Chemical Induction of Aminoglycoside Uptake Overcomes Antibiotic Tolerance and Resistance in Staphylococcus aureus

Abstract

Aminoglycoside antibiotics require proton motive force (PMF) for bacterial internalization. In non-respiring populations, PMF drops below the level required for drug influx, limiting the utility of aminoglycosides against strict and facultative anaerobes. We recently demonstrated that rhamnolipids (RLs), biosurfactant molecules produced by Pseudomonas aeruginosa, potentiate aminoglycoside activity against Staphylococcus aureus. Here, we demonstrate that RLs induce PMF-independent aminoglycoside uptake to restore sensitivity to otherwise tolerant persister, biofilm, small colony variant, and anaerobic populations of S. aureus. Furthermore, we show that this approach represses the rise of resistance, restores sensitivity to highly resistant clinical isolates, and is effective against other Gram-positive pathogens. Finally, while other membrane-acting agents can synergize with aminoglycosides, induction of PMF-independent uptake is uncommon, and distinct to RLs among several compounds tested. In all, small-molecule induction of PMF-independent aminoglycoside uptake circumvents phenotypic tolerance, overcomes genotypic resistance, and expands the utility of aminoglycosides against intrinsically recalcitrant bacterial populations.

Keywords: Staphylococcus aureus; aminoglycosides; antibiotics; biofilm; persisters; resistance; rhamnolipids; tolerance.

Figure 1.. Rhamnolipids synergize with aminoglycosides against tolerant S. aureus populations.

A. Chemical structure of a representative di-rhamnolipid congener, di-rhamnolipid C10C10. B. S. aureus strain HG003 was grown to mid-exponential phase and challenged with 20x the MIC (15.6µg/mL) of tobramycin +/− 10 or 30µg/ml RLs. Survivors were enumerated at the indicated time points. C. S. aureus HG003 membrane potential was measured by calculating the red/green ratio using the population mean fluorescence intensities (MFI) for HG003 incubated with 30µM DiOC₂(3) for 30 minutes. Prior to staining, cells were grown to mid-exponential phase, then treated for 30 minutes with the indicated concentration of RLs or for 5 minutes with 5µM CCCP as a control. *p<0.05 (One-way ANOVA with Tukey’s multiple comparison post test). D. Mid-exponential phase HG003 was challenged with the C_max concentration of tobramycin (58µg/mL) +/− 30µg/ml RLs under anaerobic conditions. Survivors were enumerated at the indicated time points. E. Biofilm-associated S. aureus HG003 was challenged with 58µg/mL tobramycin +/− 30µg/ml RLs for 24 hours prior to CFU enumeration. F. S. aureus SCV strain HG003 menD::tn was grown to mid-exponential phase and challenged with 15.6µg/mL tobramycin +/− 10 or 30µg/ml RLs. CFU enumeration occurred at the indicated time points. All experiments were performed in biological triplicate. **p<0.005 (Student’s t-test). Error bars represent mean +/− SD. Limit of detection is indicated by the horizontal dashed line. See also Figure S1 and Figure S2.

Figure 2.. Rhamnolipid/aminoglycoside combinational therapy targets S. aureus persisters.

A. Mid-exponential phase S. aureus strain HG003 was treated with 5mM AsKO₂ for 30 min prior to addition of 58µg/mL tobramycin. Intracellular ATP was measured using a BacTiter-Glo cell viability assay immediately prior to antibiotic challenge (black bars). An aliquot of each culture was removed after 24hr, washed, and plated to enumerate survivors (grey bars). ***p<0.005 (Student’s t-test, calculated relative to cultures treated with tobramycin alone). B, C. Exponential phase populations of HG003 were exposed to B. 30µg/mL chloramphenicol or C. 1µM CCCP for 30 min prior to challenge with 58µg/mL tobramycin +/− 30µg/ml RLs. At the indicated time points an aliquot was removed, washed and plated to enumerate survivors. All experiments were performed in biological triplicate. Error bars represent mean +/− SD. Limit of detection is indicated by the horizontal dashed line. See also Figure S3.

Figure 3.. Rhamnolipids repress the rise of tobramycin resistance and restore sensitivity to resistant isolates.

A. Six independent lineages of HG003 were passaged daily in subinhibitory concentrations of tobramycin +/− 30µg/mL RLs and monitored for the spontaneous occurrence of tobramycin resistant mutants through changes in MIC. B, C. Minimum tobramycin concentration necessary to inhibit the growth of B. resistant isolates from passaged strain HG-2 (Figure 3A, black arrows) or C. CF clinical isolates, +/− 30µg/mL RLs.

Figure 4.. Rhamnolipids specifically induce PMF-independent aminoglycoside uptake to resensitize tolerant S. aureus.

A. S. aureus was grown to mid-exponential phase then challenged with 58µg/mL tobramycin alone, or in combination with 30µg/mL RLs, 40µg/mL GML, 3.2µg/mL adarotene, or 0.5µg/mL oxacillin (black bars). An aliquot of each culture was removed after 24hr, washed, and plated to enumerate survivors. White bars represent S. aureus survivors following 24hr treatment with each cell envelope-acting agent without tobramycin. B. Texas Red-conjugated tobramycin was added to S. aureus cultures with our without each compound. Following 1hr, Texas Red-tobramycin uptake was measured by flow cytometry. C, D. S. aureus HG003 was C. treated with 1µM CCCP or D. grown anaerobically prior to treatment with 58µg/mL tobramycin +/− each cell envelope acting compound individually. Survivors were enumerated at the indicated time points. E. S. aureus strain SH1000 harboring an inducible PzapA::gfp cell division reporter was grown to OD₆₀₀=0.5, then treated with 30 RLs, 40µg/mL GML 3.2µg/mL adarotene, or 0.5µg/mL oxacillin for 1 hr. Cells were washed, then treated with the membrane dye, FM4–64. Changes to membrane morphology and ZapA localization relative to control cultures were visualized using a GE Applied Precision DeltaVision Elite de-convolution fluorescence microscope equipped with a Photometrics CoolSnap HQ2 camera. Scale bar: 1µm. *p<0.05 (Student’s t-test). All experiments were performed in biological triplicate. Error bars represent mean +/− SD. See also Figure S4.