Abstracts. Janne Lunden 2

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Journal of Food Microbiology, Volume 82, Issue 3 , 15 May 2003, Pages 265-272

Adaptive and cross-adaptive responses of persistent and non-persistent Listeria monocytogenes strains to disinfectants

Janne Lundén, Tiina Autio, Annukka Markkula, Sanna Hellström and Hannu Korkeala

Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, University of Helsinki, P.O. Box 57, Helsinki FIN-00014, Finland

Received 24 February 2002; accepted 20 June 2002. ; Available online 28 August 2002.

ABSTRACT

Persistent and non-persistent Listeria monocytogenes strains were tested for initial resistance and adaptive and cross-adaptive responses towards two quaternary ammonium compounds, alkyl-benzyl-dimethyl ammonium chloride and n-alkyldimethyl ethylbenzyl ammonium chloride, one tertiary alkylamine, 1,3-propanediamine-N-(3-aminopropyl)N-dodecyl, sodium hypochlorite and potassium persulphate. The initial resistance of two persistent and two non-persistent L. monocytogenes strains was observed to differ. Both types of strains adapted after a 2-h sublethal exposure to the quaternary ammonium compounds and the tertiary alkylamine, the highest increase in the minimum inhibitory concentration (MIC) being 3-fold. Progressively increasing disinfecting concentrations at 10 and 37 °C resulted in adaptation of L. monocytogenes to all disinfectants except potassium sulphate. The highest observed increase in MIC was over 15-fold, from 0.63 to 10 g/ml of n-alkyldimethyl ethylbenzyl ammonium chloride. All strains reached approximately similar MICs. Stability of the increased resistance was tested by measuring MICs every seventh day for 28 days. The increased resistance to sodium hypochlorite disappeared in 1 week, but the quaternary ammonium compounds and the tertiary alkylamine showed increased resistance for 28 days. These results suggest that cellular changes due to adaptive responses continue to have an effect on the resistance some time after the exposure. All disinfectants were shown to cause cross-adaptation of L. monocytogenes, the highest increase in MIC being almost 8-fold. The only agent that L. monocytogenes could not be shown to cross-adapt to was potassium persulphate which did, however, cause cross-adaptation to the other disinfectants. The mechanism behind these adaptive responses seemed to be non-specific as cross-adaptation was observed not only between related but also unrelated disinfectants. These findings suggest that sustaining high disinfectant effectiveness may be unsuccessful by rotation, even when using agents with different mechanisms of action.

Author Keywords: Listeria monocytogenes; Disinfectants; Adaptation; Cross-adaptation

1. INTRODUCTION

Disinfectants are widely used in food processing plants in the elimination of Listeria monocytogenes. However, elimination of L. monocytogenes has proven difficult despite of regular cleaning and disinfection treatments. Especially some L. monocytogenes strains seem to persist causing prolonged contamination (R; Unnerstad; Autio and Miettinen). Reasons leading to persistent contamination are unclear, but it has been proposed that adaptation (Aase et al., 2000) and resistance of L. monocytogenes to disinfectants could influence survival of the organism in food processing plants (Lema; Aase and Mereghetti).

L. monocytogenes has been shown to adapt to quaternary ammonium compounds (QAC) which are widely used in the food processing industry (Aase et al., 2000). Adaptive responses develop when the bacteria are exposed to sublethal concentrations of disinfectants (Gandhi and Aase). Conditions leading to adaptive responses occur probably easily in food processing plants, e.g. as a result of disturbances in cleaning and disinfection as proposed by Aase et al. (2000). Adaptation of L. monocytogenes to QACs and possible adaptation of L. monocytogenes to other disinfectants used in the food processing industry could lead to increased survival of the organism.

Adaptation to a disinfectant may lead to cross-adaptation to other disinfectants, enhancing the survival of the bacteria (Jones and Gandhi). Cross-adaptation arises due to specific or non-specific cellular changes and results in reduced efficiency of related or unrelated disinfectants (McDonnell and Russell, 1999). Rotation of disinfectants has been suggested to be beneficial in cleaning to prevent the development of resistant strains (Lema and Mereghetti). Unfortunately, very little information is available on how the rotation should be performed and which disinfectants to use. Knowledge of possible cross-adaptation of L. monocytogenes to disinfectants would be valuable when designing rotation of disinfectants in food processing plants.

The focus of this study was to investigate the occurrence and magnitude of adaptive and cross-adaptive responses of persistent and non-persistent L. monocytogenes strains to disinfectants used in the food processing industry. A further aim was to assess the applicability of the disinfectants for rotation in food processing plants.

MATERIALS AND METHODS

2.1. L. monocytogenes isolates

L. monocytogenes isolates were obtained in earlier performed surveys in an ice cream plant (Miettinen et al., 1999) and a poultry plant (Miettinen et al., 2001). The isolates were characterized by serotyping with commercial Listeria antisera (Denka Seiken, Tokyo, Japan) and pulsed-field gel electrophoresis typing (Miettinen et al., 1999). Two persistent (41 and 2904) and two non-persistent (35 and 2919) strains of different pulsotypes were included in the study (Table 1). A strain was considered to be persistent if it was found repeatedly in the plant during a period of several months or years. Persistent strains were not only found in the environment and equipment but also in products. A strain found sporadically was considered to be non-persistent.

Table 1. Characterization, source and persistence of L. monocytogenes strains

2.2. Disinfecting agents

Two quaternary ammonium compounds, alkyl-benzyl-dimethyl ammonium chloride (QAC 1) (Goldschmidt, Pandino, Italy) and n-alkyldimethyl ethylbenzyl ammonium chloride (QAC 2) (Pointing Chemicals, Huddersfield, UK), one tertiary alkylamine, 1,3-propanediamine-N-(3-aminopropyl)N-dodecyl (Lonza, Basle, Switzerland), sodium hypochlorite (active chlorine 10%) (Finnish Chemicals, Äetsä, Finland) and potassium persulphate (Degussa, Hanau, Germany) were included in the study.

2.3. Stock solutions

Stock solutions were prepared by dissolving the disinfecting agents in sterile water to attain a concentration of 10% (w/w). A stock solution of sodium hypochlorite with a concentration of 25% (w/w) was also prepared. The stock solutions were filter-sterilized (Minisart 0.45 m, Sartorius, Göttingen, Germany) and stored at 2 °C. Further dilutions were freshly prepared before each experiment. Possible deterioration of the stock solutions was monitored with susceptibility testing (minimum inhibitory concentrations [MIC]) using L. monocytogenes strains 35 and 41 as control strains.

2.4. Minimum inhibitory concentrations

MICs were determined with the microdilution broth method according to the NCCLS standard (Anonymous, 1999). Briefly; Mueller–Hinton (MH) broth (Difco, Detroit, MI, USA) was inoculated with five colonies grown on blood agar and incubated at 37 °C until the concentration was approximately 1×10⁸ cells/ml. The inoculum was diluted to give a final concentration in the well of 5×10⁵ cfu/ml. The concentration of the inoculum was confirmed by plating. Serial 2-fold dilutions of the disinfecting agents in four parallel wells were used to determine the MIC. The microwell plates were incubated at 37 °C for 20 h, and the MIC was determined as the lowest concentration of the disinfecting agent to prevent growth.

2.5. Adaptation following short sublethal exposure

L. monocytogenes strains 35, 41, 2904 and 2919 were tested for their ability to adapt to disinfectants after a 2-h sublethal exposure. An inoculum in MH broth with a final concentration of 5×10⁵ cfu/ml was prepared for each strain in the same manner as described for determination of MIC. Each L. monocytogenes strain was pre-exposed to sublethal concentrations (MIC/8 and MIC/4) of disinfectants at 37 °C for 2 h. Pre-exposed cells were challenged with the disinfectants at concentrations of 1×MIC, 2×MIC or 3×MIC at 37 °C for 24 h, and these are referred to as pre-exposed challenged cells. Cells that were challenged with disinfectants at concentrations of 1×MIC, 2×MIC or 3×MIC at 37 °C for 24 h without pre-exposure are referred to as not pre-exposed challenged cells. Control cells were not subjected to disinfectants at any time. Growth was monitored with a Bioscreen C analyser (Labsystems, Helsinki, Finland), which measures turbidity by vertical pathway spectrophotometry. The test was performed twice in four parallel wells.

2.6. Adaptation and cross-adaptation to disinfectants

Persistent strain 41 and the non-persistent strain 35 were tested for their ability to adapt to disinfectants by exposing the strains to increasing concentrations of a disinfectant. The test was performed in microtitre plates. An inoculum was prepared in the same manner as described for determination of MIC. The starting concentration of the disinfectant was below the MIC (MIC/2) of the disinfectant and the L. monocytogenes strain. The total volume of the suspension in the well was 200 l. When growth was observed, 20 l of the suspension was transferred to the next well, which contained after the transfer a 1.5 times higher concentration of the disinfectant than the previous well. This procedure was continued until no growth was observed after 3 days of incubation. The test was performed twice, both at 10 and 37 °C. The suspension of the last well with recorded growth was centrifuged, and the pellet was washed with phosphate-buffered saline to remove the disinfectant. The pellet was resuspended in MH broth and incubated at 37 °C to achieve a concentration of about 10⁸ cfu/ml. Purity of the culture was controlled by streaking the suspension on a blood agar plate. MICs of all disinfectants were measured for determining the level of adaptation and possible cross-adaptation of L. monocytogenes strains.

2.7. Stability of resistance

The adapted strains were subcultivated in MH broth every 48 h for 28 days, both at 37 and 10 °C. The stability of resistance was tested by measuring MICs every seventh day

RESULTS

The initial MICs of the QACs and the tertiary alkylamine for L. monocytogenes strains varied between 0.63 and 5.0 g/ml (Table 2). Persistent strain 41 showed the highest MICs of the QACs, with MICs 4-fold those of non-persistent strain 35. Persistent strain 2904 showed similar or higher MICs of the QACs and the tertiary alkylamine than non-persistent strain 2919. The MICs of potassium persulphate for all strains were 2500 g/ml, and the MICs of sodium hypochlorite were in the range of 2500–5000 g/ml.

Table 2. Minimum inhibitory concentrations (MIC) and adaptation of L. monocytogenes strains to disinfecting agents following a 2-h sublethal exposure

Two different sublethal concentrations (MIC/8 and MIC/4) were used.

L. monocytogenes strains adapted as a result of a 2-h sublethal exposure to disinfectants QAC 1, QAC 2 and the tertiary alkylamine (Table 2). Differing sublethal exposures (MIC/8 and MIC/4) resulted in similar adaptive responses. Adaptation to potassium persulphate and sodium hypochlorite was not observed after the 2-h sublethal exposure. The adaptive responses were greatest for the strains that showed low initial MICs, the highest increases in MIC being 3-fold. Low or no adaptation was observed for strains with high initial MICs. No growth of not pre-exposed challenged cells was observed during a 24-h period, whereas pre-exposed challenged cells initiated growth after a prolonged lag phase (Fig. 1).

Fig. 1. Growth of disinfectant challenged L. monocytogenes strains pre-exposed or not pre-exposed to disinfectants in Mueller–Hinton broth at 37 °C was monitored for 24 h. The cells were exposed to a sublethal concentration (MIC/4) of a disinfectant for 2 h, after which they were challenged with a disinfectant concentration equal to MIC. Cells referred to as not pre-exposed challenged cells were exposed to a disinfectant concentration equal to MIC without previous contact with the disinfectant. Control cells were not exposed to disinfectants at any time. (A) L. monocytogenes strain 2919 challenged with a tertiary alkylamine (1,3-propanediamine-N-(3-aminopropyl)N-dodecyl). (B) L. monocytogenes strain 2904 challenged with a quaternary ammonium compound (alkyl-benzyl-dimethyl ammonium chloride).

Progressively increasing disinfecting concentrations at 10 and 37 °C resulted in adaptation of L. monocytogenes to all disinfectants except potassium sulphate (Table 3). Adaptive responses were greatest when the initial MIC for the strain was low. The highest observed increase in MIC was over 15-fold, from 0.63 to 10 g/ml of QAC 2. Persistent strain 41 and non-persistent strain 35 reached similar MICs at 37 °C, but persistent strain 41 showed a 2-fold MIC of QAC 2 and of the tertiary alkylamine at 10 °C compared with non-persistent strain 35.

Table 3. Minimum inhibitory concentrations (MIC) of L. monocytogenes strains before and after repeated exposure to disinfectants

The concentration of the disinfectant was increased until no growth was observed. Tests were performed at 10 and 37 °C.

All disinfectants were observed to cause cross-adaptation of L. monocytogenes to various disinfectants (Table 4). The only disinfectant that L. monocytogenes did not cross-adapt to was potassium persulphate. Potassium persulphate did, however, cause cross-adaptation to all other disinfectants. The MICs of disinfectants due to cross-adaptive responses were similar to or smaller than the MICs of disinfectants resulting from adaptive responses.

Table 4. Cross-adaptation of L. monocytogenes strains following adaptation to disinfectants at 10 and 37 °C

The increased resistance to QAC 1 was shown to persist for 28 days at both 10 and 37 °C, but for QAC 2 and the tertiary alkylamine, the increased resistance decreased from 10 to 5 g/ml in 7 days at both temperatures. However, the MICs showed no further decrease during the 28-day period. The increased resistance to sodium hypochlorite was lost in 1 week. The initial MIC of potassium persulphate showed no decrease in the 28 days.

DISCUSSION

The initial MICs of the disinfectants differed between L. monocytogenes strains, persistent strain 41 showing higher MICs of the QACs than non-persistent strains. Differences in the resistance of L. monocytogenes strains to disinfectants have been suggested to influence the survival of the bacteria in food processing plants and may contribute to persistent plant contamination(Lema; Aase and Mereghetti).

L. monocytogenes was observed to adapt to QACs and the tertiary alkylamine after a 2-h sublethal exposure, the highest increase in MIC being 3-fold. This indicates that even short-term contact with sublethal concentrations causes cellular changes, which lead to adaptive responses of L. monocytogenes cells. The level of the used sublethal concentration (MIC/8 or MIC4/) was not observed to influence the occurrence or the magnitude of the adaptive responses.

Exposure to increasing disinfecting concentrations resulted in adaptation of L. monocytogenes to disinfectants commonly used in food processing plants. The magnitude of the adaptive responses reached similar levels in L. monocytogenes strains 41 and 35. These results are in agreement with an earlier study which showed that L. monocytogenes strains adapted to benzalkonium chloride reached approximately similar MIC values (Aase et al., 2000). The increase in MICs due to adaptive responses was observed to be up to 15-fold. The final MICs, however, did not exceed the concentrations used in working solutions in the food processing plants. Therefore, increased resistance to disinfectants due to adaptation does not appear to be of major importance in the survival of the L. monocytogenes cells when the disinfecting procedure is performed adequately. However, it has been speculated that increased resistance may have a synergistic effect with other resistance mechanisms such as biofilm-related resistance (Aase et al., 2000). Of particular interest is that persistent L. monocytogenes strains have been observed to adhere more efficiently to stainless steel surface after short contact time as compared with non-persistent strains (Lundén et al., 2000). Enhanced adherence together with increased resistance to disinfectants due to adaptation may have a synergistic effect on promoting the survival of L. monocytogenes in food processing plants. Furthermore, increased resistance may be of importance when bacteria encounter suboptimal concentrations of disinfectants. Suboptimal concentrations may occur because of inadequate distribution or dosage of disinfectants, insufficient rinsing and failure to remove organic matter known to inactivate disinfectants (Gélinas and Goulet, 1983). Particularly hard-to-reach places, such as complex processing machines of poor hygienic design, have been suggested to create good conditions for adherence and adaptive responses of L. monocytogenes (Lundén et al., 2002).

Cross-adaptation was observed in L. monocytogenes to disinfectants with similar mechanisms of action. The QACs and the tertiary amine, which are cationic surfactants (Marriott, 1989) and cause damage to the cytoplasmic membrane ( McDonnell and Russell, 1999), cross-adapted to each other. However, disinfectants with different mechanisms of action were also observed to cause cross-adaptation of L. monocytogenes, indicating that the mechanism causing adaptation of L. monocytogenes to these disinfectants is non-specific. The cross-adaptive response resulted in increased MICs, the highest increase being almost 8-fold. These results imply that rotation of disinfecting agents with differing mechanisms of action in food processing plants may not have the desired effect.

L. monocytogenes strains were not observed to adapt to potassium persulphate at 10 or 37 °C. This agent therefore seems suitable for long-term use in food processing plants. It did, however, cause non-specific cross-adaptation, which might reduce the efficiency of other disinfectants.

The stability of the increased resistance to different disinfectants varied. The highest increases in MICs for QAC 2 and the tertiary amine decreased over 7 days, but further decreases were not observed during the 28-day period. These observations suggest that cellular changes due to adaptation continue to have an effect on resistance to disinfectants some time after exposure.

This study showed that the susceptibilities of persistent and non-persistent L. monocytogenes strains to disinfectants differ, which may influence survival of the strains. Further, this study showed that persistent and non-persistent L. monocytogenes strains adapt to disinfectants commonly used in the food processing plants at production temperature. Adaptive responses were observed to occur even after short-term exposure. Maintaining high effectiveness of these disinfectants towards L. monocytogens by means of rotation may prove ineffective because persistent and non-persistent strains cross-adapted both to related and unrelated disinfectants.

ACKNOWLEDGEMENTS

We are grateful to Jari Aho for excellent technical assistance. This study was supported by the Walther Ehrström Foundation and the Finnish Veterinary Foundation.

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