Box plot of the concentration, expressed in log CFU per milliliter, of all STEC strains after 7 h of enrichment in the five enrichment media, subdivided by type of stress applied. The line within the box marks the median. The boundaries of the box represent the 25th and 75th percentiles. The upper and lower adjacent values are calculated as defined by Tukey (29). Significant differences ( P , 0.05) are indicated with a different letter. BPW, buffered peptone water; BPWp, buffered peptone water plus sodium pyruvate; mTSB, modified tryptic soy broth; SEB, Shiga toxin–producing E. coli enrichment broth . 

Contexts in source publication

Context 1

... coli is present in the natural intestinal microbiota of mammals and in a variety of habitats, including soil, water, sediment, and food. Within the species, some strains of E. coli can cause human diseases. The most severe of these E. coli are linked to strains belonging to the pathogenic group of the Shiga toxin– producing E. coli (STEC) (5, 27) . This group was first recognized in 1982 in patients with hemorrhagic colitis and was consequently named enterohemorrhagic E. coli (36) . The causative agent responsible was STEC serogroup O157:H7. This serogroup is characterized by several virulence genes, such as Shiga toxin genes stx 1 and stx 2 and the eae gene encoding an intimin. The symptoms range from nonbloody diarrhea, hemorrhagic colitis, and hemo- lytic uremic syndrome to thrombotic thrombocytopenic purpura. STEC O157:H7 remains the most common serogroup within the STEC group. The development of a detection and isolation method has therefore been targeted to this serogroup, resulting in the widely accepted standard ISO 16654:2001, which is based on the inability of most O157:H7 strains to ferment sorbitol (15) . However, non- O157 STEC strains are being increasingly recognized and reported as important foodborne pathogens worldwide. In Europe, the most frequently isolated non-O157 serotypes belong to the serogroups O26, O91, O103, O111, and O145 (9, 19, 22, 34) . Because the above-mentioned method for the detection and isolation of O157:H7 fails to detect these serogroups, a new method should be formulated to detect and isolate non-O157 STEC serogroups in a broad variety of matrices. In matrices such as foodstuffs, low numbers of STEC cells may have a large impact on public health and are often found in the presence of high numbers of closely related competitor organisms. Therefore, before any isolation step can be performed, a suitable initial enrichment step is required. This step should ensure the growth of STEC cells while inhibiting that of the competitor organisms. In addition, it should be taken into account that STEC cells are often impaired by sublethal injuries sustained during food processing and storage (e.g., exposure to salt, acid, cold, heat, and freeze stresses). Despite the common occurrence of these stresses, little information is available concerning the growth of non-O157 STEC after exposure to the various physical and chemical stresses present in foods (25) . A better understanding of the influence of stress types such as acid, cold, and freeze on the growth of non-O157 STEC strains compared with nonstressed cells is needed. Furthermore, injured cells may become susceptible to selective agents included in enrichment media. Consequently, these media might be insufficient to resuscitate injured or stressed cells during the enrichment phase, possibly resulting in false-negative results. The selection of a suitable enrichment medium that facilitates the isolation and confirmation of STEC in foods is of great importance (2, 5, 18) . Several enrichment media are now commonly in use. The International Organization for Standardization recommends the use of modified tryptic soy broth with the addition of novobiocin (16 mg/liter) or acriflavin (12 mg/liter) for the enrichment of food samples and dairy products, respectively (in TS 13136:2012). For the enrichment of stressed bacteria, buffered peptone water (BPW) is recommended (5, 16) . Although the International Organization for Standardization/ Technical Specification specifies an enrichment period of 18 to 24 h, multiple studies have proven the advantageous effect of a shorter enrichment period. This may be owing to overgrowth of competing organisms and the release of PCR- inhibiting organic components during overnight incubation (14, 17, 31) . The purpose of this study was to compare the growth dynamics of several pure cultures of non-O157 STEC strains in five enrichment media developed for the enrichment of members of the Enterobacteriaceae, including E. coli and coliforms, STEC, and other gram-negative pathogens from food samples. The following enrichment media were evaluated: modified tryptic soy broth (mTSB) as described by Doyle and Schoeni ( 6 ), which differs from TSB by the addition of bile salts and phosphate buffer; BPW with and without the addition of sodium pyruvate (35) ; and two selective enrichment media, i.e., Brila and STEC enrichment broth (SEB). In summary, the aims of the present study were to (i) determine the effect of acid, cold, and freeze stresses on strains belonging to non-O157 STEC serogroups and (ii) compare the capabilities of five enrichment media to resuscitate these stressed bacteria. conditions. The effect of plating nonstressed STEC cells on a selective isolation medium was investigated. No significant difference was observed between the counts of all strains on nonselective TSA and selective TBX media ( P . 0.05) (data not shown). The selective components in TBX did not inhibit the growth of nonstressed STEC cells. Table 2 shows that cold and freeze stresses caused lethal injuries in all strains tested, although strain variations were observed. Furthermore, the percentage of lethally injured cells was, on average, higher after freeze stress compared with cold stress. In contrast to cold and freezing, acid stress did not cause lethal injury for the majority of strains. Considering the percentage of sublethally (stressed) cells, acid stress generally had the least influence. Freeze stress caused, on average, the most sublethally injured cells (89.1% 6 8.8%) (Table 2). Growth in different enrichment media. After an enrichment period of 7 h, the highest counts were observed in BPW, regardless of the type of stress applied ( P , 0.05) (Fig. 1). All stressed strains grew in this 7-h period above 3 log CFU/ml, except for a few freeze-stressed STEC strains grown on mTSB, Brila, and SEB. After 24 h of enrichment, regardless of the enrichment medium, strain, or type of stress, all counts varied between 8.26 and 9.11 log CFU/ml. Enrichment in BPW and BPWp showed no significant differences in duration of the lag phase for nonstressed, acid- stressed, and freeze-stressed cells ( P . 0.05) (Fig. 2). However, resuscitation of cold-stressed cells in BPWp significantly prolonged the lag phase ( P , 0.01) compared with the lag phase in BPW. A significantly shorter lag phase was demonstrated after freeze stress in BPW and BPWp compared to that in the enrichment media mTSB, Brila, and SEB ( P , 0.001). A significantly lower maximum growth rate was observed during enrichment on BPWp after cold ( P , 0.05) and freeze ( P , 0.05) stresses than on BPW (Fig. 3). After acid stress, the SEB enrichment showed a significant longer lag phase than all other media ( P , 0.05) and a significantly lower maximum growth rate compared with BPW and BPWp ( P , 0.05). Moreover, the maximum growth rate was significantly lower on SEB during enrichment of nonstressed cells compared with rates on BPW and Brila ( P , 0.05). The effect of stress on the growth of E. coli O157:H7 is well documented; however, it cannot be assumed that non- O157 STEC strains will behave similarly to O157:H7 strains when exposed to the same stress conditions. The acid tolerance observed in the present study has already been described in multiple studies on O157:H7 and non-O157 serogroups (25, 32) . To take advantage of this feature, an acid treatment procedure to inhibit the growth of competitor organisms can be used as a preliminary selective step before the isolation. Multiple studies have shown the usefulness of this procedure (10, 11, 28) . The observed acid tolerance in the present study was comparable to that in the study of Jasson et al. (18) using the same stress conditions, but their work focused on O157:H7 strains. In our study, some strain variations were observed within the STEC serogroups. These findings support the statement of Benjamin and Datta (3) that the ability of STEC to survive in an acidic environment depends on the strain and not on a ...

Context 2

... in different enrichment media. After an enrichment period of 7 h, the highest counts were observed in BPW, regardless of the type of stress applied (P , 0.05) (Fig. 1). All stressed strains grew in this 7-h period above 3 log CFU/ml, except for a few freeze-stressed STEC strains grown on mTSB, Brila, and SEB. After 24 h of enrichment, regardless of the enrichment medium, strain, or type of stress, all counts varied between 8.26 and 9.11 log ...

Context 3

... might imply that these pathogens do not all succumb to the freeze stress to which they were exposed. A sufficient number of cells remain viable enough to pose a health threat (1, 8). Therefore, resuscitation in a suitable enrichment medium before isolation is essential to avoid false-negative test results (18). ...