Assessment of Cold Atmospheric Pressure Plasma (CAPP) Treatment for Degradation of Antibiotic Residues in Water.

The presence of antibiotic residues in water is linked to the emergence of antibiotic resistance globally and necessitates novel decontamination strategies to minimize antibiotic residue exposure in both the environment and food. A holistic assessment of cold atmospheric pressure plasma technology (CAPP) for ß-lactam antibiotic residue removal is described in this study. CAPP operating parameters including plasma jet voltage, gas composition and treatment time were optimized, with highest ß-lactam degradation efficiencies obtained for a helium jet operated at 6 kV. Main by-products detected indicate pH-driven peroxidation as a main mechanism of CAPP-induced decomposition of ß-lactams. No in vitro hepatocytotoxicity was observed in HepG2 cells following exposure to treated samples, and E. coli exposed to CAPP-degraded ß-lactams did not exhibit resistance development. In surface water, over 50% decrease in antibiotic levels was achieved after only 5 min of treatment. However, high dependence of treatment efficiency on residue concentration, pH and presence of polar macromolecules was observed.

Cold atmospheric pressure plasma-antibiotic synergy in Pseudomonas aeruginosa biofilms is mediated via oxidative stress response.

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.

Atmospheric pressure non-thermal plasma exposure reduces Pseudomonas aeruginosa lipopolysaccharide toxicity in vitro and in vivo.

Lipopolysaccharide (LPS) is an endotoxin composed of a polysaccharide and lipid component. It is intrinsically responsible for the pathogenicity of Gram-negative bacteria and is involved in the development of bacterial sepsis. Atmospheric pressure non-thermal plasma is proposed as a potential new approach for the treatment of infected tissue such as chronic wounds, with both antibacterial and wound-healing activities extensively described. Using both the RAW264.7 murine macrophage cell line in vitro assays and the Galleria mellonella insect in vivo toxicity model, the effect non-thermal plasma exposure on LPS-mediated toxicity has been characterised. Short (60 s) non-thermal plasma exposures of Pseudomonas aeruginosa conditioned growth media, membrane lysates and purified P. aeruginosa LPS, resulted in a substantial detoxification and reduction of LPS-induced cytotoxicity in RAW264.7 murine macrophages. Non-thermal plasma exposure (60 s) of purified P. aeruginosa LPS led to a significant (p < 0.05) improvement in the G. mellonella health index (GHI) score, a measure of in vivo toxicity. These findings demonstrate the ability of short plasma exposures to significantly reduce LPS-induced cytotoxicity both in vitro and in vivo; attenuating the toxicity of this important virulence factor intrinsic to the pathogenicity of Gram-negative bacteria.

Cold Plasmas for Biofilm Control: Opportunities and Challenges.

Bacterial biofilm infections account for a major proportion of chronic and medical device associated infections in humans, yet our ability to control them is compromised by their inherent tolerance to antimicrobial agents. Cold atmospheric plasma (CAP) represents a promising therapeutic option. CAP treatment of microbial biofilms represents the convergence of two complex phenomena: the production of a chemically diverse mixture of reactive species and intermediates, and their interaction with a heterogeneous 3D interface created by the biofilm extracellular polymeric matrix. Therefore, understanding these interactions and physiological responses to CAP exposure are central to effective management of infectious biofilms. We review the unique opportunities and challenges for translating CAP to the management of biofilms.

Evolutionary clade affects resistance of Clostridium difficile spores to Cold Atmospheric Plasma.

Clostridium difficile is a spore forming bacterium and the leading cause of colitis and antibiotic associated diarrhoea in the developed world. Spores produced by C. difficile are robust and can remain viable for months, leading to prolonged healthcare-associated outbreaks with high mortality. Exposure of C. difficile spores to a novel, non-thermal atmospheric pressure gas plasma was assessed. Factors affecting sporicidal efficacy, including percentage of oxygen in the helium carrier gas admixture, and the effect on spores from different strains representing the five evolutionary C. difficile clades was investigated. Strains from different clades displayed varying resistance to cold plasma. Strain R20291, representing the globally epidemic ribotype 027 type, was the most resistant. However all tested strains displayed a ~3 log reduction in viable spore counts after plasma treatment for 5 minutes. Inactivation of a ribotype 078 strain, the most prevalent clinical type seen in Northern Ireland, was further assessed with respect to surface decontamination, pH, and hydrogen peroxide concentration. Environmental factors affected plasma activity, with dry spores without the presence of organic matter being most susceptible. This study demonstrates that cold atmospheric plasma can effectively inactivate C. difficile spores, and highlights factors that can affect sporicidal activity.

Non-thermal Plasma Exposure Rapidly Attenuates Bacterial AHL-Dependent Quorum Sensing and Virulence.

The antimicrobial activity of atmospheric pressure non-thermal plasma has been exhaustively characterised, however elucidation of the interactions between biomolecules produced and utilised by bacteria and short plasma exposures are required for optimisation and clinical translation of cold plasma technology. This study characterizes the effects of non-thermal plasma exposure on acyl homoserine lactone (AHL)-dependent quorum sensing (QS). Plasma exposure of AHLs reduced the ability of such molecules to elicit a QS response in bacterial reporter strains in a dose-dependent manner. Short exposures (30-60 s) produce of a series of secondary compounds capable of eliciting a QS response, followed by the complete loss of AHL-dependent signalling following longer exposures. UPLC-MS analysis confirmed the time-dependent degradation of AHL molecules and their conversion into a series of by-products. FT-IR analysis of plasma-exposed AHLs highlighted the appearance of an OH group. In vivo assessment of the exposure of AHLs to plasma was examined using a standard in vivo model. Lettuce leaves injected with the rhlI/lasI mutant PAO-MW1 alongside plasma treated N-butyryl-homoserine lactone and n-(3-oxo-dodecanoyl)-homoserine lactone, exhibited marked attenuation of virulence. This study highlights the capacity of atmospheric pressure non-thermal plasma to modify and degrade AHL autoinducers thereby attenuating QS-dependent virulence in P. aeruginosa.

Eradication and phenotypic tolerance of Burkholderia cenocepacia biofilms exposed to atmospheric pressure non-thermal plasma.

Chronic lung infection with bacteria from the Burkholderia cepacia complex (BCC), and in particular B. cenocepacia, is associated with significant morbidity and mortality in patients with cystic fibrosis (CF). B. cenocepacia can spread from person to person and exhibits intrinsic broad-spectrum antibiotic resistance. Recently, atmospheric pressure non-thermal plasmas (APNTPs) have gained increasing attention as a novel approach to the prevention and treatment of a variety of hospital-acquired infections. In this study, we evaluated an in-house-designed kHz-driven plasma source for the treatment of biofilms of a number of clinical CF B. cenocepacia isolates. The results demonstrated that APNTP is an effective and efficient tool for the eradication of B. cenocepacia biofilms but that efficacy is highly variable across different isolates. Determination of phenotypic differences between isolates in an attempt to understand variability in plasma tolerance revealed that isolates which are highly tolerant to APNTP typically produce biofilms of greater biomass than their more sensitive counterparts. This indicates a potential role for biofilm matrix components in biofilm tolerance to APNTP exposure. Furthermore, significant isolate-dependent differences in catalase activity in planktonic bacteria positively correlated with phenotypic resistance to APNTP by isolates grown in biofilms.

Bactericidal efficacy of atmospheric pressure non-thermal plasma (APNTP) against the ESKAPE pathogens.

The emergence of multidrug-resistant pathogens within the clinical environment is presenting a mounting problem in hospitals worldwide. The 'ESKAPE' pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) have been highlighted as a group of causative organisms in a majority of nosocomial infections, presenting a serious health risk due to widespread antimicrobial resistance. The stagnating pipeline of new antibiotics requires alternative approaches to the control and treatment of nosocomial infections. Atmospheric pressure non-thermal plasma (APNTP) is attracting growing interest as an alternative infection control approach within the clinical setting. This study presents a comprehensive bactericidal assessment of an in-house-designed APNTP jet both against biofilms and planktonic bacteria of the ESKAPE pathogens. Standard plate counts and the XTT metabolic assay were used to evaluate the antibacterial effect of APNTP, with both methods demonstrating comparable eradication times. APNTP exhibited rapid antimicrobial activity against all of the ESKAPE pathogens in the planktonic mode of growth and provided efficient and complete eradication of ESKAPE pathogens in the biofilm mode of growth within 360s, with the exception of A. baumannii where a >4log reduction in biofilm viability was observed. This demonstrates its effectiveness as a bactericidal treatment against these pathogens and further highlights its potential application in the clinical environment for the control of highly antimicrobial-resistant pathogens.