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Aim: Bacterial biofilms can form on surfaces in hospitals, clinics, farms, and food processing plants, representing a possible source of infections and cross-contamination. This study investigates the effectiveness of new commercial wipes against Staphylococcus aureus and Pseudomonas aeruginosa biofilms (early attachment and formed biofilms), assessing LH SALVIETTE wipes (Lombarda H S.r.l.) potential for controlling biofilm formation., Methods and Results: The wipes efficacy was studied against the early attachment phase and formed biofilm of S. aureus ATCC 6538 and P. aeruginosa ATCC 15442 on a polyvinyl chloride (PVC) surface, following a modified standard test EN 16615:2015, measuring Log10 reduction and cell viability using live/dead staining. It was also evaluated the wipes anti-adhesive activity over time (3 h, 2 4h), calculating CFU.mL-1 reduction. Data were analyzed using t-student test. The wipes significantly reduced both early phase and formed S. aureus biofilm, preventing dispersion on PVC surfaces. Live/dead imaging showed bacterial cluster disaggregation and killing action. The bacterial adhesive capability decreased after short-time treatment (3 h) with the wipes compared to 24 h., Conclusions: Results demonstrated decreased bacterial count on PVC surface both for early attachment phase and formed biofilms, also preventing the bacterial biofilm dispersion., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Aims: The aim of this work was to evaluate the efficacy of an organosilicon-based, commercially available antimicrobial formulation in the My-shield® product line against bacterial surface contamination., Methods and Results: The antimicrobial product was tested in vitro for its long-term persistence on surfaces and effectiveness against Staphylococcus aureus biofilms in comparison to 70% ethanol and 0.1% or 0.6% sodium hypochlorite. Field testing was also conducted over 6 weeks at a university athletic facility. In vitro studies demonstrated the log reductions achieved by the test product, 70% ethanol, and 0.1% sodium hypochlorite were 3.6, 3.1, and 3.2, respectively. The test product persisted on surfaces after washing and scrubbing, and pre-treatment with this product prevented S. aureus surface colonization for up to 30 days. In comparison, pre-treatment with 70% ethanol or 0.6% sodium hypochlorite was not protective against S. aureus biofilm formation after seven days. The field test demonstrated that weekly applications of the test product were more effective at reducing surface bacterial load than daily applications of a control product., Conclusions: The test product conferred greater long-term protection against bacterial growth and biofilm formation by S. aureus than ethanol and sodium hypochlorite. Even with less frequent applications, the test product maintained a high level of antimicrobial activity., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Aims: This study aimed to assess the effects of chlorine dioxide (ClO2) in water on whiteleg shrimp Penaeus vannamei, evaluating its impact on the stomach microbiota, gill transcriptome, and pathogens., Methods and Results: ClO2 was added to the aquarium tanks containing the shrimp. The application of ClO2 to rearing water was lethal to shrimp at concentrations above 1.2 ppm. On the other hand, most of the shrimp survived at 1.0 ppm of ClO2. Microbiome analysis showed that ClO2 administration at 1.0 ppm significantly reduced the α-diversity of bacterial community composition in the shrimp stomach, and this condition persisted for at least 7 days. Transcriptome analysis of shrimp gill revealed that ClO2 treatment caused massive change of the gene expression profile, including stress response genes. However, after 7 days of the treatment, the gene expression profile was similar to that of shrimp in the untreated control group, suggesting a recovery to the normal state. This 1.0-ppm ClO2 significantly reduced shrimp mortality in artificial challenges with an acute hepatopancreatic necrosis disease-causing Vibrio parahaemolyticus and white spot syndrome virus, which were added to rearing water., Conclusions: The use of ClO2 at appropriate concentrations effectively eliminates a significant portion of the bacteria in the shrimp stomach and pathogens in the water. The results of this study provide fundamental knowledge on the disinfection of pathogens in water using ClO2 and the creation of semi germ-free shrimp, which has significantly decreased microbiome in the stomach., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Aims: We aimed to identify mechanisms underlying the tolerance of Proteus mirabilis-a common cause of catheter associated urinary tract infection-to the clinically used biocides chlorhexidine (CHD) and octenidine (OCT)., Methods and Results: We adapted three clinical isolates to grow at concentrations of 512 µg ml-1 CHD and 128 µg ml-1 OCT. Genetic characterization and complementation studies revealed mutations inactivating the smvR repressor and increasing smvA efflux expression were associated with adaptation to both biocides. Mutations in mipA (encoding the MltA interacting protein) were less prevalent than smvR mutations and only identified in CHD adapted populations. Mutations in the rppA response regulator were exclusive to one adapted isolate and were linked with reduced polymyxin B susceptibility and a predicted gain of function after biocide adaptation. Biocide adaptation had no impact on crystalline biofilm formation., Conclusions: SmvR inactivation is a key mechanism in both CHD and OCT tolerance. MipA inactivation alone confers moderate protection against CHD, and rppA showed no direct role in either CHD or OCT susceptibility., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Aims: Airborne transmission of diseases presents a serious threat to human health, so effective air disinfection technology to eliminate microorganisms in indoor air is very important. This study evaluated the effectiveness of a non-thermal plasma (NTP) air disinfector in both laboratory experiments and real environments., Methods and Results: An experimental chamber was artificially polluted with a bioaerosol containing bacteria or viruses. Additionally, classroom environments with and without people present were used in field tests. Airborne microbial and particle concentrations were quantified. A 3.0 log10 reduction in the initial load was achieved when a virus-containing aerosol was disinfected for 60 min and a bacteria-containing aerosol was disinfected for 90 min. In the field test, when no people were present in the room, NTP disinfection decreased the airborne microbial and particle concentrations (P < 0.05). When people were present in the room, their constant activity continuously contaminated the indoor air, but all airborne indicators decreased (P < 0.05) except for planktonic bacteria (P = 0.094)., Conclusions: NTP effectively inactivated microorganisms and particles in indoor air., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Aims: This study investigated the antimicrobial efficacy of ultrasound technology (US) in combination with two different disinfectants (Disinfectant A and Disinfectant B), containing peracetic acid (PAA) and quaternary ammonium compounds (QACs), respectively, against two sporigenic pathogens, Aspergillus brasiliensis and Bacillus subtilis., Methods and Results: The microbicidal activity of the coupled treatment was compared with the use of the disinfectants alone, and the efficacy of the disinfection strategies was evaluated by the log reduction of the population of the microorganism inoculated onto stainless-steel surface. The combination treatment resulted in a log reduction of 5.40 and 3.88 (Disinfectant A + US) against A. brasiliensis and B. subtilis, at 850 and 500 ppm PAA, compared to 265 and 122 (Disinfectant A only). For Disinfectant B, in combination with US, showed a logarithmic reduction of 5.04 and 4.79 against A. brasiliensis and B. subtilis at 078% v v-1 and 392% v v-1 QACs, respectively, vs. 1.58 and 1.64 (Disinfectant B only). Moreover, no colonies or not statistically significant growth was observed within the US bath containing the disinfectant., Conclusions: The antimicrobial efficacy of the two disinfectants was greatly enhanced when used in combination with US, and this also makes it possible to avoid the overuse of chemicals for disinfection., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Aims: Nucleic acids, particularly antibiotic resistance genes, are commonly found on surfaces within healthcare environments, with levels not reducing following cleaning. Within the UK, there are no regulations for testing disinfectants against nucleic acids., Methods and Results: A series of commonplace in vitro methods were used to determine disinfectant-induced physical and functional damage to various nucleic acids; RNA (10 μg), genomic DNA (2 μg), and plasmids (1 μg). Using these methods, the optimal residence time (10 minutes) and working concentration (10%) were determined for a new disinfectant. Furthermore, comparison of disinfectants with different active ingredients including lactic acid (LA), sodium hydroxide (NaOH), chloroxylenol (PCMX), and quaternary ammonium compounds (QACs), were compared to controls. All disinfectants showed greater degradation by gel electrophoresis of genomic DNA and RNA than of purified plasmids. Functional analysis using quantitative polymerase chain reaction (qPCR) and polymerase chain reaction (PCR) demonstrated that no disinfectant tested (apart from control) could damage DNA to the level where PCR amplification was not possible, and only the NaOH reagent could achieve this for RNA., Conclusions: The set of methods described herein provides a platform for future standardization and potential regulation regarding monitoring cleaning solutions for their activity against nucleic acids., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

Aims: Disinfectants such as benzalkonium chloride (BC), extensively used in animal farms and food-processing industries, contribute to the development of adaptive and cross-resistance in foodborne pathogens, posing a serious threat to food safety and human health. The purpose of this study is to explore whether continuous exposure of Salmonella enterica serovar 1,4,[5],12:i:- (S. 1,4,[5],12:i:-) to sublethal concentrations of BC could result in acquired resistance to this agent and other environmental stresses (e.g. antibiotics, heat, and acid)., Methods and Results: BC tolerance increased in all tested strains after exposure to gradually increasing concentrations of BC, with increases in minimum inhibitory concentrations between two and sixfold. The survival rate of BC-adapted strains was significantly (P < 0.05) higher than that of their wild-type (non-adapted) counterparts in lethal concentrations of BC. In addition, significant reductions (P < 0.05) in zeta potential were observed in BC-adapted strains compared to wild-type ones, indicating that a reduction in cell surface charge was a cause of adaptative resistance. More importantly, two BC-adapted strains exhibited increased antibiotic resistance to levofloxacin, ceftazidime, and tigecycline, while gene mutations (gyrA, parC) and antibiotic efflux-related genes (acrB, mdsA, mdsB) were detected by genomic sequencing analysis. Moreover, the tolerance of BC-adapted strains to heat (50, 55, and 60°C) and acid (pH 2.0, 2.5) was strain-dependent and condition-dependent., Conclusions: Repeated exposure to sublethal concentrations of BC could result in the emergence of BC- and antibiotic-resistant S. 1,4,[5],12:i:- strains., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

Chlorine dioxide (ClO2) is a disinfectant gas with strong antifungal, antibacterial, and antiviral activities. Applied on hard, non-porous surfaces as an aqueous solution or gas, the ClO2 exerts antimicrobial activity through its interaction and destabilization of cell membrane proteins, as well as through DNA/RNA oxidation, triggering cell death. As for viruses, the ClO2 promotes protein denaturalization mechanisms, preventing the union between the human cells and the viral envelope. Currently, ClO2 has been pointed out as a potential anti-SARS-CoV-2 clinical treatment for use in humans with the ability to oxidize the cysteine residues in the spike protein of SARS-CoV-2, inhibiting the subsequent binding with the Angiotensin-converting enzyme type 2 receptor, located in the alveolar cells. Orally administered ClO2 reaches the gut tract and exacerbates the symptoms of COVID-19, generating a dysbiosis with gut inflammation and diarrhea as side effects, and once absorbed, produces toxic effects including methemoglobinemia and hemoglobinuria, which can trigger respiratory diseases. These effects are dose-dependent and may not be entirely consistent between individuals since the gut microbiota composition is highly heterogeneous. However, to support the use of ClO2 as an anti-SARS-CoV-2 agent, further studies focused on its effectiveness and safety both in healthy and immunocompromised individuals, are needed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

Aims: The COVID-19 pandemic has heightened awareness of the need for novel surface disinfectants and hand-hygiene modalities. Ozone gas is an effective surface disinfectant, but toxicity limits its use in human applications. Ozonated water is a safer means to use ozone for disinfection, especially for human antisepsis. However, there are little data available regarding the effectiveness of ozonated water in eliminating severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)., Methods and Results: This study utilizes a novel hand hygiene device that produces a stable ozone concentration of 0.5 +/-0.1 ppm in water and applies it using a proprietary spray that controls droplet size, velocity, and direction. The Device was used to apply ozonated water to a known quantity of SARS-CoV-2 Delta Variant viral particles on a non-porous surface (glass) for seven seconds. Post-exposure growth was compared to the unexposed matched control utilizing the Spearman-Karber method. Compared to control, ozonated water decreased SARS-CoV-2 viral growth by a mean log10 reduction of 4.33, or >99.99% reduction., Conclusions: These results suggest that the ozonated water, when applied by a spray hand hygiene device, is highly effective at surface disinfection of SARS-CoV-2., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

Aims: The aim of this study was to determine the biofilm-forming ability of a strain belonging to the Pseudomonas fluorescens group isolated from the dairy environment under food-relevant conditions. Moreover, the effects of commercial sanitizers against preformed biofilms were assessed both in terms of viability and structure., Methods and Results: The biofilms were formed on polystyrene, stainless steel (SS), and polytetrafluoroethylene (PTFE) in a wide range of temperatures (4-25°C) and were subjected to the action of 10 different sanitizers. The strain under study showed to be a strong biofilm-former regardless of temperature, particularly on polystyrene. The biofilms were mostly sensitive to chlorine and peracetic acid-based sanitizers. For some sanitizers (e.g. amphoteric), a relationship was observed between the material and the tolerance, while the temperature was not statistically significant. The formation of long-term biofilms on SS was also structurally affected by the temperature, showing microcolonies more irregular in shape and with lower cellularity at 4°C compared to 15°C, where the biofilm was more compact and with a high presence of EPS., Conclusions: The strain belonging to the P. fluorescens group was shown to quickly adhere and form mature biofilm at temperatures and on materials relevant to the food sector; however, biofilms formed under different conditions were differently tolerant to disinfectants., Significance and Impact of the Study: Findings from this study could provide a basis for developing targeted sanitation protocols in food plants., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

Aim: To investigate the effect of adapting Klebsiella pneumoniae clinical isolates harboring ESBL genes to cetrimide (CT) in terms of subsequent cross-resistance to other biocides and antibiotics, and to investigate changes to virulence markers, such as biofilm formation and efflux activity. The changes between phenotypic extended spectrum β-lactamases (pESBL) expressing isolates and non-phenotypic ESBL (npESBL) isolates was compared., Methods and Results: Kl. pneumoniae isolates (14 pESBL and 17 npESBL) were adapted to increasing concentrations of CT, until 4 × MIC was reached. The MIC of the adapted isolates was tested against chloroxylenol (CX) and chlorhexidine. Disk diffusion techniques were used to determine the susceptibility of the isolates to different antibiotics. Biofilm formation was assessed for the isolates using the crystal violet method and efflux pump activity was studied using the ethidium bromide assay. After CT adaptation, 100% of npESBL isolates and 85.7% of pESBL isolates showed increase in CT MIC after CT adaptation. While 41.2% of npESBL and 57.1% of the pESBL isolates showed a cross-resistance with chlorhexidine. CT adaptation resulted in a significant decrease in the susceptibility of npESBL isolates to aztreonam and cefotaxime compared to pESBL isolates, which could be linked to the increase in efflux activity of npESBL compared to pESBL. Biofilm formation was significantly increased after CT adaptation regardless of the type of isolate., Conclusions: The extensive use of biocides in the environment can induce cross-resistance to other biocides and antibiotics, and can increase the ability of bacteria to form biofilms. The response of bacteria to biocide adaptation differs between pESBL and npESBL isolates, although the effect is strain specific., (© The Author(s) 2022. Published by Oxford University Press on behalf of Applied Microbiology International.)

Aims: The purpose of this study was to evaluate decontamination efficacy, within three soil types, against Yersinia pestis, Burkholderia pseudomallei, and the Venezuelan Equine Encephalitis virus (VEEV)., Methods and Results: One of three liquid disinfectants (dilute bleach, Virkon-S or Klozur One) was added to three soil types (sand, loam, or clay) and allowed contact for four pre-spike durations: 0, 15, 30 and 60 min. Y. pestis, B. pseudomallei, or VEEV was then spiked into the soil (10 microliters or approx. 1 × 10 7  CFU or PFU into 1 g soil) and decontamination efficacy assessed at post-spike contact times of 10 or 60 min at ambient environmental conditions. Across all soil types, sandy soil resulted in the least quenching to all three disinfectants tested as shown by sustained decontamination efficacy across all pre-spike and post-spike timepoints. Clay and loam soil types exhibited quenching effects on the hypochlorite and peroxygen based disinfectants (dilute bleach and Virkon S) and in general resulted in decreased efficacy with increased pre-spike contact time. The sodium persulfate (Klozur One) performance was the most consistent across all soil types and pre-spike contact times, resulting in greater efficacy with increased post-spike time., Conclusions: Liquid disinfectants can provide high levels of decontamination in soil for both viral and non-spore-forming bacterial select agents. Hypochlorite and peroxygen based disinfectants used in soils containing higher organic content (loam or clay) may require extended contact times or re-application of liquid disinfectant, in as little as 15 min of application, to achieve a 6-log reduction., Significance and Impact of the Study: These results provide information for the performance of three disinfectants in soil against non-spore-forming select agents. These data may aid response decision makers following a biological contamination incident by informing the selection of disinfectant as well as the re-application time to achieve effective site remediation., (© 2022 Society for Applied Microbiology. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Aims: To determine the antimicrobial potency of a surface-anchored quaternary ammonium salt (SAQAS)-based biocide during in vitro wet and dry fomite assays and to determine the mechanism of killing bacteria on the surface., Methods and Results: Wet and dry fomite assays were established in vitro for a commercially available biocide (SAQAS-A) applied to glass and low-density polyethylene (LDPE) surfaces. Both wet and dry fomite tests showed the active killing of Gram-positive and Gram-negative bacteria but not endospores. Assays measuring membrane permeability (ATP and DNA release), bacterial membrane potential and bacterial ROS production were correlated with the time-to-kill profiles to show SAQAS-A activity in suspension and applied to a surface., Conclusions: SAQAS-A is an effective biocide against model strains of vegetative bacteria. The killing mechanism for SAQAS-A observed minimal membrane depolarization, a surge in ROS production and assessment of membrane permeability supported the puncture of cells in both suspension and surface attachment, leading to cell death., Significance and Impact of the Study: SAQAS represents effective surface biocides against single challenges with bacteria through a mechanical killing ability that supports real-world application if their durability can be demonstrated to maintain residual activity., (© 2022 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

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., (© 2022 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

Aims: To investigate the priming effects of sub-inhibitory concentrations of biocides on antibiotic resistance in bacteria., Methods and Results: Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus were exposed to sub-inhibitory concentrations of biocides via a gradient plate method. Minimum inhibitory concentration (MIC) and antibiotic susceptibility were determined, and efflux pump inhibitors (thioridazine and chlorpromazine) were used to investigate antibiotic resistance mechanism(s). Escherichia coli displayed a twofold increase in MIC (32-64 mg l -1 ) to H 2 O 2 which was stable after 15 passages, but lost after 6 weeks, and P. aeruginosa displayed a twofold increase in MIC (64-128 mg l -1 ) to BZK which was also stable for 15 passages. There were no other tolerances observed to biocides in E. coli, P. aeruginosa or S. aureus; however, stable cross-resistance to antibiotics was observed in the absence of a stable increased tolerance to biocides. Sixfold increases in MIC to cephalothin and fourfold to ceftriaxone and ampicillin were observed in hydrogen peroxide primed E. coli. Chlorhexidine primed S. aureus showed a fourfold increase in MIC to oxacillin, and glutaraldehyde-primed P. aeruginosa showed fourfold (sulphatriad) and eightfold (ciprofloxacin) increases in MIC. Thioridazine increased the susceptibility of E. coli to cephalothin and cefoxitin by fourfold and twofold, respectively, and both thioridazine and chlorpromazine increased the susceptibility S. aureus to oxacillin by eightfold and fourfold, respectively., Conclusions: These findings demonstrate that sub-inhibitory concentrations of biocides can prime bacteria to become resistant to antibiotics even in the absence of stable biocide tolerance and suggests activation of efflux mechanisms may be a contributory factor., Significance and Impact of the Study: This study demonstrates the effects of low-level exposure of biocides (priming) on antibiotic resistance even in the absence of obvious increased biocidal tolerance., (© 2022 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

Aims: To determine the efficacy of a panel of nine EPA-registered disinfectants against two human norovirus (HuNoV) surrogates (feline calicivirus [FCV] and Tulane virus [TuV]) and Clostridioides difficile endospores., Methods and Results: Nine EPA-registered products, five of which contained H 2 O 2 as active ingredient, were tested against infectious FCV, TuV and C. difficile endospores using two ASTM methods, a suspension and carrier test. Efficacy claims against FCV were confirmed for 8 of 9 products. The most efficacious product containing H 2 O 2 as ingredient achieved a >5.1 log reduction of FCV and >3.1 log reduction of TuV after 5 min, and >6.0 log reduction of C. difficile endospores after 10 min. Of the five products containing H 2 O 2 , no strong correlation (R 2  = 0.25, p = 0.03) was observed between disinfection efficacy and H 2 O 2 concentration. Addition of 0.025% ferrous sulphate to 1% H 2 O 2 solution improved efficacy against FCV, TuV and C. difficile., Conclusion: Disinfectants containing H 2 O 2 are the most efficacious disinfection products against FCV, TuV and C. difficile endospores. Product formulation, rather than the concentration of H 2 O 2 in a product, impacts the efficacy of a disinfection product., Significance and Impact of Study: H 2 O 2 -based disinfectants are efficacious against surrogate viruses for HuNoV and C. difficile endospores., (© 2022 Society for Applied Microbiology.)

Aims: Spread of carbapenem-resistant Enterobacterales have become a global problem. We characterized extended-spectrum β-lactamase (ESBL)-producing Enterobacterales from urinary tract infections cases from Allied Hospital Faisalabad, Pakistan., Methods and Results: Eleven (22%, 11/50) ESBL-producing Enterobacterales (Escherichia coli; n = 10 and Enterobacter hormaechei; n = 1) were recovered and processed through VITEK-2, PCR, rep-PCR followed by whole-genome sequencing (WGS) of ESBL-producing Ent. hormaechei and carbapenem-resistant E. coli isolates. Plasmid transferability of bla NDM-1 -producers was assayed by conjugation experiments. All ESBL strains carried the bla CTX-M-15 gene. Of these bla CTX-M-15 producing E. coli, four also carried bla NDM-1 located on transferable plasmids. All E. coli strains belonged to ST448 and displayed similar genetic features including genes for antimicrobial resistance, heavy metal, biocides and virulence. Genomic features of a multidrug-resistant (MDR) Ent. hormaechei were also reported for the first time in Pakistan., Conclusion: Our findings indicate that bla NDM-1 producing E. coli ST448 is a multidrug, heavy metals and biocides-resistant strain. Therefore, the screening of these isolates may be effective in limiting the MDR bacteria spread in hospitalized patients and within the community., Significance and Impact of This Study: Spread of multi-drug-resistant ESBL-producing bacteria in the clinical settings of Pakistan is a serious challenge and further limiting treatment options in the country. WGS could be used as a tool in the nationwide antibiotic surveillance programme to explore insights of spread and outbreak., (© 2022 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

Aim: To evaluate the anti-noroviral efficacy of PURELL® surface sanitizer and disinfectant spray (PSS, an alcohol-based formulation) using human norovirus GII.4 Sydney [hNoV, by RT-qPCR and human intestinal enteroid (HIE) infectivity assay] and its cultivable surrogate, Tulane virus (TuV, infectivity assay), compared to sodium hypochlorite (NaOCl) solutions., Methods and Results: PSS efficacy was evaluated in suspension and on surfaces [stainless steel (SS)] using ASTM methods. Results were expressed as log 10 reduction (LR) of genome equivalent copy number (GEC, for hNoV, assayed by RT-qPCR) and plaque forming units (PFU, for TuV, per infectivity assay). In suspension, PSS achieved a 2.9 ± 0.04 LR hNoV GEC irrespective of contact time (30 or 60 s) and soil load (2.5% or 5%). Under all treatment conditions, infectious TuV could not be recovered following exposure to PSS, corresponding to the assay limit of detection (3.1-5.2 log 10 PFU). Infectious hNoV could not be detected in the HIE model after exposure to PSS. On SS and 2.5% soil, PSS produced a 3.1 ± 0.1 LR hNoV GEC, comparable to 500 ppm NaOCl for 60 s. With 5.0% soil, PSS produced a 2.5 ± 0.2 LR hNoV GEC, which was similar to 1000-5000 ppm NaOCl for 60 s., Conclusions: PSS showed high anti-hNoV efficacy by RT-qPCR and in in vitro (TuV) and ex vivo (HIE) infectivity assays and performed similar to 1000-5000 ppm NaOCl for a 60-s contact time on SS with added soil., Significance and Impact of Study: hNoV remains a significant cause of morbidity globally, partly due to its resistance to numerous surface disinfectants. RT-qPCR results from this study indicate PSS efficacy against hNoV is comparable to NaOCl efficacy. Infectivity assays leveraging TuV and the HIE model for hNoV support and confirm loss of virus infectivity. Collectively, these results indicate the product's ability to inactivate hNoV quickly, which could be beneficial in settings having elevated risk for hNoV transmission., (© 2022 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

Aim: During several local COVID-19 outbreaks in China in 2020, SARS-CoV-2 or its RNA was isolated or detected from frozen food or packages, revealing the lack of effective disinfection measures in the frozen food chain and risk of transmission. We explored the possibility that disinfectant plus antifreeze could be delivered as thermal fog to realize effective disinfection at subzero temperatures., Methods and Results: We selected two disinfectant-antifreeze combinations, didecyl dimethyl ammonium bromide (DDAB) - propylene glycol (PPG) and peracetic acid (PAA) - triethylene glycol (TEG), and each combination is used with a custom-optimized thermal fogging machine. The two fogs were tested in -20°C freezer warehouses for their disinfection efficacy against a coronavirus porcine epidemic diarrhoea virus (PEDV) field strain, a swine influenza virus (SIV) field strain, and three indicator bacteria, Escherichia coli, Staphylococcus aureus and Bacillus subtilis endospores. At -20°C, the DDAB-PPG or PAA-TEG thermal fogs settle within 3.5 to 4.5 h and effectively inactivated PEDV with median tissue culture infective dose of 10 -3.5 0.1 ml -1 and SIV-H1N1 with hemagglutination titre of 2 6  ml -1 within 15-60 min. DDAB-PPG could inactivate S. aureus and E. coli vegetative cells (10 6  cfu ml -1 ) within 15-60 min but not effective on B. subtilis spores, while PAA-TEG could disinfect B. subtilis spores more effectively than for S. aureus and E. coli., Conclusions: We showed that a practical subzero temperature disinfection technology was effective in killing enveloped viruses and vegetative bacteria or bacterial spores. DDAB-PPG or PAA-TEG thermal fogging may be a practical technology for cold-chain disinfection., Significance and Impact of the Study: This subzero temperature disinfection technology could help to meet the urgent public health need of environmental disinfection in frozen food logistics against pandemic and other potential pathogens and to enhance national and international biosecurity., (© 2021 The Society for Applied Microbiology.)

Aims: This work aimed to determine the occurrence, virulence, antibiogram, carbapenem resistance genes and susceptibility to disinfectants of Pseudomonas aeruginosa isolated from animals, environment and workers in intensive farms., Methods and Results: A total of 610 samples from intensive beef cattle and sheep farms in Kafr El Sheikh Governorate, Egypt were screened for the presence of P. aeruginosa using bacteriological assays. The isolates were characterized by PCR and tested for susceptibility to antibiotics using disk diffusion method and disinfectants by quantitative suspension test. In all, 60 P. aeruginosa isolates were recovered in this study and all isolates harboured at least one of the virulence genes tested. Human P. aeruginosa isolates were highly resistant to cephalosporins, fluroquinolones, aminoglycosides, carbapenems and penicillins+β-lactamase inhibitors than non-human isolates. Colistin resistance was higher in non-human than human P. aeruginosa isolates, whereas low resistance to aztreonam was observed in non-human and human isolates. Carbapenem-resistant P. aeruginosa (CRPA) strains were recovered from workers (56.5%), sheep (8.3%) and cattle (8.3%). All CRPA harboured at least one of the carbapenem resistance genes tested and most of them showed multidrug resistance (MDR) or extensive drug resistance (XDR) phenotypes. Glutaraldehyde 1% and hydrogen peroxide 3% eliminated P. aeruginosa completely in the absence and presence of organic matter within short contact time compared with other disinfectants., Conclusions: This study reported the occurrence of CRPA in animals and workers in intensive farms. Glutaraldehyde and hydrogen peroxide were the most effective disinfectants against P. aeruginosa., Significance and Impact of the Study: The occurrence of CRPA in intensive livestock farms is a serious challenge that threatens animal and human health and increases the risk of P. aeruginosa infection in the community. Therefore, it is vital to control the spread of CRPA by banning or restricting the use of antibiotics and applying proper cleaning and disinfection protocols in livestock farms., (© 2021 The Society for Applied Microbiology.)

Aim: To evaluate generally recognized as safe (GRAS) liquid wash formulations against hepatitis A virus-contaminated strawberries and blackberries in order to identify a formulation suitable for reducing virus contamination., Methods and Results: Formulations included the surfactant sodium dodecyl sulfate (SDS; 0·5% w/v) by itself, and in combination, with lactic acid (LA; 0·5% v/v), levulinic acid (LVA; 0·5% v/v) and 3 ppm aqueous chlorine dioxide (ClO 2 ). After contamination and drying overnight, the average total extracted contamination for both untreated strawberries and blackberries was 4·4 log PFU. Three successive distilled H 2 O only treatments reduced total contamination by up to 1·8 log PFU for both strawberries and blackberries, while wash formulations showed significant (P ≤ 0·05) total reductions ranging from 2·1 to 2·9 log PFU., Conclusions: Considering results for both berry types, the combination of ClO 2 and SDS was the most effective. Overall results indicate that adding surfactant and several types of sanitizers to berry wash can enhance HAV reduction on berries., Significance and Impact of the Study: This study indicates that industry could enhance the virologic safety of ready-to-eat berries by the combined use of surfactant and sanitizer., (© Published 2021. This article is a U.S. Government work and is in the public domain in the USA.)

Aims: The presence of foodborne pathogens in preharvest agricultural water has been identified as a potential contamination source in outbreak investigations, driving markets and auditing bodies to begin requiring water treatment for high-risk produce. Therefore, it is essential that we identify water treatment methods which are effective as well as practical in their application on farm., Methods and Results: In this work, we evaluated two sanitizers which are most prominent in preharvest agricultural water treatment (calcium hypochlorite (free chlorine: 3-5 ppm) and peracetic acid (PAA: 5 ppm)), an EPA registered antimicrobial device (ultraviolet light (UV)), in addition to a combination approach (chlorine + UV, PAA + UV). Treatments were evaluated for their ability to inactivate total coliforms and generic Escherichia coli and consistency in treatment efficacy over 1 h of operation. Physicochemical variables were measured along with microbial populations at 0, 5, 15, 30, 45 and 60 min of operation. Escherichia coli and coliform counts showed a significant (P < 0·05) reduction after treatment, with combination and singular treatments equally effective at inactivating E. coli and coliforms. A significant increase (P < 0·05) in oxidation-reduction potential was seen during water treatment (Chlorine; UV + Chlorine), and a significant reduction (P < 0·05) in pH was seen after PAA and PAA + UV treatments (60 min)., Conclusion: Overall, the results indicate that all treatments evaluated are equally efficacious for inactivating E. coli and coliforms present in surface agricultural water., Significance and Impact of the Study: This information when paired with challenge studies targeting foodborne pathogens of interest can be used to support grower decisions when selecting and validating a preharvest agricultural water treatment programme., (© 2021 The Society for Applied Microbiology.)

Aims: An effective decontamination procedure of personnel wearing personal protective equipment is required by CBRN responders and healthcare workers when dealing with biological warfare agents or natural outbreaks caused by highly contagious pathogens. This study aimed to identify critical factors affecting the efficacy of peracetic acid (PAA)-based disinfectants and products containing either hydrogen peroxide or sodium hypochlorite under the same conditions., Methods and Results: The influence of concentration, application (contact) time, erroneous human behaviour, interfering substance, technical assets and weather conditions on disinfection efficacy against Bacillus subtilis spores were assessed in 14 experimental groups. Residual contamination of protective suits was measured to provide responders with readily understandable information (up to 100 colony forming units classified a suit as disinfected). Weather conditions, short application time and erroneous human behaviour substantially affected the effectiveness of PAAs (P < 0·05). Non-PAA-based disinfectants (either liquid or foam) did not reach comparable efficacy (P < 0·001)., Conclusions: Peracetic acid was effective at a concentration of 6400-8200 ppm and an application time of 4 min., Significance and Impact of the Study: The study provides operationally relevant data for the use of PAA-based disinfectants in preparedness planning and management of biological incidents and natural outbreaks., (© 2021 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

Aims: This study aimed to investigate the antibacterial ability and action mechanism of dithiocyano-methane against Aeromonas hydrophila, so as to provide a reference for its application in farm disinfection., Methods and Results: After exposing the bacteria to dithiocyano-methane, the minimum inhibitory concentration (MIC), minimum bactericide concentration (MBC), activities of alkaline phosphatase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase and electric conductivity in bacterial suspensions were determined, transmission electron microscope images on cellular structure and SDS-PAGE profile of bacterial proteins were analysed and the expression of genes related to the above experimental observations was confirmed by real-time quantitative PCR. The MIC and MBC of dithiocyano-methane against three tested strains was 1·46 and 2·93 mg l -1 respectively. The results showed that dithiocyano-methane significantly damaged bacterial cell structure, inhibited the biosynthesis of bacterial proteins and changed the integrity and permeability of bacterial cell wall and cell membrane., Conclusions: Dithiocyano-methane showed remarkable antibacterial ability against three tested strains, indicating it is a potential effective bactericidal agent for preventing animal diseases resulted from Aer. hydrophila., Significance and Impact of the Study: To our best knowledge, this is the first report to examine the antibacterial ability and action mechanism of dithiocyano-methane against bacteria. The results demonstrate the great potential of dithiocyano-methane as a disinfectant against Aer. hydrophila in settings such as aquaculture ponds and livestock farms., (© 2020 The Society for Applied Microbiology.)

Aims: The efficacy of a novel photochemical method for generating chlorine dioxide (photoClO 2 ) was evaluated against human noroviruses (HuNoV) surrogate, bacteriophage MS2, and Clostridium difficile endospores., Methods and Results: Chlorine dioxide was generated by mixing 1% sodium chlorite with 10 parts-per-million (ppm) Eosin Y and irradiating with a photo-activator-excitable light. PhotoClO 2 efficacy was assessed against bacteriophage MS2 and C. difficile endospores in suspension, on hard surfaces and greenhouse conditions under soiled and unsoiled conditions. The estimated effective photoClO 2 produced and consumed was 20·39 ± 0·16 ppm at a rate of 8·16 ppm per min in a 1% sodium chlorite solution. In suspension, MS2 phage was reduced by 3·35 and >5·10 log 10 PFU per ml in 120 and 90 min, with and without soil, respectively. At the same time, when dried on stainless steel surface, MS2 phage was reduced by >4·53 log 10 PFU per carrier in 30 min under both conditions. On the other hand, C. difficile endospores in suspension were reduced by 2·26 and 3·65 log 10 CFU per ml in 120 min with and without soiling, respectively. However, on stainless steel surface, maximal reductions of the C. difficile endospores were 0·8 and 1·5 log 10 CFU per carrier with and without soiling, respectively, and a maximal reduction of 2·97 log 10 CFU per carrier under greenhouse conditions at 24 h., Conclusions: Overall, photoClO 2 showed promise as a technology to control HuNoV contamination on environmental surfaces but requires further optimization and testing against C. difficile endospores., Significance and Impact of the Study: Results from this investigation will serve as a model for how to generate and quantify photoClO 2 and how to appropriately evaluate this new class of disinfectants against environmentally resilient pathogens: viruses and bacterial endospores., (© 2020 The Society for Applied Microbiology.)

Aims: The aim of this study was to test the efficacy of new and currently used biocides in the mushroom industry for inactivating Listeria monocytogenes biofilm., Methods and Results: A laboratory-scale study was initially carried out to test the efficacy of eleven biocidal products against a cocktail of five L. monocytogenes strains that were grown to 3-day biofilms on stainless steel coupons. Biocidal efficacy was then tested under clean and dirty conditions based on the EN 13697:2015 method. The results for the biocides tested ranged between 1·7-log and 6-log reduction of biofilm, with only the efficacy of the sodium hypochlorite-based biocide being significantly reduced in dirty conditions. A pilot-scale trial was then carried out on a subset of biocides against L. monocytogenes on concrete floors in a mushroom growing room and it was found that biocide efficacy in laboratory-scale did not translate well in pilot-scale., Conclusions: Biocides that are used in the mushroom industry and potential alternative biocides were determined to be effective against L. monocytogenes biofilm in both laboratory-scale and pilot-scale experiments., Significance and Impact of the Study: This study has direct impact for the industry as it provides information on the efficacy of currently used biocides and other biocidal products against L. monocytogenes, an added benefit to their primary use., (© 2020 The Society for Applied Microbiology.)

Aims: This study aimed to determine the toxin genotypes, virulence determinants and antibiogram of Clostridium perfringens isolated from poultry, animals and humans. Biofilm formation and the efficacy of disinfectants on C. perfringens biofilms were studied., Methods and Results: Thirty C. perfringens isolates (20 clinical and 10 from chicken carcasses) were genotyped by PCR and all isolates were genotype A (cpa+). The overall prevalence of cpe, cpb2, netB and tpeL virulence genes was 6·7, 56·7, 56·7 and 36·7% respectively. Twenty-one isolates (70%) were multidrug-resistant, 8 (26·7%) were extensive drug-resistant and one isolate (3·3%) was pan drug-resistant. The average multiple antibiotic resistance index was 0·7. Biofilms were produced by 63·3% of C. perfringens isolates and categorized as weak (36·7%), moderate (16·7%) and strong (10%). Sodium hypochlorite caused significant reduction in C. perfringens biofilms (P < 0·0001)., Conclusions: All C. perfringens strains in this study were type A, resistant to multiple antibiotics and most of them were biofilm producers. Sodium hypochlorite showed higher efficacy in reducing C. perfringens biofilms., Significance and Impact of the Study: This study reported the efficacy of disinfectants in reducing C. perfringens biofilms of economic and public health concern and recommends application on surfaces in farms, food processing plants and slaughterhouses., (© 2020 The Society for Applied Microbiology.)

Aims: The study aimed to investigate the inactivation efficacy and mechanisms of plasma activated water (PAW) on selected bacteria in planktonic state., Methods and Results: Plasma activated water was generated using an atmospheric cold plasma jet at 15, 22 and 30 kV for 5 min. Escherichia coli, Listeria innocua, Staphylococcus aureus, Aeromonas hydrophila, Pseudomonas fluorescens and Shewanella putrefaciens were selected as the representative bacterial species. Each bacterial suspension was inoculated into PAW immediately after generation, and the viable counts at different exposure times of 0·5, 1, 3, 5 and 24 h during 4°C storage were measured to determine the inactivation efficacy. Scanning electron microscopy images of the bacteria were conducted to examine the structural changes. Physicochemical properties of PAW, including pH, conductivity, oxidation reduction potential (ORP), and reactive species of H 2 O 2 , NO 2 - and NO 3 - were measured. The results demonstrated that inactivation efficacy was in positive correlation with voltage and exposure time. Gram-negative bacteria were more susceptible to PAW than Gram-positive bacteria. Morphology damage was observed for all the bacterial species. PAW was significantly acidified, conductivity and ORP were significantly increased, and reactive species were detectable after 48 h., Conclusions: This study offered a better understanding of the inactivation mechanisms of PAW, and the inactivation efficacy can be affected by voltage, exposure time and bacterial species., Significance and Impact of the Study: This study demonstrated the potential usage of PAW as an alternative disinfectant., (© 2020 The Society for Applied Microbiology.)

Aims: Develop an effective laboratory method to consistently recover viral loads from porous concrete coupons sufficient for disinfectant efficacy testing. Investigate the role of concrete matrix pH on the recovery of foot-and-mouth disease virus (FMDV) and African Swine Fever virus (ASFV) from porous concrete. Compare parameters off FMDV and ASFV inactivation on porous and nonporous surfaces in quantitative carrier tests of a liquid chemical disinfectant., Methods and Results: Concrete test coupons were fabricated from commercial and industrial sources and carbonated by exposure to 5% CO 2 in a humidified incubator, lowering the matrix pH. Neither dried FMDV nor ASFV were recovered from high-pH concrete control coupons. Recovery of infectious virus from lower pH carbonated concrete was similar to stainless steel coupon controls. Exposure to the liquid disinfectant Virkon™ S inactivated FMDV and ASFV on porous concrete., Conclusions: Concrete matrix pH had a greater impact than surface porosity on the ability to recover viable virus from unsealed concrete., Significance and Impact of the Study: Concrete is commonly found in environments where virus decontamination is required. This study demonstrates a reproducible method to recover sufficient viral loads from porous concrete coupons to facilitate quantitative carrier testing. This method provides a basis for evidence-based validation testing of chemical disinfectants to inactivate pH-sensitive viruses on unsealed concrete., (© 2020 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

Aim: This research aimed to monitor pharmaceutical water system by sampling water from all treatment stages, identify bacterial isolates from each phase and determine the most suitable methods to control them., Methods and Results: Water samples were collected and examined from pharmaceutical water system in a pharmaceutical factory in Giza, Egypt during 12 months, once per month (from December 2017 to November 2018) from 15 points covering all stages of the treatment process starting from wells, pre-treatment points; treatment points ending with purified points which are the main source of water used in all pharmaceutical process. In all, 216 water samples were collected and examined, 156 isolates were selected according to morphological characteristics. VITEK system 2 (BioMérieux) was used for identification of all isolates resulting in 24 different identified bacteria. Antibiotic assay test using disc diffusion methods were carried out using seven antibiotics from different groups. Several disinfectants were also examined for efficacy against the isolates to control micro-organisms in water treatment stage and manufacturing area. The effect of different preservatives (parabens, acids and alcohols) in various pharmaceutical formulas was also tested on bacterial isolates, 63% of formulas were effective against all bacterial isolates., Conclusion: Ciprofloxacin was the most effective antibiotic, mixture of 0·45% peracetic acid plus 2·2% of hydrogen peroxide (Minncare 1%) was maximally effective disinfectant, and Cronobacter sakazakii was the most resistant micro-organism against 22·7% of tested preservatives., Significance and Impact of the Study: Controlling pharmaceutical manufacturing operation from pathogenic bacteria that affect the quality of drugs., (© 2020 The Society for Applied Microbiology.)

Aim: Following previous research on improving the cleaning of crates used to transport broiler chickens from the farm to the abattoir, a demonstration project was undertaken to investigate improvements in crate washing on a commercial scale., Methods and Results: The soak tank of a conventional crate washing system was replaced with a high-performance washer fitted with high-volume, high-pressure nozzles. The wash water could be heated, and a greatly improved filtration system ensured that the nozzles did not lose performance or become blocked. Visual cleanliness scores and microbial counts were determined for naturally contaminated crates which had been randomly assigned to different cleaning protocols., Conclusions: When a combination of mechanical energy, heat and chemicals (i.e. detergent and disinfectant) was used, the results showed significant improvements to crate cleaning. Reductions of up to 3·6 and 3·8 log 10 CFU per crate base were achieved for Campylobacter and Enterobacteriaceae, respectively, along with a marked improvement in visual cleanliness., Significance and Impact of the Study: Broiler transport crates may become heavily contaminated with faeces and this may contribute to the spread of disease between farms. The results of this trial may be of use in reducing the spread of zoonotic pathogens in the poultry meat supply chain., (© 2020 The Society for Applied Microbiology.)

Aims: In case of biological hazards and pandemics, personal protective equipment of rescue forces is currently manually decontaminated with harmful disinfectants, primarily peracetic acid. To overcome current drawbacks regarding supply, handling and disposal of chemicals, the use of plasma processed air (PPA) represents a promising alternative for surface decontamination on site. In this study, the sporicidal efficiency of a portable plasma system, designed for field applications, was evaluated., Methods and Results: The developed plasma device is based on a dielectric barrier discharge (DBD) and operated with ambient air as process gas. PPA from the plasma nozzle was flushed into a treatment chamber (volume: 300 l) and bacterial endospores (Bacillus subtilis and Bacillus atrophaeus) dried on different surfaces were treated under variable conditions. Reductions in spores by more than 4 log 10 were found within 3 min of PPA exposure. However, the presence of endospores in agglomerates or in an organic matrix as well as the complexity of the respective surface microstructure negatively affected the inactivation efficiency. When endospores were embedded in a dried protein matrix, mechanical wiping with swabs during exposure to PPA increased the inactivation effect significantly. Gaseous ozone alone did not provide a sporicidal effect. Significant spore inactivation was only obtained when water vapour was injected into the PPA stream., Conclusion: The results show that endospores dried on surfaces can be reduced by several orders of magnitude within few minutes in a treatment chamber which is flushed with PPA from of a DBD plasma nozzle., Significance and Impact of the Study: Plasma processed air generated on site by DBD plasma nozzles could be a suitable alternative for the disinfection of various surfaces in closed rooms., (© 2019 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

Aims: This study investigated the efficacy of hydrogen peroxide vapour (HPV) at inactivating hazard group 3 bacteria that have been presented dried from their growth medium to present a realistic challenge., Methods and Results: Hydrogen peroxide vapour technology (Bioquell) was used to decontaminate a class III microbiological safety cabinet containing biological indicators (BIs) made by drying standard working suspensions of the following agents: Bacillus anthracis (Ames) spores, Brucella abortus (strain S99), Burkholderia pseudomallei (NCTC 12939), Escherichia coli O157 ST11 (NCTC 12079), Mycobacterium tuberculosis (strain H37Rv) and Yersinia pestis (strain CO92) on stainless steel coupons. Extended cycles were used to expose the agents for 90 min. The HPV cycle completely inactivated B. anthracis spores, B. abortus, B. pseudomallei, E. coli O157 and Y. pestis when BIs were processed using quantitative and qualitative methods. Whilst M. tuberculosis was not completely inactivated, it was reduced by 4 log 10 from a starting concentration of 10 6 colony-forming units., Conclusions: This study demonstrates that HPV is able to inactivate a range of HG3 agents at high concentrations with associated organic matter, but M. tuberculosis showed increased resistance to the process., Significance and Impact of the Study: This publication demonstrates that HPV can inactivate HG3 agents that have an organic load associated with them. It also shows that M. tuberculosis has higher resistance to HPV than other agents. This shows that an appropriate BI to represent the agent of interest should be chosen to demonstrate a decontamination is successful., (© 2019 The Society for Applied Microbiology.)

Aims: (i) To develop an analytical method for recovery and quantification of bacteriophage MS2-as a surrogate for foot-and-mouth disease virus-from complex porous surfaces, with and without the presence of laboratory-developed agricultural grime; (ii) to evaluate, with a 4-log dynamic range, the virucidal activity of common biocides for their ability to decontaminate surfaces and hence remediate facilities, following a foreign animal disease contamination incident., Methods and Results: An analytical method was developed and optimized for MS2 recovery from simulated agricultural surfaces. The addition of Dey-Engley neutralizing broth to an extraction buffer improved MS2 viability in liquid extracts, with optimal analytical holding times determined as <8 to ≤24 h, depending on matrix. The recovery of MS2 from surface materials decreased in the order: nonporous reference material >grimed porous materials >nongrimed porous materials. In disinfectant testing, two spray applications of pAB were effective against MS2 (≥4-log reduction) on all operational-scale materials. Two per cent citric acid had limited effectiveness, with a ≥4-log reduction observed on a selected subset of grimed concrete samples., Conclusions: Decontamination efficacy test results can be affected by surface characteristics, extraction buffer composition, analytical holding time and surface-specific organism survivability. Efficacy should be evaluated using a test method that reflects the environmental characteristics of the intended application., Significance and Impact of the Study: The results of this study demonstrate the importance of analytical method verification tests for disinfectant testing prior to application in complex environments., (© 2019 The Society for Applied Microbiology.)

Aims: The current study aimed to assess the potential of a new high dose ultraviolet (UV) disinfection device to inactivate methicillin-resistant Staphylococcus aureus (MRSA), Clostridium difficile and a norovirus surrogate on handheld mobile devices, and to compare the efficacy of the UV-C device to hydrogen peroxide disinfection wipes., Methods and Results: Suspensions of MRSA, C. difficile spores and a surrogate for norovirus (MS2) were inoculated onto glass or plastic coupons, with or without organic contamination and were exposed to continuous UV-C light for 15-60 s (165-646 mJ cm -2 ) in a self-contained UV-C chamber or treated with hydrogen peroxide wipes. Increasing the UV-C dose from 310 to 650 mJ cm -2 did not result in greater levels of inactivation. UV-C light inactivated all three micro-organisms, in the absence of organic contamination, by >2·9 log. Treatment of MRSA, C. difficile spores or MS2, in the presence of organic contamination, with UV-C light (310-646 mJ cm -2 ) resulted in 2·3-3·7 log reductions. Treatment of MRSA with UV-C light provided levels of inactivation comparable to treatment with hydrogen peroxide wipes used following the manufacturer's instructions., Conclusions: UV-C light and hydrogen peroxide wipes had strong antimicrobial activity against MRSA, C. difficile spores and a norovirus surrogate, in the presence or absence of organic contamination., Significance and Impact of the Study: Chemical disinfection wipes are widely used in healthcare facilities, but they are not recommended for use on handheld mobile devices which may harbour pathogenic micro-organisms. The powerful bactericidal, sporicidal and virucidal activity of this high dose UV-C light device, shows that this technology is a promising alternative to chemical disinfectants, particularly for control of MRSA., (© 2019 The Society for Applied Microbiology.)

Aims: To investigate effects of the cationic surfactant cetyltrimethylammonium bromide (CTAB), a disinfectant, on spores of Bacillus species., Methods and Results: The ability of CTAB to trigger release of Bacillus spores' large depot of dipicolinic acid (DPA) in a 1 : 1 chelate with Ca 2+ (CaDPA), and to kill spores was investigated. CTAB-triggered CaDPA release from spores of Bacillus subtilis, Bacillus cereus and Bacillus megaterium, but was not followed by completion of germination. CaDPA release triggered by CTAB increased at higher temperatures, and was optimal for B. subtilis spores at pH 9·4 and 30 μg ml -1 CTAB. CTAB also killed Bacillus spores as shown by plate counts and vital staining of treated dormant spores, and after their germination. However, B. cereus and B. megaterium spores were more CTAB-sensitive than were B. subtilis spores. CaDPA release from and killing of CTAB-treated spores of isogenic B. subtilis mutants lacking germination proteins was also examined, and compared with effects of the well-known germinant dodecylamine on spores, to determine how CTAB exerts its effects on spores., Conclusions: The results of this investigation showed that CTAB kills spores of three Bacillus species, perhaps by damaging the spore inner membrane, although it is also possible that some killing by this agent follows its triggering of spore germination., Significance and Impact of the Study: The results of this work indicate that CTAB is also a disinfectant, but also a sporicide, and may be a useful adjunct in spore decontamination, especially at higher temperatures., (© 2019 The Society for Applied Microbiology.)

Biocides (antiseptics and disinfectants) are widely used in hospitals and pharmaceutical industries for contamination control. The emergence of reduced susceptibility to biocides is the major concern and this is caused by various factors, among which plasmid-mediated resistance is common. Many publications describe the antibiotic resistance and mechanisms in a clinical setting. However, there are only limited studies available worldwide addressing the molecular mechanisms of biocide resistance in the pharmaceutical sector. In addition, there is a considerable lack of scientific reports regarding minimum inhibitory concentration (MIC) values of typical biocides against pharmaceutical cleanroom environmental isolates. This review analyses the plasmid-mediated resistance in typical pharmaceutical micro-organisms and prevalence of biocide-resistant genes among common clinical and pharmaceutical isolates. This review discusses the MIC values of biocides in pharmaceutical environmental isolates, indicating the importance of the correlation between the presence or absence of biocide-resistant genes and reduced susceptibility of MIC values. This review recommends that pharmaceutical organizations adopt policies and test methodologies to examine the MICs of common cleanroom biocides against the most common types of cleanroom environmental isolates., (© 2018 The Society for Applied Microbiology.)

Aims: The assessment of the bacterial viability of chlorine- and quaternary ammonium compounds (QACs)-treated Lactobacillus cells by culture-dependent and -independent methods., Methods and Results: Lactobacillus isolates (Lactobacillus plantarum G1, Lactobacillus plantarum B1, Lactobacillus brevis S1 and Lactobacillus paracasei W1) in biofilm and planktonic cell suspensions were treated with chlorine-based (0·018 and 0·18%) and QACs-based (0·2 and 2·0%) disinfectants for 5 min and then analysed by plate counting, flow cytometry (FCM) and fluorescence activated cell sorting (FACS). The reaction of sessile cells to disinfectants was assessed with the confocal laser scanning microscopy (CLSM). Plate counts revealed L. paracasei W1 to be substantially inactivated by both disinfectants, while counts of the other isolates to be significantly reduced only by QACs, with L. plantarum B1 and L. brevis S1 showing a greater difference between QACs concentrations and cell types. In several cases, the disinfectants caused slightly higher inactivation of planktonic than biofilm cells, with L. plantarum B1 being significantly less sensitive to QACs in biofilm cells (P < 0·05). Following FCM with a Syto ® 9/PI assay, which addresses cell membrane integrity, the emergence of damaged (Syto ® 9 - PI + ) and injured (Syto ® 9 + PI + ) subpopulations was often observed in cells when they were treated with QACs, whereas intact (Syto ® 9 + PI - ) and unstained (Syto ® 9 - PI - ) subpopulations were mostly encountered in chlorine-treated cells. Except Syto ® 9 - PI + , all subpopulations were recovered on agar plates following FACS, with biofilm cells showing higher culturability irrespective of conditions, probably because of the residues of the biofilm matrix which serve as a protective cover for the bacteria. The CLSM revealed a substantial cell membrane damage within the QACs-treated biofilms, however, some cells deep in the biofilm were still intact and thus remained protected against this disinfectant., Conclusion: We found that FCM/FACS proved useful in the analysis of lactobacilli membrane integrity in disinfection experiments as well as in recovery evaluation of planktonic-biofilm cell subpopulations. In turn, CLSM was particularly useful in investigating the resistance mechanism when Lactobacillus cells were embedded in biofilms., Significance and Impact of the Study: This study highlights the need for treatment optimization on a case-by-case basis to avoid the emergence of cells in intermediate states with recovery potential and to reach and, thus, kill all bacteria in already developed lactobacilli biofilms., (© 2019 The Society for Applied Microbiology.)

Aims: Infectious health risks are associated with handling human cadavers and to decrease such risks, cadavers are embalmed using different chemicals. The aim of this study is to quantify the amount of micro-organisms present in different regions of human cadavers before embalming, after embalming and over a period of 8 months., Methods and Results: Human cadavers were embalmed using Thiel, formalin, Genelyn and the Imperial College London soft-preservation (ICL-SP) solution with two cadavers per technique. Sterile swabs were used to collect samples from different regions. Samples were collected every 2 months. All cadavers had a high number of microbial colonies before embalming. While no colonies were detected on formalin and Genelyn embalmed cadavers post-embalming, the number of colonies decreased significantly in Thiel-embalmed cadavers and stayed relatively the same in ICL-SP-embalmed cadavers., Conclusions: Formalin-embalmed cadavers showed the strongest disinfecting abilities followed by Thiel-embalmed cadavers, then Genelyn-embalmed cadavers and finally by ICL-SP cadavers., Significance and Impact of the Study: This study highlights how under researched this area is and the evident variation in the antimicrobial abilities of different embalming solutions on the cadaver as a whole and within different regions of the same cadaver., (© 2018 The Society for Applied Microbiology.)

Aims: Investigate the interactions of organic acids (OAs), acetic, butyric, citric, formic, lactic and propionic acid against 50 Gram-positive vancomycin-resistant Enterococcus faecium (VRE) strains to determine whether pH, undissociated or dissociated acid forms correlate with bacterial inhibition., Methods and Results: Concentrations of undissociated and dissociated OAs at the molar minimum inhibitory concentrations (MIC M s) of the VRE were calculated using the Henderson-Hasselbalch equation. The pH at the MIC M s of all VRE strains against acetic, butyric, formic and propionic acids was similar, 4·66 ± 0·07, but there was a 1·1 pH unit difference for all six OAs. Inhibition of VRE by all six OAs did not appear to be solely dependent on pH or on the undissociated OA species. The inhibition of VRE by all six dissociated acids was within Δ = 3·1 mmol l -1 ., Conclusions: Vancomycin-resistant Enterococcus faecium inhibition correlated with the dissociated OA species. A small decrease in the concentration of the dissociated OAs from optimum may result in allowing VRE strains to escape disinfection., Significance and Impact of the Study: When an OA is used to disinfect VRE strains, the concentration of the dissociated OA should be carefully controlled. A concentration of at least 20 mmol l -1 dissociated OA should be maintained when disinfecting VRE., (© 2018 The Society for Applied Microbiology.)

Objectives: In our previous research, we have developed a new combination disinfectant, glutaraldehyde-didecyldimethylammonium bromide (GD). It was verified that GD had a strong effect on both Escherichia coli and Staphylococcus aureus. In this work, Candida albicans was selected as an object, and it could be killed by GD. We aimed to investigate the cellular and molecular mechanism of GD effecting on C. albicans., Methods and Results: The results of sterilization experiment indicated that GD was effective on C. albicans. Flow cytometry and atomic absorption spectrometry were applied to detect cell membrane damage of C. albicans. Luciferase reaction and Bradford method were carried out to detect ATP content and protein quantitation. Transmission electron microscopy was used for intracellular organelles morphological observation. In order to study changes in mitochondrial membrane potential, Rh 123 was used as an indicator. DNA conformation analysis was performed by molecular modelling and circular dichroism. The results indicated that membrane permeability was increased rapidly owing to GD effect, and the leaked K + and Mg 2+ were about 12·1 and 12·4 times those of the control, respectively, at 10 min after GD treatment. Simultaneously, ATP and protein also leaked rapidly out of the cell. Mitochondrial membrane potential was destroyed, succinic dehydrogenase activity was significantly decreased and DNA conformation was changed because of GD action., Conclusions: Glutaraldehyde-didecyldimethylammonium bromide disinfected C. albicans through distorting cell membrane integrity and permeability, disturbing the intracellular homeostasis by intracellular substances leakage, especially K + , Mg 2+ , ATP and protein, causing electrolyte imbalance of mitochondria, changing DNA structure, which finally led to cell death., Significance and Impact of the Study: This study focused on the cellular and molecular mechanism of GD as a disinfectant against C. albicans. It is important to provide theoretical support to GD against Candida albicans in practical application., (© 2018 The Society for Applied Microbiology.)

Aims: To investigate the potent control of microbial surface contamination of an innovative process which consists in incorporating metal oxide microspheres homogeneously into materials., Methods and Results: Spherical microspheres containing zinc and magnesium oxides synthesized via a one-step manufacturing process (Pyrolyse Pulvérisée ® ) and incorporated into different plastic matrices were evaluated for their antimicrobial activity according to JIS Z 2801 standard. A significant activity was observed for microsphere-added polyethylene coupons with a reduction of all tested bacteria populations, including Gram negative and Gram positive even expressing acquired antibiotic resistance (Escherichia coli ESBL, Staphylococcus aureus metiR). An antiviral activity higher than 2 log of reduction was also observed on H1N1 and HSV-1 viruses. This antimicrobial effect was dose-dependent and time-dependent for both polyethylene and polypropylene matrices. Antimicrobial activity was maintained after exposition to disinfectants and totally preserved 50 months after the preparation of the coupons., Conclusions: Incorporated into plastic matrices, metal oxide microspheres showed significant antibacterial and antiviral activities., Significance and Impact of Study: This is, to our knowledge, the first report on an original process incorporating metal oxide microspheres, which have specific physico-chemical and antimicrobial properties, into materials that could be used for surface contamination prevention., (© 2018 The Society for Applied Microbiology.)

Aims: The effects of a new glutaraldehyde-didecyldimethylammonium bromide combination disinfectant (GD) on the cell surface of Staphylococcus aureus, a representative Gram-positive bacterium, were investigated in this study., Methods and Results: Results of bacterial surface structural analysis showed that GD significantly changed the bacterial morphology. The membrane fluidity decreased and outer membrane permeabilization increased after contact with GD. Furthermore, the integrity of the cytoplasmic membrane was destroyed by over 99% after exposure to GD for a short time. Bacterial ATPase activity correlated negatively with the treatment of GD over time, and proteins were degraded. Assays of intracellular component leakage indicated that GD caused the rapid leakage of K + , Mg 2+ , ATP molecules, and proteins into the extracellular environment., Conclusions: The effects of GD against S. aureus are probably attributable to the removal of the permeability barrier, changes in the S. aureus morphology, changes in the structures and functions of the cell membrane, leakage of intracellular substances and disturbance of the intracellular homeostasis. As a result, this irreversible damage accelerated the death of S. aureus., Significance and Impact of the Study: In an earlier study, the bactericidal mechanism of GD against Escherichia coli was investigated. Hence, this study focused on the action of mechanism of GD against S. aureus. It is important to clarify the disinfectant bactericidal mechanisms of GD against bacterium, in general, and this study provides theoretical support to the prevention of bacterial resistance., (© 2018 The Society for Applied Microbiology.)

Aims: Preanalytic aspects can make results of hygiene studies difficult to compare. Efficacy of surface disinfection was assessed with an evaluated swabbing procedure., Methods and Results: A validated microbial screening of surfaces was performed in the patients' environment and from hands of healthcare workers on two intensive care units (ICUs) prior to and after a standardized disinfection procedure. From a pure culture, the recovery rate of the swabs for Staphylococcus aureus was 35%-64% and dropped to 0%-22% from a mixed culture with 10-times more Staphylococcus epidermidis than S. aureus. Microbial surface loads 30 min before and after the cleaning procedures were indistinguishable., Conclusions: The quality-ensured screening procedure proved that adequate hygiene procedures are associated with a low overall colonization of surfaces and skin of healthcare workers. Unchanged microbial loads before and after surface disinfection demonstrated the low additional impact of this procedure in the endemic situation when the pathogen load prior to surface disinfection is already low., Significance and Impact of the Study: Based on a validated screening system ensuring the interpretability and reliability of the results, the study confirms the efficiency of combined hand and surface hygiene procedures to guarantee low rates of bacterial colonization., (© 2017 The Society for Applied Microbiology.)

Bacterial spores resist environmental extremes and protect key spore macromolecules until more supportive conditions arise. Spores germinate upon sensing specific molecules, such as nutrients. Germination is regulated by specialized mechanisms or structural features of the spore that limit contact with germinants and enzymes that regulate germination. Importantly, germination renders spores more susceptible to inactivating processes such as heat, desiccation, and ultraviolet radiation, to which they are normally refractory. Thus, germination can be intentionally induced through a process called germination-induction and subsequent treatment of these germinated spores with common disinfectants or gentle heat will inactivate them. However, while the principle of germination-induction has been shown effective in the laboratory, this strategy has not yet been fully implemented in real-word scenarios. Here, we briefly review the mechanisms of bacterial spore germination and discuss the evolution of germination-induction as a decontamination strategy. Finally, we examine progress towards implementing germination-induction in three contexts: biodefense, hospital settings and food manufacture., Significance and Impact: This article reviews implementation of germination-induction as part of a decontamination strategy for the cleanup of bacterial spores. To our knowledge this is the first time that germination-induction studies have been reviewed in this context. This article will provide a resource which summarizes the mechanisms of germination in Clostridia and Bacillus species, challenges and successes in germination-induction, and potential areas where this strategy may be implemented., (Published 2017. This article is a U.S. Government work and is in the public domain in the USA.)

Aims: The work aimed at developing and evaluating practically relevant methods for testing of disinfectants on contaminated personal protective equipment (PPE)., Methods and Results: Carriers were prepared from PPE fabrics and contaminated with Bacillus subtilis spores. Peracetic acid (PAA) was applied as a suitable disinfectant. In method 1, the contaminated carrier was submerged in PAA solution; in method 2, the contaminated area was covered with PAA; and in method 3, PAA, preferentially combined with a surfactant, was dispersed as a thin layer. In each method, 0·5-1% PAA reduced the viability of spores by a factor of ≥6 log 10 within 3 min. The technique of the most realistic method 3 proved to be effective at low temperatures and also with a high organic load. Vaccinia virus and Adenovirus were inactivated with 0·05-0·1% PAA by up to ≥6 log 10 within 1 min. The cytotoxicity of ricin was considerably reduced by 2% PAA within 15 min of exposure., Conclusions: PAA/detergent mixture enabled to cover hydrophobic PPE surfaces with a thin and yet effective disinfectant layer., Significance and Impact of the Study: The test methods are objective tools for estimating the biocidal efficacy of disinfectants on hydrophobic flexible surfaces., (© 2017 The Society for Applied Microbiology.)

Aims: This study aimed to evaluate the biofilm formation and disinfectant resistance of Salmonella cells in mono- and dual-species biofilms with Pseudomonas aeruginosa, and to investigate the role of extracellular polymeric substances (EPS) in the protection of biofilms against disinfection treatment., Methods and Results: The populations of Salmonella in mono- or dual-species biofilms with P. aeruginosa on stainless steel (SS) coupons were determined before and after exposure to commercial disinfectant, 50 μg ml -1 chlorine or 200 μg ml -1 Ecolab ® Whisper™ V (a blend of four effective quaternary ammonium compounds (QAC)). In addition, EPS amount from biofilms was quantified and biofilm structures were observed using scanning electron microscopy (SEM). Antagonistic interactions between Salmonella and P. aeruginosa resulted in lower planktonic population level of Salmonella, and lower density in dual-species biofilms compared to mono-species biofilms. The presence of P. aeruginosa significantly enhanced disinfectant resistance of S. Typhimurium and S. Enteritidis biofilm cells for 2 days, and led to an average of 50% increase in polysaccharides amount in dual-species biofilms than mono-species biofilms of Salmonella. Microscopy observation showed the presence of large microcolonies covered by EPS in dual-species biofilms but not in mono-species ones., Conclusion: The presence of P. aeruginosa in dual-species culture inhibited the growth of Salmonella cells in planktonic phase and in biofilms, but protected Salmonella cells in biofilms from disinfection treatment, by providing more production of EPS in dual-species biofilms than mono-species ones., Significance and Impact of the Study: This study provides insights into inter-species interaction, with regard to biofilm population dynamics and disinfectant resistance. Thus, a sanitation protocol should be designed considering the protective role of secondary species to pathogens in biofilms on SS surface which has been widely used at food surfaces and manufacturers., (© 2017 The Society for Applied Microbiology.)

Aims: The spread of bacteria that are simultaneously resistant to disinfectants and antimicrobials would constitute an unsettling scenario. In order to explore an association between antimicrobial resistance and reduced susceptibility to biocides/microbicides (disinfectants) in agriculture, we investigated Escherichia coli (n = 438) and enterococci (n = 120) isolated from six different flocks of the same poultry farm with known history of antimicrobial treatment., Methods and Results: Susceptibility to disinfectants (formic acid and a quaternary ammonium compound (QAC), didecyldimethylammoniumchloride-DDAC) was assessed by macrodilution according to guidelines of the German Veterinary Society. Escherichia coli, Enterococcus faecalis and Enterococcus faecium were screened (i) for reduced biocide susceptibility and (ii) for an association of biocide susceptibility and antimicrobial resistance including the production of extended-spectrum beta-lactamases (ESBL) and the hyperproduction of AmpC-type beta-lactamases. DDAC inhibited ESBL/AmpC(hyper)-producing E. coli (n = 53) from poultry at similar or slightly lower inhibitory concentrations, compared with non-ESBL/AmpC strains (median MIC = 0·36 vs 1·44 mg l -1 ). In contrast, DDAC-MICs were positively correlated with several other antibiotic MICs (e.g. piperacillin and sulphamethoxazole + trimethoprim in E. coli, chloramphenicol in E. faecalis) and increased DDAC-MICs were statistically linked to high-level aminoglycoside resistance in enterococci (streptomycin high level). DDAC-MICs did not correlate with the presence of the integron marker qacEDelta1., Conclusions: This study provides indication that residual disinfectant might be able to select antimicrobial-resistant enterococci, but not ESBL-/AmpC (hyper)producing E. coli from poultry., Significance and Impact of the Study: While ESBL-/AmpC-E. coli were inhibited at disinfectant concentrations comparable to or lower than wildtype values, low concentrations of QACs might be able to select other antimicrobial-resistant E. coli or enterococci-a finding with special significance for the food processing industry, where QACs are regularly used., (© 2017 The Society for Applied Microbiology.)