International Journal of Food Microbiology 189 (2014) 106–112

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

The influence of subminimal inhibitory concentrations of benzalkonium chloride on biofilm formation by Listeria monocytogenes Sagrario Ortiz a, Victoria López b, Joaquín V. Martínez-Suárez a,⁎ a b

Departamento de Tecnología de Alimentos, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Madrid, Spain Departamento de Bioinformática y Salud Pública, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain

a r t i c l e

i n f o

Article history: Received 24 April 2014 Received in revised form 19 July 2014 Accepted 2 August 2014 Available online 11 August 2014 Keywords: L. monocytogenes Quaternary ammonium compounds Benzalkonium chloride Resistance Biofilm SubMIC

a b s t r a c t Disinfectants, such as benzalkonium chloride (BAC), are commonly used to control Listeria monocytogenes and other pathogens in food processing plants. Prior studies have demonstrated that the resistance to BAC of L. monocytogenes was associated with the prolonged survival of three strains of molecular serotype 1/2a in an Iberian pork processing plant. Because survival in such environments is related to biofilm formation, we hypothesised that the influence of BAC on the biofilm formation potential of L. monocytogenes might differ between BAC-resistant strains (BAC-R, MIC ≥ 10 mg/L) and BAC-sensitive strains (BAC-S, MIC ≤ 2.5 mg/L). To evaluate this possibility, three BAC-R strains and eight BAC-S strains, which represented all of the molecular serotype 1/2a strains detected in the sampled plant, were compared. Biofilm production was measured using the crystal violet staining method in 96-well microtitre plates. The BAC-R strains produced significantly (p b 0.05) less biofilm than the BAC-S in the absence of BAC, independent of the rate of planktonic growth. In contrast, when the biofilm values were measured in the presence of BAC, one BAC-R strain (S10-1) was able to form biofilm at 5 mg/L of BAC, which prevented biofilm formation among the rest of the strains. A genetic determinant of BAC resistance recently described in L. monocytogenes (Tn6188) was detected in S10-1. When a BAC-S strain and its spontaneous mutant BAC-R derivative were compared, resistance to BAC led to biofilm formation at 5 mg/L of BAC and to a significant (p b 0.05) stimulation of biofilm formation at 1.25 mg/L of BAC, which significantly (p b 0.05) reduced the biofilm level in the parent BAC-S strain. Our results suggest that the effect of subminimal inhibitory concentrations of BAC on biofilm production by L. monocytogenes might differ between strains with different MICs and even between resistant strains with similar MICs but different genetic determinants of BAC resistance. For BAC-R strains similar to S10-1, subminimal inhibitory BAC may represent an advantage, compensating for the weak biofilm formation level that might be associated with resistance. Biofilm formation in the presence of increased subminimal inhibitory concentrations of the disinfectant may represent an important attribute among certain resistant and persistent strains of L. monocytogenes. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The survival and persistence of Listeria monocytogenes after cleaning and disinfection in food processing plants represent a major food safety concern (Ferreira et al., 2014). L. monocytogenes strains vary in their ability to persist within food environments (Holah et al., 2002; Keto-Timonen et al., 2007; López et al., 2008; Lundén et al., 2003); however, the factors governing persistence in L. monocytogenes remain unclear (Carpentier and Cerf, 2011). The ability to produce biofilms has been investigated as a characteristic favouring the persistence of L. monocytogenes strains (Møretrø and

Abbreviations: BAC, benzalkonium chloride; BAC-R, BAC-resistant strains (MIC ≥ 10 mg/L); BAC-S, BAC-sensitive strains (MIC ≤ 2.5 mg/L); MIC, minimal inhibitory concentration. ⁎ Corresponding author. Tel.: +34 91 3474027; fax: +34 91 3572293. E-mail address: [email protected] (J.V. Martínez-Suárez).

http://dx.doi.org/10.1016/j.ijfoodmicro.2014.08.007 0168-1605/© 2014 Elsevier B.V. All rights reserved.

Langsrud, 2004). However, the ability of L. monocytogenes to form true biofilms is not clear (Ferreira et al., 2014), as the more common method used to quantify L. monocytogenes biofilm formation (crystal violet staining of adhered cells) simply reflects cell adherence to a surface rather than a true biofilm (Ferreira et al., 2014). Few studies have employed techniques for characterisation of biofilm structure and architecture, e.g. microscopic methods, and analytical methods for the investigation of extracellular polymeric substances (EPS), e.g. carbohydrate-binding dyes. In addition, biofilm formation assays are difficult to correlate with persistence, most likely due to the influence on biofilm formation of the different experimental conditions tested (Folsom et al., 2006; Nilsson et al., 2011; Pan et al., 2010) and of the different strains assayed in different reports (Djordjevic et al., 2002; Folsom et al., 2006). In spite of this variation, biofilms provide many outstanding characteristics that enable bacteria to resist harmful environmental conditions, including disinfectants (Bridier et al., 2011; Møretrø and Langsrud,

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2004). However, resistance to disinfectants of cells grown in biofilms and the resistance of planktonic cells are different phenomena (Kastbjerg and Gram, 2009; Pan et al., 2006; Stopforth et al., 2002). The resistance of planktonic cells might be dependent on intrinsic attributes of the cells, such as the minimal inhibitory concentration (MIC) value of the strain. In several reports, no association between increased MICs of disinfectants and persistence in strains of L. monocytogenes have been demonstrated (Earnshaw and Lawrence, 1998; Heir et al., 2004; Holah et al., 2002; Kastbjerg and Gram, 2009; Lourenço et al., 2009). Disinfectants commonly used in the food industries include quaternary ammonium compounds (QACs), the most common of which is benzalkonium chloride (BAC). The presence of intrinsic and acquired QAC resistance in several foodborne pathogens may result in reduced efficacy of that disinfectant class (Buffet-Bataillon et al., 2012). For L. monocytogenes, resistance to BAC has been observed in different countries (Aase et al., 2000; Mereghetti et al., 2000; Mullapudi et al., 2008; Müller et al., 2013; Ortiz et al., 2014b; To et al., 2002). Although the final MIC values of these resistant strains are substantially lower than the concentrations at which QACs are used in food production facilities (Kastbjerg and Gram, 2012), a similar low level of resistance to QACs has been associated with the environmental persistence of L. monocytogenes in different food supply chains (Aase et al., 2000; Fox et al., 2011; Lundén et al., 2003; Ortiz et al., 2014b). In addition, it has been observed that L. monocytogenes strains which express increased resistance to BAC are generally less susceptible to antibiotics due to overexpression of different multidrug efflux pumps (Rakic-Martinez et al., 2011; Romanova et al., 2006). However, the resistance to BAC does not necessarily imply increased resistance to antibiotics at a level that could be relevant for clinical practice (Ortiz et al., 2014a). According to Carpentier and Cerf (2011), the presence of growth niches is the main factor that allows for the establishment of L. monocytogenes persistence; nevertheless, some strains may possess certain characteristics that increase their chances of becoming persistent (Ferreira et al., 2014). One of these properties might be biofilm formation in the presence of increased subminimal inhibitory concentrations (subMICs) of disinfectants by disinfectant-resistant strains (Nilsson et al., 2011), a foreseeable scenario in certain locations of the food plants (Carpentier and Cerf, 2011). More specifically, QACs are considered to have poor biodegradability, meaning that contact between bacteria and QACs may be prolonged and, as a consequence, microbial communities are exposed to subinhibitory concentrations (Buffet-Bataillon et al., 2012; Tezel and Pavlostathis, 2012). Subinhibitory concentrations of QACs can change biofilm morphology and architecture (Dynes et al., 2009), and even the effect of QACs at subinhibitory doses on biofilm formation can be stimulatory (Houari and Di Martino, 2007). These effects of subinhibitory QAC on biofilm formation could be different in QAC-resistant or -susceptible strains, similar to the different effect of subinhibitory BAC on the virulence of L. monocytogenes observed in strains with different susceptibilities to BAC (Pricope et al., 2013). Thus, the current study reports the influence of both subminimal inhibitory concentrations of BAC and the MIC values of the strain on biofilm formation by L. monocytogenes. We hypothesised that the exposure to subminimal inhibitory concentrations of BAC would have distinct effects on biofilm formation by BAC-resistant strains (BAC-R, MIC ≥ 10 mg/L) and BAC-sensitive strains (BAC-S, MIC ≤ 2.5 mg/L). 2. Materials and methods 2.1. Strains isolated from the Iberian pork processing plant Using pulsed-field gel electrophoresis (PFGE), 29 PFGE types were previously identified at an Iberian pork processing plant during a three-year period (Ortiz et al., 2010). The PFGE typing results were analysed in accordance with the optimised PulseNet standardised protocol (Halpin et al., 2010). For the molecular serotyping of isolates of

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L. monocytogenes, a multiplex PCR assay was performed as described by Doumith et al. (2004). The assay differentiated the most common disease-associated conventional serotypes (1/2a, 1/2b, 1/2c, and 4b) into four distinct molecular serotypes. An additional PCR assay based on amplification of the flaA gene allowed 1/2a (and 3a) strains to be confirmed and differentiated from atypical 1/2a, 3a, and 1/2c strains (Kérouanton et al., 2010). After the three-year study, the isolation rate of L. monocytogenes decreased, and only four of the 29 PFGE types were found in the following year (Ortiz et al., 2014b). These four “surviving” types included three from molecular serotype 1/2a, which were the only BAC-R subtypes found in the plant (Ortiz et al., 2014b). The first isolate with a unique PFGE type was considered the PFGE type strain of each PFGE type. In the present study, a subset of 11 PFGE type strains, which represented all of the serotype 1/2a PFGE types (three BAC-R and eight BAC-S), was selected for the characterisation of the biofilm formation. Only one molecular serotype was selected to assess the differences among strains, as there have been some reports of a correlation between serotype and the ability to form biofilms (Borucki et al., 2003; Nilsson et al., 2011; Pan et al., 2010). 2.2. Additional strains of L. monocytogenes L. monocytogenes EGD-e (ATCC BAA-679), which is a wild-type serotype 1/2a strain, was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and included in all tests as a BAC-S reference strain. A BAC-R laboratory mutant (S2BAC) of BAC-S strain S2 (Table 1) was obtained by selecting colonies grown on MIC plates inoculated with high inocula (109 CFU/mL, ca. 2 × 106 CFU per spot) after an incubation for 48 h at 37 °C. The resistant subculture was prepared from the plate containing the highest concentration of BAC, which resulted in a stable increase in MIC after subculture without BAC. The following strains of L. monocytogenes of serotype 1/2a were used as positive controls in the PCR screening of genetic determinants of BAC resistance: 4423 (qacH from Tn6188 and the flanking radC gene) and CDL 69 (bcrABC) (Müller et al., 2013). 2.3. Standard culture methods and inoculum preparation Tryptic soy yeast extract broth (TSYEB) and tryptic soy yeast extract agar (TSYEA), both obtained from Biolife (Milano, Italy), were used for routine growth of the strains. A fixed inoculum was prepared and used in all the experiments. Briefly, single colonies from fresh agar plates were transferred to sterile 0.1% (w/v) peptone water (PW, Biolife). The bacterial suspension was adjusted to an absorbance at 600 nm (A600 nm) of 0.85 (corresponding to 109 CFU/mL, as determined from a standard measurement relating A600 nm to plate counts). 2.4. Biofilm formation assay The assessment of biofilm formation by L. monocytogenes was performed using a microplate assay with crystal violet (CV, SigmaAldrich, St. Louis, MO, USA) staining and using the A580 nm reading of destained CV (Pan et al., 2010). Inoculum and polystyrene microtitre plates (Greiner Bio-One, Frickenhausen, Germany) were employed as described by Pan et al. (2010). The experimental culture medium (TSYEB supplemented with 1% glucose and 2% sodium chloride, s-TSYEB) and temperature (37 °C) conditions were selected based on a previous study (Pan et al., 2010) of the optimal conditions for biofilm formation in L. monocytogenes of serotype 1/2a. The incubation time was extended from two days (Pan et al., 2010; Ortiz et al., 2014b) to seven days based on findings from preliminary experimentation to address the assay's variability due to low absorbance measurements. Biofilm production was also assessed in a s-TSYEB medium with different concentrations of BAC (from 0.07 to 40 mg/L). For each strain, six wells (rows) of each concentration of BAC (columns) were inoculated.

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Table 1 Characteristics of Listeria monocytogenes strains of the molecular serotype 1/2a as used in this study. Strain

Characteristic

Origin

MIC of BAC (mg/L)a

Biofilmb

PCR screening for BAC resistance genesc Tn6188

S1 S2 S3 S6 S10-1 S10-2 S11 S14 S15 S16 S19 EGD-e S2BAC 4423 CDL 69

“Surviving” and BAC-resistant

“Surviving” and BAC-resistant “Surviving” and BAC-resistant

Reference strain BAC-resistant derivative of S2 Control strain for Tn6188 Control strain for bcrABC

Ortiz et al. (2014b) Ortiz et al. (2014b) Ortiz et al. (2014b) Ortiz et al. (2014b) Ortiz et al. (2014b) Ortiz et al. (2014b) Ortiz et al. (2014b) Ortiz et al. (2014b) Ortiz et al. (2014b) Ortiz et al. (2014b) Ortiz et al. (2014b) American Type Culture Collection This study Müller et al. (2013) Müller et al. (2013)

10 2.5 2.5 10 20 2.5 2.5 2.5 2.5 2.5 2.5 1.25 20 20 20

0.25 0.68 0.68 0.24 0.23 0.41 0.37 0.44 0.34 0.45 0.52 0.28 0.74 NP e NP

± ± ± ± ± ± ± ± ± ± ± ± ±

0.03F 0.06A 0.07A 0.05F 0.04F 0.06CD 0.07DE 0.07C 0.05E 0.05C 0.08B 0.05 0.11

bcrABC

qacH

radC

+

+

+

+

d

+

a

MIC, minimal inhibitory concentration; BAC, benzalkonium chloride. b The values represent the average A580 nm results for 18 replicates plus/minus the standard deviation corresponding to three independent assays, with six results each. The values of the 11 strains from the same origin with different uppercase letters (A–F) were significantly different (p b 0.05). c All results were negative unless indicated by +. d Flanking Tn6188. e NP, not performed.

2.5. Growth measurements in bioscreen C microwell plates

2.7. PCR screening for genetic determinants of BAC resistance

The growth of each strain in the biofilm formation medium (s-TSYEB) was individually monitored using an automated turbidity reader (Bioscreen C, Oy Growth Curves AB Ltd., Helsinki, Finland). The cultures were prepared as for the biofilm formation assay and inoculated in 200-well Bioscreen C microplates, which were incubated at 37 °C. The cell density was estimated as the final A600 nm reading taken after 24 h. The maximum specific growth rate [μ(A600 nm)] of the change in A600 nm with time was estimated as previously described (Baranyi and Roberts, 1994) using the DMFit 2.0 software (www.ifr.ac.uk/safety/ DMfit).

The presence of two different determinants for BAC resistance (qacH and bcrABC) was assayed in all strains, according to Müller et al. (2013). Screening for qacH was performed using two PCR assays, with primers targeting the qacH gene from Tn6188 and the flanking radC gene, into which Tn6188 is integrated, respectively (Müller et al., 2013). The third PCR assay used primers targeting the bcrABC genes (Elhanafi et al., 2010).

2.6. Susceptibility testing to BAC 2.6.1. MIC of BAC MIC determinations of BAC (Sigma-Aldrich) were performed as previously described (Ortiz et al., 2014b), using an agar dilution assay on Mueller Hinton agar (Biolife) plates (CLSI, 2008). The MIC was defined as the lowest concentration of BAC that completely prevented growth after incubation at 37 °C for 24 h.

2.6.1.1. Definition of resistant strain. Resistance in the current study refers to the resistance of planktonic cells, not to the resistance of biofilms. This term is used to describe the strains in which the MIC of BAC is clearly higher than that of the reference strains from the culture collections, i.e., L. monocytogenes EGD-e, which were tested in parallel (Ortiz et al., 2014b).

2.6.2. Viability of L. monocytogenes cells in the presence of BAC To determine the viability of L. monocytogenes cells in the presence of BAC, dilutions of the standardised inocula prepared in PW were exposed to BAC (from 0.6 to 10 mg/L) for 24 h at 37 °C. This was followed by QAC neutralisation (Langsrud and Sundheim, 1998) and the determination of the number of viable microorganisms.

2.8. Statistical analysis The biofilm formation was determined in replicates of 6, and these experiments were repeated on three different occasions (n = 18). The biofilm formation results were expressed as the means ± the standard deviation (SD) of the A580 nm reading of destained CV or as the percentages of biofilm reduction due to BAC, compared to the control without BAC. All other experiments involving the growth of L. monocytogenes were performed at least with duplicate samples and in three biological independent replicates. The differences between means were tested using the two sample Student's t-test. A one-way analysis of variance with Tukey's multiple comparison test was used at the 5% significance level to test for significant differences among the strains. Calculations were performed using Microsoft Excel 2007 (Microsoft Corporation, Redmond, WA, USA) and Minitab 16 statistical software (Minitab Inc., State College, PA, USA). 3. Results and discussion 3.1. Biofilm formation with no added BAC All strains were able to form biofilm; however, there was inter-strain variability. Among the 11 food-related strains (Table 1), the highest biofilm level was observed in strains S2 and S3 (0.68), and the lowest biofilm level was observed in S10-1 (0.23). The three BAC-R strains, S1, S6, and S10-1, showed a lower level of biofilm compared with the eight BAC-S strains (Table 1). Thus, there was a statistically significant association between reduced biofilm formation and BAC resistance

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3.2. Monitoring growth of L. monocytogenes strains individually in the biofilm formation medium without BAC Crystal violet has been found to stain both bacterial cells and biofilm matrix material (Pan et al., 2010), and, consequently, the different growth rates of strains may confound data interpretation. The growth in the biofilm formation medium without BAC of the different strains of L. monocytogenes was evaluated at 37 °C from absorbance measurements by assessing the final cell density based on the A600 nm at 24 h and maximum specific growth rate [μ(A600 nm)]. There were significant differences (p b 0.05, Tukey's test) in the final cell densities among several strains. For example, the BAC-R strain S10-1 showed a higher level of A600 nm at 24 h (0.36) compared with most of the strains, including BAC-R strains S1 and S6 (0.29 and 0.28, respectively) (Table S1). Thus, cell density could not be associated with the level of biofilm formed, and low biofilm did not necessarily imply low cell density. Although these values were relatively low, the absorbance of a sample in the Bioscreen is dependent on the volume used, and an A600 nm of 0.3 for the volume of 0.2 mL used in the wells can represent a standard A600 nm of 0.5 measured in the spectrophotometer (Mytilinaios et al., 2012). There were no significant differences (p N 0.05, Tukey's test) in the μ(A600 nm) of the different strains of L. monocytogenes when BAC was not present (Table S1). Although viable count data are generally believed to be more reliable than absorbance data for the estimation of maximum growth rate, absorbance measurements also may be used reliably for the estimation of maximum growth rates, in combination with time-to-detection measurements (Baranyi and Pin, 1999; Dalgaard et al., 1994). The comparison of the growth rates of low and

high biofilm-producing strains suggested that the observed effect of BAC resistance on biofilm production was independent of the rate of planktonic growth (Table S1). This result supports the findings in previous studies that the growth rate under optimal conditions is not directly correlated with biofilm formation (Djordjevic et al., 2002; Pan et al., 2010).

3.3. Biofilm formation in the presence of BAC Only three concentrations of BAC (1.25, 2.5, and 5 mg/L) had different effects on the biofilm formation by this group of strains of L. monocytogenes (Table S2), and only four different patterns of variation in the biofilm levels were observed, represented by strains S1, S11, S10-1, and S2BAC. Fig. 1 shows the percentage of the biofilm formed in the presence of BAC relative to the biofilm formed without BAC by these four representative strains. At 1.25 mg/L of BAC, all strains except S10-1 and S2BAC significantly reduced the biofilm levels (p b 0.05, Student's t-test) relative to controls without BAC (Fig. 1 and Table S2). The behaviour of the BAC-R strains S1 and S6 was similar to that of the BAC-S strains. Strain S10-1 maintained a 97% production of the biofilm, whereas S2BAC increased its biofilm production significantly, up to 123% (p b 0.05, Student's t-test), compared with the control without BAC. Therefore, the resistance of S2BAC led to the stimulation of biofilm formation at a concentration of BAC that significantly reduced biofilm levels in the parent strain S2 (Table S2). At 2.5 mg/L of BAC, i.e., the MIC of most BAC-S strains (Table 1), an additional and significant reduction of the biofilm was observed in most strains (p b 0.05, Student's t-test), except for S10-1 and S2BAC (Fig. 1). However, for a group of five strains (S11, S14, S15, S16, and S19), the level of biofilm at 2.5 mg/L of BAC increased, albeit not significantly (p N 0.05, Student's t-test), compared to the levels observed at 1.25 mg/L (Table S2). At 5 mg/L of BAC, a complete inhibition of biofilm formation was observed in all strains (Table S2) except S10-1 and S2BAC (Fig. 1). These two strains were able to form biofilm at a concentration of BAC that was inhibitory for most strains, although the biofilm levels were significantly reduced (p b 0.05, Student's t-test) relative to controls without BAC (Fig. 1). In L. monocytogenes, exposure to subinhibitory BAC has been shown to change the cell morphology (To et al., 2002) and the composition of

150

0 mg/L 1.25 mg/L

125

Percentage relative biofilm

(p = 0.011, Tukey's test). Because the three BAC-R strains belonged to long-term “surviving” PFGE types (Ortiz et al., 2014b), reduced biofilm formation was also associated with prolonged survival in the food plant (p = 0.011, Tukey's test). The biofilm formation value of EGD-e (0.28) can be considered intermediate compared with that of the BAC-R (0.23–0.25) and BAC-S (0.34–0.68) strains. The MIC of BAC for S2BAC (20 mg/L) was eight-fold higher than the MIC of parent strain S2 and similar to that of the naturally occurring BAC-R strain S10-1 (Table 1). The biofilm of L. monocytogenes S2BAC (0.74) was similar to the biofilm of its parent strain S2 and higher than those of natural BAC-R strains (Table 1). The 11 food-related strains studied were isolated from the same processing plant. The strains had been previously tested for BAC susceptibility (Ortiz et al., 2014b), and the results were correlated with the sanitation procedures employed in that plant (Ortiz et al., 2010). In addition, the study focused on molecular serotype 1/2a because the three BAC-R strains previously described belonged to that molecular serotype (Ortiz et al., 2014b) and because the results of biofilm formation by the same serotype might be more homogeneous and comparable (Borucki et al., 2003; Nilsson et al., 2011; Pan et al., 2010). The biofilm assay conditions were not intended to mimic food processing environments, but rather, to address the assay variability due to low absorbance measurements of BAC-R strains. Biofilm formation was measured only by crystal violet staining, and the number of strains was very limited; therefore, the biological relevance of the low biofilm formation by BAC-R strains should be confirmed with additional strains and conditions. Further, a high MIC of BAC was not always related to a low biofilm level in the absence of BAC, as observed with the high biofilm value of the artificial BAC-R strain S2BAC (Table 1). In this case, laboratory selection of the resistance to BAC of S2 did not affect biofilm formation potential, indicating that natural BAC-R strains might contain additional changes/mutations, compared with single-step artificially selected BAC-R mutants. Nevertheless, and in agreement with our results, Nilsson et al. (2011) found that most isolates of L. monocytogenes recovered as persistent food factory contaminants produced less biofilm than sporadic strains.

109

2.5 mg/L

**

5 mg/L

100

* *

75

* 50

*

*

*

25

0

Strain S1

Strain S11

Strain S10-1

Strain S2BAC

Fig. 1. Effect of benzalkonium chloride (BAC) on biofilm formation by four representative Listeria monocytogenes strains (S1, S11, S10-1, and S2BAC). The results are presented as the average percentages of biofilm formed with the addition of BAC concentrations of 1.25 mg/L (clear grey bar), 2.5 mg/L (dark grey bar) and 5 mg/L (black bar), compared with the control without the addition of BAC (100%, white bar). Reduced (*) or increased (**) percentages of biofilm differed significantly from the controls without BAC (p b 0.05, Student's t-test). The numerical results for all 13 strains included in this study are presented in Table S2.

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cellular phospholipids and fatty acids (Bisbiroulas et al., 2011). Subinhibitory concentrations of the QAC benzethonium chloride also can induce complex peptidoglycan biosynthesis responses in L. monocytogenes (Casey et al., 2014; Fox et al., 2011). Additionally, Romanova et al. (2006) showed that strains of L. monocytogenes resistant to BAC had cross-resistance to other QACs or QAC combinations such as myristalkonium chloride and clinicide (60% didecyl dimethyl ammonium chloride and 40% BAC). Therefore, it could be assumed that the same effect of subinhibitory BAC on L. monocytogenes applies to other disinfectants based on QACs. However, the effect might be specific to both the strain and the compound used. According to our results, the effect of BAC at 1.25 mg/L on biofilm formation differed between strains with different MICs (i.e., BAC-S strains or BAC-R strains S10-1 and S2BAC, Fig. 1) and even between BAC-R strains with similar MICs but likely different mechanisms of resistance (i.e., BAC-R strains S1 and S10-1, Fig. 1) (Ortiz et al., 2014b). Further work is needed to address the different mechanisms involved in the resistance and the response to BAC at concentrations allowing for the growth of the different BAC-R strains. Similar to the effects on biofilm formation, the effect of BAC at 1.25 mg/L on the virulence of L. monocytogenes was recently shown to be different in strains with different susceptibilities to BAC (Pricope et al., 2013).

A

3.4. Assessment of additional differences among the resistant strains To further compare the BAC-R strains, viability data were obtained from suspensions of three representative strains incubated for 24 h in BAC concentrations ranging from 0.6 to 10 mg/L. The viable count values of these strains were compared with those obtained from the same strains by the biofilm formation assay in the presence of BAC at the same concentrations (Fig. 2). Three L. monocytogenes strains were chosen, including two BAC-R strains, S1 and S10-1, and one representative BAC-S strain, S2. The exposure of L. monocytogenes to subminimal inhibitory concentrations of BAC resulted in the reduction of viability in all strains, although the pattern of reduction was strain-dependent. The incubation of strain S2 with 1.25 mg of BAC per litre (50% below MIC) resulted in less than 1 log CFU reduction (Fig. 2). However, the incubation of resistant strains S1 and S10-1 with 5 and 10 mg of BAC per litre (the corresponding 50% below MIC) resulted in viability reductions above 3 and 5 log CFU, respectively (Fig. 2). Thus, the bactericidal effect of BAC at the level of 50% below MIC was more pronounced among BAC-R strains. QACs are bactericidal agents (Tezel and Pavlostathis, 2012), but the bactericidal activity of BAC might be influenced by the different MICs and different types of BAC resistance that can exist. Nevertheless, results have shown that the effect of BAC on CFU at 24 h correlated

D

8

0.60

B

A580 nm

log10 CFU/mL

6 4

0

0.6

1.25 2.5 BAC (mg/L)

5

0.00

10

E

8

C

4 2

0

0.6

1.25 2.5 BAC (mg/L)

5

1.25 2.5 BAC (mg/L)

5

10

0

0.6

1.25 2.5 BAC (mg/L)

5

10

0

0.6

1.25 2.5 BAC (mg/L)

5

10

0.80

0.40

0.00

10

F

0.80 0.60

A580 nm

6 log10 CFU/mL

0.6

0.20

8

4

0.40 0.20

2 0

0

0.60 A580 nm

log10 CFU/mL

6

0

0.40 0.20

2 0

0.80

0

0.6

1.25 2.5 BAC (mg/L)

5

10

0.00

Fig. 2. Reduction of viability in peptone water (A, B, and C) and biofilm formation in nutrient-rich medium (D, E, and F) in the presence of benzalkonium chloride (BAC) by three L. monocytogenes strains (BAC-sensitive strain S2 [A and D], and BAC-resistant strains S10-1 [B and E], and S1 [C and F]). The graphs show the average results of three biological independent replicates using two replicates each. Error bars indicate the standard deviation.

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with the results of the seven-day old biofilm formation assay (Fig. 2), confirming that different BAC-R L. monocytogenes phenotypes have different abilities to develop biofilm in the presence of subinhibitory BAC. All 11 strains were screened for the presence of the two known genetic markers conferring BAC resistance in L. monocytogenes, Tn6188 or the bcrABC resistance cassette. Only one L. monocytogenes strain harbouring Tn6188 was detected (S10-1), and no strain was detected to possess the BAC-resistance cassette bcrABC (Table 1). We used primers targeting qacH or the flanking radC gene, which suggest that Tn6188 is integrated in the radC locus in S10-1 (Müller et al., 2013). In a panel of 91 strains of L. monocytogenes, Müller et al. (2013) found Tn6188 in 10 of the analysed strains, whereas bcrABC was found in 5 strains of the same panel. The strains harbouring Tn6188 were predominantly serotype 1/2a strains isolated from food or food processing environments (Müller et al., 2013), which is similar to strain S10-1. Dutta et al. (2013) analysed a group of 116 strains and found, however, that most BAC-R isolates of L. monocytogenes harboured bcrABC. This finding suggested a widespread dissemination of bcrABC across BAC-R L. monocytogenes strains, regardless of the serotype and source (Dutta et al., 2013). However, we found BAC-R strains which were negative for both genetic determinants, such as S1 and S6 (Table 1). Compared to Tn6188-harbouring strain S10-1, strains S1 and S6 showed a multidrug resistance (MDR) phenotype (Ortiz et al., 2014b). Mutations in endogeneous efflux pumps (Rakic-Martinez et al., 2011; Romanova et al., 2006) or modifications in the cell wall (To et al., 2002) resulting in increased MICs of BAC have already been described. These two mechanisms might explain the resistance phenotypes of strains S1 and S6, and of laboratory mutant S2BAC. In the case of S2BAC, the first mechanism might be associated with increased production of biofilm, as efflux pumps and biofilm production seem to be interrelated (Zhu et al., 2011). An increase in efflux pump activity could have several effects on biofilm formation through an increase in the extrusion or intrusion of signalling molecules. For example, in L. monocytogenes an ABC transporter involved in negative regulation of biofilm formation has been identified (Zhu et al., 2011), and a mutant in one of the components of this ABC transporter causes a strong increase in the capacity to form biofilm. 4. Conclusions The present findings suggest that the effect of subinhibitory concentrations of BAC on the biofilm production by L. monocytogenes might differ between strains with different MICs and even between resistant strains with similar MICs but different resistance genes. For BAC-R strains harbouring Tn6188, similar to the long-term persistent strain S10-1, subinhibitory BAC may represent an advantage, compensating for the weak biofilm formation level that might be associated with resistance. Biofilm formation in the presence of increased subinhibitory disinfectants may be an important attribute among certain resistant and persistent strains of L. monocytogenes. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ijfoodmicro.2014.08.007. Conflict of interest None declared. Acknowledgements The authors thank very much Pilar López for technical assistance. This work was supported by Research Project grant RTA2011-00098C02 (INIA FEDER) from the Spanish Ministry of Economy and Competitiveness. We also greatly acknowledge Dr. Stephan Schmitz-Esser (Institute for Milk Hygiene, Vienna, Austria), for providing strains 4423 and CDL 69.

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