Shin Sata

Modification of Sorbitol MacConkey Medium Containing Cefixime and Tellurite for Isolation of Escherichia coli O157:H7 from Radish Sprouts

ABSTRACT A modified version of sorbitol MacConkey medium containing cefixime and tellurite (CT-SMAC medium) was produced by adding salicin and 4-methylumbelliferyl-β-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. coliO157:H7 grown on CT-SMAC medium, 92 (91%) formed colonies that were red to pink or were β-galactosidase negative and colorless on CT-SSMAC medium. On the other hand, colonies of E. coliO157:H7 strains were colorless and β-galactosidase positive on CT-SSMAC medium. Our results suggest that CT-SSMAC medium is more selective than CT-SMAC medium for isolating E. coliO157:H7.

Evaluation of sorbitol-salicin MacConkey medium containing cefixime and tellurite (CT-SSMAC medium) for isolation of Escherichia coli O157:H7 from raw vegetables.

The utility of CT-SSMAC medium (sorbitol-salicin MacConkey medium containing cefixime and tellurite) for the isolation of Escherichia coli O157:H7 from raw vegetables was investigated. The colonies of all E. coli O157:H7 and O157:NM strains tested were colorless and beta-galactosidase-positive on CT-SSMAC medium. Furthermore, the number of colorless colonies on the CT-SSMAC medium was less than that on the sorbitol MacConkey medium containing cefixime and tellurite (CT-SMAC medium) from several raw vegetable samples. All colorless colonies grown on CT-SSMAC medium from raw vegetable samples were beta-galactosidase-negative. These findings suggest that the CT-SSMAC medium is useful for the isolation of E. coli O157:H7 from raw vegetable samples.

An Improved Enrichment Broth for Isolation of Escherichia coli O157, with Specific Reference to Starved Cells, from Radish Sprouts

ABSTRACT An enrichment broth was developed for the efficient isolation of Escherichia coli O157 from radish sprouts. The broth was buffered peptone water containing 0.5% sodium thioglycolate (STG-BPW), which was designed to allow growth of E. coli O157 in starved and unstarved states. However, this medium suppressed the growth of non-carbohydrate-fermenting obligate aerobes whose colonial appearance on sorbitol MacConkey agar containing cefixime and tellurite (CT-SMAC) resembled that of E. coli O157. Both starved and unstarved cells of E. coli O157 experimentally inoculated into radish sprouts were successfully recovered with STG-BPW enrichment in all cases, most of which showed marked disappearance of E. coli O157-like colonies on CT-SMAC.

Growth of Starved Escherichia coli O157 Cells in Selective and Non‐Selective Media

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.

Identification of Shiga Toxin-Producing Escherichia coli Possessing Insertionally Inactivated Shiga Toxin Gene

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, i.e., the insertion can be reflected in the size of amplicon.

Isolation of Aeromonas Species from Patients with Sporadic Diarrhea and Characterization of Aeromonas hydrophila Isolates

A total of 16 strains of Aeromonas species were isolated from feces of 348 patients with sporadic diarrhea in western Kanagawa, Japan from 1996 to 1998. Of the 16 isolates, 7 were Aeromonas hydrophila, 1 was A. sobria and 8 were A. caviae. The strains of A. hydrophila were examined for hemolytic activities, hemolysin gene types and O-serogroups. Although all 7 strains of A. hydrophila showed hemolytic activities on sheep blood agar, in the test for hemolytic activities in culture supernatant, only 1 of the these strains showed no hemolytic activity against sheep erythrocytes. From the results of PCR assay, the tested strains of A. hydrophila were grouped into 2 hemolysin gene types of [ahh1 + ahh3 + aerA] (n = 6) and [ahh1 + aerA] (n = 1) both of which are recognized to be enteropathogenic. Five of the 7 strains of A. hydrophila belonged to serogroup O11. These results suggest that 7 strains of A. hydrophila isolates are recognized to be enteropathogenic strains and serogroup O11 is the major O-serogroup of enteropathogenic A. hydrophila in humans.