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.     

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。     

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.     

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...     

Disinfection and hygiene in the veterinary field and disinfection of animal houses and transport vehicles

Disinfection in animal houses means always a combination between cleaning and disinfection because the high amount of organic material present in such an environment will neutralize rapidly each disinfectant brought to the surface if no cleaning step had been done before. Depending on the kind of material, cleaning gives a 3 log reduction of total bacterial count on the surface and disinfection another 3 log reduction. This means that under practical conditions generally 10³ cfu of bacteria remain per cm² of surface, mostly sporeformers. The choice of disinfectant depends on the purpose of disinfection. In the case of notifiable diseases, it must be active against a defined pathogen. In the case of prophylactic disinfection, it must be active against a broad spectrum of microorganisms.Only disinfectants which are tested for their activity on surfaces which are representative for animal houses e.g. wood should be used, otherwise the failure under practical conditions can be predicted and work, as well as money, will be wasted. The optimal temperature for the liquids used in cleaning and disinfection is 40°C and the optimal temperature of surfaces is 20°C. Colder surfaces require higher concentrations of active substances in the solution, below 10°C the effect of practical disinfection is incomplete. Low air humidity and high air velocity have a negative influence on the action of most disinfectants on surfaces. The amount of disinfectant necessary to disinfect a surface in an animal house is at least 0.4 l/m². Transport vehicles are very difficult to disinfect, especially in winter-time. They should be cleaned with hot water, the remaining liquid should be removed by a suitable vacuum cleaner and the concentration of the disinfectant should be elevated at least for the factor 3. Mechanic action will improve the disinfecting effect in vehicles as well as preliminary disinfection prior cleaning.     

Efficacy of a variety of disinfectants against Listeria spp.

The efficacy of 14 disinfectants against Listeria innocua and two strains of Listeria monocytogenes in the presence of organic matter was studied. Quantitative efficacy tests were used. Many of the disinfectants tested were not as effective on Listeria spp. when the test organisms were dried onto the surface of steel disks (carrier tests) as they were when the organisms were placed in suspension (suspension test). The presence of whole serum and milk (2% fat) further reduced the disinfectant capacities of most of the formulations studied. Only three disinfectants (povidone-iodine, chlorhexidine gluconate, and glutaraldehyde) were effective in the carrier test in the presence of serum; however, all three were ineffective when challenged with milk (2% fat). Only one solution, sodium dichloroisocyanurate, was effective in the presence of milk. All but four formulations (chloramine-T, phosphoric acid, an iodophor, and formaldehyde) were effective in the suspension tests, regardless of the organic load. L. monocytogenes was observed to be slightly more resistant to disinfection than L. innocua was. There was no difference in disinfectant susceptibility between the two strains of L. monocytogenes. These findings emphasize the need for caution in selecting an appropriate disinfectant for use on contaminated surfaces, particularly in the presence of organic material.     

Efficacy of a variety of disinfectants against Listeria spp

The efficacy of 14 disinfectants against Listeria innocua and two strains of Listeria monocytogenes in the presence of organic matter was studied. Quantitative efficacy tests were used. Many of the disinfectants tested were not as effective on Listeria spp. when the test organisms were dried onto the surface of steel disks (carrier tests) as they were when the organisms were placed in suspension (suspension test). The presence of whole serum and milk (2% fat) further reduced the disinfectant capacities of most of the formulations studied. Only three disinfectants (povidone-iodine, chlorhexidine gluconate, and glutaraldehyde) were effective in the carrier test in the presence of serum; however, all three were ineffective when challenged with milk (2% fat). Only one solution, sodium dichloroisocyanurate, was effective in the presence of milk. All but four formulations (chloramine-T, phosphoric acid, an iodophor, and formaldehyde) were effective in the suspension tests, regardless of the organic load. L. monocytogenes was observed to be slightly more resistant to disinfection than L. innocua was. There was no difference in disinfectant susceptibility between the two strains of L. monocytogenes. These findings emphasize the need for caution in selecting an appropriate disinfectant for use on contaminated surfaces, particularly in the presence of organic material.     

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.     

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。     

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.     

A comparison of the efficacy of different disinfection methods in eliminating Salmonella contamination from turkey houses

Aims: This study aimed to compare the efficacy of different disinfection methods in eliminating Salmonella contamination from turkey houses. Methods and Results: Fifty depopulated turkey houses which had all housed Salmonella‐positive flocks were visited after cleaning and disinfection. A minimum of 45 swab samples from different surfaces were taken per house and analysed for the presence of Salmonella. The sampled surfaces included intact floor surfaces, floor cracks, walls, feeders, drinkers, anteroom, nestboxes and miscellaneous items. Houses were grouped according to the disinfectant which had been used and the efficacy of the different groups of disinfectants was compared. Sixty‐eight % of houses tested positive for Salmonella after C&D. Out of 4440 samples, 207 tested positive for Salmonella, giving an overall sample prevalence of 4·7%. There was no significant difference in the level of residual contamination between breeding, rearing and finishing houses. Products containing a mixture of formaldehyde, glutaraldehyde and quaternary ammonium compounds (QAC) performed significantly better than products containing hydrogen peroxide and peracetic acid. Cleaning and disinfection was least effective in nestboxes and anterooms. Conclusions: Thorough cleaning and the choice of a suitable disinfectant are crucial if Salmonella contamination of turkey houses is to be eliminated. Significance and impact of the study: This study shows that disinfectants containing a mixture of formaldehyde, glutaraldehyde and QAC perform significantly better under field conditions than oxidising products and should therefore be the first choice for disinfection of turkey premises where Salmonella is present.     

医院公用电话病原菌污染调查及消毒模式

目的:调查医院公用电话污染情况,比较两种不同消毒液对公用电话的消毒效果。方法:分别对公用电话听筒、话筒及手柄(打接电话时手握部位)在未消毒和A组用复方新洁灵、B组用"84"消毒液消毒后分别涂抹采样做细菌培养。结果:未消毒前电话听筒、话筒及手柄细菌污染率A组为96.7%、B组为93.3%,A组用复方新洁灵、B组用"84"消毒液消毒后做细菌培养均符合卫生学标准要求,经x2检验,P<0.01,差异具有统计学意义。结论:未消毒前电话听筒、话筒及手柄细菌污染率90%以上,而复方新洁灵和"84"消毒液对医院公用电话均可以用来消毒,两种消毒剂的消毒效果无明显差异,临床可根据具体情况选用。     

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.     

Effect of biocides on Pseudomonas aeruginosa phage F116

Pseudomonas aeruginosa phage F116 is of interest in understanding the virucidal mechanisms of disinfectant action. Phage F116 has been used to test several disinfectants. The bacteriophage was relatively resistant to several biocides commonly used in disinfection processes. Only 0.05% cetylpyridinium chloride, 0.1% peracetic acid and 2% phenol were highly active against the phage.     

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 rapid method for assessing the suitability of quenching agents for individual biocides as well as combinations

AIMS: To develop a novel, rapid method for testing the ability of quenching agents to neutralize disinfectants. METHODS AND RESULTS: Tests were performed to determine the suitability of different neutralizers for a range of disinfectants, using a new method based on the Bioscreen optical density analyser. Results showed that during disinfection tests, efficacy could be over-estimated due to poor, or no, neutralization of the disinfectant after a specified time of exposure to the bacteria. The failure to distinguish adequately between bacteriostatic and bactericidal effects can lead to false results during disinfectant testing. Experiments also showed that dilution of the disinfectant, following exposure to the bacteria, was not always sufficient to stop the activity of the disinfectant for chemicals with low dilution coefficients. CONCLUSIONS: The quench test proved to be very quick and easy to perform, with results being available within 18 h. Using the Bioscreen, the test is automated and determines whether dilution into a particular neutralizer is able to inactivate a disinfectant within 30 s. SIGNIFICANCE AND IMPACT OF THE STUDY: This new approach allows the efficacy of quenching agents to be determined, prior to undertaking each disinfection study, and can help in the development of more suitable quenching solutions. The test has also been used to find suitable neutralizers for mixtures of disinfectants which might be used during studies on synergistic biocide combinations.     

Known data on the effectiveness of silver nano particles on root canal disinfection

The goal of endodontic treatment is the debridement and removal of the microbial ecosystem associated with the disease process. The need for root canal disinfectants increases especially in those cases where infection is resistant to the regular treatment and the outcome of endodontic therapy is often compromised. Therefore, it is of interest to document the known effectiveness of silver nanoparticle based root canal disinfectants with other root canal disinfectants on microbial load reduction during root canal disinfection. Known data shows that the overall risk of bias for the selected studies was moderate. Silver nanoparticle based root canal disinfectants showed superior reduction of microbial counts in majority of the studies. This data is limited to vitro studies with no clinical information to validate the use of antimicrobial properties of silver nanoparticles used as root canal disinfectant.     

A conductance-based surface disinfection test for food hygiene

A surface disinfectant test method for food hygiene is described in which biocidal action was assessed using bacterial biofilms developed on stainless steel. The viability of control and surface-bound treated biofilms was assessed, using a Malthus microbiological growth analyser. The surface test was compared with the European Suspension Test on three bacteria, Pseudomonas aeruginosa, Staphylococcus aureus and Proteus mirabilis , for 12 commonly used food surface disinfectants. In all cases the bacteria were shown to be up to 10 times more resistant to biocides, and in seven cases up to 100 times more resistant, when surface-attached. Disinfectants that performed well against bacteria in suspension did not necessarily perform well against the same bacteria when attached to surfaces.     

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.     

Characterization of micro-organisms isolated from dairy industry after cleaning and fogging disinfection with alkyl amine and peracetic acid

AIMS: To characterize micro-organisms isolated from Norwegian dairy production plants after cleaning and fogging disinfection with alkyl amine/peracetic acid and to indicate reasons for survival. METHODS AND RESULTS: Microbial samples were collected from five dairy plants after cleaning and fogging disinfection. Isolates from two of these production plants, which used fogging with alkylamino acetate (plant A), and peracetic acid (plant B), were chosen for further characterization. The sequence of the 16S ribosomal DNA, fatty acid analysis and biochemical characteristics were used to identify isolates. Three isolates identified as Rhodococcus erythropolis, Methylobacterium rhodesianum and Rhodotorula mucilaginosa were isolated from plant A and one Sphingomonas sp. and two M. extorquens from plant B. Different patterns of resistance to seven disinfectants in a bactericidal suspension test and variable degree of attachment to stainless steel were found. The strains with higher disinfectant resistance showed lower degree of attachment than susceptible strains. CONCLUSIONS: The study identifies and characterizes micro-organisms present after cleaning and fogging disinfection. Both surface attachment and resistance were shown as possible reasons for the presence of the isolates after cleaning and disinfection. SIGNIFICANCE AND IMPACT OF THE STUDY: These results contribute to the awareness of disinfectant resistance as well as attachment as mechanisms of survival in dairy industry. It also strengthens the argument of frequent alternation of disinfectants in the food processing industry to avoid the establishment of resistant house strains.