Staphylococcus aureus Small-Colony Variants from Airways of Adult Cystic Fibrosis Patients as Precursors of Adaptive Antibiotic-Resistant Mutations

Abstract

Prototypic Staphylococcus aureus and their small-colony variants (SCVs) are predominant in cystic fibrosis (CF), but the interdependence of these phenotypes is poorly understood. We characterized S. aureus isolates from adult CF patients over several years. Of 18 S. aureus-positive patients (58%), 13 (72%) were positive for SCVs. Characterization included genotyping, SCCmec types, auxotrophy, biofilm production, antibiotic susceptibilities and tolerance, and resistance acquisition rates. Whole-genome sequencing revealed that several patients were colonized with prototypical and SCV-related clones. Some clonal pairs showed acquisition of aminoglycoside resistance that was not explained by aminoglycoside-modifying enzymes, suggesting a mutation-based process. The characteristics of SCVs that could play a role in resistance acquisition were thus investigated further. For instance, SCV isolates produced more biofilm (p < 0.05) and showed a higher survival rate upon exposure to ciprofloxacin and vancomycin compared to their prototypic associated clones. SCVs also developed spontaneous rifampicin resistance mutations at a higher frequency. Accordingly, a laboratory-derived SCV (ΔhemB) acquired resistance to ciprofloxacin and gentamicin faster than its parent counterpart after serial passages in the presence of sub-inhibitory concentrations of antibiotics. These results suggest a role for SCVs in the establishment of persistent antibiotic-resistant clones in adult CF patients.

Keywords: MRSA; Staphylococcus aureus; antibiotic resistance; cystic fibrosis; persister cells; resistance mutation; small-colony variant.

Figure 1

Phylogenetic tree of SCVs based upon SNP comparison. The phylogeny of the SCVs was assessed upon comparison of the presence/absence of high-impact SNPs (annotated by SNPeff) within each sequenced strain. A heatmap illustrating the dispersion of SNPs was also generated. Each vertical bar represents the presence of an SNP.

Figure 2

Biofilm production of prototypical and SCV S. aureus isolates collected from CF patients. The mean biofilm production for each isolate is indicated on the graph. Blue circles represent prototypic isolates and yellow circles represent SCVs. X symbols represent MRSA isolates. The horizontal bars represent the median relative biofilm production for each group, with the boxes showing the interquartile range and the whiskers indicating the minimum and maximum values (excluding outliers). The mean biofilm production (i.e., each dot) was calculated from at least three independent experiments in which each isolate’s biofilm formation was measured in four replicates. SCVs produced significantly more biofilm than prototypical S. aureus isolates (p-value = 0.0015; Wilcoxon test).

Figure 3

Ciprofloxacin and vancomycin kill kinetics in clinical prototype–SCV pairs. Blue symbols represent prototypic isolates (-L) and yellow symbols represent SCV (-S) isolates. Circles report the control condition (no antibiotic) and triangles report the kill kinetics with the indicated antibiotic. Standard deviations are also indicated. (A) represents CF39A-L and CF39A-S viable counts (CFU/mL); (B) represents CF54A-L and CF54A-S; (C) represents CF78A-L and CF78A-S. The detection limit was 2 log10 CFU/mL. Each condition was tested in triplicates and each symbol represents the mean.

Figure 4

Comparison of rifampicin-induced mutation frequencies between prototypic isolates and SCVs. Ten prototypic isolates and eleven related SCVs were included in this analysis. Mutation frequency was determined by normalizing the number of resistant CFUs growing on the rifampicin plate by the initial inoculum. Blue circles represent prototypes and yellow circles are SCVs. The horizontal bars represent the mutation frequency median for each group, with the boxes showing the interquartile range, and the whiskers indicating the minimum and maximum values (excluding outliers). Results demonstrate that the mutation frequency leading to rifampicin resistance for SCVs is significantly higher than that measured for prototypic isolates (p-value = 0.0079; Wilcoxon test).

Figure 5

Selection of resistance using sequential passages on sub-inhibitory concentrations of antibiotics. S. aureus Newbould (blue bars) and the SCV NewbouldΔhemB (yellow bars) were sequentially exposed to sub-inhibitory concentrations of gentamicin (A) or ciprofloxacin (B) for a total of 30 passages. The bars represent MIC values (the highest value is >128 and >8 µg/mL, respectively, for gentamicin and ciprofloxacin).