Evaluation of a non-radioactive chemiluminescent method for using oligonucleotide and polynucleotide probes to identify enterotoxigenic Escherichia coli.

We compared the applicability of an enhanced chemiluminescent (ECL) method for using gene probes with that of radioactive probes to identify enterotoxigenic Escherichia coli (ETEC) in stools of Bangladeshi children with diarrhoea. Colony blots of E. coli isolates were hybridized using both [alpha-32P]-dCTP labelled and fluorescein-11-dUTP labelled polynucleotide and oligonucleotide gene probes for heat-labile enterotoxin (LT), and heat-stable enterotoxin (ST). Analysis of 1,620 isolates obtained from 540 patients gave similar results by both radioactive and chemiluminescent probes. The ECL method was faster than the radioactive method. Both polynucleotide and oligonucleotide probes could be used by the ECL method. Hybridization and detection by the ECL method appeared to be a convenient alternative to radioactive probes for screening E. coli isolates for ETEC.

Differentiation of Shigella flexneri strains by rRNA gene restriction patterns.

We studied the restriction endonuclease cleavage patterns of rRNA genes (ribotypes) of 72 clinical isolates of Shigella flexneri representing eight serotypes to determine whether ribotyping could be used to distinguish S. flexneri strains and to compare the discriminating ability of the method with that of serotyping. By using a cloned Escherichia coli rRNA operon as the probe, Southern blot hybridization of restriction endonuclease-digested total DNA was carried out. Ribotyping of the isolates with each of the five restriction endonucleases BamHI, EcoRI, HindIII, PstI, and SalI generated reproducible restriction patterns. However, HindIII produced the optimum digestion pattern of the rRNA genes and was more useful than the other enzymes used in differentiating strains. Analysis of the 72 isolates showed 11 different HindIII cleavage patterns of their rRNA genes. Four of these HindIII-generated ribotypes could be further differentiated into two to four subribotypes by using PstI. The results indicate that ribotyping has an application for differentiation of S. flexneri strains and can complement serotyping. Definition of strains in terms of both serotype and ribotype may be of greater use in epidemiological studies.

Evaluation of a DNA probe to identify enteroaggregative Escherichia coli from children with diarrhoea in Bangladesh.

Six hundred and seventy-five Escherichia coli isolates obtained from 225 diarrhoeal children less than five years of age were tested for adherence to HeLa cells and for hybridisation with DNA probes for genes conferring aggregative adherence (AggA), localised adherence (LA) and diffuse adherence (DA) to assess the usefulness of a recently developed DNA probe for AggA of enteroaggregative E. coli (EAggEC). The strains were further analysed with the DNA probes for heat--labile enterotoxin (LT), heat--stable enterotoxin (ST), Shiga--like toxins (SLT I and SLT II) and for enteroinvasiveness and adherent strains were all negative for these properties. The HeLa cell assay and DNA probe assays showed excellent agreement in identifying LA and DA positive isolates. However, significant disparities occurred in the case of AggA positive isolates, and the DNA probe failed to identify 31.9% (15 of 47) of the EAggEC identified by the HeLa cell adherence assay. The failure of the DNA probe to identify all the EAggEC indicated that there may be a high degree of genetic heterogeneity for the expression of AggA, and development of more DNA probes is necessary to detect all the possible genetic variants of EAggEC.

A comparative study of specific gene probes and standard bioassays to identify diarrhoeagenic Escherichia coli in paediatric patients with diarrhoea in Bangladesh.

We compared the usefulness of gene probes with standard bioassays to identify diarrhoeagenic Escherichia coli amongst isolates from Bangladeshi children under 1 year of age with diarrhoea. E. coli isolates were analysed with specific gene probes for localised adhesiveness (LA), diffuse adhesiveness (DA), heat-labile toxin (LT), heat-stable toxin (ST), Shiga-like toxins (SLT I and SLT II), and enteroinvasiveness, and in bioassays for production of enterotoxins and cytotoxins, and for cell adherence. With 1136 isolates from 387 patients, there was general agreement between the two assay methods. When there was disparity, gene-probe-positive isolates gave negative results in the corresponding bioassay. In the HeLa cell adherence assay, 94% of the LA probe-positive isolates and 91.6% of the DA probe-positive isolates gave positive bioassay results for LA and DA respectively. Thirty-six of 39 LT probe-positive isolates and 73 of 86 ST probe-positive isolates gave positive results in the bioassays. Of 28 isolates that gave negative results in the suckling mouse assay but were initially positive with the probe for ST, 15 were later found to hybridize with the cloning vector for the ST probe. Addition of denatured vector DNA at a concentration of 10 micrograms/ml in the hybridisation solution eliminated these false positive results. None of the other probe-positive isolates hybridised with any of the cloning vectors used. The DNA hybridisation assay appeared to be a convenient alternative to bioassays for screening large numbers of isolates in epidemiological investigation.

Electropherotyping of plasmid DNA of different serotypes of Shigella flexneri isolated in Bangladesh.

One hundred and twenty-five Shigella flexneri strains, isolated during January-December 1984, at the Dhaka treatment centre of the International Centre for Diarrhoeal Disease Research, Bangladesh, were serotyped using absorbed rabbit antisera specific for all type- and group-factor antigens, as well as a group of ten mouse and rat monoclonal antibodies. Electropherotypes of the plasmid deoxyribonucleic acid (DNA) were also determined. S. flexneri 2a was the predominant serotype followed by 3b, 1a, and 2b. The recently described E1037 antigen was also found in three strains of S. flexneri serotype 6. Electropherotyping of the plasmid DNA showed that three plasmids of approximately 140, 2.7, and 2 megadalton (MDa) were present, respectively, in 97, 97 and 94% of the 125 strains. Additional plasmids of various other sizes were also present in different serotypes except in serotype 2a. The additional plasmids again appeared to be specific for that particular serotype. For example, all 12 strains of S. flexneri 2b harboured an additional plasmid of approximately 1 MDa. Thus, electropherotyping of plasmid DNA of different serotypes of S. flexneri might be useful to differentiate S. flexneri from other species of Shigella and in identifying different serotypes of S. flexneri. Therefore, the common plasmids, plus the additional plasmids, could be used to identify epidemic, as well as sporadic, subclones of S. flexneri strains.

In vitro and in vivo cholera toxin production by classical and El Tor isolates of Vibrio cholerae.

A comparative study was carried out on the in vitro production of cholera toxin by 19 Vibrio cholerae El Tor isolates from patients with cholera in South Africa, one El Tor isolate from a patient in Malawi (a country approximately 1000 km north-northeast of South Africa), 6 El Tor and 12 classical type isolates from patients in Bangladesh, and 5 culture collection classical strains. Identical phage types and indistinguishable toxigenicities among the South African and Malawi V. cholerae, representing isolations obtained over a 10-year period, indicated that essentially a single strain was involved in the cholera of these regions. Similarly, phage typing and toxin profiles indicated that the 12 classical and 6 El Tor V. cholerae cultures in Bangladesh, all isolated in November 1983, represented just two strains. As assessed by titrations in Y-1 mouse adrenal and Chinese hamster ovary cell lines, the general order of toxigenicities was Bangladesh and culture collection classical greater than Bangladesh El Tor greater than southern African El Tor. The African isolates consistently gave rise to very low titers. Their relative reluctance to produce the toxin in vitro compared with the culture collection classical strains, particularly strain 569B, was confirmed by rocket electrophoresis. In somewhat of a contrast, maximum in vivo titers in rice water stools from cholera patients in South Africa and from both classical and El Tor type cholera patients in Bangladesh were essentially equal. It is postulated that under the continuous culture conditions that occur in vivo, cholera toxin concentrations can accumulate to a maximum level, depending on the rate of purging by the diarrheal fluid rather than the toxigenicity of the infecting stain. The relevance of these findings to the relative severities of classical and El Tor types of cholera is discussed.