Targeting bioenergetics is key to counteracting the drug-tolerant state of biofilm-grown bacteria.

Embedded in an extracellular matrix, biofilm-residing bacteria are protected from diverse physicochemical insults. In accordance, in the human host the general recalcitrance of biofilm-grown bacteria hinders successful eradication of chronic, biofilm-associated infections. In this study, we demonstrate that upon addition of promethazine, an FDA approved drug, antibiotic tolerance of in vitro biofilm-grown bacteria can be abolished. We show that following the addition of promethazine, diverse antibiotics are capable of efficiently killing biofilm-residing cells at minimal inhibitory concentrations. Synergistic effects could also be observed in a murine in vivo model system. PMZ was shown to increase membrane potential and interfere with bacterial respiration. Of note, antibiotic killing activity was elevated when PMZ was added to cells grown under environmental conditions that induce low intracellular proton levels. Our results imply that biofilm-grown bacteria avoid antibiotic killing and become tolerant by counteracting intracellular alkalization through the adaptation of metabolic and transport functions. Abrogation of antibiotic tolerance by interfering with the cell's bioenergetics promises to pave the way for successful eradication of biofilm-associated infections. Repurposing promethazine as a biofilm-sensitizing drug has the potential to accelerate the introduction of new treatments for recalcitrant, biofilm-associated infections into the clinic.

Towards individualized diagnostics of biofilm-associated infections: a case study.

Organized within biofilm communities, bacteria exhibit resistance towards a broad spectrum of antibiotics. Thus, one might argue that bacteria isolated from biofilm-associated chronic infections should be subjected to resistance profiling under biofilm growth conditions. Various test systems have been developed to determine the biofilm-associated resistance; however, it is not clear to what extent the in vitro results reflect the situation in vivo, and whether the biofilm-resistance profile should guide clinicians in their treatment choice. To address this issue, we used confocal microscopy in combination with live/dead staining, and profiled biofilm-associated resistance of a large number (>130) of clinical Pseudomonas aeruginosa isolates from overall 15 cystic fibrosis patients. Our results demonstrate that in addition to a general non-responsiveness of bacteria when grown under biofilm conditions, there is an isolate-specific and antibiotic-specific biofilm-resistance profile. This individual resistance profile is independent on the structural properties of the biofilms. Furthermore, biofilm resistance is not linked to the resistance profile under planktonic growth conditions, or a mucoid, or small colony morphology of the tested isolates. Instead, it seems that individual biofilm structures evolve during biofilm-associated growth and are shaped by environment-specific cues. In conclusion, our results demonstrate that biofilm resistance profiles are isolate specific and cannot be deduced from commonly studied phenotypes. Further clinical studies will have to show the added value of biofilm-resistance profiling. Individualized diagnosis of biofilm resistance might lead to more rational recommendations for antimicrobial therapy and, thus, increased effectiveness of the treatment of chronically infected patients.