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Typhoid Fever Bacteria Sequencing Reveals Drug Resistance Genes

NEW YORK (GenomeWeb) – The Salmonella enterica serovar Typhi strain behind a current outbreak has acquired a plasmid that renders it resistant to five classes of antibiotics, according to an analysis from an international team of researchers.

A typhoid fever outbreak emerged in Pakistan in November 2016, with a number of cases, especially in Sindh Province, resistant to ceftriaxone, a third-generation cephalosporin, and to the first-line drugs chloramphenicol, ampicillin, and trimethoprim-sulfamethoxazole. The strains were also resistant to fluoroquinolones, which are used to treat multidrug resistant cases.

A Wellcome Trust Sanger Institute-led team of researchers sequenced 80 S. enterica serovar Typhi isolates from this outbreak to find that they harbored resistance genes that are embedded both in chromosomal regions and on a plasmid. As the team reported in mBio this week, it found that the plasmid conferring fluoroquinolone resistance appears to have been acquired from Escherichia coli.

"We have used genetic sequencing to uncover how this particular strain of typhoid became resistant to several key antibiotics," senior author Gordon Dougan from the Sanger Institute said in a statement. "Sporadic cases of typhoid with these levels of antimicrobial resistance have been seen before, but this is the first time we've seen an ongoing outbreak — which is concerning."

The researchers used the Illumina HiSeq 2500 platform to sequence 87 isolates — mostly from Hyderabad and Karachi — that exhibited a pattern of being extensively drug resistant (XDR). For one sample, the researchers also generated a complete genome using a combination of Oxford Nanopore and Pacific Biosciences long-read sequencing approaches. The final assembled genome was 4.73 million base pairs in length with an 84,492-base-pair long plasmid.

At the same time, the researchers analyzed 12 isolates that were susceptible to ceftriaxone.

All the XDR and 11 of the 12 ceftriaxone-susceptible samples belong to the S. Typhi H58 clade.

Further phylogenetic analysis with these and additional H58 strains indicated that the XDR samples and four of the ceftriaxone-susceptible samples belonged to their own branch, which was separated other members of the clade by 17 SNPs, six of which were specific to the XDR samples.

Dougan and his colleagues noted that isolates from this branch — though without the plasmid — have been circulating in Pakistan since 2010, suggesting the XDR clone is likely a local Pakistani strain that since acquired ceftriaxone resistance.

By combing through the samples' genomes for antibiotic resistance genes, the researchers found that the XDR isolates contained a transposon, which has been observed previously in multidrug-resistant H58 strains. Genes within the transposon provide resistance to chloramphenicol, ampicillin, trimethoprim-sulfamethoxazole, and streptomycin.

But, the XDR isolates were also resistant to ceftriaxone and ciprofloxacin. The researchers traced that resistance to genes residing on a plasmid. The blaCTX-M-15 extended-spectrum-lactamase gene provides resistance to ceftriaxone, while the combination of a mutation in gyrA and the acquisition of a qnrS gene give a high tolerance to ciprofloxacin. The blaCTX-M-15 extended-spectrum-lactamase gene and the qnrS gene are on a lncY plasmid specific to the XDR isolates of this branch, which they dubbed p60006.

Since plasmids can pass from species to species, the researchers compared the p60006 plasmid to other resistance plasmids to find it is highly similar to a plasmid previously found in a Nigerian E. coli isolate as well as in a number of other E. coli samples.

This led the researchers to propose that an endemic multidrug resistant strain of S. Typhi in Pakistan acquired this plasmid, likely from E. coli, to develop extensive drug resistance.

Dougan and his colleagues noted that during their analysis, a patient in the UK who had been in Pakistan presented with typhoid fever identical to this strain, which they said underscores how drug resistance in one region can affect the health of another.