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. Proteus mirabilis is one of the most common human pathogens found in catheter-associated urinary tract infections (CAUTIs) (1, 2). Although long-term CAUTIs are usually polymicrobial, P. mirabilis is considered to play a unique and important role in both CAUTI establishment and catheter blockage, due to its strong ability to induce mineral precipitation (3, 4). P. mirabilis produces extensive urease, which hydrolyzes urea to ammonia, increasing pH and inducing mineral precipitation. This process is commonly referred to as ureolytic biomineralization (5). In CAUTIs, P. mirabilis forms distinctive crystalline biofilms through ureolytic biomineralization (6). Typical minerals produced by P. mirabilis in CAUTIs are struvite (magnesium ammonium phosphate), apatite (calcium phosphate), and calcite (calcium carbonate) (7-9).The treatment of CAUTIs is challenging. Although antimicrobials such as ciprofloxacin and ampicillin are still the most commonly recommended therapeutic agents for urinary tract infections (UTIs) (10), crystalline biofilms are generally recalcitrant to antimicrobials (11). As a result, many patients suffer recurrent biofilm-based infections and catheter encrustation and blockage (12). Alternative therapies, including irrigation with antibioticcontaining solutions and the use of antibiotic-impregnated catheters, also show limited efficacy in preventing biof...

Proteus mirabilis and Pseudomonas aeruginosa are common pathogens that often form biofilms together in catheter-associated urinary tract infections (CAUTI). However, the interactions between these two species in biofilms are largely unknown. P. mirabilis induces ureolytic biomineralization that substantially modifies key biofilm properties including morphology, persistence, and recalcitrance to antimicrobial therapy. These processes are well known to complicate CAUTI, but the consequences for colonization and persistence of P. mirabilis in polymicrobial biofilms have not been explored. Here we characterized the role of biomineralization in regulating the development of P. mirabilis and P. aeruginosa dual-species biofilms. Time-series observations revealed that the dominance of P. mirabilis was synchronized with mineral formation in the biofilm. After 24 hours of development, the dual-species biofilm was dominated by P. mirabilis, and the distribution of P. mirabilis biomass was strongly correlated with the mineral fraction of the biofilm. Conversely, dual-species growth without biomineralization yielded strikingly different patterns in the biofilm, with P. aeruginosa dominating the biofilm biomass. These results show that biomineralization is responsible for the increased success of P. mirabilis in the polymicrobial biofilm. Since biofilm biomineralization commonly occurs in diverse clinical, natural and engineered systems, these findings imply that biomineralization could broadly influence the microbial ecology of multispecies biofilms.