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 microg/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 microg/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.

FIG. 1.

Growth profiles of S. Enteritidis wild-type strains (104 and 5408), their isogenic ciprofloxacin-resistant mutants (104-cip and 5408-cip), and the reverted mutant (104-revert) (A) and colony morphology of parents [B (1)] and isogenic mutants [B (2)] (B). Both 104-cip and 5408-cip formed small colonies on agar. 104-revert formed colonies similar to those of the parent strains. For purposes of clarity, 1A-revertC2 and the S. Enteritidis reference strain (S100) are not included in the growth curve below. Both showed growth profiles similar to those of 104, 5408, and 104-revert.

FIG. 2.

Comparison of the widths and lengths of wild-type S. Enteritidis strains (104 and 5408) and their isogenic ciprofloxacin-resistant mutants (104-cip and 5408-cip) as determined by electron microscopy. Results are expressed as means ± SD for 400 measurements for individual Salmonella rods from 6 different fields. Statistical comparisons were made with unpaired Student t tests. **, P < 0.005; ***, P < 0.0005.

FIG. 3.

Adherence (A) and invasion (B) of Caco-2 monolayers by S. Enteritidis wild-type strains (104 and 5408), their isogenic ciprofloxacin-resistant mutants (104-cip and 5408-cip), and the reverted mutant (104-revert). Results are expressed as mean ± SD log numbers of CFU invaded in 12 wells from three separate experiments. Statistical comparisons were made with unpaired Student t tests. ***, P < 0.0005.

FIG. 4.

The detection of porins was carried out using the polyclonal antibodies directed against denatured OmpF porin (A) or the F4 polyclonal antibody directed against the L3 internal loop of E. coli porins (B). Lanes 1, 104; lanes 2, 104-revert; lanes 3, 104-cip; lanes 4, 5408; lanes 5, 5408-cip; lanes 6, S. Enteritidis NCTC 13349. Arrows indicate the migration of F and D porins, respectively.