Abstract
Many bacteria enter the viable but non-culturable (VBNC) state to maximize resources and increase their tolerance to harmful conditions to cope with environmental stress, which has been described for a plethora of important human and foodborne pathogens. VBNC pathogens can potentially present a serious risk to human health as they are invisible to routine microbiological culture-based methods. Of high importance is the increased tolerance to antibiotics or disinfectant measures while in the VBNC state. The greatest remaining challenge for such investigations is the lack of an appropriate, cost-effective multi-species screening method due to experimental constraints. In this study, we investigated if de novo ATP production of cells in the VBNC state is a suitable indicator for overall cell viability that can be utilized to determine the minimum ATP inhibitory concentration (MAIC) of antibiotics and other antimicrobials. To validate this approach, heat-stress time-kill experiments were performed with both culturable and VBNC cells. We developed a comprehensive experimental setup and demonstrated the applicability of this VBNC–MIC assay for testing the tolerance of 12 strains of 4 important bacterial species (Escherichia coli, Bacillus cereus, Pseudomonas aeruginosa, and Listeria monocytogenes) in the VBNC state to eight important antimicrobials including four different antibiotics. We confirmed that bacteria in the VBNC state were resistant to all tested antibiotics (ampicillin, imipenem, ciprofloxacin, and gentamicin) and additionally insensitive to disinfectants (benzalkonium chloride and trioctylmethylammonium chloride) and preservatives (bronopol and sodium azide). These data emphasize the need for further research regarding the characteristics of bacterial pathogens in the VBNC state and present the advantages and high-throughput capabilities of ATP determinations to investigate tolerance of VBNC pathogens to antimicrobials. The presented method should be helpful in order to identify appropriate countermeasures, treatments, or disinfectants when confronted with bacterial pathogens in the VBNC state.
Highlights
Bacteria are exposed to environments with constantly changing conditions
Only exemplary results for one representative strain per bacterial species are depicted in Figure 2, due to the fact that no differences between the respective strains were found
Results clearly show that viable but non-culturable (VBNC) pathogens were resistant to all the antibiotics used, while culturable cells completely lost their culturability during overnight incubation in the presence of Amp, imipenem, ciprofloxacin, and gentamicin
Summary
Bacteria are exposed to environments with constantly changing conditions. To cope with these environmental stresses, many bacteria enter a viable but non-culturable (VBNC) state that results in higher tolerances to harmful conditions (Oliver, 2010; Zhang et al, 2015; Lee and Bae, 2017). The VBNC state was already discovered in 1982 by Xu et al but has been a controversial issue and along the years it was accepted as a survival strategy of bacteria and recently has become an important part in microbial research (Pinto et al, 2015; Ayrapetyan et al, 2018). The cost of this transition is that VBNC cells lose their ability to grow on routine media, they retain intact membranes, undamaged genetic material, and metabolic activity and can potentially revert to the active state (Oliver, 2005; Lin et al, 2017). The VBNC state of pathogens has relevance to human and animal health
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