Variability in biofilm production by Listeria monocytogenes correlated to strain origin and growth conditions

International Journal of Food Microbiology 150 (2011) 14–24

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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Variability in biofilm production by Listeria monocytogenes correlated to strain origin and growth conditions Rolf E. Nilsson ⁎, Tom Ross, John P. Bowman Food Safety Centre, Tasmanian Institute of Agricultural Research, School of Agricultural Science, University of Tasmania, Hobart, 7001, Tasmania, Australia

a r t i c l e

i n f o

Article history: Received 6 January 2011 Received in revised form 18 April 2011 Accepted 4 July 2011 Available online 23 July 2011 Keywords: Biofilm Environmental selection Stress Temperature pH

a b s t r a c t This study aimed to identify factors that influence the development of biofilm by Listeria monocytogenes strains and to determine the extent to which biofilm production protects against quaternary ammonium compound (QAC) disinfectant challenge. A total of 95 L. monocytogenes strains were studied and biofilm production was assessed as a function of incubation temperature, media pH, strain origin, serotype, and environmental persistence status. Attachment and biofilm development (inferred by the level of attached biomass) were measured in vitro using a colourimetric 96-well microtitre plate method in nutritive media (Brain–Heart Infusion). Increased biofilm production correlated with increasing temperature and the most acidic, or most alkaline, growth conditions tested. Clinical and environmental (food factory) strains were observed to increase biofilm production at higher and lower incubation temperatures respectively, independent of their rate of planktonic growth. Serotype 1/2a strains produced significantly more biofilm. Biofilm maturity, rather than strain, was correlated with resistance to QAC. Carbohydrate containing exopolymeric material could not be detected in the biofilm of representative strains, and no correlation between strains recovered as persistent food factory contaminants and biofilm production was identified. Although limited to in vitro inference based on the assay system used, our results suggest that environmental conditions determine the level of biofilm production by L. monocytogenes strains, independent of the rate of planktonic growth, and that this may manifest from selection pressures to which a given strain grows optimally. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Biofilms formed by Listeria monocytogenes can adhere to materials commonly used in food industries, and can enable the pathogen to contaminate food production facilities for extended periods of time, thereby increasing the risk of cross-contamination of foods (Lunden et al., 2000; Møretrø and Langsrud, 2004). Biofilms allow essential chemical transfer processes to occur between cells and the external environment while providing protection to the resident microbes from potentially harmful environmental conditions, e.g. disinfectants (Costerton et al., 1995; Pan et al., 2006). This potentially is a problem for food industries, as the complexity of both equipment and operations can mean that disinfectant doses may be below optimal in hard to reach places, resulting in harbourage sites within a facility. Furthermore, exposure to non-lethal disinfectant doses can increase an organism's resistance to subsequent, otherwise lethal, disinfectant concentrations, and stress resistance has been associated with virulence in a number of L. monocytogenes strains (Gray et al., 2006; ⁎ Corresponding author at: Food Safety Centre, Tasmanian Institute of Agricultural Research, School of Agricultural Science, University of Tasmania, Private Bag 54, Hobart, 7001, Tasmania, Australia. Tel.: +61 3 6226 6690; fax + 61 3 6226 2642. E-mail address: [email protected] (R.E. Nilsson). 0168-1605/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2011.07.012

Kallipolitis and Ingmer, 2006; Pan et al., 2006; Van der Veen et al., 2010). L. monocytogenes is a common contaminant of wet and refrigerated food processing plants, and persistent contamination and biofilm formation in such facilities has been reported (Holah et al., 2004; Lunden et al., 2002; Møretrø and Langsrud, 2004). Biofilms produced by L. monocytogenes are structurally simple in comparison to those produced by many other microorganisms, and a mature biofilm community can be established after 24 h (Chae and Schraft, 2000; Kalmokoff et al., 2001; Mafu et al., 1990; Rieu et al., 2008). Biofilm formation by L. monocytogenes varies among strains, with, however, the reasons for this variation remaining unclear (Borucki et al., 2003; Chae and Schraft, 2000; Harvey et al., 2007). It is known, however, that environmental stress influences both attachment and biofilm development in L. monocytogenes (Begley et al., 2009; Folsom et al., 2006; Norwood and Gilmour, 2001; Pan et al., 2006; Smoot and Pierson, 1998a, b). Increased knowledge of factors that contribute to variation in biofilm formation by L. monocytogenes strains is needed to optimise preventative measures and minimise the risk that biofilm production by L. monocytogenes presents to food industries. This study was designed to identify factors that influence the development of biofilm by L. monocytogenes strains in nutritive media, and to investigate whether biofilm production protects against

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disinfectant challenge that could contribute to persistent contamination of food processing environments. 95 L. monocytogenes strains, of diverse serotype and origins, were investigated to (i) assess the effect of incubation temperatures on attachment and biofilm formation and identify subgroups among those strains with increased propensity to form biofilms under these conditions, (ii) assess the effect of acidic and alkaline pH on L. monocytogenes strains biofilm formation and identify subgroups with increased propensity to form biofilm under these conditions, (iii) determine whether prolific biofilm producers have greater disinfectant resistance, and (iv) determine if environmentally “persistent” L. monocytogenes strains (i.e. strains recovered on more than one sampling occasion over a period of 12 months or greater) produce more biofilm under the different temperature and pH experimental conditions than other strains. 2. Materials and methods

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production were as described above with the following alterations: four pH levels were tested (pH 4.7, 5.7, 7.3 and 8.5 ± 0.1) at 25 °C. The pH was adjusted by the addition of sterile concentrated 1 N HCl and 1 M NaOH. 2.2.3. Statistical analysis The significance of differences in the relative amount of biofilm produced by each of the L. monocytogenes strains under each test condition, and as a function of the origin, serotype, and environmental persistence status was assessed by Univariate Analysis of Variance (ANOVA) coupled with t-test least significant difference (t-LSD) posthoc testing. Significance was assigned at P ≤ 0.05. Principal Component Analysis (PCA, based on a correlation matrix; Quinn and Keough, 2002) was performed to determine which of the qualitative traits (origin, serotype and environmental persistence) and time/temperature conditions had the greatest influence on biofilm production. Correlation was considered significant at P ≤ 0.05.

2.1. Bacterial strains 2.3. Estimation of growth rate Ninety-five L. monocytogenes isolates obtained from food factories, animals, foods and human clinical cases were studied. Environmentally “persistent” (recovered on successive occasions from the same environments over a sampling period; minimum 12 months, maximum 4 years) and presumed sporadic (recovered only once from the same plant environment over a sampling period; minimum 12 months, maximum, 4 years) strains were represented. Details of all strains are presented in Table 1. 2.2. Microtitre plate biofilm production assay 2.2.1. Effect of time and temperature on biofilm production Biofilm production at each of four temperatures was measured in triplicate (biological replicates) for each of the 95 L. monocytogenes isolates using the colorimetric 96-well microtitre plate method described by Djordjevic et al. (2002) with the modifications recommended by Borucki et al. (2003) (i.e. 0.1% crystal violet solution and inclusion of wells containing sterile water to minimise evaporative losses). All isolates were recovered from frozen storage by culture on Brain–Heart Infusion (BHI) agar (Oxoid CM0225B, Oxoid Australia, Adelaide, with 1.5% agar) and incubated at 25 °C for 24 h. Following this, each strain was transferred into four 10 mL BHI broths (pH 7.3 ± 0.1) and incubated for 24 h at 10 °C, 20 °C, 25 °C or 37 °C. After incubation, 100 μL of each culture was added to fresh 9.9 mL BHI broths, gently mixed, and 200 μL aliquots were transferred to wells of two 96-well (300 μL total volume) polystyrene (PS) microtitre plates (Greiner Scientific, Sigma-Aldrich, Australia). The duplicate plates were incubated statically at their corresponding treatment temperature (10, 20, 25 and 37 °C) for 10 h (20 h for the 10 °C incubation) to allow attachment and growth to occur (incubation times were selected based on findings from preliminary experimentation), after which the culture media were discarded. The plates were gently washed using phosphate buffered saline (PBS, pH7.3 ± 0.2), and 200 μL of fresh BHI broth was added to each well. A row of control wells containing 200 μL of sterile BHI broth was also added, and the plates were incubated at 10 °C, 20 °C, 25 °C and 37 °C. Optical density at 595 nm (OD595) was measured (as two independent experiments) at 24 and 48 h (120 and 144 h at 10 °C because biofilm could not be reliably detected prior to 120 h) to compare a newly formed and mature (older, established) biofilm respectively. 2.2.2. Effect of pH on biofilm formation A subset of 16 L. monocytogenes isolates (4 persistent and 10 sporadic contaminant strains recovered from a single Australian food processing facility, and two controls; Supplementary Table 1) were assessed for biofilm production under different pH culture conditions at 25 °C. Isolate recovery, culture and assessment of biofilm

Growth rate was compared for representative strains (strains observed to produce either high (n= 6) or low (n= 6) amounts of biofilm; Supplementary Table 1) that produced low or high OD595 measurements (least and most biofilm) at 10 or 37 °C incubation temperatures, and in acidic and alkaline culture preparations. A 100-μL inoculum collected from diluted overnight cultures was added to 10 mL BHI broths (with pH adjusted to 4.7, 5.7, 7.3 and 8.5± 0.1 as previously described) to attain an initial concentration of ≈ 1 × 103 cfu/mL. Twohundred microlitres of each cell suspension was added to triplicate wells in a 96-well microtitre plate. An uninoculated row of 12-wells containing 200 μL of sterile BHI was also prepared. The plates were incubated statically at 10 or 37 °C and OD540 recorded half-hourly using a Bio-Rad Benchmark microplate reader until the cultures entered stationary phase. Growth rates were predicted using the method described by Mellefont et al. (2003). 2.4. Attachment assay Attachment by the 12 representative strains (see Section 2.3) observed to produce low and high amounts of biofilm at 10 or 37 °C was determined using the microtitre plate biofilm production assay with the following adjustment. Twenty-four well (3.3 mL) PS microtitre plates were used (Greiner Scientific, Sigma-Aldrich, Australia) to increase the binding surface area and compensate for the decrease in measurable biomass. OD595 was measured at 1, 3, and 5 h (5, 10 and 24 h for the 10 °C incubation temperature). Incubation times were selected based on findings from preliminary experiments. 2.5. Susceptibility to a quaternary ammonium based disinfectant Disinfectant resistance in biofilms of the representative strains (see Section 2.3) that produced low and high amounts of biofilm at10 and 37 °C, and in acidic and alkaline culture media, was determined by exposing the biofilms to a quaternary ammonium compound (QAC) at 50 and 150 ppm for 60 s. Biofilm cultures were prepared as described previously and incubated statically at 10 or 37 ° C for 24, 48 and 72 h. Responses of biofilms grown at pH 4.7, 5.7, 7.3 and 8.5 ± 0.1 were assessed. After incubation, the cell suspensions were discarded and the plates were carefully washed three times using PBS to remove unattached cells. The plates were allowed to air dry for 5-min and 200 μL of a 50 or 150 ppm QAC solution (Supermix Sanitiser, Applied, Victoria, Australia; prepared from 600 ppm concentrated disinfectant solution using sterile distilled water) was applied for 60 s at room temperature (22 °C ± 2 °C), after which the plate was immediately flooded with PBS to dilute and remove the QAC. The biofilm cells were harvested by swabbing the entire well surface with sterile swabs

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R.E. Nilsson et al. / International Journal of Food Microbiology 150 (2011) 14–24 Table 1 (continued)

Table 1 The Listeria monocytogenes strains used in the current study. Strain

Origin/serotype origin

L1 (S2542)* 114-830-S7-154*‡§ Joyce* FW04-25*† 87-1599* 102-265-S3-352*‡§ Liver* FW04-19* 114-997-S7-63*‡§ L2 (S2657)* FW04-17* 102-195-S1-242*‡§ LO28* FW03-32* 102-265-S3-745*‡ ATCC 19114*† ScottA* ATCC 19115* 102-241-S1-349*‡§ FW03-35* FW04-20* 102-231-S7-566*‡§ FW04-21* 102-231-S7-232*‡§ 70-1700* 64-1495* FW06-22* 69-1793* 69-577* FW06-17* 83-2795* FW06-19* FW06-23* FW06-20* 62-2853* 70-0421* FW06-11* FW06-34* FW06-35* FW06-40* FW06-25* FW06-12* FW06-50* FW06-7* FW03-37* FW06-13* FW06-9* FW06-41* FW06-38* FW06-39* FW06-16* FW06-24* FW06-15* FW06-36* FW06-14* FW06-46* FW06-8* FW06-47* FW06-10* FW06-21* FW06-18* FW06-31* FW06-30* FW06-29* FW06-27* FW06-28* FW06-26* FW06-33* FW06-43* FW06-45* FW06-44* FW06-32* FW06-49* FW06-5* FW06-42*

Smoked salmon FPP3 Ovine Food product Bovine FPP1,4 + product Liver tissue HumanΩ FPP1 + food product Smoked salmon ClinicalΩ FPP1,2,3,4 + product Human Food product FPP1,4 + product Bovine Human Human FPP1 + food product Food product Clinical FPP1,2 + food product Clinical FPP1,2 + food product Ovine Ovine Human Bovine Bovine Human Ovine Human Human Human Bovine Wallaby Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human

1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2b 1/2b 1/2b 1/2b 1/2c 1/2c 3a 3a 4a 4b 4b 4b 4b 4b 4b 4b 4b 4e 1/2a 1/2. 4b 4b 1/2. 4a 1/2. 4/b 1/2. 1/2a 1/2b 1/2. 1/2. 4b 1/2. 1/2. 1/2. 1/2. 1/2a 1/2a 1/2. – 1/2. 4b 1/2. 3 4b 1/2. 1/2. – 4e 1/2. 1/2. 1/2. 1/2. 1/2. 1/2. 1/2. 1/2. 3 1/2. 1/2. 1/2. 3 1/2. 4b 1/2. 3 4b 4b

Strain

Origin/serotype origin

FW06-6* FW06-1* FW06-48* FW06-4* FW06-2* FW06-3* DS_14*†** DS_85*†** DS_25*†** DS_88*†** DS_31*†** DS_PRD5*†** DS_53*†** DS_B2L*†‡** DS_63*†** DS_68*†‡** DS_80*†‡** DS_81*†‡** DS_82*†** DS_84*†**

Human Human Human Human Human Human FPP5 FPP5 FPP5 FPP5 FPP5 FPP5 FPP5 FPP5 FPP5 FPP5 FPP5 FPP5 FPP5 FPP5

1/2. 1/2. 3 1/2. 1/2. 3 – – – – – – – – – – – – – –

* Assessed for ability to form biofilm at different temperatures. † Assessed for ability to form biofilm at different pH. ‡ This strain was recovered on all occasions, from multiple samples, during an Australian food factory survey conducted twice over a 12 month period (data not shown), and defined as a persistent contaminant. §These strains were recovered on multiple occasions, from multiple samples and sites, during a 21 month food factory survey and are defined as a persistent contaminant (Holah et al., 2004). Kindly provided by Dr John Holah and colleagues from the Campden and Chorleywood Food Research Association, Gloucestershire, United Kingdom. **These strains were all recovered by the authors from an Australian Food Factory sampling, conducted over a 12 month period (data not shown). FPP—Food production plant. Integers correspond to the food production plant/s from which the strain was recovered (1–4 correspond to the four food production plants sampled by Holah et al. (2004); 5 corresponds to the Australian food production plant sampled‡). Ω Strain origins are detailed exactly as recorded within the Food Safety Centre laboratory culture collection. Where a strain was not specifically recorded to have been recovered from a human its origin was assigned as “clinical”.

(MWE, UK) wetted with PBS. The swabs were placed in a sterile screw-cap tube (15 mL, Corning, Technolab, Australia) containing 5 mL of PBS and vortexed for 10 s. The resuspended biofilm cells were serially diluted in BHI broth and surviving cells were enumerated by spread plating on BHI-agar and incubation at 37 °C for 48 h. 2.6. Carbohydrate assay The presence of carbohydrate containing exopolymeric material in the biofilms of the12 representative strains (see Section 2.3; Supplementary Table 1) observed to produce low and high amounts of biofilm at 10 or 37 °C was assessed. Biofilm cultures were prepared as previously described (Section 2.4) and incubated statically at 10 or 37 °C for 24, 48 and 72 h. After incubation, the plates were gently washed 5 times using PBS, air dried for 5 min, and the presence of carbohydrate was assessed using a standard phenol-sulphuric acid colorimetric method (Dubois et al., 1956). 2.7. Scanning electron microscopy (SEM) Cell morphologies and attachment to stainless steel under stress conditions likely to be encountered in food processing environments were visualised by SEM using environmentally “persistent” strain DS_81 and environmentally “sporadic” strain DS_14. Three pH levels were tested (pH 4.7, 7.3 and 8.5 ± 0.1). The pH was adjusted as previously described. Cultures were grown on food-grade stainless steel coupons (20 mm × 20 mm × 1 mm, type-304, finish no. 4) suspended in BHI broth and incubated at 25 °C for 48 h. After incubation, the stainless steel coupons were removed from the cultures and rinsed 5 times with 10 mL of PBS. The coupons were

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immersed in 0.2 M cacodylate buffer (pH 7.4 ± 0.1) containing 25 mL/ L glutaraldehyde for 24 h for primary fixation, followed by two 5-min washes in 10 mL of 0.1 M cacodylate buffer (pH 7.4 ± 0.2), and then immersed in 1% osmium tetroxide for 20 min. The specimens were then dehydrated in a graded series of ethanol washes (15 min each at 40, 50, 70, 90 and 100% ethanol). Immediately after the final wash, the coupons were freeze-dried using a vacuum freeze dryer (Dynavac, Australia) for 5 h. The dried coupons were gold sputter coated. The fixed, coated, specimens were viewed (≥10 fields each) using an FEI Quanta 600 MLA environmental scanning electron microscope operated at an accelerating voltage of 15 kV. 3. Results High inter-strain variation in biofilm formation by L. monocytogenes was observed. Stationary growth phase was reached after an average of 108, 20.5, 19.6 and 16.7 h at incubation temperatures of 10, 20, 25 and 37 °C respectively. Increased biofilm production was observed as incubation time (post stationary growth phase) and temperature increased for all of the L. monocytogenes strains tested (Fig. 1), and temperature and incubation time were identified as the principal components contributing the most variation by PCA (eigenvalue N 1, Supplementary Fig. 1; df = 94, r ≥ 0.264, P b 0.05; Norman and Streiner, 1994; Supplementary Table 2). The greatest difference in biofilm production was observed between incubation temperatures of 10 (A595 0.01–0.48) and 20 °C (A595 0.01–0.86), and between 25 (A595 0.02–0.89) and 37 °C (A595 0.02–1.68) (Fig. 1). The observed increase was not, however, significantly different and resulted, largely, from increases in the absolute amount of biofilm production as incubation temperature increased, leading to greater absolute differences between prolific and poor biofilm producing strains (those strains observed to produce the

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highest and lowest A595 values; Fig. 2). Comparison of the growth rates of prolific and poor biofilm producing strains incubated at 10 and 37 °C, and under the range of pH studied, suggested that the observed temperature effect on biofilm production was independent of the rate of planktonic growth (Supplementary Table 3). Strains that were prolific biofilm producers at 10 or 37 °C adhered to the PS substrate faster than poor biofilm producers under the same conditions (Fig. 3). Strains exhibiting high biofilm production did not show increased survival when subjected to QAC disinfectant challenge up to 150 ppm for 60 s at either 10 or 37 °C, nor at any pH, but resistance of all strains was dependent on biofilm maturity (Fig. 4). Finally, carbohydrate containing exopolymeric material was not detected in the biofilms of either prolific or poor biofilm producing strains. The source of the L. monocytogenes isolate (Table 1) influenced the level of biofilm production, with clinical (n = 53) and food factory environmental (n = 19) isolates producing significantly more biofilm at higher and lower incubation temperatures respectively as indicated by both ANOVA (P b 0.05; Supplementary Table 4) and by post-hoc testing (t-LSD, P b 0.05; Table 2). The L. monocytogenes strains that produced significantly more biofilm at low temperatures were recovered, predominantly, from a single food production facility, and were found to produce similar levels of biofilm by principle component analysis relative to all other strains (Fig. 5). Furthermore, these strains have been identified as non-clonal by multilocus sequence typing (Nilsson, 2010). Importantly, in most cases (6 out of 8) isolates recovered as persistent food factory contaminants (three separate factories; Table 1) produced less biofilm than sporadic strains, and none of the persistent strains produced significantly greater levels of biofilm when compared against all other strains, while those recovered as sporadic contaminants did. Grouping the

Fig. 1. Biofilm production by L. monocytogenes strains (n = 95) grown in BHI medium and incubated at 10 °C (A), 20 °C (B), 25 °C (C) or 37 °C (D) measured at 24 and 48 h (120 and 144 h for the 10 °C incubation).

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Fig. 2. Mean absorbance data (●) and coefficient of variation (○) for all of the tested L. monocytogenes strains (n = 95) grown in BHI medium at 10 °C, 20 °C, 25 °C or 37 °C at measurement time one (24 or 120 h; A) and two (48 or 144 h; B).

“sporadic” (strain DS_14) food factory contaminant from this strain subset, at a range of pH levels after 48 h incubation, was visualised by SEM. Significantly increased biofilm production was observed under highly acidic (pH4.7) and alkaline (pH8.5) culture conditions after 48 h incubation (ANOVA, P b 0.05; Fig. 6; Supplementary Table 5). SEM of attachment to food-grade stainless steel after 48 h incubation supported the observations made using PS as the substratum, with increased cell numbers attached under the most acidic (pH4.7) and alkaline (pH8.5) conditions relative to pH7.3. No differences in the size or concentration of attached cells of the “persistent” and “sporadic” strains were evident (Fig. 7). 4. Discussion

Fig. 3. Attachment assay (PS substrate) results for L. monocytogenes strains (n= 12) observed to be prolific and poor biofilm producers cultured in BHI medium at 10 or 37 ° C (see Supplementary Table 1). (A) 10 ° C (B) 37 ° C. Strains DS_63 (●), DS_14 (○), DS_31 (▲), FW06-41 (■), FW06-12 (□), FW06-18 (♦) and 102-695-S1-154 (◊). Only strains that adhered during the test period are shown. The mean of three independent measurements is presented. Error bars indicate the standard deviation. Some error bars were omitted for figure clarity: A (24 h): ● 0.007, ▲0.005, ○ 0.006; B (3 h): □ 0.006, ♦ 0.005; B (5 h): ■ 0.008, □ 0.006, ♦ 0.005, ◊ 0.002.

results by serotype revealed, in most cases (5 out of 8), significantly greater biofilm production among serotype 1/2a strains (t-LSD, P b 0.05; Table 2). To assess further the single food factory strain subset (DS-strains, n = 14; see Table 1), their biofilm production was measured at a range of pH levels at 25 ° C for 24 and 48 h. In addition, attachment to foodgrade stainless steel at 25 °C by a “persistent” (strain DS_81) and

A number of studies have used variants of the crystal violet assay to measure biofilm produced by L. monocytogenes, and the validity of the assay has been questioned (Lu et al., 2009). High assay reproducibility was produced when the modifications recommended by Borucki et al. (2003) were applied (e.g. using 0.1% crystal violet solution). However, we found that assay variability increased with the low absorbance measurements obtained in the attachment assay. This was addressed by using plates with an increased surface area (17.6 mm 2 increased to 76.1 mm 2) to increase the amount of attached (measurable) biomass. We found the assay to be a reproducible measure of cellular attachment and biofilm production when used within the parameters of the original application of the assay, namely, indirect comparative assessment of irreversible bacterial attachment (biofilm production; Sauer et al., 2002) and, subsequently, in vitro biofilm measurement (Djordjevic et al., 2002). Our results are consistent with findings from a number of other studies (Borucki et al., 2003; Di Bonaventura et al., 2008; Folsom et al., 2006; Harvey et al., 2007; Møretrø and Langsrud, 2004; Pan et al., 2009; Smoot and Pierson, 1998a, b), who observed high inter-strain variation in biofilm formation by L. monocytogenes, and that environmental conditions, including the nutritive medium in which it is grown, influence biofilm production by this species. Although an

Fig. 4. Disinfectant (quaternary ammonium compound) challenge of L. monocytogenes biofilms after 24, 48 and 72 h incubation at 10 (A–D) or 37 ° C (E–H) in BHI medium with pH adjusted to 4.7 (A, E), 5.7 (B, F), 7.3 (C, G) and 8.5 (D, H). Mean viable counts recovered from each well (CFU/200 μL total well volume) from three independent experiments are presented. Error bars indicate the standard deviation. Strains DS_14 (prolific biofilm producer at 10 ° C; shaded bar, A–D), FW06-20 (poor biofilm producer at 10 ° C, white bar, A–D), L1 (poor biofilm producer at 37 ° C; shaded bar, E–H) and FW06-41 (prolific biofilm producer at 37 ° C; white bar, E–H) are shown. Minimum detection limit was 2 log CFU/well (CFU/200 μL total well volume).

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Table 2 Means of log10 absorbance (595 nm) under four incubation temperatures in BHI medium by L. monocytogenes strains (n = 95) defined by the qualitative characteristics⁎,† origin, serotype and persistent food factory contaminant. Comparisons were performed on means from a same column.

Incubation temperature 10° C

20° C

25° C

37° C

Qualitative assignment

120 h

144 h

24 h

48 h

24 h

48 h

24 h

48 h

Origin

Clinical (n = 53) Environmental (n = 22) Food production plant (n = 15) Wallaby (n = 1) Human (n = 52) Bovine (n = 5) Food production plant andproduct (n = 3) Ovine (n = 4) Food product (n = 4)

-1.82a -1.79a -1.63a -1.76a,b -1.82a,b -2.08b -2.09b -2.13b -2.17b

-1.72a -1.42b -1.21a -1.21a -1.73b -1.90b -1.87b -1.70b -1.85b

-1.40a -1.56a -1.38a -2.00b -1.39a -2.00b -1.84a,b -1.94b -2.02b

-1.18a -1.36a -1.07a -1.85c -1.17a,b -1.83c -1.89c -1.84c -2.06c

-1.49a -1.75a -1.58b -0.73a -1.48b -1.85b -2.07b -1.79b -2.15b

-0.92a -1.42b -1.95a,b -1.32a,b,c -0.90a -1.79b,c -1.85b,c -1.80b,c -1.96c

-0.94a -1.26b -1.17a,b -0.46a -0.93a -1.21a,b -1.35b -0.95a,b -1.53b

-0.69a -1.03b -0.85a,b -1.28b,c -0.68a -1.27b,c -1.28b,c -1.10a,b,c -1.52c

Persistence

Persistent (n = 11) Sporadic (n = 10)

-1.94b -1.59a

-1.60b -1.15a

-1.76b -1.31a

-1.70b -0.95a

-1.90a -1.55a

-1.63b -1.14a

-1.38a -1.33a

-1.23b -0.79a

Serotype

1/2a (n = 43) 1/2b (n = 5) 1/2c (n = 2) 3a (n = 8) 4a (n = 2) 4b (n = 17) 4e (n = 2)

-1.88a -1.96a -2.05b -1.82a -1.96a -1.97a -2.15b

-1.71a,b -1.52a -1.97b,c -1.75a,b,c -1.65a,b -1.83a,b,c -2.11c

-1.44a -1.99c -1.88c -1.47a -2.06c -1.72a,b -1.66a,b

-1.27a -1.89d -1.94d -1.39b -1.71c -1.59c -1.72c

-1.48a -1.76a -2.05a -1.50a -1.76a -1.85a -1.97a

-0.95a -1.67c -1.70c -1.06b -1.76c -1.58b -1.60b

-0.87a -1.16a -1.26a -1.14a -1.09a -1.29a -1.20a

-0.77a -1.31b -1.48c -0.84a -1.10b -1.07b -0.95b

* The values represent the log10 mean statistic for that group and significance was assigned using the Least Significant Difference method. Significantly increased levels of biofilm production for each comparison are shaded. Homogeneous groups within each qualitative assignment were assigned the same subscript letter within the table. Values with a unique subscript letter differ significantly at p ≤ 0.05. † t-Critical value for the origin, persistence and serotype post-hoc comparisons was 1.986, 1.988, 1.986 and 1.988 respectively.

Fig. 5. Principle component analysis scaling plot of biofilm production L. monocytogenes strains (n = 95) grown in BHI medium based on a correlation matrix of association between incubation times and temperatures. The isolates have been grouped by the origins of each strain; human/clinical (h), food production plant /food production plant and/or food product (f), ovine (O), bovine (b), wallaby (w) and unknown (u). Values represent standardised z-scores derived from the eigenvectors of the correlation matrix. Distances between objects are indicative of similarity in terms of the original variables; close = more similar. The circled isolates were obtained from a single food processing facility.

increase in biofilm production was detected between the two incubation times tested, this was not significant, suggesting that maximum attachment had already occurred by measurement time one. However, biofilm production significantly increased with temperature and under acidic, or alkaline, growth conditions, a finding consistent with that reported by Belessi et al. (2011) and Pan et al. (2009). Interestingly, in our studies this was neither a general

nor random response, with clinical and environmental (food factory) strains observed to increase biofilm production at higher and lower incubation temperatures respectively, independent of their rate of planktonic growth. Variability in biofilm production by L. monocytogenes is reported (Borucki et al., 2003; Harvey et al., 2007). In particular, the nutritive media used can affect biofilm growth by different L. monocytogenes strains (Pan et al., 2009). However, in the current study, a range of different strains increased relative biofilm production under the same culture conditions, all grown in highly nutritive media (BHI), and this was correlated with the origin of the strain (e.g. clinical, n = 53, or food factory environment, n = 19). Furthermore, some of the patterns in biofilm production observed differed to that described under the same nutritive growth conditions reported previously (e.g. serotype 1/2a strains producing more biofilm in dilute media; Pan et al., 2009). Our results indicate that defined sets of strains may produce biofilm at similar levels under specific (e.g. incubation temperature, media pH) conditions in highly nutritive media. At elevated incubation temperature (37 ° C) groups of strains, predominantly of clinical origin, showed significantly increased attachment to the PS substrate and increased biofilm production. This contrasts with previous studies in which clinical strains are poor biofilm formers (Harvey et al., 2007; Kalmokoff et al., 2001). Several studies have demonstrated that the cell surface properties of L. monocytogenes may be affected by incubation temperature (Briandet et al., 1999a,b; Chavant et al., 2002; Di Bonaventura et al., 2008). In addition, at temperatures above 30 °C, flagellin is repressed in most L. monocytogenes strains (Way et al., 2004). Although flagellum mediated attachment is a demonstrated means of biofilm initiation (Lemon et al., 2007), L. monocytogenes can adhere to inert surfaces through a passive attachment process independent of flagella (Tresse et al., 2009). Thus, it could be postulated that the observed strain variation may be due to altered cell surface properties that are induced in the clinical strains at 37 ° C, and to this end, further work is warranted.

Fig. 6. Biofilm production by a subset of L. monocytogenes strains (14 from a single food production facility and two clinical isolates; Table 1) following 24 h (A) and 48 h (B) incubation at 25 °C in BHI medium with pH adjusted to 4.7, 5.7, 7.3 and 8.5. Values represent the mean of three repeat measurements. Error bars indicate the standard deviation.

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Fig. 7. Scanning electron micrograph of substrate-attached L. monocytogenes cells. Strains DS_14 (sporadic food factory contaminant) and DS_81 (persistent food factory contaminant) are shown attached to a food-grade stainless steel coupon under acidic (pH 4.7; A, B, G), alkaline (pH 8.5; E, F, H) and pH 7.3 (C, D) culture conditions after 48 h incubation at 25 °C in BHI medium. A–F: 3500× magnification. G–H: 31332× magnification.

At 10 °C, a subset of L. monocytogenes strains recovered from food production environments produced significantly more biofilm than the other strains. As was observed at 37 °C, that subset of strains also showed increased number of attached cells to the PS substratum after short contact (24 h) times at low incubation temperature (10 °C). In particular, the DS_n strains (see Table 1) recovered from a single food production facility produced biofilm at a similar level at identical culture temperature. This group was found to be non-clonal by multilocus sequence typing (MLST), suggesting that common environmental signals induced biofilm production in a variety of different strains recovered from a common environment. This supports the notion that environmental cues can induce biofilm production based on the environmental conditions to which the strains had the opportunity to become adapted. The response of the factory strains upon exposure to pH stress was consistent with results obtained at 10 °C (produced biofilm at a similar level under identical culture conditions). This group of strains, including both persistent and sporadic food factory contaminants, all produced significantly more biofilm than all other strains tested under conditions of alkaline and acidic stress after 48 h; however no significant difference was observed at 24 h. This observation was supported by SEM of biofilms produced by a persistent and a sporadic strain from this group on stainless steel coupons (Fig. 7). The biofilm production was homogeneous both quantitatively and temporally, and may constitute an adaptive response reflecting the conditions that optimally induce this response in these strains. L. monocytogenes is flagellated and motile at temperatures ≤30 C, and generally non-flagellated and non-motile at N 30 °C (Grundling et al., 2004). At low temperatures, flagella production by L. monocytogenes may increase and has long been demonstrated as a means of attachment (Leifson and Palen, 1955; Tresse et al., 2009). From our results it might be postulated that multiple modes of biofilm initiation are able to be induced, e.g. by a specific stress such as acid or alkaline growth conditions (Tresse et al., 2006), low temperature (e.g. flagella-mediated attachment and biofilm initiation), and high temperature (e.g. reduced

motility, passive attachment processes, altered cell surface), and that each is favoured based on the environmental conditions that optimally induce this response in a given strain. Variable planktonic and biofilm growth has been described in L. monocytogenes (Begot et al., 1997; Borucki et al., 2003), and adaptation by some strains to growth in harsh environments is reported (Begot et al., 1997). The increase in biofilm production in stressful environments represents a form of survival response, and has largely been attributed to stress induced physiological adjustment in the cells (e.g., production of surface binding structures) resulting in an increased ability of the organism to attach to surfaces (Briandet et al., 1999a, b; Giovannacci et al., 2000; Tresse et al., 2006; Tresse et al., 2009). In addition, the chemical and physical parameters of the attachment surface and composition of the liquid medium influence biofilm production, and are also affected by the prevailing environmental conditions (Cuncliffe et al., 1999; Folsom et al., 2006). In combination these factors are suggested to dictate the rate, level and structure of biofilm assembly (Cuncliffe et al., 1999; Tresse et al., 2009). Based on our results, the ability of groups of microorganisms to produce biofilm may be subject to selection pressures based on specific combinations of chemical and physical parameters. We also observed three consistent patterns in biofilm production in this study that were independent of temperature, pH level or strain origin but were correlated with serotype and resistance to QAC. The first pattern appeared to involve serotype, with 5 out of 8 serotype 1/2a strains producing significantly more biofilm than other serotypes, irrespective of incubation temperature or media pH. Increased biofilm production by this serogroup has been reported before (Borucki et al., 2003) and warrants further investigation given the high association with food and the suggested clinical significance of strains belonging to this serotype (Gilmour et al., 2010). The second pattern appeared to involve resistance to QAC disinfectant. When representative strains (high and low biofilm producers; see Supplementary Table 1) were challenged with QAC disinfectant for 60 s, increased survival was only observed in mature biofilms (N48 h

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incubation), irrespective of whether the strain produced high or low amounts of biofilm. This is consistent with the findings of previous studies (Bremer et al., 2002; Pan et al., 2006), and suggests that a component of the mature biofilm, potentially an extra-cellular polymeric substance (EPS), may provide protection against QACs. Notably, although a small increase was evident, CFU data obtained for all strains in the control group of the QAC resistance assay (0 ppm QAC) was not significantly increased at acidic and alkaline pH after 48 h (Fig. 4). This contradicted observation made using the colourimetric assay alone (Fig. 6). To investigate this further, CFU data was obtained from 24 and 48 h biofilm cultures of representative strains (n = 12; Supplementary Table 1) at each pH tested in this study, and at low and high incubation temperatures (performed as described in Section 2.5), and these were compared with absorbance values obtained from the same cultures by the crystal violet assay (performed as described in Section 2.2). Results showed that the CFU present within a biofilm culture at acidic and alkaline pH was lower than the values indicated using the colourimetric assay; however CFU correlated with the colourimetric assay at low and high incubation temperatures at both 24 and 48 h (Fig. 8). It's possible that when grown at acidic or alkaline pH the crystal violet assay may be measuring total biomass within the biofilm including live, unculturable and dead cells, and possibly EPS. All of these components are, however, important parts of the biofilm mass, although it is the number of surviving cells within a biofilm that poses the greatest risk

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within a food factory environment. The extent to which any unculturable population contributes to the total biofilm mass is important and requires further investigation. It may be that the increase in total biofilm biomass, including EPS, may protect the surviving cells within the biofilm at acidic and alkaline pH. This has serious implications, as a number of studies have shown that L. monocytogenes can persist in hard to reach places within food factory environments where it can escape the action of mechanical cleaning, and, given sufficient nutriment, the unculturable population residing in these harbourage sites may be able to proliferate (Carpentier and Cerf, 2011). The composition of EPS produced by L. monocytogenes remains to be determined but is non-polysaccharide in nature since carbohydrate containing polymeric material could not detected in the current study, and genes corresponding to known exopolysaccharide and their transporters are not present on any of the sequenced genomes of L. monocytogenes (Deng et al., 2010). This is in contrast to reports claiming to have detected them (Borucki et al., 2003). Nwaiwu et al. (2010) suggested that L. monocytogenes produces a poly-γ-glutamate extra-cellular polymer, and demonstrated a role for this substance in desiccation tolerance, indicating that a protective non-carbohydrate EPS component is present in biofilms produced by L. monocytogenes. Additionally, Van der Veen and Abee (2011) observed increased resistance to both benzalkonium chloride and peracetic acid in a mixed species biofilm (L. monocytogenes and Lactobacillus plantarum) relative to a monospecies biofilm. Research is needed to establish whether the combination of biofilm maturity and mixed bacterial species, with associated increased total biomass including EPS, would further increase the resistance to disinfecting agents. Finally, no correlation between strains recovered as persistent food factory contaminants and high biofilm production was observed. While biofilm production could potentially augment persistent contamination by generating a reservoir and increasing resistance to sanitising agents, our results suggest that the resistance is dictated by the maturity, and possibly non-cellular material such as EPS, of the biofilm rather than the amount. This emphasizes the need for regular, comprehensive physical cleaning with disinfectants to prevent L. monocytogenes biofilms. Furthermore, our results suggest that properties other than biofilm production alone are needed to characterise true persistent L. monocytogenes cells, and recent studies suggest that these properties could, in part, be plasmid mediated (Elhanafi et al., 2010; Kuenne et al., 2010). To conclude, our results indicate that defined sets of L. monocytogenes strains may produce biofilm at similar levels under specific (e.g. incubation temperature, media pH) conditions in highly nutritive media. The work shows that the strain, strain origin and environmental conditions can determine the level of biofilm production by L. monocytogenes strains, independent of the rate of planktonic growth, and that the amount of biofilm produced and the environmental cue may represent more complex selection pressures to which the given strain has been exposed. Furthermore, this study suggests that biofilm production does not characterise the persistent L. monocytogenes phenotype, and that studies investigating environmental persistence in this species should be directed at other properties in addition to biofilm production. Finally, given the influence that the nutritive media can have on biofilm production by L. monocytogenes strains, extension of the work to investigate how this may contribute to the current findings is warranted.

Acknowledgments Fig. 8. Comparison of biofilm production after 24 h (filled circle/triangle) and 48 h (unfilled circle/triangle) incubation by L. monocytogenes strain FW06-46 measured using the crystal violet colorimetric method and colony counts recovered by swabbing after growth in BHI media. (A) Biofilm production in BHI with the pH adjusted to 4.7, 5.7, 7.3 and 8.5. (B) Biofilm production after incubation at 10, 20, 25 and 37 °C. Colony counts and absorbance measurements (595 nm) are indicated by triangles and circles respectively.

This work was supported by the Food Safety Centre, University of Tasmania, the Australian National Food Industry Strategy, and the Australian Postgraduate Award scheme. We thank Professor David Ratkowsky for his assistance with statistical analysis and Professor Tom McMeekin for critical review of the manuscript.

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