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The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link Aims: This study aimed to establish the mechanisms of action (MOA) of a novel surface functionalised polyacrylonitrile (PAN) catalyst, which was previously shown to have potent antimicrobial activity in conjunction with hydrogen peroxide (H2O2). Methods and Results: Bactericidal activity was determined using a disinfectant suspension test. The MOA was investigated by measuring loss of 260nm absorbing material, membrane potential and permeability assays, analysis of intra- and extracellular ATP and pH, and tolerance to sodium chloride and bile salts. The catalyst lowered sub-lethal concentrations of H2O2 from 0.2% to 0.09%. H2O2 ± 3 g PAN catalyst significantly (p≤0.05) reduced sodium chloride and bile salt tolerance, suggesting the occurance of sublethal cell membrane damage. The catalyst significantly increased (p≤0.05) N-Phenyl-l-napthylamine uptake (1.51-fold) and leakage of nucleic acids, demonstrating increased membrane permeability. A significant (p≤0.05) loss of membrane potential (0.015 a.u.), coupled with pertubation of intracellular pH homeostasis and depletion of intracellular ATP, suggests potentiation of H2O2-mediated cell membrane damage. Conclusions: This is the first study to investigate the catalyst’s antimicrobial mechanism of action, with the cytoplasmic membrane being a target for cellular injury. Significance and Impact of the Study: Understanding the MOA by which the catalyst potentiates H2O2 could lead to optimisation of concentrations and contact times needed for disinfection in a wide range of settings.

open access article Background Novel antimicrobials with new mechanisms of action are critical to circumvent emerging antimicrobial resistant microorganisms (AMR), such as vancomycin resistant Enterococcus faecium (VanREF). Previous research demonstrated, with transcriptomic analysis and phenotypic assays, that the essential oil components (EOCs) carvacrol (CARV) (1.98 mM) and cuminaldehyde (CA) (4.20 mM) with the antibiotic vancomycin (Van) (0.022 µM), restored the susceptibility of VanREF. This finding suggested that an envelope damage has occurred. Several transport channel-related genes were also differentially expressed including bcr, ecfA1, ecsA-1, yloB and nhaC_2, indicating they could contribute to the mechanism of action of the triple combination CARV-CA-Van. Purpose The aim of this study was to elucidate the role of transport channels in the antimicrobial mechanism of action of CARV and CA with Van. Methods The expression levels of bcr, ecfA1, ecsA-1, yloB and nhaC_2 were established using qPCR under the effect of the triple combination, and in the presence of 1 mM calcium or 0.1 mM EDTA (channel blocker) over time. Results Significant (p ≤ 0.05) changes in expression of yloB and bcr genes were observed at 10 and 30 min, and of ecfA1, ecsA-1 and nhaC_2 at later time points (120 and 360 min). Adding Ca2+ to the combination induced significant changes in the expression of yloB (from -5.67 to -50-fold, +3.68 to -4.4-fold, -1.8 to +6.6-fold and +1.32 to +3.08-fold at 10, 30, 60 and 120 min, respectively). Instead, the addition of EDTA significantly changed the expression of bcr (from -13.5 to -1.11-fold, +15.3 to -1.06-fold, +15.71 to -10-fold at 10, 120 and 360 min, respectively). Since that Ca2+ participates in the stability of bacterial cell wall and EDTA blocks efflux pumps, the current findings suggest the involvement of the efflux system in the mechanism of action of the three-drug combination. Conclusion Overall, the findings of this study suggest the involvement of transportation channels in the mechanism of action of the CARV-CA-Van combination against VanREF.

Aims: This study aimed to investigate the stability of HCoV-OC43 on leather and transfer to other surfaces and to determine the antiviral activity of a silver-based leather coating. Methods and Results: The infectivity of HCoV-OC43 (6.6 log10 TCID50) on patent, nubuck, full-grain calf and corrected grain leathers (untreated and silver-coated) was measured over 72 hours by titration on BHK-21 cells. Recovery from pig skin (8-9 log10 TCID50) was also assessed. Transfer of infectious HCoV-OC43 from leather onto cardboard and stainless steel (0-48 hours post-inoculation) was quantified. HCoV-OC43 remained infectious for 6-48 hours on patent, finished and calf leathers; no infectious HCoV-OC43 was recovered from nubuck at 0 hours. Silver coating of full-grain calf and corrected grain leathers significantly reduced HCoV-OC43 infectivity (p≤0.05) after 2 hours, where no infectious virus was recovered. Transfer of HCoV-OC43 (≤3.1-5.5 log10) was detected from calf, finished and patent leather onto stainless steel and carboard up to 2 hours after inoculation, while no transfer was detected for silver-coated leathers at 2 hours. Leather has also been utilised as a skin surrogate for investigating fomite transmission. HCoV-OC43 was reduced by 4.71 log10 on pig skin at 0 hours, in a similar trend to highly absorbent nubuck leather. Conclusions: Human coronaviruses remain infectious on leather for up to 48 hours and transfer onto cardboard and stainless steel up to 2 hours post-inoculation. Absorbency contributes to the recovery/persistence of HCoV-OC43 on surfaces. A silver-based leather coating demonstrated antiviral activity and limited viral transfer onto other surfaces. Significance of Study: This investigation suggests that leather could pose a risk of indirect transmission of human coronaviruses; this is of significance for settings where there is close contact with leathers such as in manufacturing, retail and domestic environments. A silver-based leather coating demonstrated antiviral activity and limited viral transfer onto other surfaces, reducing the potential for indirect transmission from leathers.

Background Disease outbreaks in healthcare facilities (e.g., Bacillus cereus) have been linked to contaminated linen, highlighting the importance of decontamination of healthcare textiles and infection control measures to prevent recontamination. The COVID-19 pandemic has raised further concerns around textile hygiene. Methods for monitoring microbial kill during healthcare laundering and post-laundering contamination are not standardised across Europe, leading to potential variation in validation outputs. For example, EN14065 accreditation requires microbiological monitoring, however no standard method is prescribed. Microbial kill is commonly validated by laundering inoculated textiles. Post-laundering hygiene monitoring tests can be destructive (elution) or non-destructive (e.g., swabbing). This study aims to evaluate common methods for detection of microbial contamination on textiles and measuring kill during industrial laundering processes. Methods Recovery of Escherichia coli, Staphylococcus aureus, Enterococcus faecium or Bacillus cereus spores (108, 102 and 101 CFU/ml) from 100% cotton was evaluated using different elution buffers (phosphate buffered saline or maximum recovery diluent with and without polysorbate 80), agitation methods (vortexing, stomaching, shaking by hand or with glass beads) and recovery agar (selective or non-selective). Recovery was compared to swabbing, RODAC plates and dip slides. Survival of E. faecium during a cold industrial wash with and without detergent was compared between loose swatches and those enclosed within cloth bags. Results Recovery of all test species was significantly (p≤0.05) greater using elution methods than non-destructive methods, while RODAC plates and dip slides were unquantifiable at 108 CFU/ml. Recovery was not significantly different between elution buffers or recovery agar. Shaking by hand was generally marginally more precise and efficient in microbial recovery. E. faecium was significantly (p≤0.05) reduced by industrial laundering with and without detergent; there was no significant difference when enclosed in cloth bags. Conclusions Surface testing methods have limited sensitivity compared to elution methods, suggesting that destructive methods are most appropriate for hygiene monitoring of laundered healthcare textiles. Swatch testing is confounded by a loss of microorganisms to dilution and agitation in the wash; further tests need to be developed to monitor the kill of microorganisms in the wash in order to develop a standardised test methodology to be used throughout global industrial laundries.

Aims The aim of this investigation was to determine the persistence of human coronaviruses on a range of textiles both in the environment and during laundering. Methods and Results The stability of human coronaviruses HCoV-OC43 and HCoV-229E on cotton, polycotton and polyester textiles was determined up to 72 hours. The transfer of HCoV-OC43 from textiles to plastic or another textile was quantified. Persistence of HCoV-OC43 on cotton during domestic and industrial laundering was also investigated in the presence and absence of detergents and temperature (40-75°C). Infectious virus was quantified by titration on mammalian cells. Infectious HCoV-OC43 was detectable for 6 hours on polycotton, 24 hours on cotton and ≥72 hours on polyester. HCoV-229E was less stable, where it was detectable for 2 hours on polycotton, 6 hours on cotton and 24 hours on polyester. HCoV-OC43 transferred from polyester to PVC and polyester up to 72 hours post-inoculation, whereas no transfer was detected from cotton or polycotton. Domestic and industrial laundering without temperature and detergent completely removed HCoV-OC43 from cotton (≥ 4.58 log10 reduction) under clean conditions. Under dirty conditions, HCoV-OC43 was detected on cotton (≤1.78 log10) after domestic laundering without temperature and detergent. However, HCoV-OC43 was removed by domestic laundering with temperature (40°C) and detergent, or industrial laundering without temperature and detergent. Conclusions Human coronaviruses can persist on textiles for up to 3 days and readily transfer from polyester to other surfaces. HCoV-OC43 were removed from cotton during both domestic and industrial laundering. Significance of Study Polyester could potentially act as fomites for the transmission of coronaviruses, demonstrating the importance of infection control procedures for handling of contaminated textiles. Human coronaviruses are removed from textiles during common domestic and industrial wash programmes, indicating that typical healthcare washing procedures are likely to be suitable for decontaminating textiles.

Previous research indicates that SARS-CoV-2 persists on stainless steel and plastic for 72 hours to 7 days and appears to be less stable on porous surfaces. However, there is limited research on the stability of coronaviruses on a range of textiles, of which the composition and construction could have an effect on its persistence. Determination of the persistence of coronaviruses on textiles is required to evaluate the potential risk of fomite transmission via textiles; this is of particular importance in healthcare settings to inform laundering policies for the adequate decontamination of hospital linens and staff uniforms. The aim of this study is to determine the stability of model human coronaviruses for SARS-CoV-2 on a range of textile fibres and how best to decontaminate them. Human coronavirus (HCoV) OC43 was cultured on HCT-8 cells and HCoV-229E was cultured on MRC-5 cells. The optimal recovery method of virus from textiles was first determined by comparing the recovery efficiency of HCoV-OC43 from 100% cotton using differing diluents (cell culture media, phosphate buffered saline (PBS) and maximum recovery diluent (MRD)) and recovery methods (vortexing, stomaching and shaking by hand). The stability of HCoV-OC43 and HCoV-229E was then determined on 100% cotton, polyester/cotton blend, 100% polyester and calf leather up to 48 hours. A no virus (culture media only) control was included. Infectious virus was quantified by titration of the supernatant on BHK-21 cells in 96-well plates. The recovery of HCoV-OC43 from 100% cotton was comparable between PBS and culture media as diluents, whereas recovery was reduced using MRD. Shaking by hand was the most efficient recovery method used, with 98.56% of the inoculum being recovered. The stability of HCoV-OC43 was greatest on polyester, where it remained infectious for at least 6 hours. Investigations into the decontamination of model human coronaviruses under wash parameters are ongoing. Investigations on the survival of coronaviruses is required to evaluate the infection control risk of contaminated textiles and to identify laundering parameters required to adequately decontaminate linen. This study demonstrates that model coronaviruses survive on textiles, indicating that there may be a risk within the healthcare and domestic environments.

Background: In the UK nurses’ uniforms are domestically laundered, posing a threat of cross contamination of healthcare-associated infections. The UK Department of Health recommends washing uniforms at 60ºC, however nurses most commonly wash them at 40ºC. Bacteria survive on textiles washed at low temperatures; 4.28-4.62 log10 Escherichia coli and Staphylococcus aureus survived washing at 40ºC and cross contaminated other textiles in the wash. Antimicrobial laundry supplements could sanitise textiles and limit cross-contamination. A supplement that deposits an antibacterial coating onto textiles could also reduce contamination between washes. This study aimed to investigate a silver-based antimicrobial textile coating product (Micro-Fresh 1911) as a dual-use antimicrobial laundry supplement and textile coating. Materials/methods: Polycotton inoculated with 108 CFU/mL E. coli or S. aureus (type and clinical isolates) were washed with 2% Micro-Fresh 1911 in a domestic washing machine (40°C) and surviving microorganisms enumerated. Washes were conducted with and without soiling and biological detergent. Control washes were water alone. The antimicrobial activity of polycotton washed with Micro-Fresh 1911 at 40°C was assessed against S. aureus and E. coli using ISO 20645 and ISO 20743 methods. Controls were polycotton washed in water and textile padded with Micro-Fresh 2611. Results: Washing with Micro-Fresh 1911 reduced E. coli and S. aureus by 7.14-8.08 log10. No cells were recovered from sterile textile, whereas washing with water alone reduced E. coli and S. aureus by 2.21-4.25 log10 and resulted in 3.13-4.01 log10 CFU cross-contamination. Combining Micro-Fresh 1911 with biological detergent reduced E. coli and S. aureus by 5.56-6.65 log10. Fabric washed with Micro-Fresh 1911 was antimicrobial against E. coli and S. aureus type and clinical isolates according to ISO 20645 and ISO 20743. The antimicrobial coating remained after washing the fabric in water once at 40°C and 73°C. Conclusions: Micro-Fresh 1911 exibits antimicrobial activity in a 40ºC domestic wash and deposits an antimicrobial layer onto polycotton. Micro-Fresh could be employed as an antimicrobial laundering product to reduce microbial contamination of healthcare laundry washed at low temperatures.

Background: New antimicrobials to treat Vancomycin Resistant Enterococcus faecium (VRE) infections are considered a high priority. Essential oil compounds have been shown to interact synergistically with antibiotics, and so could be used as adjuvants to preserve the antibiotic repertoire. A combination of carvacrol, cuminaldehyde and vancomycin was found to synergistically inhibit VRE, potentially extending the utility of vancomycin against VRE. This study aimed to investigate the mechanism of action of the carvacrol, cuminaldehyde and vancomycin combination against VRE using transcriptomic analysis. Materials/methods: The antimicrobial activity of the combination in 1% DMSO was determined by a time-kill assay. Transcriptomic response of VRE to the combination was determined by microarray analysis. VRE was treated with either 0.031 mg/L vancomycin, 1.98 mM carvacrol, 4.20 mM cuminaldehyde or the ternary combination for 60 minutes. A control of 1% DMSO only was included. RNA was extracted, converted to cDNA labelled with Cy5 and hybridised onto a custom microarray and scanned. Significant (p2-fold change). Genes with the greatest change in expression mainly related to carbohydrate metabolism; the phosphotransferase system (PTS)-associated genes mtlD, mtlF and agaC6 were downregulated 2.86-2.28-fold and ulaA3 was upregulated 4.07-fold. Glutamine-fructose-6-phosphate aminotransferase (glmS), which is associated with amino sugar biosynthesis was downregulated 2.67-3.83-fold. Vancomycin resistance-associated genes were not altered by the combination or individual components. Conclusions: Carvacrol and cuminaldehyde synergistically enhance the antimicrobial activity of vancomycin against VRE, and so could be useful to preserve the antibiotic repertoire. The combination affected carbohydrate metabolism and biosynthetic processes, indicating that the combination did not directly modulate antibiotic resistance genes. Further research will investigate significantly changed genes to enhance understanding of the synergistic mechanism of action of the combination.

Acne vulgaris is a chronic skin condition associated with overgrowth of Propionibacterium acnes and Staphylococcus epidermidis. Antibiotics are commonly prescribed to control acne; however, resistance has emerged, indicating the need for new antimicrobial therapies1. Several essential oils (EOs) are antimicrobial, including against antibiotic-resistant isolates, indicating that antimicrobial formulations may be developed using EOs2. The aim of this study was to develop a topical preparation incorporating EOs to control acne-associated bacteria, and compare its efficacy with the topical antibiotic formulations Dalacin T® and Stiemycin®. The antimicrobial activity of litsea (Litsea cubeba), rosewood (Aniba rosaeadora) and clove bud (Syzigum aromaticum) EOs was identified using the Kirby-Bauer disc diffusion method3 and the minimum inhibitory concentrations (MICs) were determined using a broth microdilution assay3.The MICs of clove bud and rosewood were 1.38 mg/ml against S. epidermidis and P. acnes. The MIC of litsea was 1.11 mg/mL against S. epidermidis and 2.23 mg/mL against P. acnes. The antimicrobial activity of a ternary combination of these EOs was further investigated using the checkerboard method with calculation of fractional inhibitory concentration index (FICI)4. Synergy was observed against both test species (FICI < 0.5). The EOs were characterised by both GC-MS and NMR. The antimicrobial efficacy of a cosmetic preparation containing the ternary EO combination was then compared to that of the current prescription medicines Dalacin T® and Stiemycin®. A combination of 0.53 mg/mL litsea, 0.11 mg/mL rosewood and 0.11 mg/mL clove bud EOs was formulated into a neroli herbal distillate with 0.5% (v/v) ethanol. Survival of P. acnes and S. epidermidis (types and clinical isolates) after a 24 h contact with the formulations was determined and compared to Dalacin T® and Stiemycin®. The formulation completely inhibited P. acnes (7 log10 reduction) and S. epidermidis (6 log10 reduction), and was equivalent in activity to Dalacin T® and Stiemycin®, including erythromycin-resistant isolates. The EO combination alone was either more antimicrobial (p ≤ 0.05) or equivalent in activity to the active ingredients of Dalacin T® and Stiemycin® (10 mg/mL clindamycin phosphate and 20 mg/mL erythromycin, respectively). The formulation may be useful as an alternative to antibiotic preparations for the control of acne-associated bacteria.

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link. Acne vulgaris, a chronic condition associated with overgrowth of Propionibacterium acnes and Staphylococcus epidermidis, is commonly treated with antibiotics. However, the emergence of antibiotic resistance has resulted in a need for alternative therapies. The aim of this study is to develop a topical preparation incorporating Essential Oils (EOs) for use against acne-associated bacteria and assess its efficacy against prescription therapies Dalacin T and Stiemycin. Antimicrobial screening of rosewood, clove bud and litsea EOs was conducted and interactions between binary and ternary combinations determined against P. acnes and S. epidermidis (type and clinical isolates) using Minimum Inhibitory Concentrations (MICs) and Fractional Inhibitory Concentrations (FICs). The EOs were characterised by both GC-MS and NMR. A combination of 0.53 mg/ml litsea, 0.11 mg/ml rosewood and 0.11 mg/ml clove bud were formulated into herbal distillates and compared to Dalacin T and Stiemycin against antibiotic sensitive and resistant isolates (erythromycin). The distillate with EO had synergistic activity against P. acnes (7log10 reduction) and indifferent activity against S. epidermidis, (6log10 reduction), antimicrobial activity was either significantly (p ≤ 0.05) more antimicrobial or equivalent to that of Dalacin T and Stiemycin. This formulation may serve as a valuable alternative for the control of acne vulgaris-associated bacteria.