Salmonella enterica serovar Typhimurium was isolated from the intestinal contents of Rattus rattus and Rattus norvegicus house rats captured at two buildings, designated buildings J and YS, in Yokohama City, Japan. From October 1997 to September 1998, 52 of 339 (15.3%) house rats were found to carry Salmonella serovar Typhimurium definitive phage type 104 (DT104). In building J, 26 of 161 (16.1%) house rats carried DT104 over the 1-year study period, compared to 26 of 178 (14.6%) rats in building YS. The isolation rates of DT104 from R. rattus and R. norvegicus were similar in the two buildings. Most DT104 strains from building J (24 of 26) showed resistance to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline and contained both the 1.0-and 1.2-kbp integrons, carrying genes pse1, pasppflo-like, aadA2, sulI, and tet(G). All DT104 strains from building YS were resistant to ampicillin and sulfisoxazole, and had the 1.2-kbp integron carrying pse1 and sulI. Cluster analysis of pulsed-field gel electrophoresis patterns of BlnI-digested DT104 DNAs showed that 22 of 26 DT104 strains from building J and 24 of 26 strains from building YS could be grouped into separate clusters each specific for the building origin. These results indicated that DT104 strains were prevalent in house rat colonies in each building and suggest that house rats may play an important role in the epidemiology of DT104.
Shiga toxin-producing Escherichia coil (STEC) O157 were isolated from processed salmon roe which had been a suspected food item in sporadic infections which occurred in Japan in 1998. A total of 45 samples of the processed salmon roe were pre-enriched in trypticase soy broth (TSB) at 36 degrees C for 6 h and novobiocin-supplemented modified EC broth (mEC-NB) at 42 degrees C for 18 h. After the pre-enrichments, the cultures were examined for possible occurrence of STEC O157, using an immunomagnetic separation (IMS) method. From the examination, a total of 84 strains of STEC O157:H7 that were positive for both stx 1 and stx 2 genes were isolated. By applying the most-probable-number technique, it was estimated that the number of STEC O157 was in the range of 0.73-1.5 per 10 g of the processed salmon roe. Subsequent analysis of the isolates by a pulsed-field gel electrophoresis (PFGE) revealed a pattern commonly seen in 82 isolates and another pattern in two isolates. Clinical isolates from 7 patients also showed an identical pattern to those of the 82 isolates and one isolate from a patient showed the other pattern identical to those of the two isolates. The isolates were found to belong to the phage type 14.
A modified version of sorbitol MacConkey medium containing cefixime and tellurite (CT-SMAC medium) was produced by adding salicin and 4-methylumbelliferyl--D-galactopyranoside to CT-SMAC medium; this medium was designated CT-SSMAC medium and was used to isolate Escherichia coli O157:H7 from radish sprouts. Of 101 non-E. coli bacteria isolated from radish sprouts that produced colorless colonies similar to colonies of E. coli O157:H7 grown on CT-SMAC medium, 92 (91%) formed colonies that were red to pink or were -galactosidase negative and colorless on CT-SSMAC medium. On the other hand, colonies of E. coli O157:H7 strains were colorless and -galactosidase positive on CT-SSMAC medium. Our results suggest that CT-SSMAC medium is more selective than CT-SMAC medium for isolating E. coli O157:H7.Sorbitol MacConkey medium (SMAC medium) (11) and sorbitol MacConkey medium containing cefixime and tellurite (CT-SMAC medium) (1, 14) were described as media that can be used for isolation of Escherichia coli O157. Recently, an optimized method for detecting verocytotoxigenic E. coli in food by using CT-SMAC medium was introduced. However, it seems that modification of CT-SMAC medium is necessary to isolate E. coli O157:H7 from radish (Raphanus sativus) sprouts. During a study of the occurrence of E. coli O157:H7 in radish sprouts grown hydroponically, many colorless colonies similar to E. coli O157:H7 colonies, which do not produce acid from sorbitol, grew on CT-SMAC medium. Sata et al. (13) found that using modified E. coli broth supplemented with novobiocin or modified Trypticase soy broth supplemented with novobiocin as a liquid enrichment medium for E. coli O157:H7 is not suitable for isolating injured E. coli O157:H7 cells from water systems. Therefore, these authors recommended that a nonselective liquid enrichment medium should be used for isolation of E. coli O157:H7 (13). From these viewpoints, the role of selective agar plates in isolating E. coli O157:H7 is very important. In this study, we investigated using a modification of CT-SMAC medium for isolation of E. coli O157:H7 from radish sprouts.The strains of E. coli O157:H7 and O157:NM (nonmotile) used in this study are listed in Table 1. Five clinical isolates from patients, six bovine fecal isolates, and eight food or environmental isolates were used. Nine strains (NIID 2, NIID 457, NIID 23, NIID 42, NIID 437, NIID 1124, NIID 1856, NIID 1646, and NIID 1496 were provided by H. Watanabe (National Institute of Infectious Diseases, Tokyo, Japan). Details concerning the E. coli O157:H7 and E. coli O157:NM strains have been described previously (13). The 101 gram-negative aerobic and facultatively anaerobic rod-shaped strains that were not E. coli strains were isolated from radish sprouts in our laboratory (Table 2). Ten lots of radish sprouts marketed in Japan were used. Samples (25 g) were incubated in 225 ml of buffered peptone water (Oxoid, Basingstoke, Hampshire, England) at 36°C for 18 h. After incubation, one loopful of each culture was spread onto CT-SMA...
Escherichia coli O157 strains starved in sterile deionized water (SDW) and filter‐sterilized natural river water (SRW) were investigated with specific reference to their culturability in selective and non‐selective media. Growth of the strains starved in both SDW and SRW were markedly suppressed with time in selective liquid media such as modified trypticase soy broth supplemented with novobiocin (mTSB+n) and modified E. coli broth supplemented with novobiocin (mEC+n). This suppression was more pronounced when incubated at 42 C than at 37 C, especially with mEC+n. By contrast, such growth suppression was seldom observed when cultured at 37 C in non‐selective liquid media such as trypticase soy broth (TSB) and buffered peptone water. In mEC+n at 42 C, the non‐starved cells from overnight cultures with an initial density of less than 103 colony‐forming units (CFU)/ml grew to the density of over 107 CFU/ml after 24 hr incubation, whereas those starved for 6 weeks in SRW were only to maintain their initial density or died off after 24 hr incubation under the same culturing conditions. These results indicated that the isolation of starved cells of E. coli O157 from water samples would be most difficult with selective enrichment or direct plating on the selective plate media. It is thus highly recommended that a “resuscitation” of the cells with non‐selective enrichment should be performed as a routine practice for maximum recovery of E. coli O157 from water systems.
Abstract:We have investigated the Shiga toxin genes of Shiga toxin-producing Escherichia coli (STEC) strains, using polymerase chain reaction (PCR) amplifying the full lengths of these genes. As a result, we found the Shiga toxin 2 gene which was insertionally inactivated by an insertion sequence (IS). This IS element was identical to IS1203v which has been also found in inactivated Shiga toxin 2 genes, and was inserted at the same site as in the previous paper. On the other hand, both Shiga toxin 2 genes were different (98.3% identity). These suggested that IS1203v independently inserted into each Shiga toxin 2 genes, and STEC strains possessing the insertionally inactivated Shiga toxin genes are most likely to have a wide distribution. Amplification of the full length of the Shiga toxin gene is one of the effective methods to detect the gene no matter where the IS element is included, Le., the insertion can be reflected in the size of amplicon. Key words: Shiga toxin-producing Escherichia coli, Insertion sequence, PCRShiga toxin-producing Escherichia coli (STEC) has been shown to be closely associated with diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (5). Isolates of STEC are known to produce either one or both of at least two immunologically distinct toxins referred to as Shiga toxin I (Stx 1) (also known as Verotoxin I) and Shiga toxin 2 (Stx2) (also known as Verotoxin 2). Stx consists of A and B subunits responsible for the inhibition of protein synthesis in the target cell and binding to the cell receptor, respectively (10). For identification of STEC, the techniques of reversed passive latex agglutination (RPLA) (4) and polymerase chain reaction (PCR) are generally used. In recent years, various methods based on PCR have been developed and successfully used on STEC. Although RPLA detects Stx proteins produced by STEC, PCR detects the stx genes encoding Stx. With respect to the detection of STEC using PCR, relations between the possession of the stx genes and the Stx productivities should be explained. In a recent study, Kusumoto et al (6) found novel stx2 genes from STEC strains of serotype 0157:H7 using *Address correspondence to Dr. Tadayuki Okitsu, Department of Bacteriology and Pathology, Kanagawa Prefectural Public Health Laboratory, 1-1-1 Nakao, Asahi-ku, Yokohama, Kanagawa 241-0815, Japan. Fax: 045-363-1037. E-mail: xh6t-okt@asahi-net.or.jp 319 PCR. The B subunit of those stx2 genes were insertionally inactivated by a l.3-kb insertion sequence (IS), designated IS1203v. IS elements are a large group of bacterial transposable DNA elements and cause various kinds of genetic rearrangements, such as deletions, inversions, duplications, and replicon fusions, by their ability to transpose (3, II). Accordingly, existence of the other STEC strains, possessing the stx genes with insertion of some IS elements, was expected. In the present study, we have investigated the stx genes of STEC strains isolated from various sources, and found an IS element in the stx2 gene of an STEC strain.The 274 s...
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