Evaluation of a new chromogenic agar medium for detection of Shiga toxin-producing Escherichia coli (STEC) and relative prevalences of O157 and non-O157 STEC in Manitoba, Canada.

This study assesses the detection performance of CHROMagar STEC medium relative to a reference cytotoxin assay and describes the current relative prevalence of O157 and non-O157 Shiga toxin-producing Escherichia coli (STEC) serotypes within the province of Manitoba, Canada. Over a 10-month period, 205 nonfrozen routine stool submissions to Cadham Provincial Laboratory (CPL) were used to assess the performance of CHROMagar STEC. Of the 205 stools, 14 were identified as true positives by a cytotoxin assay, with resultant CHROMagar STEC sensitivity, specificity, and positive predictive and negative predictive values of 85.7%, 95.8%, 60.0%, and 98.9%, respectively. Using a separate panel of 111 STEC strains, CHROMagar STEC was shown to support the growth of 96 (86.5%) isolates. To assess relative prevalence, attempts were made to isolate by any means all STEC strains identified at CPL over a 17-month period. Of 49 isolates (representing 86.0% of all STEC infections detected), only 28.6% were O157 STEC strains. Of the 35 non-O157 STEC strains, 29 were subjected to further molecular analysis. In contrast to earlier results from our area, carriage of stx(2) appears to have increased. Overall, although CHROMagar STEC is not recommended as a primary screen, our results indicate that it is an effective supplemental medium for the isolation of probable STEC strains. Increased isolation of these serotypes is warranted to better understand their prevalence, clinical characteristics, and epidemiology and aid in the development or enhancement of food safety control programs targeting all STEC serotypes.

Combining social network analysis and cluster analysis to identify sexual network types.

Increases in the rates of sexually transmitted infections (STIs) suggest that control programmes may not be effectively targeting diverse subpopulations. The objective of this investigation was to examine STI transmission within different groups, using both social network analysis and cluster analysis. Routine partner notification data were analysed from individuals diagnosed with, or exposed to an STI in Manitoba. Groups were identified and characterized. Three different clusters of groups were identified, comprised of demographically and clinically distinct individuals. A greater understanding of disease transmission patterns within these groups will aid in the development of targeted education and prevention programmes for all STIs.

Gonorrhoea and chlamydia core groups and sexual networks in Manitoba.

This paper summarises the results of the R0 equation in sexually transmitted infection (STI) repeaters in Manitoba, Canada, in the early 1990s, with both concurrent and more recent descriptions of sexual networks in the same population. The research presented provides empirical network and sex partner data to refine definitions of sexual networks and core groups in phase IV epidemics. New challenges for both practice and research are also discussed.

Sexual networks and sexually transmitted infections: a tale of two cities.

Research on risk behaviors for sexually transmitted infections (STIs) has revealed that they seldom correspond with actual risk of infection. Core groups of people with high-risk behavior who form networks of people linked by sexual contact are essential for STI transmission, but have been overlooked in epidemiological studies. Social network analysis, a subdiscipline of sociology, provides both the methods and analytical techniques to describe and illustrate the effects of sexual networks on STI transmission. Sexual networks of people from Colorado Springs, Colorado, and from Winnipeg, Manitoba, Canada, infected with chlamydia during a 6-month period were compared. In Winnipeg, 442 networks were identified, comprising 571 cases and 663 contacts, ranging in size from 2 to 20 individuals; Colorado Springs data yielded 401 networks, comprising 468 cases and 700 contacts, ranging in size from 2 to 12 individuals. Taking differing partner notification methods and the slightly smaller population size in Colorado Springs into account, the networks from both places were similar in both size and structure. These smaller, sparsely linked networks, peripheral to the core, may form the mechanism by which chlamydia can remain endemic, in contrast with larger, more densely connected networks, closer to the core, which are associated with steep rises in incidence.

Sampling individuals with large sexual networks: an evaluation of four approaches.

BACKGROUND: Methods for accessing large sexual networks are essential for investigating the mechanisms for the spread of sexually transmitted infections. GOAL: Four samples of cases were compared with the total population to determine which identified the largest networks. STUDY DESIGN: Individuals with positive test results for chlamydia during a 6-month period were selected from a laboratory database and linked with sex partner information from a notifiable disease registry. Sexual networks were constructed for a random sample, people with positive results from two or more tests for chlamydia, people with positive tests results for both gonorrhea and chlamydia, and the preceding two groups combined. RESULTS: The coinfected people combined with the repeaters yielded the highest proportion (47.8%) of large networks (>10 people), followed by the coinfected people, the repeaters, and finally the random sample. CONCLUSIONS: People coinfected with chlamydia and gonorrhea and those with repeated chlamydial infection present ideal opportunities for both research and prevention.

Patterns of chlamydia and gonorrhea infection in sexual networks in Manitoba, Canada.

BACKGROUND: The use of sexual network analysis has the potential to further our understanding of sexually tranmitted disease (STD) epidemics and contribute to the development of more effective targeted control strategies. GOAL: To use sexual network analysis to study transmission patterns of chlamydia and gonorrhea in Manitoba, Canada. STUDY DESIGN: Routinely collected case/contact information gathered by public health nurses was used to construct the sexual network. RESULTS: Components within the sexual network ranged in size from 2 to 82 people. Two types of components, designated radial and linear, were described. Large linear components resembled the theoretical structure of STD core groups. Geographic analysis of the largest components demonstrated the potential for STD transmission between isolated rural communities and within different areas of an urban center. CONCLUSIONS: The application of sexual network analysis on a provincial basis demonstrated the importance of a centralized, coordinated approach to STD control. The analysis highlights the need for a greater understanding of the causative factors promoting the formation of different component types, the homogeneity and heterogeneity of behaviors within and between components, and the temporal stability of these patterns.

Comparative evaluation of chlamydiazyme, PACE 2, and AMP-CT assays for detection of Chlamydia trachomatis in endocervical specimens.

We conducted a comparative evaluation of the Chlamydiazyme (Abbott Laboratories), PACE 2 (Gen-Probe), and AMP-CT (Gen-Probe) assays for the detection of Chlamydia trachomatis in endocervical samples. Specimens from 787 females were included in the study. The sensitivities of the PACE 2 and Chlamydiazyme assays in comparison to the results of the AMP-CT assay were 79.3 and 63.4%, respectively. The specificities of the Chlamydiazyme and PACE 2 assays were 100%. All of the positive specimens detected in this study were positive by the AMP-CT assay. On the basis of the final results of the comparison, the prevalence of C. trachomatis in the population was 10.4%. Retesting of specimens whose results were in the intermediate zone by the PACE 2 assay by a probe competition assay identified some additional true-positive specimens. Amplification assay testing of such specimens did not significantly increase the yield. The majority of specimens which tested positive by the AMP-CT assay only were not in the intermediate zone by the PACE 2 assay. We were unable to identify demographic or clinical factors which could predict those individuals who tested positive by amplified tests but not by nonamplified tests. The Gen-Probe PACE 2 assay proved to be superior to the Chlamydiazyme assay for the screening and diagnosis of C. trachomatis infections in female endocervical specimens.

Host cell phospholipids are trafficked to and then modified by Chlamydia trachomatis.

There is little information on the trafficking of eukaryotic lipids from a host cell to either the cytoplasmic membrane of or the vacuolar membrane surrounding intracellular pathogens. Purified Chlamydia trachomatis, an obligate intracellular bacterial parasite, contains several eukaryotic glycerophospholipids, yet attempts to demonstrate transfer of these lipids to the chlamydial cell membrane have not been successful. In this report, we demonstrate that eukaryotic glycerophospholipids are trafficked from the host cell to C. trachomatis. Phospholipid trafficking was assessed by monitoring the incorporation of radiolabelled isoleucine, a precursor of C. trachomatis specific branched-chain fatty acids, into host-derived glycerophospholipids and by monitoring the transfer of host phosphatidylserine to chlamydiae and its subsequent decarboxylation to form phosphatidylethanolamine. Phospholipid trafficking to chlamydiae was unaffected by brefeldin A, an inhibitor of Golgi function. Furthermore, no changes in trafficking were observed when C. trachomatis was grown in a mutant cell line with a nonfunctional, nonspecific phospholipid transfer protein. Host glycerophospholipids are modified by C. trachomatis, such that a host-synthesized straight-chain fatty acid is replaced with a chlamydia-synthesized branched-chain fatty acid. We also demonstrate that despite the acquisition of host-derived phospholipids, C. trachomatis is capable of de novo synthesis of phospholipids typically synthesized by prokaryotic cells. Our results provide novel information on chlamydial phospholipid metabolism and eukaryotic cell lipid trafficking, and they increase our understanding of the evolutionary steps leading to the establishment of an intimate metabolic association between an obligate intracellular bacterial parasite and a eukaryotic host cell.

Identification, characterization, and developmental regulation of Chlamydia trachomatis 3-deoxy-D-manno-octulosonate (KDO)-8-phosphate synthetase and CMP-KDO synthetase.

The kdsA and kdsB genes from Chlamydia trachomatis encoding 3-deoxy-D-manno-octulosonate (KDO)-8-phosphate synthetase and CMP-KDO synthetase were identified by functional complementation of temperature-sensitive Salmonella typhimurium mutants, homology to known KDO-8-phosphate synthetase and CMP-KDO synthetase proteins, and in vitro enzyme activity. The kdsA gene was transcribed as part of a polycistronic mRNA with two downstream open reading frames (ORFs). One of these ORFs appeared to encode a membrane-anchored protein, while the second encoded a protein showing homology to the ATP-binding component of periplasmic binding protein-dependent ABC transporters. Transcription of kdsA and kdsB in C. trachomatis was evident within 4 h of initiation of the C. trachomatis infection process and continued throughout the chlamydial life cycle.

Chlamydia trachomatis CTP synthetase: molecular characterization and developmental regulation of expression.

Chlamydia trachomatis is a nucleotide parasite, being entirely dependent on its host eukaryotic cell for a supply of ATP, GTP, and UTP. Chlamydiae are not, however, auxotrophic for CTP, as they are able both to transport CTP from the host and synthesize CTP de novo via a chlamydial CTP synthetase. This study addresses the developmental regulation of CTP synthetase over the course of the C. trachomatis life cycle. Given the distinct life stages of C. trachomatis, analysis of temporal changes in gene expression and regulation of protein activity is the key to unravelling the mechanism of pathogenesis of this bacterium. The results of immunodetection analysis indicate that CTP synthetase is present in C. trachomatis elementary bodies and reticulate bodies and that it is widespread in other chlamydial strains. Reverse transcriptase-polymerase chain reaction (RT-PCR) and metabolic labelling experiments show that CTP synthetase is transcribed and translated primarily during the mid- and late stages of the chlamydial growth cycle. In addition, C. trachomatis CTP synthetase was transcribed with the CTP utilizing enzyme CMP-2-keto-3-deoxy-octanoic acid synthetase (CMP-KDO synthetase) as part of a polycistronic mRNA. The co-expression of these two enzymes suggests a role for CTP synthetase in lipopolysaccharide biosynthesis, potentially channelling CTP directly to CMP-KDO synthetase. The ability of the intact operon to complement CTP synthetase and CMP-KDO deficiencies in mutant Escherichia coli strains indicates that both enzymes are efficiently translated from a single messenger RNA. Kinetic analysis revealed that the C. trachomatis CTP synthetase possessed co-operativity patterns typical of both prokaryotic and eukaryotic CTP synthetases. However, the K(m) of the enzyme for UTP was lower than that of E. coli CTP synthetase, presumably in response to the low intracellular concentration of this nucleotide in C. trachomatis.

A single point mutation in CTP synthetase of Chlamydia trachomatis confers resistance to cyclopentenyl cytosine.

A Chlamydia trachomatis strain (L2/CPEC) resistant to the cytotoxic effects of cyclopentenyl cytosine (CPEC) was isolated by a stepwise selection procedure. This strain showed an approximate 350-fold increase in resistance to CPEC. Sequencing of the gene encoding CTP synthetase from this resistant strain revealed a single point mutation, resulting in a change of amino acid 149 from Asp to Glu. This appeared to be the only mutation in L2/CPEC, because no changes in CTP transport, CTP synthetase expression, or incorporation of CPEC into DNA or RNA could be detected. The mutation in the chlamydial CTP synthetase resulted in a loss of CTP feedback inhibition. This was demonstrated both in vivo using Escherichia coli cells carrying the cloned gene, and an in vitro assay using partially purified preparations of CTP synthetase. As a result of the loss of feedback inhibition, E. coli cells carrying the CPECR CTP synthetase showed a 22-fold increase in their CTP pools. However, examination of the CTP pools of L2/CPEC revealed no change in CTP levels when compared with wild type C. trachomatis.

The OprB porin plays a central role in carbohydrate uptake in Pseudomonas aeruginosa.

Using interposon mutagenesis, we have generated strains of Pseudomonas aeruginosa which lack or overexpress the substrate-selective OprB porin of this species. A marked decrease or increase in the initial uptake of glucose by these strains verified the role of OprB in facilitating the diffusion of glucose across the outer membrane of P. aeruginosa. However, we also demonstrated that the loss or overexpression of OprB had a similar effect on the uptake of three other sugars able to support the growth of this bacterium (mannitol, glycerol, and fructose). This effect was restricted to carbohydrate transport; arginine uptake was identical in mutant and wild-type strains. These results indicated that OprB cannot be considered strictly a glucose-selective porin; rather, it acts as a central component of carbohydrate transport and is more accurately described as a carbohydrate-selective porin.

Cloning and nucleotide sequence of the Pseudomonas aeruginosa glucose-selective OprB porin gene and distribution of OprB within the family Pseudomonadaceae.

OprB is a glucose-selective porin known to be produced by Pseudomonas aeruginosa and Pseudomonas putida. We have cloned and sequenced the oprB gene of P. aeruginosa and obtained expression of OprB in Escherichia coli. The mature protein consists of 423 amino acid residues with a deduced molecular mass of 47597 Da. Several clusters of amino acid residues, potentially involved in the structure or function of the protein, were identified. An area of regional homology with E. coli LamB was also identified. Carbohydrate-inducible proteins, potentially homologous to OprB, were identified in several rRNA homology-group-I pseudomonads by sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis, Western immunoblotting and N-terminal amino acid sequencing. These species also contained DNA that hybridized to a P. aeruginosa oprB gene probe.

Biophysical characterization of OprB, a glucose-inducible porin of Pseudomonas aeruginosa.

OprB, a glucose-inducible porin of P. aeruginosa, was characterized by black lipid bilayer analysis and circular dichroism spectroscopy. Black lipid bilayer analysis of OprB revealed a single-channel conductance of 25 pS, the presence of a glucose binding site with a Ks for glucose of 380 +/- 40 mM, and the formation of channels with a strong selection for anions. Analysis of P. aeruginosa OprB circular dichroism spectra revealed a high beta sheet content (40%) which is within the range of that determined for other porins. Values obtained from black lipid bilayer analysis were compared to those previously obtained for OprB of P. putida [Saravolac et al. (1991). J. Bacteriol. 173, 4970-4976] and indicated extensive similarities in the single-channel conductance and glucose-binding properties of these two porins. Immunological and amino terminal sequence analysis revealed a high degree of homology. Of the first 14 amino terminal residues, 12 were identical. A major difference between the two porins was found in their ion selectivity. Whereas P. aeruginosa OprB is anion selective, P. putida OprB and other carbohydrate selective porins are known to be cation selective.

Substrate specificity of the high-affinity glucose transport system of Pseudomonas aeruginosa.

Specificity of the high-affinity glucose transport system of Pseudomonas aeruginosa was examined. At a concentration of [14C]glucose near the Vmax of the system, inhibition by maltose, galactose, and xylose was detected. This inhibition is similar to that detected in earlier in vivo studies and correlates with the known specificity of OprB, a glucose-specific porin of P. aeruginosa. At a level of [14C]glucose 100 times lower, only unlabelled glucose inhibited uptake to any extent. This matches the known in vitro specificity of the periplasmic glucose binding protein. These findings were used to explain the discrepancy between earlier in vivo and in vitro results reported in the literature.