Reviewing the evidence of antimicrobial activity of glycols.

In the 1940s and 1950s, researchers seeking safe and novel ways to eliminate airborne pathogens from enclosed spaces, investigated glycol vapours as a method of disinfection. More recently, the COVID-19 pandemic highlighted the need for a non-toxic aerial disinfectant that can be used in the presence of people. This scoping review is intended to analyse the early and more recent literature on glycol disinfection, scrutinizing the methodologies used, and to determine if the use of glycols as modern-day disinfectants is justified PRISMA-ScR guidelines were used to assess the 749 articles retrieved from the Web of Science platform, with 46 articles retained after the search strategy was applied. Early studies generally demonstrated good disinfection capabilities against airborne bacteria and viruses, particularly with propylene glycol (PG) vapour. Vapour pressure, relative humidity, and glycol concentration were found to be important factors affecting the efficacy of glycol vapours. Contact times depended mainly on the glycol application method (i.e. aerosolization or liquid formulation), although information on how glycol efficacy is impacted by contact time is limited. Triethylene glycol (TEG) is deemed to have low toxicity, carcinogenicity, and mutagenicity and is registered for use in air sanitization and deodorization by the US Environmental Protection Agency. Glycols are also used in liquid formulations for their antimicrobial activity against a wide range of microorganisms, although when used as a non-active excipient in products, their contribution to antimicrobial efficacy is rarely assessed. The appropriate use of liquid glycol-containing formulations was found to positively impact the antimicrobial capabilities of disinfectants when used at temperatures <0, food preservatives, and dental medicaments. Providing modern delivery technology can accurately control environmental conditions, the use of aerosolized glycol formulations should lead to successful disinfection, aiding infection prevention, and control regimens.

Disinfectants and antiseptics: mechanisms of action and resistance.

Chemical biocides are used for the prevention and control of infection in health care, targeted home hygiene or controlling microbial contamination for various industrial processes including but not limited to food, water and petroleum. However, their use has substantially increased since the implementation of programmes to control outbreaks of methicillin-resistant Staphylococcus aureus, Clostridioides difficile and severe acute respiratory syndrome coronavirus 2. Biocides interact with multiple targets on the bacterial cells. The number of targets affected and the severity of damage will result in an irreversible bactericidal effect or a reversible bacteriostatic one. Most biocides primarily target the cytoplasmic membrane and enzymes, although the specific bactericidal mechanisms vary among different biocide chemistries. Inappropriate usage or low concentrations of a biocide may act as a stressor while not killing bacterial pathogens, potentially leading to antimicrobial resistance. Biocides can also promote the transfer of antimicrobial resistance genes. In this Review, we explore our current understanding of the mechanisms of action of biocides, the bacterial resistance mechanisms encompassing both intrinsic and acquired resistance and the influence of bacterial biofilms on resistance. We also consider the impact of bacteria that survive biocide exposure in environmental and clinical contexts.

How biofilm changes our understanding of cleaning and disinfection.

Biofilms are ubiquitous in healthcare settings. By nature, biofilms are less susceptible to antimicrobials and are associated with healthcare-associated infections (HAI). Resistance of biofilm to antimicrobials is multifactorial with the presence of a matrix composed of extracellular polymeric substances and eDNA, being a major contributing factor. The usual multispecies composition of environmental biofilms can also impact on antimicrobial efficacy. In healthcare settings, two main types of biofilms are present: hydrated biofilms, for example, in drains and parts of some medical devices and equipment, and environmental dry biofilms (DSB) on surfaces and possibly in medical devices. Biofilms act as a reservoir for pathogens including multi-drug resistant organisms and their elimination requires different approaches. The control of hydrated (drain) biofilms should be informed by a reduction or elimination of microbial bioburden together with measuring biofilm regrowth time. The control of DSB should be measured by a combination of a reduction or elimination in microbial bioburden on surfaces together with a decrease in bacterial transfer post-intervention. Failure to control biofilms increases the risk for HAI, but biofilms are not solely responsible for disinfection failure or shortcoming. The limited number of standardised biofilm efficacy tests is a hindrance for end users and manufacturers, whilst in Europe there are no approved standard protocols. Education of stakeholders about biofilms and ad hoc efficacy tests, often academic in nature, is thus paramount, to achieve a better control of biofilms in healthcare settings.

Risk of organism acquisition from prior room occupants: An updated systematic review.

BACKGROUND: Evidence from a previous systematic review indicates that patients admitted to a room where the previous occupant had a multidrug-resistant bacterial infection resulted in an increased risk of subsequent colonisation and infection with the same organism for the next room occupant. In this paper, we have sought to expand and update this review. METHODS: A systematic review and meta-analysis was undertaken. A search using Medline/PubMed, Cochrane and CINHAL databases was conducted. Risk of bias was assessed by the ROB-2 tool for randomised control studies and ROBIN-I for non-randomised studies. RESULTS: From 5175 identified, 12 papers from 11 studies were included in the review for analysis. From 28,299 patients who were admitted into a room where the prior room occupant had any of the organisms of interest, 651 (2.3%) were shown to acquire the same species of organism. In contrast, 981,865 patients were admitted to a room where the prior occupant did not have an organism of interest, 3818 (0.39%) acquired an organism(s). The pooled acquisition odds ratio (OR) for all the organisms across all studies was 2.45 (95% CI: 1.53-3.93]. There was heterogeneity between the studies (I2 89%, P < 0.001). CONCLUSION: The pooled OR for all the pathogens in this latest review has increased since the original review. Findings from our review provide some evidence to help inform a risk management approach when determining patient room allocation. The risk of pathogen acquisition appears to remain high, supporting the need for continued investment in this area.

Klebsiella pneumoniae survives on surfaces as a dry biofilm.

BACKGROUND: Dry surface biofilms (DSB) are widespread in healthcare settings presenting a challenge to cleaning and disinfection. Klebsiella pneumoniae has been a focus of attention due to antibiotic resistance and the emergence of hypervirulent strains. Few studies have demonstrated K pneumoniae survival on surfaces following desiccation. METHODS: DSB were formed over 12 days. Bacterial culturability and transfer were investigated following DSB incubation up to 4 weeks. Bacterial viability in DSB was investigated with live/dead staining using flow cytometry. RESULTS: K pneumoniae formed mature DSB. After 2 and 4 weeks of incubation, transfer from DSB was low (<55%) and reduced further (<21%) following wiping. Culturability at 2 and 4 weeks varied although viability remained high indicating viable but non culturable state (VBNC). DISCUSSION: K pneumoniae was removed from surfaces by mechanical wiping as shown with DSB of other species. Although culturability was reduced over time, bacteria remained viable up to 4 weeks incubation, proving the need for robust cleaning regimens. CONCLUSIONS: This is the first study confirming K pneumoniae survival on dry surfaces as a DSB. The presence of VBNC bacteria indicated that K pneumoniae can for extended periods, raising questions about its persistence on surfaces.

Determining the potential use of biosurfactants in preventing endodontic infections.

Microbial biofilms play a dominant role in the failure of endodontic therapies. Bacterial adhesion is the first step in the establishment of biofilms, activating the host immune response leading to tissue damage. Biosurfactants are microbe-derived tensioactive molecules with latent anti-adhesive and anti-microbial activity. This study reports the extraction and characterization of a biosurfactant from Lactobacillus (L.) plantarum (Lp-BS) and investigates its anti-microbial and anti-adhesive properties compared to rhamnolipid, a commercially available biosurfactant. Lp-BS, extracted from L. plantarum during the growth phase, was characterized as a glycoprotein, able to reduce surface tension and emulsify non-polar liquids. Proteomic analysis of Lp-BS identified three bacterial adhesin-like proteins, suggesting roles in hindering bacterial adhesion. Lp-BS did not show significant anti-microbial activity against endodontic pathogens from the Streptococcus (Strep.) anginosus group or Enterococcus (Ent.) faecalis at 50 mg/ml. However, anti-adhesive activity on abiotic surfaces was observed against both Strep. anginosus and Strep. intermedius. Rhamnolipid exhibited strong anti-microbial activity, with minimum inhibitory concentrations of 0.097 mg/ml against Strep. anginosus, and 0.048 mg/ml against Strep. constellatus and Strep. intermedius, in addition to a marked anti-adhesive activity. These findings offer preliminary evidence for the potential application of biosurfactants as an anti-microbial and/or anti-adhesive pharmacotherapy in endodontics.

Responses of Salmonella biofilms to oxidizing biocides: Evidence of spatial clustering.

The spatial organization of biofilm bacterial communities can be influenced by several factors, including growth conditions and challenge with antimicrobials. Differential survival of clusters of cells within biofilms has been observed. In this work, we present a variety of methods to identify, quantify and statistically analyse clusters of live cells from images of two Salmonella strains with differential biofilm forming capacity exposed to three oxidizing biocides. With a support vector machine approach, we showed spatial separation between the two strains, and, using statistical testing and high-performance computing (HPC), we determined conditions which possess an inherent cluster structure. Our results indicate that there is a relationship between biocide potency and inherent biofilm formation capacity with the tendency to select for spatial clusters of survivors. There was no relationship between positions of clusters of live or dead cells within stressed biofilms. This work identifies an approach to robustly quantify clusters of physiologically distinct cells within biofilms and suggests work to understand how clusters form and survive is needed. SIGNIFICANCE STATEMENT: Control of biofilm growth remains a major challenge and there is considerable uncertainty about how bacteria respond to disinfection within a biofilm and how clustering of cells impacts survival. We have developed a methodological approach to identify and statistically analyse clusters of surviving cells in biofilms after biocide challenge. This approach can be used to understand bacterial behaviour within biofilms under stress and is widely applicable.

Raman Microscopic Analysis of Dry-Surface Biofilms on Clinically Relevant Materials.

Moist/hydrated biofilms have been well-studied in the medical area, and their association with infections is widely recognized. In contrast, dry-surface biofilms (DSBs) on environmental surfaces in healthcare settings have received less attention. DSBs have been shown to be widespread on commonly used items in hospitals and to harbor bacterial pathogens that are known to cause healthcare-acquired infections (HAI). DSBs cannot be detected by routine surface swabbing or contact plates, and studies have shown DSBs to be less susceptible to cleaning/disinfection products. As DSBs are increasingly reported in the medical field, and there is a likelihood they also occur in food production and manufacturing areas, there is a growing demand for the rapid in situ detection of DSBs and the identification of pathogens within DSBs. Raman microspectroscopy allows users to obtain spatially resolved information about the chemical composition of biofilms, and to identify microbial species. In this study, we investigated Staphylococcus aureus mono-species DSB on polyvinylchloride blanks and stainless steel coupons, and dual-species (S. aureus/Bacillus licheniformis) DSB on steel coupons. We demonstrated that Raman microspectroscopy is not only suitable for identifying specific species, but it also enables the differentiation of vegetative cells from their sporulated form. Our findings provide the first step towards the rapid identification and characterization of the distribution and composition of DSBs on different surface areas.

Impact of benzalkonium chloride, benzethonium chloride and chloroxylenol on bacterial antimicrobial resistance.

This review examined 3655 articles on benzalkonium chloride (BKC), benzethonium chloride (BZT) and chloroxylenol (CHO) aiming to understand their impact on antimicrobial resistance. Following the application of inclusion/exclusion criteria, only 230 articles were retained for analysis; 212 concerned BKC, with only 18 for CHO and BZT. Seventy-eight percent of studies used MIC to measure BKC efficacy. Very few studies defined the term 'resistance' and 85% of studies defined 'resistance' as <10-fold increase (40% as low as 2-fold) in MIC. Only a few in vitro studies reported on formulated products and when they did, products performed better. In vitro studies looking at the impact of BKC exposure on bacterial resistance used either a stepwise training protocol or exposure to constant BKC concentrations. In these, BKC exposure resulted in elevated MIC or/and MBC, often associated with efflux, and at time, a change in antibiotic susceptibility profile. The clinical relevance of these findings was, however, neither reported nor addressed. Of note, several studies reported that bacterial strains with an elevated MIC or MBC remained susceptible to the in-use BKC concentration. BKC exposure was shown to reduce bacterial diversity in complex microbial microcosms, although the clinical significance of such a change has not been established. The impact of BKC exposure on the dissemination of resistant genes (notably efflux) remains speculative, although it manifests that clinical, veterinary and food isolates with elevated BKC MIC carried multiple efflux pump genes. The correlation between BKC usage and gene carriage, maintenance and dissemination has also not been established. The lack of clinical interpretation and significance in these studies does not allow to establish with certainty the role of BKC on AMR in practice. The limited literature and BZT and CHO do not allow to conclude that these will impact negatively on emerging bacterial resistance in practice.

Dual species dry surface biofilms; Bacillus species impact on Staphylococcus aureus survival and surface disinfection.

AIMS: Dry surface biofilms (DSB) survive on environmental surfaces throughout hospitals, able to resist cleaning and disinfection interventions. This study aimed to produce a dual species DSB and explore the ability of commercially available wipe products to eliminate pathogens within a dual species DSB and prevent their transfer. METHODS AND RESULTS: Staphylococcus aureus was grown with two different species of Bacillus on stainless steel discs, over 12 days using sequential hydration and dehydration phases. A modified version of ASTM 2967-15 was used to test six wipe products including one water control with the Fitaflex Wiperator. Staphylococcus aureus growth was inhibited when combined with Bacillus subtilis. Recovery of S. aureus on agar from a dual DSB was not always consistent. Our results did not provide evidence that Bacillus licheniformis protected S. aureus from wipe action. There was no significant difference of S. aureus elimination by antimicrobial wipes between single and dual species DSB. B. licheniformis was easily transferred by the wipe itself and to new surfaces both in a single and dual species DSB, whilst several wipe products inhibited the transfer of S. aureus from wipe. However, S. aureus direct transfer to new surfaces was not inhibited post-wiping. CONCLUSIONS: Although we observed that the dual DSB did not confer protection of S. aureus, we demonstrated that environmental species can persist on surfaces after disinfection treatment. Industries should test DSB against future products and hospitals should consider carefully the products they choose. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first study reporting on the production of a dual species DSB. Multispecies DSB have been identified throughout the world on hospital surfaces, but many studies focus on single species biofilms. This study has shown that DSB behave differently to hydrated biofilms.

Antimicrobial stewardship of antiseptics that are pertinent to wounds: the need for a united approach.

Long before the nature of infection was recognized, or the significance of biofilms in delayed healing was understood, antimicrobial agents were being used in wound care. In the last 70 years, antibiotics have provided an effective means to control wound infection, but the continued emergence of antibiotic-resistant strains and the documented antibiotic tolerance of biofilms has reduced their effectiveness. A range of wound dressings containing an antimicrobial (antibiotic or non-antibiotic compound) has been developed. Whereas standardized methods for determining the efficacy of non-antibiotic antimicrobials in bacterial suspension tests were developed in the early twentieth century, standardized ways of evaluating the efficacy of antimicrobial dressings against microbial suspensions and biofilms are not available. Resistance to non-antibiotic antimicrobials and cross-resistance with antibiotics has been reported, but consensus on breakpoints is absent and surveillance is impossible. Antimicrobial stewardship is therefore in jeopardy. This review highlights these difficulties and in particular the efficacy of current non-antibiotic antimicrobials used in dressings, their efficacy, and the challenges of translating in vitro efficacy data to the efficacy of dressings in patients. This review calls for a unified approach to developing standardized methods of evaluating antimicrobial dressings that will provide an improved basis for practitioners to make informed choices in wound care.

TCF-ALP: a fluorescent probe for the selective detection of Staphylococcus bacteria and application in "smart" wound dressings.

Alkaline phosphatase (ALP) is an important enzyme-based biomarker present in several bacterial species; however, it is currently undervalued as a strategy to detect pathogenic bacteria. Here, we explore our ALP-responsive colorimetric and fluorescent probe (TCF-ALP) for such applications. TCF-ALP displayed a colorimetric and fluorescence response towards Staphylococcus aureus (S. aureus), with a limit of detection of 3.7 × 106 CFU mL-1 after 24 h incubation. To our surprise, TCF-ALP proved selective towards Staphylococcus bacteria when compared with Enterococcus faecalis (E. faecalis), and Gram-negative P. aeruginosa and E. coli. Selectivity was also seen in clinically relevant S. aureus biofilms. Owing to the high prevalence and surface location of S. aureus in chronic wounds, TCF-ALP was subsequently encapsulated in polyvinyl alcohol (PVA)-based hydrogels as a proof-of-concept "smart" wound dressing. TCF-ALP hydrogels were capable of detecting S. aureus in planktonic and biofilm assays, and displayed a clear colour change from yellow to purple after 24 h incubation using ex vivo porcine skin models. Overall, TCF-ALP is a simple tool that requires no prior knowledge, training, or specialist equipment, and has the potential to overcome issues related to invasive swabbing and tissue biopsy methods. Thus, TCF-ALP could be used as a tool to monitor the early development of infection in a wound and allow for the rapid provision of appropriate treatment for Staphylococcal bacterial infections.

Electropositive Nanodiamond-Coated Quartz Microfiber Membranes for Virus and Dye Filtration.

Electropositive membranes demonstrating high flux at low pressure differentials show great promise as universal separation platforms for viruses and other charged entities when centralized systems of water and power are scarce. However, the fabrication of a suitably stable membrane with optimal electrostatic characteristics remains a challenge. Here, hydrogenated detonation nanodiamond was loaded onto a quartz microfiber support membrane and coupled to the membrane surface under a high vacuum annealing process. The fabricated membranes display a zeta potential of +45 mV at pH 7 and an isoelectric point around pH 11. We show that the nanodiamond coating is robust to prolonged periods of pressurized water flow by performing extensive zeta potential measurements over time, and water filtration tests demonstrated excellent membrane retention for the electronegative dye molecule acid black 2, and at least a 6.2 log10 reduction in MS2 bacteriophage from feed waters (>99.9999%).

Is a reduction in viability enough to determine biofilm susceptibility to a biocide?

OBJECTIVE: The abundance and prevalence of dry-surface biofilms (DSBs) in hospitals constitute an emerging problem, yet studies rarely report the cleaning and disinfection efficacy against DSBs. Here, the combined impact of treatments on viability, transferability, and recovery of bacteria from DSBs has been investigated for the first time. METHODS: Staphylococcus aureus DSBs were produced in alternating 48-hour wet-dry cycles for 12 days on AISI 430 stainless steel discs. The efficacy of 11 commercially available disinfectants, 4 detergents, and 2 contactless interventions were tested using a modified standardized product test. Reduction in viability, direct transferability, cross transmission (via glove intermediate), and DSB recovery after treatment were measured. RESULTS: Of 11 disinfectants, 9 were effective in killing and removing bacteria from S. aureus DSBs with >4 log10 reduction. Only 2 disinfectants, sodium dichloroisocyanurate 1,000 ppm and peracetic acid 3,500 ppm, were able to lower both direct and cross transmission of bacteria (<2 compression contacts positive for bacterial growth). Of 11 disinfectants, 8 could not prevent DSB recovery for >2 days. Treatments not involving mechanical action (vaporized hydrogen peroxide and cold atmospheric plasma) were ineffective, producing <1 log10 reduction in viability, DSB regrowth within 1 day, and 100% transferability of DSB after treatment. CONCLUSIONS: Reduction in bacterial viability alone does not determine product performance against biofilm and might give a false sense of security to consumers, manufacturers and regulators. The ability to prevent bacterial transfer and biofilm recovery after treatment requires a better understanding of the effectiveness of biocidal products.

Ethylzingerone, a Novel Compound with Antifungal Activity.

Preservatives increase the shelf life of cosmetic products by preventing growth of contaminating microbes, including bacteria and fungi. In recent years, the Scientific Committee on Consumer Safety (SCCS) has recommended the ban or restricted use of a number of preservatives due to safety concerns. Here, we characterize the antifungal activity of ethylzingerone (hydroxyethoxyphenyl butanone [HEPB]), an SCCS-approved new preservative for use in rinse-off, oral care, and leave-on cosmetic products. We show that HEPB significantly inhibits growth of Candida albicans, Candida glabrata, and Saccharomyces cerevisiae, acting fungicidally against C. albicans Using transcript profiling experiments, we found that the C. albicans transcriptome responded to HEPB exposure by increasing the expression of genes involved in amino acid biosynthesis while activating pathways involved in chemical detoxification/oxidative stress response. Comparative analyses revealed that C. albicans phenotypic and transcriptomic responses to HEPB treatment were distinguishable from those of two widely used preservatives, triclosan and methylparaben. Chemogenomic analyses, using a barcoded S. cerevisiae nonessential mutant library, revealed that HEPB antifungal activity strongly interfered with the biosynthesis of aromatic amino acids. The trp1Δ mutants in S. cerevisiae and C. albicans were particularly sensitive to HEPB treatment, a phenotype rescued by exogenous addition of tryptophan to the growth medium, providing a direct link between HEPB mode of action and tryptophan availability. Collectively, our study sheds light on the antifungal activity of HEPB, a new molecule with safe properties for use as a preservative in the cosmetic industry, and exemplifies the powerful use of functional genomics to illuminate the mode of action of antimicrobial agents.

Potential Role of Oral Rinses Targeting the Viral Lipid Envelope in SARS-CoV-2 Infection.

Emerging studies increasingly demonstrate the importance of the throat and salivary glands as sites of virus replication and transmission in early COVID-19 disease. SARS-CoV-2 is an enveloped virus, characterized by an outer lipid membrane derived from the host cell from which it buds. While it is highly sensitive to agents that disrupt lipid biomembranes, there has been no discussion about the potential role of oral rinsing in preventing transmission. Here, we review known mechanisms of viral lipid membrane disruption by widely available dental mouthwash components that include ethanol, chlorhexidine, cetylpyridinium chloride, hydrogen peroxide, and povidone-iodine. We also assess existing formulations for their potential ability to disrupt the SARS-CoV-2 lipid envelope, based on their concentrations of these agents, and conclude that several deserve clinical evaluation. We highlight that already published research on other enveloped viruses, including coronaviruses, directly supports the idea that oral rinsing should be considered as a potential way to reduce transmission of SARS-CoV-2. Research to test this could include evaluating existing or specifically tailored new formulations in well-designed viral inactivation assays, then in clinical trials. Population-based interventions could be undertaken with available mouthwashes, with active monitoring of outcome to determine efficacy. This is an under-researched area of major clinical need.

Surface Wiping Test to Study Biocide -Cinnamaldehyde Combination to Improve Efficiency in Surface Disinfection.

Disinfection is crucial to control and prevent microbial pathogens on surfaces. Nonetheless, disinfectants misuse in routine disinfection has increased the concern on their impact on bacterial resistance and cross-resistance. This work aims to develop a formulation for surface disinfection based on the combination of a natural product, cinnamaldehyde, and a widely used biocide, cetyltrimethylammonium bromide. The wiping method was based on the Wiperator test (ASTM E2967-15) and the efficacy evaluation of surface disinfection wipes test (EN 16615:2015). After formulation optimization, the wiping of a contaminated surface with 6.24 log10 colony-forming units (CFU) of Escherichia coli or 7.10 log10 CFU of Staphylococcus aureus led to a reduction of 4.35 log10 CFU and 4.27 log10 CFU when the wipe was impregnated with the formulation in comparison with 2.45 log10 CFU and 1.50 log10 CFU as a result of mechanical action only for E. coli and S. aureus, respectively. Furthermore, the formulation prevented the transfer of bacteria to clean surfaces. The work presented highlights the potential of a combinatorial approach of a classic biocide with a phytochemical for the development of disinfectant formulations, with the advantage of reducing the concentration of synthetic biocides, which reduces the potentially negative environmental and public health impacts from their routine use.

Mapping the Efficacy and Mode of Action of Ethylzingerone [4-(3-Ethoxy-4-Hydroxyphenyl) Butan-2-One] as an Active Agent against Burkholderia Bacteria.

Burkholderia cepacia complex (Bcc) bacteria are intrinsically antimicrobial-resistant opportunistic pathogens and key risk species in the contamination of nonfood industrial products. New agents and formulations to prevent growth of Burkholderia in home care (cleaning agents) and personal-care (cosmetics and toiletries) products are required. We characterized how ethylzingerone [4-(3-ethoxy-4-hydroxyphenyl) butan-2-one] (HEPB) acts as a preservative with activity against Burkholderia species encountered in industry. Burkholderia (n = 58) and non-Burkholderia (n = 7) bacteria were screened for susceptibility to HEPB, and its mode of action and resistance were determined for a model Burkholderia vietnamiensis strain using transposon mutagenesis, transcriptomics, and genome resequencing analysis. The susceptibility of Burkholderia spp. to HEPB (MIC = 0.45% ± 0.11% [wt/vol]; MBC = 0.90% ± 0.3% [wt/vol]) was characterized, with limited inter- and intraspecies differences. HEPB (1% [wt/vol]) was rapidly bactericidal, producing a 6-log reduction in viability within 4 h. Spontaneous resistance to HEPB did not develop, but transient phenotypes with altered growth characteristics and susceptibility to antibiotics were identified after prolonged exposure to sublethal HEPB concentrations. Transposon mutagenesis and RNA-sequencing analysis identified multiple genetic pathways associated with HEPB exposure, including stress response mechanisms, altered permeability, regulation of intracellular pH, damage and repair of intracellular components, and alteration and repair of lipopolysaccharides. Key pathways included the stringent response, homeostasis of intracellular pH by the kdp operon, protection against electrophiles by KefC, and repair of oxidized proteins by methionine sulfoxide reductase enzymes. In summary, we show that HEPB has potent, targeted efficacy against Burkholderia bacteria without promoting wider stable antimicrobial resistance. The mode of action of HEPB against Burkholderia is multifactorial, but killing by intracellular oxidation is a key mechanism of this promising agent.IMPORTANCEBurkholderia bacteria are opportunistic pathogens that can overcome preservatives used in the manufacture of nonsterile industrial products and occasionally cause contamination. Consequently, new preservatives to prevent the growth of key risk Burkholderia cepacia complex bacteria in nonfood industrial products are urgently required. Here, we show that ethylzingerone is active against these problematic bacteria, killing them via a multifactorial mode of action which involves intracellular oxidation.