Virulence Gene Profiles and Clonal Relationships of Escherichia coli O26:H11 Isolates from Feedlot Cattle as Determined by Whole-Genome Sequencing.

UNLABELLED: Escherichia coli O26 is the second most important enterohemorrhagic E. coli (EHEC) serogroup worldwide. Serogroup O26 strains are categorized mainly into two groups: enteropathogenic (EPEC) O26, carrying a locus of enterocyte effacement (LEE) and mostly causing mild diarrhea, and Shiga-toxigenic (STEC) O26, which carries the Shiga toxin (STX) gene (stx), responsible for more severe outcomes. stx-negative O26 strains can be further split into two groups. One O26 group differs significantly from O26 EHEC, while the other O26 EHEC-like group shows all the characteristics of EHEC O26 except production of STX. In order to determine the different populations of O26 E. coli present in U.S. cattle, we sequenced 42 O26:H11 strains isolated from feedlot cattle and compared them to 37 O26:H11 genomes available in GenBank. Phylogenetic analysis by whole-genome multilocus sequence typing (wgMLST) showed that O26:H11/H(-) strains in U.S. cattle were highly diverse. Most strains were sequence type 29 (ST29). By wgMLST, two clear lineages could be distinguished among cattle strains. Lineage 1 consisted of O26:H11 EHEC-like strains (ST29) (4 strains) and O26:H11 EHEC strains (ST21) (2 strains), and lineage 2 (36 strains) consisted of O26:H11 EPEC strains (ST29). Overall, our analysis showed U.S. cattle carried pathogenic (ST21; stx1 (+) ehxA(+) toxB(+)) and also potentially pathogenic (ST29; ehxA(+) toxB(+)) O26:H11 E. coli strains. Furthermore, in silico analysis showed that 70% of the cattle strains carried at least one antimicrobial resistance gene. Our results showed that whole-genome sequence analysis is a robust and valid approach to identify and genetically characterize E. coli O26:H11, which is of importance for food safety and public health. IMPORTANCE: Escherichia coli O26 is the second most important type of enterohemorrhagic E. coli (EHEC) worldwide. Serogroup O26 strains are categorized into two groups: enteropathogenic (EPEC) carrying LEE, causing mild diarrhea, and Shiga toxigenic (STEC) carrying the stx gene, responsible for more severe outcomes. However, there are currently problems in distinguishing one group from the other. Furthermore, several O26 stx-negative strains are consistently misidentified as either EHEC-like or EPEC. The use of whole-genome sequence (WGS) analysis of O26 strains from cattle in the United States (i) allowed identification of O26 strains present in U.S. cattle, (ii) determined O26 strain diversity, (iii) solved the misidentification problem, and (iv) screened for the presence of antimicrobial resistance and virulence genes in the strains. This study provided a framework showing how to easily and rapidly use WGS information to identify and genetically characterize E. coli O26:H11, which is important for food safety and public health.

Genomic diversity and virulence profiles of historical Escherichia coli O157 strains isolated from clinical and environmental sources.

Escherichia coli O157:H7 is, to date, the major E. coli serotype causing food-borne human disease worldwide. Strains of O157 with other H antigens also have been recovered. We analyzed a collection of historic O157 strains (n = 400) isolated in the late 1980s to early 1990s in the United States. Strains were predominantly serotype O157:H7 (55%), and various O157:non-H7 (41%) serotypes were not previously reported regarding their pathogenic potential. Although lacking Shiga toxin (stx) and eae genes, serotypes O157:H1, O157:H2, O157:H11, O157:H42, and O157:H43 carried several virulence factors (iha, terD, and hlyA) also found in virulent serotype E. coli O157:H7. Pulsed-field gel electrophoresis (PFGE) showed the O157 serogroup was diverse, with strains with the same H type clustering together closely. Among non-H7 isolates, serotype O157:H43 was highly prevalent (65%) and carried important enterohemorrhagic E. coli (EHEC) virulence markers (iha, terD, hlyA, and espP). Isolates from two particular H types, H2 and H11, among the most commonly found non-O157 EHEC serotypes (O26:H11, O111:H11, O103:H2/H11, and O45:H2), unexpectedly clustered more closely with O157:H7 than other H types and carried several virulence genes. This suggests an early divergence of the O157 serogroup to clades with different pathogenic potentials. The appearance of important EHEC virulence markers in closely related H types suggests their virulence potential and suggests further monitoring of those serotypes not implicated in severe illness thus far.

Draft Genome Sequences of Two O104:H21 Escherichia coli Isolates Causing Hemorrhagic Colitis during a 1994 Montana Outbreak Provide Insight into Their Pathogenicity.

We sequenced the genomes of two strains of O104:H21 enterohemorrhagic Escherichia coli (EHEC) isolated during an outbreak of hemorrhagic colitis in Montana in 1994. These strains carried a plasmid that contains several virulence genes not present in pO157. The genome sequences will improve phylogenetic analysis of other non-O157 E. coli strains in the future.

Genetic characterization of Escherichia coli O104 isolates from different sources in the United States.

Escherichia coli O104 isolates collected from different sources in the United States were examined for virulence genes typical of enterohemorrhagic E. coli and those identified in the O104:H4 isolate associated with the 2011 German outbreak. The unexpected presence of virulence markers in these isolates highlights the importance of screening unusual and potentially pathogenic Shiga toxin-producing E. coli serotypes.

Prevalence, distribution and evolutionary significance of the IS629 insertion element in the stepwise emergence of Escherichia coli O157:H7.

BACKGROUND: Insertion elements (IS) are known to play an important role in the evolution and genomic diversification of Escherichia coli O157:H7 lineages. In particular, IS629 has been found in multiple copies in the E. coli O157:H7 genome and is one of the most prevalent IS in this serotype. It was recently shown that the lack of O157 antigen expression in two O rough E. coli O157:H7 strains was due to IS629 insertions at 2 different locations in the gne gene that is essential for the O antigen biosynthesis. RESULTS: The comparison of 4 E. coli O157:H7 genome and plasmid sequences showed numerous IS629 insertion sites, although not uniformly distributed among strains. Comparison of IS629s found in O157:H7 and O55:H7 showed the presence of at least three different IS629 sub-types. O157:H7 strains carry IS629 elements sub-type I and III whereby the ancestral O55:H7 carries sub-type II. Analysis of strains selected from various clonal groups defined on the E. coli O157:H7 stepwise evolution model showed that IS629 was not observed in sorbitol fermenting O157 (SFO157) clones that are on a divergent pathway in the emergence of O157:H7. This suggests that the absence of IS629 in SFO157 strains probably occurred during the divergence of this lineage, albeit it remains uncertain if it contributed, in part, to their divergence from other closely related strains. CONCLUSIONS: The highly variable genomic locations of IS629 in O157:H7 strains of the A6 clonal complex indicates that this insertion element probably played an important role in genome plasticity and in the divergence of O157:H7 lineages.

Different IS629 transposition frequencies exhibited by Escherichia coli O157:H7 strains in the stepwise evolutionary model.

The insertion sequence IS629, which is highly prevalent in Escherichia coli O157:H7 genomes, was found to be absent in O157:H- strains, which are on a divergent pathway in the emergence of O157:H7. Although O157:H- is deficient in IS629, it permits IS629 transposition, with an excision frequency higher than that of ancestral O55:H7 strains but significantly lower than that of pathogenic O157:H7 strains.

Comparison of commercial RNA extraction kits for preparation of DNA-free total RNA from Salmonella cells.

BACKGROUND: The isolation of DNA-free RNA is a crucial step in the reverse transcription PCR (RT-PCR). Every RNA extraction procedure results in RNA samples contaminated with genomic DNA, which can cause false-positive outcomes in highly sensitive applications, including a recently developed quantitative real-time PCR (RT-qPCR) assay that targets invA mRNA for the detection of live Salmonella cells. The assay of this specific mRNA can be used to indicate the presence of live, as opposed to dead, cells of Salmonella enterica in a food matrix. FINDINGS: We evaluated the ability of five RNA extraction kits to produce RNA preparations from exponentially growing Salmonella cells. The acceptability of the preparations for use in downstream applications such as RT-qPCR was judged in terms of the total amount of RNA recovered, the integrity of the RNA molecules, and minimal content of DNA. The five kits produced RNA preparations that differed markedly in yield, integrity of the Salmonella RNA and the amount of contaminant DNA. The greatest RNA recovery was achieved with the MasterPure kit; however, the preparation contained high levels of genomic DNA. The UltraClean extraction kit gave a low level of RNA recovery with a poor level of integrity. The RNeasy Mini, RiboPure and PureLink extraction kits produced high-quality, DNA-free RNA suitable for Salmonella detection by RT-qPCR. CONCLUSIONS: We showed that the RNeasy Mini and PureLink RNA extraction kits were the most suitable for the detection of Salmonella invA mRNA by RT-qPCR. The use of these two kits will greatly reduce the frequency of false-positive results and might allow fast RT-qPCR determination of invA mRNA produced by viable Salmonella in food samples.

Characterization of a gne::IS629 O rough:H7 Escherichia coli strain from a hemorrhagic colitis patient.

Shiga-toxigenic Escherichia coli strains that are O rough:H7 due to gne::IS629 were thought to be rare and to have unknown pathogenic potential. Recently, an O rough:H7 strain caused by gne::IS629 was isolated from a hemorrhagic colitis patient, suggesting that these strains are pathogenic and may not be as rare as anticipated.

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.

Use of multitoxin immunoaffinity columns for determination of aflatoxins and ochratoxin A in ginseng and ginger.

Conditions were optimized for the simultaneous, alkaline, aqueous methanol extraction of aflatoxins (AFL), i.e., B1 (AFB1), B2 (AFB2), G1 (AFG1), and G2 (AFG2), and ochratoxin A (OTA) with subsequent purification, isolation, and determination of the toxins in ginseng and ginger. Powdered roots were extracted with methanol-0.5% NaHCO3 solution (7 + 3). After shaking and centrifugation, the supernatant was diluted with 100 mM phosphate buffer containing 1% Tween 20 and filtered through glass microfiber filter paper. The filtrate was then passed through an immunoaffinity column, and the toxins were eluted with methanol. The AFL were separated and determined by reversed-phase liquid chromatography (RPLC) with fluorescence detection after postcolumn UV photochemical derivatization. OTA was separated and determined by RPLC with fluorescence detection. Recoveries of AFL added at 2-16 ng/g and OTA added at 1-8 ng/g to ginseng were 72-80 and 86-95%, respectively. Recoveries of AFL and OTA added to ginger were similar to those for ginseng. A total of 39 commercially available ginger products from 6 manufacturers were analyzed. Twenty-six samples were found to be contaminated with AFL at 1-31 ng/g and 29 samples, with OTA at 1-10 ng/g. Ten samples contained no AFL or OTA. Ten ginseng finished products were also analyzed; 3 contained AFL at 0.1 ng/g and 4 contained OTA at levels ranging from 0.4 to 1.8 ng/g. LC/tandem mass spectrometry with multiple-reaction monitoring of 3 collisionally induced product ions from the protonated molecular ions of OTA, AFB1, and AFG1 was used to confirm the identities of the toxins in extracts of the finished products.