Detection of Hemolysin Variants of Shiga Toxin-Producing Escherichia coli by PCR and Culture on VancomycinCefixime-Cefsulodin Blood Agar

Abstract

The presence of a hemolysin-encoding gene, elyA orhlyA, from Shiga toxin-producing Escherichia coli (STEC) was detected by PCR in each of 95 strains tested. PCR products of elyA from human STEC isolates of serovars frequently detected in Germany, such as O157:H−, O103:H2, O103:H−, O26:H11, and O26:H−, showed nucleotide sequences identical to previously reported ones for O157:H7 and O111:H− strains. Compared to them, four elyA amplicons derived from human isolates of rare STEC serovars showed identity of about 98% but lacked anAluI restriction site. However, the nucleotide sequence of an amplicon derived from a porcine O138:K81:H− STEC strain was identical to the corresponding region of hlyA, encoding alpha-hemolysin, from E. coli. This hlyAamplicon showed 68% identity with the nucleotide sequence of the corresponding elyA fragment. It differed from theelyA PCR product in restriction fragments generated byAluI, EcoRI, and MluI. Of the 95 representative STEC strains, 88 produced hemolysin on blood agar supplemented with vancomycin (30 mg/liter), cefixime (20 μg/liter), and cefsulodin (3 mg/liter) (BVCC). The lowest added numbers of two to six STEC CFU per g of stool or per ml of raw milk were detectable on BVCC plates after seeding of the preenrichment broth, modified tryptic soy broth (mTSB) supplemented with novobiocin (10 mg/liter), with 16 STEC strains. These strains represented the seven prevailing serovars diagnosed from German patients. However, with ground-beef samples, PCR was essential to identify the lowest added numbers of two to six STEC CFU among colonies of hemolyzing Enterobacteriaceae, such as Serratia spp. and alpha-hemolysin-producing E. coli. We conclude that preenrichment of stool and food samples in mTSB for 6 h followed by overnight culturing on BVCC is a simple method for the isolation and presumptive identification of STEC.

The detection of Shiga toxin-producing Escherichia coli in diagnostic bovine faecal samples using vancomycin-cefixime-cefsulodin blood agar and PCR

“…As well as detecting isolates with ehxA it was also useful in detecting STEC as 14 (51.9%) of the 27 samples containing ehxA on primary culture contained Shiga toxin. This study supports the ¢ndings of Lehmacher [3] who reported that BVCCA facilitates the detection of haemolysin variants of STEC. There were some BVCCA positive colonies which did not contain ehxA.…”

“…There were some BVCCA positive colonies which did not contain ehxA. These false positives may occur when E. coli isolates produce a narrow and turbid halo because of a low expression of alpha-haemolysin or when vancomycin enhances the haemolysis of some non-STEC which may show weak haemolysis [3]. It is also possible that the faecal multiplex PCR primers [7] may not have detected some haemolysin variants [4] or the mEC broth may have had an inhibitory e¡ect on the PCR assay.…”

The isolation of enterohaemorrhagic Escherichia coli O111:H‐from Australian cattle

“…The CAV isolate used in this study (CAU269/7) was obtained from D O'Rouke and T Bagust (Faculty of Veterinary Science, The University of Melbourne) and was originally isolated from a commercial breeder flock in Australia. 7 The virus was cultured in the Marek's disease virus transformed lymphoblastoid cell line, MDCC-MSB1, 2 based on the method described by McNulty et al 8 Inoculation of CAU269/7 into MDCC-MSB1 cells produced cytopathic effects consistent with that of other reported CAV isolates 9 and was characterised by the appearance of enlarged, misshapen cells within 40 h. Total cell degeneration was apparent within 96 h after infection. Infected cells were stained for the CAV-specific protein, VP3, in an indirect immunofluorescence assay (IFA) following the method of Renshaw et al 10 using the mouse derived monoclonal antibody JCU/CAV/1C1 (JCU TropBio, Townsville, Queensland).…”

“…All samples were cultured for salmonella using Muller-Kaufmann Tetrathionate and Rappaport-Vassiliadis Enrichment broths (Oxoid, Basingstoke), for yersinia using Yersinia Selective agar (Oxoid, Basingstoke), and were also cultured directly onto Vancomycin-Cefixime-Cefsulodin Blood agar (BVCCA) to facilitate detection of haemolysin variants of Shiga toxinproducing E coli. 8 The faecal sample from the cow yielded one BVCCA positive E coli colony (that is, a colony with a narrow and turbid zone of haemolysis, Figure 1). Two of the faecal samples from calves 1 and 2 yielded two and one BVCCA positive E coli colonies respectively and the two samples from calves 3 and 4 were negative.…”

“…The O84: H2 serotype has previously been reported from healthy dairy cattle in the USA and has also been associated with human disease. 14 Over the last few years there has been increased awareness of the importance of non-O157 EHEC in human disease; there have been reports from Germany 8,[15][16][17] Belgium, 18 Canada 19 and USA. 4 This was the main pathogen isolated from the first reported and largest EHEC outbreak in Australia, which occurred in South Australia in 1995, which was associated with the consumption of contaminated mettwurst.…”