Focuson:Hygieneindicatorsin

FoodMicrobiology
June2014

HygieneIndicatorsinFood
Microbiology

Definitions

A hygiene microbiological indicator (or hygiene process criteria) is a single microorganism/group of

microrganisms(whichmay/maynotbepathogenic)orametabolicproduct,revealsapotentialproblem
ofquality,hygieneandevensafetygivenaminimumlevelofcontamination(ordose).
Itmaycomprisethefollowing:
potential presence of a consumer health hazard : risk of presence of a pathogen germ or a toxic
metabolite(toxins);
noncompliance to Good Hygienic / Manufacturing Practices (GHPs/GMPs), of storage (cold chain,
warmchain)anddistribution;
initialcontaminationofrawmaterials;
commercial risk: rapid spoilage and/or alteration/deterioration of organoleptic quality of food
products.

Safety microbiological criteria define if a food product is fit for human consumption or not. Unlike
them, an out of specifications Hygiene process criterion does not necessarily mean that the food
productisunfittohumanconsumptionanddoesnotleadautomaticallytoitswithdrawaloritsrecall,
butrathertotheimplementationofappropriatecorrectiveactions,asforexamplethereviewofGMPs,
HACCPsystemortheprocessitself.

Another term, Index, is employed suggesting ahealth risk,gives an indication of the likelihood of the
presenceofpathogens.Forexample,E.coliisanindexforSalmonellaorotherpathogenicgermsoffecal
origin in drinking water. As hygiene process criteria, an out of specifications result does not allow to
concludethatthefoodproductisunfittohumanconsumption.

Somemicroorganismscanplayamultiplerole:bothasanindex(presenceofahealthriskforhuman)
and as an indicator of poorly controlled process, an issue during the process (too long storage at
inappropriatetemperatures,insufficientheattreatment,etc).

Microbiological indicators are naturally present in food products. For purposes of process validation,
indicators may be artificially introduced as inocula. Called surrogates, indicators usually replace
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pathogens in order to study the efficiency of treatments, processes, etc. For example, Clostridium
botulinumarefrequentlyreplacedbyClostridiumsporogenes(historicallyknowntobenonpathogenic)
inlowacidcanningindustrytovalidatethethermaltreatmentprocess.

Whyindicators?
InEurope,accordingtothecurrentregulation(EC2073/2005),manufacturing,storageanddistribution
processes have to be assessed. Indicators can be used as a tool for process assessment as part of
preventiveapproacheswithinHACCPplansorGMPs.

Conversely, some pathogenic microorganisms show a low prevalence and/or a low concentration in
foodproductsduetotheefficiencyofproceduresimplementedbyindustrialsforhygienecontrol.The
effectoflowprevalenceresultsin:
difficultytodetectthepathogen(effectofsampling);
lackofsensitivityofcurrentmethodsforsomemicroorganisms;
absenceofdetectionmethods,especiallyemergingmicroorganisms;
noenumerativedatatoexploit.

Betterdetectionmethodscanalsobetooexpensiveortechnicallycomplicatedtobeusedroutinely.

So,hygieneindicatorsarereallycomplementarytosafetycriteria.

Howtochoosethem?

Some process hygiene indicators are established by the regulations (e.g., Regulation EC 2073/2005
modifiedby1441/2007).

Other process hygiene indicators not established by the regulations are defined by specific industry
sectors.InFrance,thesecriteriaareavailableonDGAl(DirectionGnraledelAlimentation)websitefor
meatproductsanddelicatessen,poultry,cateringandretailersectorsasGoodpracticeguides.

Butitisforeachmanufacturingsitetodefinerelevantindicatorsaccordingtoestablishedcriticalpoints,
producttype,processes,andhistory.
Theapplicationstagehasalsotobedefined,sothattheindicatortakesonfullsignificance,givesareal
information.

Inpractice,theidealindicatorhasthefollowingfeatures:
itisusuallynonpathogenic;
itmustbesystematicallypresent(evenifonlyatlowlevel)andcanbedetected;
itmustbedetectable,preferablycountableinasimple,fastandinexpensiveway;
itshouldhavethesamehabitatasthetargetpathogenicagent.

Once chosen, it is generally regarded as good practice tomaintain the same indicator organism(s) for
purposesofevaluatingprocesscontrol.
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TypesanduseofIndicators

Theapproachtouseindicatorsmaybedifferentdependingonthecountryofuse.InEurope(Regulation
EC2073/2005)andinUS(Legislation9CFR,),forexample,commonprocessindicatorscomprisethe
following:

Totalaerobiccountat30C/Totalviablecount:Indicatoroftheglobalhygieneleveloftheproduct,
and its history from manufacturing to the distribution, may also be an indicatorof the contamination
level incoming raw material. An incubation at 30C facilitates the growth of psychrophilic
microorganismspreferringcoolenvironments.oThetechnologicalflora,intentionallyaddedorpartof
thenaturalfloraoftheproduct,shouldnotbetakenintoaccount.

E.coli: understanding glucuronidase positive E.coli, growing at 44C, give a more precise
information about potential contamination with animal fecal material and potential presence of
pathogensoffecalorigin.

Salmonella:pathogengramnegativebacteria,nonlactosefermenting,belongtoEnterobacteriaceae
family.Indicatorusedespeciallyforcattle,sheep,goats,equineandpoultrycarcassestoassesshygiene
duringtheslaughter.However,forUS,Salmonellacanbeanindicatorbutisfirstasafetyindicator:if
Salmonella is found in a product that is linked to illnesses, then the product is not sold or must be
recalled.

SpecificindicatorsinUS:

Coliforms: Particularly those of fecal origin, gram negative bacteria, lactose fermenting, belong to
Enterobacteriaceaefamily.

Listeriaspp:Grampositiverodshapedbacteria,abletogrowatrefrigeratedtemperatures,areused
asindicatorsfor:
thepotentialpresenceofListeriamonocytogenes,thepathogenspecies;
thelevelofcontaminationofthefoodprocessingenvironment:cleanlinessofequipment,surfaces

SpecificindicatorsinEurope

WhileUScontinuetousecoliformsasindicators,thecurrenttrendinEuropeistousetheentirefamily
Enterobacteriaceae.

Enterobacteriaceae:familygatheringalargenumberofGramnegativebacteria,ubiquitousinthe
environment,inwhichtakepartcoliforms(lactosefermenters),fecalcoliformsandE.coli,areindicators
of:

Fecalcontaminationofhumanoranimalorigin(foundinintestinaltracts);
Environmental contamination (soil, dust, water, insects) and contaminated equipment (surfaces
inadequatelycleanedorsanitized).
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Thisfamilyistheindicatorthemostcommonlyusedbecauserelativelysensitivetostandardtreatments
implementedtoinactivatemicroorganismsinfood(heat,ionizingradiation,freezing,drying,extremes
ofpH,andwateractivity)andintheenvironment(destroyedbymostofdisinfectants).

Coagulasepositivestaphylococci:includingbutnotlimitedtoS.aureus.Indicatorsofgoodhandling
proceduresandcontaminationfromworkers(e.g.,dirtyhands,skin,noseorthroatinfections).Itshould
benotedthathealthyworkersmayalsobecarriersofstaphylococci.Highnumbersofstaphylococciin
foodsmayindicateapotentialpresenceofenterotoxinsresponsibleforfoodpoisoning.RegulationEC
2073/2005requiresinthiscasethedetectionofstaphylococcalenterotoxins.

Bacillusofthecereusgroup3:theseubiquitousbacteria,oftelluricorigin(soil),areindicatorsforthe
following:
rawmaterialhygiene:sporesarefoundalmostinallfoodsandespeciallyindryordehydratedproducts
such as spices, aromatic herbs, some vegetables, cereals, flours. These raw materials component of
finishedproductsareallpotentialsourcesofcontamination;
efficiency of cleaning processes: spores of Bacillus cereus have strong capacities of adhesion to
stainless surfaces and can accumulate in manufacturing equipment which then becomes spores
reservoirs;
insufficientthermaltreatmentorstorageatinappropriatetemperaturesthatallowtheirgrowthafter
thermaltreatment:themostcasesreportedwereforrice,potatoes,pastas,milkpowder.

Insectors,somespecificmicroorganismscanbeusedasspoilageindicators,suchas:

Lacticacidbacteriaindelimeats
Yeastsandmoldsinyogurt,
Pseudomonasinfishes,poultry,dairyproducts
Alicyclobacillusinbeveragesandfruitjuices
SRB (sulfate Reducing bacteria) and/or Clostridium perfringens in cooked products (readycooked
dishes,cookedmeats)

Thesemicroorganismsarespecifictogivenmatricesbecauseinsomefoodproductstheytakepartof
thenormalfloraoftheproduct.Forexample,lacticacidbacteriaarenotrelevantindairyproductsnor
inreadycookeddishescontainingcheese,andfermentedfood(sauerkraut).

Sourcesandtolearnmore:

1. AFSSA(ANSES)AvisdelAgencefranaisedescuritsanitairedesalimentsrelativelademandedecrationdedocumentsde
rfrenceconcernantdesfloresmicrobiennesutilisablesentantquindicateursdhyginedesprocds.2007

2. AFSSA(ANSES)Recommandationspourllaborationdecritresmicrobiologiquesdhyginedesprocds.2008

3. ANSESFichesdedescriptiondedangertransmissibleparlesalimentsBacilluscereusSeptembre2011

4. EFSATechnicalspecificationsonharmonizedepidemiologicalindicatorsforbiologicalhazardstobecoveredbymeatinspectionof
poultry.2012

5. RglementCE2073/2005delaCommissiondu15Novembre2005concernantlescritresmicrobiologiquesapplicablesauxdenres
alimentaires.http://eurlex.europa.eu/homepage.html

6. DGAl(DirectionGnraledelAlimentation)Critresdhyginedesprocdshttp://agriculture.gouv.fr/Criteresmicrobiologiques
hygienedesprocedes

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 7. FDA(FoodandDrugAdministration)GradeAPasteruizedmilkordinancehttp://www.fda.gov/downloads/Food/GuidanceRegulation

8. CodeofFederalRegulations9CFR310.25Contaminationwithmicroorganisms;Processcontrolverificationcriteriaandtesting;
pathogenreductionstandards.http://www.gpo.gov/

9. http://www.foodsafetymagazine.com/magazinearchive1/februarymarch2011/indicatororganismassayschaosconfusionandcriteria/


RapidmethodsBIOMERIEUXcertifiedaccordingtoISO16140standard:

(NonexhaustivelistofsolutionsdedicatedtoHygieneindicators)

BIOMERIEUXBACARA(BacilluscereusRapidAgar)AFNORCertificationnAES10/1007/10
ChromogenicandselectivemediumfortheenumerationofpresumptiveBacilluscereusinallhumanfoodand
animalfeedingstuffs(endofvalidity:02/07/2014)

BIOMERIEUXchromIDColiagarAFNORCertificationnBIO12/2012/06Chromogenicandselective
mediumfortheenumerationofcoliformsandGlucuronidasepositiveE.colifromfoodsamplesValidatedfor
theenumerationofColiformsat37C(endofvalidity:14/12/2014)

BIOMERIEUXchromIDColiagarAFNORCertificationnBIO12/1912/06Chromogenicandselective
mediumfortheenumerationofcoliformsandGlucuronidasepositiveE.colifromfoodsamplesValidatedfor
theenumerationofGlucuronidasepositiveE.coliat37C(endofvalidity:14/12/2014)

BIOMERIEUXchromIDColiagarAFNORCertificationnBIO12/501/99Chromogenicandselective
mediumfortheenumerationofcoliformsandGlucuronidasepositiveE.colifromfoodsamplesValidatedfor
theenumerationofGlucuronidasepositiveE.coliat44C(endofvalidity:14/12/2014)

BIOMERIEUXREBECCATMBASEOU/ORREBECCATM+EBAFNORCertificationnAES10/0601/08
ChromogenicandselectivemediumfortheenumerationofE.colifromallhumanandanimalfoodproducts
Renewal01/12/2011(endofvalidity:17/01/2016)

BIOMERIEUXREBECCATM+EBAFNORCertificationnAES10/0701/08Chromogenicandselectivemedium
fortheenumerationofEnterobacteriaceaefromallhumanandanimalfoodproducts(endofvalidity:
17/01/2016)

BIOMERIEUXALOAOnedayAFNORCertificationnBIO10/0309/00Chromogenicandselectivemedium
forthedetectionofListeriasppandListeriamonocytogenesfromallhumanfoodproductsandenvironmental
samples(endofvalidity:27/09/2016)

BIOMERIEUXVIDASListeria(LIS)AFNORCertificationnBIO12/206/94DetectionofListeriasppfromall
humanfoodproductsandenvironmentalsamples.Immunoenzymatictest.(endofvalidity:09/06/2018)

BIOMERIEUXVIDASListeriaspeciesXpress(LSX)AFNORCertificationnBIO12/1207/04Detectionof
Listeriasppfrommilkproducts,meatproducts,vegetableproductsandenvironmentalsamples.Immuno
enzymatictest.(endofvalidity:01/07/2016)

BIOMERIEUXVIDASListeriaLDUOAFNORCertificationnBIO12/1803/06DetectionofListeriasppand
Listeriamonocytogenesfromallhumanfoodproductsandenvironmentalsamples.Immunoenzymatictest.(end
ofvalidity:09/03/2018)

BIOMERIEUXVIDASUPLISTERIAAFNORCertificationnBIO12/3305/12DetectionofListeriasppfrom
allhumanfoodproductsandproductionenvironmentalsamples.Immunoenzymatictest.(endofvalidity:
10/05/2016)

bioMrieux Industry - 2014


BIOMERIEUXTEMPOECAFNORCertificationnBIO12/1302/05EnumerationofE.colifromallhuman
foodandpetfoodproducts,withtheexceptionofbeveragesandcattlefeedproducts.(endofvalidity:
04/02/2017)

BIOMERIEUXTEMPOEBAFNORCertificationnBIO12/2112/06EnumerationofEnterobacteriaceaefrom
allhumanfoodandanimalfoodproductsexceptbeveragesandcattlefood.(endofvalidity:15/12/2014)

BIOMERIEUXTEMPOACAFNORCertificationnBIO12/3505/13Enumerationofmesophilicaerobicflora
fromallhumanfoodandpetfoodsandenvironmentalsamples.(endofvalidity:23/05/2017)

BIOMERIEUXTEMPOTVCAFNORCertificationnBIO12/1509/05Enumerationoftotalviablecountfrom
allhumanandanimalfoodproducts(exceptbeveragesandcattlefeed)(endofvalidity:19/09/2017)

BIOMERIEUXTEMPOTCAFNORCertificationnBIO12/1712/05EnumerationoftotalColiformsinfoodand
petfoodproducts,withtheexceptionofbeveragesandcattlefeed(endofvalidity:09/12/2017)

BIOMERIEUXTEMPOSTAAFNORCertificationnBIO12/2804/10Enumerationofcoagulasepositive
staphylococciinallhumanfoodproductsandanimalfeeding(endofvalidity:01/04/2018)

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