New insights for rapid evaluation of bactericidal activity: a semi-automated bioluminescent ATP assay

Transcription

1 Journal of Applied Microbiology ISSN ORIGINAL ARTICLE New insights for rapid evaluation of bactericidal activity: a semi-automated bioluminescent ATP assay L. Aragonès, C. Escudé, P. Visa, L. Salvi and L. Mocé-Llivina Eurofins Biolab S.L.U., Barcelona, Spain Keywords antiseptics, Biocidal Products Directive 98/8/EC, biocides, disinfectant efficacy, disinfection, rapid methods. Correspondence Pilar Visa, Eurofins Biolab S.L.U. Parc Científic de Barcelona, Baldiri Reixac, 4-8, Barcelona, Spain. pilarvisa@eurofins.com : received 22 December 2011, revised 10 April 2012 and accepted 18 April 2012 doi: /j x Abstract Aims: A new assay, much more rapid and efficient than the existing standardized tests, is introduced for the evaluation of bactericidal activity of chemical disinfectants and antiseptics under simulated practical conditions of use. Methods and Results: The bactericidal activity of biocides was quantified using a novel semi-automated assay based on the European Norm (EN) standard suspension tests but determining bacterial cell viability by intracellular adenosine tri-phosphate (ATP) content quantification instead of traditional culturebased microbiological techniques. The new test was validated by comparison to the standard suspension tests EN 1276 and EN During the validation, the linearity of the ATP detection system, limit of detection, specificity, sensitivity, relative accuracy and precision (repeatability and reproducibility) were determined. Conclusions: The validation study showed that the new assay evaluates the activity of biocides as well as the EN standard suspension tests, but it allows a large number of test conditions to be efficiently analysed. Significance and Impact of the Study: The new test can therefore be applied to accurately establish the lowest active concentration (MBCs) of disinfectants or antiseptics under simulated practical conditions of use and to compare the susceptibility of a large number of strains and conditions via inactivation curves. This is not possible in any reasonably practicable way with the EN standards considering the time and cost required for each determination. Introduction The European Norm (EN) standards for chemical disinfectant and antiseptic efficacy testing developed by Technical Committee (TC) 216 of the European Committee for Standardization (CEN) (Anon. 2009) are the most common tests required for registration of disinfectants and antiseptics products in the European Union (EU) according to the Biocidal Products Directive 98 8 EC (BPD). In particular, the standards categorized by CEN TC 216 as Phase 2 tests were devised with the aim of establishing whether a biocidal product has a specific activity (bactericidal, fungicidal, mycobactericidal, sporicidal, virucidal, etc.) under conditions appropriate for its intended use. EN 1276 and EN are the suspension tests (phase 2 step 2) established by CEN TC 216 for the evaluation of the bactericidal activity of chemical disinfectants and antiseptics in the food industry and for medical area, respectively. In the EN suspension tests, solutions of the biocidal product at different concentrations are mixed with a suspension of micro-organisms. Organic material, for example, bovine serum albumin (BSA), at different concentrations is used in the phase 2 EN standards as an interfering substance to simulate practical (clean or dirty) conditions of use. After a designated contact time, the activity of the test product is suppressed by dilution neutralization or by membrane filtration. The method of choice is dilution neutralization but if a suitable neutralizer cannot be found, membrane filtration is used. The 114 Journal of Applied Microbiology 113, ª 2012 The Society for Applied Microbiology

2 L. Aragonès et al. ATP assay for bactericidal activity number of surviving micro-organisms in each sample is then determined by enumeration in a culture medium, and the reduction in viable micro-organisms after the contact time is calculated. Depending on the field of application of the product tested, the EN standards specify the test conditions (contact time, temperature, interfering substances and test micro-organisms) under which the biocide is to be tested. Moreover, for specific claims and practical conditions of use of the test product, its performance under additional conditions can be evaluated using the EN standards. To determine the minimal bactericidal concentration (MBC) of a substance, different methods can be used. One of the main differences between the EN standards for disinfectants and antiseptics, and the methods used for the determination of the MBC of other substances (i.e. antibiotics), which apply procedures based on determining the minimal inhibitory concentration (MIC), is that the residual biocidal or static activity of the disinfectant must be neutralized after the designated contact time between the test product and the suspension of micro-organisms. This implies that EN tests include specific procedures for neutralizing the biocidal activity of the disinfectant and several internal checks on the validity of the test, to verify the absence of toxicity of the neutralizer and the efficacy of the neutralization step. The EN standard methods use traditional culture-based microbiological techniques and can be performed in a standard microbiology laboratory: they do not require specific equipment, most of the procedures are manual and the microbial strains grow in standard culture media tryptone soya agar (TSA) or malt extract agar. One of the main advantages of the EN tests is that they can be performed by biocide manufacturers and independent testing laboratories without the need to invest in specialized equipment. The main disadvantages are related to the reproducibility of the tests and the time required for each determination. The tests include several manual steps, and their execution requires a lot of time and material, and therefore, they allow only a few sets of conditions to be tested for a given product in a single run. The activity of the test product is usually only evaluated at three different concentrations for a given contact time. For the registration of disinfectants and antiseptics, the BPD requires that the concentration of the active substance in the biocidal product is limited to the minimum necessary (Anon. 1998, 2008). To determine the lowest active concentration, the reduction in the number of micro-organisms can be plotted against biocide concentration (Johnston et al. 2000). This approach requires a large number of tests, but the design of the EN standard suspension tests does not allow for a single run to evaluate a large number of concentrations or testing conditions (contact time, strain or interfering substances). Consequently, the use of EN standard tests to determine the lowest active concentration for a specific contact time, for each strain, at a designated temperature and under clean or dirty test conditions, turns out to be very time-consuming and therefore expensive (Lambert et al. 1998). The assessment of many sets of conditions at the same time is practicable only with faster and more efficient methods (Lambert et al. 1998, Lambert and van der Ouderaa 1999). This paper reports a new semi-automated assay for the rapid evaluation of the bactericidal activity of biocides under simulated practical conditions of use, based on cell viability detection by adenosine tri-phosphate (ATP) quantification. The new test is based on the EN standard suspension tests, but after the contact time, the quantity of viable (surviving) bacterial cells is calculated by ATP quantification, through luminescence measurement. Luminescence has previously been used in high-throughput screening to identify antimicrobial molecules (Hahn and Meeker 1991; Wirtanen and Salo 2003; Junker and Clardy 2007). There are several possible applications of this new assay. By testing many conditions at the same time, active and nonactive concentrations could be determined in a single day, thus allowing optimization of the biocide formulation. During the development of a new biocidal product, this assay could be used to screen several alternative formulations and to seek synergy between the ingredients. The evaluation of the efficacy data of a disinfectant that EU member states carry out prior to product registration includes evaluation of the dose response data generated in trials. These must include doses lower than the recommended dose, to assess whether the recommended dose is the minimum necessary to achieve the desired effect (Anon. 1998, 2008). The new test based on ATP quantification allows the MBC of the disinfectant to be determined under simulated practical conditions of use, in a much more accurate way than the EN standard tests, because a large number of test results are available for different concentrations and inactivation kinetics curves can be studied for each micro-organism. In addition, the new test can be used to screen and compare the susceptibility of specific bacterial strains to different substances, to evaluate the influence on strain susceptibility of different parameters of use (i.e. contact time and temperature). It can also be used in the framework of assessing the risk of developing resistance to biocides, by monitoring changes in the inactivation curves of the target micro-organisms. Journal of Applied Microbiology 113, ª 2012 The Society for Applied Microbiology 115

3 ATP assay for bactericidal activity L. Aragonès et al. Material and methods Bacterial strains The strains used to develop and validate the method were the reference strains included in the EN standards for biocide testing; Staphylococcus aureus ATCC 6538, Enterococcus hirae ATCC 10541, Pseudomonas aeruginosa ATCC 15442, Escherichia coli ATCC and Salmonella Typhimurium ATCC Cultures and micro-organism suspensions The strains were purchased from American Type Culture Collection (ATCC) and maintained in the laboratory by following the procedures described in EN The culture medium used was TSA (Oxoid, Hampshire, UK). Stock cultures were prepared as described in the EN 1276 standard suspension test (Anon. 2009), from the previously frozen cultures maintained at temperatures lower than )70 C. Working cultures for the tests were prepared following the instructions described in EN A working culture was prepared from the stock culture by growing it on TSA at 37 C (±1 C) for 24 h. A second working culture was prepared from the first, by growing it on TSA at 37 C (±1 C) for 24 h. This second working culture was used to prepare the test suspension of micro-organisms. The test suspension was prepared by suspending cells from the second working culture in the diluent (0Æ1455 mmol l )1 NaCl + 0Æ1% tryptone). The suspension was adjusted to a concentration of between 1Æ5 and 5Æ10 8 CFU ml )1, using the reference values of absorbance obtained previously from a spectrophotometer Synergy MX (Biotek, Winooski, VT, USA) calibration curve at a wavelength of 620 nm. Test products The test products were selected on account of their being considered representatives of classes of biocides widely used in industry. The biocides used to establish and validate the new method were benzalkonium chloride (CAS no , ref. B6295; Sigma-Aldrich Co., St Louis, MO, USA), chlorhexidine digluconate (CAS no , ref. C9394; Sigma-Aldrich Co.), triclosan (Irgasan, CAS no , ref ; Sigma-Aldrich Co.) and sodium hypochlorite (CAS no , ref ; Sigma-Aldrich Co.). Test product solutions were prepared by diluting the product in hard water. Hard water (119 mg l )1 MgCl 2, 277 mg l )1 CaCl 2 and 280 mg l )1 NaHCO 3 ) was prepared as described in EN 1276 (Anon. 2009). Test product solutions at different concentrations were used within 2 h of preparation. Evaluation of bactericidal activity: EN standard suspension tests (EN 1276 EN 13727) European Norm 1276 (Anon. 2009) and EN (Anon. 2003) were considered the reference method for establishing and validating (by a comparability study) the new test based on ATP quantification to determine cell viability. EN 1276 and EN standard suspension tests are based on the same method: a test suspension of micro-organisms in a solution of an interfering substance is mixed with a solution of the biocidal product to be tested. The mixture is maintained at a specified temperature for the designated contact time. After the contact time, the activity of the test product is suppressed and the surviving micro-organisms are enumerated. All the tests performed during the development and validation of the ATP assay were performed by suppressing the biocidal activity of the test product in a dilution neutralization step. The tests were performed as follows: 1 ml of the bacterial suspension at a concentration of between 1Æ5 and CFU ml )1 was mixed with 1 ml of a solution containing an interfering substance, prepared with albumin from bovine serum Cohn V fraction (A2153; Sigma-Aldrich Co.), at a concentration of 0Æ03 g l )1. After 2 min of equilibration at 20 C (±1 C), 8 ml of the test product solution was added and mixed thoroughly. The mixture was maintained at 20 C (±1 C) for the specified contact time. After the contact time, a 1 ml aliquot from the test tube was transferred to a tube containing 8 ml of the neutralizer (3 g l )1 lecithin, 30 g l )1 polysorbate-80, 5 g l )1 sodium thiosulfate, 1gl )1 l-histidine and 30 g l )1 saponin in diluent) and 1 ml of water. After 5 min of neutralization, dilutions of the neutralized suspension were performed in diluent (0Æ1455 mmol l )1 NaCl + 0Æ1% tryptone). One millilitre of each suspension was cultured in duplicate on TSA using the pour plate technique. The plates were incubated at 37 C (±1 C) for 24 h. Plates that presented more than 330 CFU per plate were recorded and discarded. The remaining plates were incubated at 37 C (±1 C) for 24 h. After the incubation period, the colonies were counted. The calculation of log reductions and expression of results followed the provisions of the EN methods. Validity of the EN standard tests: internal controls In accordance with the EN standard suspension tests, three internal controls were performed in parallel together with each test, for it to be considered valid. As described in the EN standards, the internal controls were Control A, Control B and Control C. For these controls, a validation suspension of micro-organisms was used, which was prepared from the test suspension by diluting the cells in 116 Journal of Applied Microbiology 113, ª 2012 The Society for Applied Microbiology

4 L. Aragonès et al. ATP assay for bactericidal activity diluent to obtain a final concentration of micro-organisms of from to 1Æ CFU ml )1. Control A validation of the selected experimental conditions and or verification of the absence of any lethal effect under the test conditions. One millilitre of the validation suspension was mixed with 1 ml of the interfering substance solution. After 2 min, 8 ml of water was added. The mixture was maintained at the test temperature for the test contact time. After the contact time, 1 ml of the suspension was enumerated as described earlier, in duplicate. Control B verification of the absence of toxicity of the neutralizer. One millilitre of the validation suspension was added to a tube containing 8 ml of neutralizer and 1 ml of water. After 5 min contact time (neutralization time), 1 ml of the mixture was enumerated as described earlier, in duplicate. Control C dilution neutralization validation. One millilitre of the interfering substance solution was mixed with 1 ml of diluent. This solution was mixed with 8 ml of the test product solution. After the test contact time, 1 ml of the mixture was transferred to a tube containing 8 ml of neutralizer. After 5 min (neutralization time), 1 ml of the validation suspension was added. After 30 min, 1 ml of the suspension was enumerated as described earlier, in duplicate. According to the standard methods, the test is valid if the recovery of micro-organisms for each control is >50% of the number of microorganisms in the validation suspension. ATP assay for evaluation of bactericidal activity (suspension test) The approach followed to devise the new method was to reproduce the EN standard suspension test but replace the traditional culture-based microbiological methods for the determination of viable cells by ATP quantification via bioluminescence measurements. The same bacterial test suspension and interfering substance solution used for the EN standard suspension tests were used for the ATP assay. A volume of 0Æ8 ml of the test product was placed in a 96-well deep-well plate (ref ; Brand, Wertheim, Germany), in triplicate. A volume of 0Æ2 ml of the bacterial suspension, previously mixed with the interfering substance as described earlier, was added to each well and mixed. After the designated contact time, 1 ml of neutralizer (7Æ5 gl )1 lecithin, 75 g l )1 Tween-80, 12Æ5 gl )1 sodium thiosulfate, 2Æ5 gl )1 l-histidine and 75 g l )1 saponin in diluent) was added to each test well. After 5 min of neutralization, the multi-well plate was centrifuged at 1250 g, the supernatant was discarded and the pellet was washed with 1 ml of diluent. After a second centrifugation step, the pellet was resuspended in 0Æ1 ml of diluent and transferred to a white 96- well microplate for ATP content analysis by luminescence. The ATP was extracted from the cells and quantified using the BacTiter Glo TM Microbial Cell Viability Assay (ref. G8231; Promega, Madison, WI, USA). 0Æ1 ml of reagent (a combination of lysis reagent, luciferin and luciferase) was added to each well. After 5 min of incubation at room temperature, luminescence was measured using the Synergy Mx luminometer module (Biotek). The results were expressed as relative luminescence units (RLU). The bacterial test suspension used for the tests was quantified as follows. First, 0Æ1 ml of the bacterial cell suspension was placed in a well of a white microplate, and the ATP quantity was determined by luminescence assay as described earlier. To check that the concentration of micro-organisms in the cell suspension was within the range 1Æ CFU ml )1, bacteria were enumerated by the pour plate technique in TSA plates, as in the EN standard method. Background luminescence (blank) was evaluated for each test as follows; 0Æ1 ml of diluent was added to a well of a white microplate, and the ATP quantity was determined by luminescence assay as described earlier. The biocidal activity was calculated by subtracting the luminescence (in RLU) detected in the test wells after the designated contact time, from the luminescence (in RLU) resulting from the ATP assay without the biocide (equivalent to internal Control B described later). Validity of the new ATP assay: internal controls To verify the ATP assay, the approach described in the EN standards was followed. All the following controls were performed in triplicate for each test. Control A validation of the experimental conditions selected and or verification of the absence of any lethal effect under the test conditions. A volume of 0Æ8 ml of hard water was placed in a 96-well deep-well plate, in triplicate. A volume of 0Æ2 ml of the bacterial suspension, previously mixed with the interfering substance as described earlier, was added to each well and mixed. After the designated contact time, 1 ml of diluent was added to each test well. After 5 min, the multi-well plate was centrifuged at 1250 g, the supernatant was discarded and the pellet was washed with 1 ml of diluent. After a second centrifugation step, the pellet was resuspended in 0Æ1 ml of diluent and transferred to a white microplate for ATP content analysis by luminescence as described earlier. Journal of Applied Microbiology 113, ª 2012 The Society for Applied Microbiology 117

5 ATP assay for bactericidal activity L. Aragonès et al. Control B verification of the absence of toxicity of the neutralizer. A volume of 0Æ8 ml of hard water was placed in a 96-well deep-well plate in triplicate. A volume of 0Æ2 ml of the bacterial suspension, previously mixed with the interfering substance as described earlier, was added to each well and mixed. After the designated contact time, 1 ml of neutralizer was added to each test well. After 5 min, the multiwell plate was centrifuged at 1250 g, the supernatant was discarded and the pellet was washed with 1 ml of diluent. After a second centrifugation step, the pellet was resuspended in 0Æ1 ml of diluent and transferred to a white microplate for ATP content analysis by luminescence as described earlier. Control C dilution neutralization validation. A volume of 0Æ8 ml of the highest concentration of the test product solution was placed in a 96-well deep-well plate in triplicate. A volume of 0Æ2 ml of diluent, previously mixed with the interfering substance, was added to each well and mixed. After the designated contact time, 1 ml of neutralizer was added to each test well. After 5 min, 0Æ1 ml of bacterial test suspension was added to each well and the multi-well plate was centrifuged at 1250 g. The supernatant was discarded, and the pellet was washed with 1 ml of diluent. After a second centrifugation step, the pellet was resuspended in 0Æ1 ml of diluent and transferred to a 96-well white microplate for ATP content analysis by luminescence as described earlier. Control D validation of the interferences of the test product and the neutralizer. A volume of 0Æ8 ml of the highest concentration of the test product solution was placed in a 96-well deep-well plate in triplicate. A volume of 0Æ2 ml of diluent, previously mixed with the interfering substance, was added to each well and mixed. After the designated contact time, 1 ml of neutralizer was added to each test well. After 5 min, the multi-well plate was centrifuged at 1250 g. The supernatant was discarded, and the pellet was washed with 1 ml of diluent. After a second centrifugation step, the pellet was resuspended in 0Æ1 ml of diluent and transferred to a 96-well white microplate for ATP content analysis by luminescence as described earlier. Validity criteria for the ATP assay The criteria for considering a test valid when the ATP assay was performed were established from the results of the experiments performed during the validation of the test. Two parameters were obtained from each experiment: the average percentage luminescence of controls A, B and C, compared with the bacterial test suspension; and the coefficient of variance between the replicates. As described in Hahn and Meeker (1991), the threshold was established as one-sided 95% tolerance for the pools of results, with Grubbs test used to reject outliers. Therefore, to consider a test valid, the following criteria for the internal controls were determined. Control A: the luminescence detected in Control A wells had to be at least 36% of the luminescence detected in the bacterial test suspension wells. Control B: the luminescence detected in Control B wells had to be at least 26% of the luminescence detected in the bacterial test suspension wells. Control C: the luminescence detected in Control C wells had to be at least 49% of the luminescence detected in the bacterial test suspension wells. Each the controls (A, B and C) had to show an intraexperiment coefficient of variation (CV) of <35%. Control D: the ratio between the luminescence detected in Control D and the luminescence detected in the blank wells had to be <2. Method comparability study and validation of the ATP assay The ATP assay was validated by assessing the following parameters: linearity of the ATP detection system, specificity, sensitivity, relative accuracy, precision (repeatability and reproducibility) and limit of detection. Linearity of the ATP detection system. The correlation between luminescence and CFU ml )1 was verified by performing serial dilutions (1 2) of bacterial suspensions at known concentrations (CFU ml )1 ). The luminescence of each dilution was quantified. The slope and R 2 of the linear regression between CFU ml )1 and luminescence were calculated. The linearity of the ATP detection system was checked after execution of the assay using hard water instead of the biocidal product, to verify that the correlation between luminescence and CFU ml )1 was maintained after the procedure. Five experiments were performed applying the ATP assay as described earlier, using different bacterial test suspensions ranging from 10 3 to 10 7 CFU ml )1 (1 10 serially diluted). Each experiment was performed in triplicate. The slope and R 2 of the linear regression between luminescence and the initial concentration of bacterial cells (CFU ml )1 ) in the test tube were calculated. Comparability of the ATP assay and EN standard suspension tests. For the comparability study, the two tests, the ATP assay and the EN standard suspension test (EN 1276 or EN 118 Journal of Applied Microbiology 113, ª 2012 The Society for Applied Microbiology

6 L. Aragonès et al. ATP assay for bactericidal activity 13727), were performed in parallel, using the same test product solutions, interfering substance and bacterial test suspension. The EN standards are designed to determine whether the test product under evaluation produces a 5-log reduction or more in the number of micro-organisms in the test tube. Tests EN 1276 and EN as defined in the standard protocols are not designed to quantify the exact reduction (from 3-log to 5-log reduction) in the number of cells. To quantify the reduction in the number of cells using the EN 1276 and EN standards, serial dilutions of the test tube contents had to be performed after the neutralization step, to obtain a dilution with colonies in the countable range ( CFU). The ATP assay has no such limitation; the quantity of cells can be directly estimated from the limit of detection to 5Æ RLU, using the luminometer. Forty-two experiments were performed in parallel. To establishing the equivalence of the two methods, the following criteria were established: for the EN standard test results, the result was considered negative for bactericidal activity when the log reduction achieved was <5Æ00 log and positive when 5Æ00 log. When possible, the log reduction was quantified. For the ATP assay, the test result was considered positive if, after the contact time in the test well with the test product, the luminescence measured was below the limit of detection. The test result was considered negative when viable cells were detected (RLU above the limit of detection) in the test tube, and the reduction in the quantity of cells was evaluated. Sensitivity, specificity and relative accuracy. The sensitivity, specificity and relative accuracy of the ATP assay were calculated from the results obtained in the comparability study. The EN standards were used as the reference methods, and the results obtained in theses tests were considered the expected results. Sensitivity was calculated as the percentage of valid positive results out of the total number of EN standard positive tests. Specificity was calculated as the percentage of valid negative results out of the total number of EN standard negative tests. The relative accuracy was calculated as the percentage of valid results correctly assigned out of the total number of tests. Precision (repeatability and reproducibility). The ATP assay was performed in 146 runs, in quadruplicate, incorporating different experimental conditions, that is, modifying the strain used, the test product, the concentration of the test product and the contact time. The standard deviation of the replicates was calculated for each test, and repeatability was calculated as the square root of the sum of squares of each standard deviation obtained. The reproducibility of the ATP assay was estimated as follows: tests were performed in quadruplicate, on four different days, with four different concentrations of the test product benzalkonium chloride (10, 20, 50 and 100 mg l )1 ). The reduction values obtained for each concentration were used to calculate the standard deviation for each concentration, and an overall standard deviation. Reproducibility was calculated as the standard deviation between the results obtained on the four different days at each concentration. Limit of detection of the ATP assay. The limit of detection of the ATP assay was calculated for each test, from the Control D data (without cells), performed as described earlier. The limit of detection was calculated as the average luminescence (in RLU) plus three standard deviations (between the three wells assayed). Luminescence above the limit of detection in test and control wells was considered significant, indicating that viable cells were present in the wells, and the cells were accurately quantified. When luminescence was below the limit of detection, it was considered that the number of cells was lower than 10 3 CFU per well; quantification of the cells was not possible. Application of the ATP assay: kinetics of microbial inactivation and determination of the minimal bactericidal concentration of the disinfectant Several concentrations of the test product were evaluated for specific test conditions (micro-organism, temperature, interfering substance and contact time). The inactivation kinetics was determined for a contact time of 5 min, using BSA 0Æ3 gl )1 as the interfering substance (clean conditions in the EN standard suspension tests), and at 20 ± 1 C. The inactivation kinetics of E. coli ATCC 10536, Ent. hirae ATCC and Ps. aeruginosa ATCC was determined for benzalkonium chloride. The inactivation kinetics of Staph. aureus ATCC 6538 and Salm. Typhimurium ATCC were determined for benzalkonium chloride, triclosan, sodium hypochlorite and chlorhexidine digluconate. Furthermore, inactivation curves for Staph. aureus ATCC 6538 with benzalkonium chloride at 37 ± 1 C were also plotted, for different contact times ranging from 5 min to 20 h. The MBC for the EN standard tests is the concentration that produces a 5-log reduction in the number of Journal of Applied Microbiology 113, ª 2012 The Society for Applied Microbiology 119

7 ATP assay for bactericidal activity L. Aragonès et al. micro-organisms. This MBC can be calculated from the inactivation curve using the linear regression obtained from the range of product concentrations that produce a quantifiable reduction. Graphs were drawn using Sigma Plot 2001 ver. 7.0 for Windows (SPSS Inc, Chicago, CA, USA). Results Figure 1 shows the correlation between Log CFU ml )1 enumerated and Log of RLU detected using the BacTiter Glo TM Microbial Cell Viability Assay. The relation was established for Ps. aeruginosa ATCC 15442, Staph. aureus ATCC 6538 and Ent. hirae ATCC In all cases, the regression was linear with a slope of 1 (a 1-log increase in RLU implied a 1-log increase in CFU ml )1 ) in the range of bacterial concentration between 10 4 and 10 8 CFU ml )1. To exclude possible of interferences of the test procedure in the ATP detection, the linearity of the ATP assay was verified by performing the new test procedure with different concentrations of cells in the bacterial test suspension: ranging from 10 3 to 10 7 CFU ml )1 with blanks. The tests were performed using water (without biocidal activity) as a test product, and for each bacterial test suspension concentration, the number of cells after the designated contact time was enumerated both by culturing (log CFU ml )1 ) and using the ATP detection system. Linear regression with a slope of 0Æ969 was obtained between log CFU ml )1 and log RLU after the execution of the test with blanks. This shows that the linearity of the ATP detection system is maintained after the execution of the full test procedure. Log 10 (RLU) Log 10 (CFU) Figure 1 Linearity of the adenosine tri-phosphate detection system BacTiter Glo TM Microbial Cell Viability Assay for Pseudomonas aeruginosa ATCC (h), Staphylococcus aureus ATCC 6538 (d and s) and Enterococcus hirae ATCC (4). The limit of detection of the ATP quantification system BacTiter Glo TM Microbial Cell Viability Assay for Ps. aeruginosa ATCC was 3Æ7 log (CFU ml )1 ); the limit of detection of the ATP quantification system for Staph. aureus ATCC 6538 was 3Æ6 log (CFU ml )1 ) and for Ent. hirae ATCC 10541, it was 4Æ6 log (CFU ml )1 ). During the comparability study, the new test was compared with the EN standard suspension tests EN 1276 and EN Experiments using the new test and the EN 1276 or the EN standard suspension tests were performed in parallel. The tests were performed using the obligatory contact time established in the EN standards. For each test product concentration, the reduction in the number of cells with the new test (log reduction in RLU) and the reduction in the number of cells with the EN standard (log reduction in CFU ml )1 ) were calculated and compared (Table 1). Depending on the reduction in the number of cells, negative or positive bactericidal activity of the test product was determined. Positive bactericidal activity was determined for the EN standards when a reduction in more than 5Æ00 log was achieved, and positive bactericidal activity was determined for the new test when cells were not detected in the test well after the contact time (quantity of ATP below the limit of detection). The bactericidal activity determination was the same for the two tests in 39 of the 42 tests performed. The discordant test results (Table 1) were because of the high number of bacteria required to differentiate a viable bacterial signal from the background signal. In one case, when Ent. hirae was tested with 100 mg l )1 of Benzalkonium Chloride, the EN standard result was a 4Æ85-log reduction in CFU ml )1 (very close to the 5-log reduction, but considered negative), whereas the log reduction in RLU obtained with the new test was >2Æ86 (considered positive). In the second case, when E. coli was tested with 100 mg l )1 of benzalkonium chloride, the EN standard result was a 3Æ70-log reduction (negative) and the result of the new test was a >2Æ24-log reduction (positive). In the third case, when Staph. aureus was tested with 600 mg l )1 of triclosan, result was a 4Æ98-log reduction (very close to the 5-log reduction, but considered negative), whereas the result of the new test was a >2Æ29-log reduction (positive). From all the 42 results obtained in the comparability study, the sensitivity, specificity and relative accuracy of the method were calculated. The sensitivity was 100%, the specificity was 88% and the relative accuracy of the new test was 95%, when compared with the EN standard suspension tests. The precision of the new test was evaluated by performing four independent experiments in quadruplicate. The results (Table 2) showed the repeatability for 120 Journal of Applied Microbiology 113, ª 2012 The Society for Applied Microbiology

8 L. Aragonès et al. ATP assay for bactericidal activity Table 1 Comparison between the new test and the reference methods: EN standard suspension tests EN 1276 and EN The tests were performed using the obligatory contact time for the EN standards. For each test product concentration, the reduction in the number of cells was calculated. Depending on the reduction, negative or positive bactericidal activity of the test product was determined. The acceptance criteria for bactericidal activity in the EN standard suspension tests is a reduction in more than 5Æ00 logarithms in the number of cells (CFU ml )1 ). The acceptance criteria for bactericidal activity in the new test were the reduction in cells below the detection limit in each test Results New test Reference method Strain Reference method Contact time (min) Test product Test product concentration (mg l )1 ) Log R* relative luminescence unit Activity Log R* CFU ml )1 Activity Pseudomonas aeruginosa ATCC Enterococcus hirae ATCC Staphylococcus aureus ATCC 6538 Escherichia coli ATCC EN Product A 100% 0Æ94 ) <4Æ07 ) Product B 100% 0Æ74 ) <4Æ07 ) BZC 1 0Æ06 ) 0Æ24 ) 10 0Æ04 ) 0Æ27 ) 100 0Æ34 ) 0Æ37 ) 500 2Æ25 ) 2Æ22 ) EN NaOCl 0Æ1 0Æ01 ) <4Æ12 ) 10 0Æ00 ) <4Æ12 ) 1000 >2Æ90 + >5Æ49 + EN BZC 1 0Æ06 ) 0Æ00 ) 10 0Æ70 ) 0Æ16 ) 100 >2Æ50 + 4Æ85 ) 500 >2Æ50 + >5Æ >3Æ41 + >5Æ >3Æ41 + >5Æ15 + EN NaOCl 0Æ1 0Æ24 ) <3Æ81 ) 0Æ1 0Æ42 ) <3Æ78 ) 0Æ1 0Æ15 ) <3Æ85 ) 10 0Æ20 ) <3Æ81 ) 10 0Æ32 ) <3Æ78 ) 10 0Æ19 ) <3Æ85 ) 1000 >3Æ48 + >5Æ >3Æ13 + >5Æ >3Æ17 + >5Æ22 + EN BZC 1 0Æ04 ) 0Æ00 ) 10 0Æ16 ) 0Æ09 ) 10 0Æ39 ) <1Æ93 ) 100 >2Æ44 + >5Æ >2Æ93 + >5Æ >2Æ44 + >5Æ >2Æ93 + >5Æ >2Æ44 + >5Æ >2Æ44 + >5Æ15 + EN Triclosan 100 0Æ17 ) <1Æ93 ) 600 >2Æ29 + 4Æ98 ) 1000 >2Æ29 + 5Æ95 + EN BZC 1 0Æ00 ) 0Æ24 ) 10 0Æ00 ) 0Æ31 ) 100 >2Æ24 + 3Æ70 ) 500 >2Æ24 + >5Æ >2Æ87 + >5Æ >2Æ87 + >5Æ15 + Product A, soap without biocidal active ingredients; Product B, soap without biocidal active ingredients; NaClO, sodium hypochlorite; BZC, benzalkonium chloride; EN, European Norm. *Logarithmic reduction in the number of cells; the reduction in the number of cells in contact with the test product was calculated as log Red = log N 0 ) log N a (where N 0 is the number of cells in the test tube before the contact time and N a is the number of cells in the test tube after contact with the test product for the contact time). These concentrations are expressed in percentage of final product. Journal of Applied Microbiology 113, ª 2012 The Society for Applied Microbiology 121

9 ATP assay for bactericidal activity L. Aragonès et al. Table 2 Precision (repeatability and reproducibility) assessment for the new adenosine tri-phosphate assay. Tests were performed in quadruplicate, on four different days, using Staphylococcus aureus ATCC 6538 as the test strain and benzalkonium chloride as the test product. All tests were performed at 20 C, 5 min contact time under clean conditions (0Æ3 gl )1 bovine serum albumin). Repeatability was evaluated as the standard deviation within each test. Reproducibility was evaluated as the standard deviation between tests Test product concentration (mg l )1 ) Repeatability results (average log reduction in relative luminescence unit ± standard deviation) Test 1 Test 2 Test 3 Test 4 Standard deviation of reproducibility (SR) 100 3Æ00 ± 0Æ08 2Æ60 ± 0Æ05 2Æ80 ± 0Æ27 2Æ83 ± 0Æ16 0Æ Æ90 ± 0Æ43 2Æ50 ± 0Æ11 2Æ74 ± 0Æ22 2Æ59 ± 0Æ08 0Æ Æ70 ± 0Æ22 2Æ04 ± 0Æ28 0Æ67 ± 0Æ04 2Æ30 ± 0Æ11 0Æ Æ20 ± 0Æ08 0Æ74 ± 0Æ58 1Æ43 ± 0Æ27 0Æ39 ± 0Æ14 0Æ39 logarithmic reduction in RLU was 0Æ2 log and the reproducibility ranged from 0Æ18 log to 0Æ88 log, depending on the test product concentration. The highest value of the standard deviation was observed at the threshold active concentration. The average reproducibility was estimated to be 0Æ42 log. To establish whether the new test can be applied to a wide spectrum of biocides, the neutralization efficacy of the test was evaluated by performing the experiment described for Control C, using different test products that contained different kinds and concentrations of active biocidal molecules (0Æ1% sodium hypochlorite, 2Æ5% glutaraldehyde, 0Æ5% benzalkonium chloride, 1% chlorhexidine digluconate and 0Æ1% triclosan). Three replicates were performed for each test, and in all cases, the recovery was higher than 50%, thus showing that the neutralization procedure was effective against the range of disinfectants tested, for all the strains tested (data not shown). Using the new ATP assay, the inactivation kinetics of Ps. aeruginosa ATCC 15442, Staph. aureus ATCC 6538, Ent. hirae ATCC and E. coli ATCC were determined. Concentrations of benzalkonium chloride in the range mg l )1 were tested using the ATP assay at 20 C with a 5-min contact time and under clean conditions (BSA 0Æ3 gl )1 ). Figure 2 shows the inactivation curves obtained for each strain. Because of the limit of detection, it was not possible to directly evaluate a decrease of 5 log CFU ml )1, but the concentration of the disinfectant that produces this reduction could be calculated by extrapolation from the inactivation curve in the range where Log Reduction vs concentration was quantifiable. Therefore, the product concentration that is active according to the EN 1276 standard was estimated for each strain. These estimated active concentrations for benzalkonium chloride, under the experimental conditions described earlier, were 42 mg l )1 for Staph. aureus ATCC 6538, 803 mg l )1 for Ps. aeruginosa ATCC 15442, Log R (RLU) Log R (RLU) (a) mg/i BZC mg/i BZC 32 mg l )1 for Ent. hirae ATCC and 195 mg l )1 for E. coli ATCC To check that these primary concentrations, using the ATP assay as a preliminary test, were active in accordance with the EN 1276, the standard experimental procedure was applied and all the concentrations were confirmed. Therefore, the ATP assay can be used as a preliminary test. To evaluate the reproducibility of the estimates using the ATP assay, the active concentration for benzalkonium chloride acting on Staph. aureus was estimated from five different inactivation curves obtained from different tests. The estimates obtained ranged from 42 to 98 mg l )1, with an average of 68 mg l )1 and a standard deviation of 27 mg l )1 ; the coefficient of variance for this method was thus 40%. The inactivation kinetics of Staph. aureus ATCC 6538 and Salm Typhimurium ATCC were established for benzalkonium chloride, chlorhexidine digluconate, triclosan and sodium hypochlorite (Fig. 3). (b) (c) (d) Figure 2 Inactivation kinetics of Staphylococcus aureus ATCC 6538 (a), Pseudomonas aeruginosa ATCC (b), Enterococcus hirae ATCC (c) and Escherichia coli ATCC (d). Test conditions: benzalkonium chloride in the range from 1 mg l )1 to 2000 mg l )1 at 20 C, 5 min contact time and clean conditions (bovine serum albumin 0Æ3 gl )1 ). 122 Journal of Applied Microbiology 113, ª 2012 The Society for Applied Microbiology

10 L. Aragonès et al. ATP assay for bactericidal activity Log R (RLU) Log R (RLU) (a) (c) (d) mg/i test product mg/i test product Figure 3 Inactivation kinetics of Staphylococcus aureus ATCC 6538 (d) and Salmonella Typhimurium ATCC (s) at 20 C, 5 min contact time and clean conditions (bovine serum albumin 0Æ3 gl )1 ) using benzalkonium chloride (a), chlorhexidine digluconate (b), triclosan (c) and sodium hypochlorite (d). The inactivation kinetics of Staph. aureus ATCC 6538 was established for 20, 10 and 2Æ5 mgæl )1 of benzalkonium chloride at 20 C, using different contact times with the aim of establishing at what point the contact time is key in the bactericidal activity of the test product (Fig. 4). The bactericidal activity of benzalkonium chloride on Staph. aureus ATCC 6538 did not show a significant increase in response to contact times between 15 and 60 min. A significant increase in the bactericidal activity of the test product related to the contact time was observed for 2Æ5 mgl )1 and for 10 mg l )1 between 60 min and 20 h. In the contact time range from 15 to 60 min, the test LogR (RLU) (b) t = 5 t = 15 t = 30 t = 1 h Contact time (h) t = 20 h Figure 4 Reduction in the number of cells of Staphylococcus aureus ATCC 6538 measured using the new adenosine tri-phosphate assay, at 20 C, under clean conditions (bovine serum albumin 0Æ3 gl )1 ) when different contact times were tested for benzalkonium chloride at different concentrations; 2Æ5 mgl )1 (m), 10 mg l )1 ( ) and 20 mg l )1 (d). For each reduction result, the coefficient of variation at 95% confidence interval is shown. product concentration was more relevant than the contact time for a bactericidal effect on the tested strain under the experimental conditions applied. The inactivation kinetics of Staph. aureus ATCC 6538 was established under the same experimental conditions (5 min contact time and clean conditions) at different temperatures; 20 and 37 C (Fig. 5). The inactivation curves obtained did not differ at the different temperatures. Discussion Disinfectants and antiseptics are used to prevent nosocomial, community-acquired and food-borne infections. Their use has helped to improve the quality of life in modern societies. Concerns about the resistance of pathogens to antibiotics have been increasing for years. Some resistance mechanisms are shared by both antibiotic resistance and biocide resistance (Levy 2002; Russell 2002; Sheldon 2005). Therefore, the use of biocides may contribute to the increased occurrence of antibiotic-resistant bacteria. This is one of the reasons that have led the European Chemicals Bureau to suggest the use of the minimum necessary concentration of active substances (Anon. 2008). Furthermore, the toxicity of the disinfectant residues makes it important to use the minimum active concentration (Wirtanen and Salo 2003; Moretro et al. 2009). A new method based on the EN standard suspension tests has been devised to reduce the amount of work required to determine the minimum bactericidal concentration necessary. The main innovations of the method are its semi-automated nature and the speed of cell viability analysis through the use of an ATP quantification system. In devising the method, four internal controls were introduced to ensure that the quantified reduction is Log (RLU) mg/i BZC Figure 5 Inactivation kinetics of Staphylococcus aureus ATCC 6538 for benzalkonium chloride, at 20 C (s) and 37 C (d), 5 min contact time, under clean conditions (bovine serum albumin 0Æ3 gl )1 ). Journal of Applied Microbiology 113, ª 2012 The Society for Applied Microbiology 123

11 ATP assay for bactericidal activity L. Aragonès et al. solely because of the test product during the contact time assayed. Furthermore, performing the verification tests in replicate allowed intra-experiment variance to be assessed and the coefficient of variance for considering a test valid to be established. Acceptable limits of the verification tests were established from initial laboratory data (Hahn and Meeker 1991) and are described in the Material and Methods section of this paper. The values established are used to check the validity of each test performed. The method has been shown to be equivalent to European Normalization suspension tests and the repeatability and reproducibility of the method lie within the range obtained in traditional biocide tests (Tilt and Hamilton 1999). The testing of antimicrobial activity has been a major concern since the dawn of microbiology (Reybrouck 1998). The new ATP assay is a step forward in the evaluation of the bactericidal activity of chemical disinfectants and antiseptics, because it reduces the work and time required to determine an active concentration of the product. Testing four bacterial reference strains, three product concentrations, one contact time, one interfering substance and one test temperature with the new test requires 5 h work by one technician and 1 h work by a senior scientist, including the analysis of results and calculation and verification, instead of approximately the 24 h total required for EN standard tests. Furthermore, it reduces the time if the timeline is taken into account. Results of the EN standards are obtained after 48 h of incubation time, whereas the results using the new ATP assay are obtained in the same working day. An advantage of the new ATP assay compared with other high-throughput techniques is that the final results are obtained at the moment the test is finished, and no incubation is required to check bacterial viability (Silley and Forsythe 1996; Lambert et al. 1998). Although EN standard tests are today the reference tests, some authors have criticized them for being single endpoint tests with many parameters that may influence the test outcome, especially for biocides with nonlinear kinetics (Johnston et al. 2000). The same authors suggest carrying out a complete kinetic examination to obtain true-rate data. Whether that is necessary or not, the study of the biocide under a broad range of testing conditions (time, concentration, temperature, interfering substance, initial inoculums, etc.) helps to understand the mechanism of biocidal activity and to establish the most suitable procedures for achieving the desired level of disinfection together with the robustness of the disinfectant when applied in field conditions. Using this new rapid microbiological method, several concentrations of a test product can be assayed in the same run, giving the new method the capacity to generate inactivation curves for each strain. The inactivation curves allow the MBC of a biocidal product to be calculated for a defined set of test conditions (contact time, temperature and interfering substances), in a fast and cost-effective way. Moreover, using the new ATP assay, different variables related to disinfectant and antiseptic efficacy can be tested and the factors that really affect the bactericidal activity of the disinfectants and antiseptics can be determined. The new ATP assay makes it easier to find a balanced active concentration: not too high to pose economical and environmental issues, but not too low to compromise the disinfection capacity when the product is in use (Meyer et al. 2010). This is not possible in any reasonably practicable way with EN standards considering the time and cost required for each determination. This innovation in testing is very important in the context of BPD (Biocidal Product Directive 98 8 EC) implementation where a well-balanced trade-off between product efficacy and its toxicological and environmental safety is sought. This test could help manufacturers to adjust the concentration of the active substances in their formulations to the minimum level necessary for the required biocidal efficacy, thus minimizing the environmental and toxicological impact. It is also a useful tool for searching for synergy when combining different active substances. Acknowledgements This work was supported by the BIOHYPO project, grant number , funded by the EU Seventh Framework Programme, theme FP7-KBBE B. We thank Marina Rafols for her technical support. References Anon. (1998) Directive 98 8 EC of the European parliament and the council of 16 February of 1998 concerning the placing of biocidal products on the market. Anon. (2003) Chemical disinfectants and antiseptics quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants for instruments used in the medical area test method and requirements (phase 2, step 1). European Committee for Standardisation CEN TC 216. EN Anon. (2008) Technical notes for guidance in support of Annex VI of directive 98 8 EC of the European Parliament and the council concerning the placing of biocidal products on the market. Anon. (2009) Chemical disinfectants and antiseptics quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in 124 Journal of Applied Microbiology 113, ª 2012 The Society for Applied Microbiology