Test Method for the Evaluation of Virucidal Efficacy of Three Common Liquid Surface Disinfectants on a Simulated Environmental Surface

The Association of Official Analytical Chemists bacterial use-dilution test for evaluating liquid surface disinfectants was modified to determine the efficacy of three common environmental germicide compounds against representatives of four virus groups. Modifications were made to conform to the Environmental Protection Agency guidelines on virucidal testing procedures. Alterations included: the use of a concentrated tissue culture preparation of virus instead of a standard bacterial culture; HEp-2 tissue culture cells as a visible test system in place of standard nutrient broth; and controls to measure the virus titer end point and the toxicity of the disinfectant to the cell cultures. Comparison of control end points with results from the test proper were the measure of the effectiveness of the germicides against the viruses. Results are described which agree with those based on other methods previously reported.     

Evaluation of Disinfectants for Hospital Housekeeping Use

A use-dilution procedure and screening method are proposed to aid the hospital bacteriologist in selecting the most effective disinfectants. Sixteen disinfectants were tested with and without organic material on six different organisms. Sporadic results usually obtained with quaternaries tested by the procedure of the Association of Official Agricultural Chemists (AOAC) were eliminated by the methods of this study. The use-dilution procedure employs the material upon which the disinfectant is to be used, rather than stainless steel, as in the AOAC use-dilution test. The importance of testing each disinfectant against several organisms and in the presence of organic material is discussed.     

A suspension method to determine reuse life of chemical disinfectants during clinical use

In-use testing of disinfectants is necessary to ensure efficacy over time. The current official procedure for testing disinfectants, the Association of Official Analytical Chemists (AOAC) use-dilution method, cannot be adapted to repeated sampling techniques of use-life testing. It is therefore necessary to use an alternative method when evaluating the activity of a disinfectant under actual use. The Clinical Research Associates (CRA) suspension method was developed to fill this need. It consists of adding 0.5 ml of a standard culture to 5.0 ml of test disinfectant and sampling the mixture after 10 min for surviving bacteria. When this test was compared with the AOAC use-dilution method under a simulated use situation, the two methods were generally equivalent in identifying disinfectant inactivation. In addition, the CRA method was less time consuming, easier to perform, and less variable than the AOAC method. Use of the CRA method in a clinical study demonstrated the need for reuse claims to be based on clinical use studies rather than on laboratory testing only.     

A suspension method to determine reuse life of chemical disinfectants during clinical use.

In-use testing of disinfectants is necessary to ensure efficacy over time. The current official procedure for testing disinfectants, the Association of Official Analytical Chemists (AOAC) use-dilution method, cannot be adapted to repeated sampling techniques of use-life testing. It is therefore necessary to use an alternative method when evaluating the activity of a disinfectant under actual use. The Clinical Research Associates (CRA) suspension method was developed to fill this need. It consists of adding 0.5 ml of a standard culture to 5.0 ml of test disinfectant and sampling the mixture after 10 min for surviving bacteria. When this test was compared with the AOAC use-dilution method under a simulated use situation, the two methods were generally equivalent in identifying disinfectant inactivation. In addition, the CRA method was less time consuming, easier to perform, and less variable than the AOAC method. Use of the CRA method in a clinical study demonstrated the need for reuse claims to be based on clinical use studies rather than on laboratory testing only.     

An Examination of the Effect of Three Phenolic Disinfectants on Mycobacterium tuberculosis

The effect of a phenolic disinfectant ( o -phenylphenol 45% w/w) with linseed oil soap or with soya oil soap on Mycobacterium tuberculosis was determined by three methods. Neither the geometrical dilution test nor the modified capacity use-dilution test revealed any differences between the two disinfectants. However, paradoxically both methods proved that the highest concentration of the disinfectants tested (3·5% v/v) exhibited a very low germicidal effect on M. tuberculosis , whereas lower concentrations showed a much better effect. When determining the tuberculocidal effects of various concentrations of the two disinfectants at different exposure times, the higher concentrations showed very low effects, even after the longest exposure time. At concentrations of 2·0 and 1·0% (v/v), the disinfectants displayed the most rapid effects. In the present investigation the disinfectant with the linseed oil soap seemed to destroy the cells more quickly than that with the soya oil soap. The third disinfectant containing p -chloro- m -cresol and o -benzyl- p -chlorophenol with a total of 9·2% (w/w) phenols in a detergent system, did not display, when employing the capacity use-dilution test, the same phenomenon in the concentrations used, but the experiment showed that the recommended use-dilution concentration ought to be doubled.     

Efficacy of some commerical disinfectants against the bacterial isolates from a poultry slaughterhouse in Turkey

In this study, efficacy of seven different commercial disinfectant preparations was investigated against characteristic bacteria of a poultry slaughterhouse in Ankara (Turkey) by using paper disc-agar diffusion method and surface effectiveness test. According to paper disc-agar diffusion method, some disinfectants had wide efficacy against the test bacteria. The disinfectant effectiveness generally increased by the increasing of disinfectant concentration. However, some disinfectants were ineffective even though at their highest concentrations. The most effective disinfectant was B which contains QAC as active agent.Staphylococcus spp.,Staphyloccus aureus andEscherichia coli were the most sensitive bacteria to the disinfectants. Some pathogenic isolates, especiallySalmonella spp. andCampylobacter spp., were the most resistant ones to many of the disinfectants tested. The results of paper disc-agar diffusion method indicated the importance of characteristic bacterial strains of food plants as the test bacteria for disinfectant efficacy tests. Conversely of paper disc-agar diffusion method, all disinfectants were effective against the isolates by surface effectiveness test depending on exposure time. The disinfectants, except A and F, produced at least 3 log unit reduction during 5 min of exposure. However, all disinfectants at their lowest concentrations were effective against all tested bacteria during 15 and 30 min by this test.     

A Comparative Investigation of the Bactericidal and Fungicidal Effects of Three Phenolic Disinfectants

Two methods, a capacity use-dilution test and another employing geometrical dilution, are used when comparing the bactericidal and fungicidal effects of a phenolic disinfectant containing 45% (w/w) o -phenylphenol in an aqueous soap solution of linseed oil (soap content 6·5% w/v) with the corresponding effects of the same phenolic compound in an aqueous soya oil soap solution (soap content 3·5% w/v). The soya oil soap did not change the disinfectant capacity on the different test organisms used. For the most resistant strain ( Staphylococcus aureus ) the usedilution concentration was evaluated to be slightly above 1%, i.e. 2%, which is much lower than the recommended use-dilution concentration. However, using the same method and test organisms the capacity use-dilution testing of a third phenolic disinfectant, containing p -chloro- m -cresol and o -benzyl- p -chlorophenol with a total of 9·2 (w/w) phenols in a detergent system, indicated that the recommended use-dilution concentration should be doubled.     

Wide variation in effectiveness of laboratory disinfectants against bacteriophages

Aims: The purpose of this study was to identify an effective disinfectant for the inactivation of the bacteriophages (phages) being used in our laboratory, as published studies on phage inactivation are far from unanimous in their conclusions. Methods and Results: The phages studied were three closely related strains of Myoviridae and three strains of Siphoviridae. Three disinfectants which are used commonly in microbiology laboratories were evaluated: Virkon (1%), ethanol (75%) and sodium hypochlorite (2500 ppm available chlorine). The most effective of these was Virkon, which inactivated all six phages rapidly. Ethanol was effective against the Myoviridae but had little effect on the Siphoviridae. Sodium hypochlorite was the least effective of the disinfectants evaluated. Conclusions: The findings of this study demonstrate a wide diversity in the effectiveness of disinfectants tested for inactivation of phages. Significance and Impact of the Study: Of the disinfectants tested Virkon is the most suitable choice for those unable to carry out disinfection validation studies, or where a broad spectrum disinfectant against phages is required. All of the phages in this study showed resilience to inactivation by sodium hypochlorite, and therefore this disinfectant is an unwise choice for use against phage without first assessing its effectiveness.     

The testing of disinfectants

Although all disinfectant tests have the same final purpose, namely measuring the antimicrobial activity of a chemical substance or preparation, a large number of testing methods has been described. They are subdivided into suspension tests, carrier and surface disinfection tests and other practice-mimicking tests. The suspension tests comprise qualitative and quantitative suspension tests, and, as derived tests, the determination of the phenol coefficient and capacity tests. There is an essential difference between a carrier test and a surface disinfectant test: in the former case the carrier is submerged in the disinfectant solution during the whole exposure time, whereas in the latter case the disinfectant is applied on the carrier for the application time and thereafter the carrier is drying during the exposure.The basic principle now widely accepted is that the antimicrobial efficiency of a disinfectant is examined at three stages of testing.The first stage concerns laboratory tests in which it is verified whether a chemical compound or a preparation possesses antimicrobial activity. For these preliminary screening tests, suspension tests are considered.In the second stage of tests, disinfection procedures and not disinfectants are examined. It is determined under which conditions and at which use-dilution for a given application the preparation is active: the tests simulate real-life situations; such tests are carrier tests for the disinfection of materials by submersion and surface disinfection tests.The last stage takes place in the field, and comprises the in-loco or in-situ tests with as variants the in-use tests, which examine whether, after a normal period of use, germs are still killed by the disinfectant solution.It is the task of the European and international standardization organisations to develop new standards and to elaborate tests, which predict the effectiveness of a preparation in practice under variable circumstances.     

A Practical Approach to Evaluation of the Germicidal Efficiency of a General Purpose Military Disinfectant

The bactericidal activity of a general purpose disinfectant consisting of 25% sodium-o-phenylphenolate and 75% sodium-4- and 6-chloro-2-phenylphenolate was evaluated by a simulated in-use, surface-square dilution method. Common floor (asphalt, rubber, and unglazed tiles) and wall (stainless steel tile, ceramic tile, and painted wood) surfaces of various porosities and compositions were selected to simulate actual-use conditions.

The method used consisted of inoculating the surfaces of 1-in. square sections of floor and wall covering with a test organism, air-drying the inoculated surface, applying the disinfectant, allowing it to act for 10 min, and recovering the survivors by plating. Confirmatory results of the standard phenol coefficient and use-dilution tests indicated 700 ppm of the disinfectant to be a safe use concentration. The in-use surface-square dilution studies have shown that this is a more than adequate safe concentration for stainless steel, both glazed and unglazed ceramic tile, and nonwaxed surface of asphalt tile. However, concentrations ranging between 2,500 and 6,000 ppm for plastic-fortified rubber tile, 1,500 and 2,000 ppm for waxed asphalt tile, and 2,000 ppm for painted wood were required to achieve 99.9% reduction of either Salmonella choleraesuis or Salmonella schottmuelleri. These results indicate that a disinfectant concentration derived from the Association of Official Agricultural Chemists use-dilution test cannot always be relied upon to provide a dependable index to actual safe use-dilution when a disinfectant is supplied to certain wall or floor surfaces.

     

Pseudomonas pellicle in disinfectant testing: electron microscopy, pellicle removal, and effect on test results.

Pseudomonas aeruginosa ATCC 15442 is a required organism in the Association of Official Analytical Chemists use-dilution method for disinfectant efficacy testing. When grown in a liquid medium, P. aeruginosa produces a dense mat or pellicle at the broth/air interface. The purpose of this investigation was to examine the pellicle by scanning electron microscopy, to evaluate three pellicle removal methods, and to determine the effect of pellicle fragments on disinfectant efficacy test results. The efficacies of three methods of pellicle removal (decanting, vacuum suction, and filtration) were assessed by quantifying cell numbers on penicylinders. The Association of Official Analytical Chemists use-dilution method was used to determine whether pellicle fragments in the tubes used to inoculate penicylinders affected test results. Scanning electron micrographs showed the pellicle to be a dense mass of intact, interlacing cells at least 10 microns thick. No significant differences in pellicle removal methods were observed, and the presence of pellicle fragments usually increased the number of positive tubes in the use-dilution method significantly.     

Disinfection in Food Processing – Efficacy Testing of Disinfectants

The key to effective cleaning and disinfection of food plants is the understanding of the type of the soil to be removed from the surfaces. An efficient cleaning and disinfection procedure consists of a sequence of rinses using good quality water with application of detergents and disinfectants. Disinfection is required in food plant operations, where wet surfaces provide favourable conditions for the growth of microbes. The efficacy of disinfectants is usually determined in suspensions, which do not mimic the growth conditions on surfaces where the agents are required to inactivate the microbes. Therefore, the suspension tests do not give adequate information and reliable carrier tests, which mimic surface growth, are needed. In developing a proposal for the testing of disinfectants on surfaces to an analytical standard, it is important to identify the major sources of variation in the procedure. In response to the need for a relatively realistic, simple and reliable test for disinfectant efficacy a method for culturing laboratory model biofilms has developed. The use of artificial biofilms i.e. biofilm-constructs inoculated with process contaminants in disinfectant testing can also be used for screening the activity of various disinfectants on biofilm cells. Both biofilm carrier tests showed clearly that the biofilm protects the microbes against the disinfectants. The chemical cleanliness is also essential in food plants. The total cleanliness of the process lines is mainly based on measuring the microbial load using culturing techniques. These results can give an incorrect picture of the total cleanliness, because the viable microbes do not grow when disinfectants are left on the surface. The luminescent bacteria light inhibition method offers a useful alternative for testing chemical residue left on surfaces after cleaning and disinfection operations.     

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.     

Pseudomonas pellicle in disinfectant testing: electron microscopy, pellicle removal, and effect on test results

Pseudomonas aeruginosa ATCC 15442 is a required organism in the Association of Official Analytical Chemists use-dilution method for disinfectant efficacy testing. When grown in a liquid medium, P. aeruginosa produces a dense mat or pellicle at the broth/air interface. The purpose of this investigation was to examine the pellicle by scanning electron microscopy, to evaluate three pellicle removal methods, and to determine the effect of pellicle fragments on disinfectant efficacy test results. The efficacies of three methods of pellicle removal (decanting, vacuum suction, and filtration) were assessed by quantifying cell numbers on penicylinders. The Association of Official Analytical Chemists use-dilution method was used to determine whether pellicle fragments in the tubes used to inoculate penicylinders affected test results. Scanning electron micrographs showed the pellicle to be a dense mass of intact, interlacing cells at least 10 microns thick. No significant differences in pellicle removal methods were observed, and the presence of pellicle fragments usually increased the number of positive tubes in the use-dilution method significantly.     

Bactericidal activities of selected organic N-halamines

The bactericidal efficacies of three organic N,N'-dihalamine disinfectants in the class of compounds termed imidazolidinones were determined for combinations of pH, temperature, and water quality treatments by using Staphylococcus aureus and Shigella boydii as test organisms. The compound 1,3-dibromo-4,4,5,5-tetramethyl-2-imidazolidinone was found to be the most rapidly acting bactericide, especially under halogen-demand-free conditions. The mixed N,N'-dihalamine 1-bromo-3-chloro-4,4,5,5-tetramethyl-2-imidazolidinone was found to be intermediate in terms of rate of disinfection, while the compound 1,3-dichloro-4,4,5,5-tetramethyl-2-imidazolidinone was observed to be the slowest acting bactericide. When overall effectiveness was judged on the basis of stability of the disinfectants along with rates of disinfection, the mixed halamine was considered to exhibit great potential for use as a disinfectant in an aqueous solution.     

多药耐药大肠杆菌对不同消毒剂的抗性探究

目的：研究多药耐药大肠杆菌对不同消毒剂的抗力水平,为临床上合理选择消毒剂提供依据。方法：采用营养肉汤稀释法及悬液定量杀菌实验分别测定临床分离出的具有多重耐药性的大肠杆菌对临床常用消毒剂的最低抑茵浓度（minimum inhibitory concentration,MIC）及不同作用时间消毒剂对菌株的杀灭率,并与大肠杆菌标准株的测定结果进行比较。结果：不同消毒剂对多药耐药大肠杆菌的MIC值不同,洁芙柔消毒凝胶的MIC值为2．8±1．9,绿莎新爱尔施牌消毒片的MIC值为2583．4±1107．2,爱护佳免洗手消毒液的MIC值为144．63：86．9,安尔碘II型皮肤消毒剂的MIC值为16．1±7．7。4种常用消毒剂对多要耐药大肠杆菌的MIC值均高于标准菌,绿莎新爱尔施牌消毒片及爱护佳免洗手消毒液的MIC值与标准大肠杆菌较为接近。另外,4种常用消毒剂在常用浓度下对标准大肠杆菌和多药耐药大肠杆菌作用5min的杀灭率均能打到100％,标准大肠杆菌的被杀灭率略高于多药耐药大肠杆菌。结论：多药耐药菌株对消毒剂的抗性日益受到关注,使用时调整浓度、加强消毒剂科学管理对控制医院感染具有重要意义。     

Surface-Dried Viruses Can Resist Glucoprotamin-Based Disinfection

Touching of contaminated objects and surfaces is a well-known method of virus transmission. Once they are attached to the hands, viruses can easily get adsorbed and initiate infection. Hence, disinfection of frequently touched surfaces is of major importance to prevent virus spreading. Here we studied the antiviral activity of a glucoprotamin-containing disinfectant against influenza A virus and the model virus vaccinia virus (VACV) dried on inanimate surfaces. The efficacy of the surface disinfectant on stainless steel, polyvinyl chloride, and glass coupons was investigated in a quantitative carrier test. Vacuum-dried viruses were exposed to 0.25%, 0.5%, and 1% disinfectant for 5 min, 15 min, and 30 min without agitation, and residual infectivity was determined by endpoint titration. Although glucoprotamin was highly active against both viruses in suspension, limited antiviral activity against the surface-dried viruses was detected. Even after 30 min of exposure to 1% disinfectant, VACV was not completely inactivated. Furthermore, influenza A virus inactivation was strongly affected by the surface composition during the 5-min and 15-min treatments with 0.25% and 0.5% disinfectant. The results presented in this study highlight the relevance of practical tests to assess the antiviral activity of surface disinfectants. High virucidal activity in solution is not necessarily indicative of high antiviral activity against surface-dried viruses. In addition, we want to emphasize that the mere exposure of surfaces to disinfectants might not be sufficient for virus inactivation and mechanical action should be applied to bring attached viruses into contact with virucidal compounds.     

Comparative testing of disinfectants using proposed European surface test methods

A surface test method for disinfectants currently under development as a standard European test method is described. The test involves determining the number of viable organisms recoverable from a contaminated stainless steel surface before and after application of disinfectant. Evaluation of a range of disinfectant agents and products currently used in the UK indicated that products at recommended use concentrations produced log reductions in viable count ranging from < 1.0 to > 6.4 (i.e. no detectable survivors) after a 5 min contact period against Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecium and Candida albicans. Increased activity was observed by increasing the disinfectant contact time to 30 min. Addition of 3% w/v albumin to the test suspension used to inoculate surfaces caused a substantial reduction in activity.     

微生物消毒剂抗性机理

在物体表面和传播介质中,消毒剂能有效抑制或杀死微生物,广泛用于食品、卫生、健康、防疫等领域.在新型冠状病毒肺炎(COVID-19)疫情期间,全球消毒剂的使用量激增,对有效防控病毒传播和防止疫情扩散起到重要作用.但消毒剂的不正确使用会降低其有效性,甚至会诱导微生物产生抗性,从而增加传染性疾病的传播风险.微生物的消毒剂抗性...     

综述与专论：微生物消毒剂抗性机理

在物体表面和传播介质中,消毒剂能有效抑制或杀死微生物,广泛用于食品、卫生、健康、防疫等领域。在新型冠状病毒肺炎(COVID-19)疫情期间,全球消毒剂的使用量激增,对有效防控病毒传播和防止疫情扩散起到重要作用。但消毒剂的不正确使用会降低其有效性,甚至会诱导微生物产生抗性,从而增加传染性疾病的传播风险。微生物的消毒剂抗性基因还会通过繁殖传代增殖或在不同种属间水平转移而加剧其污染和传播风险,严重威胁到公共卫生安全。目前,抗生素抗性基因(ARG)的广泛出现引起了全球对公共卫生的关注,但对消毒剂的抗性认识非常有限。本文综述了近年来微生物对消毒剂抗性的研究,着重就微生物通过形成生物膜、降低细胞膜通透性、过量表达外排泵、产生消除或减弱消毒剂的特异性酶、改变作用靶点等方式产生抗性的机理进行综述。另外针对微生物消毒剂抗性的获得和传播,对染色体和质粒介导的抗性基因、环境中微生物消毒剂抗性与抗生素抗性的关联进行了论述。消毒剂抗性基因能通过质粒、噬菌体等可移动遗传元件,以转化、转导或接合的方式转移传播,对科学消毒提出新要求。