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To increase the rate of E. coli isolation from samples, different enrichment methods have been developed to enhance the growth of E. coli (O157 and non-O157) in samples prior to culturing on selective media. This is of particular importance when the pathogen is present in low densities compared with the background microflora in samples (e.g. faecal samples or minced beef). Commonly used enrichment media are buffered peptone water (BPW), tryptic soy broth (TSB), and E. coli broth, which can be modified with antibacterial additives, for example BPW with vancomycin, cefsulodin and cefixime (BPW + vcc) [218], TSB with novobiocin [219] or vancomycin, cefsulodin and cefixime [220], or E. coli broth with novobiocin (mEC + n) [221], to suppress the growth of competitive natural bacteria in samples.

Several studies have evaluated the efficacy of these media on various types of samples, but often providing conflicting results. For example, Seo et al. [222] reported that modified BPW with casaminoacids was the most effective enrichment for STEC O157 in ground beef, whereas Heuvelink et al. [223] found mEC + n to be the most efficacious media for selective enrichment of STEC in minced beef. Sanderson et al. [224] assessed the efficacy of enrichments for STEC O157 in bovine faeces and found no statistically significant differences between TSB modified with cefixime, vancomycin and tellurite, and TSB modified with cefixime and vancomycin. In contrast, Foster et al. [225] reported that BPW without antibacterial additives was a superior enrichment to isolate STEC O157 from bovine faecal samples, compared to BPW + vcc. Hara-Kudo et al. [226] noted that mEC + n at 42°C was the most effective enrichment method for isolation of both E. coli O157 and O26 from inoculated ground beef and radish sprout samples, whereas Catarame et al. [227] observed

optimum enrichment for E. coli O26 in beef samples using TSB modified with cefixime, vancomycin, and potassium tellurite at 41.5°C.

2.5.2 Culture

Based on the reported inability of most E. coli O157 to ferment sorbitol, sorbitol-MacConkey agar (SMAC) has been developed as a selective medium to differentiate E. coli O157 from other E. coli serogroups and to advance the isolation of E. coli O157:H7 from faecal samples by direct culture [36]. The selectivity of SMAC agar has been further improved by the supplementation with antibiotics cefixime [228] and potassium tellurite [229], creating cefixime-tellurite sorbitol-MacConkey agar (CT-SMAC) [230]. Cefixime suppresses the growth of Proteus species, while tellurite suppresses most gram-negative organisms. CT- SMAC culture plates are commonly used in diagnostic laboratories for culture isolation of E. coli O157 from faecal samples, however, despite the supplementation with cefixime and tellurite, the test specificity of these plates is not 100% and false-positive isolates are still detected. Furthermore, sorbitol fermentation does not exclude the carriage of stx genes in E. coli O157 as reported by Gunzer et al. [231]. They isolated sorbitol-fermenting, Stx2- producing E. coli O157:Hˉ from patients with HUS and diarrhoea, highlighting the need to

screen also sorbitol-fermenting colonies for potentially pathogenic STEC O157, which otherwise could be missed. In addition, Karch et al. [232] indicated that some sorbitol- fermenting toxin-producing E. coli O157:Hˉ are sensitive to the antibacterial supplements in

CT-SMAC agar, providing evidence of reduced test sensitivity of CT-SMAC plates.

Similarly, STEC O26 was reported to be unable to ferment rhamnose, in contrast to many other E. coli and some stx-negative O26 strains. Hence, to differentiate STEC O26 from other STEC serogroups, a rhamnose-MacConkey agar (RMAC) has been developed and supplemented with cefixime and potassium tellurite to create CT-RMAC agar [233]. CT- RMAC has been tested as an optimum agar for recovery of STEC O26 [227, 234]. However, Evans et al. [235] observed a test sensitivity of 97% for CT-RMAC, when used in combination with tryptone bile X-glucuronide (TBX) agar to recover STEC O26 from ovine and bovine faecal samples, indicating that some STEC O26 strains could be sensitive to the antibacterial supplements in CT-RMAC agar. Furthermore, they also tested a collection of 96 bovine E. coli O26 strains (previously screened for the presence of virulence genes stx1, stx2, eae, and ehxA) for rhamnose fermentation and noted that 5.0% (2/40) and 19.2% (5/26) of

stx-positive and stx-negative E. coli O26 isolates, respectively, fermented rhamnose. Their findings highlighted the importance to screen all colony types, as rhamnose fermentation does not exclude the carriage of stx genes in E. coli O26, which could lead to some STEC O26 being overlooked. Rhamnose fermentation is also not a phenotypic characteristic of aEPEC O26 strains as shown in a study by Aktan et al. [236]. They observed rhamnose-fermenting and non-fermenting strains among ovine and bovine aEPEC O26 isolates from the UK, providing further evidence of the phenotypic diversity of aEPEC O26 strains prevalent in ruminants.

Culture isolation of E. coli may also be affected by cells being viable but non-culturable when exposed to natural stress factors [237]. This phenomenon has been observed for E. coli and STEC and a large number of other bacterial pathogens such as Campylobacter spp., Helicobacter pylori, Legionella pneumophila, Listeria monocytogenes, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Salmonella and Shigella spp., and Vibrio cholera [237].

2.5.3 Immunomagnetic separation (IMS)

IMS is a technique applied to increase the sensitivity of E. coli isolation from enriched samples and is likely to occur through the concentration of E. coli relative to the background microbiota, which may inhibit growth of STEC on selective culture media. IMS is based on the immunological binding of the target organism followed by physical separation from the mixed enrichment culture, resulting in a concentration of the target organism. In brief, magnetic beads coated with polyclonal antibodies against surface antigens of E. coli O157 or O26 are added to the enriched sample to bind with E. coli. Using external magnets, magnetic beads are then separated from other sample material and plated on selective medium. Several studies have shown that IMS is a sensitive method for isolation of E. coli O157 and other non- O157 serogroups from artificially mixed bacterial cultures, inoculated and naturally contaminated samples of meat and bovine faeces [238-242].