Collateral Sensitivity to β-Lactam Antibiotics in Evolved Apramycin-Resistant MRSA

Abstract

Collateral sensitivity is an evolutionary trade-off for bacteria where acquiring resistance to one antibiotic results in an increased sensitivity to another antibiotic. This study was designed to evaluate the collateral sensitivity of methicillin-resistant Staphylococcus aureus (MRSA) to β-lactam antibiotics induced by the evolution of resistance to apramycin. Collateral sensitivity to ampicillin, cephazolin, ceftriaxone, cefotaxime, cefepime and cefquinome occurred after MRSA were exposed to apramycin and induced to acquire resistance. This sensitivity was associated with reduced β-lactamase activity and decreased expression of the mecA gene. We also found a decrease in the proton motive force and decreased efflux activity. These results provide new insights into collateral sensitivity-based strategies for the treatment of MRSA.

Keywords: apramycin; collateral sensitivity; methicillin-resistant Staphylococcus aureus; β-lactam antibiotics.

Figure 1

Effects of apramycin on the development of antibiotic resistance in MRSA. MICs for apramycin were determined daily. (a) Six strains rapidly developed resistance to apramycin within three days (MIC > 32 mg/L). (b) Four strains remained sensitive to apramycin over seven days.

Figure 2

Sensitivity of apramycin-resistant mutants to β-lactams. Fold change in MIC values for each antibiotic are represented as blocks. Values are listed as log₂.

Figure 3

Bacterial growth curves. Effect of apramycin resistance on in vitro growth of S. aureus isolates. The graphs depict growth of 10 susceptible (red dots) and 10 apramycin-resistant (blue squares) clinical MRSA isolates followed over 24 h at 37 °C in LB broth. Each data point shown is the mean (±SD) for three independent experiments. Significant differences were assessed by one-way analysis of variance: *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Figure 4

Biofilm formation of parental and evolved MRSA strains. Strains were evolved and sampled on days 5 and 16. For reference, biofilm formation data from isolates with no drug exposure were used as comparison (first bar on each graph). Each data point shown is the mean ± SD of three independent experiments. Significant differences were assessed by one-way analysis of variance: ns: not significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Figure 5

Transmission electron microscopy cross-section of ((a) ATCC 43300, (b) 5ZX7, (c) N5) parental strain cells and ((d) ATCC 43300, (e) 5ZX7, (f) N5) apramycin-evolved strain cells. Red arrows indicate loss of cell wall density. Green arrows indicate deformation of shape of S. aureus cells. Blue arrows indicate reduction of cytoplasm. Scale bars represent 100 nm.

Figure 6

β-Lactamase activity. (a) Comparison of three parental strains and apramycin-evolved strain designated with the prefix A. (b) Color change of β-lactamase produced in group N5. + is positive control containing 1 mg/mL of sulbactam; − is negative control containing 2% Tween 80. Each data point shown is the mean ± SD for three independent experiments. Significant differences were assessed by one-way analysis of variance: ns: not significant; **: p < 0.01; ****: p < 0.0001.

Figure 7

Expression of mecA. Fold change in gene expression normalized to 16S rRNA levels and relative to the untreated control. Values represent the mean ± SD for three independent experiments. Significant differences were assessed by one-way analysis of variance: ns: not significant; ***: p < 0.001; ****: p < 0.0001.

Figure 8

Efflux pump activity. Blue and red bars represent parental and induced evolutionary endpoint strains, respectively. (a) Bacterial membrane potential energy (Δψ) before and after the evolution of drug resistance. (b) Transmembrane proton gradient before and after the evolution of drug resistance (ΔpH). (c) Efflux functions before and after the evolution of drug resistance. Values represent the mean ± SD for three independent experiments. Significant differences were assessed by one-way analysis of variance: ns: not significant; *: p < 0.05; **: p < 0.01; ****: p < 0.0001.