Ureolytic biomineralization reduces Proteus mirabilis biofilm susceptibility to ciprofloxacin

Ureolytic biomineralization induced by urease-producing bacteria, particularly Proteus mirabilis, is responsible for the formation of urinary tract calculi and the encrustation of indwelling urinary catheters. Such microbial biofilms are challenging to eradicate and contribute to the persistence of catheter-associated urinary tract infections, but the mechanisms responsible for this recalcitrance remain obscure. In this study, we characterized the susceptibility of wild-type (ure-) and urease-negative (ure+) P. mirabilis biofilms to killing by ciprofloxacin. Ure+ biofilms produced fine biomineral precipitates that were homogeneously distributed within the biofilm biomass in artificial urine, while ure-biofilms did not produce biomineral deposits under identical growth conditions. Following exposure to ciprofloxacin, ure+ biofilms showed greater survival (less killing) than ure-biofilms, indicating that biomineralization protected biofilm-resident cells against the antimicrobial. To evaluate the mechanism responsible for this recalcitrance, we observed and quantified the transport of Cy5-conjugated ciprofloxacin into the biofilm by video confocal microscopy. These observations revealed that the reduced susceptibility of ure+ biofilms resulted from hindered delivery of ciprofloxacin into biomineralized regions of the biofilm. Further, biomineralization enhanced retention of viable cells on the surface following antimicrobial exposure. These findings together show that ureolytic biomineralization induced by P. mirabilis metabolism strongly regulates antimicrobial susceptibility by reducing internal solute transport and increasing biofilm stability.