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Applied and Environmental Microbiology, March 2003, p . 1858-1860, Vol . 69, No . 3 An Improved Enrichment Broth for Isolation of Escherichia coli O157, with Specific Reference to Starved Cells, from Radish SproutsShin Sata,1 Tomohiko Fujisawa,1 Ro Osawa,2* Atsushi Iguchi,2 Shiro Yamai,1 and Toshio Shimada3 Kanagawa Prefectural Public Health Laboratory, Asahi-ku, Yokohama 241-0815,1 Department of Bioscience, Graduate School of Science and Technology, Kobe University, Nada-ku, Kobe 657-8501,2 Department of Bacteriology, National Institute of Infectious Diseases, Toyama, Shinjuku-ku, Tokyo 162-8670, Japan3 Received 8 July 2002/ Accepted 13 December 2002
Despite these incidences of infection, radish sprouts are still commercially available in Japan, being frequently consumed by the public . It is therefore of paramount importance to establish an appropriate method by which to detect possible E . coli O157 contamination of this particular food item . In this connection, we have demonstrated that enrichment cultures using a selective medium and/or at a high temperature are unsuitable for the isolation of E . coli O157 from water samples (16) . More recently, Fujisawa et al . have reported that many bacteria that formed colorless colonies similar to those of E . coli O157 on sorbitol MacConkey agar containing cefixime and tellurite (CT-SMAC) were present in radish sprouts, causing difficulty in selecting E . coli O157 colonies on the plate (9) . Here we describe a novel enrichment culture method that is designed to facilitate the growth of both starved and unstarved cells of E . coli O157 but suppress the growth of concomitant E . coli O157-like colonies on CT-SMAC for successful isolation of E . coli O157 from radish sprouts . The bacterial strains used included 19 strains of Shiga toxin-producing E . coli O157:H7 or NM from various sources such as patient or cattle feces and foods and 15 strains of non-E . coli O157 bacteria that had been isolated from commercially available radish sprouts during our routine practice . These non-E . coli O157 bacteria were all gram-negative rods that formed colorless or slightly pinkish colonies on CT-SMAC plates, some remarkably resembling those of E . coli O157 . Of these 15 strains, 5 were glucose fermenting but not sorbitol fermenting and 10 were non-carbohydrate fermenting . Four of these non-sorbitol-fermenting strains were identified by commercially available identification kits (API 20E and API 20NE; API System, Montalieu-Vercieux, France) as Enterobacter cloacae, Aeromonas hydrophila, or Hafnia alvei . Five of the non-carbohydrate-fermenting strains were identified as Pseudomonas aeruginosa, Alcaligenes xylosoxidans, Ralstonia pickettii, or Acinetobacter lwoffii, but the identities of the other five strains could not be determined . We prepared two different enrichment media: (i) buffered peptone water (BPW; Oxoid, Basingstoke, England) and (ii) BPW supplemented with 0.5% sodium thioglycolate (STG; Wako Pure Chemical Co . Ltd., Osaka, Japan) (BPW-STG) . It should be noted that STG has a reducing process, thereby making the liquid medium anaerobic and hence unsuitable for the growth of aerobes (15) . For studies of growth on artificially contaminated radish sprouts, we used BPW, STG-BPW, and modified EC broth (Eiken Chemical Co., Ltd., Tokyo, Japan) containing novobiocin (Sigma Chemical Co., St . Louis, Mo.) at a final concentration of 20 mg/liter (mEC+n) . Starved and unstarved cells of E . coli O157 were prepared as described previously (9) . Briefly, test strains that had been cultured at 37°C for 18 h in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) were washed with sterilized deionized water and resuspended in sterilized deionized water . Bacterial cells immediately after suspension and those kept in the dark at 23°C over a 3-week period were considered unstarved and starved cells, respectively . First, an appropriately diluted suspension (0.1 ml) containing 1.1 x 103 to 2.6 x 103 starved or unstarved cells of E . coli O157 and that containing 1.0 x 103 to 6.6 x 103 unstarved cells of the non-sorbitol- and the non-carbohydrate-fermenting strains were each inoculated into 10 ml of BPW and STG-BPW and incubated aerobically or anaerobically in jars using Anaeropack Kenki (Mitsubishi Gas Chemicals Co . Ltd., Tokyo, Japan) at 36°C for 18 h . After incubation, each culture of E . coli O157 and the other strains was diluted with sterile saline and 0.1-ml volumes of serial 10-fold dilutions onto duplicate plates of heart infusion agar (Eiken) and then incubated at 36°C for 24 h . After incubation, the colonies on the agar plates were counted to evaluate growth . Regardless of the culture conditions, the growth of both starved (1.8 x 108 to 3.8 CFU/ml) and unstarved (1.8 x 108 to 4.0 CFU/ml) cells of all E . coli O157 isolates was comparable . All of the non-carbohydrate-fermenting strains had much less growth when incubated anaerobically or in the presence of STG since most are obligate aerobes . Only one of the non-sorbitol-fermenting strains showed such suppressed growth when incubated anaerobically or in the presence of STG . Second, 3 g of radish sprouts and 0.1 ml of a bacterial suspension containing 1.3 x 103 to 3.2 x 103 starved or 0.5 x 103 to 1.7 x 103 unstarved cells of six randomly selected E . coli O157 isolates were added to 27 ml of BPW, STG-BPW, or mEC+n in sterilized screw-cap centrifuge tubes in which the initial concentration of E . coli O157 cells was adjusted to approximately 10 to 100 CFU/ml of broth medium . The experimentally contaminated radish sprouts thus prepared were incubated aerobically or anaerobically at 36 or 42°C for 18 h . It should be noted that a preliminary microbiological test confirmed the original radish sprouts as negative for E . coli O157 contamination . After incubation, 0.1-ml volumes of serial 10-fold dilutions of the spent medium were spread onto CT-SMAC agar plates (sorbitol MacConkey agar no . 3 [Oxoid] containing a solution of cefixime and tellurite [Selectivial; Mast Group Ltd., Merseyside, United Kingdom]) . After incubation at 36°C for 20 to 22 h, the number of colonies that grew on the plates was determined . For differentiation of E . coli O157 colonies from others, colorless or slightly pinkish colonies on CT-SMAC were picked up and then tested by a commercial latex agglutination test kit (UNI; Oxoid) in order to determine whether they were E . coli O157 or not . The number of cases that showed a maximum rate of recovery (40 to 100%) of E . coli O157 colonies from the total number of colonies grown on CT-SMAC and a minimum rate of recovery (0 to 7%) of other colorless colonies (high recovery) and the number of cases in which E . coli O157 was not isolated from six experimentally contaminated radish sprouts (false negative) are summarized in Table 1 . For the radish sprouts contaminated with unstarved E . coli O157 cells, both aerobic and anaerobic enrichments with mEC+n at 42°C showed high recovery in three out of six cases but failed to isolate any E . coli O157 in one or two cases, yielding false-negative results . For the radish sprouts with the starved cells, aerobic enrichments with STG-BPW at 36°C showed high recovery of E . coli O157 in five out of the six cases whereas both aerobic and anaerobic enrichments with mEC+n at 36 or 42°C failed to isolate E . coli O157 in three or four cases .
We are grateful to R . A . Whiley of the Department of Oral Microbiology, St . Bartholomew's and Royal London School of Medicine and Dentistry, for valuable comments on an earlier draft of this paper .
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