Antibiotic-resistant sub-populations of the pathogenic bacterium Staphylococcus aureus confer population-wide resistance

An important clinical feature of invasive Staphylococcus aureus disease is the tendency for infection to persist or relapse despite seemingly adequate antibiotic therapy. One mechanism by which this is believed to occur is through the emergence of antibiotic-resistant small colony variants (SCVs) [1]. When SCVs are isolated from individuals with S. aureus infections, it is common to simultaneously isolate the antibiotic-sensitive parental strain [1–4]. In this work we describe how SCVs create an environment that protects wild-type S. aureus from the lethal effects of aminoglycoside antibiotics. We previously reported that growth of S. aureus in broth containing gentamicin resulted in the emergence of SCVs [5]. However, after 24-hour incubation the population became dominated by wild-type antibiotic-sensitive bacteria (66.2% of the population; 95% confidence interval, 32.8–99.5%). Thus, the gentamicin-sensitive S. aureus present in this heterogeneous population were able to multiply in the presence of a lethal antibiotic. To examine whether this resistance was conferred by bacterial factors present in the broth supernatant, wild-type bacteria and SCVs were grown in broth (from an OD 650 of 0.3 to 1) and the supernatants harvested. Fresh inocula of wild-type gentamicin-sensitive S. aureus were added either to fresh broth, wild-type supernatant, or SCV supernatant and growth inhibition by gentamicin was assayed (Figure 1A). Gentamicin inhibited growth in broth and in wild-type supernatant, but did not inhibit growth in SCV supernatant. Proteomic comparisons between wild-type, SCV and revertant S. aureus were used to examine how SCVs altered their environment to permit growth of the sensitive variant in the presence of gentamicin. Amino-terminal sequencing of the differentially expressed proteins revealed that several of these are associated with anaerobic respiration in other organisms (Figure 2). Although the changes in metabolism accompanying a shift from aerobic to anaerobic respiration are not fully understood in S. aureus, these data suggest that SCVs were respiring anaerobically despite growth in air. The downregulation of an alkaline shock protein in SCVs (Figure 2) suggests that the pH of the environment may have been changing. Uptake of gentamicin by S. aureus is dependent upon pH, in that lowering pH reduces gentamicin uptake and efficacy [6]. Changes in pH during bacterial growth were therefore examined. Fresh broth and the supernatant of wild-type S. aureus grown aerobically had a pH of 7, whereas SCV supernatant and that of wild-type S. aureus grown anaerobically had a reduced pH of 6. We examined the effect of readjusting the …