bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for Horizontal Transfer of
Ssu
DAT1I Restriction-Modification Genes to the
Streptococcus suis
Genome
Authors: Sekizaki Tsutomu , Otani Yoshiko , Osaki Makoto , Takamatsu Daisuke , Shimoji Yoshihiro
Published: July 2002

Different strains of
Streptococcus suis
serotypes 1 and 2 isolated from pigs either contained a restriction-modification (R-M) system or lacked it. The R-M system was an isoschizomer of
Streptococcus pneumoniae Dpn
II, which recognizes nucleotide sequence 5′-GATC-3′. The nucleotide sequencing of the genes encoding the R-M system in
S. suis
DAT1, designated
Ssu
DAT1I, showed that the
Ssu
DAT1I gene region contained two methyltransferase genes, designated
ssuMA
and
ssuMB
, as does the
Dpn
II system. The deduced amino acid sequences of M.
Ssu
MA and M.
Ssu
MB showed 70 and 90% identity to M.
Dpn
II and M.
Dpn
A, respectively. However, the
Ssu
DAT1I system contained two isoschizomeric restriction endonuclease genes, designated
ssuRA
and
ssuRB
. The deduced amino acid sequence of R.
Ssu
RA was 49% identical to that of R.
Dpn
II, and R.
Ssu
RB was 72% identical to R.
Lla
DCHI of
Lactococcus lactis
subsp.
cremoris
DCH-4. The four
Ssu
DAT1I genes overlapped and were bounded by purine biosynthetic gene clusters in the following gene order:
purF-purM-purN-purH-ssuMA-ssuMB-ssuRA-ssuRB-purD-purE
. The G+C content of the
Ssu
DAT1I gene region (34.1%) was lower than that of the
pur
region (48.9%), suggesting horizontal transfer of the
Ssu
DAT1I system. No transposable element or long-repeat sequence was found in the flanking regions. The
Ssu
DAT1I genes were functional by themselves, as they were individually expressed in
Escherichia coli
. Comparison of the sequences between strains with and without the R-M system showed that only the region from 53 bp upstream of
ssuMA
to 5 bp downstream of
ssuRB
was inserted in the intergenic sequence between
purH
and
purD
and that the insertion target site was not the recognition site of
Ssu
DAT1I. No notable substitutions or insertions could be found, and the structures were conserved among all the strains. These results suggest that the
Ssu
DAT1I system could have been integrated into the
S. suis
chromosome by an illegitimate recombination mechanism.
Transcription ofnhaA, the Main Na+/H+Antiporter ofEscherichia coli, Is Regulated by Na+and Growth Phase
Authors: Dover Nir , Padan Etana
Published: July 2002

The transcription ofnhaA, encoding the main Na+/H+antiporter ofEscherichia coli,is induced by Na+, regulated by NhaR, and affected by H-NS. In this work the roles of the twonhaApromoters (P1 and P2) were studied by analysis of transcription both in vivo and in vitro and promoter mutations. We found that P1 is an NhaR-dependent, Na+-induced, and H-NS-affected promoter both in the exponential and stationary phases. An in vitro transcription assay demonstrated that P1 is activated by ς70-RNA polymerase and both NhaR and H-NS increase the specificity of P1. Remarkably, in marked contrast to P1, P2 exhibits very low activity during the exponential phase but is induced in the stationary phase to become the major promoter. Furthermore, P2 is activated by ςSand is neither induced by Na+nor dependent on NhaR or affected by H-NS. Hence, this work establishes thatnhaAhas a dual mode of regulation, each involving a different promoter, and reveals that P2 and ςStogether are responsible for the survival of stationary-phase cells in the presence of high Na+, alkaline pH, and the combination of high Na+and alkaline pH, the most stressful condition.
Acetate and Formate Stress: Opposite Responses in the Proteome of
Escherichia coli
Authors: Kirkpatrick Christopher , Maurer Lisa M. , Oyelakin Nikki E. , Yoncheva Yuliya N. , Maurer Russell , Slonczewski Joan L.
Published: July 2002

Acetate and formate are major fermentation products of
Escherichia coli
. Below pH 7, the balance shifts to lactate; an oversupply of acetate or formate retards growth.
E. coli
W3110 was grown with aeration in potassium-modified Luria broth buffered at pH 6.7 in the presence or absence of added acetate or formate, and the protein profiles were compared by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Acetate increased the steady-state expression levels of 37 proteins, including periplasmic transporters for amino acids and peptides (ArtI, FliY, OppA, and ProX), metabolic enzymes (YfiD and GatY), the RpoS growth phase regulon, and the autoinducer synthesis protein LuxS. Acetate repressed 17 proteins, among them phosphotransferase (Pta). An
ackA-pta
deletion, which nearly eliminates interconversion between acetate and acetyl-coenzyme A (acetyl-CoA), led to elevated basal levels of 16 of the acetate-inducible proteins, including the RpoS regulon. Consistent with RpoS activation, the
ackA-pta
strain also showed constitutive extreme-acid resistance. Formate, however, repressed 10 of the acetate-inducible proteins, including the RpoS regulon. Ten of the proteins with elevated basal levels in the
ackA-pta
strain were repressed by growth of the mutant with formate; thus, the formate response took precedence over the loss of the
ackA-pta
pathway. The similar effects of exogenous acetate and the
ackA-pta
deletion, and the opposite effect of formate, could have several causes; one possibility is that the excess buildup of acetyl-CoA upregulates stress proteins but excess formate depletes acetyl-CoA and downregulates these proteins.
The Dimerization Function of MinC Resides in a Structurally Autonomous C-Terminal Domain
Authors: Szeto Tim H. , Rowland Susan L. , King Glenn F.
Published: July 2002

Limited proteolysis of the
Escherichia coli
cell division inhibitor MinC reveals that its dimerization function resides in a structurally autonomous C-terminal domain. We show that cytoplasmic MinC is poised near the monomer-dimer equilibrium and propose that it only becomes entirely dimeric once recruited to the membrane by MinD.
Comprehensive DNA Microarray Analysis of
Bacillus subtilis
Two-Component Regulatory Systems
Authors: Kobayashi Kazuo , Ogura Mitsuo , Yamaguchi Hirotake , Yoshida Ken-Ichi , Ogasawara Naotake , Tanaka Teruo , Fujita Yasutaro
Published: July 2002

It has recently been shown through DNA microarray analysis of
Bacillus subtilis
two-component regulatory systems (DegS-DegU, ComP-ComA, and PhoR-PhoP) that overproduction of a response regulator of the two-component systems in the background of a deficiency of its cognate sensor kinase affects the regulation of genes, including its target ones. The genome-wide effect on gene expression caused by the overproduction was revealed by DNA microarray analysis. In the present work, we newly analyzed 24 two-component systems by means of this strategy, leaving out 8 systems to which it was unlikely to be applicable. This analysis revealed various target gene candidates for these two-component systems. It is especially notable that interesting interactions appeared to take place between several two-component systems. Moreover, the probable functions of some unknown two-component systems were deduced from the list of their target gene candidates. This work is heuristic but provides valuable information for further study toward a comprehensive understanding of the
B. subtilis
two-component regulatory systems. The DNA microarray data obtained in this work are available at the KEGG Expression Database website (
http://www.genome.ad.jp/kegg/expression
).
Effect of Sequences of the Active-Site Dipeptides of DsbA and DsbC on In Vivo Folding of Multidisulfide Proteins in
Escherichia coli
Authors: Bessette Paul H. , Qiu Ji , Bardwell James C. A. , Swartz James R. , Georgiou George
Published: July 2002

We have examined the role of the active-site CXXC central dipeptides of DsbA and DsbC in disulfide bond formation and isomerization in the
Escherichia coli
periplasm. DsbA active-site mutants with a wide range of redox potentials were expressed either from the
trc
promoter on a multicopy plasmid or from the endogenous
dsbA
promoter by integration of the respective alleles into the bacterial chromosome. The
dsbA
alleles gave significant differences in the yield of active murine urokinase, a protein containing 12 disulfides, including some that significantly enhanced urokinase expression over that allowed by wild-type DsbA. No direct correlation between the in vitro redox potential of
dsbA
variants and the urokinase yield was observed. These results suggest that the active-site CXXC motif of DsbA can play an important role in determining the folding of multidisulfide proteins, in a way that is independent from DsbA's redox potential. However, under aerobic conditions, there was no significant difference among the DsbA mutants with respect to phenotypes depending on the oxidation of proteins with few disulfide bonds. The effect of active-site mutations in the CXXC motif of DsbC on disulfide isomerization in vivo was also examined. A library of DsbC expression plasmids with the active-site dipeptide randomized was screened for mutants that have increased disulfide isomerization activity. A number of DsbC mutants that showed enhanced expression of a variant of human tissue plasminogen activator as well as mouse urokinase were obtained. These DsbC mutants overwhelmingly contained an aromatic residue at the C-terminal position of the dipeptide, whereas the N-terminal residue was more diverse. Collectively, these data indicate that the active sites of the soluble thiol- disulfide oxidoreductases can be modulated to enhance disulfide isomerization and protein folding in the bacterial periplasmic space.
Inhibition of
Escherichia coli
Acetyl Coenzyme A Carboxylase by Acyl-Acyl Carrier Protein
Authors: Davis Mark S. , Cronan John E.
Published: July 2002

Escherichia coli
acetyl coenzyme A carboxylase (ACC), the first enzyme of the fatty acid biosynthetic pathway, is inhibited by acylated derivatives of acyl carrier protein (ACP). ACP lacking an acyl moiety does not inhibit ACC. Acylated derivatives of ACP having chain lengths of 6 to 20 carbon atoms were similarly inhibitory at physiologically relevant concentrations. The observed feedback inhibition was specific to the protein moiety, as shown by the inability of the palmitoyl thioester of spinach ACP I to inhibit ACC.
VpsR, a Member of the Response Regulators of the Two-Component Regulatory Systems, Is Required for Expression of
vps
Biosynthesis Genes and EPS
ETr
-Associated Phenotypes in
Vibrio cholerae
O1 El Tor
Authors: Yildiz Fitnat H. , Dolganov Nadia A. , Schoolnik Gary K.
Published: July 2002

The rugose colonial variant of
Vibrio cholerae
O1 El Tor produces an exopolysaccharide (EPS
ETr
) that enables the organism to form a biofilm and to resist oxidative stress and the bactericidal action of chlorine. Transposon mutagenesis of the rugose variant led to the identification of
vpsR
, which codes for a homologue of the NtrC subclass of response regulators. Targeted disruption of
vpsR
in the rugose colony genetic background yielded a nonreverting smooth-colony morphotype that produced no detectable EPS
ETr
and did not form an architecturally mature biofilm. Analysis of two genes,
vpsA
and
vpsL
, within the
vps
cluster of EPS
ETr
biosynthesis genes revealed that their expression is induced above basal levels in the rugose variant, compared to the smooth colonial variant, and requires
vpsR
. These results show that VpsR functions as a positive regulator of
vpsA
and
vpsL
and thus acts to positively regulate EPS
ETr
production and biofilm formation.
Analysis of
cis-
and
trans-
Acting Factors Involved in Regulation of the
Streptococcus mutans
Fructanase Gene (
fruA
)
Authors: Wen Zezhang T. , Burne Robert A.
Published: July 2002

There are two primary levels of control of the expression of the fructanase gene (
fruA
) of
Streptococcus mutans
: induction by levan, inulin, or sucrose and repression in the presence of glucose and other readily metabolized sugars. The goals of this study were to assess the functionality of putative
cis-
acting regulatory elements and to begin to identify the
trans
-acting factors involved in induction and catabolite repression of
fruA.
The
fruA
promoter and its derivatives generated by deletions and/or site-directed mutagenesis were fused to a promoterless chloramphenicol acetyltransferase (CAT) gene as a reporter, and strains carrying the transcriptional fusions were then analyzed for CAT activities in response to growth on various carbon sources. A dyadic sequence, ATGACA(TC)TGTCAT, located at −72 to −59 relative to the transcription initiation site was shown to be essential for expression of
fruA
. Inactivation of the genes that encode fructose-specific enzymes II resulted in elevated expression from the
fruA
promoter, suggesting negative regulation of
fruA
expression by the fructose phosphotransferase system. Mutagenesis of a terminator-like structure located in the 165-base 5′ untranslated region of the
fruA
mRNA or insertional inactivation of antiterminator genes revealed that antitermination was not a mechanism controlling induction or repression of
fruA
, although the untranslated leader mRNA may play a role in optimal expression of fructanase. Deletion or mutation of a consensus catabolite response element alleviated glucose repression of
fruA
, but interestingly, inactivation of the
ccpA
gene had no discernible effect on catabolite repression of
fruA
. Accumulating data suggest that expression of
fruA
is regulated by a mechanism that has several unique features that distinguish it from archetypical polysaccharide catabolic operons of other gram-positive bacteria.
Plasmid-Encoded Autolysin inBacillus anthracis: Modular Structure and Catalytic Properties
Authors: Mesnage Stéphane , Fouet Agnès
Published: July 2002

ABacillus anthracisvirulence plasmid-encoded peptidoglycan hydrolase (AmiA) with sequence similarity toN-acetylmuramoyl-l-alanine amidases hydrolyzes peptidoglycan independently of cell wall binding. Residues H341, E355, H415, and E486 are absolutely required for catalysis. Many AmiA paralogs are fused to different sorting signals, suggesting that these modular proteins result from domain shuffling.
Heterologous Production of
Clostridium cellulovorans engB
, Using Protease-Deficient
Bacillus subtilis
, and Preparation of Active Recombinant Cellulosomes
Authors: Murashima Koichiro , Chen Chyi-Liang , Kosugi Akihiko , Tamaru Yutaka , Doi Roy H. , Wong Sui-Lam
Published: July 2002

In cellulosomes produced by
Clostridium
spp., the high-affinity interaction between the dockerin domain and the cohesin domain is responsible for the assembly of enzymatic subunits into the complex. Thus, heterologous expression of full-length enzymatic subunits containing the dockerin domains and of the scaffolding unit is essential for the in vitro assembly of a “designer” cellulosome, or a recombinant cellulosome with a specific function. We report the preparation of
Clostridium cellulovorans
recombinant cellulosomes containing the enzymatic subunit EngB and the scaffolding unit, mini-CbpA, containing a cellulose binding domain, a putative cell wall binding domain, and two cohesin units. The full-length EngB containing the dockerin domain was expressed by
Bacillus subtilis
WB800, which is deficient in eight extracellular proteases, to prevent the proteolytic cleavage of the enzymatic subunit between the catalytic and dockerin domains that was observed in previous attempts to express EngB with
Escherichia coli
. The assembly of recombinant EngB with the mini-CbpA was confirmed by immunostaining, a cellulose binding experiment, and native polyacrylamide gel electrophoresis analysis.
Novel 2,4-Dichlorophenoxyacetic Acid Degradation Genes from Oligotrophic
Bradyrhizobium
sp. Strain HW13 Isolated from a Pristine Environment
Authors: Kitagawa Wataru , Takami Sachiko , Miyauchi Keisuke , Masai Eiji , Kamagata Yoichi , Tiedje James M. , Fukuda Masao
Published: July 2002

The
tfd
genes of
Ralstonia eutropha
JMP134 are the only well-characterized set of genes responsible for 2,4-dichlorophenoxyacetic acid (2,4-D) degradation among 2,4-D-degrading bacteria. A new family of 2,4-D degradation genes,
cadRABKC
, was cloned and characterized from
Bradyrhizobium
sp. strain HW13, a strain that was isolated from a buried Hawaiian soil that has never experienced anthropogenic chemicals. The
cadR
gene was inferred to encode an AraC/XylS type of transcriptional regulator from its deduced amino acid sequence. The
cadABC
genes were predicted to encode 2,4-D oxygenase subunits from their deduced amino acid sequences that showed 46, 44, and 37% identities with the TftA and TftB subunits of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) oxygenase of
Burkholderia cepacia
AC1100 and with a putative ferredoxin, ThcC, of
Rhodococcus erythropolis
NI86/21, respectively. They are thoroughly different from the 2,4-D dioxygenase gene,
tfdA
, of
R. eutropha
JMP134. The
cadK
gene was presumed to encode a 2,4-D transport protein from its deduced amino acid sequence that showed 60% identity with the 2,4-D transporter, TfdK, of strain JMP134.
Sinorhizobium meliloti
Rm1021 cells containing
cadRABKC
transformed several phenoxyacetic acids, including 2,4-D and 2,4,5-T, to corresponding phenol derivatives. Frameshift mutations indicated that each of the
cadRABC
genes was essential for 2,4-D conversion in strain Rm1021 but that
cadK
was not. Five 2,4-D degraders, including
Bradyrhizobium
and
Sphingomonas
strains, were found to have
cadA
gene homologs, suggesting that these 2,4-D degraders share 2,4-D degradation genes similar to those of strain HW13
cadABC
.
Glyoxylate Regeneration Pathway in the Methylotroph
Methylobacterium extorquens
AM1
Authors: Korotkova Natalia , Chistoserdova Ludmila , Kuksa Vladimir , Lidstrom Mary E.
Published: July 2002

Most serine cycle methylotrophic bacteria lack isocitrate lyase and convert acetyl coenzyme A (acetyl-CoA) to glyoxylate via a novel pathway thought to involve butyryl-CoA and propionyl-CoA as intermediates. In this study we have used a genome analysis approach followed by mutation to test a number of genes for involvement in this novel pathway. We show that methylmalonyl-CoA mutase, an R-specific crotonase, isobutyryl-CoA dehydrogenase, and a GTPase are involved in glyoxylate regeneration. We also monitored the fate of
14
C-labeled carbon originating from acetate, butyrate, or bicarbonate in mutants defective in glyoxylate regeneration and identified new potential intermediates in the pathway: ethylmalonyl-CoA, methylsuccinyl-CoA, isobutyryl-CoA, methacrylyl-CoA, and β-hydroxyisobutyryl-CoA. A new scheme for the pathway is proposed based on these data.
Enterococcus faecalis
Acetoacetyl-Coenzyme A Thiolase/3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase, a Dual-Function Protein of Isopentenyl Diphosphate Biosynthesis
Authors: Hedl Matija , Sutherlin Autumn , Wilding E. Imogen , Mazzulla Marie , McDevitt Damien , Lane Pamela , Burgner John W. , Lehnbeuter Kevin R. , Stauffacher Cynthia V. , Gwynn Michael N. , Rodwell Victor W.
Published: July 2002

Many bacteria employ the nonmevalonate pathway for synthesis of isopentenyl diphosphate, the monomer unit for isoprenoid biosynthesis. However, gram-positive cocci exclusively use the mevalonate pathway, which is essential for their growth (E. I. Wilding et al., J. Bacteriol. 182:4319-4327, 2000). Enzymes of the mevalonate pathway are thus potential targets for drug intervention. Uniquely, the enterococci possess a single open reading frame,
mvaE
, that appears to encode two enzymes of the mevalonate pathway, acetoacetyl-coenzyme A thiolase and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Western blotting revealed that the
mvaE
gene product is a single polypeptide in
Enterococcus faecalis
,
Enterococcus faecium
, and
Enterococcus hirae
. The
mvaE
gene was cloned from
E. faecalis
and was expressed with an N-terminal His tag in
Escherichia coli
. The gene product was then purified by nickel affinity chromatography. As predicted, the 86.5-kDa
mvaE
gene product catalyzed both the acetoacetyl-CoA thiolase and HMG-CoA reductase reactions. Temperature optima, Δ

H
a

and

K
m

values, and pH optima were determined for both activities. Kinetic studies of acetoacetyl-CoA thiolase implicated a ping-pong mechanism. CoA acted as an inhibitor competitive with acetyl-CoA. A millimolar

K
i

for a statin drug confirmed that
E. faecalis
HMG-CoA reductase is a class II enzyme. The oxidoreductant was NADP(H). A role for an active-site histidine during the first redox step of the HMG-CoA, reductase reaction was suggested by the ability of diethylpyrocarbonate to block formation of mevalonate from HMG-CoA, but not from mevaldehyde. Sequence comparisons with other HMG-CoA reductases suggest that the essential active-site histidine is His756. The
mvaE
gene product represents the first example of an HMG-CoA reductase fused to another enzyme.
Gene Transfer betweenSalmonella entericaSerovar Typhimurium inside Epithelial Cells
Authors: Ferguson Gayle C. , Heinemann Jack A. , Kennedy Martin A.
Published: July 2002

Virulence and antibiotic resistance genes transfer between bacteria by bacterial conjugation. Conjugation also mediates gene transfer from bacteria to eukaryotic organisms, including yeast and human cells. Predicting when and where genes transfer by conjugation could enhance our understanding of the risks involved in the release of genetically modified organisms, including those being developed for use as vaccines. We report here thatSalmonella entericaserovar Typhimurium conjugated inside cultured human cells. The DNA transfer from donor to recipient bacteria was proportional to the probability that the two types of bacteria occupied the same cell, which was dependent on viable and invasive bacteria and on plasmidtragenes. Based on the high frequencies of gene transfer between bacteria inside human cells, we suggest that such gene transfers occur in situ. The implications of gene transfer between bacteria inside human cells, particularly in the context of antibiotic resistance, are discussed.
TetR Family Member PsrA Directly Binds the
Pseudomonas rpoS
and
psrA
Promoters
Authors: Kojic Milan , Aguilar Claudio , Venturi Vittorio
Published: July 2002

We have previously described a
Pseudomonas
gene,
psrA
, which enhances transcription of the
rpoS
sigma factor gene at stationary phase. We present molecular data which demonstrate that in
Pseudomonas putida
PsrA binds specifically to the
rpoS
and
psrA
promoters in DNA regions having similar palindromic sequences, C/GAAAC N
2-4
GTTTG/C, where N is any nucleotide. The position of the initiation of transcription was determined for both promoters, and PsrA binds from positions −59 to −35 in the
rpoS
promoter and from −18 to +20 in the
psrA
promoter with respect to the +1 transcription site. Expression studies with a
psrA-lacZ
transcriptional fusion in wild-type and
psrA
::Tn
5
knockout mutants revealed that
psrA
was under additional control in response to growth phase. A model for the role of PsrA in the regulation of
rpoS
and
psrA
is presented.
Simultaneous Detection of Influenza Viruses A and B Using Real-Time Quantitative PCR
Authors: van Elden L. J. R. , Nijhuis M. , Schipper P. , Schuurman R. , van Loon A. M.
Published: July 2002

Since influenza viruses can cause severe illness, timely diagnosis is important for an adequate intervention. The available rapid detection methods either lack sensitivity or require complex laboratory manipulation. This study describes a rapid, sensitive detection method that can be easily applied to routine diagnosis. This method simultaneously detects influenza viruses A and B in specimens of patients with respiratory infections using a TaqMan-based real-time PCR assay. Primers and probes were selected from highly conserved regions of the matrix protein gene of influenza virus A and the hemagglutinin gene segment of influenza virus B. The applicability of this multiplex PCR was evaluated with 27 influenza virus A and 9 influenza virus B reference strains and isolates. In addition, the specificity of the assay was assessed using eight reference strains of other respiratory viruses (parainfluenza viruses 1 to 3, respiratory syncytial virus Long strain, rhinoviruses 1A and 14, and coronaviruses OC43 and 229E) and 30 combined nose and throat swabs from asymptomatic subjects. Electron microscopy-counted stocks of influenza viruses A and B were used to develop a quantitative PCR format. Thirteen copies of viral RNA were detected for influenza virus A, and 11 copies were detected for influenza virus B, equaling 0.02 and 0.006 50% tissue culture infective doses, respectively. The diagnostic efficacy of the multiplex TaqMan-based PCR was determined by testing 98 clinical samples. This real-time PCR technique was found to be more sensitive than the combination of conventional viral culturing and shell vial culturing.
Evaluation of Two Commercial Enzyme Immunoassays, Testing Immunoglobulin G (IgG) and IgA Responses, for Diagnosis of
Helicobacter pylori
Infection in Children
Authors: Kindermann Angelika , Konstantopoulos Nikolaos , Lehn Norbert , Demmelmair Hans , Koletzko Sibylle
Published: July 2002

Serological testing to diagnose
Helicobacter pylori
infection in children is still controversial, although commonly used in clinical practice. We compared the immunoglobulin G (IgG) and IgA results of two commercially available enzyme immunoassays (EIAs) (Pyloriset IgG and IgA and Enzygnost II IgG and IgA) for 175 children with abdominal symptoms divided into three age groups (0 to ≤6 years,
n
= 47; >6 to ≤12 years,
n
= 77; >12 years,
n
= 51). A child was considered
H. pylori
infected if at least two of three tests (histology, rapid urease test,
13
C-urea breath test) or culture were positive and noninfected if all results were concordantly negative. Of 175 children, 93 (53%) were
H. pylori
negative and 82 were
H. pylori
positive. With the recommended cutoff values, the overall specificity was excellent for all four EIAs (95.7 to 97.8%) regardless of age. Sensitivity varied markedly between tests and was 92.7, 70.7, 47.5, and 24.4% for Enzygnost II IgG, Pyloriset IgG, Enzygnost II IgA, and Pyloriset IgA, respectively. Sensitivity was low in the youngest age group (25 to 33.3%), except for Enzygnost II IgG (91.6%). Receiver-operating curve analyses revealed that lower cutoff values would improve the accuracy of all of the tests except Enzygnost II IgG. Measurement of specific IgA, in addition to IgG, antibodies hardly improved the sensitivity. The specificity of commercial serological tests is high in children when the cutoff values obtained from adults are used. In contrast, sensitivity is variable, with a strong age dependence in some, but not all, tests. We speculate that young children may have a different immune response to
H. pylori
, with preferable responses to certain antigens, as well as lower titers than adults. The Pyloriset test may fail to recognize these specific antibodies.
Effect of Endocervical Specimen Adequacy on Ligase Chain Reaction Detection of
Chlamydia trachomatis
Authors: Loeffelholz Mike J. , Jirsa Sandra J. , Teske Rebecca K. , Woods Juliet N.
Published: July 2002

Several studies have demonstrated that the sensitivity of a commercially available PCR test for the detection of
Chlamydia trachomatis
(Roche Diagnostics) is affected by the cellular quality of the endocervical swab specimens. The cellular adequacies of 1,633 female endocervical swab specimens were assessed and compared with the results of
C. trachomatis
detection obtained by ligase chain reaction (LCR; Abbott Laboratories). Specimen adequacy studies and LCR were performed with samples from the same swab, after demonstration of the stability of human epithelial cells in LCR transport medium. Prior to heat treatment of the swab specimen, an aliquot was removed and cytocentrifuged onto a slide. Cell spots were stained and examined at ×400 magnification for endocervical (columnar epithelial or metaplastic) cells and erythrocytes. The overall rate of positivity of the LCR was 6.5% (106 of 1,633 specimens) with pooled specimens (pools of 4 specimens each; reduced cutoff). Of the 1,633 specimens examined, 655 (40.1%) were found to contain one or more endocervical cells. The rate of positivity for
C. trachomatis
was 10.8% (71 of 655 specimens) among specimens containing endocervical cells, whereas it was 3.6% (35 of 978 specimens) among specimens lacking endocervical cells (
P
< 0.0001). There was no linear trend between the rate of positivity for
C. trachomatis
and the number of endocervical cells (
P
= 0.24). The rate of positivity for
C. trachomatis
was 5.4% (8 of 147 specimens) among specimens containing large numbers of erythrocytes (≥100 per high-power field), whereas it was 6.6% (98 of 1,486 specimens) among specimens containing less than 100 erythrocytes per high-power field (
P
= 0.59). These results show that the sensitivity of the Abbott
C. trachomatis
LCR test is affected by the presence of endocervical cells. Additionally, they indicate that the presence of a single endocervical cell is as good an indicator of specimen adequacy as the presence of many endocervical cells. The presence of a large number of erythrocytes was not associated with an increased rate of sensitivity of the LCR.
Pneumocystis carinii
f. sp.
hominis
DNA in Immunocompetent Health Care Workers in Contact with Patients with
P. carinii
Pneumonia
Authors: Miller Robert F. , Ambrose Helen E. , Wakefield Ann E.
Published: July 2002

The possible transmission of
Pneumocystis carinii
f. sp.
hominis
from patients with
P. carinii
pneumonia to asymptomatic health care workers (HCW), with or without occupational exposure to human immunodeficiency virus (HIV)-infected patients with
P. carinii
pneumonia, was examined. HCW in a specialist inpatient HIV-AIDS facility and a control group in the general medical-respiratory service in the same hospital provided induced sputum and/or nasal rinse samples, which were analyzed for the presence of
P. carinii
f. sp.
hominis
DNA by using DNA amplification (at the gene encoding the mitochondrial large subunit rRNA [mt LSU rRNA]).
P. carinii
f. sp.
hominis
DNA was detected in some HCW samples; those with the closest occupational contact were more likely to have detectable
P. carinii
DNA.
P. carinii
DNA was detected in one HCW who carried out bronchoscopy over a 2-year period.
P. carinii
-positive samples were genotyped by using DNA sequence variations at the internal transcribed spacer (ITS) regions of the nuclear rRNA operon, along with bronchoalveolar lavage samples from patients with
P. carinii
pneumonia hospitalized at the same time. Genotyping identified 31 different
P. carinii
f. sp.
hominis
ITS genotypes, 26 of which were found in the patient samples. Five of the eight ITS genotypes detected in HCW samples were not observed in the patient samples. The results suggested that HCW in close occupational contact with patients who had
P. carinii
pneumonia may have become colonized with
P. carinii
. Carriage was asymptomatic and did not result in the development of clinical disease.
Molecular Epidemiology of Rotaviruses in Nigeria: Detection of Unusual Strains with G2P[6] and G8P[1] Specificities
Authors: Adah Mohammed I. , Wade Abel , Taniguchi Koki
Published: July 2002

During an epidemiological study on rotaviruses among diarrheic children in the northeastern and middle belt regions of Nigeria, the distribution of G and P types was investigated in 127 stool specimens. By PCR G typing, the G type of rotaviruses in 97 samples was identified. Interestingly, an unusual G8 type, as well as common G1, G2, and G3 types, was detected more frequently (31 of 112; 27.7%). Eleven samples contained multiple G types, and a G9 strain (Bulumkutu) was identified for one of the probable mixed infections. In PCR P typing, P[6] was detected most frequently, P[8] being the second most common type, while the P type of 73 samples could not be identified. One rotavirus strain with a G8 type specificity could be cultivated in cell culture, and the P type of this strain was found to be P[1], which is usually carried by bovine strains. When the combinations of G and P types were examined, the unusual strains G2P[6] and G8P[1] were often identified. Sequence analysis was performed for the VP7 gene of the G9 strain Bulumkutu and the VP4 and VP7 genes of G8P[1] strain HMG035. The VP7 sequence of the Nigerian serotype G9 was more closely related to that of a Brazilian strain than to those of other African strains. The VP7 and VP4 genes of G8P[1] strain HMG035 were found to be very similar to that of a Thai bovine strain A5, suggesting that bovine strains may have been transmitted directly to humans. These results highlight an unexpected diversity among rotavirus strains in Nigeria and emphasize the need for further serological and genetic surveys on more rotavirus strains in African countries, including Nigeria.
Recombinant Flagellin A Proteins from
Borrelia burgdorferi
Sensu Stricto,
B
.
afzelii
, and
B. garinii
in Serodiagnosis of Lyme Borreliosis
Authors: Panelius Jaana , Lahdenne Pekka , Saxen Harri , Heikkilä Tero , Seppälä Ilkka
Published: July 2002

Genes for flagellin A (FlaA) proteins from European borrelial strains of
Borrelia burgdorferi
sensu stricto,
B. afzelii
, and
B. garinii
were cloned and sequenced. An identity of 92 to 93% was observed in the
flaA
sequences of the different species. Polyhistidine-tagged recombinant FlaA (rFlaA) proteins were produced in
Escherichia coli
and used as antigens in Western blotting (WB) and enzyme-linked immunosorbent assay (ELISA). In immunoglobulin G (IgG) WB, 71% (10 of 14) of the sera from neuroborreliosis and 86% (12 of 14) of those from Lyme arthritis patients reacted with one to three rFlaAs. In IgG ELISA, 74% (14 of 19) and 79% (15 of 19) of patients with neuroborreliosis and arthritis, respectively, were positive. The immunoreactivity in local European patient sera was stronger against rFlaA from
B. garinii
and
B. afzelii
than against rFlaA from
B. burgdorferi
sensu stricto. Neither IgG nor IgM ELISA was sensitive in the serodiagnosis of erythema migrans. Serum samples from patients with syphilis and systemic lupus erythematosus showed mild cross-reactivity in IgG tests. Sera from
Yersinia
enterocolitica
or beta-hemolytic
Streptococcus
infections showed only occasional responses. With IgM ELISA, 58% (11 of 19) and 37% (7 of 19) of patients with neuroborreliosis and arthritis, respectively, were positive. Cross-reactive antibodies to FlaA, especially in serum samples from patients with rheumatoid factor positivity and Epstein-Barr virus infection, reduced the specificity of IgM serodiagnosis. Therefore, rFlaA seems to have a limited role for IgM serodiagnosis, yet rFlaA might be useful in the IgG serodiagnosis of disseminated Lyme borreliosis.
Epstein-Barr Virus (EBV) DNA in Sera of Patients with Primary EBV Infection
Authors: Chan K. H. , Ng M. H. , Seto W. H. , Peiris J. S. M.
Published: July 2002

Detection of Epstein-Barr Virus (EBV) DNA by PCR in serum had a sensitivity of 80%, a specificity of 94%, and positive and negative predictive values of 95 and 79%, respectively, for the diagnosis of primary EBV infection. We suggest that this is a useful addition to the panel of tests used for this purpose.
Comparison of Automated Ribotyping to Pulsed-Field Gel Electrophoresis for Genetic Fingerprinting of
Streptococcus pneumoniae
Authors: Quale John M. , Landman David , Flores Carlos , Ravishankar Jayashree
Published: July 2002

Fifty-two isolates of
Streptococcus pneumoniae
were characterized by pulsed-field gel electrophoresis (PFGE) and automated ribotyping by using
Hin
dIII and
Pvu
II.
Hin
dIII ribotypes correlated well with PFGE.
Pvu
II produced fewer bands and was less discriminatory. Automated ribotyping with
Hin
dIII is an accurate method for genetic fingerprinting of
S. pneumoniae
and can complement PFGE.
Recovery of
Candida dubliniensis
and Other Yeasts from Human Immunodeficiency Virus-Associated Periodontal Lesions
Authors: Jabra-Rizk M. A. , Ferreira S. M. S. , Sabet M. , Falkler W. A. , Merz W. G. , Meiller T. F.
Published: July 2002

Oral and subgingival samples from periodontal lesions were collected from 54 human immunodeficiency virus (HIV)-positive and 20 HIV-negative patients and cultured for yeast species. Of the 54 samples cultured from HIV-positive patients, 44 (82%) were positive for yeast species, of which 29 (66%) were subgingival. A total of 19 (48%) patients were positive for
Candida dubliniensis
, of which 15 (79%) were colonized in subgingival sites. Seven isolates of
Candida glabrata
, two isolates of
Candida parapsilosis
, and one isolate of
Saccharomyces cerevisiae
were recovered. This study reports for the first time the recovery of
C. dubliniensis
from subgingival intraoral sites and confirms the presence of
Candida
species in sites of periodontal disease associated with HIV.
Molecular Characterization of First Human
Bartonella
Strain Isolated in Italy
Authors: Ciervo Alessandra , Petrucca Andrea , Ciarrocchi Simonetta , Pinto Antonella , Bonazzi Lucio , Fabio Anna , Farnetti Enrico , Chomel Bruno B. , Ciceroni Lorenzo
Published: July 2002

The aim of this study was to characterize a
Bartonella
strain (BA-1) isolated from a blood culture of an Italian, human immunodeficiency virus-positive patient with bacillary angiomatosis. We analyzed the isolate using molecular biology methods such as whole-cell fatty acid analysis, PCR-restriction fragment length polymorphism analysis, type-specific 16S rRNA PCRs, sequence analysis of the 16S rRNA, pulsed-field gel electrophoresis, and arbitrarily primed PCR. The BA-1 isolate turned out to be a
Bartonella quintana
strain, similar but not identical to
B. quintana
Oklahoma, which was used as a control strain.
Fatal Myocarditis Associated with Acute Parvovirus B19 and Human Herpesvirus 6 Coinfection
Authors: Rohayem Jacques , Dinger Jürgen , Fischer Rainer , Klingel Karin , Kandolf Reinhard , Rethwilm Axel
Published: July 2002

We report on the case of a healthy young boy who developed a fulminant myocarditis due to acute coinfection with erythrovirus (parvovirus B19) and human herpesvirus 6 (HHV-6) in the absence of an antiviral immune response. We suggest that the HHV-6-induced immunosuppression enhanced dissemination of parvovirus B19, which led to fatal myocarditis.
Molecular Epidemiology of Penicillin-Susceptible, Multidrug-Resistant Serotype 6B Pneumococci Isolated from Children in Greece
Authors: Syrogiannopoulos G. A. , Bogaert D. , Grivea I. N. , Beratis N. G. , De Groot R. , Hermans P. W. M.
Published: July 2002

Since January 1996, and over a 3-year time span, a significant spread of serotype 6B multidrug-resistant (MDR) pneumococci, susceptible to penicillin and resistant to erythromycin, clindamycin, tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole, was noted in young carriers living in central and southern Greece. Using restriction fragment end labeling and penicillin binding protein (PBP) genotyping, we studied 41 serotype 6B penicillin-susceptible MDR pneumococci isolated during two independent studies in Greece. Forty (98%) of these 41 isolates were strongly related, representing a single lineage (genetic relatedness, ≥91%). The Greek isolates were closely related (genetic relatedness, ∼91%) to the penicillin-resistant MDR clone of serotype 6B that spread from Spain to Iceland in the late 1980s. Moreover, the Greek group of isolates was genetically distinct (genetic relatedness, ≤83%) from other penicillin-susceptible or -resistant serotype 6B strains from various parts of the world. All serotype 6B penicillin-susceptible MDR isolates displayed a penicillin-susceptible PBP 1A-2B-2X genotype. Our findings suggest that the penicillin-susceptible MDR 6B clone that was found in Greece between the years 1996 and 1999 represents the ancestor of the pandemic penicillin-resistant MDR clone 6B.
Peritonitis Due to
Thermoascus taitungiacus
(Anamorph
Paecilomyces taitungiacus
)
Authors: Korzets Asher , Weinberger Miriam , Chagnac Avry , Goldschmied-Reouven Anna , Rinaldi Michael G. , Sutton Deanna A.
Published: July 2002

The first case of human disease due to the thermophilic ascomycete
Thermoascus taitungiacus
(the teleomorph of
Paecilomyces taitungiacus
) is presented.
T. taitungiacus
was recovered from four dialysate fluid specimens of a 57-year-old patient undergoing chronic peritoneal dialysis. Identification was based upon cylindrical conidia, reddish orange nonostiolate ascomata, lack of growth at 20°C, thermotolerance, and ascospores that appeared pale yellow, elliptical, thick walled, and predominately echinulate by light microscopy but irregularly verrucose by scanning electron microscopy.
Evaluation of a Rapid Optical Immunoassay for Influenza Viruses (FLU OIA Test) in Comparison with Cell Culture and Reverse Transcription-PCR
Authors: Boivin Guy , Hardy Isabelle , Kress Andrew
Published: July 2002

The FLU OIA test was evaluated with 146 throat swab specimens from subjects with a flu-like illness in six Canadian clinics during the 1999–2000 flu season. The rate of positivity of the FLU OIA test (41.5%) was significantly lower than that of cell culture (55.2%) or reverse transcription-PCR (55.9%) during a season in which only influenza A virus was detected.
Serology of Typhoid Fever in an Area of Endemicity and Its Relevance to Diagnosis
Authors: House Deborah , Wain John , Ho Vo A. , Diep To S. , Chinh Nguyen T. , Bay Phan V. , Vinh Ha , Duc Minh , Parry Christopher M. , Dougan Gordon , White Nicholas J. , Hien Tran Tinh , Farrar Jeremy J.
Published: July 2002

Currently, the laboratory diagnosis of typhoid fever is dependent upon either the isolation of
Salmonella enterica
subsp.
enterica
serotype Typhi from a clinical sample or the detection of raised titers of agglutinating serum antibodies against the lipopolysaccharide (LPS) (O) or flagellum (H) antigens of serotype Typhi (the Widal test). In this study, the serum antibody responses to the LPS and flagellum antigens of serotype Typhi were investigated with individuals from a region of Vietnam in which typhoid is endemic, and their usefulness for the diagnosis of typhoid fever was evaluated. The antibody responses to both antigens were highly variable among individuals infected with serotype Typhi, and elevated antibody titers were also detected in a high proportion of serum samples from healthy subjects from the community. In-house enzyme-linked immunosorbent assays (ELISAs) for the detection of specific classes of anti-LPS and antiflagellum antibodies were compared with other serologically based tests for the diagnosis of typhoid fever (Widal TO and TH, anti-serotype Typhi immunoglobulin M [IgM] dipstick, and IDeaL TUBEX). At a specificity of ≥0.93, the sensitivities of the different tests were 0.75, 0.55, and 0.52 for the anti-LPS IgM, IgG, and IgA ELISAs, respectively; 0.28 for the antiflagellum IgG ELISA; 0.47 and 0.32 for the Widal TO and TH tests, respectively; and 0.77 for the anti-serotype Typhi IgM dipstick assay. The specificity of the IDeaL TUBEX was below 0.90 (sensitivity, 0.87; specificity, 0.76). The serological assays based on the detection of IgM antibodies against either serotype Typhi LPS (ELISA) or whole bacteria (dipstick) had a significantly higher sensitivity than the Widal TO test when used with a single acute-phase serum sample (
P
≤ 0.007). These tests could be of use for the diagnosis of typhoid fever in patients who have clinical typhoid fever but are culture negative or in regions where bacterial culturing facilities are not available.
Epidemiological Usefulness of Anti-Opacity Factor Antibody Screening in Schoolchildren
Authors: Kim Sunjoo , Lee Nam Yong
Published: July 2002

The presence of the anti-opacity factor (anti-OF) antibody (Ab) in the serum used for identifying the OF antigen (Ag) type represents previous or current infection with group A streptococci (GAS) of the OF Ag type. Throat cultures were taken from 172 elementary schoolchildren in Chinju, Korea, and venous blood samples were collected at the same time to screen for the frequency of the anti-OF Ab. After isolation of GAS, the OF Ag of each GAS was identified by inhibition of the opacity reaction with recognized anti-OF sera. The anti-OF Abs in the sera were screened with the six most common OF Ag types. OF22 and OF28 were high in prevalence (28.2 and 20.5%, respectively) among OF Ag types, while anti-OF types 4, 28, and 22 were frequently identified (39.5, 29.7, and 15.7%, respectively) in the sera. Thirty-two of 39 (82.1%) OF Ag-producing GAS carriers, 25 of 34 (73.5%) GAS carriers not producing OF Ag, and 72 of 99 (72.7%) throat culture-negative children harbored the anti-OF Ab. Forty-five (26.2%) of 172 children had two different anti-OF Abs, and 11 (6.4%) had more than three anti-OF Abs. Seventy-five percent of 172 elementary schoolchildren were shown to be previously or currently infected with GAS. The percentages of children positive for the anti-OF Ab were very high regardless of the result of throat culture or OF Ag production of GAS. We could also demonstrate (i) that the prevalent strains of GAS changed according to the time span by determining the difference between the frequencies of OF Ag and anti-OF Ab and (ii) that repeated infections were not uncommon in schoolchildren, as one-third had more than two different anti-OF Abs.
Development of a Genomics-Based PCR Assay for Detection of
Mycoplasma pneumoniae
in a Large Outbreak in New York State
Authors: Waring Alfred L. , Halse Tanya A. , Csiza Charles K. , Carlyn Cynthia J. , Musser Kimberlee Arruda , Limberger Ronald J.
Published: July 2002

A genomics-based PCR method was developed and used to test specimens from patients involved in a large outbreak of
Mycoplasma pneumoniae
in a closed religious community in New York State. New P1 adhesin gene primers were designed to bind to 9 of 10 target sequences in the repetitive-element sequences obtained from the whole genome sequence of
M. pneumoniae
. This PCR method had a sensitivity of 0.006 CFU and a specificity of 100% for
M. pneumoniae
. The PCR was validated by testing a subset of patient samples by culture and comparing the results to those obtained by PCR. Of the initial 280 samples tested, 73 were positive by PCR and 22 were positive by culture. All samples positive by culture were also positive by PCR. Follow-up testing of selected patients 3 to 6 weeks after antibiotic treatment revealed that eight samples remained positive by PCR and that three samples remained positive by culture. Additionally, no nonspecific PCR inhibition was detected as a result of the specimen type, transport medium, or sample preparation methodology. The study demonstrates that the PCR described here is a rapid, sensitive, and specific method for the identification of
M. pneumoniae
and was helpful for the detection and monitoring of the outbreak.