International Journal of Food Microbiology 199 (2015) 41–46
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Short communication
Exploring the strain-specific attachment of Leuconostoc gelidum subsp. gasicomitatum on food contact surfaces Vasileios Pothakos ⁎, Yosi Ayu Aulia, Inge Van der Linden, Mieke Uyttendaele, Frank Devlieghere LFMFP, Laboratory of Food Microbiology and Food Preservation, Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University, Member of Food2Know, Coupure Links 653, B-9000 Gent, Belgium
a r t i c l e
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Article history: Received 20 November 2014 Received in revised form 7 January 2015 Accepted 8 January 2015 Available online 15 January 2015 Keywords: Leuconostoc gelidum subsp. gasicomitatum Attachment Intra-subspecies diversity Food contact surface Putative biofilm formation
a b s t r a c t The psychrotrophic lactic acid bacterium (LAB) Leuconostoc gelidum subsp. gasicomitatum has emerged as one of the most prevalent specific spoilage organisms (SSOs) of packaged, cold-stored food products in Northern Europe. The whole genome sequencing of the type strain L. gelidum subsp. gasicomitatum LMG 18811T revealed genes encoding for proteins related to adhesion. In the present study the attachment of six food and environmental isolates was monitored on stainless steel (SS) and glass surfaces incubated (7 °C for 5–9 days) in two food simulating substrates (i.e. sweet bell pepper juice and boiled eggs in brine). The selection encompassed unique genotypes, isolated from different food products or sampling sites as well as slime-forming biotypes. The evaluation of the attached cells was performed with the bead vortexing method and a viability staining assay coupled with epifluorescence microscopy. On SS surfaces the slime-formers showed the lowest attachment (3.3–4.5 log CFU/cm2), while strain L. gelidum subsp. gasicomitatum ab2, which was isolated from an acetic acid bath in a vegetable salad company, reached significantly higher populations of attached cells exceeding 7 log CFU/cm2. Strain ab2 formed dense cell aggregations on SS after 9 days of incubation in sweet bell pepper juice. The attachment ability of L. gelidum subsp. gasicomitatum on surfaces documented in the present study extends our knowledge and understanding of the spoilage potential and intra-subspecies diversity of this microbe. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The former species Leuconostoc gasicomitatum was described in 2000 as a specific spoilage organism (SSO) in packaged, marinated meat in Finland (Björkroth et al., 2000) and for one decade numerous severe spoilage cases attributed to this psychrotrophic LAB were further reported (Lyhs et al., 2004; Nieminen et al., 2011; Susiluoto et al., 2003; Vihavainen and Björkroth, 2007, 2009; Vihavainen et al., 2008). Recently, the high occurrence of this microbe was substantiated for various-packaged and refrigerated foodstuffs in Belgium highlighting the underestimation of its populations when implementing mesophilic incubation (30 °C) due to its strict psychrotrophic character, its high spoilage potential and the broad range of food products, in which it thrives (Pothakos et al., 2012, 2014a, 2014d). Raw, fresh, marinated or cooked meat, vegetable sausages and salads, ready-to-eat (RTE) meals, boiled eggs in brine and marinated herring have been commodities of isolation. Currently, incidents of spoilage alterations in packaged, retail food products have been reported to us by industries of other Northern European countries confirming the prevalence and rapid increase of L. gelidum contamination in food processing environments (personal
⁎ Corresponding author. Tel.: +32 2 629 38 63; fax: +32 2 629 27 20. E-mail address: [email protected] (V. Pothakos).
http://dx.doi.org/10.1016/j.ijfoodmicro.2015.01.008 0168-1605/© 2015 Elsevier B.V. All rights reserved.
communication). The genome sequencing of the type strain L. gelidum subsp. gasicomitatum LMG 18811T has unraveled the gene repertoires associated with fast outgrowth and spoilage manifestations (i.e. nutrient-uptake mechanisms, functional heme-mediated respiration, slime formation, emission of buttery off-odor, green discoloration) and indicated the presence of genes encoding for adhesion (Johansson et al., 2011). Studies so far have focused on the phylogeny (Rahkila et al., 2014; Vihavainen and Björkroth, 2009), spoilage potential (Pothakos et al., 2014b), metabolic patterns and growth dynamics (Jääskeläinen et al., 2013) of this subspecies. Nonetheless, it remains unclear which factors mediate contamination in the first place. A source tracking performed in a vegetable-salad company showed that the origin of psychrotrophic leuconostocs was the raw sweet bell peppers and cross-contamination of other vegetables was facilitated by air and surface-mediation (Pothakos et al., 2014c). Despite the fact that raw materials could constitute carriers of psychrotrophic LAB (Audenaert et al., 2010), a theory concerning cold processing facilities favoring L. gelidum subsp. gasicomitatum upon the basis of environmental selection (Björkroth, 2005) could explain its adaptation to production lines and premises as a member of the “house-microbiota”. The present study evaluates for the first time the attachment of L. gelidum subsp. gasicomitatum on food contact surfaces, attempting to elucidate this phenotype and outline presumptive routes facilitating the habitation of the microbe to food manufacturing installations.
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2. Materials and methods 2.1. Bacterial strains and cultivation conditions Six strains of L. gelidum subsp. gasicomitatum (i.e. HS1, HS10, HS27, 6.2.3, 10.16.3, ab2) were tested for the purposes of the study (Table 1) in the form of four cultures. Strains HS1, HS10 and HS27 (used together in co-culture as HS cocktail) represent slimeproducing biotypes isolated from food (Pothakos et al., 2014b, 2014d), while isolates 6.2.3, 10.16.3, ab2 were recovered from environmental sampling in a vegetable-salad processing environment (Pothakos et al., 2014c) and were used as single strain cultures. The isolates constitute distinct biotypes, differentiated from the type strain LMG 18811T (Fig. 1) and were selected in order to represent the intra-subspecies diversity of the taxon. All isolates were resuscitated, by transferring one glass bead from cryovials stored at − 80 °C in de Man–Rogosa–Sharpe broth (MRS, Oxoid, Hampshire, UK) and incubated at 22 °C overnight. After 24 h 100 μL were transferred in a new broth tube with 10 mL of medium and kept for another 24 h at 22 °C. In the case of the HS cocktail 33 μL of each strain were transferred to the same broth tube. 2.2. Preparation of stainless steel (SS) and glass surfaces Stainless steel (SS) coupons (2 cm × 5 cm, 1 mm thickness; type AISI-304; Baudoin, Mol, Belgium) were used for the attachment experiments, as stainless steel is the most frequently used material in blades, benches and for the construction of food-processing equipment. The coupons were initially soaked in acetone (overnight) to remove any debris and grease from the manufacturing process. Coupons were then washed in commercial detergent solution, rinsed thoroughly with distilled water and subsequently with ethanol, airdried in a laminar flow cabinet and finally sterilized by autoclaving at 121 °C for 15 min. Likewise, microscope glass slides were wiped clean with a dry piece of cloth and autoclaved at 121 °C for 15 min. 2.3. Preparation of sweet bell pepper (SBP) juice and boiled eggs in brine Food-based media (i.e. milk, meat exudate, yogurt, custard cream) are often used to simulate processing environments (Fouladkhah et al., 2013; Knight and Craven, 2010; Poimenidou et al., 2009), where food particles accumulate on food contact surfaces that are not efficiently sanitized due to unhygienic design (Carpentier and Cerf, 2011). The media of the present study were selected based on the high affinity of sweet bell peppers and brined/ marinated products to L. gelidum subsp. gasicomitatum and the fact that they provide very different nutrients for growth. The sweet bell pepper juice was prepared following a protocol previously described (Pothakos et al., 2014b). Briefly, equal amounts of green, red and yellow sweet bell peppers (cultivar Capsicum annuum) were juiced with the aid of a juice extractor and homogeneously mixed. A large volume of juice was prepared in order to avoid significant fluctuations in the composition of the medium and subsequently stored at − 20 °C. Three hundred milliliters of the mixture was poured in sterile bottles and sterilized for 2 min by boiling over the flame. After sterilization of the juice, 50 mL was aseptically
distributed in sterile containers. In order to prepare boiled eggs in brine, fresh eggs were covered in aluminum foil, placed in a glass jar and autoclaved at 121 °C for 15 min. Once they reached room temperature, they were peeled aseptically in a biosafety cabinet and cut in halves using sterile forceps and blades. The halved boiled eggs were transferred in sterile containers (one half/container) and 50 mL of brine was added. The brine consisted of: 10 g citric acid; 20 g trisodium citrate and 17 g of NaCl suspended in 1.5 L of distilled water. The initial pH of the brine was 4.5 ± 0.1 and reached 6.0 ± 0.1 when added to the egg. 2.4. Immersion of surfaces, inoculation and attachment conditions SS coupons and glass slides were immersed in SBP juice and eggs in brine and positioned in the containers ensuring that they were covered by the medium. The inoculum for each strain and the cocktail were prepared after two consecutive subcultures in MRS at 22 °C for 24 h. The second culture was kept at 7 °C for 1 h prior to inoculation in order to stimulate cold adaptation. Subsequently, a series of decimal dilutions for the second culture was prepared in peptone physiological solution (PPS: 0.85% w/v NaCl and 0.1% w/v bacteriological peptone). A hundred microliters of the appropriate dilution was used for the inoculation of each container obtaining a level of approximately 103–104 viable cells per milliliter of medium. The dilution series of the inoculum of each strain and the cocktail was plated out on MRS in order to evaluate the initial population (Day 0). The inoculated media with the surfaces were kept at 7 °C for 9 days facilitating the attachment of the cells. The experiments were performed two times in duplicate. 2.5. Detachment of the attached cells from SS coupons and enumeration of suspended and attached populations On Days 1, 2, 5, 6, 7 and 8 the population of the suspended cells was determined for all inocula in both media (SBP juice and eggs in brine). Aliquots of 1 mL were extracted from the containers, series of dilutions were prepared in PPS and spread plated on MRS in duplicate. For assessing the attached populations on SS, the bead-vortexing method was performed as previously described (Giaouris et al., 2005; Poimenidou et al., 2009) on Days 5, 6, 7 and 8. The SS coupons were removed from the containers and thoroughly washed with 25 mL of PPS in order to remove all loosely adhering cells as well as residual food particles. They were left in the biosafety cabinet until all of the liquid had dripped off, subsequently placed in 50 mL-centrifuge tubes filled with 10 mL PPS containing 24 glass beads (12 beads with diameter 3 mm, 12 with diameter 5 mm) and vortexed for 2 min at maximum intensity in order to detach the cells from the coupon. Quantification of attached populations was performed by pour plating 1 mL aliquots of the serial dilutions of the vortexed suspension on MRS in duplicate. An overlay of MRS was applied in order to achieve microaerophilic conditions. The formed colonies were counted after incubation at 22 °C for 5 days. The enumeration results of both the suspended and the attached cells were compared statistically (SPSS version 21, IBM, SPSS, Chicago, IL, USA) by paired t-test in order to assess the impact
Table 1 Description of the six L. gelidum subsp. gasicomitatum biotypes used in the present study concerning their origin of isolation and strain history. Strains of L. gelidum subsp. gasicomitatum
Isolation
Description of origin
References
HS1 HS10 HS27 6.2.3 10.16.3 ab2
Food sample
Spoiled boiled eggs in brine
Pothakos et al. (2014b, 2014d)
Surface swab Surface swab Water sample
Rotating blades of vegetable-dicer (after disinfection) Rotary pouch packaging equipment Acetic acid solution bath
Pothakos et al. (2014c)
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Fig. 1. AFLP dendrogram with the fingerprints of the L. gelidum subsp. gasicomitatum isolates used in the present study. The dendrogram was constructed using the Dice coefficient and UPGMA clustering method. (T) The type strain L. gelidum subsp. gasicomitatum LMG 18811T is also included. AFLP dendrogram adapted from Pothakos et al. (2014c, 2014d).
of medium composition and genotype on the growth and the attachment. 2.6. Microscopic observation of attached cells On Days 5, 6, 7, 8 and 9 the SS coupons and the glass slides were aseptically removed from the containers, washed thoroughly with 25 mL of PPS in order to remove all loosely adhering cells and residual food particles and transferred in empty 50 mL-centrifuge tubes to dry. Each slide was stained with 20 μL Baclight dye (Molecular Probes, Eugene, OR, USA) prepared following the instructions of the manufacturer and incubated in the dark for 5 min at room temperature. Live cells (staining Syto 9) were imaged by means of epifluorescence microscopy (Mattila, 2002) through an Axio Imager A1 microscope (Carl Zeiss NV-SA, Zaventem, Belgium). Image capturing was carried out by an Axiocam camera. 3. Results & discussion The four inoculated cultures of L. gelidum subsp. gasicomitatum showed overall the same growth dynamics suspended in both SBP juice and boiled eggs in brine without any significant statistical difference (P N 0.05). As shown in Table 2, on Day 5 or 6 they had reached the static phase of growth (8.15–9.17 log CFU/mL) and remained at densities ranging between 8.44 and 9.37 log CFU/mL until Day 8. The only difference among the four tested inocula was the formation of thick and viscous, ropy slime by the HS cocktail in both matrices. Strains 6.2.3, 10.16.3 and ab2 did not form slime. In terms of attachment capacities on SS, great variance was observed among the different strains in both media (Fig. 2). In SBP juice the HS cocktail and strain 6.2.3 showed weak attachment on Day 8, reaching a population of 4.54 and 4.77 log CFU/cm2, respectively. Strain 10.16.3 reached levels of approximately 5.3 log CFU/cm2, however strain ab2 had the highest attachment among all isolates showing adhesion of 7.0 log CFU/cm2. The same trend was observed in the simulation medium of the boiled eggs in brine. The attached cells of the HS cocktail on the SS coupons remained below 4.0 log CFU/cm2 and strain 6.2.3 did
not exceed 4.5 log CFU/cm2. On the contrary, for the environmental isolate 10.16.3 and the isolate surviving in acetic bath (i.e. strain ab2) a firm attachment was evaluated resulting in 6.0 and 7.4 log CFU/cm2 of attached cells, respectively. Through statistical analysis of the obtained results by paired t-test, with 95% level of confidence, strain L. gelidum subsp. gasicomitatum ab2 showed a significantly stronger (P ≤ 0.05) attachment capacity compared to the other environmental and food isolates. Additionally, SS and glass surfaces were stained with a live/dead dye, in order to capture the image of the viable cells. For the glass slides the adhering populations were only evaluated semiquantitatively (Pantanella et al., 2013) without applying the beadvortexing method. Overall, the images obtained by microscopy from the immersed surfaces in the suspension of the HS cocktail, 6.2.3 and 10.16.3 showed very sparse attachment of cells (data not shown). The attachment of L. gelidum subsp. gasicomitatum ab2 was very dense on both media and in the present study we demonstrate representative results. As shown in Figs. 3 & 4, the cell aggregations of strain ab2 on SS are massive and compact, while on the glass surface the cells are forming a monolayer. The surfaces incubated in the boiled eggs in brine exhibited similar densities, however the quality of the acquired images was poor due to fluorescence of the medium in several cases. Overall the quantification of attached cells through plating and the visualization of viable cells by microscopic observation confirmed the attachment potential of strain ab2 and 10.16.3, which were differentiated by the other biotypes. L. gelidum subsp. gasicomitatum has been one of the most dominant spoilage related microbes in Finland, during the last 15 years (Björkroth et al., 2000), whereas in Belgium it has been documented as the primary causative agent of spoilage in packaged and refrigerated foodstuffs, since 2010 (Pothakos et al., 2014d). Recently, the dominance of psychrotrophic LAB species at the end of shelf-life of meat and fishery products in France was substantiated, highlighting L. gelidum as a member of the core microbiota that prevail from the initial, heterogeneous microbial communities due to ecological selection by the storage conditions (Chaillou et al., 2014).
Table 2 Population of suspended cells of L. gelidum subsp. gasicomitatum cultures in sweet bell pepper juice and boiled eggs in brine, stored at 7 °C for 8 days. The data represent the average log CFU/mL and the standard deviation of two independent experiments performed in duplicate. Time (day)
Suspended cells (log CFU/mL) Sweet bell pepper juice
0 1 2 5 6 7 8
Boiled eggs in brine
HS cocktail
6.2.3
10.16.3
ab2
HS cocktail
6.2.3
10.16.3
ab2
3.60 ± 0.03 4.09 ± 0.07 5.35 ± 0.17 9.03 ± 0.01 8.94 ± 0.03 9.07 ± 0.07 8.96 ± 0.02
3.48 ± 0.05 3.75 ± 0.27 5.34 ± 0.03 8.23 ± 0.26 9.03 ± 0.03 9.02 ± 0.03 9.10 ± 0.01
3.73 ± 0.02 3.85 ± 0.07 4.96 ± 0.00 9.17 ± 0.07 8.92 ± 0.11 9.20 ± 0.03 9.35 ± 0.01
3.51 ± 0.04 4.10 ± 0.14 5.30 ± 0.03 8.82 ± 0.01 9.05 ± 0.07 9.05 ± 0.01 9.17 ± 0.01
3.60 ± 0.03 3.65 ± 0.33 5.24 ± 0.14 8.15 ± 0.27 8.72 ± 0.21 8.44 ± 0.06 8.95 ± 0.02
3.48 ± 0.05 4.14 ± 0.09 5.24 ± 0.09 8.22 ± 0.06 9.00 ± 0.18 8.95 ± 0.08 9.13 ± 0.02
3.73 ± 0.02 3.82 ± 0.06 5.15 ± 0.04 8.82 ± 0.31 9.01 ± 0.16 8.87 ± 0.04 9.37 ± 0.03
3.51 ± 0.04 3.99 ± 0.30 5.32 ± 0.09 8.97 ± 0.19 8.93 ± 0.03 8.94 ± 0.08 9.17 ± 0.01
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A
B
Fig. 2. Quantification of attachment obtained by the four L. gelidum subsp. gasicomitatum cultures on SS surfaces submerged in: A. sweet bell pepper (SBP) juice and B. boiled eggs in brine, stored at 7 °C for 8 days. The counts were generated applying the beadvortexing method and expressed per surface unit. The data represent the average log CFU/cm2 and the standard deviation of two independent experiments performed in duplicate.
L. gelidum subsp. gasicomitatum has exhibited severe spoilage potential in a wide range of food products (Lyhs et al., 2004; Nieminen et al., 2011; Pothakos et al., 2014a), persistence in food processing installations (Pothakos et al., 2014c) and competitive metabolic features (Jääskeläinen et al., 2013). Additionally, several findings concerning spoilage-related characters corroborate the intra-subspecies diversity of the taxon. Vihavainen and Björkroth (2009) demonstrated that only distinct genotypes were recovered from meat products and were distinguished from isolates originating from vegetable-based foodstuffs, suggesting affinity to substrate and processing plant. Previously, the intra-subspecies diversity of L. gelidum subsp. gasicomitatum was investigated in respect to the metabolic patterns and growth dynamics, when food isolates originating from different sources, were compared by single strain inoculation in sweet bell pepper simulating medium (Pothakos et al., 2014b). It was concluded that the tested biotypes behaved similarly but certain metabolites (i.e. ethanol and slime) were detected as strain-dependent. Additionally, the genome of type strain L. gelidum subsp. gasicomitatum LMG 18811T harbors three genes related to adhesion, encoding for a putative mucus binding protein, a serine-rich protein and a putative collagen-adhesion protein (Johansson et al., 2011). Nonetheless, Johansson et al. (2011) stated that a reference L. gelidum subsp. gasicomitatum strain, namely KG1-16, isolated from spoiled vegetable sausages (Vihavainen et al., 2008; Vihavainen and Björkroth, 2009) lacks these genes, compared to the type strain LMG 18811T, which was isolated from marinated meat. These facts underpin the diversity of L. gelidum subsp. gasicomitatum and suggest that certain accessory genomic elements are associated with the niche each strain thrives, presumably affected by lifestyle. Currently, the comparison of food and environmental isolates, showed large attachment variability confirming phenotypic diversity among strains adapted to certain habitats, resulting in different spoilage
Fig. 3. Alive cells of strain L. gelidum subsp. gasicomitatum ab2 attached on a glass slide and stained with Syto 9 (Backlight kit) after 8 days of incubation at 7 °C in SBP juice. The image was captured at magnification: A. ×40 and B. ×100. (Scale bars: A. 100 μm; B. 50 μm.).
potential. Clearly, the scope of the present study was not the investigation of the occurrence of adhesion genes in the tested biotypes because gene presence alone does not constitute solid evidence of functionality and because the function of the aforementioned genes has not been studied in relation to attachment on biotic or abiotic surfaces. Moreover no documented functionality has been reported for type strain LMG 18811T. Therefore, the study was limited on the phenotypic screening of selected strains with the aim of assessing the strain-specific attachment on food contact surfaces. Strain L. gelidum subsp. gasicomitatum ab2 demonstrated a significant ability to adhere on food contact surfaces (≥ 7 log CFU/cm2) along with the environmental isolate 10.16.3 unlike the other tested isolates (i.e. slime forming HS strains and strain 6.2.3). Another finding deriving from this study is the strain-specific formation of viscous slime observed for the food isolates (HS1, HS10 and HS27) in the boiled eggs in brine. Slime (i.e. homopolysaccharide dextran production) is associated with sucrose utilization in Leuconostoc spp. (Björkroth and Holzapfel, 2006; Holt and Cote, 1998; Kim et al., 2001; Naessens et al., 2005), while biosynthesis of heteropolysaccharides has not been encountered for members of the genus. However, in this medium no sucrose was supplemented, whereas only residual amounts of glucose were available from the eggs (Aslam et al., 2013). Slime production did not show any correlation to attachment, although it has been linked
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Acknowledgements The Ghent University “Geconcerteerde Onderzoeks Actie” (GOA project) “Fast and convenient mass spectrometry-based real-time monitoring of volatile organic compounds of biological origin” funded by the Flemish Government (BOF10/GOA/010) is acknowledged for the financial support in the present research. The Greek State Scholarships Foundation (PhD Fellowship 2010-11) is also greatly thanked for the support.
References
Fig. 4. Alive cells of strain L. gelidum subsp. gasicomitatum ab2 attached on a SS coupon and stained with Syto 9 (Backlight kit) after 9 days of incubation at 7 °C in SBP juice. The image was captured at magnification: A. ×40 and B. ×100. (Scale bars: A. 100 μm; B. 50 μm.).
to biofouling (De Vuyst and Degeest, 1999) especially in the case of L. citreum, L. gelidum and L. mesenteroides (Côté and Leathers, 2009; Leathers and Bischoff, 2011). The incubation of surfaces in food simulating media under low temperature and for an extended period has been implemented for studying the attachment of pathogenic microbes on surfaces mimicking food processing installations (Fouladkhah et al., 2013; Giaouris et al., 2005; Møretrø et al., 2013; Park et al., 2012; Poimenidou et al., 2009; Renier et al., 2011). It has been indicated that abraded or inadequately sanitized surfaces facilitate the deposition of food residues or exudates in which microorganism proliferate and remain sheltered for extended time periods (Chmielewski and Frank, 2003). These food-soiled surfaces constitute hard-to-reach or -clean sites that harbor spoilage or pathogenic biota, wherefrom they detach and subsequently contaminate the following production batches (Carpentier and Cerf, 2011). The pioneering work of Björkroth and co-workers the past two decades has depicted this arising problem for food industries and positioned psychrotrophic leuconostocs on the spotlight. At the moment we are witnessing the spreading of L. gelidum contamination in Northern Europe and we consider that in depth understanding of the function and genomic variance of this microbe as well as wide dissemination of this knowledge would benefit food industries.
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Nieminen, T.T., Vihavainen, E., Paloranta, A., Lehto, J., Paulin, L., Auvinen, P., Solismaa, M., Björkroth, K.J., 2011. Characterization of psychrotrophic bacterial communities in modified atmosphere-packed meat with terminal restriction fragment length polymorphism. Int. J. Food Microbiol. 144, 360–366. Pantanella, F., Valenti, P., Natalizi, T., Passeri, D., Berlutti, F., 2013. Analytical techniques to study microbial biofilm on abiotic surfaces: pros and cons of the main techniques currently in use. Ann. Ig. 25, 31–42. Park, S.-H., Cheon, H.-L., Park, K.H., Chung, M.-S., Choi, S.H., Ryu, S., Kang, D.-H., 2012. Inactivation of biofilm cells of foodborne pathogen by aerosolized sanitizers. Int. J. Food Microbiol. 154, 130–134. Poimenidou, S., Belessi, C., Giaouris, E.D., Gounadaki, A.S., Nychas, G.-J.E., Skandamis, P.N., 2009. Listeria monocytogenes attachment to and detachment from stainless steel surfaces in a simulated dairy processing environment. Appl. Environ. Microbiol. 75, 7182–7188. Pothakos, V., Samapundo, S., Devlieghere, F., 2012. Total mesophilic counts underestimate in many cases the contamination levels of psychrotrophic lactic acid bacteria (LAB) in chilled-stored food products at the end of their shelf-life. Food Microbiol. 32, 437–443. Pothakos, V., Nyambi, C., Zhang, B.-Y., Papastergiadis, A., De Meulenaer, B., Devlieghere, F., 2014a. Spoilage potential of psychrotrophic lactic acid bacteria (LAB) species: Leuconostoc gelidum subsp. gasicomitatum and Lactococcus piscium, on sweet bell pepper (SBP) simulation medium under different gas compositions. Int. J. Food Microbiol. 178, 120–129. Pothakos, V., Snauwaert, C., De Vos, P., Huys, G., Devlieghere, F., 2014b. Monitoring psychrotrophic lactic acid bacteria contamination in a ready-to-eat vegetable salad production environment. Int. J. Food Microbiol. 185, 7–16. Pothakos, V., Snauwaert, C., De Vos, P., Huys, G., Devlieghere, F., 2014c. Psychrotrophic members of Leuconostoc gasicomitatum, Leuconostoc gelidum and Lactococcus piscium
dominate at the end of shelf-life in packaged and chilled-stored food products in Belgium. Food Microbiol. 39, 61–67. Pothakos, V., Taminiau, B., Huys, G., Nezer, C., Daube, G., Devlieghere, F., 2014d. Psychrotrophic lactic acid bacteria associated with production batch recalls and sporadic cases of early spoilage in Belgium between 2010 and 2014. Int. J. Food Microbiol. 191, 157–163. Rahkila, R., De Bruyne, K., Johansson, P., Vandamme, P.A., Björkroth, J., 2014. Reclassification of Leuconostoc gasicomitatum into the species Leuconostoc gelidum and description of Leuconostoc gelidum subsp. gelidum subsp. nov., Leuconostoc gelidum subsp. gasicomitatum subsp. nov. and Leuconostoc gelidum subsp. aenigmaticum subsp. nov. Int. J. Syst. Evol. Microbiol. 4, 1290–1295. Renier, S., Hébraud, M., Desvaux, M., 2011. Molecular biology of surface colonization by Listeria monocytogenes: an additional facet of an opportunistic Gram-positive foodborne pathogen. Environ. Microbiol. 13, 835–850. Susiluoto, T., Korkeala, H., Björkroth, K.J., 2003. Leuconostoc gasicomitatum is the dominating lactic acid bacterium in retail modified-atmosphere-packaged marinated broiler meat strips on sell-by-day. Int. J. Food Microbiol. 80, 89–97. Vihavainen, E.J., Björkroth, K.J., 2007. Spoilage of value-added, high-oxygen modified atmosphere packaged raw beef steaks by Leuconostoc gasicomitatum and Leuconostoc gelidum. Int. J. Food Microbiol. 119, 340–345. Vihavainen, E.J., Björkroth, K.J., 2009. Diversity of Leuconostoc gasicomitatum associated with meat spoilage. Int. J. Food Microbiol. 136, 32–36. Vihavainen, E.J., Murros, A.E., Björkroth, K.J., 2008. Leuconostoc spoilage of vacuum packaged vegetable sausages. J. Food Prot. 71, 2312–2315.
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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Short communication
Exploring the strain-specific attachment of Leuconostoc gelidum subsp. gasicomitatum on food contact surfaces Vasileios Pothakos ⁎, Yosi Ayu Aulia, Inge Van der Linden, Mieke Uyttendaele, Frank Devlieghere LFMFP, Laboratory of Food Microbiology and Food Preservation, Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University, Member of Food2Know, Coupure Links 653, B-9000 Gent, Belgium
a r t i c l e
i n f o
Article history: Received 20 November 2014 Received in revised form 7 January 2015 Accepted 8 January 2015 Available online 15 January 2015 Keywords: Leuconostoc gelidum subsp. gasicomitatum Attachment Intra-subspecies diversity Food contact surface Putative biofilm formation
a b s t r a c t The psychrotrophic lactic acid bacterium (LAB) Leuconostoc gelidum subsp. gasicomitatum has emerged as one of the most prevalent specific spoilage organisms (SSOs) of packaged, cold-stored food products in Northern Europe. The whole genome sequencing of the type strain L. gelidum subsp. gasicomitatum LMG 18811T revealed genes encoding for proteins related to adhesion. In the present study the attachment of six food and environmental isolates was monitored on stainless steel (SS) and glass surfaces incubated (7 °C for 5–9 days) in two food simulating substrates (i.e. sweet bell pepper juice and boiled eggs in brine). The selection encompassed unique genotypes, isolated from different food products or sampling sites as well as slime-forming biotypes. The evaluation of the attached cells was performed with the bead vortexing method and a viability staining assay coupled with epifluorescence microscopy. On SS surfaces the slime-formers showed the lowest attachment (3.3–4.5 log CFU/cm2), while strain L. gelidum subsp. gasicomitatum ab2, which was isolated from an acetic acid bath in a vegetable salad company, reached significantly higher populations of attached cells exceeding 7 log CFU/cm2. Strain ab2 formed dense cell aggregations on SS after 9 days of incubation in sweet bell pepper juice. The attachment ability of L. gelidum subsp. gasicomitatum on surfaces documented in the present study extends our knowledge and understanding of the spoilage potential and intra-subspecies diversity of this microbe. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The former species Leuconostoc gasicomitatum was described in 2000 as a specific spoilage organism (SSO) in packaged, marinated meat in Finland (Björkroth et al., 2000) and for one decade numerous severe spoilage cases attributed to this psychrotrophic LAB were further reported (Lyhs et al., 2004; Nieminen et al., 2011; Susiluoto et al., 2003; Vihavainen and Björkroth, 2007, 2009; Vihavainen et al., 2008). Recently, the high occurrence of this microbe was substantiated for various-packaged and refrigerated foodstuffs in Belgium highlighting the underestimation of its populations when implementing mesophilic incubation (30 °C) due to its strict psychrotrophic character, its high spoilage potential and the broad range of food products, in which it thrives (Pothakos et al., 2012, 2014a, 2014d). Raw, fresh, marinated or cooked meat, vegetable sausages and salads, ready-to-eat (RTE) meals, boiled eggs in brine and marinated herring have been commodities of isolation. Currently, incidents of spoilage alterations in packaged, retail food products have been reported to us by industries of other Northern European countries confirming the prevalence and rapid increase of L. gelidum contamination in food processing environments (personal
⁎ Corresponding author. Tel.: +32 2 629 38 63; fax: +32 2 629 27 20. E-mail address: [email protected] (V. Pothakos).
http://dx.doi.org/10.1016/j.ijfoodmicro.2015.01.008 0168-1605/© 2015 Elsevier B.V. All rights reserved.
communication). The genome sequencing of the type strain L. gelidum subsp. gasicomitatum LMG 18811T has unraveled the gene repertoires associated with fast outgrowth and spoilage manifestations (i.e. nutrient-uptake mechanisms, functional heme-mediated respiration, slime formation, emission of buttery off-odor, green discoloration) and indicated the presence of genes encoding for adhesion (Johansson et al., 2011). Studies so far have focused on the phylogeny (Rahkila et al., 2014; Vihavainen and Björkroth, 2009), spoilage potential (Pothakos et al., 2014b), metabolic patterns and growth dynamics (Jääskeläinen et al., 2013) of this subspecies. Nonetheless, it remains unclear which factors mediate contamination in the first place. A source tracking performed in a vegetable-salad company showed that the origin of psychrotrophic leuconostocs was the raw sweet bell peppers and cross-contamination of other vegetables was facilitated by air and surface-mediation (Pothakos et al., 2014c). Despite the fact that raw materials could constitute carriers of psychrotrophic LAB (Audenaert et al., 2010), a theory concerning cold processing facilities favoring L. gelidum subsp. gasicomitatum upon the basis of environmental selection (Björkroth, 2005) could explain its adaptation to production lines and premises as a member of the “house-microbiota”. The present study evaluates for the first time the attachment of L. gelidum subsp. gasicomitatum on food contact surfaces, attempting to elucidate this phenotype and outline presumptive routes facilitating the habitation of the microbe to food manufacturing installations.
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2. Materials and methods 2.1. Bacterial strains and cultivation conditions Six strains of L. gelidum subsp. gasicomitatum (i.e. HS1, HS10, HS27, 6.2.3, 10.16.3, ab2) were tested for the purposes of the study (Table 1) in the form of four cultures. Strains HS1, HS10 and HS27 (used together in co-culture as HS cocktail) represent slimeproducing biotypes isolated from food (Pothakos et al., 2014b, 2014d), while isolates 6.2.3, 10.16.3, ab2 were recovered from environmental sampling in a vegetable-salad processing environment (Pothakos et al., 2014c) and were used as single strain cultures. The isolates constitute distinct biotypes, differentiated from the type strain LMG 18811T (Fig. 1) and were selected in order to represent the intra-subspecies diversity of the taxon. All isolates were resuscitated, by transferring one glass bead from cryovials stored at − 80 °C in de Man–Rogosa–Sharpe broth (MRS, Oxoid, Hampshire, UK) and incubated at 22 °C overnight. After 24 h 100 μL were transferred in a new broth tube with 10 mL of medium and kept for another 24 h at 22 °C. In the case of the HS cocktail 33 μL of each strain were transferred to the same broth tube. 2.2. Preparation of stainless steel (SS) and glass surfaces Stainless steel (SS) coupons (2 cm × 5 cm, 1 mm thickness; type AISI-304; Baudoin, Mol, Belgium) were used for the attachment experiments, as stainless steel is the most frequently used material in blades, benches and for the construction of food-processing equipment. The coupons were initially soaked in acetone (overnight) to remove any debris and grease from the manufacturing process. Coupons were then washed in commercial detergent solution, rinsed thoroughly with distilled water and subsequently with ethanol, airdried in a laminar flow cabinet and finally sterilized by autoclaving at 121 °C for 15 min. Likewise, microscope glass slides were wiped clean with a dry piece of cloth and autoclaved at 121 °C for 15 min. 2.3. Preparation of sweet bell pepper (SBP) juice and boiled eggs in brine Food-based media (i.e. milk, meat exudate, yogurt, custard cream) are often used to simulate processing environments (Fouladkhah et al., 2013; Knight and Craven, 2010; Poimenidou et al., 2009), where food particles accumulate on food contact surfaces that are not efficiently sanitized due to unhygienic design (Carpentier and Cerf, 2011). The media of the present study were selected based on the high affinity of sweet bell peppers and brined/ marinated products to L. gelidum subsp. gasicomitatum and the fact that they provide very different nutrients for growth. The sweet bell pepper juice was prepared following a protocol previously described (Pothakos et al., 2014b). Briefly, equal amounts of green, red and yellow sweet bell peppers (cultivar Capsicum annuum) were juiced with the aid of a juice extractor and homogeneously mixed. A large volume of juice was prepared in order to avoid significant fluctuations in the composition of the medium and subsequently stored at − 20 °C. Three hundred milliliters of the mixture was poured in sterile bottles and sterilized for 2 min by boiling over the flame. After sterilization of the juice, 50 mL was aseptically
distributed in sterile containers. In order to prepare boiled eggs in brine, fresh eggs were covered in aluminum foil, placed in a glass jar and autoclaved at 121 °C for 15 min. Once they reached room temperature, they were peeled aseptically in a biosafety cabinet and cut in halves using sterile forceps and blades. The halved boiled eggs were transferred in sterile containers (one half/container) and 50 mL of brine was added. The brine consisted of: 10 g citric acid; 20 g trisodium citrate and 17 g of NaCl suspended in 1.5 L of distilled water. The initial pH of the brine was 4.5 ± 0.1 and reached 6.0 ± 0.1 when added to the egg. 2.4. Immersion of surfaces, inoculation and attachment conditions SS coupons and glass slides were immersed in SBP juice and eggs in brine and positioned in the containers ensuring that they were covered by the medium. The inoculum for each strain and the cocktail were prepared after two consecutive subcultures in MRS at 22 °C for 24 h. The second culture was kept at 7 °C for 1 h prior to inoculation in order to stimulate cold adaptation. Subsequently, a series of decimal dilutions for the second culture was prepared in peptone physiological solution (PPS: 0.85% w/v NaCl and 0.1% w/v bacteriological peptone). A hundred microliters of the appropriate dilution was used for the inoculation of each container obtaining a level of approximately 103–104 viable cells per milliliter of medium. The dilution series of the inoculum of each strain and the cocktail was plated out on MRS in order to evaluate the initial population (Day 0). The inoculated media with the surfaces were kept at 7 °C for 9 days facilitating the attachment of the cells. The experiments were performed two times in duplicate. 2.5. Detachment of the attached cells from SS coupons and enumeration of suspended and attached populations On Days 1, 2, 5, 6, 7 and 8 the population of the suspended cells was determined for all inocula in both media (SBP juice and eggs in brine). Aliquots of 1 mL were extracted from the containers, series of dilutions were prepared in PPS and spread plated on MRS in duplicate. For assessing the attached populations on SS, the bead-vortexing method was performed as previously described (Giaouris et al., 2005; Poimenidou et al., 2009) on Days 5, 6, 7 and 8. The SS coupons were removed from the containers and thoroughly washed with 25 mL of PPS in order to remove all loosely adhering cells as well as residual food particles. They were left in the biosafety cabinet until all of the liquid had dripped off, subsequently placed in 50 mL-centrifuge tubes filled with 10 mL PPS containing 24 glass beads (12 beads with diameter 3 mm, 12 with diameter 5 mm) and vortexed for 2 min at maximum intensity in order to detach the cells from the coupon. Quantification of attached populations was performed by pour plating 1 mL aliquots of the serial dilutions of the vortexed suspension on MRS in duplicate. An overlay of MRS was applied in order to achieve microaerophilic conditions. The formed colonies were counted after incubation at 22 °C for 5 days. The enumeration results of both the suspended and the attached cells were compared statistically (SPSS version 21, IBM, SPSS, Chicago, IL, USA) by paired t-test in order to assess the impact
Table 1 Description of the six L. gelidum subsp. gasicomitatum biotypes used in the present study concerning their origin of isolation and strain history. Strains of L. gelidum subsp. gasicomitatum
Isolation
Description of origin
References
HS1 HS10 HS27 6.2.3 10.16.3 ab2
Food sample
Spoiled boiled eggs in brine
Pothakos et al. (2014b, 2014d)
Surface swab Surface swab Water sample
Rotating blades of vegetable-dicer (after disinfection) Rotary pouch packaging equipment Acetic acid solution bath
Pothakos et al. (2014c)
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Fig. 1. AFLP dendrogram with the fingerprints of the L. gelidum subsp. gasicomitatum isolates used in the present study. The dendrogram was constructed using the Dice coefficient and UPGMA clustering method. (T) The type strain L. gelidum subsp. gasicomitatum LMG 18811T is also included. AFLP dendrogram adapted from Pothakos et al. (2014c, 2014d).
of medium composition and genotype on the growth and the attachment. 2.6. Microscopic observation of attached cells On Days 5, 6, 7, 8 and 9 the SS coupons and the glass slides were aseptically removed from the containers, washed thoroughly with 25 mL of PPS in order to remove all loosely adhering cells and residual food particles and transferred in empty 50 mL-centrifuge tubes to dry. Each slide was stained with 20 μL Baclight dye (Molecular Probes, Eugene, OR, USA) prepared following the instructions of the manufacturer and incubated in the dark for 5 min at room temperature. Live cells (staining Syto 9) were imaged by means of epifluorescence microscopy (Mattila, 2002) through an Axio Imager A1 microscope (Carl Zeiss NV-SA, Zaventem, Belgium). Image capturing was carried out by an Axiocam camera. 3. Results & discussion The four inoculated cultures of L. gelidum subsp. gasicomitatum showed overall the same growth dynamics suspended in both SBP juice and boiled eggs in brine without any significant statistical difference (P N 0.05). As shown in Table 2, on Day 5 or 6 they had reached the static phase of growth (8.15–9.17 log CFU/mL) and remained at densities ranging between 8.44 and 9.37 log CFU/mL until Day 8. The only difference among the four tested inocula was the formation of thick and viscous, ropy slime by the HS cocktail in both matrices. Strains 6.2.3, 10.16.3 and ab2 did not form slime. In terms of attachment capacities on SS, great variance was observed among the different strains in both media (Fig. 2). In SBP juice the HS cocktail and strain 6.2.3 showed weak attachment on Day 8, reaching a population of 4.54 and 4.77 log CFU/cm2, respectively. Strain 10.16.3 reached levels of approximately 5.3 log CFU/cm2, however strain ab2 had the highest attachment among all isolates showing adhesion of 7.0 log CFU/cm2. The same trend was observed in the simulation medium of the boiled eggs in brine. The attached cells of the HS cocktail on the SS coupons remained below 4.0 log CFU/cm2 and strain 6.2.3 did
not exceed 4.5 log CFU/cm2. On the contrary, for the environmental isolate 10.16.3 and the isolate surviving in acetic bath (i.e. strain ab2) a firm attachment was evaluated resulting in 6.0 and 7.4 log CFU/cm2 of attached cells, respectively. Through statistical analysis of the obtained results by paired t-test, with 95% level of confidence, strain L. gelidum subsp. gasicomitatum ab2 showed a significantly stronger (P ≤ 0.05) attachment capacity compared to the other environmental and food isolates. Additionally, SS and glass surfaces were stained with a live/dead dye, in order to capture the image of the viable cells. For the glass slides the adhering populations were only evaluated semiquantitatively (Pantanella et al., 2013) without applying the beadvortexing method. Overall, the images obtained by microscopy from the immersed surfaces in the suspension of the HS cocktail, 6.2.3 and 10.16.3 showed very sparse attachment of cells (data not shown). The attachment of L. gelidum subsp. gasicomitatum ab2 was very dense on both media and in the present study we demonstrate representative results. As shown in Figs. 3 & 4, the cell aggregations of strain ab2 on SS are massive and compact, while on the glass surface the cells are forming a monolayer. The surfaces incubated in the boiled eggs in brine exhibited similar densities, however the quality of the acquired images was poor due to fluorescence of the medium in several cases. Overall the quantification of attached cells through plating and the visualization of viable cells by microscopic observation confirmed the attachment potential of strain ab2 and 10.16.3, which were differentiated by the other biotypes. L. gelidum subsp. gasicomitatum has been one of the most dominant spoilage related microbes in Finland, during the last 15 years (Björkroth et al., 2000), whereas in Belgium it has been documented as the primary causative agent of spoilage in packaged and refrigerated foodstuffs, since 2010 (Pothakos et al., 2014d). Recently, the dominance of psychrotrophic LAB species at the end of shelf-life of meat and fishery products in France was substantiated, highlighting L. gelidum as a member of the core microbiota that prevail from the initial, heterogeneous microbial communities due to ecological selection by the storage conditions (Chaillou et al., 2014).
Table 2 Population of suspended cells of L. gelidum subsp. gasicomitatum cultures in sweet bell pepper juice and boiled eggs in brine, stored at 7 °C for 8 days. The data represent the average log CFU/mL and the standard deviation of two independent experiments performed in duplicate. Time (day)
Suspended cells (log CFU/mL) Sweet bell pepper juice
0 1 2 5 6 7 8
Boiled eggs in brine
HS cocktail
6.2.3
10.16.3
ab2
HS cocktail
6.2.3
10.16.3
ab2
3.60 ± 0.03 4.09 ± 0.07 5.35 ± 0.17 9.03 ± 0.01 8.94 ± 0.03 9.07 ± 0.07 8.96 ± 0.02
3.48 ± 0.05 3.75 ± 0.27 5.34 ± 0.03 8.23 ± 0.26 9.03 ± 0.03 9.02 ± 0.03 9.10 ± 0.01
3.73 ± 0.02 3.85 ± 0.07 4.96 ± 0.00 9.17 ± 0.07 8.92 ± 0.11 9.20 ± 0.03 9.35 ± 0.01
3.51 ± 0.04 4.10 ± 0.14 5.30 ± 0.03 8.82 ± 0.01 9.05 ± 0.07 9.05 ± 0.01 9.17 ± 0.01
3.60 ± 0.03 3.65 ± 0.33 5.24 ± 0.14 8.15 ± 0.27 8.72 ± 0.21 8.44 ± 0.06 8.95 ± 0.02
3.48 ± 0.05 4.14 ± 0.09 5.24 ± 0.09 8.22 ± 0.06 9.00 ± 0.18 8.95 ± 0.08 9.13 ± 0.02
3.73 ± 0.02 3.82 ± 0.06 5.15 ± 0.04 8.82 ± 0.31 9.01 ± 0.16 8.87 ± 0.04 9.37 ± 0.03
3.51 ± 0.04 3.99 ± 0.30 5.32 ± 0.09 8.97 ± 0.19 8.93 ± 0.03 8.94 ± 0.08 9.17 ± 0.01
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A
B
Fig. 2. Quantification of attachment obtained by the four L. gelidum subsp. gasicomitatum cultures on SS surfaces submerged in: A. sweet bell pepper (SBP) juice and B. boiled eggs in brine, stored at 7 °C for 8 days. The counts were generated applying the beadvortexing method and expressed per surface unit. The data represent the average log CFU/cm2 and the standard deviation of two independent experiments performed in duplicate.
L. gelidum subsp. gasicomitatum has exhibited severe spoilage potential in a wide range of food products (Lyhs et al., 2004; Nieminen et al., 2011; Pothakos et al., 2014a), persistence in food processing installations (Pothakos et al., 2014c) and competitive metabolic features (Jääskeläinen et al., 2013). Additionally, several findings concerning spoilage-related characters corroborate the intra-subspecies diversity of the taxon. Vihavainen and Björkroth (2009) demonstrated that only distinct genotypes were recovered from meat products and were distinguished from isolates originating from vegetable-based foodstuffs, suggesting affinity to substrate and processing plant. Previously, the intra-subspecies diversity of L. gelidum subsp. gasicomitatum was investigated in respect to the metabolic patterns and growth dynamics, when food isolates originating from different sources, were compared by single strain inoculation in sweet bell pepper simulating medium (Pothakos et al., 2014b). It was concluded that the tested biotypes behaved similarly but certain metabolites (i.e. ethanol and slime) were detected as strain-dependent. Additionally, the genome of type strain L. gelidum subsp. gasicomitatum LMG 18811T harbors three genes related to adhesion, encoding for a putative mucus binding protein, a serine-rich protein and a putative collagen-adhesion protein (Johansson et al., 2011). Nonetheless, Johansson et al. (2011) stated that a reference L. gelidum subsp. gasicomitatum strain, namely KG1-16, isolated from spoiled vegetable sausages (Vihavainen et al., 2008; Vihavainen and Björkroth, 2009) lacks these genes, compared to the type strain LMG 18811T, which was isolated from marinated meat. These facts underpin the diversity of L. gelidum subsp. gasicomitatum and suggest that certain accessory genomic elements are associated with the niche each strain thrives, presumably affected by lifestyle. Currently, the comparison of food and environmental isolates, showed large attachment variability confirming phenotypic diversity among strains adapted to certain habitats, resulting in different spoilage
Fig. 3. Alive cells of strain L. gelidum subsp. gasicomitatum ab2 attached on a glass slide and stained with Syto 9 (Backlight kit) after 8 days of incubation at 7 °C in SBP juice. The image was captured at magnification: A. ×40 and B. ×100. (Scale bars: A. 100 μm; B. 50 μm.).
potential. Clearly, the scope of the present study was not the investigation of the occurrence of adhesion genes in the tested biotypes because gene presence alone does not constitute solid evidence of functionality and because the function of the aforementioned genes has not been studied in relation to attachment on biotic or abiotic surfaces. Moreover no documented functionality has been reported for type strain LMG 18811T. Therefore, the study was limited on the phenotypic screening of selected strains with the aim of assessing the strain-specific attachment on food contact surfaces. Strain L. gelidum subsp. gasicomitatum ab2 demonstrated a significant ability to adhere on food contact surfaces (≥ 7 log CFU/cm2) along with the environmental isolate 10.16.3 unlike the other tested isolates (i.e. slime forming HS strains and strain 6.2.3). Another finding deriving from this study is the strain-specific formation of viscous slime observed for the food isolates (HS1, HS10 and HS27) in the boiled eggs in brine. Slime (i.e. homopolysaccharide dextran production) is associated with sucrose utilization in Leuconostoc spp. (Björkroth and Holzapfel, 2006; Holt and Cote, 1998; Kim et al., 2001; Naessens et al., 2005), while biosynthesis of heteropolysaccharides has not been encountered for members of the genus. However, in this medium no sucrose was supplemented, whereas only residual amounts of glucose were available from the eggs (Aslam et al., 2013). Slime production did not show any correlation to attachment, although it has been linked
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Acknowledgements The Ghent University “Geconcerteerde Onderzoeks Actie” (GOA project) “Fast and convenient mass spectrometry-based real-time monitoring of volatile organic compounds of biological origin” funded by the Flemish Government (BOF10/GOA/010) is acknowledged for the financial support in the present research. The Greek State Scholarships Foundation (PhD Fellowship 2010-11) is also greatly thanked for the support.
References
Fig. 4. Alive cells of strain L. gelidum subsp. gasicomitatum ab2 attached on a SS coupon and stained with Syto 9 (Backlight kit) after 9 days of incubation at 7 °C in SBP juice. The image was captured at magnification: A. ×40 and B. ×100. (Scale bars: A. 100 μm; B. 50 μm.).
to biofouling (De Vuyst and Degeest, 1999) especially in the case of L. citreum, L. gelidum and L. mesenteroides (Côté and Leathers, 2009; Leathers and Bischoff, 2011). The incubation of surfaces in food simulating media under low temperature and for an extended period has been implemented for studying the attachment of pathogenic microbes on surfaces mimicking food processing installations (Fouladkhah et al., 2013; Giaouris et al., 2005; Møretrø et al., 2013; Park et al., 2012; Poimenidou et al., 2009; Renier et al., 2011). It has been indicated that abraded or inadequately sanitized surfaces facilitate the deposition of food residues or exudates in which microorganism proliferate and remain sheltered for extended time periods (Chmielewski and Frank, 2003). These food-soiled surfaces constitute hard-to-reach or -clean sites that harbor spoilage or pathogenic biota, wherefrom they detach and subsequently contaminate the following production batches (Carpentier and Cerf, 2011). The pioneering work of Björkroth and co-workers the past two decades has depicted this arising problem for food industries and positioned psychrotrophic leuconostocs on the spotlight. At the moment we are witnessing the spreading of L. gelidum contamination in Northern Europe and we consider that in depth understanding of the function and genomic variance of this microbe as well as wide dissemination of this knowledge would benefit food industries.
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