Chip-calorimetry provides real time insights into the inactivation of biofilms by predatory bacteria.

Control or removal of undesired biofilms has frequently been found to be quite difficult. In addition to biocidal or antibiotic chemicals or materials designed to prevent biofouling, biological control agents appear to be promising. Reports of bacterial predators eradicating biofilms or eliminating pathogens motivate a more systematic screening of biofilm-eliminating bacterial predators. Unfortunately, the analysis of the eradication process is demanding. In the present study, chip-calorimetry was applied to monitor the elimination of Pseudomonas sp. biofilms by Bdellovibrio bacteriovorus. The method uses metabolic heat as a real-time parameter for biofilm activity. The method is non-invasive, fast and convenient due to real-time data acquisition. In addition, heat-production data can reveal information about the energetics of the predator-prey interaction. The calorimetric results were validated by confocal laser scanning microscopy. The approach described may be useful for the screening of biofilm susceptibility to different predators.

Chip calorimetry for fast and reliable evaluation of bactericidal and bacteriostatic treatments of biofilms.

Chip calorimetry is introduced as a new monitoring tool that provides real-time information about the physiological state of biofilms. Its potential for use for the study of the effects of antibiotics and other biocides was tested. Established Pseudomonas putida biofilms were exposed to substances known to cause toxicity by different mechanisms and to provoke different responses of defense and resistance. The effects of these compounds on heat production rates were monitored and compared with the effects of these compounds on the numbers of CFU and intracellular ATP contents. The real-time monitoring potential of chip calorimetry was successfully demonstrated by using as examples the fast-acting poisons formaldehyde and 2,4-dinitrophenol (DNP). A dosage of antibiotics initially increased the heat production rate. This was discussed as being the effect of energy-dependent resistance mechanisms (e.g., export and/or transformation of the antibiotic). The subsequent reduction in the heat production rate was attributed to the loss of activity and the death of the biofilm bacteria. The shapes of the death curves were in agreement with the assumed variation in the levels of exposure of cells within the multilayer biofilms. The new monitoring tool provides fast, quantitative, and mechanistic insights into the acute and chronic effects of a compound on biofilm activity while requiring only minute quantities of the biocide.

Miniaturized calorimetry - a new method for real-time biofilm activity analysis.

The partial dissipation of Gibbs energy as heat reflects the metabolic dynamic of biofilms in real time and may also allow quantitative conclusions about the chemical composition of the biofilm via Hess' law. Presently, the potential information content of heat is hardly exploited due to the low flexibility, the low throughput and the high price of conventional calorimeters. In order to overcome the limitations of conventional calorimetry a miniaturized calorimeter for biofilm investigations has been evaluated. Using four thermopiles a heat production with spatial and temporal resolutions of 2.5 cm(-1) and 2 s(-1) could be determined. The limit of detection of the heat flow measurement was 20 nW, which corresponds to the cell density of an early stage biofilm (approx. 3x10(5) cells cm(-2)). By separating biofilm cultivation from the actual heat measurement, a high flexibility and a much higher throughput was achieved if compared with conventional calorimeters. The approach suggested allows cultivation of biofilms in places of interest such as technological settings as well as in nature followed by highly efficient measurements in the laboratory. Functionality of the miniaturized calorimeter was supported by parallel measurements with confocal laser scanning microscopy and a fiber optic based oxygen sensor using the oxycaloric equivalent (-460 kJ mol-O2(-1)).