Status of Vi gene, its expression and Salmonella Pathogenicity Island (SPI-7) in Salmonella Typhi in India.

Salmonella enterica serotype Typhi (S. Typhi) is a causative organism of typhoid fever. A number of Salmonella serovars express a capsular polysaccharide antigen known as Vi, the biosynthetic and export proteins of which are encoded within the viaB locus of Salmonella Pathogenicity Island -7 (SPI-7). SPI-7 is inserted between two partially duplicated copies of tRNA -pheU gene. We have investigated the frequency of viaB operon deletion and loss of SPI-7 due to storage of strains collected during the period 1987-2006 by PCR amplification of fliC (for confirmation of serotype Typhi), tviB (for status of viaB operon) and tRNA -pheU (for absence of SPI-7). All 111 isolates were observed with positive amplification of 495 bp amplicon for fliC. A total of 36 isolates were negative for Vi by agglutination while 39 were negative for viaB operon. Interestingly, 106 isolates were found to have SPI-7. The 5 SPI-7 negative isolates were isolated during recent years. Long-term storage and repeated culture had little or no effect on SPI-7, as none of the 18 isolates recovered from blood before 1997 lacked SPI-7. On the other hand, loss of viaB operon was directly proportional to duration of storage. Thus, it is proposed that stability of Vi gene is dependent on the presence of selection pressure.

Drug resistance patterns in Salmonella enterica subspecies enterica serotype Typhi strains isolated over a period of two decades, with special reference to ciprofloxacin and ceftriaxone.

Fluoroquinolone-resistant Salmonella enterica subspecies enterica serotype Typhi are being increasingly reported from the Asian subcontinent. This has been hypothesised to be due to a double mutation in the gyrA gene. A total of 113 S. Typhi strains isolated during 1987-2006 in a tertiary-level hospital of North India were monitored for their antibiotic susceptibility by the disk diffusion and minimum inhibitory concentration (MIC) methods. The study period was arbitrarily divided into four equal parts, each comprising 5 years. The antibiotics tested showed an extremely wide range of MICs during all four periods except for ceftriaxone, which showed no resistance during the study period. However, a gradual increase in the MIC of this drug was observed, i.e. 0.047 mg/L, 0.098 mg/L, 0.211 mg/L and 0.3652 mg/L during the four study periods. Ninety-one percent of the strains isolated in the final study period were observed to have MIC levels > or = 0.125 mg/L to ciprofloxacin. Furthermore, gyrA restriction analysis showed no mutation at the two reported sites of the gene, suggesting that the double mutation theory in the development of ciprofloxacin resistance may not be the only mechanism responsible for fluoroquinolone resistance.

ERIC PCR and RAPD based fingerprinting of Salmonella Typhi strains isolated over a period of two decades.

Salmonella enterica serotype Typhi strains (n=113) were isolated from typhoid patients over a period of 2 decades, i.e. 1987-2006. RAPD and ERIC PCR methods were used for random whole genome typing of these strains. ERIC PCR was found to be very efficient with the discriminatory index (DI) of 0.9821 with 100% reproducibility. RAPD was satisfactory in discriminating the strains (DI=0.8978) but with poor reproducibility (40%). However, composite genotypic analysis was still better with DI of 0.9981 but with inherent poor reproducibility due to RAPD. Two major clones were observed to be circulating in the community with few unrelated strains too. The dendrogram constructed based on ERIC PCR banding pattern by involving 89 Typhi strains revealed 71 patterns, indicating that the genome of the bacterium is capable of rapid changes and variations. Thus, the spectrum of biological manifestations of human infection by S. Typhi may be related to its capacity for genetic diversity underlined by its highly plastic hypermutable genome.

Drug resistance pattern and clonality in H. pylori strains.

BACKGROUND: This aim of this work was to determine the in vitro activity of clarithromycin, amoxycillin, metronidazole and tetracycline against Helicobacter pylori and clonality among resistant and sensitive strains isolated from North India. METHODOLOGY: A total of 68 H. pylori isolates from peptic ulcer disease and non ulcer dyspepsia patients were examined. These strains were subjected for determination of minimum inhibitory concentration of clarithromycin, amoxycillin, metronidazole and tetracycline. For molecular characterization of resistant and sensitive strains, enterobacterial repetitive intergenic consensus sequences (ERIC) and random amplified polymorphic DNA-PCR (RAPD-PCR) methods were used. RESULTS: All the tested isolates were found resistant to metronidazole, while 65% were resistant to amoxycillin and 4.7% were resistant to clarithromycin. However, none of the isolates were found to be resistant to tetracycline. Molecular fingerprinting and cluster analysis of resistant and sensitive strains did not give clues for clonal spread of resistant strains. CONCLUSIONS: Various chromosomal mutations were seen in the putative resistance genes of resistant strains, possibly indicating selection pressure as the major cause of high resistance.