The Influence of Antibiotics, Hypoxia and Neutrophil Proteins on the Evolution of Antibiotic Resistance in Staphylococcus aureus

Invading bacterial pathogens are confronted by numerous challenges including harsh environmental conditions at the site of infection, host immune responses and exposure to antibiotic treatments. Herein, I have studied the interactions between antibiotics, hypoxia and neutrophil-derived proteins in driving the development of antibiotic resistance in Staphylococcus aureus, using an in vitro system. Spontaneous mutations (no antibiotic selection) conferred bacterial clarithromycin, flucloxacillin and tobramycin resistance, but doxycycline and linezolid resistance did not emerge spontaneously. S. aureus evolved in each antibiotic developed mutations associated with resistance, including 50S ribosomal proteins, following S. aureus evolution in clarithromycin, tobramycin and linezolid. Doxycycline selection conferred low level cross-resistance to tobramycin even in the absence of doxycycline resistance. Acquiring the tetracycline-specific efflux pump tetL reduced competitive growth against the isogenic strain without tetL for S. aureus in hypoxia, but not in normoxia. Treatment of S. aureus carrying tetL with tetracycline or doxycycline drove enhanced resistance through mutations in an RluD-like protein and duplications of tetL, suggesting that horizontal acquisition of a resistance mechanism may accelerate the emergence of higher level resistance under antibiotic selection. By contrast, exposure of S. aureus to hypoxic conditions without antibiotics drove adaptive mutations including those in gyrB, and this was prevented by antibiotic treatment. Extensive neutrophilic inflammation was revealed by sputum proteomics from patients with chronic lung diseases, with specific neutrophil proteins upregulated in samples from participants with Pseudomonas aeruginosa infections. Sensitivity to killing by the neutrophil antimicrobial peptide LL-37 was decreased in S. aureus that had evolved clarithromycin, linezolid and tobramycin resistance but not organisms evolved in doxycycline or flucloxacillin. Together these data suggest that interactions between antibiotic treatment and environmental selective forces such as hypoxia and antimicrobial peptides, may alter pathogen adaptation and survival and the evolution of antimicrobial resistance. These important interactions remain to be tested in vivo.