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.     

Comparative testing of disinfectant and antiseptic products using proposed European suspension testing methods

The development of standard suspension test methods for disinfectants and antiseptics for adoption in Europe is described. Evaluation of a range of disinfectant agents and products currently used in the UK under conditions as proposed for these tests indicates that the majority of products diluted in water of standard hardness showed satisfactory activity producing a 4·5–5 log reduction in viable count within 5 min against Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecium, Proteus mirabilis and Candida albicans in the absence and presence of 1% albumin. All the products, when diluted with distilled water, produced greater than 5 log reduction in 60 min.     

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.     

Assessment of the efficacy of disinfectants on surfaces

The Literature on testing the efficacy of disinfectants covers a century. Most predominant and standardized are the so called suspension tests that allow for the quantitative estimation of the microbicidal activity (log reduction factors) of disinfectants on test organisms suspended in solutions of these products.Since the outcome of suspension tests might be a poor predictor for the efficacy of a disinfectant under practical circumstances, especially with regard to bacteria attached to surfaces, a variety of test procedures have been designed to mimic those conditions. Within the framework of CEN/TC 216 a quantitative surface test has been developed to assess the activity of disinfectants on bacteria or fungi attached to steenless steel surfaces. Preliminary data suggest that covering a dried inoculum with disinfectant without any further mechanical action to improve contact between organisms and disinfectant, will usually result in lower reduction factors than those obtained with suspension tests. Comparative testing further suggests that by applying mechanical action, with the effect of resuspending cells in the liquid on the surface,—similar to mopping, brushing etc.— will result in higher reduction rates. Although not unexpected these findings emphasize the importance of designing test methods based on practical applications of disinfectants.     

Use of impedance methods for testing bactericidal activity of antiseptics

Standards PN-EN 1040 and EN 12054 describe test methods and minimum requirements for bactericidal activities of antiseptics. However, standard procedures are time consuming and require 48 hours of incubation. New alternative technique based on impedimetric procedure provides possibilities to reduce this time to several hours. The Bactometer (BioMerieux, Vitek System, USA) is a fully automated impedance technology system used to microbial quantitation of products. Impedance measures microbial activity by electrical methods. The aim of the study was to adaptate the impedimetric method utilising Bactometer--system to microbiological activity control of chemical antiseptics. Eight different products were utilised throughout the study. The samples for classical and alternative method were prepared in the same way as described in standards. The method of choice was dilution-neutralization method. All procedures conducted in Bactometer were verified by plate count method. The high correlation was observed between results obtained by normative methods and impedimetric measurement. All tested products meet requirements. The procedure utilising the Bactometer, provides a rapid and accurate system for the determination of bacterial content. The results of validation carried out during this study indicate, possibility to use impedimetric method alternatively to traditional methods.     

Evaluation of efficacy of disinfectants against Salmonella from the feed industry

Aims:  To evaluate disinfectants against Salmonella under conditions relevant for the feed industry.
Materials and Results:  A survey on the use of disinfectants in feed industry showed that a range of different types was used. Nine disinfectants, reflecting the most commonly used active ingredients, were tested for bactericidal activity on Salmonella isolated from the feed industry. All disinfectants were efficient against Salmonella in suspension. The bactericidal effect varied considerably between different types of active compounds on bacteria dried on surfaces or grown as biofilm. Tenside-based disinfectants and hypochlorite were found to have low bactericidal activity and the efficiency was significantly reduced when the ratio of amount disinfectant per cell decreased. It was shown that concentrations of 70–80% ethanol were effective against Salmonella. Among the disinfectants tested a product containing 70% ethanol was most efficient followed by Virkon S.
Conclusions:  Many disinfectants had low bactericidal activity against Salmonella at surfaces while Virkon S and a product containing 70% ethanol were most effective. Another advantage of ethanol-based disinfectants is evaporation of ethanol, resulting in low residual water after use.
Significance and Impact of the Study:  Use of the disinfectants found to be efficient against surface associated Salmonella , may assist the industry in combating Salmonella .     

Evaluation of hydrogen peroxide-based disinfectants in a new resazurin microplate method for rapid efficacy testing of biocides

Aims:  Development of the resazurin microplate method (RMM) as a novel test system for the evaluation of the antimicrobial activity of antiseptics and disinfectants. The validated RMM was subsequently applied for the evaluation of hydrogen peroxide (H₂O₂) and stabilized H₂O₂ combination products.
Methods and Results:  The European Committee for Standardization prescribes the plate count challenge test (PCCT) for antiseptic and disinfectant efficacy testing. This protocol was adapted to a microplate-based assay, using resazurin as viability indicator. The RMM was as accurate as the PCCT, had an identical detection limit and showed high intermediate precision. Using the validated RMM, it was shown that H₂O₂ combined with silver possessed a higher bactericidal and fungicidal activity compared to native H₂O₂ with and without glycerol.
Conclusions:  Validation showed that the RMM may replace the PCCT. When applying the RMM, H₂O₂ combined with silver was clearly a more potent disinfectant compared to H₂O₂ in killing bacteria and fungi.
Significance and Impact of the Study:  The RMM is easier to use for antimicrobial efficacy testing of antiseptics and disinfectants. As the RMM is in accordance with the norms of the European Committee for Standardization, it may become a more cost-effective alternative to the more laborious PCCT reference method. H₂O₂ with silver may replace native H₂O₂ to increase overall disinfection efficiency.     

Fluorescent assay based on resazurin for detection of activity of disinfectants against bacterial biofilm

A new, quick method, using the resazurin dye test as a bacterial respiration indicator, has been developed to assay the antibacterial activity of various substances used as disinfectants against bacterial biofilm growth on clinical devices. Resazurin was used to measure the presence of active biofilm bacteria, after adding disinfectant, in relation to a standard curve generated from inocula in suspension of the same organism used to grow the biofilm. The biofilm was quantified indirectly by measuring the fluorescent, water-soluble resorufin product produced when resazurin is reduced by reactions associated with respiration. Four products used as disinfectants and the biofilm growth of five bacterial species on carriers made of materials commonly found in clinical devices were studied. Under test conditions, chlorhexidine, NaOCl, ethanol, and Perasafe at concentrations of 0.2, 0.01, 350, and 0.16 mg/ml, respectively, all produced 5-log reductions in biofilm cell numbers on the three different carriers. The redox-driven test depends on bacterial catabolism, for which reason resazurin reduction produces an analytic signal of the bacterial activity in whole cells, and therefore could be used for determining disinfectant efficacy in an assay based on the metabolic activity of microorganisms grown as biofilm or in suspension.     

CEN/TC 216: its role in producing current and future European disinfectant testing standards

CEN, the European Committee for Standardization, is a legal association comprising of National Standards Bodies (e.g. AFNOR, BSI, DIN) responsible for the production of European Standards (ENs) which are designed to facilitate the exchange of goods and services through the elimination of technical barriers to trade. CEN/TC 216 was conceived in the late 1980s with the scope Standardization of the terminology, requirements, test methods including potential efficacy under in-use conditions, recommendations for use and labelling in the whole field of chemical disinfection and antiseptics. Areas of activity include agriculture (but not crop protection chemicals), domestic service, food hygiene and other industrial fields, institutional, medical and veterinary applications. Following a meeting in 1990, the Technical Committee (TC) delegated its work to four Working Groups (WG); a Horizontal Working Group (HWG) and three Working Groups responsible for the Medical (WG1), Veterinary (WG2) and Food Hygiene, Domestic and Institutional (WG3) market sectors. Whilst the three WGs could develop test methods to assess bactericidal and fungicidal product activity, specialist Task Groups of the HWG have been established to provide specific guidance on viruses, spores, surface tests, ring trials and to harmonize methodology. The main objective of TC/216 is to produce test methods in a sequential, three phase mode. In Phase 1, the ability of a product to demonstrate bactericidal, fungicidal or sporicidal activity is tested. Phase 2 tests are divided into two steps. Step 1 tests are suspension tests to determine bactericidal, fungicidal, virucidal or sporicidal activity under laboratory conditions that simulate practical conditions. Step 2 tests are other laboratory tests e.g. handwash, handrub or surface tests that are more representative of in-use conditions. Phase 3 tests hope to give guidance to product users as to how to undertake suitable field trials. To date, eight standards have been produced and another 18 are in the final stages of development. WG1 has been the most prolific with 18 test methods under development whilst WG2 and 3 have six and three tests, respectively. Following production of standard test methodologies, the major issues for CEN/TC 216 are concerned with assessing the performance of the tests in practice, especially their statistical reliability. In addition, standards are being further harmonized and guidelines being developed to help product manufacturers and users select the appropriate tests for appropriate fields of use.     

Evaluation of the efficacy of electrochemically activated solutions against nosocomial pathogens and bacterial endospores

Aims: Electrochemically activated solutions (ECAS) are generated from halide salt solutions via specially designed electrolytic cells. The active solutions are known to possess high biocidal activity against a wide range of target microbial species, however, literature revealing the kill‐kinetics of these solutions is limited. The aim of the study was to identify the kill‐rate and extent of population kill for a range of target species (including endospores) using ECAS generated at the anode (anolyte). Methods and Results: Standard suspensions of methicillin‐resistant Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus atrophaeus spores and Clostridium difficile spores were treated with anolyte in a quantitative suspension assay. For vegetative cells, all concentrations of anolyte tested reduced the viable population to below the detection limit within 10 s. At a concentration of 99%, anolyte produced a log₁₀ reduction factor of greater than five in viable B. atrophaeus endospores within 90 s and reduced numbers of C. difficile endospores to below the experimental detection limit within 20 s at concentrations of 5% or greater. Conclusions: Anolyte was highly effective in killing test‐bacteria and spores. The bactericidal efficacy was retained against vegetative cells at dilutions as low as 1% and against C. difficile spores as low as 5%. Significance and Impact of Study: The results of this study demonstrate that ECAS are effective at lower concentrations and act more rapidly than previously reported. Potent bactericidal and sporicidal activity coupled with point‐of‐use generation, low production‐costs and environmental compatibility suggest that acidic ECAS has the potential to be a useful addition to the current armoury of disinfectants.     

Comparative susceptibility of planktonic and 3‐day‐old Salmonella Typhimurium biofilms to disinfectants

Aims: To compare the susceptibility of a 3‐day‐old biofilm and planktonic Salmonella to disinfectants at different exposure times. We hypothesize that Salmonella biofilms are more resilient to disinfectants compared to planktonic Salmonella. Methods and Results: The susceptibility of planktonic cells to disinfectants was tested by a modified version of the Council of Europe suspension test EN 1276. Salmonella biofilms were formed using the Calgary Biofilm Device. Results show that 3‐day‐old Salmonella biofilms are less susceptible to the disinfectants benzalkonium chloride, chlorhexidine gluconate, citric acid, quaternary ammonium compounds, sodium hypochlorite (SH) and ethanol, compared to planktonic Salmonella. Surprisingly, the results also demonstrate that low concentrations of SH were more effective against a 3‐day‐old biofilm compared to high concentrations of SH. Conclusions: While all the disinfectants evaluated were able to reduce biofilm‐associated cells at concentrations and contact times sufficient to eliminate planktonic cells, there were still sufficient viable cells remaining in the biofilm to cause further contamination and potential infection. Significance and Impact of the Study: Protocols for the use of chemical disinfectants need to include biofilm susceptibility testing. There is a requirement for an effective and standardized tool for determining the susceptibility of biofilms to disinfectants.     

Development of a standard test to assess the resistance of Staphylococcus aureus biofilm cells to disinfectants

A standardized disinfectant test for Staphylococcus aureus cells in biofilms was developed. Two disinfectants, the membrane-active compound benzalkonium chloride (BAC) and the oxidizing agent sodium hypochlorite, were used to evaluate the biofilm test. S. aureus formed biofilms on glass, stainless steel, and polystyrene in a simple system with constant nutrient flow that mimicked as closely as possible the conditions used in the current standard European disinfectant test (EN 1040). The biofilm that was formed on glass contained cell clumps and extracellular polysaccharides. The average surface coverage was 60%, and most (92%) of the biofilm cells were viable. Biofilm formation and biofilm disinfection in different experiments were reproducible. For biofilms exposed to BAC and hypochlorite the concentrations needed to achieve 4-log killing were 50 and 600 times higher, respectively, than the concentrations needed to achieve this level of killing with the European phase 1 suspension test cells. Our results show that a standardized disinfectant test for biofilm cells is a useful addition to the current standard tests.     

Evaluation of selected alcoholic antiseptics activity against clinical bacterial strains

The aim of this study was to evaluate antimicrobial activity of selected alcoholic antiseptics against clinical strains, which possessed in majority a high level of drug resistance: MRSA (7), MSSA (3), E. coli: (9): strains producing ESBL (4), P. aeruginosa: (4), E. cloacae: (3), K. pneumoniae: (3). These strains were defined by MIC value, using antibiotic agar dilution method according to NCCLS. Fourteen alcoholic antiseptics were used in this study. Beside alcohol, they contained other active substances like iodine, hydrogen peroxide, chlorhexidine. Some additional agents were included for easier application, such as: gelling, moisturizing, aromatic or coloring substances. The objective of this study was also to determine the dependence of bactericidal activity on preparations (concentration). Product undiluted and diluted two and four times in water was analyzed according to prEN 12054 standard; 30 seconds and 1 minute contact time was used. The obtained data indicate that all tested undiluted antiseptics possessed bactericidal activity described by producers. However antiseptics (dilution leads to decrease and even loss of bactericidal activity. Two-times dilution of gel almost completely inactivated the product. Antimicrobial activity after 30 seconds of contact time was not affected by presence of additional agents in the tested antiseptics.     

A review on biocide reduced susceptibility due to plasmid-borne antiseptic-resistant genes—special notes on pharmaceutical environmental isolates

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.     

Development of a Standard Test To Assess the Resistance of Staphylococcus aureus Biofilm Cells to Disinfectants

A standardized disinfectant test for Staphylococcus aureus cells in biofilms was developed. Two disinfectants, the membrane-active compound benzalkonium chloride (BAC) and the oxidizing agent sodium hypochlorite, were used to evaluate the biofilm test. S. aureus formed biofilms on glass, stainless steel, and polystyrene in a simple system with constant nutrient flow that mimicked as closely as possible the conditions used in the current standard European disinfectant test (EN 1040). The biofilm that was formed on glass contained cell clumps and extracellular polysaccharides. The average surface coverage was 60%, and most (92%) of the biofilm cells were viable. Biofilm formation and biofilm disinfection in different experiments were reproducible. For biofilms exposed to BAC and hypochlorite the concentrations needed to achieve 4-log killing were 50 and 600 times higher, respectively, than the concentrations needed to achieve this level of killing with the European phase 1 suspension test cells. Our results show that a standardized disinfectant test for biofilm cells is a useful addition to the current standard tests.     

Treatment of water-based metalworking fluids to prevent hypersensitivity pneumonitis associated with Mycobacterium spp

Aim: To prevent further outbreaks of hypersensitivity pneumonitis (HP), biocides are required which are capable of protecting water‐based coolants from proliferating mycobacteria. The aim of this study was therefore, to test different biocide preparations on their mycobactericidal activity. Methods and Results: Minimal inhibitory concentration values were determined for Mycobacterium chelonae and Mycobacterium immunogenum for triazine‐based, methyloxazolidine‐based, N/O‐formal‐based biocidal formulations. All biocides were effective already at a low dosage (<0·05%) irrespective of the presence or absence of organic soiling, except for one N/O‐formal‐based formulation containing Kathon 886 (CMI). Quenching of CMI in the presence of organic soiling was found to account for loss in efficacy as determined by high‐performance liquid chromatography measurement. Preservation tests were carried out to investigate the efficacy of the biocidal preparations under practical conditions. Conclusions: Results indicate that methyloxazolidine‐based biocidal preparations were most effective to prevent coolants from microbial contamination including rapidly growing mycobacteria. Furthermore, it could be demonstrated that common dipslides can be used to easily monitor coolants for contamination by mycobacteria. Significance and Impact of the Study: Our data does not support the hypothesis that mycobacterial proliferation is enhanced by the reduction of competitive microbial population by biocides such as triazines as decribed earlier but rather suggests a protective effect of biocides regarding mycobacteria in the presence of competitive microbial flora, thereby preventing further outbreaks of HP.     

A novel approach to mode of action of cationic biocides morphological effect on antibacterial activity

A. KANAZAWA, T. IKEDA AND T. ENDO. 1995 A new concept for the mode of action of cationic biocides is proposed in which the antibacterial activity of cationic disinfectants is ascribed essentially to molecular organizations of cations within aggregates, i.e. the activity is determined by the size of aggregates and number of active molecules comprising the aggregate. On the basis of the new concept, the morphological effect of disinfectants in aqueous solution on the antibacterial activity is discussed for low molecular weight phosphonium salts with single and double long alkyl chains (carbon number 14). The proposed new concept can be applied to all phenomena reported previously in antibacterial activity of cationic biocides and this concept is very important from the viewpoint of molecular design of more active cationic biocides.     

Sporicidal effect of disinfectants on Bacillus cereus isolated from the milk processing environment

The sporicidal efficacy of sodium hypochlorite and a combination of peracetic acid and hydrogen peroxide on Bacillus cereus spores isolated from the milk processing environment was examined using the European Suspension Test and by a surface disinfection test on stainless steel and rubber. The results of the laboratory tests were compared with field trials in a milking installation. In general, it was difficult to obtain consistent results, as the repeatability and reproducibility of the tests were found to vary according to the test strain, spore suspension preparation, disinfectant test solution, organic load, contact time and temperature. The sporulation medium used to obtain spores influenced the sporicidal effect considerably. To overcome this problem a standard method for preparation of spore suspensions should be prescribed. The various disinfectants were more effective in suspensions than on surfaces and in field trials. For the suspension tests SE values ranging from 1.0 to 3.0 were reached within 10 min at 50°C, depending on the disinfectant used. Sodium hypochlorite-based products were most effective. The activity on spores on surfaces and in field trials was limited. In surface tests reductions of 0.4–0.8 were observed within 10 min at 50°C depending on the type of surface. The SE values obtained for rubber were lower compared with stainless steel. The decrease in spore levels found in the milking installation was comparable with the surface experiments, i.e. 0.4–1.0. It is important to develop standard test procedures to assess the sporicidal efficacy of disinfectants used in food hygiene. Surface tests should be included to reflect the in-use conditions more closely and minimum standards should be determined for both suspension tests and surface tests.     

Disinfectant Evaluation by a Capacity Use-dilution Test

SUMMARY: Two testing procedures, B.S. 3286: 1960 and Kelsey & Sykes (1969), have been examined in terms of their reproducibility and ease of operation using different types of disinfectants. The capacity use-dilution test realistically simulated practical conditions and gave reasonable results. It is recommended for use-dilution assessments of general purpose disinfectants.     

Intrinsic and acquired resistance to quaternary ammonium compounds in food-related Pseudomonas spp

AIMS: To determine the sensitivity of a strain used for disinfectants testing (Pseudomonas aeruginosa ATCC 15442) and food-associated isolates to benzalkonium chloride and didecyl dimethylammonium chloride (DDAC). To determine whether the increase in bacterial resistance after adaptation to DDAC can be associated with phenotypic changes. To test the activity of alternative disinfectants to eliminate resistant Pseudomonas spp. METHODS AND RESULTS: Pseudomonas aeruginosa ATCC 15442 was among the most resistant strains tested using a bactericidal suspension test. Growth of a sensitive Ps. fluorescens in gradually higher concentrations of DDAC resulted in stable higher resistance and to some cross-resistance to several antibacterial agents, with the exception of disinfectants containing chloramine T, glutaraldehyde or peracetic acid. It was shown by microscopy that adaptation was followed by loss of flagella, and slime formation. Removal of the slime by sodium dodecyl sulphate resulted in partial loss of the acquired resistance. CONCLUSIONS: Pseudomonas spp. may adapt to survive against higher concentrations of quaternary ammonium compounds (QACs), but resistant strains can be eliminated with chemically unrelated disinfectants. SIGNIFICANCE AND IMPACT OF THE STUDY: The work supports the rotation of disinfectants in food processing environments for avoiding the development of bacterial resistance to QACs. The alternating disinfectants should be chosen carefully, because of possible cross-resistance.