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.     

Use-Dilution Test and Newcastle Disease Virus

The use-dilution test for evaluating the effectiveness of disinfectants against bacteria was modified to determine the effectiveness of disinfectants against a group of viruses. Modifications were kept to a minimum to retain the general principles of the test and thereby retain the test's familiarity among testing laboratory personnel. Modifications included the use of a standard allantoic fluid suspension of Newcastle disease virus instead of a standard bacterial culture. The only other modification was the inoculation of six embryonated chicken eggs (10 to 12 days old) with 0.1 ml of nutrient broth into which a carrier ring was transferred after a standard period in diluted disinfectant. The death or survival of 60 embryos, then, is the criterion by which a disinfectant can be judged effective at use-dilution. Experiments are described which establish the validity of the modified test procedure. The effectiveness of nine common disinfectants against Newcastle disease virus as judged by this test procedure is reported.     

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.     

Neutralization of the activity of eight disinfectants by organic matter

The effect of organic matter on the activity of eight disinfectants was evaluated. Three types of interfering substrates (whole milk powder, dried beef blood and fish meal) were tested according to the method of Whitmore and Miner adapted to the AOAC use-dilution method. Glutaraldehyde and to a certain extent, chlorhexidine acetate and the amphoteric surfactant kept their disinfecting activity after contact with high concentrations of organic matter. The quaternary ammonium compound as well as the quaternary ammonium-glutaraldehyde complex were more readily neutralized whereas anionic acid, iodophor and sodium hypochlorite did not tolerate the presence of organic matter. The neutralizing activity of powders was correlated to their solubility and composition.     

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.     

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.     

Susceptibility of clinical isolates of multiresistant Pseudomonas aeruginosa to a hospital disinfectant and molecular typing

The aim of this study was to evaluate the susceptibility of 35 resistant Pseudomonas aeruginosa clinical isolates to a quaternary ammonium hospital disinfectant. The methodology was the AOAC Use-Dilution Test, with disinfectant at its use-concentration. In addition, the chromosomal DNA profile of the isolates were determined by macro-restriction pulsed field gel electrophoresis (PFGE) method aiming to verify the relatedness among them and the behavior of isolates from the same group regarding the susceptibility to the disinfectant. Seventy one percent of the isolates were multiresistant to antibiotics and 43% showed a reduced susceptibility to the disinfectant. The PFGE methodology detected 18 major clonal groups. We found isolates with reduced susceptibility to the disinfectant and we think that these are worrying data that should be further investigated including different organisms and chemical agents in order to demonstrate that microorganisms can be destroyed by biocide as necessary. We also found strains of the same clonal groups showing different susceptibility to the disinfectant. This is an interesting observation considering that only few works are available about this subject. PFGE profile seems not to be a reliable marker for resistance to disinfectants.     

Modified quantitative association of official analytical chemists sporicidal test for liquid chemical germicides

The Association of Official Analytical Chemists (AOAC) test for sporicidal activity of disinfectants (966.04) is used in the United States as the legal criteria for classifying a liquid as a chemical sterilant and also as the indicator of the highest level of disinfectant. This qualitative test contains procedures that may cause inaccurate results. A modified, quantitative version of the AOAC sporicidal test has been developed that uses a specified minimum number of spores (2.0 x 10(sup5)) of Bacillus subtilis or Clostridium sporogenes dried onto porcelain penicylinders. This modified test was validated with three commercial sterilant chemicals tested on three separate groups of spore-labeled cylinders.     

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.     

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.     

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.     

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 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.     

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.     

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.     

Effect of methodology, dilution, and exposure time on the tuberculocidal activity of glutaraldehyde-based disinfectants

The Association of Official Analytical Chemists (AOAC) test for assessing the tuberculocidal activity of disinfectants has been shown to be variable. A modified AOAC test, which substituted Middlebrook 7H9 broth as the primary subculture medium and used neutralization by dilution, was compared with the standard AOAC method to assess the mycobactericidal activity of three glutaraldehyde-based disinfectants at 20 degrees C and various exposure times. These changes had a marked effect on results, with the modified AOAC test providing more positive penicylinders per 10 replicates in 12 of the 13 comparisons that provided positive results. These differences were observed with both Mycobacterium bovis (ATCC 35743) and a clinical isolate of Mycobacterium tuberculosis. The effects of various exposure times to and dilutions of the glutaraldehyde-based disinfectants were also examined. The minimum exposure time needed to inactivate reliably M. bovis or M. tuberculosis with 2% glutaraldehyde was 20 min at 20 degrees C. Diluting 2% glutaraldehyde caused a significant decline in mycobactericidal activity. Modification of the standard AOAC test to improve its sensitivity in detecting the failure of disinfectants to inactivate mycobacteria is indicated.     

细菌抵抗消毒剂及其对抗生素共耐药

消毒剂是一种可杀灭物体表面、器材设备、皮肤、空气和水源等传播媒介上携带的病原微生物的有机分子。它在体外能杀灭病原微生物,切断其传播途径,进而达到控制污染的目的,在生命安全防控中起着重要的作用。但是不合理地使用消毒剂导致细菌对消毒剂产生耐药。消毒剂耐药基因在不同种属间的水平转移加剧其传播风险,使消毒剂耐药情况进一步恶化。更令人担忧的是,细菌对消毒剂的耐药可能会导致对抗生素产生共耐药,给公共安全带来巨大的威胁。但目前为止,对消毒剂耐药以及共耐药的认识还不够全面。本文总结了关于细菌对消毒剂耐药的研究报道,对消毒剂的作用机制、细菌对消毒剂的耐药机制进行了论述,另外针对消毒剂耐药基因的传播以及细菌对消毒剂和抗生素的共耐药进行了综述,为减少消毒剂耐药性的产生和制定合理的消毒剂使用规范奠定基础。