Modification of the multiplex PCR for unambiguous differentiation of the El Tor & classical biotypes of Vibrio cholerae O1.

BACKGROUND & OBJECTIVES: Biotyping of Vibrio cholerae O1 using multiplex PCR (ctxA-tcpA) exploits the nucleotide sequence differences of the major subunit protein of the toxin co-regulated pilus (TCP) gene (tcpA) to differentiate between the classical and El Tor biotypes. However, the presence of classical biotype specific tcpA amplicon with the El Tor strains often complicates the interpretation. The effect of PCR variables on the amplification of biotype specific tcpA in the multiplex PCR has been investigated. METHODS: Reference strains of toxigenic V. cholerae O1 belonging to classical and El Tor biotypes were selected to optimize the PCR variables for the unambiguous biotype determination by multiplex PCR. RESULTS: In the multiplex PCR assay, a reduction in the reaction volume from 100 microliters to 25 microliters and the annealing temperature of 64 degrees C, the El Tor strain produced ctxA amplicon (302 bp) along with tcpA amplicons of 618 bp and 472 bp which are specific for classical and El Tor tcpA respectively. The simplex PCR with biotype specific tcpA primer pairs showed the amplification of either 472 bp or 618 bp tcpA amplicon with El Tor template. With the classical biotype strain, the specific primer pair yielded tcpA amplicon of the expected size. Lowering of PCR annealing temperature from 64 to 60 degrees C resulted in the elimination of the amplification of the nonspecific tcpA amplicon with El Tor strain. INTERPRETATION & CONCLUSION: A comparison of the theoretical melting temperature (Tm) values of the reacting primers, and their alignment to the biotype specific tcpA revealed the basis of unambiguous biotyping of V. cholerae O1 at a PCR annealing temperature of 60 degrees C.

Adherence & colonization properties of Vibrio cholerae & diarrhoeagenic Escherichia coli.

Bacterial adherence to host cells is the initial key step towards colonization and establishment of infection within the host. The adherence process requires the participation of two components: an 'adhesin' (adherence or colonization factor) of bacteria and a 'receptor' on the host (eucaryotic) cell surface. Many bacteria express several distinct and alternative mechanisms of cell adherence depending on the environmental conditions and nature of the adhesins as well as receptors. Bacteria causing gastrointestinal infection need to penetrate the mucous layer before attaching themselves to epithelial and other absorptive cells in the intestine. This attachment is usually mediated by fimbriae or pilus structures although other cell surface components of bacteria may also take part in the process. Adherent bacteria colonize intestinal epithelium by multiplication and initiation of a series of biochemical reactions inside the target cell through signal transduction mechanisms (with or without the help of toxins). Alternatively, adherent bacteria induce extensive rearrangement of the cytoskeletal structure of the epithelial cell thereby making more intimate contact with the cell or even forcing their entry into it. This is followed by bacterial multiplication and intercellular spread leading to eventual death of the target cell. Available information on the adherence and colonization properties of V. cholerae and E. coli, the two important causative agents of gastrointestinal illness in man, is discussed and summarized in this article.