Fitness Costs and Stability of a High-Level Ciprofloxacin Resistance Phenotype in Salmonella enterica Serotype Enteritidis: Reduced Infectivity Associated with Decreased Expression of Salmonella Pathogenicity Island 1 Genes

Abstract

The fitness costs associated with high-level fluoroquinolone resistance were examined for phenotypically and genotypically characterized ciprofloxacin-resistant Salmonella enterica serotype Enteritidis mutants (104-cip and 5408-cip; MIC, >32 g/ml). The stability of the fluoroquinolone resistance phenotype in both mutants was investigated to assess whether clones with better fitness could emerge in the absence of antibiotic selective pressure. Mutants 104-cip and 5408-cip displayed altered morphology on agar and by electron microscopy, reduced growth rates, motility and invasiveness in Caco-2 cells, and increased sensitivity to environmental stresses. Microarray data revealed decreased expression of virulence and motility genes in both mutants. Two clones, 104-revert and 1A-revertC2, with ciprofloxacin MICs of 3 and 2 g/ml, respectively, were recovered from separate lineages of 104-cip after 20 and 70 passages, respectively, on antibiotic-free agar. All fitness costs, except motility, were reversed in 104-revert. Potential mechanisms associated with reversal of the resistance phenotype were examined. Compared to 104-cip, both 104-revert and 1A-revertC2 showed decreased expression of acrB and soxS but still overexpressed marA. Both acquired additional mutations in SoxR and ParC, and 1A-revertC2 acquired two mutations in MarA. The altered porin and lipopolysaccharide (LPS) profiles observed in 104-cip were reversed. In contrast, 5408-cip showed no reversal in fitness costs and maintained its high-level ciprofloxacin resistance for 200 passages on antibiotic-free agar. In conclusion, high-level ciprofloxacin resistance in S. Enteritidis is associated with fitness costs. In the absence of antibiotic selection pressure, isolates may acquire mutations enabling reversion to an intermediate-level ciprofloxacin resistance phenotype associated with less significant fitness costs.

Antimicrobial Resistance and Virulence: a Successful or Deleterious Association in the Bacterial World?

“…In contrast, the high level of resistance to ciprofloxacin in in vitro-derived mutants of S. enterica is associated with fitness costs. In the absence of evidence of compensatory evolution, such fitness costs may account for the lack of emergence and spread (to date) of highly resistant S. enterica clones via the farm-to-fork route (120). Moreover, quinolone-resistant Salmonella spp.…”

Salmonella enterica Serovar Typhimurium Skills To Succeed in the Host: Virulence and Regulation

“…Recently, however, a relationship between acquisition of high levels of fluoroquinolone resistance and decreased cell invasion ability has been reported, and this may explain why, in general terms, a high prevalence of fluoroquinolone-resistant S. enterica strains remains rare among clinical isolates. These resistant bacteria show an impaired growth rate which may appear as a consequence of the acquisition of fluoroquinolone resistance and compromise the expression of the invasion genes (50)(51)(52). In view of these results, azithromycin and aztreonam are alternative agents that may be useful for patients with multiple allergies or for organisms with unusual resistance patterns (29).…”

Quinolone Resistance in Non-typhoidal Salmonella

“…Nevertheless, results from previous studies suggest that high-level CIP resistance mechanisms in Salmonella lead to restrictive conditions of itness costs and minimizing the emergence and spread of highly resistant clones in the absence of drug selection pressure [101,102]. As demonstrated in previous study [103], high-level CIP-resistant S. Enteritidis in vitro derived mutants in the absence of antibiotic selective pressure result in compensatory evolution favoring a reversion back to a more sensitive phenotype associated with lesser itness costs, rather than the compensatory mutations that would restore resistance. However, under in vivo conditions, a previous study has found that chromosomal mutations of S. Typhimurium that confer resistance to NAL, streptomycin, or rifampicin decrease growth rate and ability to colonize in mice rather than a reversion to the susceptible phenotype and restore virulence [104].…”