Salmonella enterica Serotype Typhi with Nonclassical Quinolone Resistance Phenotype

We report Salmonella enterica serotype Typhi strains with a nonclassical quinolone resistance phenotype (i.e., decreased susceptibility to ciprofloxacin but with susceptibility to nalidixic acid) associated with a nonsynonymous mutation at codon 464 of the gyrB gene. These strains, not detected by the nalidixic acid disk screening test, can result in fluoroquinolone treatment failure.

strains have been associated with slower clinical responses to fluoroquinolones and treatment failures, clinical laboratories should attempt to identify these isolates (3,6,7). However, despite the accumulation of clinical, microbiologic, and pharmacokietic-pharmacodynamic studies suggesting a resistance breakpoint of >0.125 μg/mL for ciprofloxacin, the clinical breakpoints published by the Clinical and Laboratory Standards Institute (CLSI) (susceptibility <1 μg/mL, resistance >4 μg/mL) and those from the antibiogram committee of the French Society for Microbiology (CA-SFM) (susceptibility <0.5 μg/mL, resistance >1 μg/mL) (www.sfm.asso.fr/nouv/general.php?pa=2) have not been reevaluated (6)(7)(8)(9). Use of these standard breakpoints has probably resulted in the underreporting of Cip DS Salmonella Typhi strains. The Nal R screening test has been proposed as an alternative since the mid-1990s and recommended since 2004 by CLSI and 2010 by CA-SFM (3,7). This screening test is based on the fact that Cip DS Salmonella Typhi isolates with nonsynonymous (NS) mutations in codons 83 or 87 of gyrA are uniformly Nal R . However, recent reports have indicated that this approach cannot identify the newly described Salmonella Typhi isolates that are Nal susceptible (Nal S )-Cip DS for which mechanisms of resistance are not linked to mutations in gyrA (7,10,11).
Recently, NS mutations in codons 464 (Ser to Phe) and 466 (Glu to Asp) of gyrB were found in 7 Nal S -Cip DS Salmonella Typhi isolates (12). We present data on the occurrence and characterization of the resistance mechanisms of Nal S -Cip DS isolates in 685 Salmonella Typhi isolates of the French National Reference Center for Salmonella (FNRC-Salm).

The Study
In France, laboratory surveillance of typhoid fever infections is performed by the FNRC- QRDRs of gyrA, gyrB, parC, and parE genes were studied on 133 isolates selected to represent diversity in terms of year of isolation, geographic origin, and MICs. To analyze the isolate characteristics, we used the following approaches: sequencing (5), denaturing high performance liquid chromatography (4), and Luminex-based genotyping assays (12). QRDR DNA sequences were compared with those of Salmonella Typhi strain Ty2 (GenBank accession no. AE014613).
In subpopulation A, 75 isolates had wild-type QRDR sequences, whereas 2 isolates had a gyrB mutation at codon 465 leading to amino acid substitution Gln to Leu. Their Nal MICs were 2 and 4 μg/mL, respectively, and those of Cip were 0.04 μg/mL and 0.08 μg/mL, respectively. Notably, both isolates were acquired in Mexico during 1998 and 2009, respectively. In subpopulation C, the lowest MIC values for Cip (0.06 μg/mL) were associated with a mutation at codon 87 of the gyrA gene, whereas MICs did not increase with the additional mutation in the parE gene.
Subpopulation D consisted of 1 isolate, highly resistant to ciprofloxacin, which was acquired by a traveler in India in 2004. This isolate contained 2 NS mutations in the gyrA gene and 1 in the parC gene.
Eleven isolates of subpopulation B were categorized as susceptible to Nal by determining MICs and by using CLSI breakpoints (susceptibility, <16 μg/mL; resistance, >32 μg/mL). Of the 11 isolates, 8 (from 7 patients) had a ciprofloxacin MIC >0.125 μg/mL and were thus classified as Cip DS isolates. We were able to review the medical records of 2 patients infected with a Nal S -Cip DS isolate. One patient (isolates 08-7675 and 09-1986) relapsed 15 days after completion of the treatment (oral ofloxacin, 200 mg 2×/d for 8 days) (13). The second patient (isolate 05-2556) was treated with extended-spectrum cephalosporins, and no fluoroquinolones. Regarding the resistance mechanisms the plasmid-mediated quinolone resistance-conferring genes qnr (qnrA, B, S, D), qepA, and aac(6′)-Ib-cr were not detected by PCR (5,14). The QRDRs of gyrA, parC, and parE genes were of a wild type, whereas an NS mutation was found in gyrB for all but 1 isolate. However, only the 8 isolates with mutations at codon 464 were Nal S -Cip DS . To assess whether these isolates were genetically related, haplotyping (4) and XbaI-pulsed-field gel electrophoresis (PFGE) subtyping (5) were performed. On the strength of the results, we concluded that the gyrB mutation was acquired independently by strains belonging to different PFGE types (Figure 2). According to a newly developed single nucleotide polymorphism assay (Y.S.), 2 of these strains belong to the current emerging H58 Asian population (4), whereas the others do not (Table 2). In our study, the Nal S -Cip DS isolates with gyrB mutations at codon 464 were most often non-multidrug-resistant and acquired mainly in India. Our first Nal S -Cip DS isolate was isolated 13 years ago, and since is rare (prevalence ≈1%.). Although Cooke et al. (10) did not characterize isolates for their resistance mechanisms, they reported that Nal S -Cip DS

Conclusions
Nal S -Cip DS Salmonella Typhi isolates originating from Asia comprise ≈1% of Salmonella Typhi isolates in France but are more prevalent in the United States and the United Kingdom. The NS gyrB mutation at codon 464 was found exclusively in Nal S -Cip DS isolates; however, the effects of this mutation need to be formally demonstrated by site-directed mutagenesis. Furthermore, the involvement of an efflux system, such as AcrAB-TolC and OqxA, or the qnrC gene, have not been investigated and cannot be excluded.
Whatever the molecular mechanism of resistance of such strains, the main concern is detection of such isolates in clinical practice to prevent fluoroquinolone treatment failures.