Improved method for tuberculocidal and mycobactericidal activity testing of disinfectants based on the European Standard EN 14348.

Safe measurements to prevent the transmission of (multidrug-resistant) mycobacteria such as disinfection are essential in healthcare settings. In Europe chemical disinfectants are tested for their tuberculocidal and mycobactericidal efficacy by the internationally accepted test procedure described in EN 14348. However, especially for amine-based disinfectants, invalid results may occur by this procedure due to insufficient neutralization. In this multi-laboratory study the procedure described in EN 14348 was optimized by a combination of chemical neutralization and membrane filtration in order to obtain a valid and secure method especially for amine-based disinfectants.

Comparison of a rapid detection fluorescence-based assay targeting tuberculocidal efficacy with EN 14348 and EN 14563.

BACKGROUND: A rapid test system using fluorescent Mycobacterium terrae to evaluate the tuberculocidal efficacy of disinfectants has recently been published. Results were obtained in a significantly shorter time than was previously possible. AIM: To compare the European Standard test system with the fluorescence assay and to validate the rapid test system, including particularly the quantitative suspension test. METHODS: Quantitative suspension tests and quantitative carrier tests were carried out according to EN 14348 and EN 14563, respectively. Quantitative carrier tests and subsequent green fluorescent protein (GFP)-based determination of germicidal efficacy were carried out as described previously. A peracetic acid-based formulation was used as a test germicide. FINDINGS: Testing of the germicide in the quantitative suspension test EN 14348 and in the quantitative carrier test EN 14563 revealed tuberculocidal efficacy at a concentration of 1% after 15 min contact time. Accordingly, data obtained from the fluorescence assay demonstrated that a germicide concentration of 1% was effective after 15 min, indicating no live mycobacteria following this treatment. Thus, identical in-use parameters for tuberculocidal efficacy were obtained either by applying the quantitative suspension and quantitative carrier tests EN 14348 and EN 14563 or by using the GFP-based rapid test system. CONCLUSION: The GFP-based rapid test system compares well with the established European Standard test procedure including both phase 2, step 1 and phase 2, step 2 tests and provides a rapid and sensitive tool for testing germicides for relevant in-use concentrations and contact times.

Impact of the amount of hand rub applied in hygienic hand disinfection on the reduction of microbial counts on hands.

Two different hand rubs were tested in order to investigate the minimum volume required for microbicidal efficacy according to the European Norm EN 1500; we also sought to determine whether there is a correlation with hand size. Eight male volunteers with big hands (mean 184 cm(2)) and eight female volunteers with significantly smaller hands (mean 148 cm(2); P<0.001) participated in our study. Application of 2 mL of both products (P) provided mean log(10) reductions significantly smaller than those of the reference disinfectant (R) (product A: P=3.34, R=4.00, P=0.001; product B: P=3.37, R=3.75, P=0.022). Higher volumes (product A: 3 and 4 mL; product B: 2.5, 3 and 4 mL) ensured that the pass criteria of the European Norm (EN) 1500 were fulfilled. For both products log(10) reductions increased with increasing product volume until a plateau was reached. For the smaller female hands, this plateau level was reached after applying 3 mL of product A and 2.5 mL of product B. The plateau level on male hands was observed after treating the hands with > or =4 mL of product A and 3 mL of product B. The increase in product volume also correlated with the decrease in the number of volunteers considering the product volume applied as insufficient. In conclusion, the applied volume for hygienic hand rub should not fall below 3 mL in order to achieve maximum benefit.

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 described earlier but rather suggests a protective effect of biocides regarding mycobacteria in the presence of competitive microbial flora, thereby preventing further outbreaks of HP.

Attitudes of general dental practitioners in Europe to the microbial risk associated with dental unit water systems.

UNLABELLED: Dental Unit Water Systems (DUWS) are used in dental practices to provide water for cooling of dental equipment and irrigation of the oral cavity. However, they have been demonstrated to be contaminated with micro-organisms. There are currently no European Union (EU) Commission guidelines for the microbial quality of water discharged by DUWS. This study was part of an EU research programme to investigate the microbial contamination of DUWS in general dental practice (GDP) in the UK, Denmark, Germany, The Netherlands, Ireland, Greece and Spain. OBJECTIVE: To undertake a questionnaire survey on the type of DUWS in use and determine the attitude of GDPs to the risk of microbial infection from DUWS. MATERIALS AND METHODS: The questionnaire was written and translated into the language of each country before being posted to each participating dentist. Dentists were asked to complete the questionnaire survey and return it by post. RESULTS AND CONCLUSIONS: The major findings were that the majority of dentists did not clean, disinfect or determine the microbial load of their DUWS, and that dentists would welcome regular monitoring and advice on maintaining their DUWS; the introduction of guidelines; and recommendations on controlling the microbial load of DUWS.

Comparison of the efficacies of disinfectants to control microbial contamination in dental unit water systems in general dental practices across the European Union.

Water delivered by dental unit water systems (DUWS) in general dental practices can harbor high numbers of bacteria, including opportunistic pathogens. Biofilms on tubing within DUWS provide a reservoir for microorganisms and should be controlled. This study compared disinfection products for their ability to meet the American Dental Association's guideline of <200 CFU x ml(-1) for DUWS water. Alpron, BioBlue, Dentosept, Oxygenal, Sanosil, Sterilex Ultra, and Ster4Spray were tested in DUWS (n = 134) in Denmark, Germany, Greece, Ireland, The Netherlands, Spain, and the United Kingdom. Weekly water samples were tested for total viable counts (TVCs) on yeast extract agar, and, where possible, the effects of products on established biofilm (TVCs) were measured. A 4- to 5-week baseline measurement period was followed by 6 to 8 weeks of disinfection (intermittent or continuous product application). DUWS water TVCs before disinfection ranged from 0 to 5.41 log CFU x ml(-1). Disinfectants achieved reductions in the median water TVC ranging from 0.69 (Ster4Spray) to 3.11 (Dentosept) log CFU x ml(-1), although occasional high values (up to 4.88 log CFU x ml(-1)) occurred with all products. Before treatment, 64% of all baseline samples exceeded American Dental Association guidelines, compared to only 17% following commencement of treatment; where tested, biofilm TVCs were reduced to below detectable levels. The antimicrobial efficacies of products varied (e.g., 91% of water samples from DUWS treated with Dentosept or Oxygenal met American Dental Association guidelines, compared to 60% of those treated with Ster4Spray). Overall, the continuously applied products performed better than those applied intermittently. The most effective products were Dentosept and Oxygenal, although Dentosept gave the most consistent and sustained antimicrobial effect over time.

Investigation of the hygienic safety of continuous positive airways pressure devices after reprocessing.

With the widespread use of continuous positive airways pressure (CPAP) therapy, the safety of CPAP devices after reprocessing is the subject of debate. In this study, the contamination of CPAP devices and the effectiveness of disinfection was investigated. A total of 122 CPAP devices were examined including 50 CPAP devices used by patients, which were examined before and after reprocessing. Seventy-two new CPAP devices that had not been in contact with patients served as controls. The results of this study show that the microbial contamination of new and used CPAP devices is only minimal. Contaminating micro-organisms were predominantly micro-organisms reflecting the normal environmental microflora such as Penicillium spp., Aspergillus spp., Micrococcaceae and Bacillaceae. Gram-negative species could only be found in rare cases. The data obtained give no indication of poor disinfection of CPAP devices.

Microbiological evaluation of dental unit water systems in general dental practice in Europe.

A range of opportunistic pathogens have been associated with dental unit water systems (DUWS), particularly in the biofilms that can line the tubing. This study therefore aimed to assess the microbiology of DUWS and biofilms in general dental practices across seven European countries, including the United Kingdom (UK), Ireland (IRL), Greece (GR), Spain (ES), Germany (D), Denmark (DK) and the Netherlands (NL). Water supplied by 51% of 237 dental unit water lines exceeded current American Dental Association recommendations of < or = 200 colony-forming units (CFU) ml(-1). Microbiological loading of the source waters was between 0 (Denmark, the Netherlands and Spain) and 4.67 (IRL) log CFU ml(-1); water line samples from the DUWS ranged from 1.52 (ES) to 2.79 (GR) log CFU ml(-1); and biofilm counts ranged from 1.49 (GR) to 3.22 (DK) log CFU.cm(-2). Opportunistic pathogens such as legionellae (DK and ES), including Legionella pneumophila SG1 (DK and GR), and Mycobacterium spp. (DK, NL, GR, D and ES) were recovered occasionally. Presumptive oral streptococci (ES and NL), oral anaerobes (GR), Candida spp. (UK, NL and ES) and blood (GR and IRL) were detected at relatively low frequencies, but their presence indicated a failure of the 3-in-1 antiretraction valve, leading to back siphonage of oral fluids into the water and biofilm phase. These findings confirm that a substantial proportion of DUWS have high levels of microbial contamination, irrespective of country, type of equipment and source water. The study emphasizes the need for effective mechanisms to reduce the microbial burden within DUWS, and highlights the risk of occupational exposure and cross-infection in general dental practice.

Inactivation of feline calicivirus, a surrogate of norovirus (formerly Norwalk-like viruses), by different types of alcohol in vitro and in vivo.

Hand disinfection is an important measure to prevent transmission of norovirus (formerly called Norwalk-like viruses) from hands or environmental surfaces to other objects. Therefore, three types of alcohol (ethanol, 1- and 2-propanol) were examined for their virus-inactivating properties against feline calicivirus (FCV) as a surrogate for norovirus. Tests were performed as quantitative suspension assays or as in vivo experiments with artificially contaminated fingertips. The in vitro experiments showed that 1-propanol was more effective than ethanol and 2-propanol for the inactivation of FCV: in tests with the 50 and 70% solutions of the different alcohols, a 10(4)-fold reduction was observed with 1-propanol after 30 s, whereas the other alcohols were effective only after 3 min contact time. The greatest efficacy did not occur at the highest concentrations (80%). The following concentrations (extrapolated data) showed the greatest virus-inactivating properties in the suspension test: ethanol 67%, 2-propanol 58% (exposure times of 1 min) and 1-propanol 60% (exposure time of 30 s). The results from fingertips experiments with 70 and 90% solutions and an application time of 30 s confirmed these findings: the 70% alcoholic solutions were more effective than the 90% solutions. In contrast to the suspension tests, 70% ethanol showed the greatest efficacy in vivo with a log(10) reduction factor (RF) of 3.78 compared with 70% 1-propanol (RF 3.58), 70% 2-propanol (RF 2.15) and hard water (RF 1.23). Ethanol and 1-propanol-based solutions with a high alcohol content thus appear most effective.

Effect of biocides on S. cerevisiae: relationship between short-term membrane affliction and long-term cell killing.

The long-term action of recommended (RC) and near-recommended concentrations of several commercial biocides (Lonzabac 12.100, Genamin CS302D, benzalkonium chloride and 2-phenoxyethanol) on cells of S. cerevisiae wild-type strain DTXII was described using plating tests while short-term effects were determined using the potentiometric fluorescent probe diS-C3(3) that detects both changes in membrane potential and impairment of membrane integrity. A 2-d plating of cells exposed to 0.5xRC of benzalkonium chloride and Genamin CS302D for 15 min showed a complete long-term cell killing, with 2-phenoxyethanol the killing was complete only at 2xRC and Lonzabac caused complete killing at RC but not at 0.5xRC. The diS-C3(3) fluorescence assay performed immediately after a 10-min biocide exposure revealed several concentration-dependent modes of action: Lonzabac at 0.5xRC caused a mere depolarization, higher concentrations causing gradually increasing cell damage; benzalkonium chloride and Genamin CS302D rapidly damaged the membrane of some cells and depolarized the rest whereas 2-phenoxyethanol, which had the lowest effect in the plating test, produced a concentration-dependent fraction of cells with impaired membranes. Cell staining slightly increased during the diS-C3(3) assay; addition of a protonophore showed that part of the remaining undamaged cells retained their membrane potential. Comparison of short-term and long-term data implies that membrane depolarization alone is not sufficient for complete long-term killing of yeast cells under the action of a biocide unless it is accompanied by perceptible impairment of membrane integrity. The results show that the diS-C3(3) fluorescence assay, which reflects the short-term effects of a biocide on cell membranes, can be successfully used to assess the microbicidal efficiency of biocides.

Comparison of two test methods for the determination of sufficient antimicrobial activity of three commonly used alcohol-based hand rubs for hygienic hand disinfection.

In Europe, the antimicrobial efficacy of alcohol-based hand rubs is determined with a quantitative suspension test (prEN 12054) and a test under practical conditions (EN 1500). Another test method has recently been published by the German Society for Hygiene and Microbiology (DGHM) with four differences to the European system in the in vitro tests: additional qualitative suspension tests with product dilutions to the ineffective range; a selection of the most resistant Gram-negative test strains in the qualitative suspension test, which should be used adjacent to Pseudomonas aeruginosa in the quantitative suspension test; a high organic load in the quantitative suspension tests (0.3% albumin and 0.3% sheep erythrocytes); and an aqueous control in the quantitative suspension test. According to DGHM, the in vitro tests should be followed by EN 1500. We have determined the antimicrobial efficacy of three commonly used alcohol-based hand rubs according to both methods. prEN 12054 was carried out without organic load. The qualitative suspension tests (DGHM) were carried out with P. aeruginosa, Escherichia coli, Proteus mirabilis, Staphylococcus aureus, Enterococcus hirae and Candida albicans. The quantitative suspension test (DGHM) was carried out with product dilutions of 75%, 50% and 25%, and a high organic load using the following test organisms: P. aeruginosa, P. mirabilis (one product only), S. aureus, E. hirae and C. albicans. All these suspension tests were carried out in quadruplicate with each product and exposure time. EN 1500 was carried out with 3 mL of each product and an application time of 30 s. All three products achieved the required bactericidal activity of prEN 12054 and the new DGHM method within 30 s, and were equally effective with the reference hand disinfection of EN 1500 within 30 s. In our study, the DGHM test method did not provide additional information for hand rubs which exhibit their bactericidal efficacy with 3 mL within 30 s (EN 1500).

Hand disinfection according to the European Standard EN 1500 (hygienic handrub): a study with gram-negative and gram-positive test organisms.

It was the aim of this study to compare the efficacy of alcohol-based hand disinfectants according to European Standard EN 1500 (hygienic handrub), using the routine test organism Escherichia coli and, additionally, Micrococcus luteus as a surrogate for Gram-positive pathogens. One ethanol-based hand disinfectant (product A) and one propanol-based hand disinfectant (product B) were used in all experiments. Product B (propanol-based) was significantly more effective against both test organisms than product A (ethanol-based) in quantitative suspension tests but not in tests simulating practical conditions. In the experiments according to EN 1500 germ reduction rates obtained with the ethanol-containing formulation A were identical for E. coli and M. luteus. Product B was slightly, but not significantly more effective against M. luteus. To conclude, using E. coli as the test organism for evaluating the antibacterial efficacy of alcoholic hand disinfectants under practical conditions even appears to be sufficient to permit the drawing of conclusions for Gram-positive pathogens. However, more alcohol-based hand disinfectants should be tested in further studies to verify the results obtained.

Investigation into the efficacy of disinfectants against MRSA and vancomycin-resistant enterococci.

In the study reported here, several disinfectants for surfaces, instruments, hands and mucous membrane were tested for their effectiveness against methicillin-resistant strains of S. aureus and vancomycin-resistant enterococci. The results show that after use of the recommended concentrations with the recommended contact times, which were determined on the basis of the criteria for disinfectants according to the DGHM guideline for the testing and evaluation of chemical disinfection procedures, an adequate effect was able to be proved. Increased resistance of the methicillin-resistant S. aureus and vancomycin-resistant E. faecium strains to the tested disinfectants was not found. Only after use of diluted solutions differences in the bacterial count reductions were observed. In further studies it is to be investigated whether these differences are due to resistance to antibiotics or to intrinsic resistance.

[Effectiveness of peroxide solutions against microorganisms in biofilms]. / Wirksamkeit von peroxidhaltigen Wirkstofflösungen gegen Mikroorganismen in Biofilmen.

Central dosing units for surface disinfectants can constitute a particular hygiene problem in hospitals if a biofilm can develop in the piping system despite the disinfectant solution that is present. In earlier studies it was able to be shown that oxygen-releasing compounds in particular were highly effective against microorganisms in generated biofilms. The purpose of the study presented was to investigate in a test simulating practical conditions whether, in addition to the described good effect, peroxide-containing solutions also have a uniform effect against all isolates from disinfectant use-solutions, or whether there are differences. The results show that a 1% H2O2 solution (C) was not sufficiently effective against all test organisms after a contact time of one hour. In contrast, the 1% test solution of A (10% perglutaric acid, 28% H2O2, < 0.5% perbenzoic acid) was highly and uniformly effective. The 3% test solution of B (10% tertiary butylhydroperoxide, 20% phenoxypropanols, 48% dipropylene glycol) exhibited weaknesses against some test organisms after the one-hour contact time. After a contact time of three hours, this solution, unlike the 1% H2O2 solution achieved a reduction in bacterial count of more than 5 log steps against all species. On the basis of these results, before cleaning piping systems it appears advisable to test the effectiveness of the solution to be used in a suitable test with the isolates.

[Biologic degradation of phenoxypropanolene. Biodegradability of phenoxypropanols]. / Biologische Abbaubarkeit von Phenoxypropanolen.

Phenoxypropanols have been used for some time as ingredients in surface and instrument disinfectants. Since the residual solutions of these preparations are discharged into the waste-water, the biodegradability of the ingredients used is of great importance. Tests by means of the Zahn-Wellens test in accordance with OECD guidelines have shown that phenoxypropanols are biodegradable. In further studies it was investigated whether unwanted intermediate products are formed in considerable amounts. Of particular interest was the question of the possible development of phenol outside the bacterial cells. To test the biodegradability of the aromatic alcohols and to ascertain whether there is extracellular formation of phenol, microorganisms were isolated from the municipal sewage plant at Stellinger Moor, Hamburg, and from water from the River Elbe. Phenoxypropanol-containing solutions were then inoculated with the bacteria and incubated at 30 degrees C. At regular intervals both the concentration of the alcohols was determined and the test for phenol was performed by means of liquid chromatography on reverse phase material (RP 18) and detection with a photodiode array detector. The breakdown of the phenoxypropanols was monitored for a period of up to 4 weeks. During this period a clear breakdown of the aromatic alcohols was observed. No phenol was detected.