Youichi Onoue

Collaborative evaluation of detection methods for Escherichia coli O157:H7 from radish sprouts and ground beef

For the evaluation of plating and immunological methods applicable to the detection of Escherichia coli O157:H7 from ground beef and radish sprouts, a collaborative study was conducted. It focused on a comparison of the efficiency of the plating and immunological methods using various plating agars and immuno-kits in combination with enrichment in modified E. coli broth supplemented with novobiocin (mEC + n), and using immunomagnetic separation. The plating media tested were sorbitol MacConkey agar (SMAC), SMAC supplemented with cefixime (0.05 mg/l) and potassium tellurite (2.5 mg/l) (CT-SMAC), and agars containing beta-glucuronidase substrates such as BCM O157 and CHROMagar O157. The immuno-kits used were Now E. coli, Path-Stick O157, VIP, EHEC-Tek ELISA System and Rapiblot E. coli O157. The 20 participating laboratories attempted to detect E. coli O157:H7 in 25 g chilled and frozen samples of ground beef uninoculated and inoculated with E. coli O157:H7 at levels of 138.9 and 23.9 cfu/25 g, and in 25 g chilled and frozen samples of radish sprouts uninoculated and inoculated at levels of 20.4 and 1.7 cfu/25 g. E. coli O157:H7 was recovered well from ground beef by all of the methods except direct plating with SMAC. For radish sprouts, the IMS-plating methods with CT-SMAC, BCM O157 and CHROMagar O157 were most efficient at detecting E. coli O157:H7 in more than 90% of the chilled samples inoculated at the level of 20.4 cfu/25 g. All the methods were less sensitive when applied to similar levels of E. coli O157:H7 in radish sprouts (20.4 cfu/25 g) compared with ground beef (23.9 cfu/25 g) especially if the sprouts were frozen. The sensitivity of the immuno-kits appeared to be similar to the IMS-plating methods, but the specificity was lower. Based on the results, we recommend the IMS-plating method using CT-SMAC and agars containing beta-glucuronidase substrate in combination with static enrichment incubation in mEC + n at 42 degrees C.

Amino acid requirements for the growth and enterotoxin production by Staphylococcus aureus in chemically defined media

The production of enterotoxins A, B, and C by five strains of Staphylococcus aureus was studied in a series of defined media, each differing from the complete defined medium in the lack of one amino acid. Valine was required for growth; arginine and cystine for growth and enterotoxin production. Omission of individual amino acid affected in different ways the yields of the toxins produced.

Comparison of enrichment procedures for isolation of Escherichia coli O157:H7 from ground beef and radish sprouts

Abstract Enrichment procedures using Tryptic soy broth (TSB), modified TSB (mTSB), modified E. coli broth with novobiocin (mEC+n), mTSB (without novobiocin) with vancomycin, cefixime and cefsulodin (mTSB-VCC), or TSB with cefixime, tellurite and vancomycin (TSB-CTV) were evaluated by determining the rate of successful isolation of fifteen Escherichia coli O157:H7 strains from inoculated broth containing ground beef or radish sprout extract. E. coli O157:H7 tended to be isolated more efficiently after enrichment with TSB, mTSB and mEC+n than with the other broths. In order to identify the most efficient enrichmemt condition using these broths, E. coli O157:H7 were inoculated into 25 g ground beef or radish sprouts, which were then homogenized in 225 ml broth and incubated static at 37°C or 42°C for 6 h or 18 h. Attempts were made to isolate the inoculated bacteria by plating method in combination with the immunomagnetic separation method. The most effective enrichment condition was incubation in mTSB or mEC+n at 42°C for 18 h for ground beef, and in mEC+n at 42°C for 18 h for radish sprouts.

Evaluation of Methods for the Isolation and Detection of Escherichia coli O157:H7 from Minced Beef

This study has evaluated enrichment and detection procedures for the isolation and detection of Escherichia coli O157:H7 inoculated into minced beef, i. e., 24hr enrichment in modified EC broth containing novobiocin (mEC+n) followed by plating onto sorbitol IPA bile salt agar (SIB), sorbitol MacConkey agar (SMAC), cefixime-tellurite SMAC (CT-SMAC) and Rainbow O157 agar, or immunomagnetic separation (IMS) followed by plating onto these four kind of media. The recovery of E. coli O157:H7 was greater on CT-SMAC than SMAC, SIB or Rainbow O157 Agar. The protocol using mEC+n incubated at 42°C, followed by IMS and inoculation onto CT-SMAC was the most succesful procedure. However, 24hr enrichment in mEC+n followed by CT-SMAC was almost as effective as the IMS method for the isolation of E. coli O157:H7.