Environmental microbiome mapping as a strategy to improve quality and safety in the food industry

Environmental

microbiome

mapping

as

a

strategy

to

improve

quality

and

safety

in

the

food

industry

Francesca

De

Filippis

,

Vincenzo

Valentino

,

Avelino

Alvarez-Ordo´n˜ez

,

Paul

D

Cotter

and

Danilo

Ercolini

Infoodindustries,anenvironmentally-adaptedmicrobiome

cancolonizethesurfacesofequipmentandtoolsandbe

transferredtothefoodproductorintermediatesofproduction.

Thesecomplexmicrobialconsortiamayincludemicrobial

spoilers,pathogens,aswellasbeneficialmicrobes.

Advancesinsequencingtechnologiesandmetagenomics

providetheopportunitytomaptheenvironmentalmicrobiome

infoodindustriesatanunprecedenteddepth,highlightingthe

importanceoftheresidentmicrobialcommunitiesininfluencing

foodqualityandsafety,aswellasthemainfactorsshapingits

compositionandactivities.However,specifictechnicalissues

mustbeconsidered.Althoughmicrobiomemappinginthefood

industryhasthepotentialtorevolutionizefoodsafetyand

qualitymanagementsystems,itsapplicationasroutine

practiceisstillchallengingandtechnicalissueslimitthe

exploitationofthepowerfulinformationthatcanbeobtainedby

theapplicationofsuchstate-of-the-artapproaches.

Addresses

1_{DepartmentofAgriculturalSciences,UniversityofNaplesFedericoII,} Portici,80055,Italy

2

TaskForceonMicrobiomeStudies,UniversityofNaplesFedericoII, Naples,80100,Italy

3_{DepartmentofFoodHygieneandTechnology,UniversidaddeLeo´n,} Leo´n,Spain

4

InstituteofFoodScienceandTechnology,UniversidaddeLeo´n,Leo´n, Spain

5_{APCMicrobiomeIreland,UniversityCollegeCork,Cork,Ireland} 6_{TeagascFoodResearchCentre,Moorepark,Fermoy,Cork,Ireland}

Correspondingauthor:Ercolini,Danilo(ercolini@unina.it)

CurrentOpinioninFoodScience2021,38:168–176

ThisreviewcomesfromathemedissueonFoodmicrobiology EditedbyAndersondeSouzaSant’Ana

https://doi.org/10.1016/j.cofs.2020.11.012

2214-7993/_ã2020TheAuthors.PublishedbyElsevierLtd.Thisisan openaccessarticleundertheCCBY-NC-NDlicense( http://creative-commons.org/licenses/by-nc-nd/4.0/).

Food

processing

facilities

are

inhabited

by

a

resident

microbiome

Microbialcontaminationinfoodprocessingenvironments influencesfoodqualityandsafety.Infoodindustries,an

environmentally-adapted microbiome can colonize the surfaces of equipment and tools and be transferred to thefood product or intermediates of production during handling, manufacture, processing and storage. Indeed, food contact surfaces often represent a good niche for microorganismstopersistand,indeed,proliferate. More-over,surfacesthatarenotindirectcontactwithfoodsare alsopotentialreservoirsofmicrobes,whichoveralonger term can be sources of food contamination. Although frequent cleaningand disinfectionproceduresareroutinely implementedin allfoodindustries, itis recognizedthat thesearenotalwayseffectiveineliminatingtheresident microbial consortiaspecific to each foodplant [1]. Such microbial populations are well-adapted to the specific environmental conditions that they are exposed to and tend to develop, often as biofilms, on surfaces that are particularlydifficulttocleanduetochallengesrelatingto access, surface irregularities or the retention of sticky materials. These microbes can then proliferate due to the availability of food residues and exudates in such micro-environmentsandcanultimatelyrepresenta possi-blesource of pathogens or spoilage-associated microbes thatcanleadtocross-contaminationoffoods.

Inthelastdecade,metagenomicshasbegunpopularfor microbiome mapping in food handling or processing facilities (Table 1). This approach has been primarily applied in dairies and, to a lesser extent, raw meat processingenvironments(e.g.butchers,facilities produc-ingfreshsausages).Allthesestudiesclearlyshowedthat foodprocessingenvironmentsareinhabitedbyaresident microbiomethatpersistdespiteroutinecleaningpractices andmaybeeasily transferredto thefinal foodproduct. Indeed,thestudiestodatesuggestthatmostofthetaxa foundinprocessingenvironmentsarealsofoundinfood productsproducedin thatfacility(Table1).

Theenvironmentalmicrobiomemayrepresentaprimary sourceofcontaminationinfacilitieswherefreshproducts are produced or handled, such as raw meat and fish [18,19,20,23],ready-to-eat,compositemeals[21]andfresh fruit[15].Forinstance,meatprocessingenvironmentsare oftencontaminatedbywell-knownmicrobialspoilers (Bro-chothrix thermosphacta, Pseudomonas spp., lactic acid bacteria) thataretransferredtotheproductandthenselectedforby thestorageconditions,forexample,temperature,gaseous atmosphereemployed.Moreover,somestudiesalsoreport

Table1

StudiesusingHTStomapmicrobialcommunitiesinfoodmanufacturingfacilities

Typeoffood industry Number of facilities sampled

Dominanttaxa(environment) Dominant taxawere foundin food?

Surfacessampled Detectionof potential pathogensin theenvironment Detection of beneficial microbes Reference African fermented milk

120 Lactobacillus,Streptococcus Yes Woodenbowls No Yes [2]

Bakery 4 Saccharomycescerevisiae,Weissella, Lactobacillus,Leuconostoc,Bacillus, Streptococcus,Pseudomonas, Staphylococcus

Yes Doughmixer,storage boxes,walls

Staphylococcus Yes [3]

Brewery 1 Saccharomycescerevisiae,Kocuria, Micrococcus,Acinetobacter, Pediococcus

Yes Fermentationtanks, drain,sink,barrels

No Yes [4]

Cheeses 1 Leuconostoccitreum,Pseudomonas, Lactococcuslactis

NA Floordrains Listeria monocytogenes Yes [5] Cheeses, pasta-filata 1 Streptococcusthermophilus, Lactobacillusdelbrueckii, Lactococcuslactis,Pseudomonas

Yes Curdvat,drainingtable, moldingandstretching machines,knives, ripeningroom No Yes [6] Cheeses, pasta-filata 1 Macrococcuscaseolyticus, Lactococcuslactis

Yes Curdvat,drainingtable, knives,briningtank, stretchingandmolding machines

No Yes [7]

Cheeses 4 Escherichiacoli,Acinetobacter johnsonii,Salmonellaenterica

Yes Curdvats,milktanks, molds,floors,sink, drains E.coli,S. enterica, antibiotic resistance genes No [8] Cheeses, smear-ripened 1 Lactobacilluskefiranofaciens, Streptococcusthermophilus, Debaryomyceshansenii, Saccharomycesunisporus

Yes Floordrains No Yes [9]

Cheeses, smear-ripened

2 Debariomyces,Lactococcus, Staphylococcus,Brevibacterium

Yes Drains,agingracks, tanks,drainingtable

Staphylococcus Yes [10] Cheeses, smear-ripened 1 Streptococcus,Staphylococcus, Lactococcus,Pseudomonas

Yes Cowteats,milktanks, molds,packaging,aging shelves No Yes [11] Cheeses, smear-ripened 1 Brevibacterium,Corynebacterium, Debariomyces,Galactomyces

Yes Woodenagingshelves No Yes [12]

Cheeses, washed rinds

2 Halomonas,Corynebacterium, Staphylococcus,Brevibacterium

Yes Agingshelvesandracks, walls,floors

Staphylococcus Yes [13]

Chineseliquor 1 Lactobacillus,Leuconostoc, Pseudomonas,Saccharomyces, Rhizopus,Rhizomucur

Yes Fermentationjar Staphylococcus Yes [14]

Fruitpacking 3 Pseudomonadaceae, Flavobacteriaceae, Xanthomonadaceae,

Aureobasidiaceae,Aspergillaceae

Yes Floors Listeria monocytogenes

No [15]

Milk 1 Lactococcus,Acinetobacter, Streptococcus,Staphylococcus, Bacillus

Yes Silos,pasteurizers, concentrators

Staphylococcus Yes [16]

Milk Streptococcus,Pseudomonas, Staphylococcus,Enterobacteriaceae

Yes Tankertucks Staphylococcus Yes [17]

Rawmeat, sausages

1 Brochothrix,Leuconostoc, Lactobacillus,Yersinia

Yes Transportbelt,meat emulsionblender,filling machine,trolleys Yersinia No [18] Rawmeat, steaks 20 Brochothrix,Pseudomonas, Psychrobacter,Streptococcus

Yes Choppingboards, knives,operatorhands

the presence of potential pathogens (e.g. Salmonella, Escher-ichiacoli,Listeriamonocytogenes,Staphylococcusspp.)or unde-sirable gene families (e.g. antimicrobial resistance genes) on foodprocessingsurfaces,whichmaycontaminatethefood product.Thesehazardousmicrobesmaythenproliferate whentheyfindtheappropriateconditions(Table1). Nev-ertheless,the environmentalmicrobiome mayalso bea reservoirofbeneficialmicrobesthatcontributetothefood manufactureprocess,especiallyinthecaseoffermented foods(Table1).Thiswas highlightedin severalstudies involvingfermenteddairyproductsorbeverages(Table1). Dairies usually harbor lactic acid bacteria and other microbesimportantforripeningof specificcheeses(e.g. Debaryomyces,Brevibacterium, Corynebacterium; relevant to smear-ripened cheese maturation), while the environments ofwineriesandbreweriescanbeasourceofSaccharomyces cerevisiae and other yeasts involved in fermentation to producealcoholicbeverages(Table1).Also, microorgan-ismsresidingonfoodcontactsurfacesmayexertan antimi-crobial activity against pathogens such as Staphylococcus aureus and L. monocytogenes, by competing for nutrients and producing bacteriocins or other antimicrobial com-pounds[25,26].However, it should bepointed outthat mostofthestudiesavailablefocusedonjust1or2different facilities. Thus,awide-scaleandsystematicanalysisoffood environmentalmicrobiomeswouldbenecessaryto encour-agethe implementationof microbiome mapping proce-dures in foodindustriesas an additionaltool to support overallqualityandsafetymanagementsystems.

Metagenomics-based

microbiome

mapping

in

food

processing

environments

Microbialcolonization of surfacesand toolsin thefood processing environments is a widespread phenomenon [27], but thestructure or composition of the microbial

communitiesmayvarysubstantiallyineachfoodplantor indifferentsites of thesamefacility, influencedbythe buildinglayout(Figure1).Moreover,severalotherfactors maycontributetothenumberandcompositionof micro-bialpopulationsonfoodcontactsurfaces,orinfluencethe microbial dynamics thereof (Figure 1). Depending on their composition and hygienic conditions, ingredients, raw materials and processing water entering the food

Table1 (Continued) Typeoffood industry Number of facilities sampled

Dominanttaxa(environment) Dominant taxawere foundin food?

Surfacessampled Detectionof potential pathogensin theenvironment Detection of beneficial microbes Reference Rawmeat, steaks 1 Brochothrix,Pseudomonas, Psychrobacter,Streptococcus

Yes Choppingboards, knives,operatorhands, cold-storewalls,beef carcass No No [20] Ready-to-eat meals 2 Leuconostoc,Lactobacillus, Streptococcaceae,Pseudomonas

Yes Mixingvessel,bench, carriervessel,mixing machine,washingtank, dicer No No [21] Japaneserice liquor(sake) 1 Saccharomycescerevisiae, Aspergillus,Leuconostoc, Staphylococcu,Bacillus, Lactobacillaceae

Yes Fermentationtanks, agingtanks,mixingtub, drains,filterpress, steamer

Staphylococcus Yes [22]

Salmonfillets 2 Pseudomonas,Shewanella Yes Seawatertanks, conveyors,gutting machine

No No [23]

Winery 1 Saccharomycescerevisiae, Hanseniasporauvarum,

Brevundimonas,Comamonadaceae, Enterobacteriaceae

Yes Grapecrusher,press, fermentor,pump, barrels,drain

No Yes [24]

Figure1

Buliding layout Raw materials

Seasons Temperature Air recycling Cleaning parctices Organic residdes Employees Waste handling

Current Opinion in Food Science

processing facilitymay introducenewmicrobial popu-lations that might be different from lot-to-lot. Also, microbial sources along the food chain may include contaminated air (bioaerosols), an incorrect handling ofindustrial wastesandfoodindustry

opera-tors (Figure 1). These populations might ultimately

becomeresidentintheenvironmentwhenappropriate niches are found, but can also change over time in response to factors such as the presence of organic residues, variations in the cleaning and disinfection practices,temperatureshifts(e.g.duringdifferent sea-sons) andother factors(Figure1).

Figure2 Amplicon sequencing Data Analysis Taxonomic composition Shotgun sequencing Data Analysis

Taxonomic composition Genome recontruction &

strain monitoring

Functional profiling Total DNA extraction

Environmental swabs in food industry

Total DNA Random fragmentation and

sequencing

Current Opinion in Food Science

High-throughputsequencingapproachesformicrobiomemapping.Differenthigh-throughputsequencingapproachesforthestudyof environmentalmicrobiomeinfoodindustry.

The developmentof high-throughput sequencing tech-nologies(HTS) in recentyearshasprovided the oppor-tunitytoexploremicrobialconsortiaatanunprecedented depth.Theseapproachescanbesuccessfullyapplied to environmental mapping activities in the food industry

(Figure 2). When preparing for HTS-based profiling,

amplicon-basedorshotgun-basedapproachescanbe con-sidered.For theformer, ageneof taxonomicrelevance, forexample,the16SrRNAgenefrombacteria,is ampli-fied through PCR from total microbial DNA directly extractedfromthesample.Inthisway,adescriptionof thetaxonomiccompositionof themicrobiota inagiven environment is obtained (Figure 2). There are some issuesassociatedwiththisapproach.Firstly,thepresence of an amplification step may lead to a bias due to the preferential amplification of some taxa, distorting the quantitative and qualitative insights gained. This has been noted to be particularly troublesome for Fungi [28,29]. In addition, different target genes must be sequencedtogaininsightsintodifferentsubpopulations of the microbiota(e.g. Bacteria, Fungi, Archaea, Proto-zoa),meaningthatobtainingquantitativedataacrossthe respective populations is not possible. Many of these problemsareovercomebyusingshotgun metagenomics (SM). In shotgun metagenomics (SM), total DNA is fragmented and sequenced without any prior selection steps.Therefore, fragmentedmicrobial genomes of the entire microbial community are sequenced and a com-pletedescriptionofthemicrobialecosystemisobtained, includingofrepresentatives fromdifferentcategories of microorganisms,andalsophage/viruses(Figure2).Inthis case, in addition to the taxonomic composition of the microbial community, its genetic potential can be retrieved, providing the means to study the potential functionsthat aspecific microbial community may har-bour.Inaddition,microbial genomesofthemost abun-dant strains can be reconstructed, allowing precious strain-levelinformationtobegathered.Bothapproaches couldbeusedbyfoodcompaniestomonitortheresident microbial populations in their facilities and to identify possibleroutes ofcontamination(Figure 2).The use of amplicon-based HTS may be useful to evaluate the efficacyofcleaningpractices,totrackmicrobial contami-nants(eitherspoilageorpathogenicmicrobes)onspecific toolsorequipmentsurfacesandevaluatehowthe proces-singplantmicrobiotachangesovertimeorinresponseto the modification of processes (e.g. the introduction of novelcleaningpractices,newsuppliersorchangesinthe process parameters; Figure 1; [30]). This approach, which is easier and cheaper but less informative than SM, may be introduced to support routine quality and safetymanagementplans.Ontheotherhand,usingSM, thecompanyhasthepotentialtogofurthertounderstand the functional potential of the microbial communities inhabiting its processing plant (to date mainly unex-plored), identifying the presence of genes responsible for potentially dangerous activities and intervening in

timetoavoid thespread ofundesirablemicrobes tothe product.Inthisway,tracingofgenesrelatedtovirulence or spoilage activities (e.g. antibiotic resistance, toxin production, biofilm production) in the resident micro-biomeinafoodfacilityispossible[4,8].Inaddition,SM isbettersuitedtodetectingphageandidentifying bacte-ria at the species level, the latter being particularly important when discriminating between pathogens (e.g.L.monocytogenes)andcloselyrelatednon-pathogenic (e.g.Listeriainnocua)species.Furthermore,sinceseveral microbialactivitiesarestrain-specific, strain-level moni-toringmaybeachievedbySMtotrackstarter-associated strains, as wellas to identifyspoilers orpathogens, and monitorstrain persistenceand/or evolutionin the plant duringtimeorinresponsetoprocesschanges(Figure2). Strain-leveltrackingcanbealsoextendedtoraw materi-als,intermediatesoffoodprocessingandfoodproductsto identifyatwhich stageduringtheprocess the contami-nationtakesplaceandalsototracebacktheoriginofthe contaminatingstrains.Therefore,theapplicationof SM for microbiome mapping in the food industry has the potential to revolutionize food safety and quality managementsystems.

In order to use these mapping approaches atindustrial level,foodindustriesshouldbefirstprovidedwith appro-priate standard operating procedures (SOPs) that, in combination,would representanentire workflow. Con-siderableeffortshavebeenmadetostandardizesampling procedures,samplestorageandthesubsequentsteps in the analyses of microbiomes from other environments (e.g. human gut microbiome, http://www.

microbiome-standards.org/). While such protocols are

notyetavailableforfoodandfood-relatedmicrobiomes, thereareconsiderablemeritsininvestingtimetoaddress thisgap.SOPscanbedevelopeddenovooradaptedfrom existingprotocols,followed bytestingand validationin thefoodindustry.Theprocedureswillhavetobe versa-tile to reflect different processing environments and foods,includingindustriesinvolvedinrawandprocessed meatandfishproducts,rawvegetables,freshandripened cheeses, fermented beverages and others. These foods willbesusceptibletodifferentpossibletypesofmicrobial contamination as well as different routes of microbiota entry and establishment in the processing plant. Once validatedSOPsareavailable,disseminationand demon-strationactivitieswill alsobeneededin ordertoleadto thewidespread application of thedeveloped SOPs and strategies by food business operators and laboratories undertakingoutsourcedenvironmentalmonitoring anal-yses.Publicinvestment isneeded topursue theseaims andspecificinnovativeinitiativesarecurrentlyongoingin Europe to achieve this goal. One of such examples is MASTER (Microbiome applications for Sustainable Food Systems through Technologies and Enterprise;

https://www.master-h2020.eu),an EU-funded

methodologiesandSOPsfromavailablemicrobiomedata in order to provide the food industry with appropriate protocolsthatcanbeusedtomapthemicrobial contami-nationintheprocessingenvironmentswiththeultimate scope of process optimization, waste reduction and improvementoffoodqualityandsafety.

Limitations

and

technical

warnings

DNAsequencingandlibrarypreparationcostshave dra-maticallydecreasedovertime[31],makingboth ampli-con-sequencing and shotgun-sequencing more afford-able, as demonstrated by the increasing number of studies in which these techniques are used. However, the actual sequencing cost of a pool of environmental samples may vary significantly, depending on the sequencing platform, on the approach used (e.g. shot-gun-based or amplicon-based)[32]and on therequired sequencingdepth[33].Therefore,thesamplingscheme andthetypeofapproachtobeusedshouldbecarefully evaluated according to the industry requirements: cheaper amplicon-based microbiome mapping can be usedforaroutinecontrol,whilemoreexpensiveshotgun sequencingapproachescanbeusefulwhenadeeperlevel of informationisneeded.

Moreover,thereareanumberofchallengesthatneedto beovercometoharnessthefullpotentialof environmen-talmicrobiomemappingtoolsatfoodprocessingfacilities. Themajortechnicalissue,especiallyforSMapplications, is the recovery of an appropriate amount of DNA, of sufficientquality.Environmentalmappingisusually car-ried out by swabbing industry equipment, tools and surfacesafterroutinecleaning.Therefore,themicrobial loads on thesesurfacesmaybevery low, thatis, below 2.5CFU/cm2inmostcases[1],thuslimitingtheamount of nucleic acids that can be obtained. For the higher amountofDNArequiredforSM,apriorwhole-genome amplificationmaybeusedtoincreasetheavailableDNA concentration.Morespecifically,amultipledisplacement amplification (MDA)canbeused, whichisanon PCR-basedtechniquethatconsistsintherandomamplification of the whole metagenomeunder isothermalconditions, using random exonuclease-resistant primers and the phi29 DNA polymerase [34]. Although this provides a meansof increasingworkable DNAamounts,itis well-documentedthatthisapproachmayrepresentasourceof bias [35]. Indeed, when comparing two popular MDA kits,Yilmazetal.[36]showedthatbothmadequantitative comparisonsunrealisticwhencomparedwithunamplified metagenomicsamples.

Another point of primary importance with respect to optimising therecovery of microbial cells isthe choice oftheswabandtheswabbingprocedure(e.g.thewidthof thesurfacetobesampled).Severalswabtypesare avail-able on the market, which differ with respect to their shapeandthematerialused(Table2).Twomaintypesof

swab categoriesexist:swab tipsorsponges.Toimprove thecollectionofmicrobialcells,theuseofspongeswabs isrecommendedasthesehaveawidersamplingsurface. Cellulose-derivedandsyntheticarethemostcommonly usedmaterials.Cellulose-derivedswabshaveacottonora rayontipthatismadeoffibreswrappedaroundaplastic rod, whereas synthetic swabs are made of various poly-mers,suchaspolyester,polyurethaneornylon.Also,some polyester and nylon swabs maybe flocked. Cottonand rayon swabstend to trapbacterial cellswithinthe fibre matrix, thus hampering the release of the cells in the recovery; in addition, some impurities may bereleased [37].Moreover,syntheticswabsarepreferablefor molec-ularanalyses,asplantDNAmaybereleasedfrom cellu-lose-based swabs, thus contaminating the extracted microbial nucleic acids (Table 2). The performance of syntheticswabsfurtherdependsonthepropertiesofthe polymeric matrix. For example, nylon flocked swabs improve cell release because of an increased capillary action[38,39],whilepolyurethaneswabsarewell-suited forsamplingporoussurfaces[37].However,experimental dataindicatethatmicrobialadhesionstronglydependson the features of the surface being sampled [40] and on factorssuchasthepresenceofexopolysaccharidesandthe frequency and intensity of cleaning procedures [41]. Moreover,Motz etal.[42]recentlyperformeda system-atic comparison between different types of swabs by samplingsurfacesspikedwithdifferentbacterialspecies, chosenfortheirdifferentadhesivecapacity.They dem-onstratedthatswabmassandsurfaceareahaveagreater influence than swab composition in retrieving microorganisms.

Nucleic acid extraction kits and protocols are also an important point to consider. Most commercial kits cur-rently available are optimized for stool, foods or soil samplesratherthanfortheextractionofmicrobialnucleic acidsfromlow-biomassswabsamplessuchasthosefrom food processing environments. Besides having usually lowmicrobialloads,thesesurfacesmaybecontaminated

Table2

Differenttypesofswabsavailableonthemarket

Swabtypes Advantages Disadvantages References Cottonand

rayonswabs

Lowcost Mayreleaseof impurities

[34]

Mostused Mayentrapbacterial cells PlantDNA contamination Swabsmadeof foam(e.g. polyurethane) Suitablefor porous surfaces Hydrophobicitymay limitbuffer absorption [34] [35] Nylonflocked swabs Highcapillarity improvescell release

Swabmaterialmay bereleasedon roughsurfaces

[36]

with detergents, disinfectants or residual food matrix materialsthat mayinhibit subsequentenzymatic steps. Forthesereasons,theoptimizationofamicrobialDNA extraction protocol for this specific type of samples is crucial.

The most recent innovations in HTS are theso-called ‘ThirdGenerationSequencing’technologies,which are basedontheuseofreal-time,highthroughput,and—in somecases—portablesequencers.Thesenovelmethods are more suitable than Next Generation Sequencing platforms for quick and on-site sequencing, providing longer reads than previous generation of sequencers [43]. Reasonably, these high-throughput and portable sequencers could be soon used directly in factory sites forreal-timemonitoringofmicrobialcommunities. Finally,oncetheDNAhasbeensequenced, bioinformat-ics and statistical skills are necessary for data analysis. Dataanalysiscanbeconsideredtherealbottleneckinthe routine application of HTS in the food industry, since personnelspecializedinbioinformaticswouldbe neces-sary. The most common steps in metagenomic data analysisconsistofqualityfilteringofthereads,taxonomic and/or functional profiling of metagenomes and the reconstruction of metagenomes-assembled genomes (MAGs).Foreachofthemetagenomicdataanalysissteps, several tools and analysis pipelines are available,and a deepknowledgeofalgorithmsandtool-specificsettingsis needed. In addition, to get meaningful results, data analysis must be followed by a statistical exploration andresultssummarizedintablesorplots.Indeed,novel data-scientistfigureswithabackgroundinfood microbi-ologywouldbeimportantin helpingfoodcompanies to get the most from metagenomics data and understand howtointegrateandexploitthesekindsofanalysisina qualityandsafetymanagementplan.Therefore, innova-tivecoursesdirectedtounderstandtheuseofthesenovel techniques in food industries should be integrated in higher education institutions for all food science pro-grams. In addition, events and demonstration activities forfoodbusinessoperatorswouldbeofutmostutilityto achieveasuccessfulknowledgeandinnovationtransfer.

Microbiome

mapping

and

EU/US

regulation

AccordingtoEUregulationNo852/2004onthehygiene of foodstuffs, the primary responsibility for food safety restswith thefoodbusinessoperators,who, followinga preventive approach, shouldestablish and operate food safetyprogrammesandproceduresbasedontheHazards AnalysisandCriticalControlPoints(HACCP)principles to ensure that food safety is not compromised. Such a preventiveapproachhasbeenadoptedintheUStoo,with thepublicationof theFDAFood SafetyModernization Act in 2011. Since then, all the facilities under FDA jurisdiction(with some exceptions)have beenrequired

toadopttheHazardAnalysisandRisk-basedPreventive Controls(HARPC)principles.

IntheEU,validationandverificationofHACCP proce-duresareaccomplishedthrough,amongothers,the com-pliancewithmicrobiologicalcriteriadefiningthe accept-abilityoftheprocessesandtheend-products,whichare definedunderEURegulationNo2073/2005.Thatpiece ofregulation highlightsthatsampling oftheproduction and processing environment can be a useful tool to identifyand preventthepresence of pathogenic micro-organisms in foodstuffs and specifically mentions that foodbusinessoperatorsmanufacturingready-to-eatfoods shallsample theprocessing areasand equipmentfor L. monocytogenesand those manufacturing dried infant for-mulae or dried foods for special medical purposes intended for infants below six months for Enterobacter-iaceaeas partof theirsamplingschemes.IntheUS,the CodeofFederalRegulationsclearlystatesthat,ifthefood businessoperatorconsiderscontaminationofready-to-eat foodahazardrequiringpreventivecontrols, environmen-talmonitoringmust becarriedoutto verifytheir effec-tiveness.Allenvironmentalsamplingactivitiescurrently undertaken by food business operators are therefore basedontracingspecific foodbornehazardsand/or indi-catorsusingclassic toolsfortheisolation and identifica-tion/confirmationof targetmicroorganisms. Thesehave numerouslimitations,includingthelongtimerequiredto obtainresults, whichdelays theimplementation of cor-rective measures when problems are encountered. In addition,accordingtotheEURegulation,environmental samplingshallbeperformedfollowingtheISOstandard 18593 on horizontal methods for surface sampling as a reference.However,these standardmethodshave been developedforthespecificaimofisolatingand enumerat-ing microorganisms from certain particular taxa. HTS-based approaches, given their properties highlighted in previoussections,havethepotentialtorevolutionizethe way food business operators approach environmental monitoring activities within their food safety manage-mentsystems.However,thefuturetransitionfrom clas-sical microbiological techniques to HTS-based micro-biome monitoring techniques will require the development of new standards, covering aspects from samplingto bioinformaticanalysesandinterpretation of results,specificallytailoredtotheneedsoffoodbusiness operators. These new standards should be robust and flexibletosupportthefastdevelopmentofcommercially availableinnovations,but alsotoleave spacetoaccount forrapid advancesin technologyallowingthenecessary updateswhenmethodsbecomeoutdated.Moreover,they shouldbeinternationallyagreedandvalidatedonaglobal scale to provide evidence of their reproducibility and accuracy [44].Nevertheless,in the long-term, the inte-grationofHTS-basedmicrobiomeanalysisinfoodsafety policies willalso requirethe translation of thecomplex outputsprovidedbymetagenomictoolsintoquantifiable

and easy to interpret microbiological process criteria allowing rapiddecision makingbythefood industry.

Conclusions

and

future

perspectives

The resident microbiome in food factories plays an important role in influencing food quality and safety. Productionactivities,environmentalandprocess param-eters shape the microbial communities inhabiting food facilities.Monitoringofthefoodindustryenvironmental microbiomebyup-to-datesequencing-basedstrategiesis a promising tool that could support overallquality and safetymanagementplans.However,despitethe decreas-ing cost of thesetechnologies, their implementationas routinepracticeswithrespecttotheenvironmental mon-itoringinthefoodprocessingindustryisstillchallenging. In thisregard, thegenerationof resultsfrombroad and structuredinitiativesthatincludethedevelopment, vali-dationanddisseminationofmicrobiomemapping strate-gies can greatly assist the food industry and related stakeholderstoadoptnextgenerationproceduresfortheir quality assessments and develop improved sustainable production chains to be better prepared for possible specific regulatorychangesinthefoodsector.

Conflict

of

interest

statement

CRediT

authorship

contribution

statement

FrancescaDeFilippis:Conceptualization,Formalanalysis, Writing-originaldraft.VincenzoValentino:Formal analy-sis, Writing - original draft. Avelino Alvarez-Ordo´n˜ez: Formalanalysis,Writing-originaldraft,Funding acquisi-tion.PaulDCotter:Resources,Writing-review&editing, Funding acquisition. Danilo Ercolini: Conceptualization, Resources, Writing - review & editing, Funding acquisition.

Acknowledgements

ThisworkwassupportedbytheprojectMASTER(Microbiome ApplicationsforSustainablefoodsystemsthroughTechnologiesand Enterprise),receivingfundingfromtheEuropeanUnion’sHorizon 2020researchandinnovationprogrammeundergrantagreementNo 818368.Thismanuscriptreflectsonlytheauthors’viewsandtheEuropean Commissionisnotresponsibleforanyusethatmaybemadeofthe informationitcontains.

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and

recommended

reading

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