Impact of a dry inoculum deposition on the efficacy of copper-based antimicrobial surfaces.

BACKGROUND: The introduction of antimicrobial surfaces into healthcare environments is believed to impact positively on the rate of healthcare-associated infections by significantly decreasing pathogen presence on surfaces. AIM: To report on a novel efficacy test that uses a dry bacterial inoculum to measure the microbicidal efficacy of antimicrobial surfaces. METHODS: An aerosolized dry inoculum of Staphylococcus aureus or Acinetobacter baumannii was deposited on copper alloy surfaces or a hospital-grade stainless-steel surface. Surviving bacteria were enumerated following incubation of the inoculated surfaces at an environmentally relevant temperature and relative humidity. Damage caused to bacteria by the aerosolization process and by the different surfaces was investigated. FINDINGS: Dry inoculum testing showed a <2-log10 reduction in S. aureus or A. baumannii on the copper alloy surfaces tested after 24 h at 20°C and 40% relative humidity. Potential mechanisms of action included membrane damage, DNA damage and arrested cellular respiration. The aerosolization process caused some damage to bacterial cells. Once this effect was taken into account, the antimicrobial activity of copper surfaces was evident. CONCLUSIONS: Our test provided a realistic deposition of a bacterial inoculum to a surface and, as such, a realistic protocol to assess the efficacy of dry antimicrobial environmental surfaces in vitro.

Evaluation of new in vitro efficacy test for antimicrobial surface activity reflecting UK hospital conditions.

BACKGROUND: Antimicrobial surfaces aim to reduce microbial bioburden and improve hygiene. The current antimicrobial surface efficacy test (ISO22196) is an initial screening test but its conditions, high temperature (37°C) and relative humidity (RH) (100%) bear little relationship to in-use conditions. AIM: To develop an antimicrobial surface efficacy test providing a realistic second-tier test, simulating in-use conditions. METHODS: Surface relative humidity, temperature and soiling were measured over one year at a UK hospital, enabling realistic parameters to be set for our surface efficacy test. A nebulizer, connected to a cascade impactor, aerosolized and uniformly deposited a Staphylococcus aureus suspension over test copper alloys and control stainless steel surfaces. Bacteria were enumerated following nebulization, and after a range of contact times, under [20°C, 50% RH] and [20°C, 40% RH] parameters reflecting in-use conditions; [37°C, 100% RH] was employed to reflect conditions used in ISO22196. FINDINGS: All copper alloys produced a >4 log10 reduction after 24h under all conditions tested. Copper alloys were more effective at [37°C, 100% RH] showing a >4 log10 reduction after 30 min than at in-use conditions [20°C, 50% RH and 20°C, 40% RH], for which 60 min was required to achieve the same level of kill, for most but not all alloys. CONCLUSION: The use of the nebulizer to deposit bacterial inocula on surfaces showed little variability in results. Our method was more discriminatory than the ISO22196 enabling distinction between the bactericidal surface activity, which allows for a more rigorous selection of antimicrobial surfaces for potential use in healthcare settings.

Assessment of the environmental microbiological cross contamination following hand drying with paper hand towels or an air blade dryer.

AIMS: This study compared the potential for cross contamination of the surrounding environment resulting from two different hand-drying methods: paper towels and the use of an air blade dryer. METHODS AND RESULTS: One hundred volunteers for each method washed their hands and dried them using one of the two methods. Bacterial contamination of the surrounding environment was measured using settle plates placed on the floor in a grid pattern, air sampling and surface swabs. Both drying methods produced ballistic droplets in the immediate vicinity of the hand-drying process. The air blade dryer produced a larger number of droplets which were dispersed over a larger area. Settle plates showed increased microbial contamination in the grid squares which were affected by ballistic droplets. Using the settle plates counts, it was estimated that approx. 1.7 × 10(5) cfu more micro-organisms were left on the laboratory floor (total area approx. 17.15 m(2)) after 100 volunteers used an air blade dryer compared to when paper towels were used. CONCLUSIONS: The two drying methods led to different patterns of ballistic droplets and levels of microbial contamination under heavy use conditions. Whilst the increase in microbial levels in the environment is not significant if only nonpathogenic micro-organisms are spread, it may increase the risk of pathogen contamination of the environment when pathogens are occasionally present on people's hands. SIGNIFICANCE AND IMPACT OF THE STUDY: The study suggests that the risk of cross contamination from the washroom users to the environment and subsequent users should be considered when choosing a hand-drying method. The data could potentially give guidance following the selection of drying methods on implementing measures to minimise the risk of cross contamination.

Assessing the efficacy of different microfibre cloths at removing surface micro-organisms associated with healthcare-associated infections.

This study investigated the ability of 10 different microfibre cloths to remove microbial contamination from three surfaces commonly found in hospital settings (stainless steel, furniture laminate and ceramic tile), under controlled laboratory conditions. Tests were conducted using organisms known to cause healthcare-associated infections, i.e. meticillin-resistant Staphylococcus aureus (MRSA), Clostridium difficile (in spore form) and Escherichia coli. For all the cloths tested, there was significant statistical evidence to suggest a difference in cleaning performance between them on first and single use (P<0.001). However, the overall performance of the nine re-useable cloths did not differ in practice with differences in log10 reductions of <1. The performance of the disposable microfibre cloth was notably worse. The performance of all cloths decreased with repeated use on a succession of contaminated surfaces. After repeated washing, re-usable cloth performance improved at 75 washes, and reduced after 150 washes, although, in most instances, performance after 150 washes was better than at first wash. For all cloths, price was not an indication of performance. Based on these laboratory findings, it is concluded that use of the microfibre cloths investigated is an effective way to reduce the levels of MRSA, E. coli and C. difficile (in spore form) on a range of surfaces found in the clinical environment and could therefore be of benefit to these environments.

Do European Standard Disinfectant tests truly simulate in-use microbial and organic soiling conditions on food preparation surfaces?

AIMS: The results from European standard disinfectant tests are used as one basis to approve the use of disinfectants in Europe. The design of these laboratory-based tests should thus simulate as closely as possible the practical conditions and challenges that the disinfectants would encounter in use. No evidence is available that the organic and microbial loading in these tests simulates actual levels in the food service sector. METHODS AND RESULTS: Total organic carbon (TOC) and total viable count (TVC) were determined on 17 visibly clean and 45 visibly dirty surfaces in two restaurants and the food preparation surfaces of a large retail store. These values were compared to reference values recovered from surfaces soiled with the organic and microbial loading, following the standard conditions of the European Surface Test for bactericidal efficacy, EN 13697. CONCLUSIONS: The TOC reference values for clean and dirty conditions were higher than the data from practice, but cannot be regarded as statistical outliers. This was considered as a conservative assessment; however, as additional nine TOC samples from visibly dirty surfaces were discarded from the analysis, as their loading made them impossible to process. Similarly, the recovery of test organisms from surfaces contaminated according to EN 13697 was higher than the TVC from visibly dirty surfaces in practice; though they could not be regarded as statistical outliers of the whole data field. No correlation was found between TVC and TOC in the sampled data, which re-emphasizes the potential presence of micro-organisms on visibly clean surfaces and thus the need for the same degree of disinfection as visibly dirty surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: The organic soil and the microbial burden used in EN disinfectant standards represent a realistic worst-case scenario for disinfectants used in the food service and food-processing areas.

The effect of an antibacterial washing-up liquid in reducing dishwater aerobic plate counts.

AIMS: To assess any significant differences in the aerobic plate count (APC) of catering dishwaters following the use of a traditional, nonantibacterial or an antibacterial washing-up liquid. METHODS AND RESULTS: A dishwashing trial was undertaken within a commercial restaurant of 6 weeks duration (3 weeks with each washing-up liquid in a randomized, weekly pattern). Five replicate samples were taken from the dishwater at the end of the washing-up operation, on three separate occasions each day corresponding to mid-morning, lunchtime and mid-afternoon meal preparations. CONCLUSIONS: The antibacterial product was shown to significantly reduce the APC by an average log10 reduction of 1.81 CFU ml(-1) (98.5%) as compared with the traditional product. APC were lower for each of the three weekly time periods for the antibacterial product. Continued use of the antibacterial product did not decrease the APC of the dishwater, though with the traditional product, dishwater counts increased throughout the trial week. SIGNIFICANCE AND IMPACT OF THE STUDY: Antibacterial washing-up liquids, with proven activity in controlling levels of microorganisms in dishwaters, could play a significant role in reducing the risk of cross-contamination between washed articles during washing-up operations.

The microbial ecology of high-risk, chilled food factories; evidence for persistent Listeria spp. and Escherichia coli strains.

AIMS: The intention of this study was to provide evidence of any Listeria spp. or Escherichia coli strain persistence, and to identify whether strains of these organisms adapt to specific environmental or product niches in food factories. METHODS AND RESULTS: A 3-year assessment of the microbial ecology of four, ready-to-eat food-processing factories was undertaken in which approx. 196 000 and 75 000 product and environmental samples were examined for Escherichia coli and Listeria spp. respectively. A total of 152 E. coli isolates (44 environmental and 108 product in 62 ribogroups) and 260 Listeria spp. isolates (174 environmental and 86 product in 30 ribogroups) were identified and ribotyped. The overall prevalence of E. coli (0.08%), all Listeria spp. (0.35%) and L. monocytogenes (0.23%) was very low. Some 10 E. coli ribogroups and 14 Listeria spp. ribogroups showed evidence for persistence, defined as the isolation of the same strain, from the same site, over a prolonged time period. The majority of E. coli strains were product niche oriented whilst the majority of Listeria spp. strains were environmental niche oriented. CONCLUSION: Current UK high-risk food factory designs, personnel hygiene and cleaning and disinfection regimes are sufficient to control Listeria spp. and E. coli to very low levels. SIGNIFICANCE AND IMPACT OF THE STUDY: Persistent strains of these organisms, however, can remain within factory high-risk production areas over considerable time periods, warranting an examination of the strain persistence mechanisms and alternative hygiene controls.

Biocide use in the food industry and the disinfectant resistance of persistent strains of Listeria monocytogenes and Escherichia coli.

AIMS: The aims of the project were threefold: to survey the use of disinfectants in the UK food industry; to assess the product and environmental microflora of selected food factories for the persistence of Listeria monocytogenes and Escherichia coli; and to determine the disinfectant resistance of any persistent strains. METHODS AND RESULTS: A survey of the use of disinfectants in the UK food industry was undertaken in which a total of 40 sites were visited and a further 77 postal questionnaires were returned from farms, food manufacture, food transport and food retail sites. Quaternary ammonium compounds (QACs) were predominantly used, applied in small volumes as a mist. Approximately 30,000 samples from the product and environment of five chilled food factories were examined for L. monocytogenes and E. coli over a 3 year period. A total of 181 L. monocytogenes and 176 E. coli isolates were ribotyped to yield 19 and 34 ribogroups, respectively. Some strains were isolated only from the product, a number only from the environment and others from both niches. Some strains were seen to be persistent for the duration of the sampling exercise (2-3 years). The most common L. monocytogenes and E. coli strains, together with two environmental L. monocytogenes strains, were assessed for any resistance to commercial disinfectants as compared with a laboratory L. monocytogenes disinfectant testing strain. The resistance of the L. monocytogenes and E. coli strains isolated from the factory were not significantly different from the laboratory control strain. CONCLUSIONS: Persistent strains of L. monocytogenes and E. coli are found in the UK food industry, though this persistence is not related to their increased susceptibility to the most commonly used disinfectants. SIGNIFICANCE AND IMPACT OF THE STUDY: The concept of a persistent microflora in food factories will have an impact on the future selection of suitable control options, including the use of biocides.

Biocide use in the food industry and the disinfectant resistance of persistent strains of Listeria monocytogenes and Escherichia coli.

AIMS: The aims of the project were threefold: to survey the use of disinfectants in the UK food industry; to assess the product and environmental microflora of selected food factories for the persistence of Listeria monocytogenes and Escherichia coli; and to determine the disinfectant resistance of any persistent strains. METHODS AND RESULTS: A survey of the use of disinfectants in the UK food industry was undertaken in which a total of 40 sites were visited and a further 77 postal questionnaires were returned from farms, food manufacture, food transport and food retail sites. Quaternary ammonium compounds (QACs) were predominantly used, applied in small volumes as a mist. Approximately 30,000 samples from the product and environment of five chilled food factories were examined for L. monocytogenes and E. coli over a 3 year period. A total of 181 L. monocytogenes and 176 E. coli isolates were ribotyped to yield 19 and 34 ribogroups, respectively. Some strains were isolated only from the product, a number only from the environment and others from both niches. Some strains were seen to be persistent for the duration of the sampling exercise (2-3 years). The most common L. monocytogenes and E. coli strains, together with two environmental L. monocytogenes strains, were assessed for any resistance to commercial disinfectants as compared with a laboratory L. monocytogenes disinfectant testing strain. The resistance of the L. monocytogenes and E. coli strains isolated from the factory were not significantly different from the laboratory control strain. CONCLUSIONS: Persistent strains of L. monocytogenes and E. coli are found in the UK food industry, though this persistence is not related to their increased susceptibility to the most commonly used disinfectants. SIGNIFICANCE AND IMPACT OF THE STUDY: The concept of a persistent microflora in food factories will have an impact on the future selection of suitable control options, including the use of biocides.

A microbiological evaluation of warm air hand driers with respect to hand hygiene and the washroom environment.

A finger rinse technique for counting micro-organisms on hands showed no significant difference in the level of recovered micro-organisms following hand drying using either warm air or paper towels. Contact plate results appeared to reflect the degree of dampness of hands after drying rather than the actual numbers of micro-organisms on the hands. In laboratory tests, a reduction in airborne count of Pseudomonas aeruginosa and Staphylococcus aureus of between 40 and 75% was achieved from 600 readings comparing inlets and outlets of warm air hand driers. In washroom trials, the number of airborne micro-organisms was reduced by between 30 and 75%. Air emitted from the outlet of the driers contained significantly fewer micro-organisms than air entering the driers. Drying of hands with hand driers was no more likely to generate airborne micro-organisms than drying with paper towels. Levels of micro-organisms on external surfaces of hand driers were not significantly different to those on other washroom surfaces. This work shows that warm air hand driers, of the type used in this study, are a hygienic method of drying hands and therefore appropriate for use in both the healthcare and food industry.

A comparison of the bactericidal efficacy of 18 disinfectants used in the food industry against Escherichia coli O157:H7 and Pseudomonas aeruginosa at 10 and 20 degrees C.

A number of proprietary disinfectant products (18) used in the food industry were tested for their bactericidal efficacy against Pseudomonas aeruginosa and Escherichia coli O157:H7 at 20 and 10 degrees C according to the BS EN 1276 (1997) quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas. At 20 degrees C, 13 products passed at their in-use concentration (under clean and dirty conditions) against Ps. aeruginosa and 15 passed against E. coli O157:H7. The number of products passing the test at 10 degrees C was 11 and 14 for Ps. aeruginosa and E. coli O157:H7, respectively. The products exhibiting reduced efficacy at the lower temperature were amphoterics and quaternary ammonium compounds although some of these types of products were effective at both temperatures. Products that passed against Ps. aeruginosa generally also passed against E. coli O157:H7. Taking all the results together, only 11 of the total of 18 products achieved a pass result under all the parameters tested. This work demonstrates the need for final verification of disinfectant efficacy by undertaking field trials in the food-processing environment in which the product is intended for use.

Fungal endophyte symbiosis and plant diversity in successional fields

Increasing evidence suggests that microbial interactions are important determinants of plant biodiversity. The hypothesis that fungal endophyte symbiosis reduces diversity in successional fields was tested by manipulating infection of tall fescue, the most abundant perennial grass in the eastern United States. Over a 4-year period, species richness declined and tall fescue dominance increased in infected plots relative to uninfected plots without differences in total productivity. A host-specific endophyte, with negligible biomass, altered plant community structure in this long-term field experiment and may be reducing plant diversity throughout its expanding range.

Effectiveness of cleaning techniques used in the food industry in terms of the removal of bacterial biofilms.

The effectiveness of cleaning was investigated through food factory trials and laboratory experiments using a naturally occurring biofilm from a food factory environment and generated biofilms. The efficacy of factory cleaning and disinfection programmes was assessed by swabbing and total viable count (TVC) analysis of surfaces before cleaning, after cleaning and after disinfection. Cleaning produced a 0.91 log reduction in the attached population. Investigation of the effectiveness of a variety of cleaning methods in the removal of a naturally occurring food factory biofilm showed that the high pressure spray and the mechanical floor scrubber, which use a high degree of mechanical action, were most effective. Cleaning trials with biofilms of Pseudomonas aeruginosa or Staphylococcus aureus showed that spraying with water at pressures of 34.5, 51.7 and 68.9 bar did not significantly increase the removal, as assessed by direct epifluorescent microscopy (DEM) and swabbing and TVC analysis, beyond the three log reduction observed at 17.2 bar. The effect of spray time at 17.2 bar showed that increasing spray time from 1 to 10 s did not significantly increase removal of Ps. aeruginosa biofilm. Investigation of the optimum distance of the spray lance from the surface at 17.2 bar was found to be between 125 and 250 mm. The use of an alkaline, acidic or neutral detergent prior to spraying with water at 17.2 bar did not significantly increase the removal of Ps. aeruginosa or Staph. aureus. However, the acidic and alkaline products significantly (P = 0.05) affected the viability of Staph. aureus and Ps. aeruginosa, respectively, thereby minimizing the potential for the spread of contamination.

Changes in the strength of attachment of micro-organisms to surfaces following treatment with disinfectants and cleansing agents.

Suspensions of Pseudomonas aeruginosa and Staphylococcus epidermidis, and biofilms established (16 h) on submerged glass and stainless steel (216 2B) coupons, were exposed to sodium hypochlorite (0.02% or 0.015% w/v), Dodigen (0.0015% w/v or 0.0006% w/v), sodium dodecylsulphate (6% w/v or 0.1% w/v) and Tween-80 (6% w/v) for 5 min at 20 degrees C. Survival was assessed by viable counts and blot succession. Biofilm bacteria were significantly less susceptible to these biocides than were planktonic cells, but their attachment to the surfaces was loosened by such treatments. Treatment with the non-ionic surfactant, Tween-80, however, strengthened the attachment of Staph. epidermidis to stainless steel. Such effects on attachment strength, which are species and surface dependent, have profound implications on post-treatment cleansing and possible re-contamination of product in clean-in-place (CIP) systems.

Kinetic aspects of ATP amplification reactions.

The accurate determination of very low concentrations of ATP is important in many areas of pure and applied biochemistry, particularly hygiene monitoring in the food industry. Two mathematical models have been used to show that a luciferase-linked ATP recycling enzyme assay can determine sample ATP concentration from the time taken for observed bioluminescence to reach half its maximum value. Such a time-based assay has the potential to detect very low ATP concentrations in less than 10 min without the need for a sensitive photometer.

Quantification of the ease of removal of bacteria from surfaces.

This paper describes a technique which reproducibly quantifies the ease of removal of microorganisms from surfaces. Tiles (22 mm x 22 mm) of various materials were colonised with Staphylococcus epidermidis NCTC 11047, Escherichia coli K12 HB101 or Pseudomonas aeruginosa PaWH, by submersion, for various times (2 min-48 h), in inoculated Tryptone Soya broth (37 degrees C). Colonised tiles were blotted onto a Tryptone Soya agar plate for 1 min and the process was repeated through a succession of agar plates. The final plate contained tetrazolium salts (0.05% w/v) and was incubated in situ with the tile. Tetrazolium plates indicated that very few organisms remained on the tiles after 15 successive blots. In all instances, the number of recovered colonies per plate decreased exponentially with plate succession number, according to the relationship, CFU = A.10-kN, where CFU is the number of colonies transferred, k is the removal exponent, A is the intercept and N is the plate succession number. Removal exponents differed significantly between organisms (P > 0.95), depended on the nature of the test surface, and decreased as the inital attachment and colonisation time was increased from 2 min-48 h. Intercept values (A) but not the gradients were dependent upon the initial numbers of bacteria in suspension. These data indicate that the gradients derived from counting recoverable viable cells from successive blots of test tiles onto agar is a measure of the strength of attachment of the organisms to the surface.

Disinfection of food production areas.

Disinfection, other than by heat, is ineffective unless all surfaces have previously been thoroughly cleaned to remove interfering materials. Cleaning is therefore extremely important as part of a two-stage cleaning and disinfection (sanitation) programme. The author describes the principles of sanitation, the chemicals and equipment involved, and the programme of events to be followed. For food products of 'low risk' (in terms of stable shelf life and safety), traditional sanitation programmes are adequate and in some cases disinfection may not be required. However, disinfection is essential for 'high-risk' food products, but this cannot be effectively undertaken without due consideration of hygienic design and possible cross-contamination. To ensure continued satisfactory performance of a sanitation programme, routine assessments should be undertaken.

Special needs for disinfectants in food-handling establishments.

Freedom from microbial and foreign body contamination is essential in food production, and the principal means for controlling the surface route of contamination is sanitation (cleaning and disinfection). After the cleaning phase, disinfection plays a crucial role in further reducing microbial numbers and viability. However, disinfectants for use in the food industry may contaminate the product. Therefore, as well as being effective and suitable for factory use, such disinfectants must also be non-toxic and non-tainting. The author describes the usage criteria and operator safety requirements of disinfectants, together with methods to determine taint potential, toxicity and efficacy. Using these parameters, a limited number of disinfectants are judged suitable for general food industry use and these are compared.

An evaluation of the repeatability and reproducibility of a surface test for the activity of disinfectants.

A collaborative study was carried out to determine the precision of a disinfectant surface test method which is currently under consideration for development as a harmonized European standard surface test. Results indicate that significant variation in microbicidal effect occurs both within and between test laboratories despite careful standardization of test conditions, but that the variability may be less than that associated with suspension tests. Indications are that much of this variability derives from random variations in the resistance of the test strains from day to day and, most particularly, from test period to test period both within as well as between laboratories. It is concluded that although the test may be sufficiently reliable to be used as a standard method, adequate replication must be specified to distinguish borderline pass from borderline fail concentrations.

The antibacterial activity of Virkon measured by colony growth and bioluminescence of lux recombinant Listeria monocytogenes.

Concentration exponents for the broad spectrum antimicrobial Virkon were determined for Listeria monocytogenes using both plate counts and bioluminescence measurements; the values of 3.15 and 2.6 indicate a close equivalence between these two measurement procedures. Virkon is an effective biocide for L. monocytogenes at the manufacturer's in-use concentration of 1%.