Microbial diversity and ecology of biofilms in food industry environments associated with Listeria monocytogenes persistence
Annette Fagerlund, Solveig Langsrud and Trond Møretrø
Contaminationoffoodproductswiththefoodbornepathogen Listeriamonocytogenesmayoccurinthefoodprocessing environment.Manybacterialspeciesco-existinthis
environmentandcaninteractinmultispeciesbiofilms.Recent studieshaveshedlightonthecompositionofmicrobial communitiespresentinthesameecologicalhabitatasL.
monocytogenes.Othershaveaimedatidentifyingcompetitive orcooperativeinteractionsbetweenL.monocytogenesand otherspeciesinmixed-speciesbiofilms.Bothmicrobial compositionandinteractionsmaybedifferentlyinfluenced evenbydifferentstrainsbelongingtothesamespecies.Novel methodologybasedonrecentadvancesinsequencing technologiespromisetoprovidenewinsightsintohowthe residentmicrobiotamayinfluencethepresenceofL.
monocytogenesinfoodindustryenvironments.
Address
Nofima-NorwegianInstituteofFood,FisheriesandAquaculture Research,A˚s,Norway
Correspondingauthor:
Fagerlund,Annette([email protected])
CurrentOpinioninFoodScience2021,37:171–178
ThisreviewcomesfromathemedissueonFoodmicrobiology EditedbyAndersonSant’Ana
For complete overview of the section, please refer to the article collection,“FoodMicrobiology”
Availableonline28thOctober2020 https://doi.org/10.1016/j.cofs.2020.10.015
2214-7993/ã2020TheAuthor(s).PublishedbyElsevierLtd.Thisisan openaccessarticleundertheCCBYlicense(http://creativecommons.
org/licenses/by/4.0/).
Introduction
Aseriousprobleminthefoodindustryisthepresenceof microbialbiofilmsthatcanharbourandtransmitspoilage andpathogenicbacteria[1].Thesebiofilmsoftenremain onsurfacesafterregularcleaninganddisinfection.Forthe pathogen Listeriamonocytogenes,themostcommon route of transfer to foodproducts isthroughcross-contamina- tionfromsurfacesinfoodprocessingplants[2,3].Several recentlargeoutbreaksoflisteriosishavebeentracedback to L. monocytogenesstrainspersisting over extendedper- iodsoftimeinfoodprocessingenvironments[4,5],where they – like most bacteria in natural or human-made environments – arelikelyto reside withinbiofilmcom- munities.However,perhapsunexpectedly,thecapability
of L. monocytogenesstrainsto formmonospecies biofilms doesnotseemtobeakeyfactordeterminingtheirability to persistin foodprocessingfacilities [6,7].
The resident microbiota in food processing plants can influencethegrowthofL.monocytogenes.Inmultispecies biofilms, interactions canbe competitive,when L. mono- cytogenesissuppressedbyothermicroorganisms;coopera- tive,whenproliferationandsurvivalofL.monocytogenesin biofilmsareincreased;orneutral[8–10].Boththecompo- sitionoftheresidentmicrobiota,thegrowthofL.mono- cytogenes,andinteractionswithinbiofilmsareaffectedby environmentalfactors,suchasthenatureofrawmaterials, nutrientavailability,temperature,humidity,pH,surface materials and roughness, and cleaning and disinfection (C&D) regimes [11,12]. Multispecies biofilms can pro- videstablenichesforL.monocytogenes,wheretheencasing extracellular matrix can shelter cells and protect them from biocidesand other stresses.The difficultiesposed by biofilms in food industry are reflected in the large numberofrecentreviewsconcerningtheuseandeffectof methods to control microbial biofilms in food related environments [1,10,13,14–20]. Further knowledge of themicrobialecologyofbiofilmsinspecificfoodproces- sing environments can increase our understanding of persistence of pathogens such as L. monocytogenes, ulti- mately improvingourabilityto managefoodsafety.
Thecurrentreviewfocusesonrecentadvancesregarding the composition and diversityof resident microbiota in food processing facilities known to harbour L.
monocytogenes.Itwill also summarize thecurrentunder- standing of how the resident microbiota found in food processing environments mayinfluenceL.monocytogenes in biofilms.Wealsohighlight thepotentialofgenomics technologiesandothernovelapproachesforunderstand- ingthesecommunities.
Microbial diversityin thefood industry
In our previous review of the microbial diversity of residentmicroorganismsoncleanedsurfacesinthefood industry[12],wefoundthat,overall,themicrobiotawas dominatedbyGram-negativebacteriasuchasPseudomo- nas,Acinetobacter,Enterobacteriaceae,Psychrobacter,andSte- notrophomonas, especially in industries with a humid production environment, such as fish, meat, and fresh produce processing plants.Gram-positive bacteria were moreprevalentindairyanddryproductionenvironments,
withlacticacidbacteria,Staphylococcus,andBacillusasthe mostcommonlyfoundgroups.Recentliteraturein gen- eral supports the major conclusions of the review [21–
25,26].Someofthestudies[23,26]highlightthepreva- lenceofyeastandmouldsonsurfaces.Theseeukaryotic microorganismsarereportedinsomestudies[12]butare oftennotinvestigated,as theyarenotdetectedinanal- ysesbased onsequencingof16SrRNA.
Microbialcommunities harbouring L.
monocytogenes
L. monocytogenes is frequently isolated from the food industry, although it is always outnumbered by other types of bacteria, and selective enrichment is in most casesneededforenvironmentaldetection.InastudyofL.
monocytogenes positive surfaces in meat, fish, and dairy processing plants, sampled before C&D [24], the total psychrotrophic count was 5–9 log CFU/cm2, while L.
monocytogenes was present in concentrations of 2–4 log CFU/cm2 in samples where it could be quantitatively detected(9outof40positivesamples);onthemajorityof thesurfacestheconcentrationswerelower.
Table 1 lists the studies (2014–2020) in which both analysisof the microbiotaand detectionof L. monocyto- genes were performed for the same surface or sample [9,23–25,26,27–31],providinginsightsinto whichtypes of bacteria are found with L. monocytogenes in the food industry.Ingeneral,thesebacteriaarethesameasthose that usually dominate in food industrial environments (Table 1).Rodrı´guez-Lo´pez et al. [24]found that Acti- nobacteriawasthemostprevalentphylum(53%) found on the same surface as L. monocytogenes in the meat industry, while Proteobacteria dominated at such sites infish(97%)anddairyplants(69%).Inastudyofthree fruitprocessingplants[26],theprocessingplantwiththe highest prevalence (100%) of L. monocytogenes positive surfaceswasuniquelydominatedbythebacteriaPseudo- monadaceaeandthe fungiDipodascaceae.Thisled tothe conclusion that the composition and diversity of the bacteriotaandmycobiotamaybeindicationofpersistent contaminationwithL.monocytogenes.
Other studies indicate that specific bacteria may be associatedwithlowprevalenceofL.monocytogenes.Janthi- nobacterium has been shown to be more prevalent in Listeria-negative than Listeria-positive drains, and to inhibitattachmentandbiofilmformationofL.monocyto- genesinlaboratory studies[9].Instudiesof woodenvats used in cheese production, the presence of a resident microbiota dominated by the fungus Geotrichum was shownto inhibitL.monocytogenes [32].
Biofilm interactionsinvolving L.monocytogenes
Correlative associations between bacteria are not the same as causal relationships, and need to beconfirmed
by experimentation. A number of studies employing laboratory tests,and in some cases in situ trialsin food industry,haveattemptedtodeterminethenatureofthe interactionsbetweenL.monocytogenesand otherbacteria within biofilms. For older studies we refer readers to previousreviews[2,10,15,20,33],while anoverview of recentpapers(2018–2020)ispresentedinTable2[31,34–
41,42,43,44,45,46,47–49].
To study interactions relevant for the behavior of L.
monocytogenes in food industry settings, model systems shouldconsistofbacteriacommonlyfoundtogetherwith L.monocytogenesinbiofilmsinfoodindustryenvironments [50]. Pseudomonas spp. bacteria match this description (Table1),thuspapersdescribingmixed-speciesbiofilms containingL.monocytogenesandPseudomonasspp.(except Pseudomonasaeruginosa)[42,44,45,46,51]arehighlyrel- evant. The studies show that L. monocytogenes can be establishedinbiofilmswithPseudomonas,asaminorpart ofthetotalbacterial population.Interestingly,thepres- ence of L. monocytogenes may induce increased matrix production in biofilms with Pseudomonas [45] and L.
monocytogenes can be protected against desiccation and disinfection[44].Othermicroorganismsfromfoodindus- tryenvironmentsrecentlystudiedin mixed-speciesbio- filmswithL.monocytogenesareBacillus,lacticacidbacteria, Escherichia coli, Vibrio, Salmonella, Staphylococcus, and yeasts[31,34,35,37,38–41,47,48](seeTable2).However, notallof thesemicroorganisms aretypically co-isolated withL.monocytogenesinfoodindustry(Table1),indicat- ingthattheymayexistinotherecologicalnichesthanL.
monocytogenesinfactories.Otherstudies,examininginter- actions between L. monocytogenes and other pathogens such as P. aeruginosa or Salmonella Typhimurium [36,43,52],haveverylimitedrelevanceforfoodindustry, sincethesepathogensarerarelyencounteredtogetherin foodindustryenvironments[12].
Both competitive and cooperative interspecies interac- tions between L. monocytogenes and other bacteria in biofilms have been described in previous reports. In recent laboratory studies (Table 2), the most common finding was that the numbers of L. monocytogenes in multispeciesbiofilmwerelowerthanin L.monocytogenes monospecies biofilms. Inhibition of L. monocytogenes in dual species biofilms with Bacillus cereus or lactic acid bacteriahasbeenexplainedbytheproductionofantago- nisticcompounds[34,39–41].Bacteriocin-producinglac- ticacid bacteriaare knownto be antagoniststo Listeria spp.,andhaveevenbeenproposedtobeusedasameans tocontrolbiofilmsinfoodproduction[15].Whetherornot thesestrainswillthriveinnicheswhereL.monocytogenesis foundisanotherquestion.
Effectofenvironmental factors
Community-intrinsicpropertiessuchasdirectinhibition of one bacterium by another can explain some
phenotypicalobservationsfromstudiesofmixed-species biofilms.Inotherstudies,however,extrinsicenvironmen- tal factors seem to play a greater role. For example, several studiesreport thatcompetitionfor nutrientscan explain thelowercounts of L. monocytogeneswithinbio- films[42,44,46,47–49].Commonforthesestudiesisthat
biofilmformationwasstudiedonsurfaces(oftenhorizon- tal) without applied shear forces and in the absenceof flow. In such systems competition for nutrients and tolerance to inhibitory compounds are likely of higher importancefortheprevalenceofaspeciesthantheability to attach to a surface and to build astrong matrix.An
Table1
MicrobialcommunitiesfoundinL.monocytogenes(Lm)positiveenvironmentalsamplingpoints
Microbiomeanalysisapproach Environment Lmpositivesamplingpointsanalysed DominantmicrobiotafoundinLm- positivesamplesorsamplingpoints
Ref.
Cultureindependent;microarray analysisusingPhyloChipplatform
Meat production facility
2Lm-positivedrains;6samplesfrom eachdraintakenovera3-dayperiod
Lachnospiraceae,Pseudomonadaceae, Rikenellaceae,
Enterobacteriaceae.Increased abundanceofEnterococcusand Rhodococcusassociatedwithpresence ofLm
[9]
Culturebased;sequencingof16S rRNAfromrandomlypicked psychrotrophiccolonies(grown at15C)
Salmon processing plant
1Lm-positiveconveyorbeltafterC&D Pseudomonas,Brochothrix, Stenotrophomonas,Serratia
[27]
Culturebased;sequencingof16S rRNAfrommorphologically differentcolonies(grownat25C)
Fishand seafood processing plants
6Lm-positivesamples(gloves,floor, sewagechannels,conveyorbelt,scale lines)
Escherichiacoli,Staphylococcus (saprophyticus,scuriandsp.),Kocuria varians,Aerococcusviridans, Microbacterium(luteolumandsp.), Corynebacteriumsp.,Enterococcus aquimarinus,Rothiaterrae [28]
Meat slaughtering andprocessing plants
6Lm-positivesamples(trolley,mincer, massagedrum,drain)
Carnobacterium(divergensandsp.), Serratiasp.,Staphylococcus (saprophyticusandvitulinus), Pseudomonassp.,Buttiauxellasp.
Cultureindependent;construction andsequencingofa16SrRNA geneclonelibrary
Fishsauceand hoisin/oyster saucefactories
8Lm-positivefloordrains Pseudomonas(psychrophilaandsp.), Klebsiella(oxytocaandsp.),Aeromonas hydrophila
[29]
Cultureindependent;
pyrosequencingof16SrRNA PCRamplicons(V1–V2regions)
Cheese production facility
3Lm-positivefloordrains;samplesof bothdrainwaterandbiofilm,taken duringproduction
Pseudomonasmucidolens,Lactococcus lactis,Acetobactertropicalis,
Gluconobacteroxydans,Leuconostoc citreum,Chryseobacteriumureilyticum
[30]
Culturebased;sequencingof16S rRNAfrommorphologically differentcolonies(grownat25/
30C)
Dairyplant 1Lm-positivefloordrain Klebsiellasp.,Escherichiacoli, Comamonassp.,Acinetobactersp.
[31]
Cultureindependent;sequencing of16SrRNAPCRampliconusing IonTorrenttechnology
Meat(bovine andporcine) slaughterhouse
2locations(drain,platform/table),each sampled7 8timesbeforeC&D;Lmwas notdetectedonalloccasions
Drain:Rhodococcus,Chryseobacterium, Microbacterium,Acinetobacter, Athrobacter,Sphingomonas, Flavobacterium,Rothia,
Pseudoclavibacter;Platform/table:
Corynebacterium,Facklamia,
Jeotgalicoccus,Psychrobacter [24,25]
Fish processing/
market
1Lm-positivesump/drain,sampled 4timesbeforeC&D;Lmwasnot detectedonalloccasions
Pseudoalteromonas,Psychrobacter, Photobacterium,Psychromonas, Flavobacterium,Carnobacterium Cheese
production facility
1Lm-positivefloorsample(undersilo), sampledoncebeforeC&D
Acinetobacter,Lactococcus, Pseudomonas,Shewanella,Yersinia Culturebased;identificationof
randomlypickedmorphologically differentcolonies(grownat30C) bybiochemical(API)tests
Meat(porcine) slaughterhouse andprocessing plant
3locations(toolcabinet,floor, transportationcart)eachsampled 16timesovera21-monthperiod;Lm detectedon1 2occasionsineach samplingpoint
Pseudomonas,Bacillus,Mannheimia haemolytica,Enterobacter, Corynebacterium,Leifsonia,
Leuconostocmesenteroides,Candida zeylanoides
[23]
Cultureindependent;sequencing of16SrRNA(V4domain)andITS2 PCRampliconsusingIllumina technology
Appleandother treefruit packinghouses
3factories,3samplinglocationsineach (floorunderconveyorsystem;wash,dry, andwaxsections),13samplesfrom eachsamplingpoint;Lmdetectedin 56%(66/117)ofsamples
Pseudomonadaceae,
Flavobacteriaceae,Xanthomonadaceae;
Fungalfamilies:Dipodascaceae, Trichosporonaceae,Aureobasidiaceae
[26]
exceptionisthestudybyPugaetal.[45]wheretherewas 1–2logmoreL.monocytogenespresentinpreformedPseu- domonas biofilms than in L. monocytogenes monospecies biofilms.Thebiofilmsweregrownonglasscoverslipsina reactorwithappliedshearforces,andL.monocytogeneswas foundtomigratetothebottomlayerofthedualspecies biofilm.Potentially,L.monocytogenesalonewasunableto formthickbiofilmsinthepresenceofshearforces,while inco-culture,thestrongbiofilm-formerPseudomonaspro- videdaprotectedbiofilminwhichL.monocytogenescould thrive.
When designing a model system aiming to investigate mechanisms of relevance for biofilm formation and L.
monocytogenesprevalence in food industry, choosingthe rightenvironmentalfactorsisequallyimportantaschoos- ingthe right microbial consortium [50].Typical niches whereL.monocytogenessurvivesinfoodproductionenvir- onmentsarescratchesorgroovesinorbetweendifferent typesof(worn)materialsorcomplexequipment,suchas drains,floors, conveyorsor slicers– locationswhich are oftendifficultto reach withsanitationand wherenutri- entsandsolidstendtobuildup –aswellas locationsat room temperature or colder [53]. For example, biofilm formationonopen smoothstainlesssteel surfacesisnot likelytobeasignificantissueinfoodprocessingfacilities.
Nevertheless, most reviewed studies employ stainless steel coupons as the solid surface material (Table 2).
Table2
StudiesreportingonbiofilminteractionsbetweenL.monocytogenes(Lm)andothermicroorganisms;2018–2020 Microbesco-culturedwithL.
monocytogenes
Biofilma Strainsb Solid surfacec
Temp. Culturenutrients Durationof experiment
Conditions Effectd Ref.
Bacilluscereus DS 3Lm+6 SS 25C BHI 7days Static 4to0 [34]
Escherichiacoli DS 1Lm+1 SS 25C BHI,reconstituted
powdermilk
60hours Shear forces (15rpm)
2to0 [35]
Escherichiacoli,SalmonellaTyphimuriumor SalmonellaEnteritidis,Pseudomonas aeruginosa,Bacilluscereus
MS 1Lm+5 SSand PP
9, 25C
TSB+eggyolk, TSB+meat extract,whole milk
10days Static 4to 2 [36]
Escherichia,Klebsiella,Comamonas, Acinetobacter
DS,MS 1Lm+4 SS 25C BHI 72hours Shear
forces (90rpm)
DS:0to +1;MS:
< 0.5
[31]
Enterococcus,Staphylococcus,Bacillus MS 3Lm+9 SS 25C BHI,wheyprotein, skimmedmilk
10days Static 2to0 [37]
Limosilactobacillus(Lactobacillus) fermentum,Ligilactobacillus (Lactobacillus)salivarius
DS 1Lm+2 Glass 37C TSBandMRS 72hours Static 2to+1 [38]
Lactobacillusdelbrueckiisubsp.lactis DS 1Lm+1 SS 25C BHIYE 5days Static 4 [39]
Lactobacillus,Lactiplantibacillus (Lactobacillus),Latilactobacillus (Lactobacillus),Leuconostoc
DS 1Lm+8 SS,MP, lettuce
10, 25, 30C
TSB,water 24hours Static 2to 1 [40]
Leuconostoc DS 2Lm+3 MP 37C BHI 24hours Static 2to 1 [41]
Listeriainnocua,Acinetobacter, Pseudomonas,Serratia, Stenotrophomonas
MS 6Lm+
11
SS 12C BHI 9days Static 3to 2 [42]
Pseudomonasaeruginosa DS 2Lm+1 MP 10,
15C
Todd-Hewittbroth 14days Static 1.5to 0.5
[43]
Pseudomonasfluorescens DS 1Lm+1 SS 15C TSB 48hours Static 0.5to0 [44]
Pseudomonasfluorescens DS 1Lm+1 Glass 20C TSB 4days Shear
forces (80rpm)
+1to+2 [45]
Pseudomonasspp.orbacteriafromrawfish juice
DS,MS 6Lm+ 5or unknown
SS 15C Fishjuice(sea bream)
10days Static DS: 1to 0;MS:
3
[46]
Vibrioparahaemolyticus DS 2Lm+2 MP 25C TSB 72hours Static 4to 3 [47]
Yeasts(Candida,Rhodotorula) DS 1Lm+4 SS 25C Applejuice 24hours Static 0to+1 [48]
Nonidentifiedbacteriafromsalmon MS 1Lm+ unknown
SS 4,
15C
1/20TSBor salmonbroth
14days Static 1to+1 [49]
aDS:dual-speciesbiofilm;MS:multispeciesbiofilm.
bTotalnumberofstrainsofL.monocytogenes(Lm)+totalnumberofstrainsforallothertestedspecies(combined).
cSS:Stainlesssteelcoupons;PP:polypropylene;MP:Microtiterplate.
dChangeinnumbersofLminmultispeciesbiofilmsrelativetomonospeciesbiofilms,givenaschangeincolonyformingunits(cfu)forLm:log(cfuin multispeciesbiofilm) log(cfuinmonospeciesbiofilm).Forthemajorityofthestudiestheeffectvarieddependingoninoculationlevels,strains, temperature,timeand/ormedium.
Furthermore, somestudiesemploy cultivationtempera- turesof30Cor37C[38,40,41,52],conditionswhichare not relevant for food industrial environments where L.
monocytogenesisachallenge.
Inthecurrentcontext,insituinvestigationofinteractions in multispeciesbiofilmsinthefoodindustryisaninter- esting approach,andaspreviouslymentioned,there are some older studiesshowing thatcertain bacteriaaffects the prevalence of L. monocytogenes in drains [9,54] and cheese vats [32]. However,we are not awareof similar studiesfromthereviewperiod.
Strain variationin biofilmphenotypes
CertaingenotypesofL.monocytogenesaremorecommonly foundtocolonizefoodprocessingequipmentthanothers.
A largenumberofstudieshaveexamined whetherspe- cific strains or variants of L. monocytogenes have special fitness traits that can explain persistence; however, no clearlinksbetweenpersistenceandinherentphenotypes havebeenidentified[3,53,55].Lianouetal.[6]recently reviewed studies examining correlations between increased ability to produce monospecies biofilm with persistence infoodindustryenvironments.The amount of biofilmformedbydistinctL.monocytogenesstrainshas been found to behighlydependenton extrinsicfactors such as temperature and nutrients, with inconsistent variationsacrossdifferentgrowthconditionsandexperi- mental designs [6,7]. However, under a given set of environmental conditions, differencesin biofilm forma- tionefficiencybetweenL.monocytogenesstrainsorgenetic lineages canbeseen.Forexample,persistentgenotypes were associatedwith highersurvivaland biofilmforma- tioncapacityinthepresenceofsublethalconcentrations of thedisinfectantbenzalkoniumchloride [56].
StudiesexamininginteractionsbetweenL.monocytogenes and otherbacteria inmixed-speciesbiofilmsrarelytake intoaccountstrain-to-strainvariationwithinL.monocyto- genes (Table 2), and vice versa. However, in one study wherebothfactorswereexamined[42],cleardifferences in thedistributionofindividualL.monocytogenesisolates was observed between monospecies and multispecies biofilms: Of six L. monocytogenes strains, one strain out- competed theothers, but only in the presence of both ListeriainnocuastrainsandamixedGram-negativemicro- biotadominatedbyPseudomonas.Thecompositionofthe biofilmreflectedthecompositionofthesuspension sur- rounding the biofilm coupons, thus the effect was not necessarily biofilm-specific [42]. However, it may be speculated that suchstrain-specific variations in growth and survival within multispecies biofilms may explain whycertaintypesofL.monocytogenespersistsinthefood industry, and highlights the significance of including more thanonestrainofeach speciesin studiesof inter- actions withinmicrobialbiofilms.
Genomics and networkanalysis
Furtherstudiesareneededtoexaminewhetherthepres- enceofcertainmembersoftheresidentmicrobiotashowsa significantcorrelation(eitherpositiveornegative)withthe occurrenceofL.monocytogenesinfoodprocessingfacilities.
Recent advances in highthroughput sequencing (HTS) haveresultedingenerationoflargevolumesofdataonthe relative composition of microbial communities, mainly through 16S rRNA gene amplicon sequencing studies [57].Themethodsaresensitiveenoughtoallowdetection ofnondominantmembersofacommunitywhichmayplay importantroleswithinagivenecosystem.Thetechnologi- cal advances and large data volumes offered by HTS methods have resulted in rapid development of more efficient data analysis methods, such as novel methods within the field of network analysis [58–60]. Microbial interactionnetworkshaveforexamplebeenusedtopredict thatinthegut,BarnesiellainhibitsClostridiumdifficileinfec- tion,aninteractionwhichwassubsequentlyconfirmedby invitroco-cultureexperiments[61].
AnotheroptionenabledbyHTStechnologyistheuseof metatranscriptomicsequencingtostudychangesingene expression profiles underlying bacterial interactions in multispeciesbiofilms.Thisapproachhasunraveledfunc- tionality and interactions in consortia such as biofilm communities from soil and oral biofilms, revealing for examplestrain-dependenteffectsofonespeciesongene expressionpatternsinothers,aswellasgiveninsightinto specificinteractionsbetweendifferentconsortiummem- bers [62,63].
Within the field of food microbiology, the majority of microbiome studies employing HTS technology have aimedtomonitorfermentativeprocessesorfoodspoilage, withrelatively fewer studies undertakento examinefactory environments,despitetheroleoftheprocessingenviron- mentsasasourceofbothspoilagemicrobiotaandpatho- genicbacteria[57,64].Microbialassociationnetworkanal- ysis hasbeen appliedto thestudyof food microbiomes [65,66,67] and for analysis of co-occurrence patterns between bacterial families found in the environmental microbiomeof afruitprocessingfacility[26].However, thisapproachisstillunderexploitedfordetectionofeco- logicalcorrelationpatternsorinteractionsbetweenmem- bersofenvironmentalbiofilmcommunitiesfoundonsur- facesinfoodindustry.Itwouldbeinterestingtoseetowhat extenttheseapproachescanshedlightonfactorsresponsi- bleforL.monocytogenespersistence,orbeusedtoidentify nicheswhereL.monocytogeneswouldbeabletopersist,if introducedto theprocessingenvironment.The elimina- tionofpotentialnicheswouldbeamoreproactivestrategy thanmonitoringforthepathogenitself.
Conclusions
The problem of persistence of L. monocytogenes in food processing factories, as well as its association with the
formationofbiofilms, hasbeenacknowledged formany years.The microbial ecology underlyingthesurvival of thispathogenintheseman-madeenvironmentsis,how- ever,stillnotwellunderstood.Inrecentyears,researchers havestartedtostudythemicrobialecosystemsassociated with the presence of L. monocytogenesin these habitats.
There is also considerable interest in examination of interactionsbetweenL.monocytogenesandotherbacteria, inpartduetothehopethatbiocontrolinterventionsmay helpimprovethecontrolofthispathogeninfoodproces- singenvironments.
Themainimpressionfromrecent studiesis thatpersis- tent L. monocytogenes share environmental niches with severalothermembersoftheresidentmicrobiotainfood factories,andthattheinteractionsaremostlycompetitive in nature. For L. monocytogenes, attempts to find single traits that can explain persistence of certain genotypes havefailed.Mostprobably,persistencerequiresamatch between each specific L. monocytogenes strain and the microbiotaand themicroenvironment whereit isintro- duced.Therearefewinsitustudiesonthemicrobiotaand microenvironment where persistent L. monocytogenes reside, and information from such studies could guide further experimental research. With that, the focus of futurestudiescouldshiftfromreductionisticapproaches tomorecomplexandrealisticlaboratorymodels,enabling furtherinvestigationintocausalrelationshipsunderlying interspeciesor interstrain interactions and theeffect of environmental factors on the composition of microbial communitiesinfactoryenvironments.Likewise,applica- tionof novelmethodology based onrecentadvances in sequencing technologies and network analysis is expected to increase our understanding of pathogen persistence. The overall impact of these insights could beashiftin managementofL.monocytogenes,wherethe current ‘seek and destroy’ strategy is replaced with a preventiveapproachinwhichenvironmentalnichespro- motingpathogengrowthcanberemoved.
Conflictofintereststatement Nothingdeclared.
Acknowledgement
ThisworkwassupportedbytheNorwegianAgricultureandFoodIndustry ResearchFunds(grantnumber262306).
References andrecommendedreading
Papersofparticularinterest,publishedwithintheperiodofreview, havebeenhighlightedas:
ofspecialinterest ofoutstandinginterest
1. BridierA,Sanchez-VizueteP,GuilbaudM,PiardJC,Naı¨taliM, BriandetR:Biofilm-associatedpersistenceoffood-borne pathogens.FoodMicrobiol2015,45:167-178.
2. GiaourisE,HeirE,He´braudM,ChorianopoulosN,LangsrudS, MøretrøT,HabimanaO,DesvauxM,RenierS,NychasGJ:
Attachmentandbiofilmformationbyfoodbornebacteriain
meatprocessingenvironments:causes,implications,roleof bacterialinteractionsandcontrolbyalternativenovel methods.MeatSci2014,97:298-309.
3. FerreiraV,WiedmannM,TeixeiraP,StasiewiczMJ:Listeria monocytogenespersistenceinfood-associated environments:epidemiology,straincharacteristics,and implicationsforpublichealth.JFoodProt2014,77:150-170.
4. Lu¨thS,HalbedelS,RosnerB,WilkingH,HolzerA,RoedelA, DieckmannR,VinczeS,PragerR,FliegerAetal.:Backtracking andforwardcheckingofhumanlisteriosisclustersidentifieda multiclonaloutbreaklinkedtoListeriamonocytogenesinmeat productsofasingleproducer.EmergMicrobesInfect2020, 9:1600-1608.
5. StephanR,AlthausD,KieferS,LehnerA,HatzC,SchmutzC, JostM,GerberN,BaumgartnerA,Ha¨chlerHetal.:Foodborne transmissionofListeriamonocytogenesviaready-to-eat salad:anationwideoutbreakinSwitzerland,2013–2014.Food Control2015,57:14-17.
6. LianouA,NychasGJ,KoutsoumanisKP:Strainvariabilityin biofilmformation:afoodsafetyandqualityperspective.Food ResInt2020,137:109424.
Reviewsummarizingexistingknowledgeonstrain-specificvariabilityin biofilm formation phenotypes, with a focus on foodborne bacterial pathogens.
7. LeeBH,ColeS,Badel-BerchouxS,GuillierL,FelixB,KrezdornN, He´braudM,BernardiT,SultanI,PiveteauP:Biofilmformationof Listeriamonocytogenesstrainsunderfoodprocessing environmentsandpan-genome-wideassociationstudy.Front Microbiol2019,10:2698.
8. CarpentierB,ChassaingD:Interactionsinbiofilmsbetween Listeriamonocytogenesandresidentmicroorganismsfrom foodindustrypremises.IntJFoodMicrobiol2004,97:111-122.
9. FoxEM,SolomonK,MooreJE,WallPG,FanningS:Phylogenetic profilesofin-housemicrofloraindrainsatafoodproduction facility:comparisonandbiocontrolimplicationsofListeria- positiveand-negativebacterialpopulations.ApplEnviron Microbiol2014,80:3369-3374.
10. GrayJA,ChandryPS,KaurM,KocharunchittC,BowmanJP, FoxEM:NovelbiocontrolmethodsforListeriamonocytogenes biofilmsinfoodproductionfacilities.FrontMicrobiol2018, 9:605.
11. ValderramaWB,CutterCN:Anecologicalperspectiveof Listeriamonocytogenesbiofilmsinfoodprocessingfacilities.
CritRevFoodSciNutr2013,53:801-817.
12. MøretrøT,LangsrudS:Residentialbacteriaonsurfacesinthe foodindustryandtheirimplicationsforfoodsafetyandquality.
ComprRevFoodSciFoodSaf2017,16:1022-1041.
13. Alvarez-Ordo´n˜ezA,CoughlanLM,BriandetR,CotterPD:Biofilms infoodprocessingenvironments:challengesand
opportunities.AnnuRevFoodSciTechnol2019,10:173-195.
An overview of microbial biofilms in food industry, including strain- specificvariation,interactiverelationshipsbetweenspecies,andmeth- odsforbiofilmcharacterizationandcontrol.
14. Rodrı´guez-Lo´pezP,Rodrı´guez-HerreraJJ,Va´zquez-Sa´nchezD, CaboML:CurrentknowledgeonListeriamonocytogenes biofilmsinfood-relatedenvironments:incidence,resistance tobiocides,ecologyandbiocontrol.Foods2018,7:85.
15. CamargoAC,TodorovSD,ChihibNE,DriderD,NeroLA:Lactic acidbacteria(LAB)andtheirbacteriocinsasalternative biotechnologicaltoolstocontrolListeriamonocytogenes biofilmsinfoodprocessingfacilities.MolBiotechnol2018, 60:712-726.
16. Galie´ S,Garcia-Gutie´rrezC,Migue´lezEM,VillarCJ,Lombo´ F:
Biofilmsinthefoodindustry:healthaspectsandcontrol methods.FrontMicrobiol2018,9:898.
17. Gonza´lez-RivasF,Ripolles-AvilaC,Fontecha-Uman˜aF,Rı´os- CastilloAG,Rodrı´guez-JerezJJ:Biofilmsinthespotlight:
detection,quantification,andremovalmethods.ComprRev FoodSciFoodSaf2018,17:1261-1276.
