Employ of atomic force microscopy for the analysis of the metabolic response of bacteria to stress conditions imposed by antibiotics

YANTORNO, OM; CATTELAN, N; VILLALBA, MI; KASAS, S; ARNAL, L; VELA, ME; STUPAR, P

Tucuman

Congreso; XII Congreso Argentino de Microbiología General SAMIGE-TUCUMAN 2017; 2018

SAMIGE

To control the rapid spread of bacterial infections, fast, sensitive, and reliable methods for quantitative assessment of antimicrobial activities are needed. Current growth-based methods are time-consuming, not capable of distinguishing between bactericidal and bacteriostatic effects and failto detect ?non growing but metabolically active? bacteria. Most antibiotics inhibit processes that are major consumers of cellular energy output, affecting metabolic activities. Fluctuations of highly sensitive atomic force microscope (AFM) cantilevers can be used to detect low concentrations of living bacteria and to characterize their metabolic activity within minutes. In the case that the microbial cells, adhered to the cantilever, are exposed to a particular stress condition such as antimicrobial agents, this interaction could produce changes in the cantilever oscillation which would let assessing the efficiency of the drug on target cells metabolism. In the present study, we optimized an assay for determine the minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) through the analysis of changes in metabolic activity of bacteria incubated under the presence of different antibiotics using ultrasensitive AFM cantilevers. Bordetella pertussisand Escherichia coliwere incubated in presence of different antibiotics (macrolides and ampicilin) concentrations. To attach bacteria to the cantilever´s surface it was previously incubated with glutaraldehyde and then with bacterial suspension. Finally the vibrational response of the cantilever was registered through the incidence of a laser, upon exposure of antibiotics mentioned. The results of AFM device werevalidated with the ones coming from traditional triphenyl tetrazolium chloride (TTC) method, and the traditional MIC and MBC analysis by the broth dilution method (CLSI). We first prove that the new device is sensitive enough to show by an analysis of variance of cantilever oscillations, the metabolic activity of cells attached to the surface of the cantilever as well as to reflect the increase of adhered cells. It was then determined that in the presence of bactericidal concentrations of antibiotics (data taken from planktonic cell assays), after only 40 min of incubation there is a significant metabolic response. Finally, MIC and MBC were determined in the presence of increasing concentrations of antibiotics. The results obtained from AFM device were tested against the traditional MIC and MBC methods according to CLSI and the derived method from the TTC. This work identifies a link between cellular respiration and cantilevers oscillations demonstrating that the metabolic state of bacteria could be evaluated by the variance of cantilevers movements. Our data show that antibiotics disturb the metabolic state of bacteria and that the answer of this device is enough sensitive and fast to characterize the metabolic state of bacteria in different environments.