Chip-calorimetric monitoring of biofilm eradication with antibiotics provides mechanistic information.

Increased antibiotic resistance of pathogenic bacteria dwelling in biofilm structures has motivated the development of various monitoring tools specifically designed for biofilm investigations. In this study, the potential of the recently emerging chip calorimetry for this purpose was analysed. The activity of biofilms of Pseudomonas putida PaW340 was monitored chip-calorimetrically and compared with counts of colony forming units (CFU), bioluminescence-based ATP measurements, and quantitative confocal laser scanning microscopy (CLSM). The biofilms were treated with antibiotics differing in their mechanisms of action (bactericidal kanamycin vs. bacteriostatic tetracycline) and referenced to untreated biofilms. For untreated biofilms, all methods gave comparable results. Calorimetric killing curves, however, reflecting metabolic responses to biofilm eradication non-invasively in real time, differed from those obtained with the established methods. For instance, heat signals increased right after addition of the antibiotics. This transient increase of activity was not detected by the other methods, since only calorimetry delivers specific information about the catabolic part of the metabolism. In case of the bactericidal antibiotic, CFU misleadingly indicated successful biofilm eradication, whereas calorimetry revealed enduring activity. Our results show that calorimetry holds promise to provide valuable mechanistic information, thereby complementing other methods of biofilm analysis.