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Diversity assessment of Listeria monocytogenes biofilm formation: Impact of growth condition, serotype and strain origin
International Journal of Food Microbiology 165 (2013) 259–264
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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Diversity assessment of Listeria monocytogenes biofilm formation: Impact of growth condition, serotype and strain origin Sachin R. Kadam a,b, Heidy M.W. den Besten b,⁎, Stijn van der Veen a,b,1, Marcel H. Zwietering b, Roy Moezelaar a,2, Tjakko Abee a,b a b
TI Food and Nutrition, P.O. Box 557, 6700 AN Wageningen, The Netherlands Laboratory of Food Microbiology, Wageningen University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands
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Article history: Received 3 April 2013 Received in revised form 28 May 2013 Accepted 29 May 2013 Available online 5 June 2013 Keywords: Biofilm Intrinsic factors Environmental selection Stress Extracellular matrix
a b s t r a c t The foodborne pathogen Listeria monocytogenes has the ability to produce biofilms in food-processing environments and then contaminate food products, which is a major concern for food safety. The biofilm forming behavior of 143 L. monocytogenes strains was determined in four different media that were rich, moderate or poor in nutrients at 12 °C, 20 °C, 30 °C and 37 °C. The biofilm formation was mostly influenced by temperature, resulting in decreased biofilm formation with decreasing temperature. Biofilm formation was enhanced in nutrient-poor medium rather than in nutrient-rich medium, and especially in nutrient-poor medium significantly enhanced biofilm production was observed early in biofilm maturation underlining the effect of medium on biofilm formation rate. Also serotype had a significant effect on biofilm formation and was influenced by medium used because strains from both serotype 1/2b and 1/2a formed more biofilm than serotype 4b strains in nutrient-rich medium at 20 °C, 30 °C and 37 °C, whereas in nutrient-poor medium the biofilm production levels of serotype 1/2a and 4b strains were rather similar and lower than serotype 1/2b strains. The strains used originated from various origins, including dairy, meat, industrial environment, human and animal, and the level of biofilm formation was not significantly affected by the origin of isolation, irrespective of medium used and temperature tested. A linear model was used to correlate crystal violet staining of biofilm production to the number of viable cells within the biofilm. This showed that crystal violet staining was poorly correlated to the number of viable cells in nutrient-poor medium, and LIVE/DEAD staining and DNase I treatment revealed that this could be attributed to the presence of non-viable cells and extracellular DNA in the biofilm matrix. The significant impact of intrinsic and extrinsic factors on biofilm production of L. monocytogenes underlined that niche-specific features determine the levels of biofilm produced, and insights in biofilm formation characteristics will allow us to further optimize strategies to control the biofilm formation of L. monocytogenes. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Listeria monocytogenes is a Gram positive foodborne pathogen responsible for listeriosis, a rare but life-threatening systemic infection in pregnant women, immunocompromised, new-borns and elderly (Kathariou, 2002). The age group over 65 years old accounted for most of the reported cases Europe-wide (EFSA, 2012), and the main transmission route to humans is believed to be through consumption of contaminated food (Swaminathan and Gerner-Smidt, 2007). L. monocytogenes is able to persist in food processing environments by formation of biofilms on food processing equipment, and
⁎ Corresponding author. Tel.: +31 317 483213; fax: +31 317 484978. E-mail address: [email protected] (H.M.W. den Besten). 1 Present address: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom. 2 Present address: DSM Food Specialties, Biotechnology Center, Alexander Fleminglaan 1, 2613 AX Delft, The Netherlands. 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.05.025
in drains and pipes (Carpentier and Cerf, 2011), and subsequently disperses and contaminates the product. A biofilm is a multicellular complex, formed of microorganisms that are attached to a surface and generally embedded in a matrix of extracellular polymeric substances (EPS) (Donlan, 2002), including exopolysaccharides, proteins and extracellular DNA (eDNA) (Combrouse et al., 2013; Harmsen et al., 2010). Biofilms allow micro-organisms to persist in the environment and resist desiccation and provide protection against disinfection treatments (Van der Veen and Abee, 2011). Biofilm formation by L. monocytogenes varies among serotype, lineage and origin of isolation, and intrinsic and extrinsic factors like the nutrient level and temperature may influence biofilm development. A possible correlation between serotype or lineage and biofilm forming capacity is still controversial. Lineage I strains showed to produce more biofilm than strains from lineage II (Djordjevic et al., 2002), while others showed that 1/2a and/or 1/2c strains (lineage II) were better biofilm formers than 4b strains (lineage I) (Borucki et al., 2003; Combrouse et al., 2013; Harvey et al., 2007; Nilsson et al., 2011; Pan
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et al., 2010). Also the origin of isolation of the strains might affect biofilm formation (Nilsson et al., 2011). Reported differences in findings might be explained by the selected strains used, the temperature tested and the medium used. The use of an extensive strain collection is therefore important to investigate and compare biofilm influencing factors and to determine possible relationships between the ability of L. monocytogenes to form biofilm in various conditions and its origin and genetic background. We used in this study 143 L. monocytogenes strains from diverse serotypes and origins. Their biofilm forming capacity was investigated in rich, moderate and poor nutritive media at different temperatures that L. monocytogenes may encounter during its life cycle. We evaluated the biofilm production by a crystal violet assay and also enumerated the number of viable cells within the biofilm. This latter information is of importance as viable cells can introduce food safety risks upon (re)contamination of foods. We also correlated both methods, and the possible correlation between high biofilm formation capacity and role of non-viable cells and eDNA was examined. 2. Materials and methods 2.1. Bacterial strains and inoculum preparation A total of 143 L. monocytogenes strains from diverse origins and genetic backgrounds were used in this study (Table S1). Although 12 serotypes of L. monocytogenes can cause disease, only three serotypes (1/2a, 1/2b and 4b) account for more than 95% of the human listeriosis cases (Kathariou, 2002), and therefore the strains used in this study were mainly from these serotypes, namely, 1/2a (n = 53), 1/2b (n = 24), 4b (n = 43), and other serotypes (n = 23). The collection contained human (epidemic and sporadic cases; n = 44), meat (n = 40), dairy (n = 18), industrial environment (n = 23), animal (n = 8) strains, and strains with unknown origin (n = 10). Stock cultures grown in brain heart infusion (BHI, Becton Dickinson) broth were stored at −80 °C in 15% (v/v) glycerol. Working cultures were maintained on plates of four different media, namely, BHI agar, nutrient agar (NA, Oxoid), tryptic soy agar (TSA, Oxoid), and defined Hsiang-Ning Tsai medium (HTM) (Tsai and Hodgson, 2003) agar, all supplemented with 15 g agar [Oxoid] per liter. From each medium single colonies were inoculated in the respective broths and incubated overnight at 30 °C with shaking at 200 rpm (Innova 4335, New Brunswick Scientific). One percent inoculum was used in further experiments.
with 8.5 g sodium chloride [VWR] per liter) to remove non-adhered cells. Biofilm cells were dispersed in 1 ml of PPS by rigorous pipetting. Decimal dilutions were made in PPS and appropriate dilutions were plated on BHI agar plates. The plates were incubated at 30 °C for 48 h, and the colonies were counted. The biofilm formation assays for CV staining and plate counting were carried out two times. 2.3. Modelling the relationship between CV staining and plate counts To investigate the relationship between the CV staining assay and the number of viable, attached cells, the following model was used to fit the data OD595
nm
¼ a:N
ð1Þ
where OD595 nm is the absorbance of solubilized CV after biofilm formation (minus the blank), N is the number of viable, attached cells (CFU/well), and a is the proportionality constant between CV staining and number of viable cells. Parameter a was estimated by fitting Eq. (1) on 10log-scale in TableCurve 2D, and parameter estimates were verified in Microsoft Excel by using the Excel Solver add-in. 2.4. Evaluation of biofilms with fluorescent microscopy Static biofilms were grown in 12-well polystyrene microtiter plates (Greiner Bio-One) in BHI broth and NB at 30 °C. After 24 h of incubation, the medium was removed carefully and the biofilm was gently washed three times with PPS. Cell viability was examined by staining cells within the biofilm with LIVE/DEAD BacLight bacterial viability staining kit according to the manufacturer's instructions. Phase contrast and fluorescence microscopy were performed using a BX41 microscope (Olympus) using U-MNBV (SYTO 9) and U-MWIG (PI) fluorescence filters. 2.5. Effect of DNase I treatment on biofilm formation Bovine pancreatic DNase I (Sigma) was added to the growth media in different concentrations, namely, 0.5, 5 and 20 μg/ml. Biofilm formation was carried out in 96-well polystyrene microtiter plates at 30 °C as described above. 2.6. Statistical analysis
2.2. Biofilm formation assay Static biofilms were grown in 96-well polystyrene microtiter plates (Greiner Bio-One) and biofilm production was measured using crystal violet (CV) staining (Djordjevic et al., 2002) with the modification recommended by Borucki et al. (2003) (i.e. 0.1% crystal violet staining solution) and biofilm production was also measured using plate counting (Wijman et al., 2007). In short, microtiter plates were filled with 200 μl BHI broth, tryptic soy broth (TSB), nutrient broth (NB), and HTM, and for each strain two wells were inoculated with one percent inoculum, precultured in the respective media, and wrapped with parafilm and placed in plastic bags to minimize evaporation. The plates were statically incubated at 12 °C, 20 °C, 30 °C and 37 °C for 24 h, 48 h, 72 h, 96 h and 120 h. Then for CV staining, wells were gently washed three times with 250 μl of sterile water to remove non-adhered cells, and subsequently the biofilm was stained with 210 μl of 0.1% (w/v) crystal violet (Merck) for 30 min and washed three times with 250 μl of sterile water to remove unbound crystal violet. After drying, crystal violet was dissolved in 225 μl of 96% ethanol (Merck), and absorbance was measured at 595 nm (SpectraMax, Molecular Devices). Absorbance was corrected for the mean absorbance value of the blank. For enumeration by plate counting, wells were gently washed three times with 250 μl of peptone physiological salt (PPS) solution (1 g neutralized bacteriological peptone (Oxoid) supplemented
The effects of medium, temperature, incubation time, serotype and origin of isolation on biofilm formation capacities of L. monocytogenes were assessed by Student t-tests using the 10log-transformed absorbance values (10log OD595 nm). P-values that were less than 0.001 were considered significantly different. To compare the various effects on biofilm formation levels a Univariate Analysis of Variance was performed with temperature and time as quantitative variables and medium, origin and serotype as qualitative variables. Also a multiple regression was performed in which all variables were treated quantitatively to verify the findings. 3. Results 3.1. Diversity in biofilm formation: effect of medium, temperature and incubation time The biofilm forming capacity of 143 L. monocytogenes strains was investigated at 37 °C, 30 °C, 20 °C and 12 °C in four media ranging from nutrient-rich to nutrient-poor (BHI, TSB, NB, HTM) (Fig. 1). All strains were able to form biofilm, but the condition and incubation time affected the levels formed. Medium had a significant impact on biofilm formation. The highest biofilm formation was observed in nutrient-poor medium and the lowest in nutrient-rich
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Fig. 1. Biofilm formation by 143 L. monocytogenes strains grown in BHI broth (◇), TSB (△), NB (□) and HTM (○). Strains were incubated at 37 °C and 30 °C for 24 h–96 h and at 20 °C and 12 °C for 72 h–120 h. Each dot represents the average experimental absorbance value (OD595 nm) per strain, corrected for the blank. The gray dash represents the overall average absorbance value (OD595 nm) for each experimental condition.
medium. The impact of temperature was even more important than medium and resulted in decreased biofilm formation with decreasing temperature. Longer incubation times than 120 h at 12 °C did not significantly increase the biofilm formation in nutrient-rich medium (data not shown), suggesting that this effect was not only growth rate dependent. However, in HTM the biofilm levels were remarkably high at 12 °C, and slightly increased with longer incubation time (data not shown), indicating that medium influenced the temperaturedependent biofilm formation rate. Differences between biofilm formation at 37 °C and 30 °C were limited, and especially in HTM
more biofilm formation was observed at 37 °C than at 30 °C. In this nutrient-poor medium and also in NB, maximum biofilm formation was already observed at 24 h at 37 °C and 30 °C, while in BHI broth and TSB increased biofilm production was observed with increasing incubation time. 3.2. Effects of serotype and origin of isolation on biofilm formation Most of the strains belonged to the serotypes 1/2a, 1/2b and 4b, and to investigate whether L. monocytogenes biofilm formation capacity
A
B
Fig. 2. Biofilm formation by L. monocytogenes grouped per serotype, namely, serotype 1/2a (n = 53), 1/2b (n = 24), and 4b (n = 43). Strains were grown in BHI broth (A) and NB (B) and incubated at 37 °C and 30 °C for 24 h–96 h and at 20 °C and 12 °C for 72 h–120 h. Each dot represents the average experimental absorbance value (OD595 nm) per strain, corrected for the blank. The gray dash represents the overall average absorbance value (OD595 nm) for each experimental condition.
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A
B
Fig. 3. Biofilm formation by 143 L. monocytogenes strains grouped per origin of isolation. Strains were grown in BHI broth (A) and NB (B) and incubated at 30 °C for 24 h–96 h. Each dot represents the average experimental absorbance value (OD595 nm) per strain, corrected for the blank. The gray dash represents the overall average absorbance value (OD595 nm) for each experimental condition.
could be correlated to serotype, the strains belonging to these serotypes were grouped per serotype for each medium at the different temperatures. The effect of serotype on biofilm formation was significant and influenced by the medium used. In BHI broth, the biofilm production of the serotype 1/2b and 1/2a strains was higher than the biofilm production of the serotype 4b strains at 37 °C, 30 °C and 20 °C, especially after the first incubation time point (Fig. 2A). A similar trend of serotype on biofilm formation at these temperatures was observed in TSB (data not shown). However, in NB (Fig. 2B), and in HTM (data not shown) the biofilm production of the serotype 1/2a and 4b strains were rather similar and lower than the biofilm production of the serotype 1/2b strains. In these nutrient-poor media higher biofilm levels of the serotype 1/2b strains were mainly observed at the first incubation time point, underlining the effect of medium on biofilm formation rate. The 143 L. monocytogenes strains were also grouped per origin, namely dairy, meat, industrial environment, human, animal, and strains with unknown origin. The origin of isolation did not significantly affect the level of biofilm formation, irrespective of medium and temperature, and to illustrate this, the biofilm formation of the 143 strains at 30 °C in BHI broth and NB is shown in Fig. 3A and B, respectively. Only for some conditions, such as in BHI broth at 30 °C (Fig. 3A), the biofilm production of the animal isolates was lower than the other isolates, although not significantly. These findings might have been influenced by the rather small sample size of this group of isolates. 3.3. Enumeration of viable biofilm cells The CV staining assay is a widely used and accepted method for screening of biofilm formation. It has to be noted that crystal violet stains not only viable cells, but also extracellular matrix components and non-viable cells, and therefore, crystal violet staining may overestimate the number of viable, attached cells. Therefore, it is important to correlate the results of the CV staining to the number
Fig. 4. The correlation between the crystal violet (CV) staining assay and plate counts for two L. monocytogenes strains (strain 18: open symbols, strain 55: closed symbols). Biofilm was formed in BHI broth (◇) and NB (□) at 30 °C for 3 h–72 h. Absorbance values from the CV assay (OD595 nm) are plotted against the log CFU/well and fitted with the linear model (strain 18: solid line, strain 55: dashed line; BHI: black line, NB: gray line).
of viable cells within the biofilm obtained by plate counting. For that, the number of viable cells was quantified in BHI and NB medium at 30 °C every 3 h up to 72 h for two strains, namely, strain 55 and strain 18, which are moderate and high biofilm formers, respectively. Fig. 4 shows the correlation between OD values obtained in the CV staining assay and the number of viable biofilm cells for the two different strains. To evaluate the correlation between CV staining and cell counts, the data sets of the strains were fitted with a linear model (Eq. 1). The mean square error of the model fitting in BHI broth was lower than in NB for both strains, indicating that the variability was lower in BHI broth than in NB. Parameter a of the linear model represents a proportionality constant, and for both strains parameter a was higher in NB (a = 1.0 × 10−8 for strain 55 and a = 2.8 × 10−8 for strain 18, respectively) than in BHI (a = 3.2 × 10−9 for strain 55 and a = 8.0 × 10−9 for strain 18, respectively). This could suggest that in NB the extracellular matrix production is higher than in BHI broth and/or more non-viable cells are present in the biofilm matrix in NB. Therefore, the viability of cells within the biofilm was assessed using LIVE/DEAD staining for strain 55 and strain 18 in both BHI broth and NB (Fig. 5). When SYTO-9 dye and PI are used in combination, intact cells are labeled green and cells with damaged membranes are labeled red. In BHI broth, most cells were stained green, indicating that these cells were viable. But interestingly, in NB higher numbers of cells were stained red, suggesting that these cells got compromised membranes at some point during biofilm formation. In addition to membrane damaged cells, the extracellular biofilm matrix can also contain extracellular (e)DNA, which recently showed to be a central component in L. monocytogenes' biofilm matrix (Harmsen et al., 2010). To confirm the presence of eDNA, DNase I was added to the microtiter plates along with the media during biofilm formation by strain 55 (Fig. 6A) and strain 18 (Fig. 6B). This showed that only in NB the CV staining was lower in the presence of DNase, underlining that the biofilm composition included eDNA that contributed to the extracellular matrix. 4. Discussion In our study we assessed the biofilm forming behavior of a wide panel of L. monocytogenes strains in four different media that were rich, moderate or poor in nutrients and at temperatures L. monocytogenes may encounter from the food processing environment to the human host. The 143 L. monocytogenes strains originated from various origins and most of the strains belonged to the serotypes 1/2a, 1/2b and 4b, which are known to be the most important serotypes in human listeriosis incidents (Kathariou, 2002). This allowed us to investigate and compare the effects of temperature, medium, serotype and origin of isolation on biofilm production of L. monocytogenes.
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Fig. 5. Representative images of biofilm production of two L. monocytogenes strains (strain 55 and strain 18) in BHI broth and NB. The biofilms were stained after 24 h using a LIVE/DEAD BacLight bacterial viability staining kit. The scale bar represents 10 μm.
All L. monocytogenes strains were able to form biofilm, and we observed high inter-strain variation in biofilm production levels, which is consistent with the findings of other studies (Borucki et al., 2003; Di Bonaventura et al., 2008; Folsom et al., 2006; Harvey et al., 2007; Nilsson et al., 2011; Pan et al., 2009). The incubation temperature was the most significant factor influencing the biofilm production levels, and also the nutritive medium used and serotype were important factors (resulting from Univariate Analysis of Variance and verified by multiple regression). Biofilm formation significantly increased with temperature, which is in accordance with the results of other studies (Di Bonaventura et al., 2008; Nilsson et al., 2011; Pan et al., 2009). At 37 °C, flagellin expression is repressed in most of the L. monocytogenes strains (Way et al., 2004), and although flagellum-mediated motility has been demonstrated in biofilm initiation (Lemon et al., 2007), L. monocytogenes can also passively attach to surfaces (Tresse et al., 2009). This was consistent with our findings that differences between biofilm formations at 37 °C and 30 °C were limited, and biofilm production was even higher at 37 °C than at 30 °C in minimal medium. In agreement with previous studies (Combrouse et al., 2013; Harvey et al., 2007; Zhou et al., 2012), we demonstrated that L. monocytogenes biofilm production was higher in minimal medium compared to nutrient-rich medium, underlining that minimal nutrient conditions may stimulate biofilm production by L. monocytogenes. We also demonstrated that the effect of serotype was influenced by medium because both serotype 1/2b and 1/2a strains formed more biofilm than 4b strains in nutrient-rich medium, especially at higher temperature, whereas in nutrient-poor medium serotype 1/2b strains formed more biofilm than serotype 1/2a and 4b strains. However, differences between individual strains might be much bigger than the overall impact of serotype, and such realizations should be taken into account when presenting generalizing conclusions. We showed that the effect of origin of isolation on
A
*
**
B ** *
*
Fig. 6. Effect of DNase I treatment on biofilm formation in BHI broth and NB for strain 55 (A) and strain 18 (B). Absorbance values (OD595 nm) were measured after 24 h–72 h of incubation at 30 °C without the addition of DNase I (white bar), and with addition of 0.5 μg/ml (white, dashed bar), 5 μg/ml (gray bar) and 20 μg/ml (black bar). Error bars represent standard deviation of duplicates. * indicates significantly different from absorbance value without addition of DNase I (P b 0.05).
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biofilm formation was not significant, irrespective of the temperature used. This contrasts with previous findings in nutrient-rich medium (BHI) (Nilsson et al., 2011) where significant effects of origin were reported at higher and lower incubation temperatures. These differences might be explained by the different sets of isolates used. Moreover, we found in our study that besides BHI broth no significant effects of origin were also observed in TSB, NB and HTM. This underlined that it is worthwhile to investigate the effect of nutrient level on biofilm production as it is conceivable that similar variations in nutrient availability will be encountered in food production environments. The CV staining assay using polystyrene microtiter plates is commonly used to analyze biofilm capacity, and presumed to stain the total biomass including live and dead cells, but does not provide information on the number of viable cells within the matrix and the relative contribution of extracellular matrix components. In the present study we combined the CV staining results with the enumeration of viable biofilm cells because this information is of relevance as viable biofilm cells can (re)contaminate foods and introduce food safety risks. Crystal violet staining showed to be poorly correlated to the number of viable cells within the biofilm in nutrient-poor medium, and LIVE/DEAD staining and DNase I treatment revealed the presence of non-viable cells and eDNA in the extracellular matrix. It is conceivable that this contributed to the better correlation between viable counts and CV staining observed in BHI broth than in NB. To date, it is not yet known through which mechanisms eDNA is released by L. monocytogenes in biofilms (Da Silva and De Martinis, 2012), and the possible central role of eDNA in initial attachment and early biofilm formation (Harmsen et al., 2010) urges to answer this question. Biofilms containing large amounts of extracellular matrix might be more difficult to eradicate during cleaning and disinfection procedures due to the protective effect of the extracellular matrix (Sutherland, 2001). But loose biofilm cells that are not embedded in a dense extracellular matrix might disperse more easily, and may contaminate food products. Moreover, other microflora present in food processing premises might affect the ability of L. monocytogenes to colonize and form biofilms (Carpentier and Chassaing, 2004). Therefore, we are currently studying biofilm forming capacity of selected poor and high biofilm formers in absence and presence of accompanying flora. To conclude, our study showed that temperature was the most important factor influencing biofilm production. In addition, also medium and serotype significantly affected biofilm levels, whereas the impact of strain origin was not significant. Furthermore, our results indicated that the poor correlation between CV staining and viable biofilm cells, especially in nutrient-poor medium, could be attributed to the presence of non-viable cells and eDNA. More information about biofilm formation characteristics of L. monocytogenes on food contact materials other than polystyrene with representative nutrient conditions, and the role of accompanying microflora in L. monocytogenes' ability to attach and produce biofilm will help to optimize strategies to control this biofilm-forming pathogen. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.ijfoodmicro.2013.05.025.
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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Diversity assessment of Listeria monocytogenes biofilm formation: Impact of growth condition, serotype and strain origin Sachin R. Kadam a,b, Heidy M.W. den Besten b,⁎, Stijn van der Veen a,b,1, Marcel H. Zwietering b, Roy Moezelaar a,2, Tjakko Abee a,b a b
TI Food and Nutrition, P.O. Box 557, 6700 AN Wageningen, The Netherlands Laboratory of Food Microbiology, Wageningen University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands
a r t i c l e
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Article history: Received 3 April 2013 Received in revised form 28 May 2013 Accepted 29 May 2013 Available online 5 June 2013 Keywords: Biofilm Intrinsic factors Environmental selection Stress Extracellular matrix
a b s t r a c t The foodborne pathogen Listeria monocytogenes has the ability to produce biofilms in food-processing environments and then contaminate food products, which is a major concern for food safety. The biofilm forming behavior of 143 L. monocytogenes strains was determined in four different media that were rich, moderate or poor in nutrients at 12 °C, 20 °C, 30 °C and 37 °C. The biofilm formation was mostly influenced by temperature, resulting in decreased biofilm formation with decreasing temperature. Biofilm formation was enhanced in nutrient-poor medium rather than in nutrient-rich medium, and especially in nutrient-poor medium significantly enhanced biofilm production was observed early in biofilm maturation underlining the effect of medium on biofilm formation rate. Also serotype had a significant effect on biofilm formation and was influenced by medium used because strains from both serotype 1/2b and 1/2a formed more biofilm than serotype 4b strains in nutrient-rich medium at 20 °C, 30 °C and 37 °C, whereas in nutrient-poor medium the biofilm production levels of serotype 1/2a and 4b strains were rather similar and lower than serotype 1/2b strains. The strains used originated from various origins, including dairy, meat, industrial environment, human and animal, and the level of biofilm formation was not significantly affected by the origin of isolation, irrespective of medium used and temperature tested. A linear model was used to correlate crystal violet staining of biofilm production to the number of viable cells within the biofilm. This showed that crystal violet staining was poorly correlated to the number of viable cells in nutrient-poor medium, and LIVE/DEAD staining and DNase I treatment revealed that this could be attributed to the presence of non-viable cells and extracellular DNA in the biofilm matrix. The significant impact of intrinsic and extrinsic factors on biofilm production of L. monocytogenes underlined that niche-specific features determine the levels of biofilm produced, and insights in biofilm formation characteristics will allow us to further optimize strategies to control the biofilm formation of L. monocytogenes. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Listeria monocytogenes is a Gram positive foodborne pathogen responsible for listeriosis, a rare but life-threatening systemic infection in pregnant women, immunocompromised, new-borns and elderly (Kathariou, 2002). The age group over 65 years old accounted for most of the reported cases Europe-wide (EFSA, 2012), and the main transmission route to humans is believed to be through consumption of contaminated food (Swaminathan and Gerner-Smidt, 2007). L. monocytogenes is able to persist in food processing environments by formation of biofilms on food processing equipment, and
⁎ Corresponding author. Tel.: +31 317 483213; fax: +31 317 484978. E-mail address: [email protected] (H.M.W. den Besten). 1 Present address: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom. 2 Present address: DSM Food Specialties, Biotechnology Center, Alexander Fleminglaan 1, 2613 AX Delft, The Netherlands. 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.05.025
in drains and pipes (Carpentier and Cerf, 2011), and subsequently disperses and contaminates the product. A biofilm is a multicellular complex, formed of microorganisms that are attached to a surface and generally embedded in a matrix of extracellular polymeric substances (EPS) (Donlan, 2002), including exopolysaccharides, proteins and extracellular DNA (eDNA) (Combrouse et al., 2013; Harmsen et al., 2010). Biofilms allow micro-organisms to persist in the environment and resist desiccation and provide protection against disinfection treatments (Van der Veen and Abee, 2011). Biofilm formation by L. monocytogenes varies among serotype, lineage and origin of isolation, and intrinsic and extrinsic factors like the nutrient level and temperature may influence biofilm development. A possible correlation between serotype or lineage and biofilm forming capacity is still controversial. Lineage I strains showed to produce more biofilm than strains from lineage II (Djordjevic et al., 2002), while others showed that 1/2a and/or 1/2c strains (lineage II) were better biofilm formers than 4b strains (lineage I) (Borucki et al., 2003; Combrouse et al., 2013; Harvey et al., 2007; Nilsson et al., 2011; Pan
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et al., 2010). Also the origin of isolation of the strains might affect biofilm formation (Nilsson et al., 2011). Reported differences in findings might be explained by the selected strains used, the temperature tested and the medium used. The use of an extensive strain collection is therefore important to investigate and compare biofilm influencing factors and to determine possible relationships between the ability of L. monocytogenes to form biofilm in various conditions and its origin and genetic background. We used in this study 143 L. monocytogenes strains from diverse serotypes and origins. Their biofilm forming capacity was investigated in rich, moderate and poor nutritive media at different temperatures that L. monocytogenes may encounter during its life cycle. We evaluated the biofilm production by a crystal violet assay and also enumerated the number of viable cells within the biofilm. This latter information is of importance as viable cells can introduce food safety risks upon (re)contamination of foods. We also correlated both methods, and the possible correlation between high biofilm formation capacity and role of non-viable cells and eDNA was examined. 2. Materials and methods 2.1. Bacterial strains and inoculum preparation A total of 143 L. monocytogenes strains from diverse origins and genetic backgrounds were used in this study (Table S1). Although 12 serotypes of L. monocytogenes can cause disease, only three serotypes (1/2a, 1/2b and 4b) account for more than 95% of the human listeriosis cases (Kathariou, 2002), and therefore the strains used in this study were mainly from these serotypes, namely, 1/2a (n = 53), 1/2b (n = 24), 4b (n = 43), and other serotypes (n = 23). The collection contained human (epidemic and sporadic cases; n = 44), meat (n = 40), dairy (n = 18), industrial environment (n = 23), animal (n = 8) strains, and strains with unknown origin (n = 10). Stock cultures grown in brain heart infusion (BHI, Becton Dickinson) broth were stored at −80 °C in 15% (v/v) glycerol. Working cultures were maintained on plates of four different media, namely, BHI agar, nutrient agar (NA, Oxoid), tryptic soy agar (TSA, Oxoid), and defined Hsiang-Ning Tsai medium (HTM) (Tsai and Hodgson, 2003) agar, all supplemented with 15 g agar [Oxoid] per liter. From each medium single colonies were inoculated in the respective broths and incubated overnight at 30 °C with shaking at 200 rpm (Innova 4335, New Brunswick Scientific). One percent inoculum was used in further experiments.
with 8.5 g sodium chloride [VWR] per liter) to remove non-adhered cells. Biofilm cells were dispersed in 1 ml of PPS by rigorous pipetting. Decimal dilutions were made in PPS and appropriate dilutions were plated on BHI agar plates. The plates were incubated at 30 °C for 48 h, and the colonies were counted. The biofilm formation assays for CV staining and plate counting were carried out two times. 2.3. Modelling the relationship between CV staining and plate counts To investigate the relationship between the CV staining assay and the number of viable, attached cells, the following model was used to fit the data OD595
nm
¼ a:N
ð1Þ
where OD595 nm is the absorbance of solubilized CV after biofilm formation (minus the blank), N is the number of viable, attached cells (CFU/well), and a is the proportionality constant between CV staining and number of viable cells. Parameter a was estimated by fitting Eq. (1) on 10log-scale in TableCurve 2D, and parameter estimates were verified in Microsoft Excel by using the Excel Solver add-in. 2.4. Evaluation of biofilms with fluorescent microscopy Static biofilms were grown in 12-well polystyrene microtiter plates (Greiner Bio-One) in BHI broth and NB at 30 °C. After 24 h of incubation, the medium was removed carefully and the biofilm was gently washed three times with PPS. Cell viability was examined by staining cells within the biofilm with LIVE/DEAD BacLight bacterial viability staining kit according to the manufacturer's instructions. Phase contrast and fluorescence microscopy were performed using a BX41 microscope (Olympus) using U-MNBV (SYTO 9) and U-MWIG (PI) fluorescence filters. 2.5. Effect of DNase I treatment on biofilm formation Bovine pancreatic DNase I (Sigma) was added to the growth media in different concentrations, namely, 0.5, 5 and 20 μg/ml. Biofilm formation was carried out in 96-well polystyrene microtiter plates at 30 °C as described above. 2.6. Statistical analysis
2.2. Biofilm formation assay Static biofilms were grown in 96-well polystyrene microtiter plates (Greiner Bio-One) and biofilm production was measured using crystal violet (CV) staining (Djordjevic et al., 2002) with the modification recommended by Borucki et al. (2003) (i.e. 0.1% crystal violet staining solution) and biofilm production was also measured using plate counting (Wijman et al., 2007). In short, microtiter plates were filled with 200 μl BHI broth, tryptic soy broth (TSB), nutrient broth (NB), and HTM, and for each strain two wells were inoculated with one percent inoculum, precultured in the respective media, and wrapped with parafilm and placed in plastic bags to minimize evaporation. The plates were statically incubated at 12 °C, 20 °C, 30 °C and 37 °C for 24 h, 48 h, 72 h, 96 h and 120 h. Then for CV staining, wells were gently washed three times with 250 μl of sterile water to remove non-adhered cells, and subsequently the biofilm was stained with 210 μl of 0.1% (w/v) crystal violet (Merck) for 30 min and washed three times with 250 μl of sterile water to remove unbound crystal violet. After drying, crystal violet was dissolved in 225 μl of 96% ethanol (Merck), and absorbance was measured at 595 nm (SpectraMax, Molecular Devices). Absorbance was corrected for the mean absorbance value of the blank. For enumeration by plate counting, wells were gently washed three times with 250 μl of peptone physiological salt (PPS) solution (1 g neutralized bacteriological peptone (Oxoid) supplemented
The effects of medium, temperature, incubation time, serotype and origin of isolation on biofilm formation capacities of L. monocytogenes were assessed by Student t-tests using the 10log-transformed absorbance values (10log OD595 nm). P-values that were less than 0.001 were considered significantly different. To compare the various effects on biofilm formation levels a Univariate Analysis of Variance was performed with temperature and time as quantitative variables and medium, origin and serotype as qualitative variables. Also a multiple regression was performed in which all variables were treated quantitatively to verify the findings. 3. Results 3.1. Diversity in biofilm formation: effect of medium, temperature and incubation time The biofilm forming capacity of 143 L. monocytogenes strains was investigated at 37 °C, 30 °C, 20 °C and 12 °C in four media ranging from nutrient-rich to nutrient-poor (BHI, TSB, NB, HTM) (Fig. 1). All strains were able to form biofilm, but the condition and incubation time affected the levels formed. Medium had a significant impact on biofilm formation. The highest biofilm formation was observed in nutrient-poor medium and the lowest in nutrient-rich
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Fig. 1. Biofilm formation by 143 L. monocytogenes strains grown in BHI broth (◇), TSB (△), NB (□) and HTM (○). Strains were incubated at 37 °C and 30 °C for 24 h–96 h and at 20 °C and 12 °C for 72 h–120 h. Each dot represents the average experimental absorbance value (OD595 nm) per strain, corrected for the blank. The gray dash represents the overall average absorbance value (OD595 nm) for each experimental condition.
medium. The impact of temperature was even more important than medium and resulted in decreased biofilm formation with decreasing temperature. Longer incubation times than 120 h at 12 °C did not significantly increase the biofilm formation in nutrient-rich medium (data not shown), suggesting that this effect was not only growth rate dependent. However, in HTM the biofilm levels were remarkably high at 12 °C, and slightly increased with longer incubation time (data not shown), indicating that medium influenced the temperaturedependent biofilm formation rate. Differences between biofilm formation at 37 °C and 30 °C were limited, and especially in HTM
more biofilm formation was observed at 37 °C than at 30 °C. In this nutrient-poor medium and also in NB, maximum biofilm formation was already observed at 24 h at 37 °C and 30 °C, while in BHI broth and TSB increased biofilm production was observed with increasing incubation time. 3.2. Effects of serotype and origin of isolation on biofilm formation Most of the strains belonged to the serotypes 1/2a, 1/2b and 4b, and to investigate whether L. monocytogenes biofilm formation capacity
A
B
Fig. 2. Biofilm formation by L. monocytogenes grouped per serotype, namely, serotype 1/2a (n = 53), 1/2b (n = 24), and 4b (n = 43). Strains were grown in BHI broth (A) and NB (B) and incubated at 37 °C and 30 °C for 24 h–96 h and at 20 °C and 12 °C for 72 h–120 h. Each dot represents the average experimental absorbance value (OD595 nm) per strain, corrected for the blank. The gray dash represents the overall average absorbance value (OD595 nm) for each experimental condition.
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A
B
Fig. 3. Biofilm formation by 143 L. monocytogenes strains grouped per origin of isolation. Strains were grown in BHI broth (A) and NB (B) and incubated at 30 °C for 24 h–96 h. Each dot represents the average experimental absorbance value (OD595 nm) per strain, corrected for the blank. The gray dash represents the overall average absorbance value (OD595 nm) for each experimental condition.
could be correlated to serotype, the strains belonging to these serotypes were grouped per serotype for each medium at the different temperatures. The effect of serotype on biofilm formation was significant and influenced by the medium used. In BHI broth, the biofilm production of the serotype 1/2b and 1/2a strains was higher than the biofilm production of the serotype 4b strains at 37 °C, 30 °C and 20 °C, especially after the first incubation time point (Fig. 2A). A similar trend of serotype on biofilm formation at these temperatures was observed in TSB (data not shown). However, in NB (Fig. 2B), and in HTM (data not shown) the biofilm production of the serotype 1/2a and 4b strains were rather similar and lower than the biofilm production of the serotype 1/2b strains. In these nutrient-poor media higher biofilm levels of the serotype 1/2b strains were mainly observed at the first incubation time point, underlining the effect of medium on biofilm formation rate. The 143 L. monocytogenes strains were also grouped per origin, namely dairy, meat, industrial environment, human, animal, and strains with unknown origin. The origin of isolation did not significantly affect the level of biofilm formation, irrespective of medium and temperature, and to illustrate this, the biofilm formation of the 143 strains at 30 °C in BHI broth and NB is shown in Fig. 3A and B, respectively. Only for some conditions, such as in BHI broth at 30 °C (Fig. 3A), the biofilm production of the animal isolates was lower than the other isolates, although not significantly. These findings might have been influenced by the rather small sample size of this group of isolates. 3.3. Enumeration of viable biofilm cells The CV staining assay is a widely used and accepted method for screening of biofilm formation. It has to be noted that crystal violet stains not only viable cells, but also extracellular matrix components and non-viable cells, and therefore, crystal violet staining may overestimate the number of viable, attached cells. Therefore, it is important to correlate the results of the CV staining to the number
Fig. 4. The correlation between the crystal violet (CV) staining assay and plate counts for two L. monocytogenes strains (strain 18: open symbols, strain 55: closed symbols). Biofilm was formed in BHI broth (◇) and NB (□) at 30 °C for 3 h–72 h. Absorbance values from the CV assay (OD595 nm) are plotted against the log CFU/well and fitted with the linear model (strain 18: solid line, strain 55: dashed line; BHI: black line, NB: gray line).
of viable cells within the biofilm obtained by plate counting. For that, the number of viable cells was quantified in BHI and NB medium at 30 °C every 3 h up to 72 h for two strains, namely, strain 55 and strain 18, which are moderate and high biofilm formers, respectively. Fig. 4 shows the correlation between OD values obtained in the CV staining assay and the number of viable biofilm cells for the two different strains. To evaluate the correlation between CV staining and cell counts, the data sets of the strains were fitted with a linear model (Eq. 1). The mean square error of the model fitting in BHI broth was lower than in NB for both strains, indicating that the variability was lower in BHI broth than in NB. Parameter a of the linear model represents a proportionality constant, and for both strains parameter a was higher in NB (a = 1.0 × 10−8 for strain 55 and a = 2.8 × 10−8 for strain 18, respectively) than in BHI (a = 3.2 × 10−9 for strain 55 and a = 8.0 × 10−9 for strain 18, respectively). This could suggest that in NB the extracellular matrix production is higher than in BHI broth and/or more non-viable cells are present in the biofilm matrix in NB. Therefore, the viability of cells within the biofilm was assessed using LIVE/DEAD staining for strain 55 and strain 18 in both BHI broth and NB (Fig. 5). When SYTO-9 dye and PI are used in combination, intact cells are labeled green and cells with damaged membranes are labeled red. In BHI broth, most cells were stained green, indicating that these cells were viable. But interestingly, in NB higher numbers of cells were stained red, suggesting that these cells got compromised membranes at some point during biofilm formation. In addition to membrane damaged cells, the extracellular biofilm matrix can also contain extracellular (e)DNA, which recently showed to be a central component in L. monocytogenes' biofilm matrix (Harmsen et al., 2010). To confirm the presence of eDNA, DNase I was added to the microtiter plates along with the media during biofilm formation by strain 55 (Fig. 6A) and strain 18 (Fig. 6B). This showed that only in NB the CV staining was lower in the presence of DNase, underlining that the biofilm composition included eDNA that contributed to the extracellular matrix. 4. Discussion In our study we assessed the biofilm forming behavior of a wide panel of L. monocytogenes strains in four different media that were rich, moderate or poor in nutrients and at temperatures L. monocytogenes may encounter from the food processing environment to the human host. The 143 L. monocytogenes strains originated from various origins and most of the strains belonged to the serotypes 1/2a, 1/2b and 4b, which are known to be the most important serotypes in human listeriosis incidents (Kathariou, 2002). This allowed us to investigate and compare the effects of temperature, medium, serotype and origin of isolation on biofilm production of L. monocytogenes.
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Fig. 5. Representative images of biofilm production of two L. monocytogenes strains (strain 55 and strain 18) in BHI broth and NB. The biofilms were stained after 24 h using a LIVE/DEAD BacLight bacterial viability staining kit. The scale bar represents 10 μm.
All L. monocytogenes strains were able to form biofilm, and we observed high inter-strain variation in biofilm production levels, which is consistent with the findings of other studies (Borucki et al., 2003; Di Bonaventura et al., 2008; Folsom et al., 2006; Harvey et al., 2007; Nilsson et al., 2011; Pan et al., 2009). The incubation temperature was the most significant factor influencing the biofilm production levels, and also the nutritive medium used and serotype were important factors (resulting from Univariate Analysis of Variance and verified by multiple regression). Biofilm formation significantly increased with temperature, which is in accordance with the results of other studies (Di Bonaventura et al., 2008; Nilsson et al., 2011; Pan et al., 2009). At 37 °C, flagellin expression is repressed in most of the L. monocytogenes strains (Way et al., 2004), and although flagellum-mediated motility has been demonstrated in biofilm initiation (Lemon et al., 2007), L. monocytogenes can also passively attach to surfaces (Tresse et al., 2009). This was consistent with our findings that differences between biofilm formations at 37 °C and 30 °C were limited, and biofilm production was even higher at 37 °C than at 30 °C in minimal medium. In agreement with previous studies (Combrouse et al., 2013; Harvey et al., 2007; Zhou et al., 2012), we demonstrated that L. monocytogenes biofilm production was higher in minimal medium compared to nutrient-rich medium, underlining that minimal nutrient conditions may stimulate biofilm production by L. monocytogenes. We also demonstrated that the effect of serotype was influenced by medium because both serotype 1/2b and 1/2a strains formed more biofilm than 4b strains in nutrient-rich medium, especially at higher temperature, whereas in nutrient-poor medium serotype 1/2b strains formed more biofilm than serotype 1/2a and 4b strains. However, differences between individual strains might be much bigger than the overall impact of serotype, and such realizations should be taken into account when presenting generalizing conclusions. We showed that the effect of origin of isolation on
A
*
**
B ** *
*
Fig. 6. Effect of DNase I treatment on biofilm formation in BHI broth and NB for strain 55 (A) and strain 18 (B). Absorbance values (OD595 nm) were measured after 24 h–72 h of incubation at 30 °C without the addition of DNase I (white bar), and with addition of 0.5 μg/ml (white, dashed bar), 5 μg/ml (gray bar) and 20 μg/ml (black bar). Error bars represent standard deviation of duplicates. * indicates significantly different from absorbance value without addition of DNase I (P b 0.05).
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biofilm formation was not significant, irrespective of the temperature used. This contrasts with previous findings in nutrient-rich medium (BHI) (Nilsson et al., 2011) where significant effects of origin were reported at higher and lower incubation temperatures. These differences might be explained by the different sets of isolates used. Moreover, we found in our study that besides BHI broth no significant effects of origin were also observed in TSB, NB and HTM. This underlined that it is worthwhile to investigate the effect of nutrient level on biofilm production as it is conceivable that similar variations in nutrient availability will be encountered in food production environments. The CV staining assay using polystyrene microtiter plates is commonly used to analyze biofilm capacity, and presumed to stain the total biomass including live and dead cells, but does not provide information on the number of viable cells within the matrix and the relative contribution of extracellular matrix components. In the present study we combined the CV staining results with the enumeration of viable biofilm cells because this information is of relevance as viable biofilm cells can (re)contaminate foods and introduce food safety risks. Crystal violet staining showed to be poorly correlated to the number of viable cells within the biofilm in nutrient-poor medium, and LIVE/DEAD staining and DNase I treatment revealed the presence of non-viable cells and eDNA in the extracellular matrix. It is conceivable that this contributed to the better correlation between viable counts and CV staining observed in BHI broth than in NB. To date, it is not yet known through which mechanisms eDNA is released by L. monocytogenes in biofilms (Da Silva and De Martinis, 2012), and the possible central role of eDNA in initial attachment and early biofilm formation (Harmsen et al., 2010) urges to answer this question. Biofilms containing large amounts of extracellular matrix might be more difficult to eradicate during cleaning and disinfection procedures due to the protective effect of the extracellular matrix (Sutherland, 2001). But loose biofilm cells that are not embedded in a dense extracellular matrix might disperse more easily, and may contaminate food products. Moreover, other microflora present in food processing premises might affect the ability of L. monocytogenes to colonize and form biofilms (Carpentier and Chassaing, 2004). Therefore, we are currently studying biofilm forming capacity of selected poor and high biofilm formers in absence and presence of accompanying flora. To conclude, our study showed that temperature was the most important factor influencing biofilm production. In addition, also medium and serotype significantly affected biofilm levels, whereas the impact of strain origin was not significant. Furthermore, our results indicated that the poor correlation between CV staining and viable biofilm cells, especially in nutrient-poor medium, could be attributed to the presence of non-viable cells and eDNA. More information about biofilm formation characteristics of L. monocytogenes on food contact materials other than polystyrene with representative nutrient conditions, and the role of accompanying microflora in L. monocytogenes' ability to attach and produce biofilm will help to optimize strategies to control this biofilm-forming pathogen. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.ijfoodmicro.2013.05.025.
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