PCR-based method for targeting 16S-23S rRNA intergenic spacer regions among Vibrio species

<p>Abstract</p> <p>Background</p> <p>The genus <it>Vibrio </it>is a diverse group of Gram-negative bacteria comprised of 74 species. Furthermore, the genus has and is expected to continue expanding with the addition of several new species annually. Consequently, it is of paramount importance to have a method which is able to reliably and efficiently differentiate the numerous <it>Vibrio </it>species.</p> <p>Results</p> <p>In this study, a novel and rapid polymerase chain reaction (PCR)-based intergenic spacer (IGS)-typing system for vibrios was developed that is based on the well-known IGS regions located between the 16S and 23S rRNA genes on the bacterial chromosome. The system was optimized to resolve heteroduplex formation as well as to take advantage of capillary gel electrophoresis technology such that reproducible analyses could be achieved in a rapid manner. System validation was achieved through testing of 69 archetypal <it>Vibrio </it>strains, representing 48 <it>Vibrio </it>species, from which an 'IGS-type' profile database was generated. These data, presented here in several cluster analyses, demonstrated successful differentiation of the 69 type strains showing that this PCR-based fingerprinting method easily discriminates bacterial strains at the species level among <it>Vibrio</it>. Furthermore, testing 36 strains each of <it>V. parahaemolyticus </it>and <it>V. vulnificus</it>, important food borne pathogens, isolated from a variety of geographical locations with the IGS-typing method demonstrated distinct IGS-typing patterns indicative of subspecies divergence in both populations making this technique equally useful for intraspecies differentiation, as well.</p> <p>Conclusion</p> <p>This rapid, reliable and efficient IGS-typing system, especially in combination with 16S rRNA gene sequencing, has the capacity to not only discern and identify vibrios at the species level but, in some cases, at the sub-species level, as well. This procedure is particularly well-suited for preliminary species identification and, lends itself nicely to epidemiological investigations providing information more quickly than other time-honoured methods traditionally used in these types of analyses.</p

Identification of a Protein Biomarker Unique to the Pandemic O3:K6 Clone of Vibrio parahaemolyticus

The present method of characterizing Vibrio parahaemolyticus strains involves serotyping or detection methods based on assessment of the presence or absence of genes thought to be markers of an organism's pathogenicity. It is unclear whether these assays detect all pathogenic V. parahaemolyticus strains since a clear correlation between the presence of a particular gene and the organism's pathogenicity has not yet been observed. We have described a proteomics-based method to distinguish individual V. parahaemolyticus strains on the basis of their protein profiles and identified a specific protein that is characteristic of the pandemic O3:K6 strain and its clonal derivatives. In the pandemic clone of V. parahaemolyticus, a histone-like DNA-binding protein, HU-α, has a C-terminal amino acid sequence different from those of other strains of V. parahaemolyticus. Upon further study, it was discovered that the gene encoding this protein has a 16-kbp insert at the 3′ terminus of the open reading frame for this protein. By using the protein sequence of the unique biomarker for the pandemic clone of V. parahaemolyticus, it was possible to rationally design specific PCR-based probes and assays that permit the rapid and precise identification of pandemic strains of V. parahaemolyticus

Virulence Characterization of Shiga-Toxigenic Escherichia coli Isolates from Wholesale Produce▿

The 13 Shiga-toxigenic Escherichia coli (STEC) strains isolated from wholesale spinach and lettuce consisted mostly of serotypes that have not been implicated in illness. Among these strains, however, were two O113:H21 that carried virulence genes common to this pathogenic serotype (stx2, ehxA, saa, and subAB), suggesting that their presence in ready-to-eat produce may be of health concern

Produce Isolates of the Escherichia coli Ont:H52 Serotype That Carry both Shiga Toxin 1 and Stable Toxin Genes

Produce isolates of the Escherichia coli Ont:H52 serotype carried Shiga toxin 1 and stable toxin genes but only expressed Stx1. These strains had pulsed-field gel electrophoresis profiles that were 90% homologous to clinical Ont:H52 strains that had identical phenotypes and genotypes. All Ont:H52 strains had identical single nucleotide polymorphism profiles that are suggestive of a unique clonal group

Genetic Analysis for the Lack of Expression of the O157 Antigen in an O Rough:H7 Escherichia coli Strain ▿

The O-antigen (rfb) operon and related genes of MA6, an O rough:H7 Shiga-toxigenic Escherichia coli strain, were examined to determine the cause of the lack of O157 expression. A 1,310-bp insertion, homologous to IS629, was observed within its gne gene. trans complementation with a functional gne gene from O157:H7 restored O157 antigen expression in MA6