Sensitive method for detecting low numbers of verotoxin-producing Escherichia coli in mixed cultures by use of colony sweeps and polymyxin extraction of verotoxin

Abstract

High titers of Verotoxin (VT) were released from cell pellets of VT-producing Escherichia coli (VTEC; corresponding to E. coli strains producing "hikh" levels of Shiga-like toxin) after incubation in polymyxin B (0.1 mg/ml) for 30 min at 37°C. Maximal titers of polymyxin-releasable VT occurred in cells obtained from 5-h Penassay broth cultures and were up to eightfold higher than the peak culture supernatant VT titers which occurred in 8-h cultures. Polymyxin-releasable cell extracts of 5-h broth cultures inoculated with mixtures of VT-positive (VT') and VT-negative strains had easily detectable VT titers when the proportion of VT' cells in the mixture was about 1.0%, but culture supernatants were negative for VT even when this proportion was 20%. The results were the same whether the initial inoculum consisted of broth culture mixtures of VT' and VT-negative strains or colony sweeps (loopfuls of confluent bacterial growth) taken from solid plate media previously inoculated with the broth mixtures. In a clinical study, 80 stool cultures from patients with hemolytic uremic syndrome and family contacts with diarrhea were tested for free fecal VT, VT in polymyxin extracts of colony sweeps (VT/PECS), and VTEC (examination of 20 separate E. coli colonies from primary media for VT production). Of the 80 samples, 40 were positive for at least one of these three tests; all 40 were positive for free fecal VT, and 20 of these were positive for VT/PECS. VTEC (as few as 1 colony out of 20) were only isolated from 14 of the 20 cultures that were positive for VT/PECS. In six cases, the VT/PECS was positive even when none of 20 colonies tested were VT+, suggesting that the procedure was able to detect a proportion of VTEC that was less than one in 20 (5%). We conclude that the VT/PECS method is highly sensitive for detecting low concentrations of VTEC in stools and provides a rapid method for screening out stools that are negative for VTEC. The technique should also be of value in epidemiological studies for detecting low numbers of VTEC in animal feces, foods, and environmental samples.

Culture conditions affect cytotoxin production by Serratia marcescens

“…Treatment with antibiotic poiymyxin B has been used to release periplasmic toxins from Shigella dysenteriae 1 (Donohue-Rolfe and Keusch, 19831, and E. coli (Karmali et al, 1985). Addition of polymyxin B to S. marcescens culture did not increase the cytotoxicity.…”

“…The assays were done according to the method of Karmali et al (1985). Bacteria were grown at 37"C, on a shaker (150 rpm) in 20 ml of TSB for 4-5 h and then centrifuged at 10000 rpm for 10 min.…”

Verotoxin 1 binding to intestinal crypt epithelial cells results in localization to lysosomes and abrogation of toxicity

“…Cells were exposed to a 10-fold dilution series of the toxin, ranging from 0 to 200 ng ml -1 , for 3 days at 37∞C, 5% CO 2 . Surviving cells were stained with crystal violet (Karmali et al, 1985), which was solubilized with 10% acetic acid for spectrophotometric quantification at 590 nm. The concentration of VT1 causing cytotoxicity in 50% of the cell monolayer (CD 50 ) after the given time period was calculated to define relative levels of cytotoxicity.…”

Detection of Escherichia coli O157:H7 and other verocytotoxin-producing E. coli (VTEC) in food

“…A considerable amount of V T is not liberated into the medium but remains cell bound. It can be released by sonication, with the use of a French press or by polymyxin treatment, and these techniques have been used for preparing large quantities of VT (Karmali et a/. 1985).…”