Tahir Ali

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.

Role of hns in the virulence phenotype of pathogenic salmonellae

A TnphoA‐generated mutant C5060, attenuated for virulence, was derived from the mouse‐virulent Salmonella typhimurium strain C5. This mutation, designated hns‐112::TnphoA, harbours the transposon in the 3 end of hns, with the alkaline phosphatase open reading frame in the opposite orientation to that of hns. Bacterial strains harbouring hns‐112::TnphoA were mucoid and had altered levels of DNA supercoiling, as monitored using pUC18 as a reporter plasmid. Transduction of hns‐112::TnphoA into mouse virulent strains, including S. typhimurium SL1344 and Salmonella enteritidis Se795, resulted in attenuation. When an independent hns mutation, harbouring a kanamycin‐resistance cassette inserted into the Kpnl site at base pair 237 of the hns gene, was introduced into S. typhimurium C5, the isolates were also attenuated. S. typhimurium C5 isolates harbouring the multicopy plasmid pGB651, which encodes the Escherichia coli hns gene, were partially attenuated in mice. Transductional analysis, using Tn10 insertions located close to the hns gene, showed that virulence could be restored In genetic crosses that eliminated the resident hns mutations. However, some hns+ transductants were stilt attenuated, suggesting that secondary attenuating lesions can accumulate in hns‐deficient strains. These studies show that the hns locus plays a role in Salmonella virulence.

Haemophilus influenzae microarrays: virulence and vaccines

In 1995 the genome sequence of the Haemophilus influenzae KW20 (Rd) strain was published, the first available for a free-living organism. The genome has been invaluable in global strategies to identify certain virulence-related genes, e.g. those involved in LPS synthesis, and also essential genes, but there is a paucity of wholegenome transcriptome studies. We have now constructed a whole-genome array consisting of genes from Rd, additional genes identified in other strains of H. influenzae and controls (from eukaryotic sources and other bacteria). We intend to use this array in studies aimed at understanding the bacterium’s basic metabolism and its response to changing environments; deciphering global regulatory networks (by comparison of wild-type and mutant strains); and identifying genes expressed in vivo. The use of H. influenzae DNA arrays combined with proteomic approaches will enhance our understanding of the metabolism and virulence of the organism. Additionally, the genome sequence of a non-typable H. influenzae strain is in progress. The sequence from this isolate will be invaluable not only in identifying potential novel antibiotic targets and putative vaccine candidates but also in the design of a microarray for genome-typing purposes.

Phage‐encoded genes and Salmonella virulence

We read with interest the recent article by Figueroa-Bossi et al. entitled ‘Unsuspected prophage-like elements in Salmonella typhimurium ’ (1997, Mol Microbiol 25:161–172) and would like to follow up several issues arising from the paper. First, we question whether the presence of these elements in the S. typhimurium chromosome is really unsuspected. The recent publication of the entire genome sequence of Escherichia coli K-12 (Blattner et al., 1997, Science 277: 1453–74) revealed that this close relative of S. typhimurium LT2 contains six previously described prophages and three novel cryptic prophages – it is thus unsurprising to find multiple prophage-like elements in the S. typhimurium genome. Furthermore, comparison of the genetic maps of E. coli K-12 and S. typhimurium (as reproduced in Neidhardt, 1996, Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd edn, Washington DC: ASM Press) in the regions occupied by the prophage-like elements Gifsy-1 (c. 57.4 Cs) and Gifsy-2 (c. 23.8 Cs) prophages shows discontinuities at both sites.