Development and Evaluation of a PCR Assay for Tracking the Emergence and Dissemination of Haitian Variant ctxB in Vibrio cholerae O1 Strains Isolated from Kolkata, India

A PCR-based assay was developed to discriminate the classical, El Tor, and Haitian types of ctxB alleles. Our retrospective study using this newly developed PCR showed that Haitian ctxB first appeared in Kolkata during April 2006, and 93.3% of strains isolated during 2011 carried the new allele. Dendrogram analysis showed a pulsed-field gel electrophoresis (PFGE) pattern of the new variant strains isolated recently that was distinct from the PFGE pattern of the strains carrying classical ctxB that closely matched the 2006 to 2007 variant strains

Multiple infection and microdiversity among Helicobacter pylori isolates in a single host in India.

Helicobacter pylori is one of the most diverse bacterial species that chronically infects more than 70% of Indian population. Interestingly, data showing microdiversity of the H. pylori strains within a particular gastric niche remained scarce. To understand the extent of genetic diversity among H. pylori strains within a given host, 30 patients with gastro-duodenal problems were subjected to endoscopy and from each patient 10 single colonies were isolated. Characterization of each of these 10 single colonies by DNA fingerprinting as well as genotyping of several important genetic markers viz. cagA, vacA, iceA, vapD, cag PAI empty site, IS605, RFLP and two other genetic segments within cag PAI revealed that all of the 30 patients were infected with more than one strain and sometimes strains with 5 to 6 types of genetic variants. Analyses of certain genetic loci showed the microdiversity among the colonies from single patient, which may be due to the recombination events during long-term carriage of the pathogen. These results suggest that most of the patients have acquired H. pylori due to repeated exposure to this pathogen with different genetic make-up, which may increase the possibility of super infections. Genetic exchanges between these unrelated H. pylori strains may support certain H. pylori variant to grow better in a given host than the parental strain and thereby increasing the possibility for the severity of the infection

Multiple strain colonization detected on the basis of HP0527 gene in <i>cag</i> PAI.

<p>Lanes 1 to 10, single colonies isolated from the patient; C, Control strain 26695. (a) Three types of colonies were identified in PG93. Lane 1 and 6 gave a higher amplicon than that of 26695; lanes 3, 4, 7 and 8 yielded same amplicon while lanes 2, 5, 9, 10 produced lower amplicon than that of 26695. (b) Mixed infections detected on the basis of <i>vapD</i> genetic locus PCR. M, 100 bp marker; lanes 1–10, single colonies isolated from PG137; 11, positive control (PCR225); 12, Negative control (<i>E. coli</i> DNA). All the colonies are positive for <i>vapD</i> except colony numbers 1, 6, 9 and 10.</p

Over all analysis regarding the colonization of multiple <i>H. pylori</i> strains in same host in this study population.

<p>Over all analysis regarding the colonization of multiple <i>H. pylori</i> strains in same host in this study population.</p

Combination of genotype and RAPD analysis for PG157 indicated multiple infections and microdiversity in a single host.

<p>M, 100 bp marker; lanes 1–10, single colonies isolated from PG157; P, pooled DNA. (a) RAPD patterns using primer 1281 showed two distinct patterns (lanes 1–8 and lanes 9–10) (b) RAPD patterns using primer 1283 also yielded two distinct patterns (lanes 1–8 and lanes 9–10) (c) Multiplex PCR for <i>vacA</i> alleles and <i>cagA</i> showed existence of s1m1<i>cagA</i>⁺ strains in lanes 1–8 and s2m2<i>cagA</i>⁻ strains in lanes 9–10. (d) Variant <i>cagA</i> subtypes detected on the basis of PCR for 3′ end of <i>cagA</i> using primers CAG1 and CAG2. This PCR assay showed existence of type A strains in lanes 4, 6–8 and existence of type B/D strains in lanes 1–3 and 5. Lanes 9–10, which were detected as <i>cagA</i>⁻, did not produce any amplicon and the pooled sample yielded amplicons for type A and type B/D strains.</p