BACTERICIDAL EFFICIENCIES OF COMMERCIAL DISINFECTANTS AGAINST LISTERIA MONOCYTOGENES ON SURFACES MARINA AARNISALO's3,S A W SALO', HANNA MIETTMEN', MAIJA-LIISA SUIHKO', GUN WIRTANEN',TIINA AUTIOZ,JANNE L U N D ~ N ~ , HANNU KORKEALA2and ANNA-MAIJA SJC)BERG1
VZTBiotechnology P.O. Box 1500 FIN-02044 VZT,Finland
Faculty of Veterinaiy Medicine Department of Food and Environmental Hygiene P.O.Box 57 FIN-00014 Helsinki University,Finland Received for Publication April 25,2000 Accepted for Publication November 1,2000
ABSTRACT The efficiencies of potassiumpersubhate, bopropanol, hydrogenperoxide and peracetic acid, quaternary ammonium compound, hypochlorite, sodium dichloroisocyanurate, ethanol and phenol derivatives, tertiary alkylamines and dimethyl alamine betaine-based disinfectantsand a hypochlorite-baseddisinfecting cleaning agent were evaluated against eight Listeria monocytogenes strains representing three different ribotypes. AN the disinfectants were effective in a suspension test with an exposure time of 30 s at the lowest concentrations recommended by the manufacturer. The efficiencies on su$aces were reduced. However, on clean surfaces all the agents were considered effective when the exposure time was 5 min and the concentration was the average recommended by the manufacturer. Five of nine disinfectants and the disinfecting cleaning agent were considered effective in soiled conditions in the suvace test. The most efficient agent was isopropanol-basedand the least effective was the disinfectant containing tertiary alkylamine and dimethyl alamine betaine. Differences in bactericidal efticiencies of disinfectants against different L. monocytogenes strains on meat soiled surfaces werefound.
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Journal of Food Safety 20 (2000) 237-250. All Rights Reserved. "Copyright 2000 by Food & Nutrition Press, Inc., Trurnbull. Connecticut.
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INTRODUCTION The safety of foods can be improved if the destruction and survival of foodborne pathogens, e.g. Listeria monocytogenes,are better understood when cleaning and disinfecting process equipment. Many commonly used disinfecting or sanitizing agents, such as quaternary ammonium compounds, chlorine and iodofors, have been shown to be effective against L. monocytogenes in suspension (Best et 41. 1990; McCarthy 1992; Sallam and Donnelly 1992; Tuncan 1993). However, organic material reduces the activity of disinfectants( El-Kest and Marth 1988a; Best et al. 1990; Van de Weyer et al. 1993; Mullerat et al. 1995). The attachment, growth and biofilm formation of L. monocytogenes on different surfacesprovides protection of the organisms from chemical agents (Mustapha and Liewen 1989; Frank and Koffi 1990; Dhaliwal et al. 1992; Mosteller and Bishop 1993). The appearence of substantial resistance to antibioticsand recently to antiseptics and disinfectantsin food-borne Listeria isolates suggests the need for more prudent use of these products. It is important to know the different resistances of microorganisms in processes and products when selecting disinfectants (Lemaitre et al. 1998). Differences between L. monocytogenes and L. innocua in their susceptibilitiesto various disinfectantsand sanitizers have been observed (Best et al. 1990; Sallam and Donnelly 1992; Jacquet and Reynaud 1994). Differences in sensitivitiesof L. monocytogenes strains to chlorine compounds have been reported (El-Kest and Marth 1988b). Eamshaw and Lawrence (1998) found significant diversity among 20 L. monocytogenes strains with respect to their resistance to 3 commercial disinfectants. In contrast, some authors have not found differences between Listeria strains (Best et al. 1990; Van de Weyer et al. 1993). Recent studies of adherence of Listeria monocytogenes strains to stainless steel coupons showed variation among serotypes (Norwood and Gilmour 1999; LundCn et al. 2000). No information is available concerning differencesbetween the sensitivities of different ribotypes to disinfectants. The purpose of the present study was to screen the antibacterial activities of nine commercial disinfectants and one disinfecting cleaning agent representing various types of chemicals commonly used in the meat processing industry. The agents were tested for their antibacterial efficiencies against L. monocytogenes isolates from the meat processing industry. The antibacterial activities against L. monocytogenes strains representing different ribotypes were compared. The effects of increasing contact time and concentration on the antibacterial activities were also investigated. Both suspension and surface tests were used. In the surface test, the antibacterial activities were calculated both in clean and in soiled conditions. A pork meat mixture in saline solution was used to represent the soiled conditions.
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MATERIALS AND METHODS Bacterial Strains Seven of the L.monocytogenes strains used in the laboratory tests were isolated fiom different meat processing factories and one human isolate was obtained from ATCC. The strains were ATCC 7644, E-981041 (product), E-981042 (environment), E-98 1043 (product), E-981045 (product), E-981046 (product), E981047 (machine) and E-991351 (environment). The strains were stored at -7OC and ribotyped with an automatic RiboPrinteP Microbial Characterization System (Qualiconm, USA) by the standard method using EcoFU as the restriction enzyme (Bruce 1996). Disinfecting Solutions The components and concentrations of the nine commercial disinfectants (A-I) and one disinfecting cleaning agent (J) in test solutions are presented in Table 1. All disinfectants were diluted with sterile distilled water according to the instructions of the manufacturer, or the working solutions were made in distilled water and sterile filtered using a filter of porosity 0.22 pm. The concentration of the disinfectants in Trials 1 and 2 (exposure time 30 s) was the lowest recommended by the manufacturer, and in Trials 3 and 4 (exposure times 5 min and 2 min, respectively) the average recommended by the manufacturer. The concentrations of agents A, C, D, E, H and I were kept the same throughout the study, since the manufacturer specified only one recommended dilution. Inactivation Solution The inactivation solution used in the tests contained 6 g/L lecithin (Sigma, St. Louis, MO), 60 mL/L Tween 80 (Fluka Chemie, Buchs, Germany); 8 g/L sodium thiosulfate (Merck, Darmstadt, Germany); 1 g/L,L-histidinmonohydrochloride (Merck); 7.2 g/L bovine albumin (Sigma); and 10 m L L 0.25M potassium dihydrogen phosphate buffer (Merck). The inactivation procedure was checked to ensure that the inactivationsolution stopped the effect of the disinfectant in the test conditions after the exposure time. A sensitive bacterium Micrococcus luteus (VTT-E-91474) was used for this test (Wirtanen et al. 2001). The disinfectant (highest concentration used in the tests) was mixed with the inactivation solution as in the test and directly after this Micrococcus luteus bacteria were added to the solution. After 5 min the solution was cultivated on agar plates and incubated. In the interpretation of the inactivation results the number of colonies should be at least half that of the control plates before inactivation is considered to have been carried out effectively.
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TABLE 1. THE ANTIMICROBIALCOMPOUNDS AND THEIR CONCENTRATIONSIN THE SOLUTIONS OF THE DISINFECTANTS TESTED IN FOUR DIFFERENT TRIALS ~~~
Disinfectant
A B
C
D
I
J
Antimicrobial Compound
Sodium dichloro-isocyanurate (active chlorine 56%) Tertiary alkylamine citric acid NaOH dimethyl alamine betaine Ethanol phenol derivatives NaOH anionic tensides Potassium persulfate citric acid anionic tensides Isopropanol quats Polyhexamethylene biguanide chloride Sodium hypochlorite (active chlorine 13.5%) Hydrogen peroxide peracetic acid acetic acid Potassium persulfate sulfonamic acid anionic tensides Sodium hypochlorite (active chlorine 13.5%) NaOH amineoxide
~
~
Concentration in the Working Solution Trials I' and 2b
Trials 3cand 4d
0.04% (w/v)
0.009% (v/v) 0.04% (v/v) 0.03% (v/v) 0.008% (v/v) 0.15% (V/V) 0.2% (vlv) 0.02% (v/v) 0.08% (v/v) 0.5% (w/v) 0.05% (w/v) 0.02% (w/v) 6O%(v/v) 4.5
0 Clean
A
B
C
D
E
F
G
H
I
J
Disinfectant FIG. 2. THE ANTIBACTERIAL ACTIVITIES (LOGloREDUCTION IN CFU/CM2)OF NINE DISINFECTANTS (A-I) AND ONE DISINFECTING CLEANING AGENT (J) AGAINST 8 L. MUNUCYTUGENES STRAINS (AVERAGE) DETERMINED BY THE SURFACE TEST DURING 30 S EXPOSURE TIME The concentrations of the disinfectants were the lowest recommendedby the manufacturer. The sign '>>" means that results of tests in which all the bacteria from the surface were destroyed are included in the average values, which may therefore actually be higher than that presented in the figure. Standard deviations are shown in the figure.
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The results of the 30 s and 5 min tests in the soiled conditions are compared in Tables 2 and 3. When the time was increased (from 30 s to 5 min), the agents reduced the amount of L. monocyfogenes bacteria by a further 1-4 log units. When evaluating the results of Trial 4 in soiled conditions (see Tables 2 and 3), a 2 min exposure time was generally sufficient for all the five disinfectants to decrease the count of L. monocytogenes by 3 log units. However, there was considerable variation between the three strains tested and the time was not sufficient for all the strains in soiled conditions. Clear differences between the three ribotypes in their sensitivities to different dismfectants were not observed. The strain of ribotype RT-3 appeared to be more resistant to the disinfectantsthan the average results of the strains representing the other two ribotypes in the 30 s test (data not shown).
TABLE 2. THE ANTIBACTERIAL ACTIVITIES (LOG,, REDUCTION IN CFU/CM2) OF SIX DISINFECTANTS (A, C, D, E, H, I) AGAINST THREE L. MONOCYTOGENE STRAINS (AVERAGE) DETERMINED BY THE SURFACE TEST DURING 30 S, 2 MIN AND 5 MIN EXPOSURE TIMES IN SOILED CONDITIONS. THE CONCENTRATION WAS CONSTANT THROUGHOUT THE STUDY. BOLD NUMBERS ARE RESULTS WHICH EXCEEDED A 3 LOG,, CFU/CM2REDUCTION Antibacterial Activity (log CFU/cm*) Disinfectant Exposure Time: 30 s 2 min 5 min (Trial 3) (Trial 2) (Trial 4) A 0.2 NT 2.2 C D
E H I NT=not tested
0.5
1.3 3.3 0.9 1.3
NT 4.6 4.2 3.0 5.4
1.5 5.0 5.3
5.1 5.0
DISCUSSION In this study, all of the disinfectants in the suspension test were effective when the exposure time was 30 s and the concentrationwas the lowest recommended by the manufacturer. This result was expected, as many studies have shown that Listeriu in suspension culture are not particularly &fficult to destroy with disinfectantsor sanitizers (Best ef ul. 1990; McCarthy 1992; Sallam and Donnelly 1992; Tuncan 1993; Wirtanen et al. 1997).
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TABLE 3. THE ANTIBACTERIAL ACTIVITIES (LOG10 REDUCTION IN CFUKM’) OF FOUR DISINFECTANTS (B, F, G, J) AGAINST THREE L.MONOCYTOGENES STRAINS (AVERAGE) DETERMINEDBY THE SURFACETEST DURING 30 S, 2 MIN AND 5 MIN EXPOSURE TIMES IN SOILED CONDITIONS. THE CONCENTRATION INCREASED FROM THE LOWEST RECOMMENDEDBY THE MANUFACTURER (EXPOSURETIME 30 S ) TO THE AVERAGE OF CONCENTRATIONSRECOMMENDED BY THE MANUFACTURER (EXPOSURE TIMES 2 MIN AND 5 MIN). BOLD NUMBERS ARE RESULTS WHICH EXCEEDED 3 LOG,, CFU/CM’ REDUCTION Disinfectant Antibacterial Activity (log CFU/cm2) Exposure Time: 30 s 2 min 5 min (Trial 2) (Trial 4) (Trial 3) B 0.1 NT 1.1 F 1.6 NT 3.8 G 0.3 NT 1.S J 2.1 3.2 5.0 NT= not tested
In the surface test, when the exposure time was the same as in the suspension test (30 s), the results were less acceptable than in the suspension test. On the soiled surface only the isopropanol-based disinfectant was effective. Previous studies have also shown that suspension tests do not indicate the susceptibility in practice (Wirtanen and Mattila-Sandholm 1992). When the exposure time was 5 min and the concentrationwas the average recommended by the manufacturer, all the agents were effective on clean surfaces in the surface test. However, differences between disinfectants were observed in soiled conditions. According to this study, the disinfectant containing 60% isopropanol, was also considered to be very effective in soiled conditions on surfaces. However, the efficiency of the disinfectant containing ethanol (0.15%) and phenol derivatives (0.2%) was not acceptable in surface tests in soiled conditions. Effective results against L. rnonocytogenes have been reported for alcohols (ethanol, propanol and isopropanol) in suspension in the presence and absence of organic material (Best et al. 1990; Van de Weyer et al. 1993; Wirtanen et al. 1997). Acceptable results for 0.5% (vh) isopropanol-containing cleaning agent in clean conditions on surfaces, but not in soiled conditions simulating dairy environments, have also been reported (Gronholm et al. 1999). Ethanol (79% v/v) with substituted phenols (0.1% v/v) and substituted phenols alone have been reported to be effective in suspension in the presence of organic material (Van de Weyer et al. 1993). The disinfectant containing tertiary alkylamines and dimethyl alamine betaine was not effectiveagainst Listeria on surfaces. Little information is available on the efficiencies of amines against Listeria. Jacquet and Reynaud (1994) reported acceptable results for an amine-based disinfectant for destroying Lisreria in
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suspension in the presence of organic material. According to Gronholm et al. (1999), a disinfectant containing 0.4% alkylamines was not effective against L. monocytogenes on clean or soiled surfaces in the presence of soil simulating soil in the dairy industry. In this study two potassium persulfate-based disinfectants were considered to be effective on both clean and soiled surfaces. Walker et al. (1992) showed that a complex peroxygen disinfectant,the primary active ingredient of which was a triple salt that provided powerful oxidizing activity, was an effective biocide against L. monocytogenes at the working concentration of 1% recommended by the manufacturer. Acceptable results for peroxide and peracetic acid-based disinfectants in suspension in the presence of organic material (Wirtanen et al. 1997), and for a peracetic acid-based disinfectant (0.01%) on surfaces (Krysinski et al. 1992), have been reported previously. The results of this study were in agreement with these earlier results. A quaternary ammonium compound-based disinfectant was also effective in suspension and surface tests in both clean and soiled conditions. In previous studies, varying results have been obtained for the efficiency of quaternary ammonium compounds against Listeria in suspension and on surfaces (Best et al. 1990; Krysinski et al. 1992; McCarthy 1992; Ronner and Wong 1993; Sallam and Donnelly 1992; Tuncan 1993; Van de Weyer et al. 1993; Wirtanen et al. 1997), depending on e.g. concentration, pH and temperature of the working solution and the type of diluent water used in the test. The results for the two hypochlorite-based agents were different. The disinfecting cleaning agent containing hypochlorite as an antibacterial agent and anionactive tensides as cleaning compounds was considered to be much more efficient than the other hypochlorite-based disinfectant. This indicates that the organic material interfered with the effect of the hypochlorite-based disinfectant. In the disinfecting cleaning agent the cleaning compounds may have assisted in the removal of organic material. According to Krysinski et al. (1992), chemical cleaners were much more effective than sanitizers in removing L. monocytogenes attached to stainless steel surfaces. There are varying results for the efficiency of chlorine compounds against Listeria spp.. Efficient chemicals for disinfecting or sanitizing against L. monocytogenes in suspension in the presence of organic material have included sodium hypochlorite (Best et al. 1990; Wirtanen et al. 1997). However, according to Jaquet and Reynaud (1994), a chlorine-based disinfectantwas not effective at the recommended concentration in the presence of organic material in suspension. According to El-Kest and Marth (1988a), the presence of peptone caused a great and rapid loss of available chlorine in a sodium hypochlorite solution. Varying results for chlorine compounds on surfaces have been obtained (Krysinski etal. 1992;McCarthy 1992; Mosteller and Bishop 1993; Ronner and Wong 1993; Wirtanen et al. 1998).
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In this study, sodium dichloroisocyanurate at a concentration of 0.04% was not effective on surfaces soiled with 2% pork meat mix (w/v) with an exposure time of 5 min. However, in the presence of milk (2% fat), it was the only disinfectant (60 pg/mL available chlorine) of 14 tested which was reported to be effective against L. monocytogenes and L. innocuu (Best ei al. 1990). It is important to know the effects of different parameters, e.g. time, concentration and components, on the efficiency of a disinfectant. The working temperature of the disinfectant is particularly important in the case of Listeria. Listeria can grow at refrigeration temperatures and some disinfectants may lose their efficiency against Listeria at cold temperatures (Tuncan 1993). According to the results of this study there are clear differences in the bactericidal efficiencies of commercial disinfectantsagainst L. monocytogenes strains in meat soil. For food manufacturers, it is important to know whether bacterial strains recovered in their factories are susceptible to the disinfectants routinely used. The choice of an effective disinfectant is highly important when destroying Listeriu-bacteria.
ACKNOWLEDGMENTS This study was supported by the Technology Development Center of Finland
(TEKES).The manufacturers of the disinfectants and Ms. Hillevi Latxalahti from the Finnish Food and Drink Industries’ Federation (FFDIF) are thanked for their contribution to this work. The technical assistance of Ms. Hanna-Leena Rissanen, Tarja Vappula, Erja Jarvinen, Raija Keijama and Niina Torttila is greatfully acknowledged. REFERENCES BEST, M., KENNEDY, M.E. and COATES, F. 1990. Efficacy of a variety of disinfectants against Listeriu spp. Appl. Environ. Microbiol. 56, 377-380. BRUCE, J. 1996. Automated system rapidly identifies and characterizes microorganisms in food. Food Technol. 50(1), 77-81. DHALIWAL, D.S., CORDIER, J.L. and COX, L.J. 1992. Impedimetric evaluation of disinfectants against biofilms. Lett. Appl. Microbiol. 15, 217-221. EARNSHAW, A.M. and LAWRENCE, L.M. 1998. Sensitity to commercial disinfectants, and the occurence of plasmids within various Listeria monocytogenes genotypes isolated from poultry products and the poultry processing environment. J. Appl. Microbiol. 84,642-648. EL-KEST, S.E. and MARTH,E.H. 1988a. Inactivation of Listeria monocytogenes by chlorine, J. Food Prot. 51, 520-524.
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