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Food Microbiology 51 (2015) 18e24

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Food Microbiology

journal homepage: www.elsevier .com/locate/ fm

Interspecies interactions result in enhanced biofilm formation byco-cultures of bacteria isolated from a food processing environment

Henriette L. Røder a, Prem K. Raghupathi a, Jakob Herschend a, Asker Brejnrod a,Susanne Knøchel b, Søren J. Sørensen a, Mette Burmølle a, *

a Section of Microbiology, Department of Biology, University of Copenhagen, Denmarkb Department of Food Science, University of Copenhagen, Denmark

a r t i c l e i n f o

Article history:Received 22 December 2014Received in revised form11 March 2015Accepted 21 April 2015Available online 30 April 2015

Keywords:BiofilmFood productionInterspecific interactionsSynergyBacterial consortia

* Corresponding author. Universitetsparken 15, BDenmark. Tel.: þ45 40220069.

E-mail address: burmolle@bio.ku.dk (M. Burmølle

http://dx.doi.org/10.1016/j.fm.2015.04.0080740-0020/© 2015 Elsevier Ltd. All rights reserved.

a b s t r a c t

Bacterial attachment and biofilm formation can lead to poor hygienic conditions in food processingenvironments. Furthermore, interactions between different bacteria may induce or promote biofilmformation. In this study, we isolated and identified a total of 687 bacterial strains from seven differentlocations in a meat processing environment and evaluated their biofilm formation capability. A diversegroup of bacteria was isolated and most were classified as poor biofilm producers in a Calgary biofilmdevice assay. Isolates from two sampling sites, the wall and the meat chopper, were further examined formultispecies biofilm formation. Eight strains from each sampling site were chosen and all possiblecombinations of four member co-cultures were tested for enhanced biofilm formation at 15 �C and 24 �C.In approximately 20% of the multispecies consortia grown at 15 �C, the biofilm formation was enhancedwhen comparing to monospecies biofilms. Two specific isolates (one from each location) were found tobe present in synergistic combinations with higher frequencies than the remaining isolates tested. Thisdata provides insights into the ability of co-localized isolates to influence co-culture biofilm productionwith high relevance for food safety and food production facilities.

© 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Biofilm is a prevailing lifestyle for bacteria in many environ-ments and some can lead to implications with regard to healthrelated concerns in areas such as e.g. food processing and health-care (Chmielewski and Frank, 2003; Hall-Stoodley et al., 2004). Theability to adhere to food processing contact surfaces and subse-quently form biofilm has been reported for several food associatedbacteria such as the food-borne pathogenic bacteria Listeria mon-ocytogenes (Møretrø and Langsrud, 2004). Also spoilage organismssuch as e.g. Pseudomonas spp., Brochothrix thermosphacta andvarious Lactobacillus spp. have been associated with biofilm pro-duction (Giaouris et al., 2014).

Biofilms can cause problems in many areas of the food industryas they enable bacteria to better withstand standard cleaningprocedures (Sinde and Carballo, 2000; Somers et al., 1994;

ygn. 1, 2100 København Ø,

).

Stopforth J.D. et al., 2002). This enhanced resistance may becaused by different mechanisms like slow penetration rate of bio-cides, reactions between extracellular polymers and biocides,changed physiology of the bacterial cells or adaptive stress re-sponses (Van Houdt and Michiels, 2010). If biofilms become resis-tant they can be a constant source of contamination and lead toeconomic loss due to accelerated spoilage or problems with foodsafety.

It has previously been shown that biofilms composed of morethan one bacterial species may lead to an overall increase inbiomass (Burmølle et al., 2006; Filoche et al., 2004; Ren et al., 2015).The interactions in such biofilms have further been shown toenhance the opportunity for pathogenic bacteria to becomeestablished members and attain increased cell numbers as seen forEscherichia coli O157:H7 (Klayman et al., 2009; Marouani-Gadriet al., 2009). Moreover, biofilms composed of multiple bacterialspecies may become more resistant towards antibacterial agentsincluding antibiotics and detergents (Burmølle et al., 2014; Leeet al., 2014; Lopes et al., 2012). This underlines the importance ofunderstanding the interspecific competition and cooperationwithin the biofilm.

H.L. Røder et al. / Food Microbiology 51 (2015) 18e24 19

Many food associated biofilm studies have been dedicated tospecific pathogenic bacteria obtaining important knowledge on thebiology, ecology and prevalence of these specific species. However,the impact of bacterial interactions in food processing environ-ments needs to be considered, which has also been emphasized byCarpentier and Chassaing (2004).

Here, we characterize a range of bacterial communities in ameat processing environment (slaughterhouse) with respect todiversity and the species' ability to form biofilm individually and inco-cultures.

2. Methods

2.1. Sampling

Samples were collected at a small slaughterhouse in Denmark inthe late winter of 2012. The slaughterhouse receives animals onceevery week for slaughter and either sells the meat directly orprocess it into a variety of products. The meat processing equip-ment was cleaned several times during the day while the wallswere cleaned once a month. However, slaughtering, cutting, furtherprocessing, and packaging were done in separate rooms.

The samples were collected from different surfaces duringproduction at seven different locations; foil packer, meat chopper(cleaned, ready for use), cutting board (cleaned and used once),wall above the cutting board and air samples from two of therooms. Samples were collected with sterile swabs (Invasive sterileEUROTUBO® collection swab). The swap was moistened in phos-phate buffered saline (PBS) (Giaouris et al., 2014) before wiping theselected location and transferring it to 1 ml PBS. Air samples werecollected by leaving open plates with tryptic soy broth medium(TSB; Merck) with 1.5% agar on selected locations.

The 1 ml PBS containing the swabs was vortexed intensely for30 s upon return to the laboratory on the same day. Aliquots of100 ml of the sample were diluted 10-fold and plated on tryptic soyagar (TSA). The plates were incubated for one day at 24 �C followedby three days at 15 �C.

A representative number of colonies (120 colonies) wererandomly selected from the plates by selecting a section of theplate. These were restreaked on TSA with cycloheximide (CYC,50 mgml�1, Sigma) to ensure that only bacteriawere growing on theplates. The pure bacterial cultures were inoculated in liquid mediaand allowed to grow for 48 h while shaking before theywere storedat �80 �C with 20% glycerol. Further incubation of the isolates wasperformed at 24 �C.

2.2. 16S rRNA gene pyrosequencing

Tag-encoded amplicon pyrosequencing was conducted for bac-teria from seven sample locations. DNA extraction was done withaliquots from 5 to 10 ml for each isolate taken from the straincollection before being stored in a freezer. The volume depended onhowmany isolates were to be combined from each location to forma single unit for that location, the final total volumes ranged from350 ml (Air1) to 600 ml (Foil). Each unit was centrifuged for 3 min at8000 g before DNA was extracted using the FastDNA® SPIN Kit forSoil (MP Biomedicals).

The variable regions V3 and V4 were used for bacterial identi-fication by primers targeting the flanking conserved regions andamplified using the primers PRK341F (50-CCT AYG GGR BGC ASCAG-30) and MPRK806R (50GGATCTACNNGGTATSTAAT-30)(Sambrook et al., 1989; Yu et al., 2005).

The first PCR amplification contained 1 ml DNA adjusted to 5 ng/ml approx. for all the samples and 1 ml of each primer using PuRe TaqReady-To-Go PCR Beads (Amersham Biosciences). The following

conditions were used for the PCR: 98 �C for 30 s, 30 cycles ofannealing at 56 �C for 20 s, elongation at 72 �C for 20 s and finallyextension at 72 �C for 5 min. After the PCR amplification the sam-ples were placed on ice. PCR products were then analyzed on 1%agarose with ethidium bromide and visualized with UV-illumination. The PCR bands were cut and purified with a DNAExtraction Kit from Milipore (Milipore Corporation).

In the second PCR amplification the PCR mixture (25 ml) waspreparedwith 5 ml 5�Phusion HF buffer (7.5mMMgCl2, Finnzymes,Finland), 0.5 ml 10 mM dNTPmixture, 0.25 ml Phusion Hot start DNAPolymerase (2 units/ml, Finnzymes). 1.25 ml of each primer wassynthesizedwith an adapter (CCTAYGGGRBCCASCAG) and barcodesof 10 nucleotides length (10 mm), 1 ml of the PCR products from thefirst step and water to a total of 25 ml. The PCR mixture was runsimilarly to the first PCR with the visualization and purificationperformed as before. Gel-extracted amplified fragments withadapters and tags were quantified with Qubit™ fluorometer (Invi-trogen). A GS FLX Titanium pyrosequencing facility was used for thesequencing in accordancewith themanufacturer's protocol (Roche).

Raw FLX sequencing data was deionised with Titanium protocolin Ampliconnoise (Bragg et al., 2012) and with UCHIME 5.2.32(Edgar et al., 2011) against the GOLD (version microbiomeutil-r20110519) reference database as recommended in the manual.The cleaned reads were processed by the Quantitative Insight IntoMicrobial Ecology (QIIME) (Caporaso et al., 2010) pipeline version1.5.0. with default settings. Briefly, operational taxonomic units(OTUs) were clustered at 97% identity and representative sequenceswere classified with the RDP classifier against the 108 Silva release(Pruesse et al., 2007) at a confidence level of 0.8.

2.3. Cultivation and identification of wall and meat chopper isolates

For further analysis of biofilm production for mixed isolateseight strains were chosen based on phenotypic and morphologicaldifferences from both the meat chopper and the wall environment.This was done on TSA and Congo Red agar plates made using 1%agar, 1% tryptone (peptone from casein, Merck), 0.5% yeast extract(Merck) complemented with 40 mg/ml Congo red (Sigma) and20 mg/ml Coomassie blue (Serva) (Dietrich et al., 2013). The 16strains used in the combinations for biofilm assessment are listed inTable 2. The strains were grown in nutrient rich TSB media andwhen solid medium was needed, 1.5% (w/v) agar was added. Thestrains were subcultured from frozen glycerol stocks onto TSAplates for 48 h at 24 �C before a single colony was inoculated into5 ml TSB media and incubated whilst shaken at 24 �C overnight.

For identification of bacterial isolates, the DNA of pure overnight(ON) cultures was extracted using Genomic Mini for UniversalGenomic DNA Isolation Kit (A&A Biotechnology) and the 16S rRNAgene sequences were amplified using the 27F and 1492R primers(Lane, 1991) with PureTaq™ ready-to-go PCR beads (GE Healthcare,UK). The final volume was adjusted with DNA-free water to 25 ml.Amplificationwas as follows: Initial denaturation at 95 �C for 2min,35 cycles at 95 �C for 20 s, at 56 �C for 30 s and at 72 �C for 90 s. Afinal primer extension reaction was performed at 72 �C for 6 min.The resulting PCR products were purified using QIAquick PCR Pu-rification Kit (Qiagen, Germany) according to the manufacturer'sprotocol. Purified DNAwas Sanger sequenced (Macrogen), resultingsequences were trimmed in CLC Workbench 6 and strain identifi-cation was made using EzTaxon (Kim et al. 2012). Sequences wereuploaded to the EMBL database, see Table 2.

2.4. Biofilm formation

The biofilm formation of 30 randomly selected isolates fromeach of the seven sample locations and of eight morphologically

Table 1Distribution of the isolates from the seven sample locations based on 16S rRNA gene sequencing.

Air1 Air2 Cut clean Cut used Wall Chop Foil

Number of isolates 35 59 119 119 119 116 120Actinobacteria Arthrobacter 2.1 1.8

Kocuria 7.2 69Micrococcus 1.1Rhodococcus 2.4 5.4Rothia 4.7 29.5Microbacteriaceae;Other 1.4 1.6 1.2

Proteobacteria Acinetobacter 18.1 1.6 1.1Aeromonas 5.1Brevundimonas 11 13.9Citrobacter 9.2Enhydrobacter 1.3Paracoccus 1.8Pseudomonas 44.8 52.9 69.7 56.9 17.1Psychrobacter 12.4 15.7 4.3 1.1 1.2 72.7Shewanella 1.2Stenotrophomonas 14.9 1.2

Firmicutes Brochothrix 2.1 1.8 7.1 5Exiguobacterium 5.1Macrococcus 1.4Staphylococcus 3.7 11.5 1.5 1.5

Bacte-roidetes Chryseobacterium 9.2 2.1 6Pedobacter 1.4Sphingobacterium 51.9 15.3 5Other* 1.5 2.4 2.2 2.3 1.9 0.3 1.2Total 100 100 100 100 100 100 100

*Bacterial genus under 1% OTU classification.

Table 2Strains isolated from wall and meat chopper environments identified using 16S rRNA gene sequencing.

Sampling site ID# Closest relative Similarity (%) Accession# of closest relativea Source Accession #b

Wall 1 Psychrobacter cibarius 100 AY639871 This study LK3915382 Arthrobacter antarcticus 99.57 AM931709 This study LK3915393 Pseudoclavibacter helvolus 99.85 X77440 This study LK3915404 Kocuria palustris 99.92 Y16263 This study LK3915415 Staphylococcus pasturi 99.86 AF041361 This study LK3915426 Microbacterium paraoxydans 98.93 AJ491806 This study LK3915437 Pseudomonas fragi 99.93 AF094733 This study LK3915448 Psychrobacter faecalis 99.86 AJ421528 This study LK391545

Meat chopper 1 Kocuria salsicia 46.86c GQ352404 This study LN5898412 Kocuria rhizophila 99.04 Y16264 This study LN5898423 Kocuria assamensis 100 HQ018931 This study LN5898434 Kocuria rhizophila 99.04 Y16264 This study LN5898445 Kocuria rhizophila 98.96 Y16264 This study LN5898456 Kocuria varians 99.77 X87754 This study LN5898467 Rothia marina 99.84 FJ425908 This study LN5898478 Pseudoclavibacter helvolus 99.84 X77440 This study LN589848

a Accession number from closet relative provided by EzTaxon.b Accession number from the uploaded 16S rRNA gene sequence to EMBL Nucleotide Sequence Database.c Score from EzTaxon, however, NCBI Blast alignment yields 99% similarity to GQ352404.1.

H.L. Røder et al. / Food Microbiology 51 (2015) 18e2420

different isolates selected from wall and meat chopper environ-ments, respectively, was quantified by a modified version of theclassic crystal violet (CV) assay described by O'Toole and Kolter(1998). The modified version, described as a Calgary biofilm de-vice (CBD), uses a lid containing 96 pegs fitting into a 96-well platewhich allows biofilm formation on each peg (Ren et al., 2014). Theaggregated biofilmwas quantified by the CBD assay, which is basedon absorbance measurements of CV that binds to all negativelycharged molecules.

2.4.1. Biofilm quantification of 30 isolates from the seven samplelocations

The biofilm formation capability of 30 randomly selected isolatesfrom each of the seven sample locations was quantified. The purebacterial strains were individually inoculated in 5 ml TSB and grownwhilst shaken for48h. TSB (145ml)wasadded to thewells of a96-well

microtiter plate (NUNC, Roskilde, Denmark) and 5 ml of the strainculturewas added towells in triplicates. The lid (TSP, NUNC, Roskilde,Denmark)was placed on top of the plates and the biofilmgrown in LBfor 48hat24 �C.After incubation, thebiofilm formationon the lidwaswashed three consecutive times by placing the lid in washing trayswith 55 ml 1�PBS for 3 s, followed by staining for 20 min in 160 mlaqueousCV solution (1%) and thenwashedfive times for 5 s eachwithfresh 1�PBS solution. CVwas released from the pegs by placing thesein a microtiter plate containing 200 ml ethanol (96%) per well for20 min. Quantification was performed by measuring the absorbanceusing an EL 340 microplate reader (Bio-Tek Instruments) at 590 nm.Samples with an absorbance above 2.5 were diluted in ethanol. Eachplate contained a negative control in triplicate. The values from thenegative control were subtracted from the measured biofilm valuesbefore biofilm average and standard errors of the triplicates werecalculated for each isolate and combination.

H.L. Røder et al. / Food Microbiology 51 (2015) 18e24 21

2.4.2. Biofilm quantification from wall and meat chopper isolatesThe eight selected strains from both meat chopper and wall

environments (Table 2) were screened for biofilm formation assingle species as well as in all possible four species combinations aspreviously described (Ren et al., 2014) with minor modifications.Serial dilutions of stationary phase cultures were performed bymixing 1ml of culturewith 29ml fresh TSBmedia, incubating ON at24 �C while shaking at 150 rpm. Exponentially growing cultureswere then selected and a total of 150 ml monospecies or four mixedspecies (37.5 ml of each species/well) cultures were added to eachwell. Plates were incubated at either 15 �C or 24 �C for 24 h. Eachplate contained the representative mono-species cultures, andbiofilm quantification was done as previously described. The bio-film assay was performed with three technical replicates in eachsetup and three independent times for the chopper and wallisolates.

2.5. Statistical analysis

One-way ANOVA was used to test the biofilm development infour species co-cultures.

3. Results

3.1. Bacterial diversity

A total of 687 strains were isolated in the slaughterhouse fromseven different sampling locations (Table 1). The purpose was toexamine the diversity of the samples and the ability of the domi-nating, individual strains to form biofilm. No attempts were madeat determining CFU/cm2 or describing the meta-communitiespresent at the locations. Anaerobes were not included due to theisolation procedures.

Pyrosequencing was applied for identification of the speciesdistribution in the strain collections; Table 1 shows the bacterialdiversity. The sample from the used cutting board was prevalentlydominated by Pseudomonas (69.7%) followed by Sphingobacterium(15.3%). The clean cutting board sample was dominated by Sphin-gobacterium (51.9%), followed by Stenotrophomonas (14.9%), Bre-vundimonas (13.9%) and Chryseobacterium (9.2%). The last threegenera constituted only 2% or less at the used cutting board. Thisindicates a shift in the distribution following cleaning procedures.The sample from the wall above the cutting boards was dominatedby Pseudomonas (56.9%), which was also themost prevalent class inboth air samples (44.8% and 52.9%). Kocuria (69%) dominated in thesample from the meat chopper, whereas Psychrobacter (72.7%)dominated the foil packer sample.

The Gram-positive genus Brochothrix was also found as a minorcomponent in some of the samples. One of the dominant species inthis genus is Brochothrix thermosphacta, which is able to spoilrefrigerated meat (Borch et al., 1996). A representative sequencefrom this cluster was analyzed using BLAST (NCBI database) toconfirm the identity of Brochothrix thermosphacta.

Samples showing a major trend in Pseudomonas dominancewere the used cutting board, the wall, air1 and air2. The foil packer,meat chopper and clean cutting board samples appeared to havemore individually distinct profiles. It should however be kept inmind that the air samples did not have as many isolates as the restof the locations (Table 1).

3.2. Biofilm formation of single species in the sampled locations

A total of 30 strains from each sampling location were analyzedfor their biofilm production capabilities in monocultures by theCBD assay. The isolates were allowed to form biofilm for 2 days at

24 �C followed by biofilm quantification, leading to classification ofeach strain into one of five groups defined by the measured ODvalues (Fig. 1). The first group (OD590, X < 0.2) was classified as poorbiofilm producers, followed by four groups with increasing biofilmforming ability. This relative classificationwas used to highlight thetendencies of the different locations (Fig. 1). The results show thatthe strains present in the air samples were better at forming biofilmin this assay compared to the bacteria isolated from the other lo-cations with the only exception being the used cutting board. In theother samples the main part of the bacteria were found to be poorbiofilm producers.

3.3. Biofilm formation from assembled multispecies consortia

The ability of individual strains to form biofilm in the CBD assaymay, however, not reflect their ability to engage in multispeciessurface associated communities in situ. To examine the influence onthe biofilm production in multispecies communities, two locations,meat chopper and wall, were chosen for a more thorough exami-nation (Table 2). The two sampling sites were chosen as they rep-resented two very different niches with different bacterial diversity(Table 1). Additionally, both locations were dominated by poor andintermediate biofilm formers when tested as monocultures,although the cleaning regime varied strongly. Eight strains differingin colony morphology were chosen from each location, and bycombining these eight strains in all possible combinations of four, atotal of 70 different combinations from each location were tested.Each combination was investigated for its biofilm productioncapability and screened for potential synergistic interactions inbiofilm formation between the mixed bacteria. In this study, syn-ergy in biofilm formation was defined as the measured absorbancefrom the CBD assay of the multispecies biofilm (MSB) being greaterthan that of the best single strain (BSS) biofilm former present inthe relevant combination when taking standard errors into ac-count, i.e. (Abs590 MSB e Std. error) > (Abs590 BSS þ Std.error) ¼ Synergy, while (Abs590 MSB þ Std. error) < (Abs590 BSS e

Std. error) ¼ No synergy (Ren et al., 2015).A higher number of consortia showed synergy in biofilm for-

mation at 15 �C compared to 24 �C for both the meat chopper andwall combinations (Table 3 and supplementary information). Thenumber of synergistic consortia from the wall isolates was highercompared to the meat chopper, at both investigated temperatures.From the four species combinations of wall isolates, we show thatat 15 �C and 24 �C, an average of 34% and 14%, respectively, of thefour-species consortia were synergistic in biofilm formation(equivalent to 23 and 10 of the 70 combinations tested). For themeat chopper 22% at 15 �C and 3% at 24 �C of the tested consortiawere synergistic (15 and 2 of 70 combinations, respectively). Thedifference observed in the prevalence of consortia classified assynergistic at 15 �C and 24 �C was statistically significant (one-wayANOVA, P-value <0.05). From the wall consortia we observed 11combinations that were consistently synergistic both at 24 �C and15 �C. No consistent trend could, however, be identifiedwith regardto synergy from the meat chopper consortia (supplementaryinformation).

Consortia showing synergy were analyzed with respect to thespecific isolates present. We were particularly interested in iden-tifying isolates present in synergistic consortia more frequentlythan others and for each specific isolate, we therefore counted thenumber of synergistic consortia in which this isolate was present.The analysis was performed across the three trials, giving amaximum count of 105 combinations per isolate. As shown inFig. 2, Psychrobacter cibarius (strain 1 from the wall) and Kocuriarhizophila (strain 2 from the meat chopper) showed synergistic

Fig. 1. Ability of biofilm formation by mono-cultures isolated at the seven sampling locations. Biofilm formation was assessed after incubation of 30 random isolates from eachlocation for 48 h at 24 �C by use of the crystal violet retention assay. The 30 isolates were classified based on biofilm formation, and the numbers of isolates in each class arepresented as percentages. Each isolate was tested in triplicates and the average was used for classification.

H.L. Røder et al. / Food Microbiology 51 (2015) 18e2422

biofilm formation in many different consortia combinations at15 �C (31 and 13, respectively).

4. Discussion

The data obtained from pyrosequencing confirm that diversebacterial strain collections were isolated from the different sam-pling sites. The species distribution found differed between thelocations, which indicate that they represent different microhabi-tats for bacteria with differing selective conditions. Whencomparing the samples from air to the remaining samples, it is clearthat the individual sample locations are not reflections of theparticular bacterial community present in the air.

The observed differences between the sample locations could bethe result of external influences likemeat contact, cleaning routinesetc., but it will also reflect the varying micro habitats found at thelocation. As expected, it is not possible to determine the composi-tion of the bacterial community in a slaughterhouse solely based onsamples from one or only a few specific locations.

The isolated strains tested were found to be mainly poor biofilmformers in single species biofilm. We had expected to find moremoderate and good biofilm formers since surfaces are expected tobe inhabited by microbial communities with strong attachmentand biofilm formation abilities. In addition, we included regularlycleaned surfaces in our sampling sites where the remaining mi-crobial community may be assumed to be able to withstandencountered stresses, partly due to their ability of biofilmformation.

Table 3Observed synergistic trends in tested multispecies consortia.

Sampling site Temp (�C) Co-cultures showing enhanced biofilm formationa

Meat Chopper 15b 31% (22/70) 13% (9/70) 21% (15/70)24b 3% (2/70) 3% (2/70) 3% (2/70)

Wall 15b 33% (23/70) 46% (32/70) 22% (15/70)24b 13% (9/70) 14% (10/70) 16% (11/70)

a Percentage of synergistic biofilm consortia observed by co-culturing the eightstrains from the selected wall and meat chopper samples in combinations of fourisolates from each location. The detection of synergistic effects was performed bythree biological replicates (experiment 1, 2 and 3) with three technical replicateseach time.

b One-way ANOVA analysis showed the difference in synergy between the twotemperatures to be significant (P-value <0.05).

In the sampling, a diverse group of bacteria was found on thesurfaces, and we examined the interactions between the differentspecies to see how they were affecting biofilm production. Syner-gistic interactions in multispecies communities may lead to an in-crease in the overall biofilm development (Ren et al., 2015); it istherefore relevant and interesting to see howbacterial isolates fromthe meat processing environment interact in terms of biofilmformation.

Fig. 2. Absolute counts of how often each individual isolate from the wall (a) and themeat chopper (b) is present in a four species combination classified as synergistic at15 �C (grey bars) and 24 �C (black bars). Counts are summarized across the three trials.Each isolate participates in 35 four species combinations/trial, thus N ¼ 105.

H.L. Røder et al. / Food Microbiology 51 (2015) 18e24 23

Our observations show approx. 20% and 30% synergy for isolatesfrom the meat chopper and the wall, respectively, at 15 �C, whichwas significantly different for both settings compared to observa-tions at 24 �C. A larger deviation was observed in the results ofexperiments conducted at 15 �C compared to those at 24 �C(Table 3). This was due to the fact that the single strains deviatedmore in their individual biofilm formation at 15 �C according toOD590 measurements during all trials (data not shown). We hy-pothesize that this temperature dependent difference in consis-tency may be because some species grow slowly at 15 �C, and theirproportion and activity in the mixed consortia may vary highlywhen growth rates are low. In addition, some physiochemical in-teractions are stronger at higher temperatures, which may lead tomore stable biofilms.

Interactions in multispecies biofilms have in several studiesbeen reported to result in enhanced biofilm biomass, protection ofless tolerant species with low ability of biofilm formation andchanged functionality of the total community (Burmølle et al.2014). The interaction networks are complicated and thiscomplexity increases with the number of species present. Studies ofmodel communities with a few species have focused on metaboliccross-feeding (syntrophy), intra- and interspecies bacterialcommunication (quorum sensing) and horizontal gene transfer associal bacterial activities that result in enhanced biofilm formationand changed functionality by co-cultures (Burmølle et al. 2014). Weintend to examine several of the synergistic consortia identified inthis study, in order to further characterize the interactions and theunderlying molecular mechanisms.

Interestingly, when examining the multispecies biofilm, certainisolates (e.g. P. cibarius and K. rhizophila, strain 2 meat chopper)appeared to promote synergistic biofilm formationmore frequentlythan others. In contrast, some strains (3 and 4 from the wall, and 1and 4 from the meat chopper) were never or only rarely present insynergistic combinations (Fig. 2). We observed conflicting ten-dencies in the species Pseudoclavibacter helvolus, of which isolateswere obtained from both investigated niches (strain 3 in the wallisolates; 8 in themeat chopper). The P. helvolus isolate from thewallsample did not participate in synergistic interactions, whereas theisolate from the meat chopper was present in various synergisticmulti-species consortia (Fig. 2). Phenotypic variation betweenisolates of the same species was also observed for the three K.rhizophila isolates at 15 �C (meat chopper strain 2, 4 and 5). In thiscase strain 2 seemed to participate in a large number of synergisticconsortia, whereas strain 4 did not participate in any synergisticfour-species consortia, see Fig. 2b. This may reflect that only minorphenotypic differences are responsible for whether the presence ofan isolate in a combination will result in synergy in biofilm for-mation, adding complexity and possibly also variability to theoverall biofilm formation of multispecies consortia.

The data presented in this study emphasize that the capability ofa bacterial strain to engage in a biofilm community and therebypersist in an environment cannot be evaluated solely based on itsability of monoculture surface attachment and biofilm formation.Knowledge about indigenous communities and their combinedinteractions in mixed species biofilm, where studies have docu-mented both synergy and reduction in biofilm production(Carpentier and Chassaing, 2004;Marouani-Gadri et al., 2009), maypave the way for new strategies on how to remove, or, in the case ofdesired cultures, enhance bacterial biofilm in industrial settings.This study shows how the interactions of different strains can leadto synergy in biofilm formation. The use of isolates collected at thesame time from industry settings rather than using laboratorycollections of model organisms can be an important asset whenmixed species combinations are to be examined since phenotypescan vary. Future studies on the persistence of food pathogens may

need to take into account the interactions occurring with the bac-teria already present as well as the target bacteria in the foodprocessing environment.

Acknowledgements

This study was partly funded by grants to SJS and MB from TheDanish Council for Independent Research; ref no: DFF-1335-00071and ref no: DFF-1323-00235 (SIMICOM).

The authors wish to thank Johanne Damgaard Hansen and DitteMikkelsen Truelsen for assistance in sampling and isolate handling,and Karin Vestberg and Anette Hørdum Løth for technicalassistance.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.fm.2015.04.008.

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