by the MB/BacT (Organon Teknika Corp., Durham, N.C.). Seventy-one bottles from clinical specimens containing
M. gordonae
and 34 containing
M. avium
, confirmed by culture, were tested by direct AccuProbe assay for both organisms after additional incubation for ≥48 h and centrifugation at 4,500 ×
g
for 15 min. Relative light unit (RLU) values were analyzed using the manufacturer's recommended cutoff of 30,000 RLU and a lower cutoff of 10,000 RLU. Using the 30,000 RLU cutoff, 55 of 71 (77.5%) specimens containing
M. gordonae
yielded positive results, whereas 28 of 34 (82.3%)
M. avium
complex specimens were correctly identified by direct probe. No specimens shown by culture to contain either
M. gordonae
or
M. avium
complex tested positive with the probe for the opposite organism (100% specificity). When the cutoff was lowered to 10,000 RLU, 67 of 71
M. gordonae
(94.4%) and 32 of 34
M. avium
complex (94.1%) specimens were correctly identified. This difference was significant for
M. gordonae
(
P
= 0.004) but not for
M. avium
complex (
P
= 0.26) compared to detection using the recommended RLU cutoff. Specificity was 100% for specimens containing
M. gordonae
that were tested with the
M. avium
complex probe using the 10,000 RLU cutoff, whereas specificity for specimens containing
M. avium
complex tested with the
M. gordonae
probe was 97%. Using a lower RLU cutoff for determining a positive result using the
M. gordonae
or
M. avium
complex probes when testing instrument-positive MB/BacT bottles directly will improve sensitivity without substantially compromising specificity.
Molecular Characterization of
fliD
Gene Encoding Flagellar Cap and Its Expression among
Clostridium difficile
Isolates from Different Serogroups
Authors: Tasteyre Albert , Karjalainen Tuomo , Avesani Véronique , Delmée Michel , Collignon Anne , Bourlioux Pierre , Barc Marie-Claude
Published: July 2002

The
fliD
gene encoding the flagellar cap protein (FliD) of
Clostridium difficile
was studied in 46 isolates belonging to serogroups A, B, C, D, F, G, H, I, K, X, and S3, including 30 flagellated strains and 16 nonflagellated strains. In all but three isolates, amplification by PCR and reverse transcription-PCR demonstrated that the
fliD
gene is present and transcribed in both flagellated and nonflagellated strains. PCR-restriction fragment length polymorphism (RFLP) analysis of amplified
fliD
gene products revealed interstrain homogeneity, with one of two major patterns (a and b) found in all but one of the strains, which had pattern c. A polyclonal monospecific antiserum raised to the recombinant FliD protein reacted in immunoblots with crude flagellar preparations from 28 of 30 flagellated strains but did not recognize FliD from nonflagellated strains. The
fliD
genes from five strains representative of the three different RFLP groups were sequenced, and sequencing revealed 100% identity between the strains with the same pattern and 88% identity among strains with different patterns. Our results show that even though FliD is a structure exposed to the outer environment, the flagellar cap protein is very well conserved, and this high degree of conservation suggests that it has a very specific function in attachment to cell or mucus receptors.
Characterization of Lyme Borreliosis Isolates from Patients with Erythema Migrans and Neuroborreliosis in Southern Sweden
Authors: Ornstein Katharina , Berglund Johan , Nilsson Ingrid , Norrby Ragnar , Bergström Sven
Published: July 2002

Southern Sweden is an area of Lyme borreliosis (LB) endemicity, with an incidence of 69 cases per 100,000 inhabitants. The most frequent clinical manifestations are erythema migrans (77%) and neuroborreliosis (16%). There was no record of humanBorreliastrains being isolated from patients in this region before the prospective study reported here.Borreliaspirochetes were isolated from skin and cerebrospinal fluid (CSF) from LB patients living in the region. A total of 39 strains were characterized by OspA serotype analysis, species-specific PCR, and signature nucleotide analysis of the 16S rRNA gene. Of 33 skin isolates, 31 (93.9%) wereBorrelia afzeliistrains and 2 (6.1%) wereBorrelia gariniistrains. Of six CSF isolates, five (83.3%) wereB. gariniiand one (16.7%) wasB. afzelii. NeitherBorrelia burgdorferisensu stricto strains nor multiple infections were observed. TheB. afzeliiisolates were of OspA serotype 2. ThreeB. gariniistrains were of OspA serotype 5, and the remaining four strains were of OspA serotype 6. All of theB. gariniistrains belonged to the same 16S ribosomal DNA ribotype class. Our findings agree with earlier findings from other geographic regions in Europe whereB. afzeliiandB. gariniihave been recovered predominately from skin and CSF cultures, respectively. To further study the possible presence in Sweden of the genotypeB. burgdorferisensu stricto, which is known to be present in Europe and to occur predominately in patients with Lyme arthritis, molecular detection ofBorrelia-specific DNA in synovial samples from Lyme arthritis patients should be performed.
New Tests for Syphilis: Rational Design of a PCR Method for Detection of
Treponema pallidum
in Clinical Specimens Using Unique Regions of the DNA Polymerase I Gene
Authors: Liu Hsi , Rodes Berta , Chen C.-Y. , Steiner Bret
Published: July 2002

A sensitive and specific PCR method to detect
Treponema pallidum
in clinical specimens was developed. PCR primers were designed based on two unique features of the DNA polymerase I gene (
polA
). The first distinctive characteristic is that the region codes for a high cysteine content and has low homology with similar regions of DNA polymerase I gene from known microorganisms. The second unique feature is the presence of four insertions in the gene. PCR tests using primers designed on the basis these regions reacted with various pathogenic
T. pallidum
subspecies but did not react with nonpathogenic treponemal species or other spirochetes. An additional 59 species of bacteria and viruses, including those that cause genital ulcers, tested negative. This PCR method is extremely robust and sensitive. The detection limit is about 10 to 25 organisms when analyzed on gel. However, the analytic sensitivity can be increased by at least 1 log, to a detection limit of a single organism, when the ABI 310 Prism Genetic Analyzer is used to detect fluorescence-labeled amplicons. We further used this test in a clinical setting and compared the results with results from a previously reported multiplex-PCR test (for
T. pallidum, Haemophilus ducreyi
, and herpes simplex virus). We tested 112 genital ulcer specimens by the
polA
PCR, obtaining a sensitivity of 95.8% and a specificity of 95.7%. These results suggest that the
polA
PCR is applicable as a routine clinical diagnostic test for syphilis.
Coinfection of Enteric
Helicobacter
spp. and
Campylobacter
spp. in Cats
Authors: Shen Z. , Feng Y. , Dewhirst F. E. , Fox J. G.
Published: July 2002

During a 6-year period, 64 of 227 commercially reared cats had microaerobic bacteria isolated from their feces. All the isolates were initially identified as
Campylobacter
-like organisms based on biochemical and phenotypic characteristics. DNA extractions from 51 of these isolates were subjected to PCR using primers specific for
Helicobacter
spp. and
Campylobacter
spp. Of the isolates, 92% (47 of 51 isolates) were positive for
Campylobacter
spp., 41% (21 of 51 isolates) were positive for
Helicobacter
spp., 33% (17 of 51 isolates) were positive for both genera, 59% (30 of 51 isolates) were positive only for
Campylobacter
spp., and 8% (4 of 51) were positive only for
Helicobacter
spp. Sixteen of the 47
Campylobacter
-positive cultures were positive for more than one
Campylobacter
spp. Based on a species-specific PCR assay, 83% of the isolates were identified as
Campylobacter helveticus
, 47% of the isolates were identified as
Campylobacter upsaliensis,
and 6% of the isolates were classified as
Campylobacter jejuni
. The 1.2-kb PCR products of the 16S rRNA genes of 19
Helicobacter
species isolates were subjected to restriction fragment length polymorphism (RFLP) analysis. Of the five different RFLP patterns obtained, two clustered with
Helicobacter
(“
Flexispira
”) taxon 8, one clustered with
Helicobacter bilis
, one clustered with
Helicobacter canis
, and the remaining pattern was closely related to a novel
Helicobacter
sp. strain isolated from a woodchuck. The sequence data for the 16S rRNA genes of 10
Helicobacter
spp. validated the RFLP-based identification of these isolates. This study demonstrated that biochemical and phenotypic characteristics of microaerobic organisms in cat feces were insufficient to characterize mixed
Helicobacter
and
Campylobacter
infections. Molecular structure-based diagnostics using genus- and species-specific PCR, RFLP analysis, and 16S rRNA sequence analysis enabled the identification of multiple microaerobic species in individual animals. The clinical relevance of enteric
Helicobacter
and
Campylobacter
coinfection in cats will require further studies.
Molecular Epidemiology Study of Exogenous Reinfection in an Area with a Low Incidence of Tuberculosis
Authors: Bandera Alessandra , Gori Andrea , Catozzi Lidia , Esposti Anna Degli , Marchetti Giulia , Molteni Chiara , Ferrario Giulio , Codecasa Luigi , Penati Valeria , Matteelli Alberto , Franzetti Fabio
Published: July 2002

In geographical areas with a low incidence of tuberculosis, recurrent tuberculosis is generally due to reactivation of the disease. However, the relative contribution of tuberculosis reinfection increases in parallel with the incidence of disease and is likely to depend on the epidemiological context: factors such as the spread of multidrug resistance, human immunodeficiency virus (HIV) infection, and immigration from developing countries could modify disease transmission in areas at low risk for tuberculosis. A molecular epidemiology study was performed in Lombardy, Northern Italy, where the incidence of tuberculosis is 17.5 cases per 100,000 persons. A total of 2,452 cases of culture-confirmed tuberculosis in 2,127 patients were studied. A group of 32 patients (1.5%), each of whom had two episodes of tuberculosis with cure as the outcome of the first episode and with more than 6 months between the two episodes, were studied by means of restriction fragment length polymorphism DNA fingerprinting analysis. For 5 of the 32 patients (16%), the DNA fingerprinting patterns of
Mycobacterium tuberculosis
strains responsible for the second episode did not match those of the corresponding isolates of the first episode, indicating exogenous reinfection. Two of these patients developed multidrug-resistant tuberculosis during the second episode, and in three cases the isolates belonged to clusters of
M. tuberculosis
strains spreading in the community. A fourfold-increased risk for reinfection was observed in immigrant patients compared to Italian subjects. In contrast, a higher risk of relapse rather than reinfection was evidenced in HIV-positive subjects and in patients infected with multidrug-resistant tuberculosis. Episodes of tuberculosis reinfection in areas with a low incidence of tuberculosis are rare compared to those in high-incidence geographical regions. In populations that have immigrated from high-risk areas, reinfection may represent a considerable contributor to the rate of recurrent tuberculosis. This finding emphasizes the importance of containing the spread of epidemic strains in close communities, in order to prevent changes in global tuberculosis trends for developed countries.
Phaeohyphomycotic Cyst Caused byColletotrichum crassipes
Authors: Martins Castro Luiz Guilherme , da Silva Lacaz Carlos , Guarro Josep , Gené Josepa , Heins-Vaccari Elisabeth Maria , de Freitas Leite Roseli Santos , Hernández Arriagada Giovana Letı́cia , Ozaki Reguera Márcia Maria , Ito Eunice Miki , Valente Neusa Yuriko Sakai , Spina Nunes Ricardo
Published: July 2002

A case of phaeohyphomycosis is reported in a male renal transplant recipient with a nodular lesion in the right leg who was treated with immunosuppressing drugs. The lesion consisted of a purulent cyst with thick walls. The cyst was excised surgically, and the patient did not receive any antifungal therapy. One year later he remains well. Histological study of the lesion showed a granulomatous reaction of epithelioid and multinucleate giant cells, with a central area of necrosis and pus. Fontana-Masson staining demonstrated the presence of pigmented hyphal elements. The fungusColletotrichum crassipeswas grown in different cultures from the cyst. The in vitro inhibitory activities of eight antifungal drugs against the isolate were tested. Clotrimazole and UR-9825 were the most active drugs. This case represents the first known reported infection caused by this rare species.
Assessment, by Transcription-Mediated Amplification, of Virologic Response in Patients with Chronic Hepatitis C Virus Treated with Peginterferon α-2a
Authors: Sarrazin Christoph , Hendricks David A. , Sedarati Farhad , Zeuzem Stefan
Published: July 2002

Transcription-mediated amplification (TMA) is an isothermal, autocatalytic target amplification method which has the potential to detect less than 50 hepatitis C virus (HCV) RNA copies/ml (10 IU/ml). The TMA assay was used to assess the presence of residual HCV RNA in plasma from patients treated with polyethylene glycol-modified interferon α-2a (peginterferon α-2a) who showed a virologic relapse after the end of therapy. Stored end-of-treatment and end-of-follow-up plasma samples from 177 of 267 patients treated with peginterferon α-2a (S. Zeuzem et al., N. Engl. J. Med. 343:1666–1672, 2000) were available for retesting by TMA. Plasma samples from patients in the same study who exhibited virologic relapse after treatment with standard interferon α-2a served as controls. Virologic response during the trial was defined as HCV RNA that was undetectable using a PCR-based test system with a sensitivity of 50 IU/mL (Cobas Amplicor HCV version 2.0) and was compared with TMA-based retesting results (VERSANT HCV RNA Qualitative Assay). Residual HCV RNA was detected in 4 of 60 cases (7%) by the TMA technology in end-of-treatment plasma samples from patients who relapsed after receiving peginterferon α-2a and in 6 of 18 patients (33%) following therapy with standard interferon α-2a. For peginterferon α-2a-treated patients with sustained virologic response, HCV RNA was detectable by TMA in end-of-treatment samples in 3 of 78 cases but in none of the end-of-follow-up samples. For all end-of-treatment and end-of-follow-up plasma samples of virologic nonresponders, a complete concordance between the PCR-based assay and TMA was observed. In conclusion, in patients with virologic relapse after the end of therapy, according to PCR, who were treated with peginterferon α-2a or standard interferon α-2a, residual HCV RNA was detectable in end-of-treatment samples by the TMA-based assay in 7 or 33% of cases, respectively. The lower rate of residual HCV RNA detection by TMA for patients treated with peginterferon α-2a than that for patients treated with standard interferon α-2a may be due to the maintained antiviral pressure of the long-acting peginterferon α-2a at the end-of-treatment visit.
Enhancing the Specificity of the COBAS AMPLICOR CT/NG Test for
Neisseria gonorrhoeae
by Retesting Specimens with Equivocal Results
Authors: Van Der Pol Barbara , Martin David H. , Schachter Julius , Quinn Thomas C. , Gaydos Charlotte A. , Jones Robert B. , Crotchfelt Kimberly , Moncada Jeanne , Jungkind D. , Turner Buffy , Peyton Cynthia , Kelly James F. , Weiss Judith B. , Rosenstraus Maurice
Published: July 2002

The COBAS AMPLICOR CT/NG test for
Neisseria gonorrhoeae
cross-reacts with certain strains of nonpathogenic
Neisseria
species. In some strains, the target sequence is identical to that of
N. gonorrhoeae
, whereas other strains have a small number of mismatches within the regions recognized by the primers or probe used in the COBAS AMPLICOR NG test. These cross-reactive strains are occasionally present in urogenital specimens, causing false-positive results in the COBAS AMPLICOR NG test. Analysis of the data generated in a large multicenter clinical trial showed that 2.9% of the specimens gave signals between
A
660
s of 0.2 and 3.5 but that one-half of these equivocal specimens did not contain
N. gonorrhoeae
. Most of these equivocal specimens were correctly classified as true positive or true negative by retesting in duplicate and defining a PCR-positive result as two of three results with an
A
660
of ≥2.0. If specimens had been classified as positive or negative based on a single test result using a cutoff of an
A
660
of 0.2, specificity would have ranged from 96.2 to 98.9% depending on specimen type, sex, and presence of symptoms. By employing the equivocal zone-retesting algorithm, specificity increased to 98.6 to 99.9% with little effect (0.1 to 4.9% decrease) on sensitivity in most specimen types, enabling the test to achieve a positive predictive value of at least 90% in populations with a prevalence of 4% or higher. In lower-prevalence populations, the test could be used to screen for presumptive infections that would have to be confirmed by an independent test.
Francisella tularensis
Strain Typing Using Multiple-Locus, Variable-Number Tandem Repeat Analysis
Authors: Farlow Jason , Smith Kimothy L. , Wong Jane , Abrams Michelle , Lytle Michael , Keim Paul
Published: July 2002

Francisella tularensis
, the etiological agent of tularemia, is found throughout the Northern hemisphere. After analyzing the
F. tularensis
genomic sequence for potential variable-number tandem repeats (VNTRs), we developed a multilocus VNTR analysis (MLVA) typing system for this pathogen. Variation was detected at six VNTR loci in a set of 56 isolates from California, Oklahoma, Arizona, and Oregon and the
F. tularensis
live vaccine strain. PCR assays revealed diversity at these loci with total allele numbers ranging from 2 to 20, and Nei's diversity index values ranging from 0.36 to 0.93. Cluster analysis identified two genetically distinct groups consistent with the current biovar classification system of
F. tularensis
. These findings suggest that these VNTR markers are useful for identifying
F. tularensis
isolates at this taxonomic level. In this study, biovar B isolates were less diverse than those in biovar A, possibly reflecting the history of tularemia in North America. Seven isolates from a recent epizootic in Maricopa County, Ariz., were identical at all VNTR marker loci. Their identity, even at a hypervariable VNTR locus, indicates a common source of infection. This demonstrates the applicability of MLVA for rapid characterization and identification of outbreak isolates. Future construction of reference databases will allow faster outbreak tracking as well as providing a foundation for deciphering global genetic relationships.
Molecular Fingerprinting of
Clostridium difficile
Isolates: Pulsed-Field Gel Electrophoresis versus Amplified Fragment Length Polymorphism
Authors: Klaassen Corné H. W. , van Haren Hanneke A. , Horrevorts Alfons M.
Published: July 2002

Two molecular fingerprinting techniques, pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP), were used to investigate the epidemiological relatedness among
Clostridium difficile
isolates from suspected outbreaks in three general hospitals. Analysis by PFGE yielded inconclusive data as a result of extensive DNA degradation. Although this degradation could be prevented to a certain extent by the inclusion of thiourea in the electrophoresis buffer, the weak DNA banding patterns obtained in this way were still far from optimal. AFLP data were obtained by using fluorescently labeled PCR primers and analysis on an ABI PRISM automated DNA analysis platform. AFLP analysis yielded high resolution and highly reproducible DNA fingerprinting patterns from which the epidemiological relatedness among the isolates could easily be determined. AFLP results could be readily obtained within 24 h, whereas 3 to 4 days were routinely required to complete the lengthy PFGE protocol. AFLP clearly proved to be a much more fail-safe fingerprinting method for
C. difficile
isolates, especially for those isolates for which a standard PFGE procedure yielded inconclusive results due to DNA degradation.
Diagnosis of Human Leptospirosis by Monoclonal Antibody-Based Antigen Detection in Urine
Authors: Saengjaruk Patcharin , Chaicumpa Wanpen , Watt George , Bunyaraksyotin Gaysorn , Wuthiekanun Vanaporn , Tapchaisri Pramuan , Sittinont Chuanpit , Panaphut Thanachai , Tomanakan Kanchana , Sakolvaree Yuwaporn , Chongsa-Nguan Manas , Mahakunkijcharoen Yuvadee , Kalambaheti Thareerat , Naigowit Pimjai , Wambangco Michael Angelo L. , Kurazono Hisao , Hayashi Hideo
Published: July 2002

Hybridomas secreting specific monoclonal antibodies (MAb) to all members of the genus
Leptospira
(clone LF9) and those that are specific only to the pathogenic species (clones LD5 and LE1) were produced. MAb LF9, which was immunoglobulin G1 (IgG1), reacted to a 38-kDa component of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated whole-cell lysates of all
Leptospira
spp., while MAb LD5 and MAb LE1, which were IgG1 and IgG2a, respectively, reacted to the 35- to 36-kDa components of all serogroups of the pathogenic species of
Leptospira.
The MAb LD5 was used in a dot blot-enzyme-linked immunosorbent assay (dot-ELISA) for detecting
Leptospira
antigen in urine samples serially collected from two groups of patients diagnosed with leptospirosis, i.e., 36 clinically diagnosed patients and 25
Leptospira
culture confirmed patients. Their serum samples were tested serologically by IgM Dipstick assay, indirect immunofluorescence assay (IFA), and/or microscopic agglutination test (MAT). Urine samples of 26 patients diagnosed with other illnesses and 120 healthy individuals served as controls. For the first group of patients, who had been ill for an average of 3.4 days before hospitalization, the IgM Dipstick test, IFA, and MAT were positive for 69.4, 70.0, and 85.7% of patients, while the
Leptospira
antigenuria tested by the MAb-based dot-ELISA was positive for 75.0, 88.9, 97.2, 97.2, and 100% of patients on days 1, 2, 3, 7, and 14 of hospitalization, respectively. All but 1 of 11 patients whose serum samples collected on the first day of hospitalization were IgM seronegative, were positive by urine antigen test on day 1. This is strong evidence that detection of antigen in urine can provide diagnostic information that could be useful in directing early therapeutic intervention. The MAT was positive in 10 of 12 patients (83.3%) of the 25 culture-positive
Leptospira
patients who had been ill for an average of 5.04 days before hospitalization, and the
Leptospira
antigen was found in 64.0, 84.0, 96.0, 100, 100, 100, and 100% of the patients' urine samples collected on days 1, 2, 3, 4, 5, 6, and 7 of hospitalization, respectively.
Leptospira
antigenuria was found in 3 of the 26 patients diagnosed with other illnesses and 1 of the 120 healthy controls. The reasons for this positivity are discussed. The detection of antigen in urine by the monoclonal antibody-based dot-ELISA has high potential for rapid, sensitive, and specific diagnosis of leptospirosis at a low cost.