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

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 10(3) colony-forming units (CFU)/ml grew to the density of over 10(7) 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.     

Stimulative effect of skipjack tuna soluble extract on pepsin-like protease in the stomach of rockfish (Sebastes schlegelii) using an in vitro perfusion method

The effect of intra-gastric infusion of skipjack tuna (Katsuwonus pelamis) muscle soluble extract and of carnosine, anserine and histidine, on pepsin-like protease activity was studied in the isolated, externally batch-cultured stomach of the rockfish (Sebastes schlegelii). Stomach was isolated from the fish, then intragastrically infused with the above solutions or with a balanced saline solution (control solution) as the stimulant at 0.1 mL/min for 6h. Intra-gastric efflux was collected for measurement of pepsin-like protease activity. Skipjack tuna soluble extract, but not carnosine, anserine or histidine alone caused significant enhancement of pepsin-like protease activity during the infusion. Pepsin-like protease activity from skipjack tuna soluble extract responded immediately after infusion and was kept for 150 min after infusion. Stomach motility at the end of infusion was also observed. These results suggest that isolated stomach can receive mucosal side stimulants in vitro. It is likely that some effective components for stomach digestion in fish exist in skipjack tuna soluble extract.     

Effector cells working for rejection of Ad12 tumors in mice

Immune spleen cells (ISC) capable of inhibiting the growth of adenovirus type 12 (Ad12) tumors were raised in C57BL/6 mice by immunization with Ad12, fractionated according to their affinity for plastic and nylon-wool substrates or treated with various antisera plus complement, and subjected to the tumor-neutralization test (Winn) to define the effector cells for the cell species. Full antitumor activity of ISC was recovered in the cell fractions nonadherent to the two substrates; the antitumor activity of ISC was abrogated entirely by anti-Thy-1,2 serum and almost entirely by anti-Lyt-2.2 ascites fluid plus complement. These results clearly indicate that T-lymphocytes, particularly those bearing Lyt-2.2 antigen, are the principal effectors in ISC against Ad12 tumors in animals.     

Production of thermostable direct hemolysin by Vibrio parahaemolyticus enhanced by conjugated bile acids.

The effects of conjugated bile acids, glycocholic acid, and taurocholic acid (TC) on production of thermostable direct hemolysin (TDH) by Vibrio parahaemolyticus were determined by a reversed passive latex agglutination assay against TDH. The amount of TDH excreted in growth medium containing either glycocholic acid or taurocholic acid (5 mM/liter) was, on a per-cell basis, 4- to 16-fold greater than that excreted in medium without the bile acids. The amounts of TDH released from lysed cells grown with the bile acids (5 mM/liter) were 4- to 32-fold greater than those from lysed cells grown without, suggesting that the bile acids enhanced synthesis of TDH within bacterial cells. These data imply that the conjugated bile acids play a key role in the pathogenicity of V. parahaemolyticus.     

Evaluation of DNA Fingerprinting by PFGE as an Epidemiologic Tool for Salmonella Infections

To evaluate DNA fingerprinting as an epidemiologic tool, pulsed-field gel electrophoresis (PFGE) was performed on isolates of Salmonella, including S. typhimurium, S. thompson, and S. enteritidis. Chromosomal DNA was digested with the restriction endonucleases Bln I and Xba I. The patterns of S. thompson and S. typhimurium isolates from various sources were different from one another. There was no correlation between the phage type and the digestion pattern of S. enteritidis isolates. Some strains belonging to one phage type were distinguished by their PFGE pattern in this study. These results suggest that the Bln I and Xba I digestion patterns of chromosomal DNA are useful for epidemiological analysis of an outbreak of Salmonella infection or food poisoning.     

Urea Hydrolysis and Suppressed Production of Thermostable Direct Hemolysin (TDH) by Vibrio parahaemolyticus Associated with Presence of TDH-Related Hemolysin Genes

A total of 18 strains of V. parahaemolyticus isolated from patients of past food poisoning cases occurring in Kanagawa Prefecture, Japan, were assayed for presence of the thermostable direct hemolysin (TDH) gene and the TDH-related hemolysin (TRH) genes (trh 1 and trh 2) with specific reference to their ability to hydrolyze urea and TDH production. A polymerase chain reaction assay revealed that all urea-hydrolyzing strains (9 strains) carried either trh 1 gene or trh 2 gene. The strains carrying the trh genes as well as the tdh gene produced TDH less by a factor of 4 to 16 than those carrying only the tdh gene, suggesting the expression of the tdh gene was suppressed by the presence of trh gene through a mechanism yet to be defined. Received: 20 September 1996 / Accepted: 6 November 1996     

In Vitro Hemolytic Characteristic of Vibrio parahaemolyticus: Its Close Correlation with Human Pathogenicity

An in vitro hemolytic characteristic of Vibrio parahaemolyticus is closely correlated with human pathogenicity.     

Reliability of the detection of meningococcal gamma-glutamyl transpeptidase as an identification marker for Neisseria meningitidis

Detection of gamma-glutamyl transpeptidase (GGT; ggt ) activity is one of the useful methods for a specific identification of Neisseria meningitidis. However, we previously happened to isolate a ggt -deficient N. meningitidis strain (NIID113) from a healthy carrier. In this study, in order to re-examine the reliability of the marker, we again investigated the GGT activity of 245 N. meningitidis human isolates and identified two other GGT-defective N. meningitidis isolates besides NIID113. The isolation frequency (1.2%) of ggt mutants among human isolates strongly confirmed the 98.8% reliability of GGT activity as the identification marker for N. meningitidis.