A Comparative Study of Enterotoxin Gene Probes and Tests for Toxin Production to Detect Enterotoxigenic Escherichia coli

Abstract

Escherichia coli isolated from children with diarrhea were tested for enterotoxin production and for hybridization with gene probes for heat-labile (LT) and heat-stable (ST-H and ST-P) enterotoxin. Fecal specimens were also examined directly for genes coding for enterotoxins. E. coli that hybridized with the cloned enterotoxin gene probes was identified by colony hybridization from 46 children, by enterotoxin production from 38 children, and by specimen hybridization from 37 of 304 children examined. Eighty-six percent (473 of 550) of E. coli that hybridized with the cloned DNA probes produced enterotoxins. Four E. coli that hybridized with the LT and 73 E. coli that hybridized with the ST-H probes were nonenterotoxigenic. These isolates were subsequently shown not to hybridize with other constructions of the same probes and did not hybridize with synthetic single-stranded oligonucleotides directed against the LT or ST genes.

Cloned polynucleotide and synthetic oligonucleotide probes used in colony hybridization are equally efficient in the identification of enterotoxigenic Escherichia coli

“…have been described recently (3,4). Furthermore, reports on minor (21) and major (3,4) inconsistencies between standard biological techniques and hybridization assays have been reported. In contrast, the findings presented here demonstrate a complete correspondence between the results obtained with oligonucleotide and polynucleotide probes.…”

“…In general, compared with the standard bioassays for detecting the phenotypic expression of these genes, the efficiency of colony hybridization with these two classes of probes has been satisfactory (16,(19)(20)(21). However, on a few occasions, the sensitivity has appeared to be suboptimal (3,4).…”

Diagnostic deoxyribonucleic acid probes for infectious diseases

“…Since the early studies of Moseley and co-workers, there have been a series of reports describing the use of probes to identify ETEC in stools (28-32, 53, 68, 85, 117, 135). Although each has followed the traditional technique of enriching the stool for the presence of ETEC by overnight growth before analysis with the probe, some investigators have successfully used nonradioactive labels on their probes in place of 32p (32). While these studies have been very successful, there appear to be three potential problems with this technology and its transfer to the clinical diagnostic laboratory.…”

“…As a result, either stools need to be examined directly or the number of subcultures of the organisms isolated needs to be kept to a minimum to prevent plasmid loss. Third, during their field studies of ETEC in Thai villages, Echeverria et al found that test results for filters containing stool samples that were fixed in the field had a much lower sensitivity than those for samples that were transported to Bangkok for processing (32). Thus, the ability to adequately process and fix stool samples onto the filters is a critical factor to consider when using this technique, particularly since at least one commercial manufacturer of ETEC probes recommends the use of the filter hybridization format.…”

Comparison of colony and stool blots for detection of enteropathogens by DNA probes

“…sensitive as testing 10 individual colonies for enterotoxin production by standard assays. However, others reported that probing stool blots was less sensitive in detecting ETEC, E. coli producing the enteropathogenic E. coli adherence factor, and enteroinvasive E. coli (EIEC) than examining isolated colonies either by colony blots or standard assays (Chatkaeomorakot et al 1987;Echeverria et al 1986;Taylor et al 1986). …”