Comparison of polymerase chain reaction systems for detection of different cdt genes in Escherichia coli strains

Abstract

Aims: Cytolethal distending toxins (CDT) are tripartite toxins encoded by three adjacent or overlapping genes (cdtA, cdtB, cdtC) and found in multiple pathogens. The present knowledge regarding heterogeneity of cdt genes and our previous study revealed that the available polymerase chain reaction (PCR) systems lack adequate specificity. The detection of various cdt genes present in Escherichia coli strains, from different geographical regions demands further assays for wide‐range coverage. On the basis of these observations, we were prompted to undertake the present study; hence the specificity of existing PCR systems was addressed using E. coli prototype strains with known cdt gene sequences. Methods and Results: A multiplex PCR designed for the detection of E. colicdt genes was found to be sensitive and specific enough for initial screening. However, for subtyping, the PCR systems yielded nonspecific products upon amplification. These primers are usually designed for sequences of the cdtB locus (the most conserved region of the gene), and since CDT‐producing E. coli strains carry different cdt genes, none of the systems are really type specific. Furthermore, PCR systems with type‐specific primers for other regions of the gene, i.e. ORF A or ORF C are found to be strain specific and their applications in different geographical regions have limitations. Conclusions: In conclusion, based on our observations, using these available primers, it seems that the existing PCR systems are not sufficiently accurate to differentiate between different types of cdt genes. Significance and Impact of the Study: The results obtained from this study revealed that so‐far reported PCR systems are short in specificity. These PCR protocols were not found to be specific enough to detect various cdt genes and have a limited range of application. Moreover, due to similarities in cdt genes the cross‐reaction between different sets of primers exists. Hence for epidemiological studies, some additional PCR protocols are required for screening clinical isolates for cdt genes.

Molecular profile and genetic diversity of cytolethal distending toxin (CDT)‐producing Escherichia coli isolates from diarrheal patients

“…The presence of cdt in different bacterial species (1–3, 11, 27) and the results of analysis of its flanking regions suggest that this gene has been acquired by horizontal gene transfer (12) or through a phage (14). PFGE analysis confirmed our previous findings (28) that these strains are heterogeneous. The data obtained by PFGE are also in accordance with tRNA insertion analysis that revealed there is no consensus pattern of insertion in the genome of CDT‐producing strains.…”

The DNA Sequence of the Escherichia coli O22 O-Antigen Gene Cluster and Detection of Pathogenic Strains Belonging to E. coli Serogroups O22 and O91 by Multiplex PCR Assays Targeting Virulence Genes and Genes in the Respective O-Antigen Gene Clusters

“…Virulence genes, including pap, sfa, afaI (afimbrial adhesin-I), aer (aerobactin), hlyA, and cnf-1 are found in uropathogenic strains (Blanco et al 1996a;Terai et al 1997;Johnson 1991). E. coli serogroup O91 has been associated with cases of HC and HUS; therefore, this serogroup is classified as an enterohemorrhagic E. coli (Lindgren et al 1993;Beutin et al 1998;Bonnet et al 1998;Keskimäki et al 1998 Twenty-two percent, 13%, 6%, and 1% of the strains were positive for the focG, hlyA, papGIII, and papGI genes, respectively (Table 4). Also, 26% and 38% of the strains were positive for the cnf-1 and sfa genes, respectively.…”

“…Enterohemorrhagic E. coli serogroup O91 strains have caused non-bloody diarrhea, hemorrhagic colitis (HC), and HUS, and E. coli O91:H21 strains were shown to be virulent in an orally infected mouse model with a 50% lethal dose of less than ten bacteria (Lindgren et al 1993;Beutin et al 1998;Bonnet et al 1998;Cantarelli et al 2000). Virulence genes in pathogenic E. coli serotype O91 strains include those that encode the Shiga toxins (stx 1 , stx 2 ), hemolysin (ehxa/hlyA), STEC autoagglutinating adhesion (saa), and intimin (eae; Beutin et al 1998Beutin et al , 2004Cid et al 2001;Paton et al 2001;Blanco et al 2004).…”