pVir and Bloody Diarrhea in Campylobacter jejuni Enteritis

The plasmid pVir may play a role in the virulence of Campylobacter jejuni, a leading cause of bacterial gastroenteritis. The pVir plasmid was identified in 17% of 104 C. jejuni clinical isolates studied and was significantly associated with the occurrence of blood in patient stool, a marker of invasive infection. The pVir plasmid was not associated with greater occurrence of diarrhea, fever, pain, vomiting, or need for patient hospitalization. Isolates containing pVir were also associated with the presence of a tetracycline-resistance plasmid, but pVir did not transfer with tetracycline-resistance plasmids to recipient strains of C. jejuni. The association of pVir and bloody stool suggests that pVir may be clinically relevant in C. jejuni infections

Genetic characterization of clinical and agri-food isolates of multi drug resistant Salmonella enterica serovar Heidelberg from Canada

<p>Abstract</p> <p>Background</p> <p><it>Salmonella enterica </it>serovar Heidelberg ranks amongst the most prevalent causes of human salmonellosis in Canada and an increase in resistance to extended spectrum cephalosporins (ESC) has been observed by the Canadian Integrated Program for Antimicrobial Resistance Surveillance. This study examined the genetic relationship between <it>S</it>. Heidelberg isolates from livestock, abattoir, retail meat, and clinical human specimens to determine whether there was a link between the emergence of MDR <it>S</it>. Heidelberg in chicken agri-food sources and the simultaneous increase of MDR <it>S</it>. Heidelberg in human clinical samples.</p> <p>Results</p> <p>Chromosomal genetic homogeneity was observed by pulsed-field gel electrophoresis (PFGE), DNA sequence-based typing (SBT) and DNA microarray-based comparative genomic hybridization (CGH). Sixty one percent of isolates were indistinguishable by PFGE conducted using <it>Xba</it>I and <it>Bln</it>I restriction enzymes. An additional 15% of isolates had PFGE patterns that were closely related to the main cluster. SBT did not identify DNA polymorphisms and CGH revealed only genetic differences between the reference <it>S</it>. Typhimurium strain and <it>S</it>. Heidelberg isolates. Genetic variation observed by CGH between <it>S</it>. Heidelberg isolates could be attributed to experimental variation. Alternatively, plasmid content was responsible for differences in antimicrobial susceptibility, and restriction fragment length polymorphism (RFLP) analyses followed by replicon typing identified two divergent plasmid types responsible for ESC resistance.</p> <p>Conclusion</p> <p>Due to the overall limited genetic diversity among the isolates, it was not possible to identify variable traits that would be suitable for source tracking between human and agri-food isolates of <it>S</it>. Heidelberg in Canada.</p

Limited genetic diversity in Salmonella enterica Serovar Enteritidis PT13

<p>Abstract</p> <p>Background</p> <p><it>Salmonella enterica </it>serovar Enteritidis has emerged as a significant foodborne pathogen throughout the world and is commonly characterized by phage typing. In Canada phage types (PT) 4, 8 and 13 predominate and in 2005 a large foodborne PT13 outbreak occurred in the province of Ontario. The ability to link strains during this outbreak was difficult due to the apparent clonality of PT13 isolates in Canada, as there was a single dominant pulsed-field gel electrophoresis (PFGE) profile amongst epidemiologically linked human and food isolates as well as concurrent sporadic strains. The aim of this study was to perform comparative genomic hybridization (CGH), DNA sequence-based typing (SBT) genomic analyses, plasmid analyses, and automated repetitive sequence-based PCR (rep-PCR) to identify epidemiologically significant traits capable of subtyping <it>S</it>. Enteritidis PT13.</p> <p>Results</p> <p>CGH using an oligonucleotide array based upon chromosomal coding sequences of <it>S. enterica </it>serovar Typhimurium strain LT2 and the <it>Salmonella </it>genomic island 1 successfully determined major genetic differences between <it>S</it>. Typhimurium and <it>S</it>. Enteritidis PT13, but no significant strain-to-strain differences were observed between <it>S</it>. Enteritidis PT13 isolates. Individual loci (<it>safA </it>and <it>fliC</it>) that were identified as potentially divergent in the CGH data set were sequenced in a panel of <it>S</it>. Enteritidis strains, and no differences were detected between the PT13 strains. Additional sequence-based typing was performed at the <it>fimA</it>, <it>mdh</it>, <it>manB</it>, <it>cyaA</it>, <it>citT</it>, <it>caiC</it>, <it>dmsA</it>, <it>ratA </it>and STM0660 loci. Similarly, no diversity was observed amongst PT13 strains. Variation in plasmid content between PT13 strains was observed, but macrorestriction with B<it>gl</it>II did not identify further differences. Automated rep-PCR patterns were variable between serovars, but <it>S</it>. Enteritidis PT13 strains could not be differentiated.</p> <p>Conclusion</p> <p>None of the methods identified any significant variation between PT13 strains. Greater than 11,300 base pairs of sequence for each of seven <it>S</it>. Enteritidis PT13 strains were analyzed without detecting a single polymorphic site, although diversity between different phage types of <it>S</it>. Enteritidis was observed. These data suggest that Canadian <it>S</it>. Enteritidis PT13 strains are highly related genetically.</p

Genetic Determinants and Polymorphisms Specific for Human-Adapted Serovars of Salmonella enterica That Cause Enteric Fever

Salmonella enterica serovars Typhi, Paratyphi A, and Sendai are human-adapted pathogens that cause typhoid (enteric) fever. The acute prevalence in some global regions and the disease severity of typhoidal Salmonella have necessitated the development of rapid and specific detection tests. Most of the methodologies currently used to detect serovar Typhi do not identify serovars Paratyphi A or Sendai. To assist in this aim, comparative sequence analyses were performed at the loci of core bacterial genetic determinants and Salmonella pathogenicity island 2 genes encoded by clinically significant S. enterica serovars. Genetic polymorphisms specific for serovar Typhi (at trpS), as well as polymorphisms unique to human-adapted typhoidal serovars (at sseC and sseF), were observed. Furthermore, entire coding sequences unique to human-adapted typhoidal Salmonella strains (i.e., serovar-specific genetic loci rather than polymorphisms) were observed in publicly available comparative genomic DNA microarray data sets. These polymorphisms and loci were developed into real-time PCR, standard PCR, and liquid microsphere suspension array-based molecular protocols and tested for with a panel of clinical and reference subspecies I S. enterica strains. A proportion of the nontyphoidal Salmonella strains hybridized with the allele-specific oligonucleotide probes for sseC and sseF; but the trpS allele was unique to serovar Typhi (with a singular serovar Paratyphi B strain as an exception), and the coding sequences STY4220 and STY4221 were unique among serovars Typhi, Paratyphi A, and Sendai. These determinants provided phylogenetic data on the genetic relatedness of serovars Typhi, Paratyphi A, and Sendai; and the protocols developed might allow the rapid identification of these Salmonella serovars that cause enteric fever

Functional and mutational analysis of conjugative transfer region 2 (Tra2) from the IncHI1 plasmid R27

The transfer 2 region (Tra2) of the conjugative plasmid drR27 (derepressed R27) was analyzed by PSI-BLAST, insertional mutagenesis, genetic complementation, and an H-pilus assay. Tra2 contains 11 mating-pair formation (Mpf) genes that are essential for conjugative transfer, 9 of which are essential for H-pilus production (trhA, -L, -E, -K, -B, -V, -C, -P, and -W). TrhK has similarity to secretin proteins, suggesting a mechanism by which DNA could traverse the outer membrane of donors. The remaining two Mpf genes, trhU and trhN, play an auxiliary role in H-pilus synthesis and are proposed to be involved in DNA transfer and mating-pair stabilization, respectively. Conjugative transfer abilities were restored for each mutant when complemented with the corresponding transfer gene. In addition to the essential Mpf genes, three genes, trhO, trhZ, and htdA, modulate R27 transfer frequency. Disruption of trhO and trhZ severely reduced the transfer frequencies of drR27, whereas disruption of htdA greatly increased the transfer frequency of wild-type R27 to drR27 levels. A comparison of the essential transfer genes encoded by the Tra2 and Tra1 (T. D. Lawley, M. W. Gilmour, J. E. Gunton, L. J. Standeven, and D. E. Taylor, J. Bacteriol. 184:2173-2183, 2002) of R27 to other transfer systems illustrates that the R27 conjugative transfer system is a chimera composed of IncF-like and IncP-like transfer systems. Furthermore, the Mpf/type IV secretion systems encoded by IncH and IncF transfer systems are distinct from that of the IncP transfer system. The phenotypic and ecological significance of these observations is discussed

Recommended Immunological Assays to Screen for Ricin-Containing Samples

Ricin, a toxin from the plant Ricinus communis, is one of the most toxic biological agents known. Due to its availability, toxicity, ease of production and absence of curative treatments, ricin has been classified by the Centers for Disease Control and Prevention (CDC) as category B biological weapon and it is scheduled as a List 1 compound in the Chemical Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection and quantification capabilities of 17 expert laboratories. In this exercise one goal was to analyse the laboratories’ capacity to detect and differentiate ricin and the less toxic, but highly homologuous protein R. communis agglutinin (RCA120). Six analytical strategies are presented in this paper based on immunological assays (four immunoenzymatic assays and two immunochromatographic tests). Using these immunological methods “dangerous” samples containing ricin and/or RCA120 were successfully identified. Based on different antibodies used the detection and quantification of ricin and RCA120 was successful. The ricin PT highlighted the performance of different immunological approaches that are exemplarily recommended for highly sensitive and precise quantification of ricin

Effects of the Campylobacter jejuni CJIE1 prophage homologs on adherence and invasion in culture, patient symptoms, and source of infection.

Prophages of enteric bacteria are frequently of key importance for the biology, virulence, or host adaptation of their host. Some C. jejuni isolates carry homologs of the CJIE1 (CMLP 1) prophage that carry cargo genes potentially involved in virulence. Possible role(s) of CJIE1 homologs in the biology and virulence of C. jejuni were therefore investigated by using in vitro cell culture assays and by assessing the association of C. jejuni isolates with and without these prophages with patients' symptoms, with source, and with clonal lineages within the C. jejuni population. Four C. jejuni isolates, three carrying the CJIE1-like prophage and one without, were tested in cell culture assays for adherence and invasion. Both adherence and invasion of C. jejuni to cells in culture were increased by the presence of the CJIE1-family prophage. Differences in motility and growth rate did not appear to be responsible. The CJIE1 prophage was present in 23% of isolates from human and non-human sources combined that were obtained through sentinel-site surveillance, and the distribution of CJIE1 in this population showed modest clonal associations. There was no correlation between the presence of the CJIE1 prophage in C. jejuni and patient symptoms, although there was some statistical support for lower rates of abdominal pain and fever when the prophage was present. Little evidence was found for a role of the prophage in host adaptation or host specificity. These biological effects suggest that the presence of the prophage may be a marker for differential virulence of some C. jejuni isolates. Ongoing research into the effects of the prophage on protein expression may provide additional insights into the roles the prophage may play in the biology of its host bacterium

Rapid detection of Vibrio species using liquid microsphere arrays and real-time PCR targeting the ftsZ locus

The development of rapid and sensitive molecular techniques for the detection of Vibrio species would be useful for the surveillance of sporadic infections and management of major outbreaks. Comparative sequence analysis of the ftsZ gene in the predominant Vibrio species that cause human disease revealed distinct alleles for each examined species, including Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus. Light Upon extension (LUX) real-time PCR assays were developed to target these species-specific polymorphisms, and were successful in rapidly differentiating the major pathogenic Vibrio species. Luminex liquid microsphere array technology was used to develop a comprehensive assay capable of simultaneously detecting V. cholerae, V. parahaemolyticus and V. vulnificus. These assays permitted the identification of a presumptive V. parahaemolyticus isolate as Vibrio alginolyticus, which was verified using additional molecular characterization