Antibiotic's target site affects the potentiation of Lactiplantibacillus plantarum inhibition and inactivation by electroporation.

Abstract

Antibiotic resistance represents a growing global threat, and thus the motivation to develop novel and combined methods of bacterial inactivation is increasing. Electroporation is a technique in which electric pulses of sufficient strength are applied to permeabilize cells, including bacteria. Combining antibacterials with electroporation is a promising strategy to potentiate their bactericidal and bacteriostatic effectiveness. This approach has already proved useful for increasing bacterial inactivation, yet most studies so far have mainly focused on the maximal achievable effects, and less on the underlying mechanisms. We recently demonstrated that in the Gram-negative (G-) bacterium Escherichia coli, electroporation potentiates antibacterials targeting the peptidoglycan wall more than those with intracellular targets. However, in Gram-positive (G+) bacteria, the wall is directly accessible from the outside, and thus the dependence of potentiation on the antibacterial's target may be rather different. Here, we compare the inactivation and growth inhibition of the G+ bacterium Lactiplantibacillus plantarum for two antibiotics with different modes of action: ampicillin (inhibits cell-wall synthesis) and tetracycline (inhibits intracellular protein synthesis). We used antibiotic concentrations ranging from 0 to 30 × MIC (minimum inhibitory concentration that we predetermined for each antibiotic), a single 1-ms electric pulse with an amplitude from 0 to 20 kV/cm, and post-pulse pre-dilution incubation of 24 h or 1 h. Electroporation increased the inhibition and inactivation efficiency of both antibiotics, but this was more pronounced for tetracycline, with statistical significance mostly limited to 24-h incubation. In general, both inhibition and inactivation grew stronger with increasing antibiotic concentration and electric field amplitude. Our results indicate that electroporation potentiates inactivation of G+ bacteria to a larger extent for antibiotics that inhibit intracellular processes and require transport into the cytoplasm, and to a smaller extent for antibiotics that inhibit cell-wall synthesis. This is the inverse of the relation observed in G- bacteria, and can be explained by the difference in the envelope structure: in G- bacteria the outer membrane must be breached for wall-inhibiting antibiotics to access their target, whereas in G+ bacteria the wall is inherently accessible from the outside and permeabilization does not affect this access.