The longitudinal effect of disseminating handwashing public health education to children in India via co-created, culturally relevant resources.

Infectious diseases are a leading cause of death for children from low- and middle-income countries (LMICs), often due to inadequate hand-hygiene. This study evaluates culturally relevant educational resources as a vehicle to disseminate the importance of handwashing amongst children in India. Employing a participatory action research (PAR) model and mixed methods, this follow-up longitudinal study evaluates a set of innovate educational handwashing resources and workshops specifically co-created for use in the State of Gujarat, and how they aid teachers in the teaching of hand-hygiene over a 3 year period. Working alongside local NGOs on-the-ground, teacher questionnaires (n=58) and focus groups including a brief questionnaire with teachers (n=35) were conducted to assess the impact of trainer workshops. In addition, pre- and post-workshop worksheets were conducted with children (n=98). Percentage change was calculated between children's pre-and post-worksheet scores and a cumulative frequency of responses to each questionnaire criterion was measured. Data from the focus group found that the resources had been used in over 200 schools by more than 5000 children. In addition, 92.28 % of teachers said they would use the resources within their classrooms in India, with 58.16 % of pupils having an increased understanding of germs/handwashing directly after the workshop. Teachers reported that they are able to teach microbiology and handwashing more effectively. Furthermore, following a focus group, 100 % of teachers noted a reduction in childhood vomiting and diarrhoeal illnesses linked with insufficient hand-hygiene across 46 schools in the State of Gujarat since using the Germ's Journey resources.

Heterogeneous Fenton's-like catalyst potentiation of hydrogen peroxide disinfection: an investigation into mechanisms of action.

AIMS: This study aimed to establish the mechanisms of action (MOA) of a novel surface-functionalized 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 the loss of 260 nm absorbing material, membrane potential, 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.

Porous surfaces: stability and recovery of coronaviruses.

The role of indirect contact in the transmission of SARS-CoV-2 is not clear. SARS-CoV-2 persists on dry surfaces for hours to days; published studies have largely focused on hard surfaces with less research being conducted on different porous surfaces, such as textiles. Understanding the potential risks of indirect transmission of COVID-19 is useful for settings where there is close contact with textiles, including healthcare, manufacturing and retail environments. This article aims to review current research on porous surfaces in relation to their potential as fomites of coronaviruses compared to non-porous surfaces. Current methodologies for assessing the stability and recovery of coronaviruses from surfaces are also explored. Coronaviruses are often less stable on porous surfaces than non-porous surfaces, for example, SARS-CoV-2 persists for 0.5 h-5 days on paper and 3-21 days on plastic; however, stability is dependent on the type of surface. In particular, the surface properties of textiles differ widely depending on their construction, leading to variation in the stability of coronaviruses, with longer persistence on more hydrophobic materials such as polyester (1-3 days) compared to highly absorbent cotton (2 h-4 days). These findings should be considered where there is close contact with potentially contaminated textiles.

Health care worker knowledge and attitudes towards uniform laundering during the COVID-19 pandemic.

BACKGROUND: The COVID-19 pandemic raised concerns towards domestic laundering of healthcare worker (HCW) uniforms; this is common practice in countries such as the United Kingdom (UK) and United States. Previous research suggested 4-32% of nurses did not adhere to laundry policies, which could be an infection control risk. This study aimed to investigate the knowledge and attitudes of UK healthcare workers towards domestic laundering of uniforms during the COVID-19 pandemic. METHODS: Online and paper questionnaires were distributed to HCWs and nursing students who regularly wear uniforms. Differences in knowledge between HCWs were analyzed by Chi-squared tests and attitudes were examined using exploratory factor analysis. RESULTS: About 86% of participants (n = 1099 of 1277) laundered their uniforms domestically. Respondents were confident in laundering their uniforms appropriately (71%), however 17% failed to launder at the recommended temperature (60°C). Most participants (68%) would prefer their employer launder their uniforms, with mixed negative emotions towards domestic laundering. Limited provision of uniforms and changing and/or storage facilities were a barrier to following guidelines. CONCLUSION: Most HCWs domestically launder their uniforms, despite a preference for professional laundering. One-fifth of HCWs deviated from the UK National Health Service uniform guidelines; onsite changing facilities were the most significant barrier towards adherence.

Antiviral plant-derived natural products to combat RNA viruses: Targets throughout the viral life cycle.

There is a need for new effective antivirals, particularly in response to the development of antiviral drug resistance and emerging RNA viruses such as SARS-CoV-2. Plants are a significant source of structurally diverse bioactive compounds for drug discovery suggesting that plant-derived natural products could be developed as antiviral agents. This article reviews the antiviral activity of plant-derived natural products against RNA viruses, with a focus on compounds targeting specific stages of the viral life cycle. A range of plant extracts and compounds have been identified with antiviral activity, often against multiple virus families suggesting they may be useful as broad-spectrum antiviral agents. The antiviral mechanism of action of many of these phytochemicals is not fully understood and there are limited studies and clinical trials demonstrating their efficacy and toxicity in vivo. Further research is needed to evaluate the therapeutic potential of plant-derived natural products as antiviral agents.

Investigation of the stability and risks of fomite transmission of human coronavirus OC43 on leather.

Limited research exists on the potential for leather to act as a fomite of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or endemic coronaviruses including human coronavirus (HCoV) OC43; this is important for settings such as the shoe manufacturing industry. Antiviral coating of leather hides could limit such risks. This study aimed to investigate the stability and transfer of HCoVOC43 on different leathers, as a surrogate for SARS-CoV-2, and assess the antiviral efficacy of a silver-based leather coating. The stability of HCoV-OC43 (6.6 log10) on patent, full-grain calf, corrected grain finished and nubuck leathers (silver additive-coated and uncoated) was measured by titration on BHK-21 cells. Transfer from leather to cardboard and stainless steel was determined. HCoV-OC43 was detectable for 6 h on patent, 24 h on finished leather and 48 h on calf leather; no infectious virus was recovered from nubuck. HCoV-OC43 transferred from patent, finished and calf leathers onto cardboard and stainless steel up to 2 h post-inoculation (≤3.1-5.5 log10), suggesting that leathers could act as fomites. Silver additive-coated calf and finished leathers were antiviral against HCoV-OC43, with no infectious virus recovered after 2 h and limited transfer to other surfaces. The silver additive could reduce potential indirect transmission of HCoV-OC43 from leather.

The Stability of Model Human Coronaviruses on Textiles in the Environment and during Health Care Laundering.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) persists on stainless steel and plastic for up to 7 days, suggesting that coronavirus disease 2019 (COVID-19) could be spread by fomite transmission. There is limited research on the stability of SARS-CoV-2 on textiles, with the risk of textiles acting as fomites not being well understood. To date, there does not appear to be any published research on the stability of coronaviruses during laundering, which is required to determine the efficacy of current laundering policies in the decontamination of health care textiles. The aim of this study was to investigate the environmental stability of human coronaviruses HCoV-OC43 and HCoV-229E on different textile fiber types and the persistence of HCoV-OC43 on textiles during domestic and industrial laundering. This study demonstrated that human coronaviruses (5 log10 50% tissue culture infective doses [TCID50]) remain infectious on polyester for ≥72 h, cotton for ≥24 h, and polycotton for ≥6 h; HCoV-OC43 was also able to transfer from polyester to PVC or polyester after 72 h. Under clean conditions, HCoV-OC43 was not detectable on cotton swatches laundered with industrial and domestic wash cycles without temperature and detergent (≥4.57-log10-TCID50 reduction), suggesting that the dilution and agitation of wash cycles are sufficient to remove human coronaviruses from textiles. In the presence of interfering substances (artificial saliva), ≤1.78 log10 TCID50 HCoV-OC43 was detected after washing domestically without temperature and detergent, unlike industrial laundering, where the virus was completely removed. However, no infectious HCoV-OC43 was detected when washed domestically with detergent.IMPORTANCE Synthetic textiles such as polyester could potentially act as fomites of human coronaviruses, indicating the importance of infection control procedures during handling of contaminated textiles prior to laundering. This study provides novel evidence that human coronaviruses can persist on textiles for up to 3 days and are readily transferred from polyester textile to other surfaces after 72 h of incubation. This is of particular importance for the domestic laundering of contaminated textiles such as health care uniforms in the United Kingdom and United States, where there may be a risk of cross-contaminating the domestic environment. It was demonstrated that human coronaviruses are removed from contaminated textiles by typical domestic and commercial wash cycles, even at low temperatures without detergent, indicating that current health care laundering policies are likely sufficient in the decontamination of SARS-CoV-2 from textiles.

Improving young children's handwashing behaviour and understanding of germs: The impact of A Germ's Journey educational resources in schools and public spaces.

CONTEXT: Effective handwashing can prevent the spread of germs, including Covid-19. However, young children can lack a fundamental understanding of germ transfer. A Germ's Journey educational resources were designed to support young children in learning about germs and handwashing. These resources include a book, website, song, online games and glo-gel activities that are informed by a behaviour change model. RESEARCH GAP: Prior research has not evaluated the impacts of these resources on behavioural outcomes. PURPOSE OF THE STUDY: Two intervention studies evaluated the impacts of these resources on both knowledge and behavioural outcomes. METHOD: In Study 1, children (n = 225) were recruited from four schools and randomly assigned by classrooms to participate in a multicomponent intervention (vs. control). In Study 2, children (n = 104) were recruited from a museum and randomly assigned to participate in a song intervention (vs. control). Trained observers recorded participants' engagement in six handwashing behaviours and their understanding of germs. These behavioural and knowledge outcomes were analysed using regression and related analyses. RESULTS: In Study 1, significant improvements were observed between baseline and follow up in the intervention group for both behavioural scores (Est = 0.48, SE = 0.14, t = 3.30, p = 0.001) and knowledge scores (Est = 2.14, SE = 0.52, z = 4.11, p < 0.001), whereas these improvements were not observed in the control group (ts < 1). In Study 2, the intervention group had significantly higher behavioural scores compared to the control group (Est. = -0.71, SE = 0.34, t = -2.07, p = 0.04). CONCLUSION: This research demonstrates that specifically designed hand hygiene educational resources can improve handwashing practice and understanding in young children, and could lead to the reduction of the transmission of disease within this group.

Evaluating approaches to designing effective Co-Created hand-hygiene interventions for children in India, Sierra Leone and the UK.

Effective and culturally appropriate hand-hygiene education is essential to promote health-related practices to control and prevent diseases such as Diarrhoea, Ebola and COVID-19. In this paper we outline and evaluate the Co-Creation processes underpinning a handwashing intervention for young children (A Germ's Journey) developed and delivered in India, Sierra Leone and the UK, and consider the implications surrounding Imperialist/Colonial discourse and the White Saviour Complex. The paper focuses both on the ways Co-Creation was conceptualised by our collaborators in all three countries and the catalysts and challenges encountered. Qualitative data have been drawn from in-depth interviews with five key stakeholders, focus group data from 37 teachers in Sierra Leone and responses to open-ended questionnaires completed by teachers in India (N = 66) and UK (N = 63). Data were analysed using thematic analysis and three themes, each with three constituent subthemes are presented. In the theme 'Representations of and Unique Approaches to Co-Creation' we explore the ways in which Co-Creation was constructed in relation to teamwork, innovative practice and more continuous models of evaluation. In 'Advantages of Co-Creation' we consider issues around shared ownership, improved outcomes and more meaningful insights alongside the mitigation of risks and short-circuiting of problems. In 'Challenges of Co-Creation' we discuss issues around timing and organisation, attracting and working with appropriate partners and understanding the importance of local context with inherent social, economic and structural barriers, especially in low-and-middle-income countries. We consider how theoretical elements of Co-Creation can inform effective international public health interventions; crucial during a global pandemic in which handwashing is the most effective method to control the transmission of COVID-19. Finally we reflect on some of the methodological challenges of our own work and in managing the potentially conflicting goals of the ethical and participatory values of Co-Creation with pragmatic considerations about ensuring an effective final 'product'.

The role of textiles as fomites in the healthcare environment: a review of the infection control risk.

BACKGROUND: Infectious diseases are a significant threat in both healthcare and community settings. Healthcare associated infections (HCAIs) in particular are a leading cause of complications during hospitalisation. Contamination of the healthcare environment is recognised as a source of infectious disease yet the significance of porous surfaces including healthcare textiles as fomites is not well understood. It is currently assumed there is little infection risk from textiles due to a lack of direct epidemiological evidence. Decontamination of healthcare textiles is achieved with heat and/or detergents by commercial or in-house laundering with the exception of healthcare worker uniforms which are laundered domestically in some countries. The emergence of the COVID-19 pandemic has increased the need for rigorous infection control including effective decontamination of potential fomites in the healthcare environment. This article aims to review the evidence for the role of textiles in the transmission of infection, outline current procedures for laundering healthcare textiles and review studies evaluating the decontamination efficacy of domestic and industrial laundering. METHODOLOGY: Pubmed, Google Scholar and Web of Science were searched for publications pertaining to the survival and transmission of microorganisms on textiles with a particular focus on the healthcare environment. RESULTS: A number of studies indicate that microorganisms survive on textiles for extended periods of time and can transfer on to skin and other surfaces suggesting it is biologically plausible that HCAIs and other infectious diseases can be transmitted directly through contact with contaminated textiles. Accordingly, there are a number of case studies that link small outbreaks with inadequate laundering or infection control processes surrounding healthcare laundry. Studies have also demonstrated the survival of potential pathogens during laundering of healthcare textiles, which may increase the risk of infection supporting the data published on specific outbreak case studies. CONCLUSIONS: There are no large-scale epidemiological studies demonstrating a direct link between HCAIs and contaminated textiles yet evidence of outbreaks from published case studies should not be disregarded. Adequate microbial decontamination of linen and infection control procedures during laundering are required to minimise the risk of infection from healthcare textiles. Domestic laundering of healthcare worker uniforms is a particular concern due to the lack of control and monitoring of decontamination, offering a route for potential pathogens to enter the clinical environment. Industrial laundering of healthcare worker uniforms provides greater assurances of adequate decontamination compared to domestic laundering, due to the ability to monitor laundering parameters; this is of particular importance during the COVID-19 pandemic to minimise any risk of SARS-CoV-2 transmission.

From formulation to in vivo model: A comprehensive study of a synergistic relationship between vancomycin, carvacrol, and cuminaldehyde against Enterococcus faecium.

Vancomycin-resistant Enterococcus faecium (VRE) has become endemic in healthcare settings, reducing treatment options for enterococcal infections. New antimicrobials for VRE infections are a high priority, but the development of novel antibiotics is time-consuming and expensive. Essential oils (EOs) synergistically enhance the activity of some existing antibiotics, suggesting that EO-antibiotic combinations could resensitise resistant bacteria and maintain the antibiotic repertoire. The mechanism of resensitisation of bacteria to antibiotics by EOs is relatively understudied. Here, the synergistic interactions between carvacrol (1.98 mM) and cuminaldehyde (4.20 mM) were shown to reestablish susceptibility to vancomycin (0.031 mg/L) in VRE, resulting in bactericidal activity (4.73 log10 CFU/ml reduction). Gene expression profiling, coupled with ß-galactosidase leakage and salt tolerance assays, suggested that cell envelope damage contributes to the synergistic bactericidal effect against VRE. The EO-vancomycin combination was also shown to kill clinical isolates of VRE (2.33-5.25 log10 CFU/ml reduction), and stable resistance did not appear to develop even after multiple passages. The in vivo efficacy of the EO-vancomycin combination was tested in a Galleria mellonella larvae assay; however, no antimicrobial action was observed, indicating that further drug development is required for the EO-vancomycin combination to be clinically useful for treatment of VRE infections.

Characterisation and screening of antimicrobial essential oil components against clinically important antibiotic-resistant bacteria using thin layer chromatography-direct bioautography hyphenated with GC-MS, LC-MS and NMR.

INTRODUCTION: The antimicrobial activity of many essential oils (EOs) is well established, indicating that EOs may be a source of compounds for antimicrobial drug development. Thin layer chromatography-direct bioautography (TLC-DB) can quickly identify antimicrobial components in complex mixtures and can be applied to the screening of EOs for lead compounds. OBJECTIVES: This study aimed to identify antimicrobial components of oregano, rosewood and cumin EOs against antibiotic-sensitive and -resistant bacteria using TLC-DB and a multi-faceted approach of GC-MS, LC-MS and NMR techniques to characterise bioactive compounds. The study also aimed to quantify the antimicrobial activity of bioactive compounds in order to evaluate their potential for the development of therapies against antibiotic-resistant bacteria. MATERIALS AND METHODS: EOs were eluted on TLC plates and sprayed with a suspension of Staphylococcus aureus, Enterococcus faecium, Escherichia coli or Pseudomonas aeruginosa (antibiotic-sensitive and -resistant isolates). Zones of inhibition, visualised with iodonitrotetrazolium chloride, were subject to GC-MS, LC-MS and NMR to characterise the bioactive compounds. RESULTS: Seven compounds were identified from the three EOs using GC-MS, while LC-MS and NMR failed to detect the presence of any further non-volatile or heat labile compounds. Carvacrol was most antimicrobial compound identified, with minimum inhibitory concentrations ranging 0.99-31.62 mM. CONCLUSION: The identified antimicrobial compounds present in oregano, rosewood and cumin EOs including carvacrol may be candidates for the development of novel antimicrobial therapies against antibiotic-resistant bacteria.

From ward to washer: The survival of Clostridium difficile spores on hospital bed sheets through a commercial UK NHS healthcare laundry process.

OBJECTIVE: To quantify the survival of Clostridium difficile spores on hospital bed sheets through the United Kingdom National Health System (UK NHS) healthcare laundry process (Health Technical Memorandum (HTM) 01-04) in vitro and on bed sheets from patients with C. difficile through the commercial laundry. METHODS: Clostridium difficile spores were inoculated onto cotton sheets and laundered through a simulated washer extractor cycle using an industrial bleach detergent with sodium hypochlorite 15% and peracetic acid sour 14% (acetic acid and hydrogen peroxide; pH, 2-4). Spore survival on hospital sheets naturally contaminated with C. difficile was also assessed using a washer extractor plus drying and finishing cycles at a commercial laundry.PatientsNaturally contaminated C. difficile bed sheets were taken from beds of patients that had previously been diagnosed with C. difficile infection (CDI) and had received care on an isolated C. difficile ward. RESULTS: The simulated washer extractor cycle, with an industrial detergent, demonstrated survival of 2 strains of C. difficile NCTC 11209 (0-4 colony-forming units [cfu] per 25 cm2) and ribotype 001/072 (0-9 cfu per 25 cm2). Before laundering, naturally contaminated bed sheets had an average spore load of 51 cfu per 25 cm2, and after washing, drying, and finishing, the spore load was 33 cfu per 25 cm2. Before and after washing, the C. difficile strain was identified as ribotype 001/072. Both the simulated and in-situ laundering processes failed the microbiological standards of no pathogenic bacteria remaining. CONCLUSIONS: Clostridium difficile spores are able to survive laundering through a commercial washer extractor and may be contributing to sporadic outbreaks of CDI. Further research to establish exposure of laundry workers, patients, and the hospital environment to C. difficile spores from bed sheets is needed.

Synchronous application of antibiotics and essential oils: dual mechanisms of action as a potential solution to antibiotic resistance.

Antibiotic resistance has increased dramatically in recent years, yet the antibiotic pipeline has stalled. New therapies are therefore needed to continue treating antibiotic resistant infections. One potential strategy currently being explored is the use of non-antibiotic compounds to potentiate the activity of currently employed antibiotics. Many natural products including Essential Oils (EOs) possess broad spectrum antibacterial activity and so have been investigated for this purpose. This article aims to review recent literature concerning the antibacterial activity of EOs and their interactions with antibiotics, with consideration of dual mechanisms of action of EOs and antibiotics as a potential solution to antibiotic resistance. Synergistic interactions between EOs and their components with antibiotics have been reported, including several instances of antibiotic resensitization in resistant isolates, in support of this strategy to control antibiotic resistance. However, a lack of consistency in methods and interpretation criteria makes drawing conclusions of efficacy of studied combinations difficult. Synergistic effects are often not explored beyond preliminary identification of antibacterial interactions and mechanism of action is rarely defined, despite many hypotheses and recommendations for future study. Much work is needed to fully understand EO-antibiotic associations before they can be further developed into novel antibacterial formulations.

Structure-activity modelling of essential oils, their components, and key molecular parameters and descriptors.

Many essential oil components are known to possess broad spectrum antimicrobial activity, including against antibiotic resistant bacteria. These compounds may be a useful source of new and novel antimicrobials. However, there is limited research on the structure-activity relationship (SAR) of essential oil compounds, which is important for target identification and lead optimization. This study aimed to elucidate SARs of essential oil components from experimental and literature sources. Minimum Inhibitory Concentrations (MICs) of essential oil components were determined against Escherichia coli and Staphylococcus aureus using a microdilution method and then compared to those in published in literature. Of 12 essential oil components tested, carvacrol and cuminaldehyde were most potent with MICs of 1.98 and 2.10 mM, respectively. The activity of 21 compounds obtained from the literature, MICs ranged from 0.004 mM for limonene to 36.18 mM for α-terpineol. A 3D qualitative SAR model was generated from MICs using FORGE software by consideration of electrostatic and steric parameters. An r2 value of 0.807 for training and cross-validation sets was achieved with the model developed. Ligand efficiency was found to correlate well to the observed activity (r2 = 0.792), while strongly negative electrostatic regions were present in potent molecules. These descriptors may be useful for target identification of essential oils or their major components in antimicrobial/drug development.

A Multifactorial Comparison of Ternary Combinations of Essential Oils in Topical Preparations to Current Antibiotic Prescription Therapies for the Control of Acne Vulgaris-Associated Bacteria.

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 before interactions between binary and ternary combinations were determined against P. acnes and S. epidermidis (type and clinical isolates) using minimum inhibitory concentrations and fractional inhibitory concentrations. The EOs were characterised by both gas chromatography-mass spectrometry and nuclear magnetic resonance. A combination of 0.53 mg/mL litsea, 0.11 mg/mL rosewood and 0.11 mg/mL clove bud was formulated into herbal distillates and compared with 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. Copyright © 2017 John Wiley & Sons, Ltd.

Washing uniforms at home: adherence to hospital policy.

Infection control is a priority for all hospitals to reduce the spread of healthcare-associated infections (HCAIs). Textiles especially uniforms, are a possible route of HCAI transmission. There are protocols to ensure hospital laundry services meet accepted standards, however healthcare uniforms are laundered by staff at home and variations in practice occur. A questionnaire was used to conduct a service evaluation at four hospitals in different NHS trusts to determine how closely healthcare staff followed hospital guidelines on laundering and aftercare of uniforms at home. Responses showed that not all staff followed these guidelines; 44% of staff washed their uniforms below the recommended temperature of 60°C, which presents a potential route for cross-contamination and infection.

Reduction of Legionella spp. in water and in soil by a citrus plant extract vapor.

Legionnaires' disease is a severe form of pneumonia caused by Legionella spp., organisms often isolated from environmental sources, including soil and water. Legionella spp. are capable of replicating intracellularly within free-living protozoa, and once this has occurred, Legionella is particularly resistant to disinfectants. Citrus essential oil (EO) vapors are effective antimicrobials against a range of microorganisms, with reductions of 5 log cells ml(-1) on a variety of surfaces. The aim of this investigation was to assess the efficacy of a citrus EO vapor against Legionella spp. in water and in soil systems. Reductions of viable cells of Legionella pneumophila, Legionella longbeachae, Legionella bozemanii, and an intra-amoebal culture of Legionella pneumophila (water system only) were assessed in soil and in water after exposure to a citrus EO vapor at concentrations ranging from 3.75 mg/liter air to 15g/liter air. Antimicrobial efficacy via different delivery systems (passive and active sintering of the vapor) was determined in water, and gas chromatography-mass spectrometry (GC-MS) analysis of the antimicrobial components (linalool, citral, and ß-pinene) was conducted. There was up to a 5-log cells ml(-1) reduction in Legionella spp. in soil after exposure to the citrus EO vapors (15 mg/liter air). The most susceptible strain in water was L. pneumophila, with a 4-log cells ml(-1) reduction after 24 h via sintering (15 g/liter air). Sintering the vapor through water increased the presence of the antimicrobial components, with a 61% increase of linalool. Therefore, the appropriate method of delivery of an antimicrobial citrus EO vapor may go some way in controlling Legionella spp. from environmental sources.

Identification and quantification of the antimicrobial components of a citrus essential oil vapor.

The anti-bacterial components of a citrus essential oil vapor were identified as linalool, citral and beta-pinene using a bioautography method and quantified by GC-MS. Essential oil vapor release, monitored in real-time with Atmospheric Pressure Chemical Ionization - MS (APCI-MS), showed differences in the vapor release profile oflimonene, beta-pinene and linalool over 24 hours, while Solid Phase Micro-extraction (SPME) GC-MS demonstrated changes in composition of the vapor at 35 degrees C. Fourteen isolates were tested in vitro for their susceptibility to the EO vapor and to linalool, citral and beta-pinene vapors, both separately and in a mixture containing the three components in the amounts at which they occur in the EO vapor. All eleven Gram-positive strains tested were susceptible to the EO vapor, linalool, citral and beta-pinene vapors separately and the mixture with zones of inhibition of 4.34 cm, 5.32 cm, 5.58 cm, 4.86 cm and 4.68 cm, respectively. Of the three Gram-negative strains tested, Pseudomonas aeruginosa 10145 was resistant to all the vapors. When bacteria inoculated onto stainless steel surfaces were exposed to either the EO vapor or a linalool/citral/beta-pinene vapor mixture there was no significant difference in reduction for the Gram-positive isolates, while the Gram-negative isolates were resistant to both EO vapor and the linalool/citral/beta-pinene mixture.