Molecular subtyping scheme for Salmonella panama

Abstract

We describe a genotyping scheme for Salmonella panama. Defined probes specific for the 16S rRNA gene and the DNA insertion element IS200 were generated by PCR from S. panama and were used to probe genomic Southern blots made with enzymes selected to cut within and outside the probed sequences. Plasmid profiles were determined. The typeability and discriminatory power of the individual methods were compared. Ribotyping with 16S rRNA gene probe alone was slightly more discriminatory than phage typing, but unlike the latter, ribotyping was able to type all strains. IS200 profiling was the single most discriminatory method for S. panama, having an index of discrimination (D) of 0.8 and 100% typeability. Plasmid profiling, which had moderate discriminatory power but only 50% typeability, was valuable as an adjunct technique. The use of all three methods together or simply the combination of IS200 profiling with the two most discriminatory enzymes and plasmid profiling yielded a molecular typing scheme whose discriminatory power (D ‫؍‬ 0.97) approached the maximum theoretical value. This should prove both useful and robust for epidemiological investigations of S. panama.

Evolutionary origin of a monophasic Salmonella serovar, 9,12:l,v:-, revealed by IS200 profiles and restriction fragment polymorphisms of the fljB gene

“…1. S. panama had six IS200 bands, compatible with the results of a recent large study (18). S. zaiman exhibited nine IS200 bands, of which three were shared with S. panama.…”

“…Therefore, S. panama would have been one obvious candidate to give rise to a monophasic variant through the loss of expression of its 1,5 phase 2 flagellar antigen. An extensive set of S. panama strains has recently been analyzed for IS200 distributions, and on the basis of those results (18), S. panama can be excluded as a possible ancestor of Salmonella 9,12:l,v:Ϫ. In 1984, Sechter and Cahan (15) tried to elucidate the origin of Salmonella 9,12:l,v:Ϫ by studying the sensitivity of this variant to a large battery of typing phages active on serogroup D1 salmonellae, including those sharing the phase 1 flagellar antigen l,v.…”

“…Recent molecular typing methods permit the direct testing of assumptions made in conventional serotyping. For example, analysis of the insertion sequence IS200 distribution among serogroup D1 salmonellae was able to delineate clonal relationships among Salmonella serovars (18,20,21). In the present study we address this question by analyzing IS200 profiles and the flagellar gene operon structure.…”

Assessment of genetic diversity of Bacillus spp. isolated from eutrophic fish culture pond

“…The 16S rDNA gene from individual bacterial isolates was amplified by polymerase chain reaction (Stanley et al 1995 ). The forward primer was 5′ AAG AGT TTG ATC CTG GCT CAG 3′ and the reverse primer was 5′ GGT TAC ATT GTT ACG ACT T. The PCR reaction mixture (50 μl) contained, dNTPs each 100 μmol; 1X PCR buffer (10 mMTrisCl, 50 mMKCl, 2.5 mM MgCl 2 and 0.01 % gelatin); each primer 20 pmol; Taq DNA polymerase (Genei, India) 0.75U and bacterial DNA 100 ng.…”

Molecular typing of Salmonella serotype Thompson strains isolated from human and animal sources

“…DNA (c. 2 µg) from each of the 113 Thompson isolates was digested for 18 h with PvuII, as recommended by the manufacturer (Promega, Southampton, UK) ; in addition, DNA from 70 of these isolates was digested with HincII. These restriction enzymes were chosen because their target sites are outwith the IS200 sequence [18,19]. DNA restriction fragments and digoxigenin (DIG)-labelled lambda DNA digested with EcoRI and HindIII (Boehringer-Mannheim, DIG-labelled molecularweight marker III), were separated by electrophoresis through 0n8 % agarose gels (Ultra Pure agarose from Life Technologies, Paisley, UK) with ethidium bromide 0n5 µg\ml in TBE buffer (0n089  Tris-HCl, 0n089  boric acid, 0n002  EDTA) at 2 V\cm for 16-20 h. Gels were viewed on a UV transilluminator (312 nm) and photographed on Polaroid 667 film.…”