Assembling the puzzle of antimicrobial resistance in staphylococcal biofilms

Multiple mechanisms underpinning biofilm antimicrobial resistance (AMR) have been studied individually. This study aimed to integrate these mechanisms, to understand their contributions to staphylococcal biofilm AMR, as a part of a whole, and to elucidate key hurdles hindering effective biofilm eradication by antimicrobial agents. Nine antibiotics were selected against microplate-based biofilms formed by <i>Staphylococcus aureus</i> ATCC 25923 and <i>Staphylococcus epidermidis</i> RP62A. Four mechanisms, including repressed bacterial metabolism, the barrier effect of the biofilm extracellular polymeric substances (EPS) matrix, the acidic inner-biofilm pH, and inoculum effects associated with high-cell-density biofilm growth were studied. The impact of individual mechanism on biofilm AMR was quantitated by determining the fold increase of concentration that allows antibiotics to overcome the mechanism. Antibiotic concentrations were then incrementally increased from minimum bactericidal concentration (MBC) to sequentially address all four mechanisms, ultimately aiming to kill at least 99.9% of biofilm cells. A simplified method was developed to evaluate the dependence of antibiotics on bacterial metabolic states for the lethality. Gentamicin, tobramycin and ciprofloxacin at 1024 µg/mL overcame all four mechanisms and successfully killed <i>S. aureus</i> ATCC 25923 biofilms by at least 3 log units. Ciprofloxacin at 1024 µg/mL effectively killed <i>S. epidermidis</i> RP62A biofilms. The contribution of each mechanism to biofilm AMR was strain- and drug-dependent, with low-cell metabolism being the most important factor. This study underscores the individual contributions of each mechanism to staphylococcal biofilm AMR and highlights the necessity of targeting all four mechanisms to achieve effective biofilm eradication.