bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of the Tat Transport System in Nitrous Oxide Reductase Translocation and Cytochrome
cd
1
Biosynthesis in
Pseudomonas stutzeri
Authors: Heikkilä Mari P. , Honisch Ulrike , Wunsch Patrick , Zumft Walter G.
Published: July 2002

By transforming N
2
O to N
2
, the multicopper enzyme nitrous oxide reductase provides a periplasmic electron sink for a respiratory chain that is part of denitrification. The signal sequence of the enzyme carries the heptameric twin-arginine consensus motif characteristic of the Tat pathway. We have identified
tat
genes of
Pseudomonas stutzeri
and functionally analyzed the unlinked
tatC
and
tatE
loci. A
tatC
mutant retained N
2
O reductase in the cytoplasm in the unprocessed form and lacking the metal cofactors. This is contrary to viewing the Tat system as specific only for fully assembled proteins. A C618V exchange in the electron transfer center Cu
A
rendered the enzyme largely incompetent for transport. The location of the mutation in the C-terminal domain of N
2
O reductase implies that the Tat system acts on a completely synthesized protein and is sensitive to a late structural variation in folding. By generating a
tatE
mutant and a reductase-overproducing strain, we show a function for TatE in N
2
O reductase translocation. Further, we have found that the Tat and Sec pathways have to cooperate to produce a functional nitrite reductase system. The cytochrome
cd
1
nitrite reductase was found in the periplasm of the
tatC
mutant, suggesting export by the Sec pathway; however, the enzyme lacked the heme D
1
macrocycle. The NirD protein as part of a complex required for heme D
1
synthesis or processing carries a putative Tat signal peptide. Since NO reduction was also inhibited in the
tatC
mutant, the Tat protein translocation system is necessary in multiple ways for establishing anaerobic nitrite denitrification.
Glucan Synthase Complex of
Aspergillus fumigatus
Authors: Beauvais A. , Bruneau J. M. , Mol P. C. , Buitrago M. J. , Legrand R. , Latgé J. P.
Published: July 2002

The glucan synthase complex of the human pathogenic mold
Aspergillus fumigatus
has been investigated. The genes encoding the putative catalytic subunit Fks1p and four Rho proteins of
A. fumigatus
were cloned and sequenced. Sequence analysis showed that AfFks1p was a transmembrane protein very similar to other Fksp proteins in yeasts and in
Aspergillus nidulans
. Heterologous expression of the conserved internal hydrophilic domain of AfFks1p was achieved in
Escherichia coli
. Anti-Fks1p antibodies labeled the apex of the germ tube, as did aniline blue fluorochrome, which was specific for β(1–3) glucans, showing that AfFks1p colocalized with the newly synthesized β(1–3) glucans.
AfRHO1
, the most homologous gene to
RHO1
of
Saccharomyces cerevisiae
, was studied for the first time in a filamentous fungus. AfRho proteins have GTP binding and hydrolysis consensus sequences identical to those of yeast Rho proteins and have a slightly modified geranylation site in AfRho1p and AfRho3p. Purification of the glucan synthase complex by product entrapment led to the enrichment of four proteins: Fks1p, Rho1p, a 100-kDa protein homologous to a membrane H
+
-ATPase, and a 160-kDa protein which was labeled by an anti-β(1–3) glucan antibody and was homologous to ABC bacterial β(1–2) glucan transporters.
Twelve-Transmembrane-Segment (TMS) Version (ΔTMS VII-VIII) of the 14-TMS Tet(L) Antibiotic Resistance Protein Retains Monovalent Cation Transport Modes but Lacks Tetracycline Efflux Capacity
Authors: Jin Jie , Guffanti Arthur A. , Beck Catherine , Krulwich Terry A.
Published: July 2002

A “Tet(L)-12” version of Tet(L), a tetracycline efflux protein with 14 transmembrane segments (TMS), was constructed by deletion of two central TMS. Tet(L)-12 catalyzed Na
+
/H
+
antiport and antiport with K
+
as a coupling ion as well as or better than wild-type Tet(L) but exhibited no tetracycline-Me
2+
/H
+
antiport in
Escherichia coli
vesicles.
Characterization of thed-Xylulose 5-Phosphate/d-Fructose 6-Phosphate Phosphoketolase Gene (xfp) fromBifidobacterium lactis
Authors: Meile Leo , Rohr Lukas M. , Geissmann Thomas A. , Herensperger Monique , Teuber Michael
Published: July 2002

Ad-xylulose 5-phosphate/d-fructose 6-phosphate phosphoketolase (Xfp) from the probioticBifidobacterium lactiswas purified to homogeneity. The specific activity of the purified enzyme withd-fructose 6-phosphate as a substrate is 4.28 Units per mg of enzyme.Kmvalues ford-xylulose 5-phosphate andd-fructose 6-phosphate are 45 and 10 mM, respectively. The native enzyme has a molecular mass of 550,000 Da. The subunit size upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (90,000 Da) corresponds with the size (92,529 Da) calculated from the amino acid sequence of the isolated gene (namedxfp) encoding 825 amino acids. Thexfpgene was identified on the chromosome ofB. lactiswith the help of degenerated nucleotide probes deduced from the common N-terminal amino acid sequence of both the native and denatured enzyme. Comparison of the deduced amino acid sequence of the cloned gene with sequences in public databases revealed high homologies with hypothetical proteins (26 to 55% identity) in 20 microbial genomes. The amino acid sequence derived from thexfpgene contains typical thiamine diphosphate (ThDP) binding sites reported for other ThDP-dependent enzymes. Two truncated putative genes,ptaandguaA, were localized adjacent toxfpon theB. lactischromosome coding for a phosphotransacetylase and a guanosine monophosphate synthetase homologous to products of genes inMycobacterium tuberculosis. However,xfpis transcribed inB. lactisas a monocistronic operon. It is the first reported and sequenced gene of a phosphoketolase.
Sequencing of Flagellin Genes from
Natrialba magadii
Provides New Insight into Evolutionary Aspects of Archaeal Flagellins
Authors: Serganova Inna , Ksenzenko Vladimir , Serganov Alexander , Meshcheryakova Irina , Pyatibratov Michael , Vakhrusheva Olesya , Metlina Antonina , Fedorov Oleg
Published: July 2002

We have determined the nucleotide sequence of a flagellin gene locus from the haloalkaliphilic archaeon
Natrialba magadii
, identified the gene products among proteins forming flagella, and demonstrated cotranscription of the genes. Based on the sequence analysis we suggest that different regions of the genes might have distinct evolutionary histories including possible genetic exchange with bacterial flagellin genes.
Regulation of themetC-cysKOperon, Involved in Sulfur Metabolism inLactococcus lactis
Authors: Fernández María , Kleerebezem Michiel , Kuipers Oscar P. , Siezen Roland J. , van Kranenburg Richard
Published: July 2002

Sulfur metabolism in gram-positive bacteria is poorly characterized. Information on the molecular mechanisms of regulation of genes involved in sulfur metabolism is limited, and no regulator genes have been identified. Here we describe the regulation of the lactococcalmetC-cysKoperon, encoding a cystathionine β-lyase (metC) and cysteine synthase (cysK). Its expression was shown to be negatively affected by high concentrations of cysteine, methionine, and glutathione in the culture medium, while sulfur limitation resulted in a high level of expression. Other sulfur sources tested showed no significant effect onmetC-cysKgene expression. In addition we found thatO-acetyl-l-serine, the substrate of cysteine synthase, was an inducer of themetC-cysKoperon. Using a random mutagenesis approach, we identified two genes,cmbRandcmbT, involved in regulation ofmetC-cysKexpression. ThecmbTgene is predicted to encode a transport protein, but its precise role in regulation remains unclear. Disruption ofcmbTresulted in a two- to threefold reduction ofmetC-cysKtranscription. A 5.7-kb region containing thecmbRgene was cloned and sequenced. The encoded CmbR protein is homologous to the LysR family of regulator proteins and is an activator of themetC-cysKoperon. In analogy to CysB fromEscherichia coli, we propose that CmbR requires acetylserine to be able to bind the activation sites and subsequently activate transcription of themetC-cysKoperon.
Differential Regulation of Twitching Motility and Elastase Production by Vfr in
Pseudomonas aeruginosa
Authors: Beatson Scott A. , Whitchurch Cynthia B. , Sargent Jennifer L. , Levesque Roger C. , Mattick John S.
Published: July 2002

Vfr, a homolog of
Escherichia coli
cyclic AMP (cAMP) receptor protein, has been shown to regulate quorum sensing, exotoxin A production, and
regA
transcription in
Pseudomonas aeruginosa
. We identified a twitching motility-defective mutant that carries a transposon insertion in
vfr
and confirmed that
vfr
is required for twitching motility by construction of an independent allelic deletion-replacement mutant of
vfr
that exhibited the same phenotype, as well as by the restoration of normal twitching motility by complementation of these mutants with wild-type
vfr
. Vfr-null mutants exhibited severely reduced twitching motility with barely detectable levels of type IV pili, as well as loss of elastase production and altered pyocyanin production. We also identified reduced-twitching variants of quorum-sensing mutants (PAK
lasI
::Tc) with a spontaneous deletion in
vfr
(S. A. Beatson, C. B. Whitchurch, A. B. T. Semmler, and J. S. Mattick, J. Bacteriol., 184:3598-3604, 2002), the net result of which was the loss of five residues (EQERS) from the putative cAMP-binding pocket of Vfr. This allele (VfrΔEQERS) was capable of restoring elastase and pyocyanin production to wild-type levels in
vfr
-null mutants but not their defects in twitching motility. Furthermore, structural analysis of Vfr and VfrΔEQERS in relation to
E. coli
CRP suggests that Vfr is capable of binding both cAMP and cyclic GMP whereas VfrΔEQERS is only capable of responding to cAMP. We suggest that Vfr controls twitching motility and quorum sensing via independent pathways in response to these different signals, bound by the same cyclic nucleotide monophosphate-binding pocket.
Penicillin-Binding Proteins 1a and 1b Form Independent Dimers in
Escherichia coli
Authors: Charpentier Xavier , Chalut Christian , Rémy Marie-Hélène , Masson Jean-Michel
Published: July 2002

We report here that PBP1a can dimerize but does not interact with PBP1b to form PBP1a/PBP1b heterodimers in
Escherichia coli
. These findings support the idea of a relevant involvement of dimerization of both PBP1a and PBP1b during murein synthesis and suggest the existence of different peptidoglycan synthesis complexes.
Genome Sequence and Analysis of the Oral Bacterium
Fusobacterium nucleatum
Strain ATCC 25586
Authors: Kapatral Vinayak , Anderson Iain , Ivanova Natalia , Reznik Gary , Los Tamara , Lykidis Athanasios , Bhattacharyya Anamitra , Bartman Allen , Gardner Warren , Grechkin Galina , Zhu Lihua , Vasieva Olga , Chu Lien , Kogan Yakov , Chaga Oleg , Goltsman Eugene , Bernal Axel , Larsen Niels , D'Souza Mark , Walunas Theresa , Pusch Gordon , Haselkorn Robert , Fonstein Michael , Kyrpides Nikos , Overbeek Ross
Published: July 2002

We present a complete DNA sequence and metabolic analysis of the dominant oral bacterium
Fusobacterium nucleatum
. Although not considered a major dental pathogen on its own, this anaerobe facilitates the aggregation and establishment of several other species including the dental pathogens
Porphyromonas gingivalis
and
Bacteroides forsythus
. The
F. nucleatum
strain ATCC 25586 genome was assembled from shotgun sequences and analyzed using the ERGO bioinformatics suite (
http://www.integratedgenomics.com
). The genome contains 2.17 Mb encoding 2,067 open reading frames, organized on a single circular chromosome with 27% GC content. Despite its taxonomic position among the gram-negative bacteria, several features of its core metabolism are similar to that of gram-positive
Clostridium
spp.,
Enterococcus
spp., and
Lactococcus
spp. The genome analysis has revealed several key aspects of the pathways of organic acid, amino acid, carbohydrate, and lipid metabolism. Nine very-high-molecular-weight outer membrane proteins are predicted from the sequence, none of which has been reported in the literature. More than 137 transporters for the uptake of a variety of substrates such as peptides, sugars, metal ions, and cofactors have been identified. Biosynthetic pathways exist for only three amino acids: glutamate, aspartate, and asparagine. The remaining amino acids are imported as such or as di- or oligopeptides that are subsequently degraded in the cytoplasm. A principal source of energy appears to be the fermentation of glutamate to butyrate. Additionally, desulfuration of cysteine and methionine yields ammonia, H
2
S, methyl mercaptan, and butyrate, which are capable of arresting fibroblast growth, thus preventing wound healing and aiding penetration of the gingival epithelium. The metabolic capabilities of
F. nucleatum
revealed by its genome are therefore consistent with its specialized niche in the mouth.
Properties of the Glucose-6-Phosphate Transporter from
Chlamydia pneumoniae
(HPTcp) and the Glucose-6-Phosphate Sensor from
Escherichia coli
(UhpC)
Authors: Schwöppe Christian , Winkler Herbert H. , Neuhaus H. Ekkehard
Published: July 2002

The amino acid sequence of the proposed glucose-6-phosphate (Glc6P) transporter from
Chlamydia pneumoniae
(HPTcp; hexose phosphate transporter [
Chlamydia pneumoniae
]) exhibits a higher degree of similarity to the
Escherichia coli
Glc6P sensor (UhpC) than to the
E. coli
Glc6P transporter (UhpT). Overexpression of His-UhpC in a UhpT-deficient
E. coli
strain revealed that the sensor protein is also able to transport Glc6P and exhibits an apparent

K
m

 
(Glc6P)
of 25 μM, whereas His-HPTcp exhibits an apparent

K
m

 (Glc6P)
of 98 μM. His-HPTcp showed a four-times-lower specific activity than His-UhpT but a 56-times-higher specific activity than His-UhpC. Like His-UhpT and His-UhpC, the carrier His-HPTcp performs a sugar-phosphate/inorganic-phosphate antiporter mode of transport. Surprisingly, while physiological concentrations of inorganic phosphate competitively inhibited transport mediated by the
E. coli
proteins His-UhpT and His-UhpC, transport mediated by His-HPTcp was not inhibited. Interestingly, C
3
-organophosphates stimulated His-HPTcp activity but not His-UhpT- or His-UhpC-catalyzed Glc6P transport. In contrast to His-UhpC, the His-HPTcp protein does not act as a Glc6P sensor in the
uhp
regulon.
Characterization of the Interaction of
Bacillus subtilis
PyrR with
pyr
mRNA by Site-Directed Mutagenesis of the Protein
Authors: Savacool Heather K. , Switzer Robert L.
Published: July 2002

The
Bacillus subtilis
PyrR protein regulates transcriptional attenuation of the pyrimidine nucleotide (
pyr
) operon by binding in a uridine nucleotide-dependent manner to specific sites on
pyr
mRNA and stabilizing a secondary structure of the downstream RNA that favors termination of transcription. The high-resolution structure of unliganded PyrR was used to guide site-directed mutagenesis of 12 amino acid residues that were thought likely to be involved in the binding of RNA. Missense mutations were constructed and evaluated for their effects on regulation of
pyr
genes in vivo and their uracil phosphoribosyltransferase activity, which is catalyzed by wild-type PyrR. A substantial fraction of the mutant PyrR proteins did not have native structures, but eight PyrR mutants were purified and characterized physically, for their uracil phosphoribosyltransferase activity and for their ability to bind
pyr
RNA in vitro. On the basis of these studies Thr-18, His-22, Arg-141, and Arg-146 were implicated in RNA binding. Arg-27 and Lys-152 were also likely to be involved in RNA binding, but Gln substitution mutations in these residues may have altered their subunit-subunit interactions slightly. Arg-19 was implicated in
pyr
regulation, but a specific role in RNA binding could not be demonstrated because the R19Q mutant protein could not be purified in native form. The results confirm a role in RNA binding of a positively charged face of PyrR previously identified from the crystallographic structure. The RNA binding residues lie in two sequence segments that are conserved in PyrR proteins from many species.
Comparison of the Vitek Gram-Positive Susceptibility 106 Card and the MRSA-Screen Latex Agglutination Test for Determining Oxacillin Resistance in Clinical Bloodstream Isolates of
Staphylococcus aureus
Authors: Yamazumi T. , Marshall S. A. , Wilke W. W. , Diekema D. J. , Pfaller M. A. , Jones Ronald N.
Published: July 2002

The Vitek automated susceptibility testing system with a modified Gram-Positive Susceptibility (GPS) 106 Card (bioMerieux Vitek, Inc., Hazelwood, Mo.) and a rapid slide latex agglutination test (MRSA-Screen; Denka Seiken Co., Ltd., Tokyo, Japan) were evaluated for their ability to detect oxacillin resistance in
Staphylococcus aureus
. The oxacillin-salt agar screen (OS) test, the reference broth microdilution method, and the detection of the
mecA
gene by PCR were compared with the commercial products. A total of 200 contemporary (1999) bloodstream infection isolates were collected from the SENTRY Antimicrobial Surveillance Program, representing diverse geographic areas throughout the world. Among the 99
mecA
-positive isolates, 3 isolates were found negative by the MRSA-Screen. Another two isolates did not grow on OS plates and had MICs of 0.5 and 2 μg/ml with the Vitek GPS card. All 101
mecA
-negative isolates were also found negative by the MRSA-Screen and were categorized as susceptible by the GPS card. Overall, the MRSA-Screen, GPS card, and OS test had sensitivities of 96.9, 98.0, and 98.0% and specificities of 100.0, 100.0, and 98.0%, respectively. MRSA-Screen was a rapid (≤15 min) and simple test to perform, and the GPS card provided results in <8 h. Both methods were sensitive and specific for detecting staphylococcal oxacillin resistance in the clinical microbiology laboratory.
Quantitative Detection of Hepatitis B Virus DNA by Real-Time Nucleic Acid Sequence-Based Amplification with Molecular Beacon Detection
Authors: Yates Sol , Penning Maarten , Goudsmit Jaap , Frantzen Inge , van de Weijer Bert , van Strijp Dianne , van Gemen Bob
Published: July 2002

We have developed a hepatitis B virus (HBV) DNA detection and quantification system based on amplification with nucleic acid sequence-based amplification (NASBA) technology and real-time detection with molecular beacon technology. NASBA is normally applied to amplify single-stranded target RNA, producing RNA amplicons. In this work we show that with modifications like primer design, sample extraction method, and template denaturation, the NASBA technique can be made suitable for DNA target amplification resulting in RNA amplicons. A major advantage of our assay is the one-tube, isothermal nature of the method, which allows high-throughput applications for nucleic acid detection. The homogeneous real-time detection allows a closed-tube format of the assay, avoiding any postamplification handling of amplified material and therefore minimizing the risk of contamination of subsequent reactions. The assay has a detection range of 10
3
to 10
9
HBV DNA copies/ml of plasma or serum (6 logs), with good reproducibility and precision. Compared with other HBV DNA assays, our assay provides good sensitivity, a wide dynamic range, and high-throughput applicability, making it a viable alternative to those based on other amplification or detection methods.
Alternative Identification Test Relying upon Sexual Reproductive Abilities of
Candida
lusitaniae
Strains Isolated from Hospitalized Patients
Authors: François Fabienne , Noël Thierry , Pépin Régis , Brulfert Annie , Chastin Christiane , Favel Anne , Villard Jean
Published: July 2002

The in vitro mating ability of
Candida
lusitaniae
(teleomorph
Clavispora
lusitaniae
) clinical isolates has been investigated. Studying the effects of culture conditions, we showed that ammonium ion depletion in the medium is a major trigger of the sexual cycle. Moreover, a solid support is required for mating, suggesting a role for adhesion factors in addition to the mating type gene recognition function. Monitoring of mating and meiosis efficiency with auxotrophic strains showed great variations in ascospore yields, which appeared to be strain and temperature dependent, with an optimal range of 18 to 28°C. The morphogenetic events taking place from mating to ascospore release were studied by scanning and electron microscopy, and the ultrastructure of the conjugation canal, through which intercellular nuclear exchanges occur, was revealed. Labeling experiments with a lectin-fluorochrome system revealed that the nuclear transfer was predominantly polarized, thus allowing a distinction between the nucleus donor and the nucleus acceptor strains. The direction of the transfer depended on the strain combination used, rather than on the genotypes of the strains, and did not appear to be controlled by the mating type genes. Finally, we demonstrated that all of the 76 clinical isolates used in this study were able to reproduce sexually when mated with an opposite mating type strain, and we identified a 1:1
MAT
a/
MAT
α ratio in the collection. These results support the idea that there is no anamorph state in
C. lusitaniae
. Accordingly, the mating type test, which is easy to use and can usually be completed within 48 h, is a reliable alternative identification system for
C. lusitaniae
.
Molecular Evidence of Male-to-Female Sexual Transmission of Hepatitis C Virus after Vaginal and Anal Intercourse
Authors: Halfon Philippe , Riflet Hervé , Renou Christophe , Quentin Yves , Cacoub Patrice
Published: July 2002

Hepatitis C virus (HCV) was transmitted from a chronic carrier to his female partner during unprotected anal and vaginal intercourse. Based on HVR1 and phylogenetic tree analysis, the couple had closely related isolates. These findings confirm sexual transmission of HCV without other risk factors.
Evaluation of Etest for Direct Antifungal Susceptibility Testing of Yeasts in Positive Blood Cultures
Authors: Chang Hsein Chang , Chang Jui Jung , Chan Shih Huang , Huang Ay Huey , Wu Tsu Lan , Lin Miao Chu , Chang Tsung Chain
Published: July 2002

The performance of the Etest (AB BIODISK, Solna, Sweden) for direct antifungal susceptibility testing of yeasts in positive blood cultures was compared with that of the macrodilution method for determining the MICs of five antifungal agents. Culture broths with blood from bottles positive for yeasts were inoculated directly onto plates for susceptibility testing with the Etest, and the MICs were read after 24 and 48 h of incubation. A total of 141 positive blood cultures (72 cultures of
Candida albicans
, 31 of
Candida tropicalis
, 14 of
Candida glabrata
, 11 of
Candida parapsilosis
, 3 of
Candida krusei
, and 3 of
Cryptococcus neoformans
, 4 miscellaneous yeast species, and 3 mixed cultures) were tested, and the rates of MIC agreement (±1 log
2
dilution) between the direct Etest (at 24 and 48 h, respectively) and macrodilution methods were as follows: amphotericin B, 81.8 and 93.5%; flucytosine, 84.8 and 87.7%; fluconazole, 89.4 and 85.5%; itraconazole, 69.7 and 63.8%; ketoconazole, 87.9 and 79.0%. By a large-sample
t
test, the difference in log
2
dilution between the direct Etest and the macrodilution method was found to be small (
P
< 0.05). The lone exceptions were ketoconazole at 48 h of incubation and itraconazole at both 24 and 48 h of incubation (
P
> 0.05). By Tukey's multiple comparisons, the difference between the direct Etest (48 h) and reference methods among different species was found to be less than 1 log
2
dilution. When the MICs were translated into interpretive susceptibility, the minor errors caused by the direct Etest (at 24 and 48 h, respectively) were as follows: flucytosine, 2.3 and 1.4%; fluconazole, 3.0 and 3.6%; itraconazole, 21.2 and 21.3%. Itraconazole also produced an additional 3.0 and 3.6% major errors as determined by the direct Etest at 24 and 48 h, respectively. It was concluded that, except for itraconazole, the Etest method was feasible for direct susceptibility testing of blood cultures positive for yeasts. The method is simple, and the results could be read between 24 and 48 h after direct inoculation, whenever the inhibition zones were discernible.
Dynamics of a Nosocomial Outbreak of Multidrug-Resistant
Pseudomonas aeruginosa
Producing the PER-1 Extended-Spectrum β-Lactamase
Authors: Luzzaro Francesco , Mantengoli Elisabetta , Perilli Mariagrazia , Lombardi Gianluigi , Orlandi Viviana , Orsatti Alessandra , Amicosante Gianfranco , Rossolini Gian Maria , Toniolo Antonio
Published: July 2002

From November 1998 to August 1999, a large outbreak occurred in the general intensive care unit of the Ospedale di Circolo in Varese (Italy), caused by
Pseudomonas aeruginosa
producing the PER-1 extended-spectrum β-lactamase. A total of 108 clinical isolates of
P. aeruginosa
resistant to broad-spectrum cephalosporins were recovered from 18 patients. Epidemic isolates were characterized by synergy between clavulanic acid and ceftazidime, cefepime, and aztreonam. Isoelectric focusing of crude bacterial extracts detected two nitrocefin-positive bands with pI values of 8.0 and 5.3. PCR amplification and characterization of the amplicons by restriction analysis and direct sequencing indicated that the epidemic isolates carried a
bla
PER-1
determinant. The outbreak was of clonal origin as shown by pulsed-field gel electrophoresis analysis. This technique also indicated that the epidemic strain was not related to three other PER-1-positive isolates obtained at the same hospital in 1997. Typing by enterobacterial repetitive intergenic consensus-PCR showed that minor genetic variations occurred during the outbreak. The epidemic strain was characterized by a multiple-drug-resistance phenotype that remained unchanged over the outbreak, including extended-spectrum cephalosporins, monobactams, aminoglycosides, and fluoroquinolones. Isolation of infected patients and appropriate carbapenem therapy were successful in ending the outbreak. Our report indicates that the
bla
PER-1
resistance determinant may become an emerging therapeutic problem in Europe.
Identification ofEhrlichiaspp. andBorrelia burgdorferiinIxodesTicks in the Baltic Regions of Russia
Authors: Alekseev Andrey N. , Dubinina Helen V. , Van De Pol Ingrid , Schouls Leo M.
Published: July 2002

The presence and distribution ofEhrlichiaspp. andBorrelia burgdorferisensu lato was demonstrated among ixodid ticks collected in the Baltic regions of Russia, where Lyme borreliosis is endemic. A total of 3,426Ixodes ricinusand 1,267Ixodes persulcatusspecimens were collected, and dark-field microscopy showed that 265 (11.5%)I. ricinusand 333 (26.3%)I. persulcatusticks were positive. From these samples, 472 dark-field-positive and 159 dark-field-negative ticks were subjected to PCR and subsequent reverse line blot hybridization. Fifty-four ticks (8.6%) carriedEhrlichiaspecies, and 4 (0.6%) carried ehrlichiae belonging to theEhrlichia phagocytophilacomplex, which includes the human granulocytic ehrlichiosis agent. TheE. phagocytophilacomplex and anEhrlichia-like species were detected only inI. ricinuswhereasEhrlichia muriswas found exclusively inI. persulcatus, indicating a possible vector-specific infection.Borrelia gariniiwas found predominantly inI. persulcatus, butBorrelia afzeliiwas evenly distributed among the two tick species. Only twoI. ricinusticks carriedB. burgdorferisensu stricto, whileBorrelia valaisianaand a newly identifiedB. afzelii-like species were found in 1.7 and 2.5% of all ticks, respectively. Of the dark-field-positive ticks, only 64.8% yielded aBorreliaPCR product, indicating that dark-field microscopy may detect organisms other thanB. burgdorferisensu lato. These observations show that the agent of human granulocytic ehrlichiosis may be present in ticks in the Baltic regions of Russia and that clinicians should be aware of this agent as a cause of febrile disease.
Contamination of Catheter-Drawn Blood Cultures
Authors: Everts Richard J. , Vinson Emily N. , Adholla Paul O. , Reller L. Barth
Published: July 2002

To assess the risk of contamination, we reviewed retrospectively 1,408 matched pairs of simultaneous catheter-drawn and venipuncture blood cultures. Catheter-drawn cultures were equally likely to be truly positive (14.4 versus 13.7%) but more likely to be contaminated (3.8 versus 1.8% [
P
= 0.001]). Direct venipuncture cultures are preferred.
Species-SpecificSerodiagnosis of Lyme Arthritis and Neuroborreliosis Due to
Borreliaburgdorferi
Sensu Stricto,
B. afzelii
, and
B.garinii
by Using Decorin Binding ProteinA
Authors: Heikkilä Tero , Seppälä Ilkka , Saxen Harri , Panelius Jaana , Yrjänäinen Heta , Lahdenne Pekka
Published: July 2002

The antigenic potential of decorin binding protein A
(DbpA) was evaluated in serodiagnosis of human Lyme borreliosis (LB). The
dbpA
was cloned and sequenced from the three pathogenic
Borrelia
species common in Europe. Sequence analysis revealed high
interspecies heterogeneity. The identity of the predicted amino acid
sequences was 43 to 62% among
Borrelia burgdorferi
sensu stricto,
B. afzelii
, and
B. garinii
. The respective recombinant
DbpAs (rDbpAs) were produced and tested as antigens by Western blotting and
enzyme-linked immunosorbent assay (ELISA). One hundred percent of patients
with neuroborreliosis (NB) and 93% of patients with Lyme arthritis (LA)
reacted positively. Sera from the majority of patients reacted with one
rDbpA only and had no or low cross-reactivity to other two variant
proteins. In patients with culture-positive erythema migrans (EM), the
sensitivity of rDbpA immunoglobulin G (IgG) or IgM ELISA was low. The DbpA
seems to be a sensitive and specific antigen for the serodiagnosis of LA or
NB, but not of EM, provided that variants from all three pathogenic
borrelial species are included in the combined set of
antigens.
Rapid Identification of
Staphylococcus aureus
Directly from Blood Cultures by Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes
Authors: Oliveira Kenneth , Procop Gary W. , Wilson Deborah , Coull James , Stender Henrik
Published: July 2002

A new fluorescence in situ hybridization (FISH) method with peptide nucleic acid (PNA) probes for identification of
Staphylococcus aureus
directly from positive blood culture bottles that contain gram-positive cocci in clusters (GPCC) is described. The test (the
S. aureus
PNA FISH assay) is based on a fluorescein-labeled PNA probe that targets a species-specific sequence of the 16S rRNA of
S. aureus.
Evaluations with 17 reference strains and 48 clinical isolates, including methicillin-resistant and methicillin-susceptible
S. aureus
species, coagulase-negative
Staphylococcus
species, and other clinically relevant and phylogenetically related bacteria and yeast species, showed that the assay had 100% sensitivity and 96% specificity. Clinical trials with 87 blood cultures positive for GPCC correctly identified 36 of 37 (97%) of the
S. aureus
-positive cultures identified by standard microbiological methods. The positive and negative predictive values were 100 and 98%, respectively. It is concluded that this rapid method (2.5 h) for identification of
S. aureus
directly from blood culture bottles that contain GPCC offers important information for optimal antibiotic therapy.
Direct Detection and Characterization of Shiga Toxigenic
Escherichia coli
by Multiplex PCR for
stx
1
,
stx
2
,
eae
,
ehxA
, and
saa
Authors: Paton Adrienne W. , Paton James C.
Published: July 2002

We recently described a novel megaplasmid-encoded adhesin produced by certain Shiga toxigenic
Escherichia coli
(STEC) strains that lack the locus for enterocyte effacement (LEE) pathogenicity island. This adhesin, designated Saa (STEC autoagglutinating adhesin), may be a marker for a subset of LEE-negative STEC strains capable of causing severe gastrointestinal and systemic diseases in humans. In this study, we developed a pentavalent PCR assay for the detection of
saa
as well as other proven and putative STEC virulence genes (
stx
1
,
stx
2
,
eae
, and
ehxA
). The five primer pairs used in the assay do not interfere with each other and generate amplification products of 119, 180, 255, 384, and 534 bp.
Emergence of Penicillin-Nonsusceptible
Streptococcus pneumoniae
Invasive Clones in Canada
Authors: Greenberg D. , Speert D. P. , Mahenthiralingam E. , Henry D. A. , Campbell M. E. , Scheifele D. W. ,   the CPS/LCDC IMPACT Monitoring Network
Published: July 2002

Distinctive international clones of penicillin-nonsusceptible and multidrug-resistant
Streptococcus pneumoniae
are increasingly being reported. We investigated the spread of these clones in Canada through an active surveillance that was carried out at 11 Canadian pediatric tertiary care centers from 1991 to 1998. All penicillin-nonsusceptible isolates were serotyped, tested for antibiotic susceptibility, and genotyped by pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD). Forty-five penicillin-nonsusceptible
S. pneumoniae
isolates were evaluated. Eleven serotype 9V isolates and six serotype 14 isolates displayed identical RAPD and PFGE fingerprint profiles. Twelve (70%) of these isolates were encountered in Quebec. The 9V/14 clone and the Spanish-French clone had similar PFGE fingerprint patterns. Eight isolates of serotype 23F and two isolates of serogroup 14 had the same fingerprint profiles and displayed resistance to three or more antibiotic drug classes. This clone was first detected in Calgary (Alberta) and in 1996 appeared simultaneously in various regions of Canada. This clone showed a PFGE fingerprint pattern similar to that of the Spanish-U.S. 23F clone. Our data show the emergence across Canada of two international clones of penicillin-nonsusceptible
S. pneumoniae
: (i) serotypes 9V and 14 related to the Spanish-French clone and (ii) the 23F Spanish-U.S. clone. The source of the first clone was in Quebec and the second international clone was probably originated from the United States. The exact reasons for the successful spread of these clones within Canada and their contribution to increased resistance to antibiotics have yet to be explored.
Sensitive PCR-Restriction Fragment Length Polymorphism Assay for Detection and Genotyping ofGiardia duodenalisin Human Feces
Authors: Amar C. F. L. , Dear P. H. , Pedraza-Díaz S. , Looker N. , Linnane E. , McLauchlin J.
Published: July 2002

An assay that uses heminested PCR-restriction fragment length polymorphism analysis for the detection and genotyping ofGiardia duodenalison the basis of polymorphism in the triose phosphate isomerase (tpi) gene was developed. This assay was evaluated with DNA extracted from purified parasite material, bacterial cultures, whole human feces containingG. duodenalisand other parasites, and their corresponding immunofluorescence-stained fecal smears on glass microscope slides. The assay was specific and discriminated betweenG. duodenalisassemblages A and B. RFLP analysis further distinguished two groups (designated groups I and II) within assemblage A. Among 35 DNA samples extracted from whole feces from patients with confirmed sporadic giardiasis, thetpigene was amplified from 33 (94%). Of these, nine (27%) samples contained assemblage A group II, 21 (64%) contained assemblage B, and 3 (9%) contained a mixture of assemblage A group II and assemblage B. Thetpigene ofG. duodenalisassemblage B was amplified from 21 of 24 (88%) DNA samples extracted from whole feces from patients with confirmed cases of infection in a nursery outbreak. No amplification was detected from the remaining three DNA samples. Overall, analysis of DNA extracted from material recovered from stained microscope slides identified identicalG. duodenalisgenotypes in 35 (65%) of the 54 samples for which a genotype was established with DNA from whole feces. The heminested PCR method developed is sensitive, simple, and rapid to perform and is applicable for the analysis of other intestinal pathogens.
Evaluation of Dry and Wet Transported Intravaginal Swabs in Detection of
Chlamydia trachomatis
and
Neisseria gonorrhoeae
Infections in Female Soldiers by PCR
Authors: Gaydos Charlotte A. , Crotchfelt Kimberly A. , Shah Nina , Tennant Marie , Quinn Thomas C. , Gaydos Joel C. , McKee Kelly T. , Rompalo Anne M.
Published: July 2002

Screening women for sexually transmitted diseases (STD) in nonclinic settings is highly desirable because many infections are asymptomatic. This is especially true for military women, for whom logistical, social, and other job-related obstacles present barriers to accessing medical care. We assessed the accuracy of intravaginal swabs transported by mail in a wet versus a dry state for PCR (Amplicor CT/NG test) detection of chlamydia and gonorrhea infections in a cross-sectional study of 793 active-duty military women attending an STD clinic. PCR tests of vaginal swabs (wet and dry) were compared to local clinical methods used on cervical swabs. Standard wet vaginal swab PCR testing detected more chlamydia (11.6%) than cervical enzyme immunoassay (9.3%). For detection of chlamydia using wet swabs, the sensitivity and specificity compared with adjudicated true positives were 94.6% (87 of 92) and 99.3% (696 of 701), respectively. Comparing dry swabs to true-positives for chlamydia, the sensitivity was 91.3% (84 of 92) and the specificity was 99.3% (696 of 701). Standard wet vaginal swab PCR detected more gonorrhea (3.3%) than routine cervical culture (2.1%). The sensitivity and specificity of PCR testing of wet swabs compared to true-positives (infected patients) were 96.3% (26 of 27) and 98.2% (752 of 766) for gonorrhea, respectively. For gonorrhea, the sensitivity and specificity of dry swabs compared to true-positives (infected patients) were 88.9% (24 of 27) and 98.3% (753 of 766), respectively. PCR testing of wet and dry transported intravaginal swabs to detect chlamydia and gonorrhea infections was an accurate diagnostic method for military women.
Enterococcus faecium
-Related Outbreak with Molecular Evidence of Transmission from Pigs to Humans
Authors: Lu Hong-Zhou , Weng Xin-Hua , Li Haijing , Yin You-Kuan , Pang Mao-Yin , Tang Yi-Wei
Published: July 2002

Between 24 July and 31 August 1998, thousands of domestic pigs died of hemorrhagic shock in three adjunct counties along the YangZi River in Jiangshu Province, China. From 28 July to 6 September 1998, 40 local farmers (36 males and 4 females, ages 23 to 78 years) were hospitalized with severe illness characterized by high fever, erythematous rash or petechiae, and profound lethargy after contact with sick pigs. Twelve (30%) of these patients died of respiratory failure and shock. Eleven bacterial isolates recovered from 11 blood and cerebrospinal fluid specimens collected from seven patients and two pigs were identified as
Enterococcus faecium
based on biochemical reactions and 16S rRNA gene sequence analysis. Both pig and human
E. faecium
isolates displayed indistinguishable antibiotic susceptibility and pulsed-field gel electrophoresis patterns. These data strongly suggest the spread of an outbreak of
E. faecium
-related sepsis from pigs to humans.
Transmission Studies of
Babesia microti
in
Ixodes ricinus
Ticks and Gerbils
Authors: Gray Jeremy , von Stedingk Lars Victor , Gürtelschmid Mikael , Granström Marta
Published: July 2002

In order to investigate the possible role of
Ixodes ricinus
as a vector of zoonotic
Babesia microti
infection in Europe, a European rodent isolate (HK) and a zoonotic American isolate (GI) were studied in transmission experiments. PCR detected
B. microti
in the blood and spleens of infected gerbils (
Meriones unguiculatus
) and also in laboratory-induced infections of
I. ricinus
ticks.
B. microti
DNA was detected by PCR in all pooled samples of nymphs and the majority of adults that had fed as larvae and nymphs, respectively, on gerbils with acute infection of the European isolate, confirming that
I. ricinus
could serve as a vector in Europe. The American isolate, GI, proved to be equally infective for larval and nymphal
I. ricinus
as the HK strain, despite a very different appearance in gerbil erythrocytes. Nymphs infected with the HK and GI strains readily infected gerbils. In contrast to the finding in acute infections, ticks that fed on gerbils with chronic infections of HK and GI did not become infected. It was also found that the HK strain was not transmitted transovarially. The finding that a
B. microti
strain (GI) from a distant geographical region (United States) can infect and be transmitted by
I. ricinus
suggests that other European
B. microti
strains, in addition to the HK strain used here, are probably infective for
I. ricinus
, supporting the view that infection of humans with European
B. microti
may be a regular occurrence.
Usefulness of Multilocus Sequence Typing for Characterization of Clinical Isolates of
Candida albicans
Authors: Bougnoux M.-E. , Morand S. , d'Enfert C.
Published: July 2002

Molecular characterization of
Candida albicans
isolates is essential for understanding the epidemiology of nosocomial infections caused by this yeast. Here, we investigated the potential value of multilocus sequence typing (MLST) for characterizing epidemiologically related or unrelated
C. albicans
strains of various clinical origins. Accordingly, we sequenced the internal regions (loci) of six selected housekeeping genes of 40
C. albicans
clinical isolates and 2 reference strains. In all, 68 polymorphic nucleotide sites were identified, of which 65 were found to be heterozygous in at least one isolate. Ten to 24 different genotypes were observed at the different loci, resulting, when combined, in 39 unique genotype combinations or diploid sequence types (DSTs). When MLST was applied to 26 epidemiologically unrelated isolates and the 2 reference strains, it allowed the identification of 27 independent DSTs, thus demonstrating a discriminatory power of 99.7. Using multidimensional scaling together with the minimum spanning tree method to analyze interstrain relationships, we identified six groups of genetically related isolates on the basis of bootstrap values of greater than 900. Application of MLST to 14 epidemiologically related isolates showed that those recovered from patients in the same hospital ward during the same 3 months had specific DSTs, although 73% of these isolates were genetically very close. This suggests that MLST can trace minute variations in the sequences of related isolates. Overall, MLST proved to be a highly discriminatory and stable method for unambiguous characterization of
C. albicans
.
Reovirus Nonstructural Protein μNS Binds to Core Particles but Does Not Inhibit Their Transcription and Capping Activities
Authors: Broering Teresa J. , McCutcheon Aimee M. , Centonze Victoria E. , Nibert Max L.
Published: July 2002

Previous studies provided evidence that nonstructural protein μNS of mammalian reoviruses is present in particle assembly intermediates isolated from infected cells. Morgan and Zweerink (Virology 68:455–466, 1975) showed that a subset of these intermediates, which can synthesize the viral plus strand RNA transcripts in vitro, comprise core-like particles plus large amounts of μNS. Given the possible role of μNS in particle assembly and/or transcription implied by those findings, we tested whether recombinant μNS can bind to cores in vitro. The μNS protein bound to cores, but not to two particle forms, virions and intermediate subvirion particles, that contain additional outer-capsid proteins. Incubating cores with increasing amounts of μNS resulted in particle complexes of progressively decreasing buoyant density, approaching the density of protein alone when very large amounts of μNS were bound. Thus, the μNS-core interaction did not exhibit saturation or a defined stoichiometry. Negative-stain electron microscopy of the μNS-bound cores revealed that the cores were intact and linked together in large complexes by an amorphous density, which we ascribe to μNS. The μNS-core complexes retained the capacity to synthesize the viral plus strand transcripts as well as the capacity to add methylated caps to the 5′ ends of the transcripts. In vitro competition assays showed that mixing μNS with cores greatly reduced the formation of recoated cores by stoichiometric binding of outer-capsid proteins μ1 and ς3. These findings are consistent with the presence of μNS in transcriptase particles as described previously and suggest that, by binding to cores in the infected cell, μNS may block or delay outer-capsid assembly and allow continued transcription by these particles.
Differential Pathogenicity of Two Feline Leukemia Virus Subgroup A Molecular Clones, pFRA and pF6A
Authors: Phipps Andrew J. , Chen Hang , Hayes Kathleen A. , Roy-Burman Pradip , Mathes Lawrence E.
Published: July 2002

F6A, a molecular clone of subgroup A feline leukemia virus (FeLV) is considered to be highly infectious but weakly pathogenic. In recent studies with a closely related subgroup A molecular clone, FRA, we demonstrated high pathogenicity and a strong propensity to undergo recombination with endogenous FeLV (enFeLV), leading to a high frequency of transition from subgroup A to A/B. The present study was undertaken to identify mechanisms of FeLV pathogenesis that might become evident by comparing the two closely related molecular clones. F6A was shown to have an infectivity similar to that of FRA when delivered as a provirus. Virus load and antibody responses were also similar, although F6A-infected cats consistently carried higher virus loads than FRA-infected cats. However, F6A-infected cats were slower to undergo de novo recombination with enFeLV and showed slower progression to disease than FRA-infected cats. Tumors collected from nine pF6A- or pFRA-inoculated cats expressed lymphocyte markers for T cells (seven tumors) and B cells (one tumor), and non-T/B cells (one tumor). One cat with an A-to-A/C conversion developed erythrocyte hypoplasia. Genomic mapping of recombinants from pF6A- and pFRA-inoculated cats revealed similar crossover sites, suggesting that the genomic makeup of the recombinants did not contribute to increased progression to neoplastic disease. From these studies, the mechanism most likely to account for the pathologic differences between F6A and FRA is the lower propensity for F6A to undergo de novo recombination with enFeLV in vivo. A lower recombination rate is predicted to slow the transition from subgroup A to A/B and slow the progression to disease.