A novel ciprofloxacin-resistant subclade of H58 Salmonella Typhi is associated with fluoroquinolone treatment failure

Abstract

The interplay between bacterial antimicrobial susceptibility, phylogenetics and patient outcome is poorly understood. During a typhoid clinical treatment trial in Nepal, we observed several treatment failures and isolated highly fluoroquinolone-resistant Salmonella Typhi (S. Typhi). Seventy-eight S. Typhi isolates were genome sequenced and clinical observations, treatment failures and fever clearance times (FCTs) were stratified by lineage. Most fluoroquinolone-resistant S. Typhi belonged to a specific H58 subclade. Treatment failure with S. Typhi-H58 was significantly less frequent with ceftriaxone (3/31; 9.7%) than gatifloxacin (15/34; 44.1%)(Hazard Ratio 0.19, p=0.002). Further, for gatifloxacin-treated patients, those infected with fluoroquinolone-resistant organisms had significantly higher median FCTs (8.2 days) than those infected with susceptible (2.96) or intermediately resistant organisms (4.01)(pS. Typhi clade internationally, but there are no data regarding disease outcome with this organism. We report an emergent new subclade of S. Typhi-H58 that is associated with fluoroquinolone treatment failure.

Keywords: s. enterica serovar typhi; H58; epidemiology; fluoroquinolones; global health; human; infectious disease; microbiology; nepal; randomised controlled trial; treatment failure; typhoid fever.

Figure 1.. The phylogenetic structure of 78 Nepali Salmonella Typhi isolated during a gatifloxacin versus ceftriaxone randomised controlled trial.

Maximum likelihood phylogeny based on core-genome SNPs of 78 Salmonella Typhi RCT isolates with the corresponding metadata, including the presence of mutations (dark grey) in gyrA (S83F, D87V and D87N), parC (S80I) and parC (A364V) and susceptibility to ciprofloxacin (susceptible, light blue; intermediate, mid-blue and non-susceptible, dark blue) by Minimum Inhibitory Concentration (MIC). The reference strain CT18 was used for context and highlighted by the black boxes. Red lines linking to metadata show isolates belonging to the Salmonella Typhi H58 lineage (with H58 triple mutants highlighted), other lineages (non-H58) are shown with black lines. The scale bar indicates the number of substitutions per variable site (see methods). Asterisks indicate ≥85% bootstrap support at nodes of interest. DOI: http://dx.doi.org/10.7554/eLife.14003.003

Figure 2.. The association of Salmonella Typhi lineage and ciprofloxacin susceptibility with treatment failure and fever clearance time in patients randomised to gatifloxacin.

(A) Kaplan-Meier curve for time to treatment failure by H58 and non-H58 Salmonella Typhi. (B) Kaplan-Meier curve for time to treatment failure by Salmonella Typhi susceptibility group (susceptible, intermediate, resistant to ciprofloxacin). (C) Non-parametric maximum likelihood estimators for interval-censored fever clearance time (see methods) by H58 and non-H58 Salmonella Typhi. (D) Non-parametric maximum likelihood estimators for interval-censored fever clearance time by Salmonella Typhi susceptibility group (susceptible, intermediate, resistant to ciprofloxacin). DOI: http://dx.doi.org/10.7554/eLife.14003.006

Figure 3.. Minimum Inhibitory Concentrations of Nepali Salmonella Typhi and Salmonella Paratyphi against ciprofloxacin and gatifloxacin over ten years.

Minimum Inhibitory Concentrations (μg/ml) for 568 Nepali Salmonella Typhi (blue) and 269 Nepali Salmonella Paratyphi A (grey) against (A) ciprofloxacin and (B) gatifloxacin collected from four randomised controlled trials conducted between 2005–2014 at Patan Hospital in Kathmandu, Nepal (Pandit et al., 2007; Koirala et al., 2013; Arjyal et al., 2011). The smoothed line derived from the generalized additive model showing a non-linear increase in Minimum Inhibitory Concentrations over time, with shading representing the 95% confidence interval. Lower and upper horizontal lines represent the current CLSI cut-offs for susceptible/intermediate and intermediate/resistant, respectively (CLSI, 2012). DOI: http://dx.doi.org/10.7554/eLife.14003.008

Figure 4.. The phylogenetic structure of fluoroquinolone resistant Salmonella Typhi in a regional context.

Maximum likelihood phylogeny based on core-genome SNPs of 136 (78 from the RCT) Salmonella Typhi isolates from Nepal and neighbouring India (Supplementary file 1). Main tree shows the overall phylogenetic structure and the presence of specific combinations of mutations in gyrA (S83F, D87V and D87N), parC (S80I) and parE (A364V). The inset shows a magnified view of the fluoroquinolone-resistant Salmonella Typhi H58 triple mutants from Nepal and their close association with similarly fluoroquinolone-resistant Salmonella Typhi H58 triple mutants from India (Wong et al., 2015). The scale bar on the primary tree indicates the number of substitutions per variable site, while that in the inset indicates genetic distance in number of SNPs (see methods). Asterisks indicate ≥85% bootstrap support at nodes of interest. DOI: http://dx.doi.org/10.7554/eLife.14003.009