Molecular analysis of incHI1 antimicrobial resistance plasmids from Salmonella serovar Typhi strains associated with typhoid fever

Abstract

The first outbreak of multidrug-resistant (MDR) typhoid fever in Vietnam was in 1993, and by 1995 nearly 90% of cases were MDR. Plasmid HCM1, sequenced in full, is an incHI1 plasmid from Salmonella enterica serovar Typhi strain CT18, isolated in Vietnam in 1993. Restriction analysis shows that pHCM1 shares a restriction fragment length polymorphism (RFLP) pattern with plasmids isolated from the first outbreak and 10 of 17 MDR plasmids isolated from sporadic cases occurring at the same time in Vietnam. A core region of pHCM1 has significant DNA sequence similarity to plasmid R27, isolated in 1961 from S. enterica in the United Kingdom. There are five regions of DNA in pHCM1 which are not present in R27. Two of these are putative acquisition regions; the largest is 34.955 kbp in length and includes sequences of several antibiotic resistance genes and several insertion sequences. The borders of this region are defined by two identical IS10 left elements, associated with an inversion of DNA or with a truncated Tn10 element. The second, smaller region is 14.751 kbp and carries a trimethoprim resistance gene dfr14A cassette associated with a class 1 integrase. In 1993 to 1994, restriction analysis revealed some variations in the structures of Salmonella serovar Typhi MDR plasmids which were mapped to the two putative acquisition regions and three smaller variable regions. In 1996 a single RFLP type, RFLP7, was found to carry the dfrA7 and sul-1 genes, which were not present on R27 or pHCM1. This plasmid type appears to have a selective advantage over other plasmids with the same resistance phenotype.

FIG.1.

Regions of pHCM1 which are not present in plasmid R27. (A) Large insertion region (bp 149592 to 184546). (B) Small insertion region (bp 113600 to 128350). (C) pHCM.1 bp 7963 to 10458. This region includes five CDSs, HCM1.25 to HCM1.29. (D) pHCM1 bp 77107 to 81205. This region includes three CDSs, HCM1.102 to HCM1.104. (E) pHCM1 bp 141607 to 142983. This region includes CDSs HCMI.192 and HCM1.193. The lines on the outer ring indicate cut sites for HindIII. The insert of region A begins at bp 143085 and ends at bp 184438 on the Salmonella serovar Typhi genome of CT18. The insert of region B begins at bp 13598 and ends at bp 128164. IS10-L, IS10 left element.

FIG. 2.

Plasmids from representative MDR Salmonella serovar Typhi strains isolated in 1993 and 1996. Lane 1 contains E. coli 39R size markers of 98, 24, 24, and 4.5 MDa. (This gives a predicted mass of around 140 MDa; however, the gel indicates that the plasmids have masses of around 110 MDa.) Lanes 2 to 7 contain MDR Salmonella serovar Typhi from 1993. Lanes 8 to 13 contain MDR Salmonella serovar Typhi from 1996. Lane 14 contains E. coli 39R.

FIG. 3.

Restriction patterns of MDR plasmids cleaved with HindIII to show differences between RFLP1, the most common pre-1994 pattern, and RFLP7, the predominant post-1994 pattern. Lanes 1, 2, and 4, pattern 1 Salmonella serovar Typhi strains isolated in 1993; lane 3, pattern 2 Salmonella serovar Typhi strain isolated in 1993; lanes 5 and 8, pattern 7 Salmonella serovar Typhi strains isolated in 1996; lane 6, pattern 7′ plasmid isolated in 1996; lane 7, pattern 7′′ plasmid isolated in 1996; lane λ, DNA markers at 23, 9.4, 6.5, 4.3, 2.3, and 2.0 kb.