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Cold atmospheric-pressure plasma (CAP) has emerged as a potential alternative or adjuvant to conventional antibiotics for the treatment of bacterial infections, including those caused by antibiotic-resistant pathogens. The potential of sub-lethal CAP exposures to synergise conventional antimicrobials for the eradication of Pseudomonas aeruginosa biofilms is investigated in this study. The efficacy of antimicrobials following or in the absence of sub-lethal CAP pre-treatment in P. aeruginosa biofilms was assessed. CAP pre-treatment resulted in an increase in both planktonic and biofilm antimicrobial sensitivity for all three strains tested (PAO1, PA14, and PA10548), with both minimum inhibitory concentrations (MICs) and minimum biofilm eradication concentrations (MBECs) of individual antimicrobials, being significantly reduced following CAP pre-treatment of the biofilm (512-fold reduction with ciprofloxacin/gentamicin; and a 256-fold reduction with tobramycin). At all concentrations of antimicrobial used, the combination of sub-lethal CAP exposure and antimicrobials was effective at increasing time-to-peak metabolism, as measured by isothermal microcalorimetry, again indicating enhanced susceptibility. CAP is known to damage bacterial cell membranes and DNA by causing oxidative stress through the in situ generation of reactive oxygen and nitrogen species (RONS). While the exact mechanism is not clear, oxidative stress on outer membrane proteins is thought to damage/perturb cell membranes, confirmed by ATP and LDH leakage, allowing antimicrobials to penetrate the bacterial cell more effectively, thus increasing bacterial susceptibility. Transcriptomic analysis, reveals that cold-plasma mediated oxidative stress caused upregulation of P. aeruginosa superoxide dismutase, cbb3 oxidases, catalases, and peroxidases, and upregulation in denitrification genes, suggesting that P. aeruginosa uses these enzymes to degrade RONS and mitigate the effects of cold plasma mediated oxidative stress. CAP treatment also led to an increased production of the signalling molecule ppGpp in P. aeruginosa, indicative of a stringent response being established. Although we did not directly measure persister cell formation, this stringent response may potentially be associated with the formation of persister cells in biofilm cultures. The production of ppGpp and polyphosphate may be associated with protein synthesis inhibition and increase efflux pump activity, factors which can result in antimicrobial tolerance. The transcriptomic analysis also showed that by 6 h post-treatment, there was downregulation in ribosome modulation factor, which is involved in the formation of persister cells, suggesting that the cells had begun to resuscitate/recover. In addition, CAP treatment at 4 h post-exposure caused downregulation of the virulence factors pyoverdine and pyocyanin; by 6 h post-exposure, virulence factor production was increasing. Transcriptomic analysis provides valuable insights into the mechanisms by which P. aeruginosa biofilms exhibits enhanced susceptibility to antimicrobials. Overall, these findings suggest, for the first time, that short CAP sub-lethal pre-treatment can be an effective strategy for enhancing the susceptibility of P. aeruginosa biofilms to antimicrobials and provides important mechanistic insights into cold plasma-antimicrobial synergy. Transcriptomic analysis of the response to, and recovery from, sub-lethal cold plasma exposures in P. aeruginosa biofilms improves our current understanding of cold plasma biofilm interactions.

Quorum Sensing (QS) is a well-studied intercellular communication mechanism in bacteria, regulating collective behaviors such as biofilm formation, virulence, and antibiotic resistance. However, cell–cell signaling in haloarchaea remains largely unexplored. The coexistence of bacteria and archaea in various environments, coupled with the known cell–cell signaling mechanisms in both prokaryotic and eukaryotic microorganisms and the presence of cell–cell signaling mechanisms in both prokaryotic and eukaryotic microorganisms, suggests a possibility for haloarchaea to possess analogous cell–cell signaling or QS systems. Recently, N-acylhomoserine lactone (AHL)-like compounds were identified in haloarchaea; yet, their precise role—for example, persister cell formation—remains ambiguous. This study investigated the capacity of crude supernatant extract from the haloarchaeon Halorubrum saccharovorum CSM52 to stimulate bacterial AHL-dependent QS phenotypes using bioreporter strains. Our findings reveal that these crude extracts induced several AHL-dependent bioreporters and modulated pyocyanin and pyoverdine production in Pseudomonas aeruginosa. Importantly, our study suggests cross-domain communication between archaea and bacterial pathogens, providing evidence for archaea potentially influencing bacterial virulence. Using Thin Layer Chromatography overlay assays, lactonolysis, and colorimetric quantification, the bioactive compound was inferred to be a chemically modified AHL-like compound or a diketopiperazine-like molecule, potentially involved in biofilm formation in H. saccharovorum CSM52. This study offers new insights into putative QS mechanisms in haloarchaea and their potential role in interspecies communication and coordination, thereby enriching our understanding of microbial interactions in diverse environments.