Overcoming antibiotic resistance: non-thermal plasma and antibiotics combination inhibits important pathogens

Abstract Antibiotic resistance (ATBR) is increasing every year as the overuse of antibiotics (ATBs) and the lack of newly emerging antimicrobial agents lead to an efficient pathogen escape from ATBs action. This trend is alarming and the World Health Organization warned in 2021 that ATBR could become the leading cause of death worldwide by 2050. The development of novel ATBs is not fast enough considering the situation, and alternative strategies are therefore urgently required. One such alternative may be the use of non-thermal plasma (NTP), a well-established antimicrobial agent actively used in a growing number of medical fields. Despite its efficiency, NTP alone is not always sufficient to completely eliminate pathogens. However, NTP combined with ATBs is more potent and evidence has been emerging over the last few years proving this is a robust and highly effective strategy to fight resistant pathogens. This minireview summarizes experimental research addressing the potential of the NTP-ATBs combination, particularly for inhibiting planktonic and biofilm growth and treating infections in mouse models caused by methicillin-resistant Staphylococcus aureus or Pseudomonas aeruginosa. The published studies highlight this combination as a promising solution to emerging ATBR, and further research is therefore highly desirable.


Introduction
Antimicr obial r esistance (AMR), often r eferr ed to as the "silent pandemic", impacts the treatment of infections less ob viousl y than "visible" health crises, leading to higher healthcare costs, longer hospital stays and increased mortality.Its impact extends beyond healthcare, also affecting agriculture and ecology (Schnall et al. 2019 ).In this minir e vie w, we focus on a novel approach to tackling the emergence of antibiotic resistance (ATBR) and m ultidrug-r esistant (MDR) bacteria.We discuss factors contributing to the emergence of ATBR and MDR pathogens and introduce the favor able pr operties of non-thermal plasma (NTP) and its potential for use in medicine, particularly in treating infections caused by resistant pathogens.We summarize experimental studies combining NTP pr e-tr eatment and antibiotics (ATBs) to enhance inhibition efficacy, primarily against typically used model r epr esentativ es of gr am-negativ e and gr am-positiv e bacteria, Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA), r espectiv el y.A gr owing body of e vidence shows that NTP has real potential for mitigating ATBR and restoring the sensitivity of MDR pathogens in medical practice and infection treatment.

Antibiotic resistance: a worldwide threat
ATBR, a global health problem of the 21st century, is caused by the rise of MDR pathogens and the lack of novel treatment appr oac hes.If no action is taken, MDR micr oor ganisms will become the leading cause of death worldwide by 2050 (WHO 2021 ; Tang et al. 2023 ).ATBR increases the global health problems rate, mortality and healthcare costs because of the failure of ATBs to eliminate common infections (Botelho et al. 2019, Gajdács et al. 2021 ).In addition, high costs and lengthy pr ocedur es slo w do wn the de v elopment of novel ATBs, further exacerbating the ATBR crisis (Daikos et al. 2021, Gajdács et al. 2021 ).

Emergence of ATBR
The ATBR crisis is primarily caused by the overuse of ATBs in healthcar e, a gricultur e and animal husbandry.Misuse in prescribing ATBs for viral infections and poor patient compliance with prescribed dosing contribute to ATBR development (WHO 2021).In animal husbandry, misuse of ATBs leads to the emergence of MDR bacteria potentiall y tr ansmittable to humans .T he A TBR emergence is exacerbated by additional factors, such as improper dis-posal of A TBs, A TBs treatment discontinuation, ATBs being available without medical prescription, as well as inconsistent global regulations (Polianciuc et al. 2020 ).To tackle this crisis, stricter r egulations, better dia gnostics, gr eater public awareness and inv estments in ne w tr eatment a ppr oac hes, as well as r esearc h, ar e r equir ed.
The European Union summary report, issued by the European Food Safety Authority (EFSA) and the European Centre for Disease Pr e v ention and Contr ol (ECDC), tr ac ks AMR tr ends in bacteria isolated from animals, food of animal origin and humans in 2019-2020(EFSA 2022 ) ) and shows an alarming increase in AMR.These trends of increasing ATBR in common bacteria highlight the need for continued surv eillance, r esearc h and collaboration at European and global levels.

Current status of MDR pathogens
According to recent estimates, ∼4.95 million deaths were associated with bacterial AMR in 2019 (Lancet 2022 ).Although in 2017, the World Health Organization (WHO) list of "priority pathogens" included the ESKAPE pathogens-Enterococcus faecium , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumannii , P. aeruginosa and Enterobacter spp.(WHO 2017, De Oliv eir a et al. 2020, Mancuso et al. 2021, Teng et al. 2023 )-pathogens such as Neisseria gonorrhoeae , Mycobacterium tuberculosis and Candida spp.are becoming incr easingl y r esistant as well, r eflecting a gener al tr end to w ar ds AMR (Ventola 2015 ).The resistance of important pathogens to conv entionall y used ATBs is summarized in Table 1 .
The search for alternatives to tackle AMR typically employs MDR strains of MRSA and P. aeruginosa .MRSA alone was reported to cause more than 100 000 deaths across 204 countries in 2019 (Wu et al. 2019, Guo et al. 2020 ), and the treatment of its infections is a challenge due to ATBR (Wu et al. 2019 ).Altered penicillin-binding pr oteins gr ant MRSA str ains r esistance to methicillin and other β-lactam ATBs (Gajdács 2019 ) as well as vancomycin (Haseeb et al. 2019, Guo et al. 2020 ).Mutations in the rpoB gene, associated with impair ed membr ane permeability, as well as increased biofilm formation and r eactiv e oxygen species production, led to rifampicin resistance (Portelli et al. 2020, Zhang et al. 2023a ).Resistance to trimethoprim-sulfamethoxazole, used for MRSA treatment, has also been reported (Nurjadi et al. 2022, Ham et al. 2023 ).Similarl y, r esistance to ampicillin and kanamycin, partiall y r e v ersible by metallic micr onutrients, has been r eported for MRSA (Garza-Cervantes et al. 2020 ).Mutations in the mecA gene have been linked to oxacillin-resistance (Goering et al. 2019 ).More pronounced ATBR in MRSA biofilm compared with the planktonic form has been r eported (Cr aft et al. 2019, Guo et al. 2020, Gajdács et al. 2021 ).
P. aeruginosa typically infects immunocompromised individuals , for example , patients after in v asiv e sur geries , with diabetes , or with cystic fibrosis (Botelho et al. 2019, Qin et al. 2022 ).Its ability to m utate r a pidl y and tr ansfer genes contributes to ATBR, especiall y to aminoglycosides, quinolones and β-lactams, making treatment difficult (Wang et al. 2020, Qin et al. 2022 ).Efflux pumps and alter ed cell membr anes wer e r eported as the MDR mec hanism of P. aeruginosa to chlorhexidine (Cieplik et al. 2019, Wang et al. 2020, Tag ElDein et al. 2021 ).The PhoP-PhoQ two-component regulatory system contributes to the resistance to polymyxin B, the last treatment option typically used against MDR gram-negative bacteria (Yang et al. 2021a ).Biofilm formation also increases ATBR in this bacterium.Data indicate a correlation between the occurrence of MDR bacteria, biofilm formation and the expression of virulence, underlining the role of P. aeruginosa as a k e y model in the study of biofilm-related ATBR (Eladawy et al. 2021, Gajdács et al. 2021, Silva et al. 2023 ).

NTP: a powerful multi-purpose tool
Plasma is a partially or fully ionized gas that can exhibit highly v ariable pr operties in terms of temper atur e (millions of K elvins to room temperature), pressure and composition, depending on its source.NTP has found application in many fields, for example, modification and functionalization in material science , en vironmental science and a gricultur e, food industry or biological and medical applications (Murugesan et al. 2020, Barjasteh et al. 2021, Asl et al. 2022, Moszczy ńska et al. 2023 ).
The following sections focus on NTP, also known as cold atmospheric plasma, generated at atmospheric pressure.Its ambient temper atur e is gentle to the treated material, making it well suited for medical applications, for example, as a novel non-ATB antimicr obial tr eatment (Mor eau et al. 2008, Brun et al. 2018 ).In addition, NTP can be used to treat the surfaces of various materials, which then have antibacterial or self-cleaning properties (Mozeti č 2019 ).

Medical fields benefitting from NTP treatment
Plasma medicine is a new field that was founded ∼25 years ago, and that successfully implemented the use of NTP-generated reacti ve o xygen and nitrogen species (RONS) for medical targets, benefitting from their antimicrobial effect (Laroussi 2020 ), and also yielding a clinical trial on NTP-mediated wound healing in 2010 with encour a ging r esults (Isbary et al. 2010 ).In addition to wound healing, NTP has applications in dermatology, particularly for inflammatory skin irritations (Heinlin et al. 2011, Emmert et al. 2013, Wirtz et al. 2018 ), skin infections (e .g. mycosis , on yc homycosis, acne-pr one skin) (Chutsirimongk ol et al. 2014, Lux et al. 2020 ), abscesses, burns or the r emov al of scars and skin growths, among others (Bernhardt et al. 2019 ).Furthermore, NTP has been successfully used in ophthalmology (Reitberger et al. 2018 ), in dentistry (Pan et al. 2013, Dong et al. 2014, Delben et al. 2016, Gherardi et al. 2018 ), where the AMR of dental biofilm bacteria is becoming an ur gent pr oblem, and in orthopedics, for tr eating post-sur gical infections (Nguyen et al. 2018 ).NTP might have applications in regener ativ e medicine and medical engineering due to its influence on stem cell pr olifer ation (Mileti ć et al. 2013, Park et al. 2016, Alemi et al. 2019, Xiong et al. 2019 ).NTP-mediated decontamination of medical instruments and other equipment benefits from the antimicr obial pr operties of NTP, and its efficiency against biofilms and MDR pathogens, as demonstrated by NTP-mediated biofilm r emov al fr om endotr ac heal tubes with possible a pplication for other endoscopes ( İbi ş and Ercan 2020 ).
Finely tuned doses of NTP have been shown to selectively kill cancer cells without harming healthy cells, opening a new field of plasma oncology being tested for leukemia, carcinoma, breast cancer , brain cancer , prostate cancer , colorectal cancer and others (Laroussi 2014, Yan et al. 2021 ).Preliminary studies in Germany used NTP in palliativ e ther a py in pain mitigation of ulcerations for head and neck cancer patients (Metelmann et al. 2015 ).The efforts described above eventually culminated in the US Food and Drug Administr ation a ppr oving the first American oncological clinical trial in 2019.Mor eov er, NTP a pplication may not r emain limited to surfaces, as miniaturized microplasma devices for subcutaneous and internal application are currently under development.For example, NTP was successfully used for nasal m ucosal r egener ation in vitro and in vivo (Won et al. 2018 ) and the r egener ation of se v er al nerve cell types isolated from animal models (Katiyar et al. 2019 ).An important implication of plasma medicine is the efficient inactiv ation of danger ous r esistant pathogens, with ESKAPE being a primary target (Scholtz et al. 2021 ).In vivo and ex vivo experiments pr ovide e vidence for NTP-mediated inhibition of S. aureus , E. coli , P. aeruginosa and A. baumannii (Li et al. 2023 ), as well as promoted healing of local burns, accompanied by good biological acceptance, only mild adverse reactions and overall shortening of the course of tr eatment.NTP tr eatment of experimentally wounded and MRSA-infected rabbits led to a decrease in cytokine secretion, inflammatory response and immune cell pr olifer ation, and to acceler ated r e-epithelialization and wound healing (Li et al. 2021a ).NTP-mediated decrease of P. aeruginosa load and biofilm formation was demonstrated in wounds of diabetic mice (Cooley et al. 2020 ) and in a human skin wound model in a recent preclinical study of burns healing (Bagheri et al. 2023 ).
Se v er al certified NTP-gener ating de vices for the treatment of surface infections are currently on the market.The kINPen ® MED is the first CE-certified class IIa medical device for the treatment of c hr onic wounds and pathogen-induced skin disorders.PlasmaDerm ® VU-2010 (CINOGY Technologies GmbH, Duderstadt, Germany; CE-certified in Germany by MEDCERT, ISO 13485) and SteriPlas (Adtec Ltd., London, UK) are certified for the activation of c hr onic and acute wound healing by changing its microen-vir onment and r educing the micr obial load, as well as of MDR pathogens .T he J ett Plasma devices (COMPEX, s.r.o , Brno , Czech Republic) are certified for dermatology, aesthetic medicine and ophthalmology.

Gener a tion and favorable properties of NTP
NTP is typically generated by supplying ionization energy to gas using, for example, an electrical disc har ge.Its macr oscopic temper atur e is ambient or slightly higher (typically not more than 40 • C), but highl y ener getic electr ons induce a rich mixture of differ ent r eactiv e particles (light electr ons hav e significantl y higher energy than heavy ions and neutr al particles).The c hemical composition of NTP depends on its generation parameters, including a pplied volta ge and feeding gas.Ambient air is commonl y used, but other gases like Ar, He or their mixture with O 2 are used for special applications (Tendero et al. 2006 ).RONS are generated in NTP either dir ectl y fr om O 2 and N 2 in air, or from particles (e.g.He, Ar) interacting with biological material.The most effective antimicr obial RONS ar e hydr oxyl r adical OH •, atomic oxygen O •, singlet o xygen 1 O 2 , supero xide radical O 2 − , atomic nitrogen N and excited states of N 2 and NO x .These active species interact with living cells, which is also the reason for the pr ov en antimicr obial efficacy of NTP (Gr av es 2012 ).
The adv anta ge of NTP is its antimicrobial activity against MDR bacteria, while not inducing primary or acquir ed r esistance (Zimmermann et al. 2012 ).This is due to the mechanism of NTP action, primarily o xidati ve stress induction b y R ONS, leading to cell membr ane ruptur e, cytoplasmic leaka ge and degr adation of intr acellular components (Kartasc he w et al. 2016, Ma et al. 2022 ).Therefor e, NTP is unlikel y to act with different efficiency on MDR and non-r esistant bacterial str ains (Sakudo and Misawa 2020 ).Furthermore, NTP-mediated inhibition of conjugative transfer of resistance genes, and thus inhibition of the very emergence of ATBR, has been reported (Li et al. 2021 b).Gener all y, the antimicr obial effect of NTP is mediated by a combination of se v er al mec hanisms that have not yet been fully elucidated.The specific impact and importance of each mechanism also depends on the type of NTPgener ating de vice used (Tender o et al. 2006 (Moisan et al. 2002, Zhang et al. 2023b ).Most of these mechanisms target functional cellular pathways that are potentially susceptible to escape mutations gr anting r esistance.Ho w e v er, the etc hing (at higher NTP doses) additionally causes physical damage to the membrane, eventually leading to its rupture .T his is very interesting and important as cells are unable to cope with physical damage using their usual defense mechanisms, including the emergence of resistance.Certain bacterial cell forms, such as endospores or biofilms, are cover ed by pol ysacc haride-pr otectiv e layers efficient a gainst envir onmental thr eats, typicall y the imm une system.Although gr anting some protection, these layers do not provide resistance to NTPmediated damage (Julák et al. 2020, Paldrychová et al. 2020, Khosravi et al. 2021, Das et al. 2022, Liu et al. 2022 ).
NTP treatment can be applied indirectly as well, when applied to a liquid (water, saline or others), whic h accum ulates r eactive particles and can be used for applications at a later time or different place .T his phenomenon of plasma-activated water (PAW) or plasma-activated saline (PAS) has already been described (Machala et al. 2018, Zhou et al. 2020 ).Ne v ertheless, the dir ect physical damage and membrane etching mentioned above are onl y v ery limited upon the a pplication of P AW or P AS.

Combination of NTP pr e-tr eatment and ATBs action
To date, ther e ar e v ery fe w publications addr essing the use of a NTP-ATBs combination for overcoming the ATBR of dangerous pathogens.With one exception, all the e vidence r egarding the NTP enhancement of conventional ATBs, mainly in the treatment of infections caused by MRSA and P. aeruginosa , has only emerged since 2020.Because the published r esearc h addr essing the combination is still very limited, to date, the mechanisms of the syner gy hav e not been the focus.Ho w e v er, we can speculate that NTP capable of damaging the cell by a combined mechanism of action leaves the bacterium exhausted and unable to counter the ATBmediated pr essur e .At the same time , NTP causes the release of free planktonic cells from resistant biofilm structures, leading to increased sensitivity to ATBs action.While individual ATBs target specific pathways in cells, the br oadl y acting NTP is unlikely to do that.This is crucial for the synergistic effects on MDR bacteria.
ATBR is metabolically and energetically demanding and encourages the bacterium to invest resources in escaping the respectiv e a gent, leaving insufficient r esources to tac kle NTP-induced damage.Ho w ever, given that each ATB specifically targets specific pathways in cells, it is impossible to anticipate a common mechanism of action without detailed experimental studies, as it may be different for each ATB used in combination with NTP.Mor eov er , A TBR metabolic pathways do not increase resistance to NTP and thus NTP and ATBs can act as completely independent a gents.NTP is sufficientl y gentle for live tissue applications, but due to their sensitivity, a synergy of two different approaches is beneficial.Suc h a syner gistic combination could pr ovide benefits to the treated tissue, reduce the environmental and health impact of ATB use and also increase the efficacy of ATBs against MDR bacteria, thus overcoming the ATBR problem.
As mentioned abo ve , the antimicrobial effect of NTP is highly dependent on the parameters of its generation.T herefore , detailed NTP properties, as well as ATB concentrations used in existing experimental studies and the resulting antimicrobial/antibiofilm effects, are summarized in Table 2 .

NTP-ATBs combination targeting MRSA in vitro
The very first mention, to the best of our knowledge, of combining NTP pr e-tr eatment with ATB action was published in 2013 (Bayliss et al. 2013 ).This important pioneering report was published in the form of a Letter to the Editor and ther efor e contained very limited information.Nevertheless, its success made the authors propose the combination ther a py for post-oper ativ e infections, burns or leg and foot ulcers.They tested NTP pr e-tr eatment of MRSA (undefined str ain) cultur es on Tryptone So y a Broth (TSB) agar follo w ed b y application of ATB test strips with trimethoprim, kanam ycin, o xacillin or norflo xacin.Regained ATB sensiti vity, demonstrated by a clear inhibition zone, was detected after 10 s of NTP pr e-tr eatment combined with trimethoprim, and after 30 s of NTP combined with the other ATBs.Although the inhibition was not complete, as demonstrated by se v er al isolated bacterial colonies within the inhibition zones, the results sho w ed that NTP pr e-tr eatment is capable of reversing resistance to certain ATBs.
An enhanced antibiofilm effect of NTP pr e-tr eatment follo w ed by ATBs (rifampicin, ciprofloxacin, norfloxacin and vancomycin) against MRSA was reported (Guo et al. 2021 ).MRSA ATCC 33591 biofilm formed on TSB agar plates was treated with NTP for 2, 4 or 6 min and subsequently with ATBs at concentrations of 625 (rifampicin) and 1250 mg/L (other ATBs), r espectiv el y.It w as sho wn that NTP treatment enhanced the effect of rifampicin on the reduction of MRSA biofilm.
PAS demonstr ated a syner gistic effect with ATB action a gainst MRSA biofilm (Yang et al. 2021b ).When MRSA was incubated in the presence of PAS for 30 min and combined with vancomycin (1.25 and 0.625 g/L for 24 h) or rifampicin (0.625 and 0.3125 g/L for 24 h), PAS reduced MRSA ATCC 33591 biofilm in vitro by at least six orders of magnitude colony forming units per milliliter (CFU/mL), while PAS, vancomycin and rifampicin alone only reached 1.2, 1.2 and 3.6 orders of ma gnitude, r espectiv el y.

Pseudomonas aeruginosa in vitro
Impr ov ed ATB action and e v en er adication of P. aeruginosa biofilms upon NTP treatment was reported by Paldrychová et al. ( 2020 ).Four strains of P. aeruginosa (DBM 3081 and 3777, ATCC 10145 and 15442) were exposed to NTP (15-60 min) and cultured in subinhibitory doses of gentamicin, ceftazidime and polymyxin B. The Table 2. Detailed summary of information reported in experimental studies describing the use of a combination of non-thermal plasma and antibiotics in inhibiting important pathogens.susceptibility of individual strains to NTP, ATBs and their combination differed a lot, thus the right set-up needs to be determined for eac h str ain; ho w e v er, in gener al, NTP induced a higher ATB susceptibility, with gentamicin requiring the lo w est concentrations (4-9 mg/L) for inhibition.A complete eradication of mature P. aeruginosa ATCC 15442 biofilm from Ti-6Al-4 V orthopedic allo y w as ac hie v ed after 15 min of NTP and 8.5 mg/L gentamicin combination treatment, as sho wn b y scanning electron microscopy (SEM).The effect of ciprofloxacin against P. aeruginosa PAO1 planktonic cells and biofilm can be boosted with NTP pr e-tr eatment (Mur aca et al. 2021 ).The ciprofloxacin MIC of biofilm (200 mg/L) was reduced by one-half upon NTP pr e-tr eatment (50-100 mg/L).SEM visualization sho w ed that despite considerable inhibition, complete eradication of biofilm was not ac hie v ed after exposur e to NTP for 3 min follo w ed b y ciprofloxacin.As residual cells and the risk of re-infection are not acceptable in medical practice, conditions yielding complete eradication should be determined first.This study also addr essed nanostructur ed lipid carriers delivery of ciprofloxacin.Although the efficacy of cipr ofloxacin a gainst P. aeruginosa biofilm was enhanced in this formulation, no synergistic effect was ac hie v ed when combined with NTP.

Bacteria
Ciprofloxacin was also used by Khosravi et al. ( 2022 ), who investigated NTP pr e-tr eatment of MDR P. aeruginosa (isolated fr om clinical specimens) biofilm follo w ed b y subinhibitory concentration of the ATB (16 mg/L).While ciprofloxacin inhibited biofilm biomass and cell viability by ∼70%, 90 s of NTP treatment led to ∼85% inhibition.Mor eov er, SEM visualization sho w ed that bacterial cells in the biofilm lysed and mostly only cell debris and extracellular pol ymers r emained on the surface of carrier; ther e wer e no intact viable bacterial cells .T he presence of cell debris can bias conventional biofilm quantification assa ys , and indeed, fluorescence microscop y sho w ed a substantial reduction in the biofilm biomass, with only a few residual cells still on the carrier surface .T hese results are very promising, even although complete inhibition will r equir e mor e stringent conditions.
A recent study that emplo y ed NTP in combination with ciprofloxacin, as well as other ATBs (gentamicin and tobramycin compared with disinfectant chlorhexidine) (Maybin et al. 2023 ), also reported that NTP pre-treatment increases the susceptibility of both planktonic and biofilm cells of P. aeruginosa (strains PAO1, P A14 and P A10548) to subinhibitory concentrations of ATBs.In addition to standard methods like CFU/mL counting and metabolic activity determination (isothermal microcalorimetry), a number of methods (e.g.transcriptomic analysis, signaling molecules tr ac king with hyper phosphorylated guanosine, and detection of extracellular ATP and LDH) were used to address the mechanism of action.The highest enhancement of ATB action by NTP pretreatment was exhibited for tobramycin (biofilm-eradicating concentr ation dr opping impr essiv el y fr om 256 to 2 mg/L after 90 s of NTP), follo w ed b y gentamicin and ciprofloxacin, and the lo w est enhancement efficacy was observed for the disinfectant chlorhexidine.NTP enhanced ATB action in terms of cell metabolic activity inhibition in all tested cases.Tr anscriptomic anal ysis sho w ed activation of pathways mitigating NTP-mediated oxidative stress (discussed in c ha pter Gener ation and favorable properties of NTP), for example, per oxide dism utase, oxidases, catalases, peroxidases and denitrification genes .Moreo ver, a s witch from biofilm to planktonic cells was detected 6 h after exposure, as ribosome modulation factor, involved in the formation of persistent cells, w as do wnregulated.This finding is consistent with pr e viousl y published (Kašpar ov á et al. 2022 ) NTP-mediated release of P. aeruginosa cells from biofilm to their planktonic form.Overall, Maybin et al. ( 2023 ), similar to the other studies discussed in this section, concluded that NTP pr e-tr eatment can be an effectiv e str ategy for restoring the susceptibility of P. aeruginosa biofilms to antimicrobial agents.

NTP-ATBs combination targeting other dangerous bacterial pathogens in vitro
The pioneering publication describing the NTP and ATBs combination against biofilm (Julák et al. 2020 ) used ATB-resistant bacteria Staphylococcus epidermidis and Esc heric hia coli , and a yeast Candida albicans .The biofilms were treated with NTP (15 and 30 min) and subsequently with erythromycin (10 mg/L), polymyxin B (15 mg/L) and amphotericin B (2.5 mg/L), r espectiv el y.While fluor escence micr oscop y sho w ed a reduction of biofilm area in all cases, only the bacteria sho w ed an enhanced reduction in metabolic activity upon the combination tr eatment.Importantl y, the exposure to NTP and ATBs resulted in an efficient prevention of biofilm r e-de v elopment fr om persistent cells, which makes this combination ther a py a pr omising str ategy in the treatment of pathogens.
A unique stud y re ported a synergy between NTP treatment and an antimicrobial peptide bacteriocin (Lactocin C-M2) used against putr efactiv e bacteria Morganella spp.wf-1 isolated from aquatic foods (Shan et al. 2020 ).The middle-tested concentration (0.3 g/L) of Lactocin C-M2 combined with 90 s of NTP exposure synergisticall y r eac hed a decr ease in Morganella spp.cells by ∼6-fold.Transmission electr on micr oscopy of bacteria tr eated with the combination r e v ealed disruption of cell membranes, cytoplasmic condensation, DNA relaxation, abnormal septation, irregular crosswall formation and e v en cellular lysis of greater magnitude than when treated with Lactocin C-M2 or NTP alone.In addition, the combination treatment led to a higher leakage of K + , phosphates, DN A/RN A, proteins and enzymes than after Lactocin C-M2 or NTP alone.
Food decontamination with NTP and nisin, a natural bacteriocin produced by lactic acid bacteria, was tested using Listeria innocua grown planktonically or on the surface of xanthan gum gel (Costello et al. 2021 ).Four different arrangements of bacterial cells wer e tested: gr own in a liquid food pr oduct; gr own in water used to wash a solid food pr oduct; gr own on the surface of a food product; and grown on one solid product and tr ansferr ed to another product.For nisin treatment, 50 μL of 35 IU/ml solution was dropped onto the surface of the culture disc and follo w ed b y NTP treatment for 30 min.The combination was more effective than individual treatments, but only when nisin was applied prior to NTP.The study also provided insights into the environmental stress response and adaptation of L. innocua grown in structured systems to natur al antimicr obials and nov el antimicr obial tec hnologies, and r epr esents a step to w ar ds application of food-decontamination methods in the food industry.

NTP-ATBs combination targeting dangerous pathogens in vivo and its possible clinical applications
The studies described in the pr e vious section demonstrated synergy between NTP and ATBs in vitro .Given the novelty of this appr oac h in both plasma medicine and ATBR pr e v ention, onl y limited data have demonstrated synergy in vivo .Ho w ever, tw o studies performed in mouse models support the in vitro findings and highlight the potential for implementing this technology in clinical practice.
One of the above-mentioned studies (Guo et al. 2021 ) addressed not only the in vitro effect, but also the treatment of MRSAinfected wounds in a mouse model.Shaven and disinfected mice w ere w ounded under anesthesia and infected with MRSA.Over 3 days thereafter, posterior parts of the infected mice were treated with NTP for 6 min once a day, and rifampicin (30 mg/kg) was administer ed intr a gastricall y e v ery 12 h.The number of bacteria counted in vitro as well as from sacrificed mice clearly sho w ed that NTP treatment enhanced the effect of rifampicin on the reduction of MRSA biofilm.Mor eov er, blood and histoc hemical analyses demonstrated a favorable biosafety profile of the combined treatment.
In the second study, PAS demonstrated a synergistic effect with ATB action also against MRSA for systemic infection studies.Mice were inoculated with 100 μL of MRSA suspension into the lateral tail vein, inducing systemic infection but not death during the experimental c ycle.F rom da y 2 to da y 4 post infection, 150 μL of PAS was administered intraperitoneally and 30 mg/kg rifampicin was administered intragastrically once a da y.On da y 5, blood was sampled from an eye vein, mice were sacrificed and their guts tested for bacteria presence.PAS combined with rifampicin synergistically and effectively reduced the MRSA infection (as opposed to non-combination ther a py) inv estigated b y histology, as w ell as impr ov ed hematological and bioc hemical par ameters of infected mice.
For clinical applications of NTP, a number of obvious problems need to be addressed (e.g. the treatment of systemic infections).Superficial infections can be r eadil y tac kled with the combination of surface NTP application and systemic or topical ATB administr ation.In suc h cases, NTP can be specificall y and pr ecisel y targeted onto infected tissue and the treatment enhanced by ATB application.Ne v ertheless , the abo ve-discussed study using indirect application of NTP in the form of PAS, which can be administered systemically, suggests a way of addressing systemic infections in the future.Ho w ever, a detailed discussion of this a ppr oac h is, considering the single published study to date, beyond the scope of our speculations.
Unlike the systemic administration, the topical external application of the NTP-ATBs combination in clinical practice, for example, against bacterial infections such as acne, atopy, abscesses, wound infections, military wounds and burns, is clearly a promising idea.Our study of mycotic skin infections sho w ed a significant ther a peutic effect of the sim ultaneous administr ation of systemic antifungal agents with external application of NTP (Lux et al. 2020 ).A similar success can be expected when the same appr oac h is applied to MDR bacteria.In our opinion, the treatment of non-systemic infections with a NTP-ATBs combination has major ther a peutic potential, whic h should be tr anslated into clinical trials as well.
Another promising application is in bacterial bladder infections.As an internal infection, this r equir es a more complex appr oac h, and could benefit from the promising results with PAS.PAS of compositions harmless to the bladder epithelium could be supplemented dir ectl y into the bladder, which would r estor e the ATBs sensitivity of infecting MDR bacteria.Alternativ el y, dir ect administration of NTP via a catheter comes into consideration.It is worth noting that electr ocoa gulation, a method used in clinical practice for a long time, has a very similar generation principle to NTP.Ho w e v er, pr eliminary studies ma pping the effect of PAS on the bladder epithelium, as well as in vitro or in vivo testing of PAS and NTP effects on MDR bacteria responsible for bladder infections , ha ve yet to be conducted.
The two visions discussed above appear to be best suited for initial use, as other complex or systemic infections are likely to be more difficult to treat.Treatment of internal infections of a known origin may resemble the treatment of cancer, for which the application of NTP has been studied for a long time (P artec ke et al. 2012(P artec ke et al. , Bekeschus 2023 ).Ho w ever, clinical evidence of successful gas NTP application in cancer patients is r ar e and no malignancy treatment involving gas NTP has been established in clinical practice to date.
Additionall y, NTP a pplication to mitigate contamination during implant placement may also be a promising possibility.We addressed this option by in vitro studies (Paldrychová et al. 2020 ) of biofilm elimination from Ti-6Al-4 V alloy used in orthopedics using a NTP-ATBs combination.During implantation placement, an a ppr opriate exposur e to NTP could r educe the bacteria load or increase bacteria susceptibility to ATB prophylaxis.Implant materials are generally highly resistant and therefore NTP could be applied in much higher doses than typically used for sensitive tissues.Even this ex vivo preventive application of NTP could significantl y r educe the risk of subsequent infection.

Conclusion
AMR and ATBR r epr esent a global health pr oblem of major importance.MDR pathogens ar e emer ging acr oss bacterial str ains, ATBR to most medically used ATBs has been reported and novel ATB de v elopment is not k ee ping up with the pace.Tackling the complex AMR/ATBR challenge requires a coordinated global effort that includes impr ov ed surv eillance, enhanced infection control, prudent use of ATBs in human and veterinary medicine, as well as increased investment in research into alternative therapies and innov ativ e str ategies.We belie v e that NTP r epr esents a promising tool to either combat MDR pathogens directly or to restore their ATB susceptibility and regain control over the treatment.NTP alone is capable of inactivating bacteria and the specificities of its action minimize the risk of inducing resistance.Howe v er, syner gy between NTP and ATBs ac hie v es m uc h better r esults, as demonstrated by a number of in vitro and two in vivo mouse model studies.Medical applications of NTP are being introduced, highlighting its great potential and hopefully helping to reverse worst-case scenarios.

Table 1 .
Summary of resistance of important pathogens to conventionally used antibiotics (ATBs).
, Ehlbec k et al. 2010 , Šimon čicová et al. 2019 ), but the following are typically present (Gr av es 2012 , Sc holtz et al. 2021 ): lipid per oxidation and pr otein denaturation both in membranes and in cytoplasm, triggering of metabolic and apoptotic pathways by RONS ( Čtvrte čková et al. 2019 , Akter et al. 2020 ), accum ulation of c har ged particles on the cell surface, UV radiation (only marginal for most plasma sources) (Machala et al. 2010 ) and etching