Evaluation of molecular and culture methods to determine the optimum testing strategy for verotoxigenic Escherichia coli in faecal specimens.

We evaluated 5 methods for the detection of verotoxogenic Escherichia coli (VTEC) from faecal specimens to determine the most sensitive and specific method(s) and to advise an optimum testing strategy. A total of 681 stool specimens were examined using up to 5 diagnostic molecular and phenotypic methods that are used routinely in the VTEC Reference laboratory, Dublin. A testing strategy incorporating a 2-step approach that included a single Real Time-PCR and 1 culture-based method yielded the highest sensitivity, specificity, positive predictive value, and negative predictive value of 98.21%, 100%, 100%, and 99.43%, respectively.

Characterization of farm, food, and clinical Shiga toxin-producing Escherichia coli (STEC) O113.

Thirty-nine Shiga toxin-producing Escherichia coli (STEC) O113 Irish farm, abattoir, and clinical isolates were analyzed in conjunction with eight Australian, New Zealand, and Norwegian strains for H (flagellar) antigens, virulence gene profile (eaeA, hlyA, tir, espA, espB katP, espP, etpD, saa, sab, toxB, iha, lpfA(O157/OI-141,) lpfA(O113,) and lpfA(O157/OI-154)), Shiga toxin gene variants (stx(1c), stx(1d), stx(2), stx(2c), stx(2dact), stx(2e), stx(2f,) and stx(2g)) and were genotyped using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). All of the Irish strains were O113:H4, regardless of source, while all non-Irish isolates carried the H21 flagellar antigen. The stx(1) gene was present in 30 O113:H4 strains only, whereas the stx(2d) gene was common to all isolates regardless of source. In contrast, eaeA was absent, while hlyA was found in the Australian, New Zealand, Norwegian, and two of the Irish human clinical isolates. saa was present in the O113:H21 but not in the O113:H4 serotype. To the best of the author's knowledge, this is the first report of clinically significant STEC lacking both the eaeA and saa genes. PFGE analysis was inconclusive; however, MLST grouped the strains into three sequence types (ST): ST10, ST56, and ST223. Based on our findings, it was concluded that the stx(2d) gene is common in STEC O113, which are generally eaeA negative. Furthermore, STEC O113:H4 is a new, emerging bovine serotype of human clinical significance.

Multilocus sequence typing methods for the emerging Campylobacter Species C. hyointestinalis, C. lanienae, C. sputorum, C. concisus, and C. curvus.

Multilocus sequence typing (MLST) systems have been reported previously for multiple food- and food animal-associated Campylobacter species (e.g., C. jejuni, C. coli, C. lari, and C. fetus) to both differentiate strains and identify clonal lineages. These MLST methods focused primarily on campylobacters of human clinical (e.g., C. jejuni) or veterinary (e.g., C. fetus) relevance. However, other, emerging, Campylobacter species have been isolated increasingly from environmental, food animal, or human clinical samples. We describe herein four MLST methods for five emerging Campylobacter species: C. hyointestinalis, C. lanienae, C. sputorum, C. concisus, and C. curvus. The concisus/curvus method uses the loci aspA, atpA, glnA, gltA, glyA, ilvD, and pgm, whereas the other methods use the seven loci defined for C. jejuni (i.e., aspA, atpA, glnA, gltA, glyA, pgm, and tkt). Multiple food animal and human clinical C. hyointestinalis (n = 48), C. lanienae (n = 34), and C. sputorum (n = 24) isolates were typed, along with 86 human clinical C. concisus and C. curvus isolates. A large number of sequence types were identified using all four MLST methods. Additionally, these methods speciated unequivocally isolates that had been typed ambiguously using other molecular-based speciation methods, such as 16S rDNA sequencing. Finally, the design of degenerate primer pairs for some methods permitted the typing of related species; for example, the C. hyointestinalis primer pairs could be used to type C. fetus strains. Therefore, these novel Campylobacter MLST methods will prove useful in differentiating strains of multiple, emerging Campylobacter species.

Laboratory-based surveillance of human verocytotoxigenic Escherichia coli infection in the Republic of Ireland, 2002-2004.

The aim of this study was to examine the frequency and distribution of human verocytotoxigenic Escherichia coli (VTEC) O157 and non-O157 in the Republic of Ireland, and also to examine the presence of virulence genes in these isolates. This genetic information combined with phenotypic tests was used to produce a complete laboratory-based surveillance of human clinical VTEC infection in the Republic of Ireland between 2002 and 2004. Between January 2002 and December 2004 a total of 207 VTEC isolates were studied (one isolate per patient), 185 (89 %) of these were E. coli O157. The remaining 22 (11 %) were non-O157 E. coli, made up of 15 (7.2 %) E. coli O26, one (0.5 %) E. coli O103, one (0.5 %) E. coli O146, one (0.5 %) E. coli O145, two (1 %) E. coli O111 and two (1 %) ungroupable VTEC. These isolates originated from the eight health boards in the Republic of Ireland and represented over 90 % of the clinical cases of VTEC in the Republic of Ireland during this period. The results showed that VTEC O157 was the predominant serogroup and had a predominant toxin genotype of VT2 alone. Phage type 32 was the most common phage type of E. coli O157 identified. Non-O157 VTEC was a small proportion of all VTEC (10 % in 2002, 8 % in 2003, 15.5 % in 2004). In 2004 it was noted that there was an increase in the number and variety of non-O157 VTEC strains; however, this requires further monitoring in the future to see if this trend is sustained. It was also noted throughout the study period that the incidence of VTEC was higher in rural areas. Implementation of real-time PCR for the detection and subtyping of VTEC has aided outbreak investigations and is important for enhanced surveillance of VTEC in the Republic of Ireland.