Complete Genome Sequences of 17 Clinical Campylobacter jejuni Strains from Chile.

Campylobacter jejuni is the leading cause of bacterial foodborne disease worldwide. Here, we report the complete annotated genomes and plasmid sequences of 17 Campylobacter jejuni strains isolated from patients with gastroenteritis in Santiago, Chile.

Simultaneous Presence of Insertion Sequence Excision Enhancer and Insertion Sequence IS629 Correlates with Increased Diversity and Virulence in Shiga Toxin-Producing Escherichia coli.

Although new serotypes of enterohemorrhagic Escherichia coli (EHEC) emerge constantly, the mechanisms by which these new pathogens arise and the reasons emerging serotypes tend to carry more virulence genes than other E. coli are not understood. An insertion sequence (IS) excision enhancer (IEE) was discovered in EHEC O157:H7 that promoted the excision of IS3 family members and generating various genomic deletions. One IS3 family member, IS629, actively transposes and proliferates in EHEC O157:H7 and enterotoxigenic E. coli (ETEC) O139 and O149. The simultaneous presence of the IEE and IS629 (and other IS3 family members) may be part of a system promoting not only adaptation and genome diversification in E. coli O157:H7 but also contributing to the development of pathogenicity among predominant serotypes. Prevalence comparisons of these elements in 461 strains, representing 72 different serotypes and 5 preassigned seropathotypes (SPT) A to E, showed that the presence of these two elements simultaneously was serotype specific and associated with highly pathogenic serotypes (O157 and top non-O157 Shiga toxin-producing Escherichia coli [STEC]) implicated in outbreaks and sporadic cases of human illness (SPT A and B). Serotypes lacking one or both elements were less likely to have been isolated from clinical cases. Our comparisons of IEE sequences showed sequence variations that could be divided into at least three clusters. Interestingly, the IEE sequences from O157 and the top 10 non-O157 STEC serotypes fell into clusters I and II, while less commonly isolated serotypes O5 and O174 fell into cluster III. These results suggest that IS629 and IEE elements may be acting synergistically to promote genome plasticity and genetic diversity among STEC strains, enhancing their abilities to adapt to hostile environments and rapidly take up virulence factors.

Complete DNA sequence analysis of enterohemorrhagic Escherichia coli plasmid pO157_2 in ß-glucuronidase-positive E. coli O157:H7 reveals a novel evolutionary path.

Strains of enterohemorragic Escherichia coli (EHEC) O157:H7 that are non-sorbitol fermenting (NSF) and ß-glucuronidase negative (GUD(-)) carry a large virulence plasmid, pO157 (>90,000 bp), whereas closely related sorbitol-fermenting (SF) E. coli O157:H(-) strains carry plasmid pSFO157 (>120,000 bp). GUD(+) NSF O157:H7 strains are presumed to be precursors of GUD(-) NSF O157:H7 strains that also carry pO157. In this study, we report the complete sequence of a novel virulence plasmid, pO157-2 (89,762 bp), isolated from GUD(+) NSF O157:H7 strain G5101. PCR analysis confirmed the presence of pO157-2 in six other strains of GUD(+) NSF O157:H7. pO157-2 carries genes associated with virulence (e.g., hemolysin genes) and conjugation (tra and trb genes) but lacks katP and espP present in pO157. Comparative analysis of the three EHEC plasmids shows that pO157-2 is highly related to pO157 and pSFO157 but not ancestral to pO157. These results indicated that GUD(+) NSF O157:H7 strains might not be direct precursors to GUD(-) NSF O157:H7 as previously proposed but rather have evolved independently from a common ancestor.

Genome sequence of the clinical O4:K12 serotype Vibrio parahaemolyticus strain 10329.

Vibrio parahaemolyticus is the leading cause of food-borne illnesses worldwide. Here, we report a draft genome of V. parahaemolyticus strain 10329 of the O4:K12 serotype. It belongs to the main U.S. West Coast clonal complex of V. parahaemolyticus (sequence type 36 [ST36]) causing oyster-associated human illness. It contains the virulence determinants tdh and trh but appears to infect at much lower doses than V. parahaemolyticus strains with these same determinants from other areas, such as the U.S. Gulf and Atlantic coasts.

Draft genome sequences of six Escherichia coli isolates from the stepwise model of emergence of Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) of serotype O157:H7 has been implicated in food-borne illnesses worldwide. An evolutionary model was proposed in which the highly pathogenic EHEC O157:H7 serotype arose from its ancestor, enteropathogenic E. coli (EPEC) O55:H7 (sorbitol fermenting [SOR(+)] and ß-glucuronidase positive [GUD(+)]), through sequential gain of virulence, phenotypic traits, and serotype change. Here we report six draft genomes of strains belonging to this evolutionary model: two EPEC O55:H7 (SOR(+) GUD(+)) strains, two nonmotile EHEC O157:H(-) strains (SOR(+) GUD(+)) containing plasmid pSFO157, one EHEC O157:H7 (SOR(-) GUD(+)) strain, and one O157:H7 strain containing plasmid pSFO157 (SOR(+) GUD(+)).

A versatile internal control for use as DNA in real-time PCR and as RNA in real-time reverse transcription PCR assays.

AIMS: To develop and evaluate a TaqMan-based internal amplification control (IAC) that can be used as DNA in real-time PCR (qPCR) or as RNA in reverse transcription real-time PCR (qRT-PCR) to identify the presence of assay inhibition and to evaluate its incorporation into existing qPCR and qRT-PCR methods for bacterial detection. METHODS AND RESULTS: A DNA IAC was constructed by generating a 198-bp random sequence that was synthesized and inserted into a pZErO-2 vector and transformed into Escherichia coli. The RNA IAC was generated through in vitro transcription of the DNA IAC. Both IAC formats were tested individually in singleplex TaqMan reactions and also included in existing multiplex assays. The DNA IAC was incorporated in a Shigella spp. detection qPCR assay (targeting ipaH). The RNA IAC was successfully evaluated in a Salmonella spp. detection qRT-PCR (using invA mRNA as target). CONCLUSIONS: A highly versatile IAC that can be supplemented to qPCR and qRT-PCR pathogen detection methods was developed, greatly reducing the confounding effects of false negatives because of PCR inhibitors without affecting pathogen detection. SIGNIFICANCE AND IMPACT OF THE STUDY: The frequency of false negatives associated with qPCR analyses is prevalent in certain matrices, particularly those involving complex foods. Hence, the IAC presented here provides a solution to unforeseen false-negative reactions in PCR.

Genetic relatedness among tdh+ and trh+ Vibrio parahaemolyticus cultured from Gulf of Mexico oysters (Crassostrea virginica) and surrounding water and sediment.

Pathogenic Vibrio parahaemolyticus (Vp) (tdh(+)/trh(+)) represent a small percentage of environmental Vp populations, and very little is known about this subpopulation. Repetitive extragenic palindromic PCR and multilocus sequence analysis revealed heterogeneity among 41 Vp containing thermostable direct hemolysin (tdh) and tdh-related hemolysin (trh) that were isolated from Mississippi coastal environments from October 2006 to April 2007. There was no source-specific sequestering in oysters, water, or sediment.