RESUMEN
Introduction: 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). Methods: 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. Results: 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. Discussion: 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.
RESUMEN
Antibiotic resistance is a global health threat, and there is ample motivation for development of novel antibacterial approaches combining multiple strategies. Electroporation is among the promising complementary techniques - highly optimizable, effective against a broad range of bacteria, and largely impervious to development of resistance. To date, most studies investigating electroporation as an efficacy potentiator for antibacterials used substances permissible in food industry, and only few used clinical antibiotics, as acceptable applications are largely limited to treatment of wastewaters inherently contaminated with such antibiotics. Moreover, most studies have focused mainly on maximal achievable effect, and less on underlying mechanisms. Here, we compare Escherichia coli inactivation potentiation rates for three antibiotics with different modes of action: ampicillin (inhibits cell wall synthesis), ciprofloxacin (inhibits DNA replication), and tetracycline (inhibits protein synthesis). We used concentrations for each antibiotic from 0 to 30× its minimum inhibitory concentration, a single 1-ms electric pulse with amplitude from 0 to 20 kV/cm, and post-pulse pre-dilution incubation either absent (â²1 min) or lasting 60 min, 160 min, or 24 h. Our data show that with incubation, potentiation is significant for all three antibiotics, increases consistently with pulse amplitude, and generally also with antibiotic concentration and incubation time. With incubation, potentiation for ampicillin was rather consistently (although with weak statistical significance) superior to both ciprofloxacin and tetracycline: ampicillin was superior to both in 42 of 48 data points, including 7 with significance with respect to both, while at 60- and 160-min incubation, it was superior in 31 of 32 data points, including 6 with significance with respect to both. This suggests that electroporation potentiates wall-targeting antibiotics more than those with intracellular targets, providing motivation for in-depth studies of the relationship between the mode of action of an antibiotic and its potentiation by electroporation. Identification of substances permissible in foods and targeting the cell wall of both Gram-negative and Gram-positive bacteria might provide candidate antibacterials for broad and strong potentiation by electroporation applicable also for food preservation.