A Study on Selective Broths and Agar Media for the Isolation of Escherichia coli O157:H7 Serotype

Modified tryptic soy broth, lauryl sulfate tryptose broth, modified EC broth, sorbitol MacConkey agar and Fluorocult E. coli O157 agar were tested for the recovery of 3 strains of Escherichia coli O157:H7 against E. coli type 1 and Citrobacter freundii as common competitive microflora of raw meat products. Two-stepped trials showed that modified EC broth and lauryl sulfate tryptose broth were superior to modified tryptic soy broth as selective enrichment media while sorbitol MacConkey agar was clearly better than Fluorocult E. coli O157 agar as a selective agar medium. Recovery of E. coli O157:H7 was difficult when selective enrichment broth-growing cohabitant microflora were present at more than 100 folds of E. coli O157. One percent E. coli O157:H7 in the cohabitant flora was the minimum level for recovery. This is probably more important than the minimum recovery rate of culture media and/or conventional culture methods when E. coli O157:H7 exists without competitive microorganisms. Key Words: E. coli O157:H7, mTS Broth, mEC Broth, LST Broth, SMAC Agar. Escherichia coli O157:H7 Serotipinin Üzolasyonunda Selektif Broth ve Agar Besiyerleri †zerine Bir ‚alÝßma …zet : Bu alÝßmada, modifiye tryptic soy broth, lauryl sulphate broth, modifiye EC broth, sorbitol MacConkey agar ve Fluorocult E. coli O157 agar besiyerleri E. coli O157:H7 serotipinin Ÿ ßahit sußunun, iÛ et ŸrŸnlerinin tipik refakati florasÝ olan E. coli tip 1 ve Citrobacter freundii 'nin ikißer sußuna karßÝ geri alÝnmasÝnda denenmißtir. Üki aßamalÝ denemeler modifiye EC broth ve lauryl sulphate brothun modifiye tryptic soy brotha ve sorbitol MacConkey agarÝn Fluorocult E. coli O157 agara gšre daha iyi sonu verdiÛini gšstermißtir. Selektif besiyerlerinde gelißebilen refakati flora sayÝsÝ E. coli O157 serotipi sayÝsÝndan 100 mislinden fazla ise E. coli O157:H7' nin geri alÝnmasÝ zorlaßmaktadÝr. Bir diÛer deyiß ile, selektif besiyerlerinde gelißebilen refakati flora iinde E. coli O157:H7 oranÝ en dŸßŸk olarak %1 olmalÝdÝr. Bu oran, selektif besiyerleri ve/veya geleneksel izolasyon yšntemlerinde verilen minimum geri alÝnabilme sayÝsÝndan muhtemelen daha šnemlidir. Anahtar SšzcŸkler : E. coli O157:H7, mTS broth, mEC broth, LST broth, SMAC agar. cohabitant flora (5, 9, 10). While other E. coli serotypes are sorbitol positive, E. coli O157:H7 serotype is sorbitol negative and sorbitol MacConkey (SMAC) agar contains sorbitol instead of lactose. Therefore, while sorbitol positives produce pink to red colonies, sorbitol negatives produce colorless colonies on SMAC agar (11). The second difference between the O157 serotype and other serotypes is the β-glucuronidase (β-GUR) activity. While others are β−GUR positive, E. coli O157:H7 is β−GUR negative, i.e., other serotypes split 4methylumbelliferone glucuronide (MUG) and give fluorescence under 366 nm long-wave UV light whereas E. coli O157 cannot hydrolyze MUG. Some solid culture media have been developed on the basis of this difference, e.g., Fluorocult E. coli O157 agar and Fluorocult HC agar (5). In addition 5-bromo-4-chloro-3-indoxyl-§-Dglucuronic acid cyclohexyl-ammonium salt (BCIG) plus SMAC agar, phenol red sorbitol agar plus MUG, HC agar, SMAC agar supplemented with rhamnose and cefixime (RC) or potassium tellurite and cefixime (TC) have been developed and used in some investigations (4, 5, 12-14). The main disadvantage of conventional culture methods is the possibility of obtaining false negative results. Although E. coli O157:H7 may be differentiated mainly on the basis of its sorbitol and β−GUR negative properties, it is difficult to distinguish and isolate the suspected colonies among cohabitant colonies. A general laboratory practice is that 1 different colored colony (of the same size) can be easily separated among 100 colonies (150 colonies is the maximum level) on a 9 cm standard petri dish. In other words, 1% of well-grown colonies can be separated depending on their color and/or shape. The importance of the existence of a competitive flora in detection of E. coli O157 in screening studies has been described (11, 14). Hitchins et al. (15) reported difficulties due to high levels of contaminating coliforms which may mask the presence of E. coli O157:H7 strains when grown on SMAC agar. When a food (or environmental) specimen contains E. coli O157:H7, it is most probable that a high level of contaminating microflora is present. Many of the species present are suppressed at the selective enrichment but others, for example, E. coli type 1, can grow well in many of the culture media selective for E. coli O157:H7. Some cultural practices such as alteration of the incubation temperature and/or higher levels of bile salts inhibit the growth of E. coli O157:H7, whereas E. coli type 1 grows well under these conditions also (16, 17). When isolating E. coli O157:H7 by conventional cultural methods, the level of E. coli O157:H7 in a competitive microflora grown in selective enrichment broth must be at least 1% and our study is based on this hypothesis. The purpose of this study was the recovery of E. coli O157:H7 strain vs. E. coli type 1 and C. freundii, which are common cohabitants in animal-originated foods (18), by using different types of media. Materials and Methods Bacterial cultures: Three strains of E. coli O157:H7 serotype were collected from 3 different sources, i.e., Food Engineering Department of Hacettepe University (Turkey), Institute of Refik Saydam Public Health Center (Turkey) and Landbouw University (the Netherlands). For distinguishing the strains, their names were suffixed by (h1), (h2), and (h3) respectively. Two strains each of E. coli type 1 [suffixed by (c1) and (c2)] and C. freundii [suffixed by (f1) and (f2)] were isolated and identified by conventional biochemical tests (19). All 7 bacterial strains were maintained on nutrient agar slants and activated in nutrient broth at 37 ¡C for 18 h prior to analysis. None of the 7 bacterial strains was certified and/or categorized by national collections. Culture media: Among many selective media, 3 selective enrichment broths and 2 selective agar media were tested for recovery of E. coli O157:H7 in 2-stepped trials. The compositions of the media are as follows. mEC broth: Base medium was composed of tryptone (Oxoid) 2 %; lactose (Oxoid) 0.5 %; bile salts #3 (Difco) 0.112 %; K2HPO4 (Merck) 0.4 %; KH2PO4 (Merck) 0.15 %; and NaCl (Merck) 0.2 %. 20 mg/l filter sterilized novobiocin (Merck) was added to the autoclaved base medium after cooling to room temperature (10). mTS broth: Base medium containing tryptic soy broth (Merck) 3.0 %; bile salts #3 (Difco) 0.15 % and K2HPO4 (Merck) 0.15 % was prepared and 20 mg/l filter sterilized novobiocin (Merck) was added to the autoclaved base medium after cooling to room temperature. Composition of the tryptic soy broth was peptone (casein) 0.17 %; peptone (soy meal) 0.3 %; D-glucose 0.25 %; K2HPO4 0.25 % (9). LST broth: Commercially available dehydrated medium (Merck) was used (35.5 g/l) (20). A Study on Selective Broths and Agar Media for The Isolation of The Escherichia coli O157:H7 Serotype