Acetic acid exerts bactericidal activity against the common burn wound pathogens Staphylococcus aureus and Pseudomonas aeruginosa without generating resistance

Acetic acid exerts bactericidal activity against the common burn wound pathogens Staphylococcus aureus and Pseudomonas aeruginosa without generating resistance | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Acetic acid exerts bactericidal activity against the common burn wound pathogens Staphylococcus aureus and Pseudomonas aeruginosa without generating resistance Sara Henderson, Callum Clark, Thomas Robinson, Liam Grover, Joan Geoghegan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6663847/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Staphylococcus aureus and Pseudomonas aeruginosa are two of the most common pathogens colonising burn wounds in hospitals. Resistance or tolerance to antimicrobials, due to biofilm formation, complicates treatment and increases morbidity, highlighting that current approaches to burn wound care are insufficient. Therefore, novel antimicrobial treatments are needed. In this study we characterise the antimicrobial activity of acetic acid against S. aureus and P. aeruginosa. Our results demonstrate that acetic acid exerts potent antimicrobial activity against both pathogens, including those growing within biofilms, and bacteria that are not actively dividing. The concentrations of acetic acid required to achieve antimicrobial effects were well below the therapeutically tolerated range. We also found that resistance to acetic acid did not develop in vitro , suggesting that the likelihood of resistance emerging in a single step is low. Additionally, application of acetic acid, within a Gellan hydrogel-based dressing, effectively reduced bacterial burden in a porcine ex vivo model of skin colonisation. Together, our findings support the potential of acetic acid as a promising topical antimicrobial agent for preventing burn wound infections by demonstrating that acetic acid is active against bacteria in various growth states and showing for the first time that resistance does not develop easily in vitro . These results suggest that could be a suitable candidate for the clinical management of burn wounds in the future. Biological sciences/Microbiology/Antimicrobials Biological sciences/Microbiology/Bacteriology Biological sciences/Microbiology/Biofilms Biological sciences/Microbiology/Pathogens Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Skin is one of the first barriers to infection. When this barrier is broken, for example following a burn injury, microorganisms from the host’s own resident microbiota and the environment can colonise the wound, leading to infection. Bacterial infection, followed by an inability or failure of the immune system to clear infection, often traps the wound in an inflammatory state whereby it fails to heal, and subsequently predisposes the individual to systemic infections ( 1 , 2 ). Indeed, between 30% and 52% of individuals with burn wound infections go on to develop sepsis ( 3 – 5 ), which represents the leading cause of death after the first 24 hours following injury, with a 28–65% mortality rate ( 6 – 9 ). Burn wound infections at the point of injury are sterilised, leading to a microbially deprived environment which can be re-colonised without competition from the resident skin microbiota ( 10 ). Staphylococcus aureus and Pseudomonas aeruginosa are amongst the most common bacteria to be isolated from burn wounds, and their presence increases inflammation, slows wound healing and increases morbidity ( 11 – 13 ). The human commensal S. aureus typically colonises the wound within hours of injury, from the hosts own skin or nasal microbiota ( 14 – 16 ). P. aeruginosa colonises the wound later, often from environmental sources such as contaminated water ( 17 – 20 ). Treatment of burn wounds typically consists of application of a broad-spectrum topical antimicrobial, upon admission, followed by surgical debridement of dead or severely damaged tissue, and administration of oral (systemic) antibiotics ( 21 – 24 ). The treatment of burn wound infection is complicated by a reduced permeation and perfusion of immune cells into the wound site, in combination with a repressed local and systemic immune response soon after injury ( 1 ), reduced bioavailability of antimicrobial drugs at the site, and antimicrobial resistance ( 19 , 25 ). Treatment of bacterial infection may also be hindered by the presence of bacteria that are not actively growing, and those that are contained within biofilms. Biofilms, which are multispecies communities of bacteria surrounded by a carbohydrate and protein-rich extracellular matrix, are not susceptible to the actions of some antibiotics, due to their charged nature prohibiting the diffusion of other, like-charged, drugs into the biofilm matrix ( 26 , 27 ). Biofilm formation also reduces clearance by the host immune response, leading to further inflammation and infection ( 27 – 29 ). Topical antimicrobial agents have also been trialled for the treatment of burn wound infection, including silver nitrate, silver sulfadiazine, and honey ( 30 ) (reviewed in ( 31 – 33 )). However, toxicity in some silver-based compounds has resulted in delayed healing, whilst resistance to some silver-based compounds has also emerged ( 34 – 38 ), leading to poor resolution of infection. Thus, there is an unmet need for effective, topical antimicrobial therapies for the reduction of burn wound colonisation and infection. One potential, alternative, topical antimicrobial therapy is acetic acid. The antimicrobial qualities of acetic acid, a main component of vinegar, has been known for centuries where, historically, vinegar was used for preservation of food products. Acetic acid has also been used historically, since the age of Hippocrates in 420 BC, for the sterilisation and treatment of wounds ( 39 , 40 ). More recently, acetic acid has been demonstrated to be effective against a wide range of microorganisms, including fungi ( 41 – 44 ) and bacteria, including those commonly associated with multidrug resistance, such as Pseudomonas spp. and Acinetobacter spp. ( 40 , 45 – 49 ) and within biofilms ( 45 , 46 , 50 ). Acetic acid has also been trialled in the treatment of wound infections ( 51 , 52 ). However, questions around the tolerability, dosage and application of acetic acid for the treatment of bacterially colonised wounds remains. One potential route of administration/application is the incorporation of acetic acid, or other antimicrobial or anti-inflammatory drugs, into wound dressings, including hydrogel-type dressings. Within this study, acetic acid is incorporated into a Gellan dressing. This dressing is formed by dehydrating a Gellan hydrogel to form a thin film, which can then be rehydrated at the point of care in an aqueous solution, in this case acetic acid. The Gellan dressings are optically clear, allowing clinicians to inspect the wound without having to remove the dressings. In this study, the antimicrobial activity of acetic acid against two common burn wound pathogens, S. aureus and P. aeruginosa , was characterised. The influence of concentration on the antimicrobial properties of acetic acid was determined, highlighting that low concentrations of acetic acid were required for antimicrobial activity. Furthermore, the potential for resistance to acetic acid to be selected for over 20 serial passages was assessed, with results indicating that resistance to acetic acid treatment did not emerge in vitro . Additionally, the application of acetic acid within a hydrogel-based dressing effectively reduced bacterial load in a porcine ex vivo model of bacterial colonisation. Collectively our findings support the potential of acetic acid for future clinical application. METHODS Bacterial Strains Staphylococcus aureus ATCC29213 (Source: American Type Culture Collection), Staphylococcus aureus USA300 LAC*(53) , and Pseudomonas aeruginosa PAO1, a wildtype ATCC15692 strain (Source: American Type Culture Collection), were used in this study. Bacterial strains were grown in Mueller-Hinton broth (MHB) (Oxoid) or on Mueller-Hinton agar (MHA) (MHB supplemented with 1.5% bacteriological agar, Millipore) unless otherwise stated. Antibiotics, tobramycin and vancomycin, were purchased from Sigma-Aldrich, unless otherwise specified. Time of Kill Single colonies of S. aureus USA300LAC* or P. aeruginosa PAO1 were inoculated into 10 ml MHB and grown to stationary phase at 37°C, 180 rpm, for approximately 16-18 hours. Stationary phase cultures were diluted 1:100 into 5 ml fresh MHB and grown to early exponential phase (OD 600 =0.3). Cultures were adjusted to the 0.5 McFarland standard and diluted to a starting CFU/ml of 5x10 5 . Acetic acid was added to a final concentration of 0.5 or 2% (v/v). Control antibiotics were used at a concentration at least 5 times higher than the MIC (vancomycin: 5 mg/L, tobramycin: 1.25 mg/L). Samples were taken at 0, 30, 60, 120 and 300 minutes, serially diluted and plated onto MHA. Colonies were enumerated after a 16–18-hour incubation at 37°C to give the CFU/ml values. Stationary Phase Killing Assay Single colonies were used to inoculate 10 ml MHB and grown to stationary phase at 37°C, 180 rpm, for 16-18 hours. Optical density (600 nm) was measured and adjusted to a final OD 600 = 1.0 in 4 ml either MHB or PBS. Acetic acid was added to final concentrations 0, 0.1, 0.2, 0.5, 1, 2 or 4% (v/v). Control antibiotics were added at concentrations at least 5 times higher than the (vancomycin: 5 mg/L, tobramycin: 1.25 mg/L), or at a final concentration of 2 mg/L for lysostaphin (Antibodies.com). Initial CFU counts were taken, and cultures were incubated for 5 hours at 37°C, 180 rpm. The cultures were serially diluted and plated onto MHA. Colonies were enumerated after a 16–18-hour incubation at 37°C to give the CFU/ml values. Minimum Biofilm Inhibitory Concentration (MBIC) Single colonies were used to inoculate 5 ml MHB and grown to stationary phase (as above). Optical density (600 nm) was measured and adjusted to a final OD 600 = 0.025 in 2 ml MHB ( P. aeruginosa PAO1) or MHB + 4% NaCl (ThermoFisher Scientific) ( S. aureus USA300LAC*) in 24-well tissue culture-treated plates (Costar). Acetic acid at 0.01% - 5% (v/v) final concentration was used to inhibit growth. Biofilms were grown at 37°C for 72 hours. After 72 hours, the planktonic growth was removed, and the biofilm washed twice with sterile dH 2 O. The remaining biofilm was stained with 1 ml 1% (w/v) crystal violet (ProLab Diagnostics) for 1 minute. The biofilm was washed twice with sterile dH 2 O. Bound crystal violet was dissolved in 1 ml 5% (v/v) acetic acid. 100 μl was transferred to a 96-well plate and the absorbance measured at 570 nm. Minimum Biofilm Eradication Concentration (MBEC) Single colonies were used to inoculate 5 ml MHB and grown to stationary phase (as above). Optical density (600 nm) was measured and adjusted to a final OD600 = 0.01 in MHB ( P. aeruginosa PAO1) or MHB + 4% NaCl ( S. aureus USA300LAC*). 200 μl was added to Nunc 96-well plates (ThermoFisher Scientific) and Nunc TM Immuno TSP lids (ThermoFisher Scientific) were used to close the plates. The plates were grown at 37°C, 200 rpm for 24 hours to establish a biofilm. The TSP lids were washed twice in 1x PBS, and the TSP lids transferred to 0.02% - 5% (v/v) acetic acid in MHB or MHB + 4% NaCl for 5 hours at 37°C, 200 rpm. The TSP lids were washed twice in 1x PBS, and the TSP lids transferred to MHB or MHB + 4% NaCl in the absence of acetic acid for 16 hours at 37°C, 200 rpm. Optical density at 600 nm was measured. A minimum of 3 biological replicates were included. Frequency of Resistance Single colonies were used to inoculate 5 ml MHB and grown to stationary phase (as above). The culture was centrifuged and resuspended in 2 ml PBS. 100 μl aliquots were spread onto MHA containing 0.1 ,0.2 or 0.5% (v/v) acetic acid. To determine the number of colony forming units, the inoculum was serially diluted in PBS and plated on antibiotic free MHA plates. Plates were incubated for up to 48 hours (37°C) and colonies enumerated. The spontaneous frequency of resistance was determined by dividing the number of resistant colonies (CFU/ml) by the total number of viable cells (CFU/ml). Where no colonies were obtained the limit of detection was determined to be 2 x 10 1 CFU/ml and used to give the largest possible spontaneous frequency of resistance. Serial Passage at sub-inhibitory drug concentrations Overnight cultures of S. aureus USA300 LAC* and P. aeruginosa PAO1 were grown to stationary phase, for 18 hours, and subsequently diluted to an OD 600 of 0.05 in MHB containing specified concentrations of acetic acid or tobramycin. MIC values were recorded after 18 hours, prior to the addition of glycerol (ThermoFisher Scientific) (final concentration: 10%) and one freeze-thaw cycle. Subsequent passages, at a dilution of 1:30 into fresh MIC plates, were performed for 20 passages. Testing GellanAA dressing efficacy within a porcine ex vivo model of bacterial colonisation. Porcine skin was obtained commercially (www.skinforsale.com) and frozen at -20°C until required. Skin was sterilised with 70% ethanol prior to inoculation with S. aureus USA300 LAC* or P. aeruginosa PAO1 (grown as previously described) at an OD600 of 1 x 10 -6 , corresponding to a bacterial burden of approximately 1 x 10 2 CFU/ml. Bacteria were left to acclimatise on the skin for 2 hours, prior to treatment with Gellan dressing rehydrated in PBS (treatment control) or 2% acetic acid. At 1, 4 and 24 hours, skin and dressing were removed and homogenised in PBS, with the subsequent bacterial suspension serially diluted and plated for viable CFU count. Viable CFU counts were normalised to that of the PBS control to account for bacterial growth on the skin. Statistical Analysis Statistical analysis was performed using the R package multcomp v1.4-22 or Graphpad Prism version 10.0. Typically, One-way ANOVA with Dunnett’s test of multiplicity were performed against the 0% acetic acid/PBS controls. *P<0.05, **P<0.01, ***P<0.001. RESULTS Exposure to acetic acid causes rapid cell death in P. aeruginosa and S. aureus . Together, S. aureus and P. aeruginosa are responsible for more than 50% of burn wound infections in Birmingham hospitals ( 13 ). Therefore, initially the antimicrobial properties of acetic acid were assessed against two strains of Staphylococcus aureus (one MRSA strain USA300 LAC*, and one MSSA strain ATCC29213) and one strain of Pseudomonas aeruginosa (PAO1), all of which have a range of sensitivities to known antibiotic classes ( 54 – 57 ). The minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC) for acetic acid was measured for all three strains (Table 1) and found to be well below the therapeutically tolerated acetic acid concentration of 2% ( 51 ). To explore the antimicrobial activity of acetic acid in more detail, the tolerance of the bacteria to acetic acid was tested by adding various concentrations of acetic acid to washed stationary phase cultures diluted in either rich growth medium (Mueller-Hinton broth) or phosphate buffered saline (PBS). Following incubation for 5 hours in acetic acid concentrations of 0.5% or less, S. aureus ATCC29213 (Figs. 1 B & E) and S. aureus USA300 LAC* remained viable (Figs. 1 A & D). However, viability was significantly reduced following incubation in a 1% (v/v) solution of acetic acid (Figs. 1 A, B, D & E). This was the case regardless of whether growth medium or buffer was used for both S. aureus strains tested (Figs. 1 A, B, D & E). The reduction in viable cells recovered following exposure to 2% (v/v) acetic acid in PBS was dramatic, from the order of 1x10 9 CFU/ml to less than 500 CFU/ml being recovered (Figs. 1 D & E). As a control the endopeptidase lysostaphin was included as this results in rapid death by hydrolysing cell wall peptidoglycan ( 58 ). In contrast, vancomycin, a clinically relevant antibiotic for the treatment of MRSA infection ( 59 ), did not reduce the viability of the bacteria under these conditions (Figs. 1 A, B, D & E). Similarly, P. aeruginosa cells harvested in the stationary phase of growth and incubated with acetic acid were killed during the 5-hour incubation period (Fig. 1 C & F). The concentration of acetic acid required to reduce the bacterial cell viability in both rich medium (0.5% v/v) and PBS (0.2% v/v) was notably lower for P. aeruginosa PAO1 compared to S. aureus ATCC29213 and USA300 LAC*. Next, the ability of acetic acid to kill S. aureus USA300 LAC* and P. aeruginosa PAO1 was compared with a control antibiotic using bacterial cells harvested in the exponential phase of growth. The inoculum was added to a medium supplemented with acetic acid. Based on these data, a concentration of acetic acid that significantly reduced the viability of S. aureus USA300 LAC* and P. aeruginosa PAO1 (2%), and a concentration above the MIC and MBC for both (Table 1), but that did not significantly reduce S. aureus USA300 LAC* viability (0.5%), was chosen. 2% acetic acid was determined to be bactericidal after 60 minutes for S. aureus USA300 LAC* while 0.5% acetic acid inhibited growth of S. aureus USA300 LAC* but did not reduce the CFU recovery (Fig. 2 A). In comparison, at 5x MIC (5 mg/L), vancomycin treatment reduced the number of colony forming units after 5 hours (Fig. 2 A). In contrast, 0.5% acetic acid was demonstrated to be highly bactericidal against P. aeruginosa PAO1, resulting in eradication of detectable CFU at 60 minutes, with 2% causing rapid killing, resulting in no detectable colony forming units at 30 minutes (Fig. 2 B). Additionally, the sample taken immediately after addition of acetic acid contained significantly fewer CFU when compared to the one taken immediately prior to the acetic acid addition. In contrast, 5x MIC (1.25 mg/L) tobramycin caused a decline in the viable CFU/ml over time, with no detectable CFU recovered at 120 minutes (Fig. 2 B). Biofilm formation is inhibited in the presence of acetic acid Biofilms are one of the key barriers to the successful resolution of bacterial infection, by conventional antimicrobial therapies ( 60 – 64 ). Under biofilm producing conditions, biofilm formation of S. aureus USA300 LAC* was significantly reduced when the media was supplemented with 0.16% (v/v) acetic acid (Fig. 3 A). Although P. aeruginosa PAO1 biofilm formation was not significantly reduced by the presence of acetic acid, the biofilm density was reduced by 90% at 0.08% (v/v) acetic acid (Fig. 3 B). Established biofilms are eradicated significantly by acetic acid To understand if acetic acid is effective against established biofilms, biofilms of S. aureus USA300 LAC* and P. aeruginosa PAO1 were established, before exposing the biofilms to various concentrations of acetic acid for 5 hours. The biofilms were then used to seed growth in Mueller-Hinton broth for 16 hours. While acetic acid was unable to completely eradicate bacterial viability within established biofilms under the conditions tested, the viability of S. aureus USA300 LAC* within an established biofilm was significantly reduced by a 5-hour exposure to acetic acid at a concentration of 0.625% (v/v) (Fig. 3 C). Treatment of an established P. aeruginosa PAO1 biofilm with 0.31% (v/v) acetic acid significantly reduced bacterial regrowth (Fig. 3 D), suggesting that 0.31% (v/v) acetic acid significantly reduced P. aeruginosa PAO1 bacterial viability. Acetic Acid does not induce spontaneous mutations at concentrations close to the agar MIC Bacteria become resistant to an antimicrobial agent either through random genetic mutations within their genome, or through the acquisition of foreign DNA containing resistance machinery ( 65 ). Firstly, the survival of S. aureus USA300 and P. aeruginosa PAO1 in the presence of lethal concentrations of acetic acid were assessed. At a concentration of 0.2% (v/v) acetic acid, no viable bacterial colonies (limit of detection: 2 x 10 1 CFU/ml) were detected, suggesting that spontaneous resistance to acetic acid is not possible, at this limit of detection. Selection for spontaneous mutations in the presence of acetic acid, did not produce any surviving colonies even at concentrations close to the agar MIC values (e.g. PAO1 MIC value of 0.078% (v/v) with no colonies observed at 0.1%(v/v) acetic acid) for any species tested (Table 2). Repeated exposure to sub-inhibitory concentrations of acetic acid does not induce increased tolerance to acetic acid over time. The second method by which bacteria typically can evolve resistance is through mutations selected for by repeated exposure to sub-inhibitory concentrations of antimicrobial. To determine if resistance to acetic acid could evolve, S. aureus USA300 LAC* and P. aeruginosa PAO1 were serially passaged at concentrations of acetic acid representing a quarter of the MIC, over 20 days, and recorded any changes in acetic acid susceptibility. Over a period of 20 passages, there were no discernible increases in tolerance/decreases in susceptibility of S. aureus USA300 LAC* or P. aeruginosa PAO1 to acetic acid (represented by an increase in MIC value) (Fig. 4 ). In parallel, S. aureus USA300 LAC* and P. aeruginosa PAO1 passaged in a quarter of the MIC of tobramycin demonstrated an up to 300-fold increase in MIC value from approximately 4 mg/L (USA300 LAC*) and 2 mg/L (PAO1), to greater than 1024 mg/L over 20 passages (Fig. 4 ). S. aureus USA300 LAC* did not show any increase in MIC to acetic acid, when comparing the evolved strain to the ancestral strain, whilst the MBC value decreased by 2-fold (Table 3). P. aeruginosa PAO1 showed a 2-fold increase in MIC to acetic acid, compared to the ancestral strain, but the MIC was still 16-fold below the therapeutically tolerated value of 2% (Table 3). The MBC of P. aeruginosa decreased by 2-fold, compared to the ancestral strain (Table 3). Reduction of bacterial burden, via Gellan AA dressing, within a porcine ex vivo model of bacterial colonisation. To determine whether acetic acid exhibited potent antimicrobial activity within a more physiologically relevant scenario, porcine skin was inoculated with S. aureus LAC USA300* or P. aeruginosa PAO1, with the bacterial inoculum left to establish for two hours. Inoculated regions were subsequently treated with Gellan dressing, rehydrated with either PBS (control) or 2% acetic acid for 1, 4 or 24 hours, with skin explants and dressing recovered, homogenised in PBS and plated out for CFU, as a proxy for bacterial burden at each of these timepoints. At 1, 4 and 24 hours, GellanAA (Gellan dressing rehydrated with 2% acetic acid) treatment significantly reduced bacterial CFU c.f. PBS controls for S. aureus USA300 LAC* and P. aeruginosa PAO1 (Fig. 5 ). DISCUSSION The effective treatment of burn wound infections represents a significant clinical challenge, predominantly due to the increased susceptibility of the damaged skin to colonisation by environmental microorganisms ( 66 , 67 ). S. aureus and P. aeruginosa are commonly isolated pathogens, owing to their ability to form biofilms and the emergence of multidrug resistance within a clinical setting. Current approaches are often unsuccessful, requiring multiple courses of antibiotics, or failing entirely and resulting in chronic wound infection and dissemination ( 19 , 25 ). In the era of antibiotic resistance, the use of antimicrobials that are effective against multidrug resistant bacteria, have potent anti-biofilm activity and a low probability of resistance is greatly needed. In this study, the efficacy of acetic acid on the two burn wound pathogens S. aureus and P. aeruginosa was investigated and it was shown that acetic acid had potent antimicrobial activity against both pathogens, independent of their growth state, and that resistance to acetic acid does not emerge in vitro . Bacterial metabolism and cell division are required for bactericidal activity of some conventional antimicrobial drugs, for example ampicillin and ciprofloxacin ( 26 , 27 , 68 , 69 ). This study shows that acetic acid is antimicrobial against S. aureus and P. aeruginosa at exceptionally low concentrations, independent of whether the bacteria are actively growing (Fig. 2 ), or in stationary phase (Fig. 1 ), offering a clear advantage over many conventional antibiotics. The formation of biofilms hinders the treatment and successful resolution of bacterial infection, due to the reduced permeation/permeability of antimicrobial drugs and immune factors into the biofilm matrices ( 27 – 29 ). Our study provides compelling evidence that acetic acid is effective at preventing biofilm formation by S. aureus and P. aeruginosa , highlighting its potential for preventing colonisation and subsequent infection of wounds (Fig. 3 ). These findings align with previous studies reporting that acetic acid is effective at reducing biofilm formation by S. aureus ( 45 , 46 , 50 ) and P. aeruginosa ( 70 , 71 ). Reduced biofilm formation capabilities are likely due to the high potency and bactericidal activity of acetic acid, promptly killing the bacteria prior to the induction of biofilm formation. In this study, no change in the resistance of S. aureus and P. aeruginosa to acetic acid, upon repeated exposure to sub-inhibitory concentrations of acetic acid (Fig. 4 ; Table 3), was seen. This was unexpected as resistance to other weak, organic acids is well characterised ( 72 – 74 ), whereby increased expression of the F 1 -F 0 ATPase proton pump, release of ammonia through the conversion of glutamine to glutamate ( 75 , 76 ), or increased cytoplasmic urease activity, reduces acidification of the bacterial cytoplasm ( 77 ). Tolerance to acetic acid is also seen in acetic acid producing bacteria, where upregulation of alcohol dehydrogenase enzymes and molecular chaperones, such as GroEL, DnaKJ and GrpE, confer a greater tolerance to acetic acid stress ( 78 ). Acetic acid may be the exception to these common mechanisms of acid resistance, owing to its dual mechanism of action, where it diffuses across the bacterial outer membrane as a neutral molecule, before dissociating, subsequently acidifying the cytoplasm, leading to ATP depletion ( 79 ), and causing a toxic accumulation of acetate ( 80 , 81 ). It is possible that this is why there are no reported instances of acetic acid resistance, despite resistance to other weak, organic acids being well characterised. This suggests that resistance to acetic acid is more complicated and does not evolve in vitro . Acetic acid has been used for the treatment of burn wounds previously ( 51 , 52 ). However, discussions around the tolerability, dosage and application of acetic acid remain. Application of 2% acetic acid, within a Gellan hydrogel dressing, reduced bacterial burden within a porcine ex vivo model of bacterial colonisation, highlighting its suitability for the treatment of burn wound colonisation in the future. While the emergence of resistance to acetic acid during in vitro monoculture (Fig. 4 ) was not detected in this study, a human burn wound is a much more complex environment where polymicrobial interactions, the human immune system and other factors influence bacterial physiology and drug susceptibility. Therefore, further work investigating the efficacy of this dressing, and any potential resistance to acetic acid treatment in a patient setting would prove informative. In conclusion, this work builds on previous reports of the antibacterial efficacy of acetic acid, with these findings then applied to the treatment of two common burn wound pathogens S. aureus and P. aeruginosa . This study shows that acetic acid is effective against both bacteria, independent of whether they are actively growing, or contained within biofilms two common limitations of current, conventional antimicrobial drugs. This study also shows that the likelihood of resistance to acetic acid treatment emerging is extremely low. Application of acetic acid to the treatment of burn wound infection would represent a significant advancement in current treatment practices. Declarations Author Contribution S.R.H. and C.C. contributed equally to this study. C.C, J.A.G, S.R.H. wrote the main manuscript text. S.R.H. prepared figures 1-3 and tables 1-2 and C.C prepared figures 4-5 and table 3. J.A.G. supervised the work. All authors reviewed and revised the manuscript. 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Silver Sulphadiazine: a review of the evidence. WOUNDS UK Clinical Review. 2005. Qian L-W, Fourcaudot AB, Leung KP. Silver sulfadiazine retards wound healing and increases hypertrophic scarring in a rabbit ear excisional wound model. Journal of Burn Care & Research. 2017;38(1):e418-e22. Johnston CS, Gaas CA. Vinegar: medicinal uses and antiglycemic effect. MedGenMed. 2006;8(2):61. Ryssel H, Kloeters O, Germann G, Schäfer T, Wiedemann G, Oehlbauer M. The antimicrobial effect of acetic acid—An alternative to common local antiseptics? Burns. 2009;35(5):695–700. Lastauskienė E, Zinkevičienė A, Girkontaitė I, Kaunietis A, Kvedarienė V. Formic acid and acetic acid induce a programmed cell death in pathogenic Candida species. Curr Microbiol. 2014;69(3):303–10. Giannattasio S, Guaragnella N, Ždralević M, Marra E. Molecular mechanisms of Saccharomyces cerevisiae stress adaptation and programmed cell death in response to acetic acid. Frontiers in Microbiology. 2013;4. Ludovico P, Sansonetty F, Silva MT, Côrte-Real M. Acetic acid induces a programmed cell death process in the food spoilage yeast Zygosaccharomyces bailii . FEMS Yeast Res. 2003;3(1):91–6. Valenti D, Vacca RA, Guaragnella N, Passarella S, Marra E, Giannattasio S. A transient proteasome activation is needed for acetic acid-induced programmed cell death to occur in Saccharomyces cerevisiae . FEMS Yeast Res. 2008;8(3):400–4. Halstead FD, Rauf M, Moiemen NS, Bamford A, Wearn CM, Fraise AP, et al. The Antibacterial Activity of Acetic Acid against Biofilm-Producing Pathogens of Relevance to Burns Patients. PLoS One. 2015;10(9):e0136190. Halstead FD, Rauf M, Bamford A, Wearn CM, Bishop JRB, Burt R, et al. Antimicrobial dressings: Comparison of the ability of a panel of dressings to prevent biofilm formation by key burn wound pathogens. Burns. 2015;41(8):1683–94. Tsang STJ, Gwynne PJ, Gallagher MP, Simpson AHRW. The biofilm eradication activity of acetic acid in the management of periprosthetic joint infection. Bone & Joint Research. 2018;7(8):517–23. Fraise AP, Wilkinson MA, Bradley CR, Oppenheim B, Moiemen N. The antibacterial activity and stability of acetic acid. J Hosp Infect. 2013;84(4):329–31. Agrawal KS, Sarda AV, Shrotriya R, Bachhav M, Puri V, Nataraj G. Acetic acid dressings: Finding the Holy Grail for infected wound management. Indian J Plast Surg. 2017;50(3):273–80. Bjarnsholt T, Alhede M, Jensen PØ, Nielsen AK, Johansen HK, Homøe P, et al. Antibiofilm Properties of Acetic Acid. Advances in Wound Care. 2014;4(7):363–72. Nour S, Reid G, Sathanantham K, Mackie I. Acetic acid dressings used to treat pseudomonas colonised burn wounds: A UK national survey. Burns. 2022;48(6):1364–7. Imran R, Hassouna T, Sur G, Casey A, Homer V, Barton D, et al. Efficacy and optimal dose of acetic acid to treat colonised burns wounds: protocol for a pilot randomised controlled trial. BMJ Open. 2023;13(9):e058006. Boles BR, Thoendel M, Roth AJ, Horswill AR. Identification of Genes Involved in Polysaccharide-Independent Staphylococcus aureus Biofilm Formation. PLOS ONE. 2010;5(4):e10146. Hancock REW, Speert DP. Antibiotic resistance in Pseudomonas aeruginosa : mechanisms and impact on treatment. Drug Resistance Updates. 2000;3(4):247–55. Sung K, Chon J, Kweon O, Nho S, Kim S, Park M, et al. Dynamic Adaptive Response of Pseudomonas aeruginosa to Clindamycin/Rifampicin-Impregnated Catheters. Antibiotics (Basel). 2021;10(7). Enström J, Fröding I, Giske CG, Ininbergs K, Bai X, Sandh G, et al. USA300 methicillin-resistant Staphylococcus aureus in Stockholm, Sweden, from 2008 to 2016. PLoS One. 2018;13(11):e0205761. Highlander SK, Hultén KG, Qin X, Jiang H, Yerrapragada S, Mason EO, et al. Subtle genetic changes enhance virulence of methicillin resistant and sensitive Staphylococcus aureus . BMC Microbiology. 2007;7(1):99. Bastos MD, Coutinho BG, Coelho ML. Lysostaphin: A Staphylococcal Bacteriolysin with Potential Clinical Applications. Pharmaceuticals (Basel). 2010;3(4):1139–61. Choo EJ, Chambers HF. Treatment of Methicillin-Resistant Staphylococcus aureus Bacteremia. Infect Chemother. 2016;48(4):267–73. Hall CW, Mah T-F. Molecular mechanisms of biofilm-based antibiotic resistance and tolerance in pathogenic bacteria. FEMS Microbiology Reviews. 2017;41(3):276–301. Lebeaux D, Ghigo JM, Beloin C. Biofilm-related infections: bridging the gap between clinical management and fundamental aspects of recalcitrance toward antibiotics. Microbiol Mol Biol Rev. 2014;78(3):510–43. Sharma D, Misba L, Khan AU. Antibiotics versus biofilm: an emerging battleground in microbial communities. Antimicrobial Resistance & Infection Control. 2019;8(1):76. Mah T-FC, O'Toole GA. Mechanisms of biofilm resistance to antimicrobial agents. Trends in microbiology. 2001;9(1):34–9. Maslova E, Eisaiankhongi L, Sjöberg F, McCarthy RR. Burns and biofilms: priority pathogens and in vivo models. npj Biofilms and Microbiomes. 2021;7(1):73. Darby EM, Trampari E, Siasat P, Gaya MS, Alav I, Webber MA, et al. Molecular mechanisms of antibiotic resistance revisited. Nature Reviews Microbiology. 2023;21(5):280–95. Dvorak JE, Ladhani HA, Claridge JA. Review of Sepsis in Burn Patients in 2020. Surg Infect (Larchmt). 2021;22(1):37–43. Williams FN, Lee JO. Pediatric Burn Infection. Surg Infect (Larchmt). 2021;22(1):54–7. Anderl JN, Zahller J, Roe F, Stewart PS. Role of nutrient limitation and stationary-phase existence in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicrobial agents and chemotherapy. 2003;47(4):1251–6. Mascio CT, Alder JD, Silverman JA. Bactericidal action of daptomycin against stationary-phase and nondividing Staphylococcus aureus cells. Antimicrob Agents Chemother. 2007;51(12):4255–60. Feng L, Xu M, Zeng W, Zhang X, Wang S, Yao Z, et al. Evaluation of the antibacterial, antibiofilm, and anti-virulence effects of acetic acid and the related mechanisms on colistin-resistant Pseudomonas aeruginosa . BMC Microbiol. 2022;22(1):306. Tawre MS, Kamble EE, Kumkar SN, Mulani MS, Pardesi KR. Antibiofilm and antipersister activity of acetic acid against extensively drug resistant Pseudomonas aeruginosa PAW1. PLoS One. 2021;16(2):e0246020. Lund P, Tramonti A, De Biase D. Coping with low pH: molecular strategies in neutralophilic bacteria. FEMS Microbiology Reviews. 2014;38(6):1091–125. Kanjee U, Houry WA. Mechanisms of Acid Resistance in Escherichia coli . Annual Review of Microbiology. 2013;67(Volume 67, 2013):65–81. Yang H, Yu Y, Fu C, Chen F. Bacterial Acid Resistance Toward Organic Weak Acid Revealed by RNA-Seq Transcriptomic Analysis in Acetobacter pasteurianus . Front Microbiol. 2019;10:1616. Pennacchietti E, D'Alonzo C, Freddi L, Occhialini A, De Biase D. The Glutaminase-Dependent Acid Resistance System: Qualitative and Quantitative Assays and Analysis of Its Distribution in Enteric Bacteria. Front Microbiol. 2018;9:2869. Lu P, Ma D, Chen Y, Guo Y, Chen G-Q, Deng H, et al. L-glutamine provides acid resistance for Escherichia coli through enzymatic release of ammonia. Cell research. 2013;23(5):635–44. Liu Y, Tang H, Lin Z, Xu P. Mechanisms of acid tolerance in bacteria and prospects in biotechnology and bioremediation. Biotechnology advances. 2015;33(7):1484–92. Wang B, Shao Y, Chen F. Overview on mechanisms of acetic acid resistance in acetic acid bacteria. World Journal of Microbiology and Biotechnology. 2015;31(2):255–63. Ricke SC. Perspectives on the use of organic acids and short chain fatty acids as antimicrobials. Poultry Science. 2003;82(4):632–9. Russell J. Another explanation for the toxicity of fermentation acids at low pH: anion accumulation versus uncoupling. 1992. Pinhal S, Ropers D, Geiselmann J, Jong Hd. Acetate Metabolism and the Inhibition of Bacterial Growth by Acetate. Journal of Bacteriology. 2019;201(13): 10.1128/jb.00147 – 19 . Tables Table 1: The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of acetic acid (% v/v) against Staphylococcus aureus and Pseudomonas aeruginosa . Bacterial Strain Percentage of Acetic Acid (% v/v) MIC MBC Staphylococcus aureus USA300 LAC* 0.156 0.313 Staphylococcus aureus ATCC29213 0.156 0.625 Pseudomonas aeruginosa PAO1 0.078 0.156 Table 2: Frequency of Resistance of S. aureus and P. aeruginosa to acetic acid. Stationary phase cultures of S. aureus ATCC29213, S. aureus USA300 LAC* and P. aeruginosa PAO1 were grown to stationary phase, serially diluted and subsequently plated on agar plates containing indicated concentrations of acetic acid. After incubation (maximum 48 hours), The spontaneous frequency of resistance was determined by dividing the number of resistant colonies (CFU/ml) by the total number of viable cells (CFU/ml). Agar MICs were 0.078% Acetic Acid (% v/v) for all bacterial strains tested. Values shown indicate the median value of a minimum of 3 independent biological replicates. n.d. indicates not determined. Bacterial Strain Concentration of Acetic Acid (% v/v) 0.1% 0.2% 0.5% Staphylococcus aureus USA300 LAC* n.d. < 2.74 x 10 -9 < 2.74 x 10 -9 Staphylococcus aureus ATCC29213 n.d. < 3.11 x 10 -10 < 3.11 x 10 -10 Pseudomonas aeruginosa PAO1 6.73 x 10 -10 6.69 x 10 -10 n.d. Table 3: The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of acetic acid is not elevated after serial exposure to acetic acid. MIC and MBC values of evolved bacterial isolates of S. aureus USA300 LAC* and P. aeruginosa PAO1, which had been serially passaged in sub-inhibitory concentrations of acetic acid, were tested for acetic acid, and compared to the original (ancestor) strain. Shown are the median values for each of 3 independently evolved isolates, originating from 3 independent biological replicates. Staphylococcus aureus USA300 LAC* Pseudomonas aeruginosa PAO1 MIC MBC MIC MBC Ancestor (P0) 0.125% 0.250% 0.063% 0.250% Passaged (P20) 0.125% 0.125% 0.125% 0.125% Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6663847","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":458299922,"identity":"6675ccc1-8070-435c-a6dd-31b93c01de75","order_by":0,"name":"Sara Henderson","email":"","orcid":"","institution":"University of Birmingham","correspondingAuthor":false,"prefix":"","firstName":"Sara","middleName":"","lastName":"Henderson","suffix":""},{"id":458299923,"identity":"a45a45eb-ee14-4ec5-8f9b-98f3d206d97f","order_by":1,"name":"Callum Clark","email":"","orcid":"","institution":"University of Birmingham","correspondingAuthor":false,"prefix":"","firstName":"Callum","middleName":"","lastName":"Clark","suffix":""},{"id":458299924,"identity":"9ba71760-d280-47e6-bb39-d2c712203e2c","order_by":2,"name":"Thomas Robinson","email":"","orcid":"","institution":"University of Birmingham","correspondingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Robinson","suffix":""},{"id":458299925,"identity":"e1905573-12ff-4521-af36-097dabc1a41c","order_by":3,"name":"Liam Grover","email":"","orcid":"","institution":"University of Birmingham","correspondingAuthor":false,"prefix":"","firstName":"Liam","middleName":"","lastName":"Grover","suffix":""},{"id":458299926,"identity":"1c061bfd-637d-4d10-bf24-63156452f414","order_by":4,"name":"Joan Geoghegan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYBACxgYwdQDMfgzlwQQIa2E2JkoLsgo2aaK0MDewX3zw4dcdOXn3s8eqC3fYRTOwH37AzHMGn8N4ig1n9j0zNjyTl3Z75pnk3AaeNANmnht4taRJ8/YcTtzYkGN2m7eNObeBIYeBmecDXi3pv4Fa6jf2vzEr5m2rz23gf0NIC/sxZp4fhxPkJXLMmHnbDuc2SIBsweewZh5myZkNhw03SLwxlp7Zdjy3TeKZwcE5eLxv2N7+8MOHP4fl5ftzDD8XtlXn9vMnP3zw5hgeLc08BgyMbQwMBgegImwMBCJSnoH9AQPDHyCjAZ+yUTAKRsEoGNEAADqAWJ8YMQySAAAAAElFTkSuQmCC","orcid":"","institution":"University of Birmingham","correspondingAuthor":true,"prefix":"","firstName":"Joan","middleName":"","lastName":"Geoghegan","suffix":""}],"badges":[],"createdAt":"2025-05-14 11:53:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6663847/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6663847/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83157031,"identity":"03322b78-86c2-4c32-ad6f-a9199178199b","added_by":"auto","created_at":"2025-05-20 14:51:15","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":124326,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAcetic acid reduces the viability of non-actively growing \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. aeruginosa. \u003c/strong\u003e\u003c/em\u003eStationary phase cultures of \u003cem\u003eS. aureus\u003c/em\u003e USA300 LAC* (A, D), \u003cem\u003eS. aureus\u003c/em\u003e ATCC29213 (B, E) and \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 (C, F) were diluted to an OD\u003csub\u003e600\u003c/sub\u003e of 1.0 in Mueller-Hinton broth (A-C) or Phosphate buffered saline (D-F) and exposed to various concentrations of acetic acid (% v/v), or control antibiotics (5x MIC\u003cstrong\u003e; \u003c/strong\u003evancomycin: 5 µg/ml, tobramycin: 1.25 µg/ml), or 2 μg/ml lysostaphin (A, B, D, E), as indicated, for 5 hours. Colony forming units were enumerated prior to addition of antimicrobial agents, and at 5 hours. Log\u003csub\u003e10\u003c/sub\u003e(CFU/ml) values from each experiment are indicated (circles), with the mean of all biological replicates (bar) also indicated. Error bars represent standard deviation of the dataset. Statistical significance was assessed using a 1-way ANOVA with a post-hoc Dunnett’s test of multiplicity comparing to the control sample, taken prior to the addition of acetic acid or other indicated antimicrobial agents. * p\u0026lt;0.05. *** p\u0026lt;0.001. No symbol p \u0026gt; 0.05 i.e. not significant.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6663847/v1/3534d77de1093319a80ba322.jpg"},{"id":83156685,"identity":"2c3bf6ff-0c53-45c8-9c48-07aaaed1154f","added_by":"auto","created_at":"2025-05-20 14:43:15","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":62529,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAcetic acid rapidly reduces the viability of actively growing \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. aeruginosa. \u003c/strong\u003e\u003c/em\u003eEarly exponential phase (OD\u003csub\u003e600\u003c/sub\u003e = 0.3) cultures of \u003cem\u003eS. aureus \u003c/em\u003eUSA300 LAC* (A) and \u003cem\u003eP. aeruginosa \u003c/em\u003ePAO1 (B) were exposed to 0.5% (v/v) or 2% (v/v) acetic acid alongside a control antibiotic (vancomycin (A) or tobramycin (B)) at 5x MIC. Colony forming units were enumerated prior to addition of antimicrobial agents, immediately after addition (0 minutes), and at 30, 60, 120 and 300 minutes incubation. Error bars represent the standard deviation of the mean of three independent biological replicates.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6663847/v1/0f9022cd37ff30e01b05f11e.jpg"},{"id":83157032,"identity":"678be1da-7598-48f5-a72e-bd19c2bb915a","added_by":"auto","created_at":"2025-05-20 14:51:15","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":110293,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMature \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. aeruginosa \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ebiofilms are eradicated by exposure to acetic acid.\u003c/strong\u003e Biofilms of \u003cem\u003eS. aureus\u003c/em\u003e USA300 LAC* (Mueller-Hinton Broth + 4% (w/v) NaCl; A) or \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 (Mueller-Hinton Broth\u003cstrong\u003e; \u003c/strong\u003eB) were established on Nunc\u003csup\u003eTM\u003c/sup\u003e\u0026nbsp;TSP peg lids for 24 hours from an overnight culture diluted to an OD\u003csub\u003e600\u003c/sub\u003e of 0.01. Biofilms were washed twice in PBS and exposed to varying sequential dilutions of acetic acid (% v/v) for 5 hours. The biomass of the remaining biofilm was then stained with crystal violet, with the stain resolubilised and measured at an absorbance of 570nm as a proxy for biofilm biomass (A, B). Remaining biofilms were washed twice in PBS and transferred to broth for 18 hours at which the optical density (OD\u003csub\u003e600\u003c/sub\u003e) of the broth was measured (C, D). Error bars represent standard deviation from the mean of a minimum of three independent replicates. Statistical significance was assessed via a one-way ANOVA with Dunnett's multiple comparison test comparing with the 0% acetic acid control. * p\u0026lt;0.05. ** p\u0026lt;0.01. *** p\u0026lt;0.001. No symbol p \u0026gt; 0.05 i.e. not significant.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6663847/v1/ee0a4ad6b558ac6d1c473cb8.jpg"},{"id":83157033,"identity":"29bd1536-28b8-430f-a98d-c064d1563eb6","added_by":"auto","created_at":"2025-05-20 14:51:15","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":56568,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSerial passage at sub-inhibitory concentrations of acetic acid does not result in acetic acid resistance. \u003c/strong\u003e\u003cem\u003eS. aureus\u003c/em\u003e USA300 LAC\u003csup\u003e*\u003c/sup\u003e\u0026nbsp;(A) and \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 (B) were serially passaged 1:30 after 18 hours of growth, at 1/4 of the minimum inhibitory concentration (MIC), determined from the previous passage, of acetic acid (blue) or tobramycin (red) for 20 passages. All passages were performed in Mueller-Hinton broth. Data points represent the mean of 3 independent biological replicates. Error bars represent the standard deviation of the mean values.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6663847/v1/1c3e8400fd629c64f57176d3.jpg"},{"id":83156687,"identity":"b489365f-e197-410f-9f73-7b883d0faa44","added_by":"auto","created_at":"2025-05-20 14:43:15","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":84286,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGellan dressing, impregnated with 2% acetic acid, reduces bacterial burden within a porcine \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eex vivo \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emodel of bacterial colonisation. \u003c/strong\u003ePorcine skin was inoculated with \u003cem\u003eS. aureus \u003c/em\u003eUSA300 LAC* or \u003cem\u003eP. aeruginosa \u003c/em\u003ePAO1, with the bacterial inoculum left to establish for two hours. Inoculated regions were subsequently treated with a Gellan dressing rehydrated with either PBS (control) or 2% acetic acid for 1, 4 or 24 hours, with skin explants and dressing recovered, homogenised in PBS and plated out for CFU as a proxy for bacterial burden at each of these timepoints. Viable CFU counts were normalised to that of the PBS control for each timepoint. Normalised viable CFU counts were compared via One-way ANOVA, with Holm-Sidaks multiple comparison test. *** indicates significance at p \u0026lt; 0.001. **** indicates significance at p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6663847/v1/ef437a7d82467c3abee48202.jpg"},{"id":86944084,"identity":"0464824a-e55c-40f4-87d9-1be27340cddf","added_by":"auto","created_at":"2025-07-17 12:31:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1830469,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6663847/v1/34d9fdce-f258-4ef8-a252-8787e35b7cec.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Acetic acid exerts bactericidal activity against the common burn wound pathogens Staphylococcus aureus and Pseudomonas aeruginosa without generating resistance","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eSkin is one of the first barriers to infection. When this barrier is broken, for example following a burn injury, microorganisms from the host\u0026rsquo;s own resident microbiota and the environment can colonise the wound, leading to infection. Bacterial infection, followed by an inability or failure of the immune system to clear infection, often traps the wound in an inflammatory state whereby it fails to heal, and subsequently predisposes the individual to systemic infections (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Indeed, between 30% and 52% of individuals with burn wound infections go on to develop sepsis (\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), which represents the leading cause of death after the first 24 hours following injury, with a 28\u0026ndash;65% mortality rate (\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBurn wound infections at the point of injury are sterilised, leading to a microbially deprived environment which can be re-colonised without competition from the resident skin microbiota (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e are amongst the most common bacteria to be isolated from burn wounds, and their presence increases inflammation, slows wound healing and increases morbidity (\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). The human commensal \u003cem\u003eS. aureus\u003c/em\u003e typically colonises the wound within hours of injury, from the hosts own skin or nasal microbiota (\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). \u003cem\u003eP. aeruginosa\u003c/em\u003e colonises the wound later, often from environmental sources such as contaminated water (\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTreatment of burn wounds typically consists of application of a broad-spectrum topical antimicrobial, upon admission, followed by surgical debridement of dead or severely damaged tissue, and administration of oral (systemic) antibiotics (\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). The treatment of burn wound infection is complicated by a reduced permeation and perfusion of immune cells into the wound site, in combination with a repressed local and systemic immune response soon after injury (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), reduced bioavailability of antimicrobial drugs at the site, and antimicrobial resistance (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Treatment of bacterial infection may also be hindered by the presence of bacteria that are not actively growing, and those that are contained within biofilms. Biofilms, which are multispecies communities of bacteria surrounded by a carbohydrate and protein-rich extracellular matrix, are not susceptible to the actions of some antibiotics, due to their charged nature prohibiting the diffusion of other, like-charged, drugs into the biofilm matrix (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Biofilm formation also reduces clearance by the host immune response, leading to further inflammation and infection (\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTopical antimicrobial agents have also been trialled for the treatment of burn wound infection, including silver nitrate, silver sulfadiazine, and honey (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) (reviewed in (\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e)). However, toxicity in some silver-based compounds has resulted in delayed healing, whilst resistance to some silver-based compounds has also emerged (\u003cspan additionalcitationids=\"CR35 CR36 CR37\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e), leading to poor resolution of infection. Thus, there is an unmet need for effective, topical antimicrobial therapies for the reduction of burn wound colonisation and infection.\u003c/p\u003e \u003cp\u003eOne potential, alternative, topical antimicrobial therapy is acetic acid. The antimicrobial qualities of acetic acid, a main component of vinegar, has been known for centuries where, historically, vinegar was used for preservation of food products. Acetic acid has also been used historically, since the age of Hippocrates in 420 BC, for the sterilisation and treatment of wounds (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). More recently, acetic acid has been demonstrated to be effective against a wide range of microorganisms, including fungi (\u003cspan additionalcitationids=\"CR42 CR43\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e) and bacteria, including those commonly associated with multidrug resistance, such as \u003cem\u003ePseudomonas spp.\u003c/em\u003e and \u003cem\u003eAcinetobacter spp.\u003c/em\u003e (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan additionalcitationids=\"CR46 CR47 CR48\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e) and within biofilms (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Acetic acid has also been trialled in the treatment of wound infections (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). However, questions around the tolerability, dosage and application of acetic acid for the treatment of bacterially colonised wounds remains.\u003c/p\u003e \u003cp\u003eOne potential route of administration/application is the incorporation of acetic acid, or other antimicrobial or anti-inflammatory drugs, into wound dressings, including hydrogel-type dressings. Within this study, acetic acid is incorporated into a Gellan dressing. This dressing is formed by dehydrating a Gellan hydrogel to form a thin film, which can then be rehydrated at the point of care in an aqueous solution, in this case acetic acid. The Gellan dressings are optically clear, allowing clinicians to inspect the wound without having to remove the dressings.\u003c/p\u003e \u003cp\u003eIn this study, the antimicrobial activity of acetic acid against two common burn wound pathogens, \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e, was characterised. The influence of concentration on the antimicrobial properties of acetic acid was determined, highlighting that low concentrations of acetic acid were required for antimicrobial activity. Furthermore, the potential for resistance to acetic acid to be selected for over 20 serial passages was assessed, with results indicating that resistance to acetic acid treatment did not emerge \u003cem\u003ein vitro\u003c/em\u003e. Additionally, the application of acetic acid within a hydrogel-based dressing effectively reduced bacterial load in a porcine \u003cem\u003eex vivo\u003c/em\u003e model of bacterial colonisation. Collectively our findings support the potential of acetic acid for future clinical application.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cstrong\u003eBacterial Strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStaphylococcus aureus\u0026nbsp;\u003c/em\u003eATCC29213 (Source: American Type Culture Collection),\u003cem\u003e\u0026nbsp;Staphylococcus aureus\u0026nbsp;\u003c/em\u003eUSA300 LAC*(53)\u003cem\u003e,\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Pseudomonas aeruginosa\u0026nbsp;\u003c/em\u003ePAO1, a wildtype ATCC15692 strain (Source: American Type Culture Collection), were used in this study. Bacterial strains were grown in Mueller-Hinton broth (MHB) (Oxoid) or on Mueller-Hinton agar (MHA) (MHB supplemented with 1.5% bacteriological agar, Millipore) unless otherwise stated. Antibiotics, tobramycin and vancomycin, were purchased from Sigma-Aldrich, unless otherwise specified.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTime of Kill\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSingle colonies of \u003cem\u003eS. aureus\u003c/em\u003e USA300LAC* or \u003cem\u003eP. aeruginosa\u0026nbsp;\u003c/em\u003ePAO1 were inoculated into 10 ml MHB and grown to stationary phase at 37°C, 180 rpm, for approximately 16-18 hours. Stationary phase cultures were diluted 1:100 into 5 ml fresh MHB and grown to early exponential phase (OD\u003csub\u003e600\u0026nbsp;\u003c/sub\u003e=0.3). Cultures were adjusted to the 0.5 McFarland standard and diluted to a starting CFU/ml of 5x10\u003csup\u003e5\u003c/sup\u003e. Acetic acid was added to a final concentration of 0.5 or 2% (v/v). Control antibiotics were used at a concentration at least 5 times higher than the MIC (vancomycin: 5 mg/L, tobramycin: 1.25 mg/L). Samples were taken at 0, 30, 60, 120 and 300 minutes, serially diluted and plated onto MHA. Colonies were enumerated after a 16–18-hour incubation at 37°C to give the CFU/ml values.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStationary Phase Killing Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSingle colonies were used to inoculate 10 ml MHB and grown to stationary phase at 37°C, 180 rpm, for 16-18 hours. Optical density (600 nm) was measured and adjusted to a final OD\u003csub\u003e600\u003c/sub\u003e = 1.0 in 4 ml either MHB or\u0026nbsp;PBS. Acetic acid was added to final concentrations 0, 0.1, 0.2, 0.5, 1, 2 or 4% (v/v). Control antibiotics were added at concentrations at least 5 times higher than the (vancomycin: 5 mg/L, tobramycin: 1.25 mg/L), or at a final concentration of 2 mg/L for lysostaphin (Antibodies.com). Initial CFU counts were taken, and cultures were incubated for 5 hours at 37°C, 180 rpm. The cultures were serially diluted and plated onto MHA. Colonies were enumerated after a 16–18-hour incubation at 37°C to give the CFU/ml values.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMinimum Biofilm Inhibitory Concentration (MBIC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSingle colonies were used to inoculate 5 ml MHB and grown to stationary phase (as above). Optical density (600 nm) was measured and adjusted to a final OD\u003csub\u003e600\u003c/sub\u003e = 0.025 in 2 ml MHB (\u003cem\u003eP. aeruginosa\u0026nbsp;\u003c/em\u003ePAO1) or MHB + 4% NaCl\u0026nbsp;(ThermoFisher Scientific) (\u003cem\u003eS. aureus\u003c/em\u003e USA300LAC*) in 24-well tissue culture-treated plates (Costar). Acetic acid at 0.01% - 5% (v/v) final concentration was used to inhibit growth. Biofilms were grown at 37°C for 72 hours. After 72 hours, the planktonic growth was removed, and the biofilm washed twice with sterile dH\u003csub\u003e2\u003c/sub\u003eO. The remaining biofilm was stained with 1 ml 1% (w/v) crystal violet (ProLab Diagnostics) for 1 minute. The biofilm was washed twice with sterile dH\u003csub\u003e2\u003c/sub\u003eO. Bound crystal violet was dissolved in 1 ml 5% (v/v) acetic acid. 100 μl was transferred to a 96-well plate and the absorbance measured at 570 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMinimum Biofilm Eradication Concentration (MBEC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSingle colonies were used to inoculate 5 ml MHB and grown to stationary phase (as above). Optical density (600 nm) was measured and adjusted to a final OD600 = 0.01 in MHB (\u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1) or MHB + 4% NaCl (\u003cem\u003eS. aureus\u003c/em\u003e USA300LAC*). 200 μl was added to Nunc 96-well plates (ThermoFisher Scientific) and Nunc\u003csup\u003eTM\u003c/sup\u003e Immuno TSP lids (ThermoFisher Scientific) were used to close the plates. The plates were grown at 37°C, 200 rpm for 24 hours to establish a biofilm. The TSP lids were washed twice in 1x PBS, and the TSP lids transferred to 0.02% - 5% (v/v) acetic acid in MHB or MHB + 4% NaCl for 5 hours at 37°C, 200 rpm. The TSP lids were washed twice in 1x PBS, and the TSP lids transferred to MHB or MHB + 4% NaCl in the absence of acetic acid for 16 hours at 37°C, 200 rpm. Optical density at 600 nm was measured. A minimum of 3 biological replicates were included.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFrequency of Resistance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSingle colonies were used to inoculate 5 ml MHB and grown to stationary phase (as above). The culture was centrifuged and resuspended in 2 ml PBS. 100 μl aliquots were spread onto MHA containing 0.1 ,0.2 or 0.5% (v/v) acetic acid. To determine the number of colony forming units, the inoculum was serially diluted in PBS and plated on antibiotic free MHA plates. Plates were incubated for up to 48 hours (37°C) and colonies enumerated. The spontaneous frequency of resistance was determined by dividing the number of resistant colonies (CFU/ml) by the total number of viable cells (CFU/ml). Where no colonies were obtained the limit of detection was determined to be 2 x 10\u003csup\u003e1\u003c/sup\u003e CFU/ml and used to give the largest possible spontaneous frequency of resistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSerial Passage at sub-inhibitory drug concentrations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOvernight cultures of \u003cem\u003eS. aureus\u0026nbsp;\u003c/em\u003eUSA300 LAC* and \u003cem\u003eP. aeruginosa\u0026nbsp;\u003c/em\u003ePAO1 were grown to stationary phase, for 18 hours, and subsequently diluted to an OD\u003csub\u003e600\u0026nbsp;\u003c/sub\u003eof 0.05 in MHB containing specified concentrations of acetic acid or tobramycin. MIC values were recorded after 18 hours, prior to the addition of\u0026nbsp;glycerol (ThermoFisher Scientific) (final concentration: 10%) and one freeze-thaw cycle. Subsequent passages, at a dilution of 1:30 into fresh MIC plates, were performed for 20 passages.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTesting GellanAA dressing efficacy within a porcine \u003cem\u003eex vivo\u0026nbsp;\u003c/em\u003emodel of bacterial colonisation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePorcine skin was obtained commercially (www.skinforsale.com) and frozen at -20°C until required. Skin was sterilised with 70% ethanol prior to inoculation with \u003cem\u003eS. aureus\u0026nbsp;\u003c/em\u003eUSA300 LAC* or \u003cem\u003eP. aeruginosa\u0026nbsp;\u003c/em\u003ePAO1 (grown as previously described) at an OD600 of 1 x 10\u003csup\u003e-6\u003c/sup\u003e, corresponding to a bacterial burden of approximately 1 x 10\u003csup\u003e2\u003c/sup\u003e CFU/ml. Bacteria were left to acclimatise on the skin for 2 hours, prior to treatment with Gellan dressing rehydrated in PBS (treatment control) or 2% acetic acid. At 1, 4 and 24 hours, skin and dressing were removed and homogenised in PBS, with the subsequent bacterial suspension serially diluted and plated for viable CFU count. Viable CFU counts were normalised to that of the PBS control to account for bacterial growth on the skin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was performed using the R package multcomp v1.4-22 or Graphpad Prism version 10.0. Typically, One-way ANOVA with Dunnett’s test of multiplicity were performed against the 0% acetic acid/PBS controls. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u0026nbsp;\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eExposure to acetic acid causes rapid cell death in\u003c/b\u003e \u003cb\u003eP. aeruginosa\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eS. aureus\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eTogether, \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e are responsible for more than 50% of burn wound infections in Birmingham hospitals (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Therefore, initially the antimicrobial properties of acetic acid were assessed against two strains of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (one MRSA strain USA300 LAC*, and one MSSA strain ATCC29213) and one strain of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (PAO1), all of which have a range of sensitivities to known antibiotic classes (\u003cspan additionalcitationids=\"CR55 CR56\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). The minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC) for acetic acid was measured for all three strains (Table\u0026nbsp;1) and found to be well below the therapeutically tolerated acetic acid concentration of 2% (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo explore the antimicrobial activity of acetic acid in more detail, the tolerance of the bacteria to acetic acid was tested by adding various concentrations of acetic acid to washed stationary phase cultures diluted in either rich growth medium (Mueller-Hinton broth) or phosphate buffered saline (PBS). Following incubation for 5 hours in acetic acid concentrations of 0.5% or less, \u003cem\u003eS. aureus\u003c/em\u003e ATCC29213 (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB \u0026amp; E) and S. aureus USA300 LAC* remained viable (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA \u0026amp; D). However, viability was significantly reduced following incubation in a 1% (v/v) solution of acetic acid (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B, D \u0026amp; E). This was the case regardless of whether growth medium or buffer was used for both S. aureus strains tested (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B, D \u0026amp; E). The reduction in viable cells recovered following exposure to 2% (v/v) acetic acid in PBS was dramatic, from the order of 1x10\u003csup\u003e9\u003c/sup\u003e CFU/ml to less than 500 CFU/ml being recovered (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD \u0026amp; E). As a control the endopeptidase lysostaphin was included as this results in rapid death by hydrolysing cell wall peptidoglycan (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). In contrast, vancomycin, a clinically relevant antibiotic for the treatment of MRSA infection (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e), did not reduce the viability of the bacteria under these conditions (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B, D \u0026amp; E).\u003c/p\u003e \u003cp\u003eSimilarly, \u003cem\u003eP. aeruginosa\u003c/em\u003e cells harvested in the stationary phase of growth and incubated with acetic acid were killed during the 5-hour incubation period (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC \u0026amp; F). The concentration of acetic acid required to reduce the bacterial cell viability in both rich medium (0.5% v/v) and PBS (0.2% v/v) was notably lower for \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 compared to \u003cem\u003eS. aureus\u003c/em\u003e ATCC29213 and USA300 LAC*.\u003c/p\u003e \u003cp\u003eNext, the ability of acetic acid to kill \u003cem\u003eS. aureus\u003c/em\u003e USA300 LAC* and \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 was compared with a control antibiotic using bacterial cells harvested in the exponential phase of growth. The inoculum was added to a medium supplemented with acetic acid. Based on these data, a concentration of acetic acid that significantly reduced the viability of \u003cem\u003eS. aureus\u003c/em\u003e USA300 LAC* and \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 (2%), and a concentration above the MIC and MBC for both (Table\u0026nbsp;1), but that did not significantly reduce \u003cem\u003eS. aureus\u003c/em\u003e USA300 LAC* viability (0.5%), was chosen. 2% acetic acid was determined to be bactericidal after 60 minutes for S. aureus USA300 LAC* while 0.5% acetic acid inhibited growth of S. aureus USA300 LAC* but did not reduce the CFU recovery (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In comparison, at 5x MIC (5 mg/L), vancomycin treatment reduced the number of colony forming units after 5 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eIn contrast, 0.5% acetic acid was demonstrated to be highly bactericidal against \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1, resulting in eradication of detectable CFU at 60 minutes, with 2% causing rapid killing, resulting in no detectable colony forming units at 30 minutes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Additionally, the sample taken immediately after addition of acetic acid contained significantly fewer CFU when compared to the one taken immediately prior to the acetic acid addition. In contrast, 5x MIC (1.25 mg/L) tobramycin caused a decline in the viable CFU/ml over time, with no detectable CFU recovered at 120 minutes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBiofilm formation is inhibited in the presence of acetic acid\u003c/h2\u003e \u003cp\u003eBiofilms are one of the key barriers to the successful resolution of bacterial infection, by conventional antimicrobial therapies (\u003cspan additionalcitationids=\"CR61 CR62 CR63\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUnder biofilm producing conditions, biofilm formation of \u003cem\u003eS. aureus\u003c/em\u003e USA300 LAC* was significantly reduced when the media was supplemented with 0.16% (v/v) acetic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Although P. aeruginosa PAO1 biofilm formation was not significantly reduced by the presence of acetic acid, the biofilm density was reduced by 90% at 0.08% (v/v) acetic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEstablished biofilms are eradicated significantly by acetic acid\u003c/h2\u003e \u003cp\u003eTo understand if acetic acid is effective against established biofilms, biofilms of \u003cem\u003eS. aureus\u003c/em\u003e USA300 LAC* and \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 were established, before exposing the biofilms to various concentrations of acetic acid for 5 hours. The biofilms were then used to seed growth in Mueller-Hinton broth for 16 hours. While acetic acid was unable to completely eradicate bacterial viability within established biofilms under the conditions tested, the viability of \u003cem\u003eS. aureus\u003c/em\u003e USA300 LAC* within an established biofilm was significantly reduced by a 5-hour exposure to acetic acid at a concentration of 0.625% (v/v) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Treatment of an established \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 biofilm with 0.31% (v/v) acetic acid significantly reduced bacterial regrowth (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), suggesting that 0.31% (v/v) acetic acid significantly reduced \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 bacterial viability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAcetic Acid does not induce spontaneous mutations at concentrations close to the agar MIC\u003c/h2\u003e \u003cp\u003eBacteria become resistant to an antimicrobial agent either through random genetic mutations within their genome, or through the acquisition of foreign DNA containing resistance machinery (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFirstly, the survival of \u003cem\u003eS. aureus\u003c/em\u003e USA300 and \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 in the presence of lethal concentrations of acetic acid were assessed. At a concentration of 0.2% (v/v) acetic acid, no viable bacterial colonies (limit of detection: 2 x 10\u003csup\u003e1\u003c/sup\u003e CFU/ml) were detected, suggesting that spontaneous resistance to acetic acid is not possible, at this limit of detection. Selection for spontaneous mutations in the presence of acetic acid, did not produce any surviving colonies even at concentrations close to the agar MIC values (e.g. PAO1 MIC value of 0.078% (v/v) with no colonies observed at 0.1%(v/v) acetic acid) for any species tested (Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e \u003cb\u003eRepeated exposure to sub-inhibitory concentrations of acetic acid does not induce increased tolerance to acetic acid over time.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe second method by which bacteria typically can evolve resistance is through mutations selected for by repeated exposure to sub-inhibitory concentrations of antimicrobial. To determine if resistance to acetic acid could evolve, \u003cem\u003eS. aureus\u003c/em\u003e USA300 LAC* and \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 were serially passaged at concentrations of acetic acid representing a quarter of the MIC, over 20 days, and recorded any changes in acetic acid susceptibility. Over a period of 20 passages, there were no discernible increases in tolerance/decreases in susceptibility of \u003cem\u003eS. aureus\u003c/em\u003e USA300 LAC* or \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 to acetic acid (represented by an increase in MIC value) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn parallel, \u003cem\u003eS. aureus\u003c/em\u003e USA300 LAC* and \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 passaged in a quarter of the MIC of tobramycin demonstrated an up to 300-fold increase in MIC value from approximately 4 mg/L (USA300 LAC*) and 2 mg/L (PAO1), to greater than 1024 mg/L over 20 passages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eS. aureus\u003c/em\u003e USA300 LAC* did not show any increase in MIC to acetic acid, when comparing the evolved strain to the ancestral strain, whilst the MBC value decreased by 2-fold (Table\u0026nbsp;3). \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 showed a 2-fold increase in MIC to acetic acid, compared to the ancestral strain, but the MIC was still 16-fold below the therapeutically tolerated value of 2% (Table\u0026nbsp;3). The MBC of \u003cem\u003eP. aeruginosa\u003c/em\u003e decreased by 2-fold, compared to the ancestral strain (Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003cb\u003eReduction of bacterial burden, via Gellan AA dressing, within a porcine\u003c/b\u003e \u003cb\u003eex vivo\u003c/b\u003e \u003cb\u003emodel of bacterial colonisation.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine whether acetic acid exhibited potent antimicrobial activity within a more physiologically relevant scenario, porcine skin was inoculated with \u003cem\u003eS. aureus\u003c/em\u003e LAC USA300* or \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1, with the bacterial inoculum left to establish for two hours. Inoculated regions were subsequently treated with Gellan dressing, rehydrated with either PBS (control) or 2% acetic acid for 1, 4 or 24 hours, with skin explants and dressing recovered, homogenised in PBS and plated out for CFU, as a proxy for bacterial burden at each of these timepoints. At 1, 4 and 24 hours, GellanAA (Gellan dressing rehydrated with 2% acetic acid) treatment significantly reduced bacterial CFU c.f. PBS controls for \u003cem\u003eS. aureus\u003c/em\u003e USA300 LAC* and \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe effective treatment of burn wound infections represents a significant clinical challenge, predominantly due to the increased susceptibility of the damaged skin to colonisation by environmental microorganisms (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e). \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e are commonly isolated pathogens, owing to their ability to form biofilms and the emergence of multidrug resistance within a clinical setting. Current approaches are often unsuccessful, requiring multiple courses of antibiotics, or failing entirely and resulting in chronic wound infection and dissemination (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). In the era of antibiotic resistance, the use of antimicrobials that are effective against multidrug resistant bacteria, have potent anti-biofilm activity and a low probability of resistance is greatly needed.\u003c/p\u003e \u003cp\u003eIn this study, the efficacy of acetic acid on the two burn wound pathogens \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e was investigated and it was shown that acetic acid had potent antimicrobial activity against both pathogens, independent of their growth state, and that resistance to acetic acid does not emerge \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eBacterial metabolism and cell division are required for bactericidal activity of some conventional antimicrobial drugs, for example ampicillin and ciprofloxacin (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e). This study shows that acetic acid is antimicrobial against \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e at exceptionally low concentrations, independent of whether the bacteria are actively growing (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), or in stationary phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), offering a clear advantage over many conventional antibiotics.\u003c/p\u003e \u003cp\u003eThe formation of biofilms hinders the treatment and successful resolution of bacterial infection, due to the reduced permeation/permeability of antimicrobial drugs and immune factors into the biofilm matrices (\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Our study provides compelling evidence that acetic acid is effective at preventing biofilm formation by \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e, highlighting its potential for preventing colonisation and subsequent infection of wounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These findings align with previous studies reporting that acetic acid is effective at reducing biofilm formation by \u003cem\u003eS. aureus\u003c/em\u003e (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e) and \u003cem\u003eP. aeruginosa\u003c/em\u003e (\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e). Reduced biofilm formation capabilities are likely due to the high potency and bactericidal activity of acetic acid, promptly killing the bacteria prior to the induction of biofilm formation.\u003c/p\u003e \u003cp\u003eIn this study, no change in the resistance of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e to acetic acid, upon repeated exposure to sub-inhibitory concentrations of acetic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Table\u0026nbsp;3), was seen. This was unexpected as resistance to other weak, organic acids is well characterised (\u003cspan additionalcitationids=\"CR73\" citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e), whereby increased expression of the F\u003csub\u003e1\u003c/sub\u003e-F\u003csub\u003e0\u003c/sub\u003e ATPase proton pump, release of ammonia through the conversion of glutamine to glutamate (\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e), or increased cytoplasmic urease activity, reduces acidification of the bacterial cytoplasm (\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e). Tolerance to acetic acid is also seen in acetic acid producing bacteria, where upregulation of alcohol dehydrogenase enzymes and molecular chaperones, such as GroEL, DnaKJ and GrpE, confer a greater tolerance to acetic acid stress (\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e). Acetic acid may be the exception to these common mechanisms of acid resistance, owing to its dual mechanism of action, where it diffuses across the bacterial outer membrane as a neutral molecule, before dissociating, subsequently acidifying the cytoplasm, leading to ATP depletion (\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e), and causing a toxic accumulation of acetate (\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e). It is possible that this is why there are no reported instances of acetic acid resistance, despite resistance to other weak, organic acids being well characterised. This suggests that resistance to acetic acid is more complicated and does not evolve \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAcetic acid has been used for the treatment of burn wounds previously (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). However, discussions around the tolerability, dosage and application of acetic acid remain. Application of 2% acetic acid, within a Gellan hydrogel dressing, reduced bacterial burden within a porcine \u003cem\u003eex vivo\u003c/em\u003e model of bacterial colonisation, highlighting its suitability for the treatment of burn wound colonisation in the future.\u003c/p\u003e \u003cp\u003eWhile the emergence of resistance to acetic acid during \u003cem\u003ein vitro\u003c/em\u003e monoculture (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e) was not detected in this study, a human burn wound is a much more complex environment where polymicrobial interactions, the human immune system and other factors influence bacterial physiology and drug susceptibility. Therefore, further work investigating the efficacy of this dressing, and any potential resistance to acetic acid treatment in a patient setting would prove informative.\u003c/p\u003e \u003cp\u003eIn conclusion, this work builds on previous reports of the antibacterial efficacy of acetic acid, with these findings then applied to the treatment of two common burn wound pathogens \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e. This study shows that acetic acid is effective against both bacteria, independent of whether they are actively growing, or contained within biofilms two common limitations of current, conventional antimicrobial drugs. This study also shows that the likelihood of resistance to acetic acid treatment emerging is extremely low. Application of acetic acid to the treatment of burn wound infection would represent a significant advancement in current treatment practices.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.R.H. and C.C. contributed equally to this study. C.C, J.A.G, S.R.H. wrote the main manuscript text. S.R.H. prepared figures 1-3 and tables 1-2 and C.C prepared figures 4-5 and table 3. J.A.G. supervised the work. All authors reviewed and revised the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Jess Blair for kindly providing P. aeruginosa strain PAO1 and S. aureus ATCC29213.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data supporting the findings of this study are contained within the manuscript. All raw data is available from the authors upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChurch D, Elsayed S, Reid O, Winston B, Lindsay R. Burn wound infections. Clin Microbiol Rev. 2006;19(2):403\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSinger AJ, McClain SA. Persistent wound infection delays epidermal maturation and increases scarring in thermal burns. Wound Repair Regen. 2002;10(6):372\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBelba MK, Petrela EY, Belba AG. Epidemiology and outcome analysis of sepsis and organ dysfunction/failure after burns. Burns. 2017;43(6):1335\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCumming J, Purdue GF, Hunt JL, O'Keefe GE. Objective estimates of the incidence and consequences of multiple organ dysfunction and sepsis after burn trauma. 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Journal of Bacteriology. 2019;201(13):\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/jb.00147\u0026thinsp;\u0026ndash;\u0026thinsp;19\u003c/span\u003e\u003cspan address=\"10.1128/jb.00147\u0026thinsp;\u0026ndash;\u0026thinsp;19\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1: The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of acetic acid (% v/v) against \u003cem\u003eStaphylococcus aureus\u0026nbsp;\u003c/em\u003eand \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBacterial Strain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 401px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePercentage of Acetic Acid (% v/v)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMIC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMBC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u0026nbsp;\u003c/em\u003eUSA300 LAC*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e0.156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e0.313\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u0026nbsp;\u003c/em\u003eATCC29213\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e0.156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e0.625\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u0026nbsp;\u003c/em\u003ePAO1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e0.078\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e0.156\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2: Frequency of Resistance of \u003cem\u003eS. aureus\u0026nbsp;\u003c/em\u003eand \u003cem\u003eP. aeruginosa\u0026nbsp;\u003c/em\u003eto acetic acid.\u0026nbsp;\u003c/strong\u003eStationary phase cultures of \u003cem\u003eS. aureus\u0026nbsp;\u003c/em\u003eATCC29213, \u003cem\u003eS. aureus\u0026nbsp;\u003c/em\u003eUSA300 LAC* and \u003cem\u003eP. aeruginosa\u0026nbsp;\u003c/em\u003ePAO1 were grown to stationary phase, serially diluted and subsequently plated on agar plates containing indicated concentrations of acetic acid. After incubation (maximum 48 hours), The spontaneous frequency of resistance was determined by dividing the number of resistant colonies (CFU/ml) by the total number of viable cells (CFU/ml). Agar MICs were 0.078% Acetic Acid (% v/v) for all bacterial strains tested. Values shown indicate the median value of a minimum of 3 independent biological replicates. n.d. indicates not determined.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 264px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBacterial Strain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 337px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConcentration of Acetic Acid (% v/v)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.1%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.2%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.5%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 264px;\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u0026nbsp;\u003c/em\u003eUSA300 LAC*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003e\u0026lt; 2.74 x 10\u003csup\u003e-9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003e\u0026lt; 2.74 x 10\u003csup\u003e-9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 264px;\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u0026nbsp;\u003c/em\u003eATCC29213\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003e\u0026lt; 3.11 x 10\u003csup\u003e-10\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003e\u0026lt; 3.11 x 10\u003csup\u003e-10\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 264px;\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003ePAO1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003e6.73 x 10\u003csup\u003e-10\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003e6.69 x 10\u003csup\u003e-10\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3: The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of acetic acid is not elevated after serial exposure to acetic acid.\u0026nbsp;\u003c/strong\u003eMIC and MBC values of evolved bacterial isolates of \u003cem\u003eS. aureus\u0026nbsp;\u003c/em\u003eUSA300 LAC* and \u003cem\u003eP. aeruginosa\u0026nbsp;\u003c/em\u003ePAO1, which had been serially passaged in sub-inhibitory concentrations of acetic acid, were tested for acetic acid, and compared to the original (ancestor) strain. Shown are the median values for each of 3 independently evolved isolates, originating from 3 independent biological replicates.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 239px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eStaphylococcus aureus\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eUSA300 LAC*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 239px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ePseudomonas aeruginosa\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003ePAO1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMIC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMBC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMIC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMBC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eAncestor (P0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.125%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.250%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.063%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.250%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003ePassaged (P20)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.125%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.125%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.125%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.125%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6663847/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6663847/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e are two of the most common pathogens colonising burn wounds in hospitals. Resistance or tolerance to antimicrobials, due to biofilm formation, complicates treatment and increases morbidity, highlighting that current approaches to burn wound care are insufficient. Therefore, novel antimicrobial treatments are needed. In this study we characterise the antimicrobial activity of acetic acid against \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa.\u003c/em\u003e Our results demonstrate that acetic acid exerts potent antimicrobial activity against both pathogens, including those growing within biofilms, and bacteria that are not actively dividing. The concentrations of acetic acid required to achieve antimicrobial effects were well below the therapeutically tolerated range. We also found that resistance to acetic acid did not develop \u003cem\u003ein vitro\u003c/em\u003e, suggesting that the likelihood of resistance emerging in a single step is low. Additionally, application of acetic acid, within a Gellan hydrogel-based dressing, effectively reduced bacterial burden in a porcine \u003cem\u003eex vivo\u003c/em\u003e model of skin colonisation. Together, our findings support the potential of acetic acid as a promising topical antimicrobial agent for preventing burn wound infections by demonstrating that acetic acid is active against bacteria in various growth states and showing for the first time that resistance does not develop easily \u003cem\u003ein vitro\u003c/em\u003e. These results suggest that could be a suitable candidate for the clinical management of burn wounds in the future.\u003c/p\u003e","manuscriptTitle":"Acetic acid exerts bactericidal activity against the common burn wound pathogens Staphylococcus aureus and Pseudomonas aeruginosa without generating resistance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-20 14:43:10","doi":"10.21203/rs.3.rs-6663847/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"36770bc5-e063-4536-8378-ce0a3c2c4886","owner":[],"postedDate":"May 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":48691285,"name":"Biological sciences/Microbiology/Antimicrobials"},{"id":48691286,"name":"Biological sciences/Microbiology/Bacteriology"},{"id":48691287,"name":"Biological sciences/Microbiology/Biofilms"},{"id":48691288,"name":"Biological sciences/Microbiology/Pathogens"}],"tags":[],"updatedAt":"2025-07-17T12:23:36+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-20 14:43:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6663847","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6663847","identity":"rs-6663847","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}