Single-Nucleotide Polymorphism Mutation Spectra and Resistance to Quinolones in Salmonella enterica Serovar Enteritidis with a Mutator Phenotype

Abstract

Resistance to quinolone antibiotics has been associated with single-nucleotide polymorphisms (SNPs) in the quinolone resistance-determining region (QRDR) of gyrA. Mutations in the gyrA gene were compared by using mutant populations derived from wild-type Salmonella enterica serovar Enteritidis and its isogenic mutS::Tn10 mutator counterpart. Spontaneous mutants arising during nonselective growth were isolated by selection with either nalidixic acid, enrofloxacin, or ciprofloxacin. QRDR SNPs were identified in approximately 70% (512 of 695) of the isolates via colony hybridization with radiolabeled oligonucleotide probes. Notably, transition base substitution SNPs in the QRDR were dramatically increased in mutants derived from the mutS strain. Some, but not all, antibiotic-resistant mutants lacking QRDR SNPs were resistant to tetracycline and chloramphenicol, consistent with alterations in nonspecific efflux pumps or other membrane transport mechanisms. Changing the selection conditions shifted the mutation spectrum. Selection with ciprofloxacin was least likely to yield a mutant harboring either a QRDR SNP or chloramphenicol resistance. Selection with enrofloxacin was more likely to yield mutants containing Ser833Phe mutations, whereas selection with ciprofloxacin or nalidixic acid favored recovery of Asp873Gly mutants. Fluoroquinolone-resistant Salmonella strains isolated from veterinary or clinical settings frequently display a mutational spectrum with a preponderance of transition SNPs in the QRDR, the pattern found in vitro among mutS mutator mutants reported here. Both the preponderance of transition mutations and the varied mutation spectra reported for veterinary and clinical isolates suggest that bacterial mutators defective in methyl-directed mismatch repair may play a role in the emergence of quinolone and fluoroquinolone resistance in feral settings.

Long-Term Dissemination of CTX-M-5-Producing Hypermutable Salmonella enterica Serovar Typhimurium Sequence Type 328 Strains in Russia, Belarus, and Kazakhstan

“…This is also consistent with our finding of a strong mutator phenotype of studied isolates, as evidenced by an increased (up to four orders of magnitude) rate of spontaneous resistance to nalidixic acid. Numerous studies have demonstrated that hypermutability plays an important role in the development of quinolone resistance in many species, including S. enterica, and is common among nosocomial strains (37,62,63). We accordingly suggest that hypermutability facilitates emergence of quinolone resistance in S. Typhimurium ST328 and thus contributes to its adaptation to the hospital environment.…”

“…S1 in the supplemental material). By plating serial dilutions of exponentialphase cultures, we found the frequency of spontaneous nalidixic acid-resistant mutants in the studied isolates to be about 1 ϫ 10 Ϫ5 , which is four orders of magnitude higher than in normomutable S. Typhimurium strains (37,38). Therefore, the studied isolates were regarded as strong mutators.…”

Fluoroquinolone Susceptibility Testing of Salmonella enterica: Detection of Acquired Resistance and Selection of Zone Diameter Breakpoints for Levofloxacin and Ofloxacin

“…One hundred isolates of nontyphi Salmonella with or without different quinolone resistance mechanisms were included: 40 isolates with no topoisomerase mutations or PMQR mechanisms, 40 isolates with one or more mutations in the topoisomerase genes, and 20 isolates with PMQR mechanisms and no topoisomerase mutations (11,22). For the two latter groups, the presence or absence of PMQR genes and topoisomerase mutations was investigated by PCR and sequencing (11,(22)(23)(24).…”

“…For each isolate screened, genomic DNA was prepared by lysing the bacteria at 95°C and collecting the supernatant following centrifugation. The gyrA/B and parC/E genes were amplified using previously described primers (23,24). Amplicons were visualized on 1.5% agarose gels stained with GelRed nucleic acid gel stain (Biotium, Inc., Hayward, CA).…”

Comparative Genomics of 28 Salmonella enterica Isolates: Evidence for CRISPR-Mediated Adaptive Sublineage Evolution

“…These mutations are known to mediate quinolone resistance in S. enterica and E. coli (44,47). S. enterica strains of serotype Schwarzengrund were responsible for the first recognized outbreak of fluoroquinolone-resistant salmonellosis in the United States (55).…”