Prevalence of plasmid-mediated quinolone resistance (PMQR) genes in non-typhoidal Salmonella strains with resistance and reduced susceptibility to fluoroquinolones from human clinical cases in Alberta, Canada, 2009–13 Free

Sir,

Non-typhoidal Salmonella is a leading bacterial cause of foodborne illnesses around the world.¹ Although non-typhoidal salmonellosis is usually self-limiting, antimicrobial therapy is required for patients with serious illness and fluoroquinolones (FQs) are the drug of choice for the treatment of human salmonellosis. However, the emergence of FQ-resistant Salmonella results in treatment failure and adverse patient outcomes.² Besides the primary mechanisms related to spontaneous mutations in DNA gyrase subunit A (GyrA) and topoisomerase IV subunit C (ParC), horizontally transferable plasmid-mediated quinolone resistance (PMQR) genes are associated with reduced susceptibility to FQs, including the qnr genes (e.g. qnrA, qnrB, qnrC, qnrD and qnrS) encoding pentapeptide repeat proteins that protect DNA gyrase and topoisomerase IV from quinolones, aac(6′)-Ib-cr mediating the acetylation of quinolones, and qepA and oqxAB producing a drug efflux pump.³ Although the PMQR genes do not confer FQ resistance exceeding the MIC breakpoint, the reduced FQ susceptibility conferred by the PMQR genes is still important clinically.^2,3

In Canada, the prevalence of PMQR genes has been reported only in Escherichia coli, Klebsiella spp. and Proteus mirabilis⁴ and little is known about PMQR genes in Salmonella. In this study, we investigated the prevalence of nine PMQR genes, including qnrA, qnrB, qnrC, qnrD, qnrS, qepA, oqxA, oqxB and aac(6′)-Ib-cr, in 111 non-typhoidal Salmonella strains with resistance and reduced susceptibility to FQs, which belong to 31 different serotypes. The strains were isolated from human clinical cases in Alberta, Canada, by the Alberta Provincial Laboratory for Public Health during 2009–13. The isolates were serotyped using commercial antisera (SSI, Difco) and the antisera from the National Microbiology Laboratory in Canada. The Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) determined the MIC by using the broth microdilution method according to CLSI guidelines.⁵ All (n = 111) non-typhoidal Salmonella strains in the collection with a ciprofloxacin MIC ≥0.12 mg/L were used for the PCR detection of PMQR genes.

Salmonella strains were routinely grown on LB medium at 37°C under aerobic conditions. PCR was carried out with ExTaq (Takara, Japan) with primer sets for qnrA, qnrB, qnrC, qnrD, qnrS, qepA, oqxA, oqxB and aac(6')-Ib-cr.^6,7 Positive results were detected in 21 (18.9%) of 111 Salmonella isolates, including 2 (1.8%) isolates positive for qnrA, 10 (9.0%) isolates positive for qnrB, 8 (7.2%) isolates positive for qnrS, 1 (0.9%) isolate positive for oqxAB and 2 (1.8%) isolates positive for aac(6′)-Ib-cr. The isolation information and antibiotic resistance information for the 21 positive strains are provided in Table S1 (available as Supplementary data at JAC Online). Any relationship between the PMQR distribution and the Salmonella serotype was not clear. One Salmonella enterica serotype Agona strain and one S. enterica serotype Typhimurium strain were positive for both aac(6′)-Ib-cr and qnrB, and both qnrB and oqxAB, respectively, but the rest of the PMQR-positive isolates possessed only one gene (Table 1). Ten qnrB-positive isolates were distributed in eight different serotypes, including Agona, Corvallis, Enteritidis, Montevideo, Muenchen, Newport, Reading and Typhimurium, while qnrS was found in Corvalis, I 4,[5],12,[27]:d:−, I 4,[5],12:d:−, I 4,[5],12:i:−, I O Rough:d: and Typhimurium (Table 1). Although the majority (∼46%) of the tested clinical isolates were Enteritidis, only two Enteritidis strains were positive for the PMQR genes (Table 1). All tested strains were negative for qnrC, qnrD and qepA (Table 1). The positive PCR bands were sequenced by Macrogen (South Korea) and analysed by using Blastn and the qnr Numbering and Sequence database (http://www.lahey.org/qnrStudies/).⁸ All the detected qnrA and qnrS genes were identified as qnrA1 and qnrS1 and all qnrB were recognized as qnrB19, except for one qnrB from S. enterica serotype Agona that was matched with qnrB6. The ciprofloxacin MICs for the 21 positive strains were mostly 0.5 and 0.25 mg/L at a rate of 47.6% (10 of 21) and 33.3% (7 of 21), respectively (data not shown). A previous study in the USA reported that 10/335 human clinical isolates of Salmonella were positive for either qnrB or qnrS, but none of them was positive for qnrA.⁹ According to a study on the prevalence of PMQR genes in Salmonella in the EU, qnrS and qnrB were most frequently identified at a rate of 26% and 28%, respectively.¹⁰ Among 183 Salmonella human isolates with reduced FQ susceptibility collected in Finland during 2000–11, 43 were positive for qnrS. Interestingly, 19 of the qnrS-positive isolates were 4,[5],12:i:− and most of them originated from Thailand.¹¹

To the best of our knowledge, this is the first paper about the prevalence of PMQR genes in human clinical isolates of Salmonella in Canada. We show that PMQR genes have a relatively high prevalence (18.9%) in non-typhoidal clinical isolates of Salmonella with resistance and reduced susceptibility to FQs, revealing that qnrB and qnrS are the most prevalent PMQR genes in non-typhoidal Salmonella human isolates. Future WGS analysis will provide further characterization of these PMQR genes in the Salmonella clinical isolates.

Funding

This work was supported by the Natural Sciences and Engineering Research Council of Canada Discovery Grant (401843-2012-RGPIN), and the laboratory infrastructure was supported by the Canada Foundation for Innovation.

Transparency declarations

None to declare.

Acknowledgements

X. H. was a recipient of the intern fellowship from the University of Alberta Research Experience (UARE).

References

Author notes