Characterizing staphylococcus aureus properties in patient biopsies to test current concepts of antibiotic persistence

Staphylococcal infections vary from superficial skin infections to life threatening bacteraemia. They are often associated with prolonged hospital stays leading to high morbidity and mortality. Deep-seated musculoskeletal and blood stream infections are difficult to eradicate with antimicrobial therapy even if the causative S. aureus strain is susceptible in laboratory assays. Various mechanisms explain survival of antibiotic-exposed S. aureus but their relevance in humans is lacking, primarily due to difficulty associated with processing and analysing heterogeneous human tissues.
In this dissertation, we developed a microscopy based method to visualize single S. aureus in millimetre sized human tissues in a time and data efficient manner. Specifically, we used combination of novel fluorescently conjugated vancomycin, immuno-staining and adaptive high resolution microscopy reliant on trained neural networks. Further, we used single cell microfluidic assays to observe clinically relevant antibiotic derived killing in vitro.
We quantified 12,552 S. aureus and its respective niche in biopsies from musculoskeletal infected patients with different antibiotic regimens. We found ~80% S. aureus intracellularly and ~20% extracellularly. Intracellular S. aureus adopted monocyte-macrophage intermediate cells (CD14+CD16+CD68+) as their primary niche whereas macrophages, neutrophils and other eukaryotic cells contained ~30% S. aureus. All S. aureus, irrespective of their location, resided in singlets or in clusters of up to 10 cells. Large clusters, indicative of biofilm formation, were rare and typically undetectable in tissues. These data suggest location-independent inadequacy of conditions prevalent in tissues for sustaining massive S. aureus growth. Antibiotic therapy did not reduce extracellular S. aureus burden or select for particular intracellular niches but significantly increased individual cellular loads. These data fail to support specific stress tolerant niches as crucial permissive sites for observed antibiotic treatment failure. Instead, antibiotic survival seemed to be a S. aureus population-wide property in vivo. Collectively, human data also supports presence of S. aureus in a state of low toxicity, characterized by limited proliferation. This state allows for evasion from antibiotic and host derived killing of both intra- and extracellular subsets. To test this idea, we used microfluidic assay with slow growing S. aureus exposed to a clinically relevant antibiotic – flucloxacillin. In contrast to colony forming units (CFU) based assays, we observed substantial bactericidal effects after antibiotic was removed and growth resumed. This single-cell observation highlighted the complexity associated with understanding antibiotic effects, even when a single aspect of human relevance is focused upon.
Thus, human-centric studies are necessary for understanding relevant bacterial phenotypes that account for diseases. In vitro assays recapitulating multiple aspects of in-patient conditions might serve as better predictors of treatment outcome and provide a basis for identifying more effective control strategies.