Listeria Monocytogenes Virulence and Pathogenicity, a Food Safety (2024)

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Journalof Food Protection, Vol. 65, No. 11, 2002, Pages 1811– 1829 Copyright Q,InternationalAssociation for FoodProtection

Review Listeria monocytogenes Virulenceand Pathogenicity, a Food Safety Perspective

SOPHIA KATHARIOU *

FoodScience Departmentand Program in Genomic Sciences, NorthCarolina State University, 339Schaub Hall,

Raleigh,North Carolina 27695, USA Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/11/1811/1672430/0362-028x-65_11_1811.pdf by guest on 26 September 2021

MS01-481:Received 18December 2001/Accepted 23March 2002

ABSTRACT

Severalvirulence factors of Listeriamonocytogenes havebeen identiŽ ed and extensively characterized at the molecular andcell biologic levels, including the hemolysin (listeriolysin O), twodistinct phospholipases, a protein(ActA), several internalins,and others. Their study has yielded an impressive amount of information on the mechanisms employed by this facultativeintracellular pathogen to interact with mammalian host cells, escape the host cell’ s killingmechanisms, and spread fromone infected cell to others. In addition, several molecular subtyping tools have been developed to facilitate the detection ofdifferent strain types and lineages of the pathogen, including those implicated in common-source outbreaks of the disease. Despitethese spectacular gains in knowledge, the virulence of L.monocytogenes asa foodbornepathogen remains poorly understood.The available pathogenesis and subtyping data generally fail to provide adequate insight about the virulence of Želdisolates and the likelihood that a givenstrain will cause illness. Possible mechanisms for the apparent prevalence of three serotypes(1/ 2a,1/ 2b,and 4b) in human foodborne illness remain unidentiŽ ed. The propensity of certain strain lineages (epidemicclones) to be implicatedin common-source outbreaks and the prevalence of serotype4b amongepidemic-associated strainsalso remain poorly understood. This review Ž rstdiscusses current progress in understanding the general features of virulenceand pathogenesis of L.monocytogenes. Emphasisis then placed on areas of special relevance to the organism’ s involvementin human foodborne illness, including (i) the relative prevalence of different serotypes and serotype-speciŽ c featuresand genetic markers; (ii) the ability of the organism to respond to environmental stresses of relevance to the food industry(cold, salt, iron depletion, and acid); (iii) the speciŽ c featuresof the major known epidemic-associated lineages; and (iv)the possible reservoirs of the organism in animals and the environment and the pronounced impact of environmental contaminationin the food processing facilities. Finally, a discussionis provided on the perceived areas of special need for futureresearch of relevance to food safety, including (i) theoretical modeling studies of niche complexity and contamination inthe food processing facilities; (ii) strain databases for comprehensive molecular typing; and (iii) contributions from genomic andproteomic tools, including DNA microarraysfor genotyping and expression signatures. Virulence-related genomic and proteomicsignatures are expected to emerge from analysis of the genomes at the global level, with the support of adequate epidemiologicdata and access to relevant strains.

Listeriamonocytogenes istheonly species in thegenus thisbacterium in the environment, its ability to grow in the Listeria thatis of concernfor humanhealth. This facultative cold,and its pathogenic potential make this pathogen of intracellular,gram-positivebacterium is capable of causing particularconcern for thesafety of refrigerated and ready- seriousinvasive illness (listeriosis) in both humans and an- to-eat(RTE) foodsconsumed without reheating, cooking, imals (62,97, 179, 180). Thetransmission of this pathogen orboth.Several outbreaks of listeriosishave been traced to bycontaminated food was Žrst conclusivelydemonstrated contaminatedcold-stored RTE foods, including dairy, veg- byepidemiologic and laboratory investigations in 1983 etable,and meat products (179). Earlierreviews have ad- (176) andhas been shown to causeboth sporadic cases and dressedin detail the bacteriology and human epidemiology outbreaksof listeriosis.Certain segments of thepopulation, of L.monocytogenes(62, 91, 97, 179). includingthe elderly, neonates, pregnant women, human As afacultativeintracellular pathogen, L.monocyto- immunodeŽciency virus– infected individuals, and individ- genes cansurvive and grow in mammalian cells, including ualsundergoing immunosuppressive therapy, are at in- phagocytes.Protective immunity is cell mediated, as Ž rst creasedrisk of infection. In nature, the primary habitat of shownby Mackaness (119), withmany investigations hav- Listeria appearsto be soil and decaying vegetation. Unlike ingaddressed the components of thelisterial cell-mediated mosthuman pathogens, L.monocytogenes cangrow at re- immuneresponse (for reviews,see (35, 100)). Thesefun- frigerationtemperatures (78). Theubiquitous distribution of damentalattributes of thepathogen, along with the fact that itcan be grown easily in the laboratory, have rendered it *Authorfor correspondence. Tel: 919-513-2075; Fax: 919-515-7124; aneffective model system for thestudy of bacterial path- E-mail: [emailprotected]. ogenesis,intracellular survival, cell biology of host-patho- 1812 KATHARIOU J.FoodProt., Vol. 65, No. 11 geninteractions, and cell-mediated immunity. Intracellular Expressionof thevirulence genes mentioned above re- replicationof the pathogen appears to be intimately con- quiresthe transcriptional activator ,PrfA, encodedby agene nectedwith virulence as well as with induction of protec- inthe same genomic cluster .PrfA-mediatedregulation has tiveimmunity, as intracellular growth and processing of beenextensively studied (see reviewsin (56, 89, 97)). selectedantigens is required for effectiveimmune responses (19, 35). 1.1.b.Determinants involved in cell invasion, intra- Sincethe middle 1980s, an impressivebody of knowl- cellularmotility, and cell-to-cell spread. In the mouse edgehas accumulated concerning the molecular biology of model, L.monocytogenes hasbeen shown to invade enter- virulencedeterminants of this microorganism and the cell ocytesor M cellsin Peyer’ s patches (117, 166). The bac- biologyof its interactions with host cell receptors, the cy- teriumreplicates in enterocytes and in highly permissive toskeleton,and signal transduction pathways. This work has mononuclearcells in the Peyer’ s patches,in a processthat beenreviewed extensively (for recentreviews, see (40, 56, isstillbeing studied (165), anddisseminates to theprimary 76, 89, 103)).Hence,these studies will not be reviewed targetorgans (liver and spleen). Most bacteria that reach Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/11/1811/1672430/0362-028x-65_11_1811.pdf by guest on 26 September 2021 here.Furthermore, although these studies have contributed theliver are killed by resident macrophages (114, 119). The greatlyto our understanding of listerial (and general) cel- few thatsurvive are likely to be cleared by the host’ s nor- lularpathogenesis, they have not adequately addressed or malimmune system. However ,anysurvivors can go on to elucidatedthe special and numerous virulence-related con- infecthepatocytes and can eventually cause systemic infec- cernsthat the pathogen raises for foodsafety. This review tionand invasion of the secondary target organs (central detailsand discusses our current understanding of some key nervoussystem, placenta, and fetus). issuesin this regard, with special attention drawn to the Inmodel cell culture systems, adherence and invasion differentserotypes and clonal lineages found in foods and ofhostcells require several determinants. Most intensively implicatedin foodborne listeriosis; the lineages responsible studiedare the surface proteins internalin A (InlA) andin- for epidemics;and the avenues for effectivedetection of ternalinB (InlB),which are differentially required for in- pathogenicstrains in foods and in the community. fectionof different cell types and recognize different re- ceptorson the host cells (27, 127). Theassociation of L. 1.0.VIRULENCE AND PATHOGENESIS monocytogenes withthese receptors leads to phosphoryla- 1.1.Overview of virulence determinants and inter- tionof various host cell proteins and a complexsignal actions of L.monocytogenes with host cells:1.1.a. The transductioncascade that results in pathogen-mediated in- listeriolysinO region. Thegenes for severalkey virulence ternalizationof the bacteria. As willbe discussed later (see determinantsof L.monocytogenes, includingthe hemolysin section1.2.1.a), the receptor for InlA(E-cadherin) is not (listeriolysinO), twophospholipases, and a protein(ActA) functionalin murine cells. However ,transgenicmice that essentialfor intracellularmotility of the pathogen, are lo- harbora functionalE-cadherin are much more sensitive to catedin one well-deŽ ned gene cluster in the chromosome wild-type L.monocytogenes thanto isogenicInlA-deŽ cient ofthe bacterium. Upon infection of hostcells, the bacteria mutants,indicating an important role of InlA in virulence areinternalized in a vacuole.Expression of listeriolysin O in vivo (109). Severalother invasion determinants of L. promotesescape from thevacuole into the cytoplasm, monocytogenes havebeen identiŽ ed, suggesting that the wherethe microbe replicates. The intracytoplasmic bacteria celladherence and invasion process is complex and mul- usethe actin of thehost cell, in conjunction with their ActA tifactorial(for reviews,see (40,56, 76, 89, 97, 103) ). The protein,to promote their motility intracellularly, their lo- celladherence-invasion process of Listeria isstill being ac- cationin protrudingpseudopods, and the engulfment of the tivelystudied (27, 130). pseudopodsby adjacent host cells. After theiruptake by Actin-basedintracellular motility of L.monocytogenes adjacentcells, the bacteria escape the now double-mem- iscrucial to cellular pathogenesis and requires ActA, en- brane–bound vacuole by means of listeriolysin O andthe codedby a genein the listeriolysin O generegion. ActA is phospholipases,and the cycle repeats. The process, exhib- bothessential and sufŽ cient for Listeria actin-basedmotil- ited by L.monocytogenes,Shigella  exneri, and certain ity, and actA mutantsare avirulent in mice.Mechanisms of Rickettsia spp.,is as striking at the electron microscopic ActA action,along with other components of the patho- andcell biologic level today as itwas whenŽ rst described gen’s intracellularmotility and cell-to-cell spread, have (190). beenextensively studied (reviewed in (67, 89)). Theproduction of listeriolysin O inthe host cell is Geneticstudies continue to identify genes essential for understringent regulatory control. In the murine model, a thevirulence of L.monocytogenes (e.g., (12,41, 68, 109) ). geneticallyconstructed variant with a singleamino acid Moststudies, however ,haveutilized strains EGD and changethat rendered the protein more stable in the cyto- 10403S(both serotype 1/ 2a)or strainLO28 (serotype 1/ 2c) plasmof the infected cell was foundto have a decreasein andmay not effectively address virulence attributes speciŽ c virulenceof more than 3 logs (49). Clearly,this is a viru- toother serotypes that are of clinical importance, such as lencefactor that is expressed in the vacuole but not in the 1/2band 4b. Strains of serotypes 1/ 2aand 1/ 2cbelong to cytosol,thus preventing killing of thehost cell and allowing oneof two major genetic divisions in L.monocytogenes, thehost cell cytoplasm to serveas a safe havenfor bacterial foundto be quite distinct from thedivision that includes survivaland replication. Similar regulatory controls may serotypes1/ 2band 4b (21,22, 31, 156); seealso section wellexist for othervirulence factors. 2.1.3).Although the key virulence factors known to date J.FoodProt., Vol. 65, No. 11 L.MONOCYTOGENES VIRULENCE AND PATHOGENICITY 1813

(listeriolysinO, phospholipases,ActA, internalins A andB, for routinestudies and surveys of virulence, these results andothers) are present in all serotypes, their regulation of highlightthe continuing need for alternativemodels, espe- expressionmay di ffer amongserot ypes.Furth ermore, ciallyin regard to infections via the oral or i.g. route. strainsof serotypes 1/ 2band 4b may have additional vir- Inearlier studies, i.v. and i.g. infections were usedto ulencedeterminants, which will not become identiŽ ed until determinethe impact of bacterial growth temperature on andunless strains of these serotypes become included in virulence.Interestingly, low-temperature growth enhanced geneticstudies of virulence. The identiŽ cation of poten- virulencein i.v. but not in i.g. infections (44, 186), sug- tiallyunique virulence factors of thesestrains may be aided gestingthat caution should be exerted in interpreting viru- bycomparative genomic analyses (see alsosection 4.4) lencedata obtained following different routes of infection. 1.2.Laboratory determinations of virulence— ani- 1.2.1.b.Brain infection models. Efforts havebeen maland cell culture models: 1.2.1. Murine models. Mu- madeto model central nervous system invasion in food-

rinemodels have been used extensively, with virulence borneinfection, with maternal encephalitis detected in the Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/11/1811/1672430/0362-028x-65_11_1811.pdf by guest on 26 September 2021 quantitatedin termsof thepersistence of thebacteria in the pregnantmouse model, depending on the timing of infec- spleenor liver following intravenous (i.v.) infections (87, tionin regard to gestation (128). Repeatedoral dosing (5 100). Awidelyused model involves determinations of 50% 3 109 over7 to10 days) resulted in severe brain lesions lethaldose for immunocompromisedmice (185). (withhistopathology similar to thatseen in humanenceph- 1.2.1.a.Oral and intragastric infections. To better alitis)in 25% of the mice (6). Neurovirulenceof the path- simulateaspects of foodborne infection, oral and intragas- ogenafter direct brain inoculation also has been studied tric(i.g.) infection models have been used (14,158, 175). (177). Recentstudies have pursued the identiŽ cation of Certainstrains that had similar virulence in i.v. infections genesessential for bacterialmultiplication in the brain of were poorlyinfective in terms of spleen colonization fol- micefollowing i.v. infections (see alsosection 2.4), using lowingi.g. inoculation, suggesting that i.g. infection may signature-taggedmutagenesis, a methodthat allows the bea morediscriminatory model for detectingpotential vir- identiŽcation of mutantsthat are attenuated in virulence and ulencedifferences (14). Miceimmunosuppressed by cyclo- thusfail to be recovered from thetarget organ (brain). In- sporinwere moresensitive to i.g. infection than untreated terestingly,a largenumber of mutantswere foundto harbor animalsand were usedin experiments to demonstrate the lesionsin the gene gtcA, whichvaries noticeably in se- impactof intestinal bacterial  oraon the outcome of oral quencebetween serotypes 4b and1/ 2a (12). Inserotype 4b, infectionby L.monocytogenes(150). Corticosteroidtreat- gtcA isessentialfor serotype-speciŽc glycosylationof wall- mentin an oral infection model also has been used, result- associatedteichoic acid (164). ingin prolonged infections (161). Usingoral infections in 1.2.2.Other animalmodels. As mentionedearlier , pregnantmouse models, Lammerding et al. (104) and Men- mice(and rats) lacka functionalreceptor for theprotein udier et al. (128) were ableto monitor maternal and fetal InlA,which is required for listerialinvasion of human in- infection.Strains were foundto differ in termsof theirabil- testinalepithelial cells in culture. The guinea pig, which ityto causefetal infection following oral inoculation during hasa functionalreceptor ,hasbeen proposed as a better pregnancy (104). modelin this regard (108, 109). Thechick embryo model Theexperimental study of oralinfections in the murine (146) alsohas been used to compare virulence of different modelhas been hampered by theapparent innate resistance isolates (13,141, 147) onthe basis of 50% lethal dose or ofmicefollowing oral or i.g.routes of infection, a factthat percentagemortality in infected embryos. The examination 9 10 requiresthe use of high inocula (commonly 10 to 10 ofa panelof strains with the chick embryo model and the bacteria)and that likely accounts for thecommonly ob- murinemodel (i.v. infection) suggested agreement between servedlack of reproducibility of the virulence estimates. the two (146, 147). Unlikehumans, mice (and rats) lacka functionalreceptor for InlA(E-cadherin), and this may compromise efforts to 1.2.3.Cell culture models. Tobypassthe practical and evaluateenterocyte invasion following oral and i.g. infec- ethicalproblems posed by animal models, as well as to tionsusing murine and rat models. The inability of murine betteraddress speciŽ c componentsof thepathogenesis pro- andrat E-cadherin to bind InlA was localizedin a speciŽc cess,several teams have developed cell culture models. aminoacid substitution. Guinea pigs, in contrast, were Thesemodels can determine the ability of L.monocytoge- foundto harbora human-typeE-cadherin (108). These data nes toattach to cells grown in culture, invade, and dissem- mayexplain previous results, which suggested that InlA inatefrom oneinfected cell to another .Usingthe human was notessential for virulencein the murine model. Re- intestinalepithelial cell Caco-2, Pine et al. (157) showed cently,however ,itwas shownthat transgenic mice that har- that L.monocytogenes was the only Listeria spp.virulent boredthe human form ofE-cadherin in their enterocytes inthis assay and demonstrated strain-speciŽ c differences. were muchmore susceptible to oralinfections by wild-type Althoughpronounced differences were reproduciblyfound L.monocytogenes (serotype1/ 2a)than by a mutantthat amongstrains, no obvious correlations with serotype or lackedthe gene for InlA,suggesting that InlA may be a source(clinical versus food or sporadic versus epidemic) keyvirulence factor for animalsexpressing a functionalE- were identiŽed. V anLangendonck et al. (200) also used cadherinreceptor ,includinghumans (109). Althoughtrans- Caco-2cells to differentiate among strains that had differ- genicmice would represent a cumbersomeanimal model encesin virulence in the immunodeŽ cient mouse model. 1814 KATHARIOU J.FoodProt., Vol. 65, No. 11

Cellplaque-forming assays have been used to screen food growth of L.monocytogenes iniron-depleted media also andclinical isolates of L.monocytogenes(144). Inaddition, requiresa generalstress response protein (ClpC), which, if cellculture assays determining the cytotoxicity of L. mon- mutated,renders the organism avirulent in the murine mod- ocytogenes havebeen described (20). el (170, 171). Inaddition, these mutants are sensitive to heat,salt, and oxidative stresses in syntheticmedia (but not 1.3.Environmental stresses (iron deprivation, acid- incomplex media such as brain heart infusion) (171). An- ity,and osmolarity) and virulence. Infoods and the en- otherstress protein (ClpE), required for survivalduring vironment,as well as in vivo during infection, L. mono- prolongedexposure to high temperature, may also contrib- cytogenes isexposed to numerous stress signals, which may uteto virulence in the murine model (137). Inearlier stud- stronglyin uence its pathogenicity. Stresses because of re- ies,heat shock and other stresses appeared to induce viru- frigeration,dehydration, freezing and freeze-thawing, heat, lence-relatedproteins (183). acid,and salt, as wellas exposureto disinfectants and other

antimicrobialsubstances, are of special relevance to the 1.3.3.Acid toleranceand acid stress. Acidtolerance Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/11/1811/1672430/0362-028x-65_11_1811.pdf by guest on 26 September 2021 physiologicalstatus and virulence of thispathogen in foods. of L.monocytogenes isof interest, since this pathogen is Workin thisarea has involved model systems with bacteria exposedto low pH at several stages during infection, in- grownin liquid batch cultures (planktonic cells) and may cludingthe acidic environment of the stomach and the pha- notadequately re ect the state of the microbe in the food gosomalenvironment. Following phagocytosis of L. mono- processingfacilities, the foods themselves, and the tissues cytogenes bymacrophages, the phagosome is rapidlyacid- ofthe host, where the bacteria are attached to surfaces, iŽed, with this acidiŽ cation being a prerequisitefor thees- oftenas components of bioŽ lms that may involve other capeof thebacteria into the cytoplasm, where they replicate microorganisms.Recent reports have highlighted the food (50). Interestingly, L.monocytogenes isnoticeablyless tol- safetyrelevance of bioŽ lm formation in the contamination erantto low pH than Escherichiacoli O157:H7 or S.  ex- ofprocessing plants by the pathogen (191). neri (46). Thisis in agreement with epidemiological evi- dencethat links antacids and reduced stomach acidity with 1.3.1.Cold and plant-derived molecules repress vir- susceptibilityto listeric infection (75, 86) andwith similar ulencegene expression. Underlaboratory conditions, the resultsobtained from anoral infection model (175). None- productionof listeriolysin O andseveral other virulence theless, L.monocytogenes doesexhibit a degreeof acid factorsis repressed at temperatures below ca. 25 8C (99, tolerance,which is dependent on themedium and microbial 113), suggestingthat virulence genes may be repressed growthphase (46, 48), withacid-adapted bacteria having whenthe bacteria are growing in cold-stored foods or in enhancedsurvival in acidiŽed dairy products and other low coldenvironmental niches in nature. Interestingly, virulence pH foods (70). geneexpression is also repressed by plant-derived mole- Usinga biochemicalapproach that employed two-di- culessuch as cellobiose and the phenolic compound arbu- mensionalgels and mass spectrometry,Phan-Thanh and tin,both of which may be encountered by the organism in Mahouin (155) showedthat acid stress (pH 3.5)and acid soil,in nonpathogenic situations (28,152, 153). Thus, L. adaptation(pH 5.5)resulted in the induction of the expres- monocytogenes appearsto beableto detect signals speciŽ c sionof 47 and 37 proteins, respectively, including 23 pro- toatleast two fundamentally distinct habitats (warm-blood- teinsin common. Although the speciŽ c functionsof these edanimals versus soil and vegetation) and to express or proteinsand their possible impact on virulence remain to repressspeciŽ c setsof genes accordingly. bedetermined, several Ž ndingssuggest that the acid resis- Therepression of virulence gene expression at 4 8C tance of L.monocytogenes isimportant in pathogenesis.An doesnot affect proper expression of the genes following acid-sensitivemutant of L.monocytogenes was foundto be infection(in response to the body temperature of the host reducedin virulence in the mouse model, whereas an acid- andother signals in vivo). Interestingly, strains may differ tolerantmutant had increased virulence (41,122, 148). The intheir ability to resume growth at 37 8Cfollowingcold latterobservation is of interest, as it suggests that certain storage,with clinical isolates resuming growth more readily acidicconditions may select for variantsof the microbe thanstrains derived from raw meat (13), suggestingthat the withenhanced virulence. physiologyof thetransition from lowtemperature to human Interestingly,no effects on virulence were seenwhen bodytemperature may be relevantto the infectious process. a protein (sB)requiredfor thetranscription of severalgen- eralstress genes of L.monocytogenes was mutated,even 1.3.2.Response systemsto iron depletion. Recent thoughthe mutants had impaired acid resistance (208). Cer- biochemicaland molecular studies have produced novel in- taincomponents of the acid and general stress resistance formationconcerning the possible impact of environmental systemmay be redundant so that their inactivation does not stressessuch as iron deprivation and acid stress on viru- impairvirulence noticeably, at least in the model systems lence. L.monocytogenes doesnot produce siderophores to thatwere employed. sequesteriron from itsgrowth medium, but it may be able touse exogenous siderophores produced by other micro- 1.3.4.The impact of cold and salt tolerance on vir- organismsin itsvicinity (181a). Interestingly,esculetin, the ulence. Theextent to whichcold and salt stress signals may hydrolysisproduct of the plant glycoside esculin, neutral- affectvirulence gene expression has not been determined. izesthe effect of iron-chelating agents and increases the Improved,simpliŽ ed assays to detect cold-osmotic adap- virulenceof the bacteria in the murine model (42). The tation (25) andvirulence gene expression (69, 110) will be J.FoodProt., Vol. 65, No. 11 L.MONOCYTOGENES VIRULENCE AND PATHOGENICITY 1815 usefulin this regard. Although genes involved in salt and resultsshowed that the genetic structure of the species is coldtolerance have been identiŽ ed (15,16, 101, 182, 211), clonal,i.e., consists of genetically distinct lineages (156). theimpact of thesegenetic determinants on virulenceis not Theobserved clonal structure in L.monocytogenes suggests clear.Arecentstudy showed that mutants deŽ cient in oli- thathorizontal gene  owbetween the different serotype- gopeptidetransport were notonly cold sensitive but were associatedclonal lineages is limited. alsounable to survive intracellularly in macrophages,even Itis intriguingthat the H (agellin) antigens of L. mon- thoughoverall virulence was notaffected (26), suggesting ocytogenes usedin the serotyping scheme devised by See- thatinterference with metabolic processes essential for low- ligerand Hoehne (181) correlateso precisely with the ge- temperaturegrowth may have pleiotropic effects on certain neticpartitioning of the species. The genetic clusters iden- aspectsof cellular pathogenesis. tiŽed by these early MEE investigationshave been con- 2.0.EPIDEMIOLOGICAL ASPECTS Žrmedby numerous alternative typing schemes, including ribotyping (77, 209), pulsed-Želd gel electrophoresis (31, 2.1.Serotype-associated species partitioning in L. 135), and,more recently, computerized analyses of random Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/11/1811/1672430/0362-028x-65_11_1811.pdf by guest on 26 September 2021 monocytogenes :2.1.1.Incidence of different serotypesin ampliŽed polymorphic DNA (123) andampliŽ ed fragment human illness. Although12 serotypes can cause disease, lengthpolymorphisms (1, 5). Inaddition, sequencing or atleast 95% of L.monocytogenes strainsisolated from hu- restrictionfragment length polymorphism analysis of nu- manlisteriosis cases (both outbreak and sporadic) are of merous L.monocytogenes genes(including genes encoding threeserotypes: 1/ 2a,1/ 2b,and 4b (168a,179, 189, 200a). listeriolysinO andother virulence genes,  agellin,and p60, Theprevalence of these serotypes in illness has been doc- aswell as a genomicregion essential for low-temperature umentedin numerous surveys from differentcountries (9, growth)could differentiate strains of serotypes 1/ 2a,1/ 2c, 47,106, 124, 168a). Itis not clear whether the observed 3a,and 3c from thoseof serotypes 1/ 2b,3b, and 4b (167, serotypedistribution re ects potential differences in human 202,203, 212). Althoughclonal lineages are common in virulenceand other ,as-yetunidentiŽ ed, attributes of theor- bacterialpathogens (e.g., (199)), theclonal partitioning of ganism’s ecologyand physiology (including survival and thespecies along serotypic groups is not routinely seen in growthin foods) that make strains of theseserotypes more otherpathogens that possess multiple serotypes (2). These likelyto colonize food processing environments and con- Žndingsmay have substantial implications for foodsafety, taminatecold-stored RTE foods at infectious levels. In one asthey suggest that strains of certain serotype-associated study,correlations were identiŽed between certain sero- lineagesmay pose much higher risks to human health than typesand clinical presentation (e.g., prevalence of serotype 4bin pregnancy-associated cases) (124), suggestingvaria- others. tionin human virulence among serotypes. 2.1.4.Genetic structure of L.monocytogenes : addi- 2.1.2.The use ofmolecular subtyping for surveil- tionalinsights. Thetyping results have provided some ad- lanceof human listeriosis. Pulsed-Želd gel electrophoresis ditionalinteresting information about the genetic structure protocolsand data sharing via the Internet have been uti- of L.monocytogenes, particularlyas noted in the four par- lizedto implement PulseNet, a nationalnetwork coordinat- agraphsthat follow. edby the Centers for DiseaseControl and Prevention and (i) Geneticdiversity differs among serotypes. Certain currentlyinvolving 46 state laboratories, 2 localpublic serotypesare much more diverse genetically than others, healthlaboratories, and Food and Drug Administration as withserotype 1/ 2abeing the most diverse. In the study by wellas U.S. Departmentof Agriculture food safety labo- Bibb et al. (22), serotype1/ 2aexhibited 30 distinct MEE ratories (187). Canadianlaboratories now participate in types(in contrast to 10 and 11 for serotypes4b and 1/ 2b, PulseNet,and it is likely that additional international lab- respectively).This diversity of serotype 1/ 2ahasbeen con- oratorieswill join the network. The epidemiological and Žrmedby other typing investigations (1,32, 93, 135). The publichealth impact of sucha standardizedmolecular sub- relativelylow genetic diversity of serotype4b alsohas been typingnetworkcan notbeo veremphasized.C ommon- noted (77) andmay suggest that this serotype emerged rel- sourceoutbreaks can be detectedwhile still in earlystages, ativelyrecently, as has been discussed in regard to certain andclusters involving diverse strains can also be readily otheranimal pathogens (198). Furthermore,all screened identiŽed. PulseNet is expected to continuously augment strainsof serotype 1/ 2cwere geneticallyindistinguishable ourknowledge on trends in the incidence of the pathogen, withthe typing tools that were used (1,36, 138), suggesting thecontribution of different strain types to human illness, thatthis clonal lineage may be very young, may be under andthe possible emergence of differentlineages implicated strongselective pressures, or both. This Ž ndingis intrigu- insporadic or epidemic listeriosis. ing,considering that serotype 1/ 2cis often prevalent in 2.1.3.Serotype-associated clonal structure ofthe foodsand food processing environments (see section3.2). species. Over 10years ago, multilocus enzyme electropho- (ii) Food-derivedstrains have pronounced genetic di- resis(MEE) showedthat serotypes 1/ 2a,1/ 2c,3a, and 3c versity. Food-derivedstrains were moregenetically diverse belongedto a majorgenetic division that was distinctfrom thanclinical strains (21), suggestingthat variable niches thedivision that includes serotypes 1/ 2b,3b, and 4b (21, andconditions select for diversegenotypes in foods and 22, 156). However,bothdivisions contain serotypes prev- foodprocessing environments and that only certain food- alentin human illness (1/ 2a,1/ 2b,and 3b). These typing derivedlineages may become implicated in human illness. 1816 KATHARIOU J.FoodProt., Vol. 65, No. 11

Thedevelopment of tools to differentiate between clinical nichespeciŽ city for serotype4b, which would allow it to andfood isolates is discussed in a separatesection (3.4). establish*tself and become prevalent only in certainmicro- (iii) Commonstrains infect both humans and animals. habitatsin food processing facilities. Such aspects of the Thesame genotypes are commonly found in human and ecologyof this serotype as well as other serotypes of L. animalisolates (156), withthe exception of a lineagethat monocytogenes arecurrently poorly understood and in need isassociated primarily with animal listeriosis but rarely ofstudy. It is also possible that serotype 4b may be more foundamong human clinical isolates (209). sensitiveto selective enrichment protocols than other se- (iv) Strainsof serotypes other than 1/ 2aand 1/ 2care rotypes,with its presence in foods and the food processing relativelyunderstudied. Mostgenetic and immunologic environmentsbeing underestimated. The fact that different studies of L.monocytogenes haveinvolved strains of se- strains of L.monocytogenes aredifferentially sensitive to rotype1/ 2a(strains 10403S, EGD, NCTC7973, and Mack) isolationprotocols has been documented (115, 172). Alter- or1/ 2c(strain LO28), with these strains representing only nativeisolation protocols and DNA-based methodswill be oneof the two major genomic divisions of the pathogen. neededto better monitor and detect serotype 4b bacteriain Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/11/1811/1672430/0362-028x-65_11_1811.pdf by guest on 26 September 2021 StrainEGD (serotype1/ 2a),isolated from anepidemic of foodand food processing environments. listeriosisin a rabbitlaboratory colony in 1924 (136) and 2.2.c.Environmental prevalence. Twoseparate stud- usedfor numerousimmunologic, bacteriologic, and genetic iesfrom Russiaand Italy, respectively, suggest that 4b is studiesthereafter ,was chosenfor the L.monocytogenes ge- theprevalent serotype in sewage (52, 120). Thisis signif- nomesequencing project (74). Itis imperative that strains icant,considering that L.monocytogenes hasbeen repeat- representingthe other major genomic division be actively edlyshown to persist in treated sewage and sewage sludge studied,both genetically and in terms of their physiology, (3,4, 72, 116, 118, 204). Inthe study by De Lucaet al. virulence,and ecology. This is dictated not only by the (52),L. monocytogenes (with4b as the prevalent serotype) documentedgenetic distance between the two genomic di- was primarilyrecovered from activatedsludge but not from visions,but also by the fact that strains of serotypes 1/ 2b anaerobicdigesters, suggesting that the pathogen was re- and4b are of substantial epidemiologic and clinical impor- sistantto biological oxidation. The reported incidence of L. tance.From thisperspective, the choice of a serotype4b monocytogenes serotype4b in sewage is surprising and epidemic-associatedstrain for thegenome sequencing by mayre ect human carriage of thisserotype, subclinical in- theU.S. Departmentof Agriculture-The Institute for Ge- fections,or resistanceof thebacteria to environmental con- nomicResearch (www.tigr .org)is especially opportune. ditionsin activatedsludge (such as oxidation,UV light,and 2.2.Serotype 4b L.monocytogenes —epidemiology phages).In fact, serotype 4b was foundto be predominant andecology: 2.2.a. Clinical prevalence. Severalsurveys amongstrains from asymptomatichuman carriers (118). No ofclinicallisteriosis, in differentcountries, indicate that the recentstudies of L.monocytogenes insewage in theUnited overallincidence of serotype 4b is high, ranging from 50 Stateshave been reported, and human carriage studies in to 70% (9,106, 120, 124). Strainsof serotype 4b account thiscountry are similarly lacking. Important epidemiolog- for asubstantialfraction of sporadicinfections and numer- icaland public health functions will be servedby such stud- ouscommon-source outbreaks of listeriosis (91, 97), and iesand by the typing of prevalent L.monocytogenes line- thepresence of bacteria of this serotype in an RTE food agesrecovered from sewage. appearsto increase the food’ s riskof implication in liste- 2.3.Epidemiology of serotypes 1/ 2aand 1/ 2b. Even riosis (159). Strainsof serotype 4b tend to be overrepre- thoughthese two serotypes account for asubstantialportion sentedin perinatal listeriosis (124, 140), suggestingthat ofsporadic listeriosis and occasional outbreaks, virtually theymay have special virulence attributes for pregnancy nothingis known about their potentially unique features of andbreach of the blood-placenta barrier . pathogenesis,transmission, and ecology. In a studyby 2.2.b.Prevalence in foods. Itmust be emphasized that McLauchlin (124), serotype1/ 2bwas primarilyassociated currentdata on the prevalence of serotype 4b (and other withnonpregnant individuals with severe underlying illness serotypes)in RTE foods in the United States are not com- andwas foundin 10% of all cases. In a surveyin Los monlyavailable. However ,earlierstudies surveying various AngelesCounty, California, the incidence of serotype1/ 2b foodssuggested that serotype 4b was notthe leading se- was 31%among listeriosis cases (excluding those associ- rotypeamong food isolates (79, 84). Thediscrepancy be- atedwith known foodborne outbreaks) but was noticeably tweenfood incidence and prevalence in illnessmay suggest higher(65%) among human immunodeŽ ciency virus– in- thatstrains of serotype4b aremore virulent to humansthan fectedpatients, suggesting a possibleassociation of infec- otherserotypes, although other possibilities, including tionby this serotype with special dietary factors or sexual transmissionby routes other than contaminated foods, can- practices (60). Alternatively,severe types of immunosup- notbe excluded. pression(e.g., following human immunodeŽ ciency virus in- Thereasons for theapparent scarcity of serotype 4b fection)may allow infection by serotype 1/ 2bstrains that amongmost food-derived strains are unclear .Itis possible maybe relatively noninfectious for individualsin otherrisk thatother serotypes are better adapted to food and food categories,such as pregnancy. processingenvironments. Indeed, strains of serotype 1/ 2c TheseŽ ndingssuggest that the relative incidence of were foundto adhere to stainless steel surfaces more efŽ - serotype1/ 2bis likely to be variable in different surveys, cientlythan those of serotype4b (145). Theremay also be dependingon theprevalence of individualswith severe im- J.FoodProt., Vol. 65, No. 11 L.MONOCYTOGENES VIRULENCE AND PATHOGENICITY 1817 munocompromisingconditions. As discussedearlier ,atthe geneor genesfor itsincorporation in the teichoic acid may genomiclevel, serotype 1/ 2bstrains appear to be closely alsobe unique. Currently, serotype 1/ 2aor 1/ 2bmutants alignedwith serotype 4b and quite distinct from serotype thatspeciŽ cally lack rhamnose are not available, and the 1/2a.Nonetheless, the two serotype groups (1/ 2band 4b) effectsof these substituents on virulence and interactions clearlyrepresent distinct clonal lineages that differ in terms withhost cells are not known. However ,rhamnosemay be oftheir surface antigenic composition (see section2.4 be- involvedin the attachment of the Ž rst componentof com- low)and, possibly, their virulence and ecological niche in plementC1q to the cell walls of the L.monocytogenes of foodsand the environment. serotype1/ 2 (7). Thestudy of gene cassettes essential for teichoicacid 2.4.Serotype-speciŽ c genesand surface antigens: glycosylationin serotype 4b (111, 164) ledto the identiŽ - 2.4.1.Serotype 4b. Serotypedesignations in Listeria fol- cationof genomically equivalent genes in other serotypes. lowan alphanumeric system based on agellinantigens (the Interestingly,the serotype 4b gtcA genehas a divergent

letterportion of thedesignation) and somatic antigenic de- Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/11/1811/1672430/0362-028x-65_11_1811.pdf by guest on 26 September 2021 counterpartin serotype 1/ 2aand all other screened sero- terminants(the numeric portion of the designation). The typesand, in addition, is preceded by a gene( mtrA) that lattercorrespond primarily to sugar substituents of the an- lacksany hom*ologous counterpart in serotype 4b and that ioniccell wall polymer ,teichoicacid, which is covalently may,on the basis of DNA anddeduced protein sequence boundto the peptidoglycan. In serotype 4b, teichoic acid analysis,encode a glycosylationenzyme (105). hasa uniquecomposition, with both glucose and galactose Arecentstudy utilized signature-tagged mutagenesis to substituentsattached to N-acetylglucosaminein the teichoic identifymutants of strain EGD (serotype1/ 2a)that were acid chains (66,96, 195). Recently,two genomic regions unableto multiply in the brains of mice. Interestingly, a thatare required for theincorporation of galactose,glucose, largenumber of mutationswere localizedin thegene gtcA, orboth in the teichoic acid of serotype 4b bacteria were whichvaries noticeably in sequence between serotypes 4b identiŽed. Mutations in theseregions abolish reactivity with and 1/2a (12, 105). Itis currently not known whether gtcA apanelof monoclonal antibodies speciŽ c for serotype4b hassimilar virulence functions in serotype4b. The possible (andthe antigenically similar serotypes 4d and4e) (98, 112, virulencerole of the newly identiŽ ed mtrA gene,which is 164). Inboth cases, a serotype-speciŽc genecassette that absentfrom serotype4b but present in all other major se- is ankedby genes conserved among different serotypes rotypes,remains to be determined. was identiŽed (111, 164). Interestingly,in strains of other serotypes,the serotype-speciŽ c spaceis occupied by unre- 2.5.Epidemic-associated L.monocytogenes. Even latedsequences, which are also unique to serotypes other thoughmost incidences of human listeriosis are sporadic, thanserotype 4 (105, 111). Theavailability of serotype- itisfoodborne outbreaks that have earned special notoriety speciŽc sequenceswill allow the implementation of DNA- for L.monocytogenes. Implicatedfoods have included milk basedassays (e.g., by polymerase chain reaction) to more anddairy products, cold-smoked salmon and other Ž shand accuratelysurvey foods and food processing facilities for seafoodproducts, vegetables, coleslaw, and RTE meat thepresence of L.monocytogenes serotype4b. The unique products.In certain cases, the foods have been epidemio- teichoicacid composition of serotype 4b may have impor- logicallyassociated but not conŽ rmed bacteriologically, tantecologic and pathogenesis functions. The presence of whereasin many of the well-publicized outbreaks, the im- galactoseappears essential for theattachment of serotype plicationof aspeciŽc contaminatedfood was basedon both 4b–speciŽ c phagesand for theinvasion of mammalian cells epidemiologicanalysis and bacteriologic conŽ rmation (59, in culture (163). Thesugar substituents on teichoic acid are 62,90, 132, 134). immunodominantdeterminants (98, 196) andmay thus be Bacteriologiccharacterization of the strains from out- importantin pathogen-host interactions and in the genera- breakshas failed to identify discrete determinants common tionof subsequent protective immunity. Interestingly, al- toall. In Europe and North America, most publicized out- thoughthese surface antigens are very stable in laboratory breaksin the past 20 years have involved serotype 4b. culturesand in sporadic clinical strains, population-level Strainsof other serotypes, however ,arenot exempt from surveysof different outbreaks identiŽ ed several strains that thepotential to cause outbreaks, as willbe discussedbelow. lackedgalactose in their teichoic acid (38). Itis possible Severalefforts usingcell culture and animal models have thatsuch strains have been selected in the course of infec- failedto identify differences in virulence between epidem- tionas an evasion strategy toward the host immune system ic-associatedstrains and most other strains of the same se- (see alsosection 2.7). rotype (33,157, 158). 2.4.2.Serotypes other than 4b. Bothof theother pre- 2.5.1.Epidemic clone I. Inspite of theirapparent bac- dominantclinical serotypes, 1/ 2aand 1/ 2b,have teichoic teriologicconformity, many epidemic-associated serotype acidthat contains N-acetylglucosamineand rhamnose sub- 4bstrains appear to be genetically distinct from other stituents.Thus, this teichoic acid composition (common in strainsof the same serotype. Results from severalsubtyping allserogroup 1/ 2strains)is distinct from thatof serotype schemessuggest that the strains implicated in several geo- 4b.Rhamnose and N-acetylglucosaminesubstituents on the graphicallyand temporally distinct outbreaks are closely teichoicacid of serotype 1/ 2 L.monocytogenes areessential related (32,33, 90, 156), eventhough each outbreak pop- for phageadsorption (192, 205). Sincerhamnose is a tei- ulationis genetically distinct (32, 206). Thisgroup of choicacid component characteristic of serotype 1/ 2,the strainsincludes those implicated in outbreaksin NovaSco- 1818 KATHARIOU J.FoodProt., Vol. 65, No. 11 tia(19 81),Massachusetts(1 983),Ca lifornia(Jalisco epidemicclone in different outbreaks suggests that the im- cheese,1985), Switzerland (1983 to 1987),Denmark (1985 plicatedstrains have a reservoirduring the often lengthy to1987), and France (1992). intervalsbetween outbreaks. The widespread incidence and Severalof these outbreak strains (Nova Scotia, Cali- epidemiologyof ECI suggeststhat this clonal group may fornia,and Switzerland, but not Massachusetts) share a bea ubiquitousenvironmental lineage, which can become uniquerestriction fragment length polymorphism in a ge- ampliŽed inanimals and humans. No information currently nomicregion essential for low-temperature(4 8C) growth of existsas to thespeciŽ c environmentalniche or niches(e.g., L.monocytogenes(212). Inaddition, these same strains also soil,silage, sewage, and others) for thisgroup. ECI has appearto methylatecytosines at GA TCsitesin their DNA, beenfrequently isolated from foodanimals (24, 142), sug- whichrenders the DNA resistantto digestion by therestric- gestingthat farm animalsand the animal-associated envi- tion enzyme Sau3AI (213). Recently,several DNA se- ronment,including animal feed, may serve as both tem- quencesunique to these strains were identiŽed, although poraryreservoirs and a meansfor ampliŽcation. Human thefunctional roles of the genes were notdetermined (85). carriers,or subclinically infected individuals, may also Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/11/1811/1672430/0362-028x-65_11_1811.pdf by guest on 26 September 2021 Theseand other Ž ndingssuggest that these strains belong serveas reservoirs for thisclone. Routine surveys of food toadistinctlineage, designated epidemic clone I (ECI). The haveonly sporadically yielded ECI strains,suggesting that reportedgenetic similarity between the strains in ECI and thislineage, which is clearly virulent to humans and farm thestrain implicated in the French pork-tongue-in-aspic animals,does not commonly contaminate the food process- outbreakof 1992 (90) suggeststhat this strain also belongs ingenvironment. The sources and possible reservoirs of toECI. Thus, ECI appearsto be a cosmopolitanepidemic epidemicclones II andIII, whichcaused multistate out- clonallineage. In this context, the choice of an ECI strain breaksin the United States in 1998 to 1999 and 2000, re- (Jaliscooutbreak) for theserotype 4b genomesequence de- spectively,remain unidentiŽ ed. As mentionedearlier ,epi- terminationby the U.S. Departmentof Agriculture-Agri- demicclone III strainsappeared to have persisted in the culturalResearch Service seems especially appropriate. processingplant for morethan a decade. Currentdata on possible reservoirs and ampliŽ cation 2.5.2.Epidemic clone II. From 1998to 1999, a new hostsfor thispathogen need to be viewedin thecontext of genotype of L.monocytogenes serotype4b was implicated theorganism’ s potentialto contaminate food and the food ina multistateoutbreak of listeriosis in the United States processingenvironment. However ,substantialgaps remain thatinvolved contaminated hot dogs. Strains from thisout- inour knowledge of the transmission of foodborne listeri- breakhad unique ribotype and pulsed-Ž eld gel electropho- osis.The earlier conclusion of the W orldHealth Organi- resispatterns not commonly encountered in previous sur- zationworking committee on foodborne listeriosis that L. veys (132, 133). Thus,the strains from thisoutbreak appear monocytogenes shouldbe viewedas an environmentalcon- torepresenta novelepidemic-associated lineage, designated taminant (8) maystill be largely true. This view, however , epidemicclone II. mayneed to be updated to take into account other sources Theinvolvement of an ‘ ‘unusual’’ serotype4 strainin thatalso may be important, including the handling and anoutbreak has been described at leastonce previously. In cross-contaminationof food in food service settings such 1987,23 cases of listeriosis in the United Kingdom were asrestaurants, delicatessens, and salad bars and by thecon- attributedto a strainof an unusual serotypic designation, sumerin the home. More survey work is neededto clarify 4b(X) (125). Althoughan implicated food was notidenti- thecontribution of thesesegments of the farm-to-fork con- Žed,the bacteriologic and genotypic characterization of the tinuumto the burden of foodborne listeriosis. strainssuggested that they represented a uniqueepidemic clonerarely seen before or afterward. 2.7.In situ evolutionin common-source outbreaks 2.5.3.Epidemic clone III. Themost recent multistate oflisteriosis. Outbreaksare population-level events. Even outbreakof listeriosis in theUnited States involved contam- whenone common source is involved, outbreaks involve inatedturkey deli meat products and resulted in several cas- multipleinocula, multiple infection events, and multiple esand a massiveproduct recall (134). Unlikemost other hosts,each with unique immune system idiosyncrasies. It outbreaks,the implicated strain was serotype1/ 2a.An es- wouldtherefore be expected that the pathogen-host inter- peciallyinteresting Ž ndingwas thatthe outbreak strain was actionmight have different dynamics in each case within thesame genotype as a strainthat was implicatedin a hu- anoutbreak and that the pathogen might have the oppor- manlisteriosis case associated with the consumption of tunityto adapt independently in each host. Such an adap- contaminatedturkey franks in 1988 (131). Theproducts tationwill yield genetic variants, which may or maynot be implicatedin the 2000 multistate outbreak were from the easilydetectable genotypically and phenotypically. The samefood processing facility as the earlier isolate, sug- identiŽcation of such variants by necessity requires the gestingthat this strain had persisted there over several years studyof multiple strains from aspeciŽc outbreakpopula- withoutdetectable genotypic changes (188). Geneticfea- tion. turesunique to and characteristic of epidemicclone III re- Variationwithin the outbreak population has indeed mainto be identiŽ ed. beenobserved. Twenty-seven percent of thepatient-derived strainsfrom theNova Scotia outbreak lacked galactose sub- 2.6.Epidemic strains: ampliŽ cation and reservoirs. stituentsin the teichoic acid of the cell wall. Such strains Theperiodic (and often repetitive) involvement of thesame were negativewith serotype 4b– speciŽ c monoclonalanti- J.FoodProt., Vol. 65, No. 11 L.MONOCYTOGENES VIRULENCE AND PATHOGENICITY 1819 bodiesand resistant to serotype 4b– speciŽ c phages.Similar whichwould otherwise be unrecognized, will prove to be antibody-negative,phage-resistantstrains were identiŽed in causedby these serotypes. boththe California and Massachusetts outbreaks (38). Al- thoughwe cannotexclude the possibility that these phe- 2.9.Role of dairy and meat animals in the trans- notypesbecame established during passage and storage of missionof listeriosis. Inits early era, listeriosis was often thebacteria in the laboratory, an alternative possibility is describedas a zoonosis—an illness transmitted from ani- thatthe observed variants were selectedduring the infection malsto humans (180)—butzoonotic transmission of inva- oftheir human host. T eichoicacid substituents are strong siveillness has been seldom documented, and this view is immunogens (196), andglycosylation-neg ativevariants notwidely held currently. Some animal listeriosis cases are maybe atanadvantage in terms of immunesystem evasion. caused by Listeriaivanovii, wellknown as ananimalpath- Thecharacterization of multiple isolates from addition- ogenbut extremely rare inhuman infections, and by sero- aloutbreaks may show similar evolutionary, population- type4a and 4c strains of L.monocytogenes, which are levelevents. In fact, such events may account for certain foundmore frequently associated with animal than human Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/11/1811/1672430/0362-028x-65_11_1811.pdf by guest on 26 September 2021 atypicalresults reported in the literature. For instance,char- listeriosisand which constitute a uniquelineage (209). acterizationof the strains implicated in the Swiss outbreak However,moststrains from animallisteriosis are not read- (1983to 1987) showed one common MEE typebut two ilydistinguishable genetically from thoseimplicated in hu- (156, 209). closelyrelated genotypes and phage types. In addition, man illness Hence,the question arises as to identicalgenotypes were seenin strains that were either whetheranimals may transmit such strains to humans. Thetransmission from farm animalsto humans may nontypeableby phage typing or of different phage types occurunder certain circ*mstances. In a 23-year(1972 to (139). Somedifferences in phage types may correspond to 1994)survey in Denmark, Jensen et al. (92) found that strainsthat are deŽ cient in the expression of teichoic acid herdsharbored L.monocytogenes ata lowbut constant lev- substituents,which function as receptors for certainphages el(0.2 to 4.2% of herds). During the same period, 79% of in L.monocytogenes(205). Noceraet al. (139) noted that, theisolates from bovinemastitis and 48% of the human ifthe phage typing data were toexclude such strains from clinicalisolates had overlapping ribotypes, suggesting that theoutbreak designation (as indeedwas thecase), the ex- milkand other dairy products from mastiticcows accounted tentof the outbreak could be underestimated. for atleast some human cases (92). 2.8.Involvement of other L.monocytogenes sero- Thepotential role of meat animals in transmission is types(1/ 2a,1/ 2b,and 3a) in foodborne listeriosis out- lessclear and probably less signiŽ cant. Food can become breaks. Eventhough serotype 4b has been implicated in contaminatedby manurefrom animalsthat may have active mostof thewell-publicized outbreaks, several outbreaks in- infectionsor may be asymptomatic carriers. This was pre- volvingother serotypes of L.monocytogenes strainshave sumablythe case in the Nova Scotia outbreak, in which beenreported. In fact, the Ž rst recordedoutbreak of liste- manure-contaminatedcoleslaw was implicated (176). riosis,which took place in Halle (formerly East Germany) Itis likely that common environmental sources serve in1945, involved serotype 1/ 2a (151). Morerecent out- asreservoirs of strains that infect both meat animals and breaksin W esternAustralia (1978 to 1980 and 1990 to humansby potentially independent routes. The infection of 1991)and Auckland, New Zealand(1992), have involved animalsthrough contaminated feed and poor-quality silage serotypes1/ 2aand 1/ 2b (29, 193). Arecentoutbreak in iswell documented (54,63, 207). Thesame strains can Finlandwas attributedto the contamination of butter by a causehuman illness following their introduction and estab- strainof serotype 3a (116a) and,as mentioned earlier ,tur- lishmentin food processing facilities through environmen- keydeli meat products contaminated by bacteriaof serotype talcontamination from stillunknown sources or via other 1/2awere implicatedin a recentmultistate outbreak in the vehicles,including food handlers. Systematic data on the UnitedStates (134). Furthermore,in thelast decade, strains contaminationof food handlers are currently lacking. The ofserotypes1/ 2aand1/ 2bhavebeen involved in epidemics factthat certain well-studied virulent clones (e.g., ECI) are offebrile gastroenteritis in the United States and Europe generallyrare inprocessing environments and foods sug- (45,129, 162, 174). Anoutbreak of febrile gastroenteritis geststhat their entry may be a relativelyrare eventor that hasalso been caused by serotype 4b (10). theirestablishment in food processing facilities requires Mostlisteriosis cases are sporadic and frequently in- specializedconditions or environmental niches. volveserotypes 1/ 2aand 1/ 2b (179). At leastsome of the 3.0. L.MONOCYTOGENES STRAINS IN apparentlysporadic cases, however ,mayin realityrepresent FOODS AND INTHEFOOD clustersof unrecognizedoutbreaks, especially if they occur PROCESSING ENVIRONMENT overa longperiod. In fact, high-resolution strain typing has conŽrmed this hypothesis in Austria (5), withthis situation 3.1.Origin of foodcontamination. Aclearpattern has likelyto occur elsewhere as well. Similar recognition of beenemerging from numerousstudies in differentfood pro- ‘‘hidden’’ outbreakswill become more frequent in the Unit- cessingplants, primarily in Europe, during the past 10 edStates as typing data (pulsed-Ž eld gel electrophoresis) years.The primary source of food product contamination from ‘‘sporadic’’ casescontinue to enter the PulseNet da- beforerelease to consumers appears to be the processing tabase.Because many sporadic cases are serotypes 1/ 2aand environment.The incidence of L.monocytogenes in milk 1/2b,it is likely that at least several of these outbreaks, inprocessingcenters (33.3%) was substantiallyhigher than 1820 KATHARIOU J.FoodProt., Vol. 65, No. 11 insamplesfrom dairyfarms (5.3%) (81). Anextensive sur- ingplant select for theresident clones while inhibiting other veyof levels of meat contamination indicated that chilling strains. andcutting signiŽ cantly increased the contamination of Infoods and food processing facilities, L.monocyto- pork,in agreement with the high environmental prevalence genes oftencoexists with other Listeriae, especially Listeria ofthe pathogen (71 to 100%) in the chilling-cutting area innocua, withthe latter frequently outgrowing L. monocy- oftheprocessing plant (197). Verysimilar results concern- togenes incommonly used selective media (43, 154). Cur- ingthe impact of chillingrooms were reportedin anearlier rently,however ,we lackan understanding of the possible studyof mutton contamination in New Zealand (160). Al- interactionsbetween L.monocytogenes and other Listeriae thoughwhole-carcass contamination levels of sheep,cattle, insitu (e.g., in the processing environment and foods). andswine were remarkablylow, minced beef was exten- 3.3.Contribution of persistent strainsin the pro- sivelycontaminated (21 of 23samples) (65). Beforeslaugh- cessingenvironment to foodborne listeriosis. In general, ter, L.monocytogenes was notcommonly detected in poul-

straintypes derived from foodprocessing environments Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/11/1811/1672430/0362-028x-65_11_1811.pdf by guest on 26 September 2021 tryfecal samples, but poultry became heavily contaminated onlypartially overlap with those implicated in human ill- afterslaughter .Insurveys of poultry slaughterhouses, live ness.This is strongly suggested by the observed serotype birdscontributed little to listerial contamination (42a, 148a, distributionpatterns. Many resident clones are serotype 1/ 149). Similarresults have been obtained from extensive 2c,which only rarely causes human illness. Other prevalent workin smoked Ž shplants, where the genotypes in the serogroup1/ 2serotypes,however ,arenot rare amongclin- plant,or on theŽ nishedproducts, were oftendifferent from icalisolates, and additionally, serotype 4b can persist in thoseon the raw Žsh (11,143, 169). foodprocessing facilities, albeit less frequently (see below). 3.2.Types of strains found inthe processingenvi- Qualitativeand quantitative analysis of straindiversity ronment. Certainserotypes tend to be prevalent in pro- andpersistence has been most rigorously and extensively cessingenvironments and in foods. In an extensive study donein seafood and seafood processing plants, as RTE ofstrains contaminating pork slaughtering and cutting cold-smokedsalmon and other Ž shproducts have a rela- plants,Giovannacci et al. (73) foundthat the prevalent se- tivelyhigh incidence of thepathogen (18). Multiplestrains rotypeswere 1/2a,1/ 2c,3a, and 3c. Serogroup 1/ 2was appearto coexist in Ž shproducts (23, 71), andstrain clus- prevalentin several other surveys of processing environ- tersassociated with speciŽ c areasand operating procedures mentsfor poultryand processed meats, as well as in Ž nal inthe processing plant have been identiŽ ed. For instance, products (107,149, 194). onesurvey of strain types in a shrimpprocessing plant showeda discretestrain cluster associated with water and 3.2.1.Resident strainsin food processing facilities. utensils (53). Inanother study, strains associated with brin- Strainsubtyping has shown the presence of both transient ingand salting were identiŽed in a cold-smokedrainbow (sporadic)and resident (persistent) strains in theprocessing troutplant and in a plantproducing vacuum-packed, environment.Persistent strains have been found in products smoked,and cold-salted Ž shproducts (11, 94). thatoriginated from differentproducers and were processed Onseveral occasions, clones that were persistentand ina commonfacility (82, 83), suggestingthat their source prevalentin processing facilities were alsoassociated with was thefood processing environment itself or a common humanlisteriosis (29,58, 83, 148a). Areportof special ingredient.Such strains can become established in a spe- interestconcerned the only characterized outbreak of in- ciŽc facility,and the literature contains several reports of vasivelisteriosis that involved contaminated Ž sh(trout) certainstrains having been isolated repeatedly over several (58). Theimplicated strain, which was bothpersistent and years.In fact, the strain implicated in the 2000 multistate prevalentin the plant, was serotype4b. In another study, outbreakof listeriosis in the United States involving con- 1/2a,1/ 2b,and 4b strains were repeatedlydetected as con- taminateddeli turkey meats (134) appearsto havepersisted taminantsof softcheeses, and subtyping of theserotype 4b inthe processing facility for morethan 10 years (section strainsshowed that they were similarto strains implicated 2.5.3). inhuman illness, including an earlier outbreak (121). Eventhough multiple strain types can be isolated from Inconclusion, these and other data suggest complex aprocessingplant or from theŽ nalproduct, one or a few listerialcontamination patterns in the processing plants. strains(clones) frequently become established in the facil- Certainresident clones in the processing plant (e.g., those ity,with these strains often found in the Ž nishedproduct ofserotype 1/ 2c)are not commonly implicated in human as well (107,115, 148a, 149, 169). Theliterature contains listeriosis,whereas others clearly have the potential to cause numerousadditional reports on resident clones in seafood, humanillness, depending on theirserotype, strain type, and dairy,poultry, and pork processing facilities, suggesting levelof contamination of the Ž nishedproduct. thatthese strains are common in the food industry. It is possiblethat such strains get established in the processing 3.4.Comparative virulence of food-derived versus facilitiesbecause they compete effectively against other clinicalstrains of L.monocytogenes :3.4.1.Evidence bacteria,or against other L.monocytogenes strains, by fromepidemiology and subtyping. Theepidemiologic and meansof currently unknown mechanisms (including, but strainsubtyping data have prompted speculation that human notlimited to, phage resistance, bacteriocin production, bio- virulencemay be attenuated in many, if not most, strains cideresistance, competitive adherence to surfaces, and biof- contaminatingfood (88, 144). ilmformation). Possibly, certain conditions in the process- Thereis little doubt that clinical serotypes (mostly 1/ J.FoodProt., Vol. 65, No. 11 L.MONOCYTOGENES VIRULENCE AND PATHOGENICITY 1821

2a,1/ 2b,and 4b) do not accurately re ect the serotypes offood-derivedstrains (but none of the 12 screenedclinical prevalentin foods and food processing facilities (1/ 2a,1/ strains)were negativewith a panelof lectins, which nor- 2c,3a, and 3c). However ,substantialevidence indicates that mallybind to sugars on the cell surface of the pathogen. atleast a fractionof thedifferent strains in dairy,meat, and Interestingly,the lectin-negative strains were avirulentin a poultryproducts genotypically overlap with strains causing cellculture model for virulence.In a comparisonof clinical humanillness (24,64, 92, 121, 172, 193). Inthe study by andmeat-derived strains, Avery and Buncic (13) found that Ryser et al. (172), therelative recovery of ‘‘clinical’’ (i.e., freshlygrown isolates were indistinguishableintheir viru- subtypesrelated to thoseassociated with human illness) and lencein the chick embryo model; however ,signiŽcant dif- ‘‘nonclinical’’ subtypesfrom meatand poultry products ferencescould be observed after the inoculum had been was dependenton the enrichment protocol used, suggesting maintainedin buffer at 4 8C.Clinicalstrains remained vir- thatthese distinct strain types are physiologically different ulent,whereas the virulence of severalmeat-derived strains aswell. The extent to which food-derived strains, as a was noticeablyreduced. In addition, meat-derived strains group,may be genetically distinct from clinicalstrains hada longerlag phase when shifted from 4to37 8C (34). Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/11/1811/1672430/0362-028x-65_11_1811.pdf by guest on 26 September 2021 needsto be rigorously deŽ ned. A recentpolymerase chain Eventhough a relativelysmall number of strains from reaction–based typing investigation using the distribution of onesource (meat) were included,these latter results are of repeatedsequences in the genome suggested a distinctge- interest,considering that foodborne listeriosis commonly nomicgroup for food-derivedversus clinical strains of L. involvesrefrigerated foods. Although the physiology of the monocytogenes(93), butthe surveyed collection of food transitionof L.monocytogenes from thecold, starved state strainsconsisted of a rathersmall number of independent togrowthat 37 8Cinrichmedia is far from clear,interstrain isolates. differenceslikely exist. Furthermore, such studies suggest thepossible impact of environmental modulation on viru- 3.4.2.Laboratory determinations. Theevaluation of lence.It is possible that the cold environment associated thepotential food safety threat posed by many strains con- withfoods and food processing facilities, along with other taminatingfood is complicated by the absence of a labo- existingstresses, selects for specialsubpopulations of ratorystandard for humanvirulence and the fact that vir- strainswith adaptive physiology attributes that promote ulenceestimates can be obtainedonly from animaland cell bothsurvival in the food and human virulence. These sub- culturemodels. Such virulence models have not identiŽ ed populations,representing only a minorityof strainsin food, consistentdifferences between food and clinical isolates of wouldserve as inocula for humansand would eventually L.monocytogenes, althoughinterstrain variation within becomerepresented among clinical strains. eachof these groups was observed (157, 158). Using two Inconclusion, the available data indicate that some differentcell culture models that allowed quantitation of food-derivedstrains of L.monocytogenes mayhave surface invasivenessand cytotoxicity to mammalian cell cultures, antigenic,genetic, and physiologic characteristics that can DelCorral et al. (51) determinedthat no systematic differ- differentiatethem from themajority of human clinical encescould be found between 30 food and clinical strains. strainsbut do not provide conclusive evidence for differ- Ina largersurvey, Conner et al. (39) determinedthat most encesin humanvirulence between clinical and food-derived strainswere pathogenicin the immunocompromised mouse strains. modeland identiŽ ed only a smallnumber of weakly he- molyticstrain swithatt enuatedvi rulence.A rigorous 3.5.IdentiŽ cation of virulentfood strains of L. mon- screeningof a largecollection by Brosch et al. (30) re- ocytogenes:3.5.1.The role of new andimproved models. vealedthat only 2 of63foodstrains were avirulentin mice, If, indeed,certain food strains are more likely to causehu- leadingthe authors to conclude that all strains posed po- maninfection than others, the question arises as towhether tentialhealth hazards, regardless of their source, serotype, andhow such problem-prone strains can be identiŽed. Ex- orgenotype.Screening a smallernumber of strainswith the perimentalidentiŽ cation with high discriminatory potential mousebioassay as well as the chick embryo test, Noter- wouldbe extremely valuable but is fraught with problems mans et al. (147) concludedthat ‘ ‘almostall serovars pre- intrinsicto method design and the speciŽ c experimental sentin food have clear virulence properties.’ ’ Inanother modelthat is used. Animal and cell culture models may studyemploying the chick embryo model, strains of dif- neverbe completely satisfactory because not all compo- ferentMEE typesof both food and clinical origin were nentsof humanfoodborne infection can be realisticallysim- pathogenic,but death of the chick embryos occurred more ulatedin these models. The search for newand better an- rapidlyfollowing infection by clinical versus food-derived imalmodels continues. For example,work is currentlyun- strains (141). derway on newanimal models (including transgenics) that Althoughthese investigations all suggest that L. mon- aremore relevant to oral infections (e.g., (109)).Itremains ocytogenes isolatesfrom foodare basically virulent in the tobe seen if this is a cost-effectiveinvestment in terms of modelsthat were used,several other studies indicate that relevanceof the results to human foodborne disease. possibledifferences between food and clinical strains can Valuableinformation can be obtained from carefully beidentiŽ ed using other experimental designs. It is not designed,compartmentalized model systems chosen specif- clearwhether such differences are ‘ ‘indicators’’ ofvirulence icallyfor theirrelevance to human infection (e.g., the in- orrepresentative of actual virulence characteristics. Faci- fectionand activation of human vascular and brain endo- nelli et al. (61) foundthat a signiŽcant fraction (16 of 23) thelialcells, which provide indications as to the potential 1822 KATHARIOU J.FoodProt., Vol. 65, No. 11 ofthe bacterial strain to induce an in ammatory response whichis now commonly required before a newgene can andto pass through the blood-brain barrier). Such systems bedescribed in a publication.Participation must be clearly havebeen established primarily for immunologicaland ge- voluntaryfor thefood industry, with appropriate protec- neticstudies (57,80, 102, 210). Thesesystems have not tionsinstituted for conŽdentiality. yetbeen used to evaluate different strains from food,but theyhold promise in this regard. 3.6.Virulence-attenuated strains of L.monocytoge- nes infoods: possibleprotective roles? Inanimal models, 3.5.2.Biotype- and genotype-based predictive as- strainswith attenuated virulence persisted for shortperiods sessment ofvirulence. Generalpredictive assessments of inthe animal and induced protective immunity against humanvirulence can be made on the basis of certainwell- pathogenicstrains when administered in sufŽ cient doses establishedbacteriologic criteria, such as phage type and (37). Theconsumption of foods contaminated by strains of serotype.Strains of certainserotypes (e.g., 1/ 2c,4a, 4c, 4d, relativelylow virulence may cause subclinical infections

and4e) are rarely recovered from patientsand may consti- thatsimilarly protect humans against more virulent strains. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/11/1811/1672430/0362-028x-65_11_1811.pdf by guest on 26 September 2021 tutea relativelylow (but not negligible) risk for human Presumingthat the putative foodborne-attenuate dstrains listeriosis.Following the same reasoning, the presence of sharekey antigenic determinants with the virulent strains, serotype4b in an RTE food should be cause for concern thepotential for antibodiesor other immune responses to because,while not commonly prevalent in foods, such theformer toconfer protective immunity toward the latter strainshave been involved in numerousoutbreaks. The risk maybe substantial. Such cross-protection would especially associatedwith such contamination would be substantially applyfor strainsin the same serogroup, which share im- increasedif molecular subtyping data suggested that the munodominantsugar substituents on their cell surface. In- strainbelonged to a genotypecommon to oneof theknown terestingly,in each major serogroup, some serotypes are epidemic-associatedclones. morecommonly encountered in illness than others. In the Strainsof serotype1/ 2acontribute substantially to hu- caseof serogroup 4, strains other than 4b had reduced vir- manlisteriosis and are, in addition, frequently found in ulencein animal and cell culture models (184). It is pos- RTEfoods and food processing environments. Assessing siblethat subclinical infection by such strains may confer therisk posed by food contamination by bacteria of sero- resistanceto infection by serotype 4b. type1/ 2aisespeciallychallenging: serotype 1/ 2ahasa rel- Perhapsof greater interest is the observed frequent ativelyhigh genetic variation, and the identiŽ cation of 1/2a contaminationof foods by strains of serotype 1/ 2c,which lineagesthat are predominant in human illness has not yet israrely implicated in illness. All 1/ 2cstrains screened to beenachieved. The need exists to augment the typing da- dateare genetically closely related (36, 138) and harbor a tabasefor strainsof serotype 1/ 2a,both from foodsand mutationin oneof thevirulence genes (95), suggestingthat from clinicalcases. thisgroup of strains may have arisen rather recently. Con- Sucha systemis in placethrough PulseNet for clinical ceivably,the contamination of foods by serotype 1/ 2cmay isolatesand for foodisolates provided by government lab- renderhumans more resistant to infections by virulent oratories,but it must be rigorously extended to include ad- strainswith similar surface antigens, e.g., serotypes 1/ 2a ditionalfood isolates. Currently, food microbiologists in the and 1/2b. academicand industrial sectors who isolate and type L. Thepotential for L.monocytogenes tocause mild dis- monocytogenes donot have access to PulseNet. Hence, a ease,which in many cases goes undiagnosed, has been re- largeportion of the food strain genotype data is being un- ported (168). However,theextent to which humans, in- derutilized.Furthermore, the food strain genotype database cludingimmunosuppressed individuals, may be infectedby shouldnot be limited to strains derived from NorthAmer- L.monocytogenes asymptomatically,or with only mild ica.Food contamination is an international issue, and the symptoms,has not been rigorously determined. It will be incidenceof outbreaks due to ECI makesit abundantly ofinterest to determine the possible correlations between clearthat this bacterial clone knows no nationalborders. In theexposure to foodscontaminated by clinicallyrare strains addition,a reviewof the literature has revealed that most (e.g.,serotype 1/ 2c),the carriage of L.monocytogenes, and large-scale,rigorous studies on L.monocytogenes geno- theincidence of foodborne listeriosis in distinct cohorts typesisolated from foodsand food processing plants are chosenin terms of dietaryhabits, immunodeŽ ciencies, and beingconducted outside North America, especially in the otherrisk factors. The identiŽ cation of humans who have Scandinaviancountries and elsewhere in Europe. hadan immune response to L.monocytogenes iscurrently Acontinuouslyaugmented standardized international difŽcult, as antibodiesare frequently of lowtiter and com- databasebased on the PulseNet format but accessible to monlycross-react with antigens from othergram-positive scientistsfrom government,industry, and academic insti- bacteria. tutionswill greatly facilitate the identiŽ cation of food lin- 4.0.A VIEW FORFUTURE NEEDS eagesthat are also encountered in infection and will, in addition,determine the proportion of the food strains that 4.1.Theoretical studies andmodeling. Almostall re- mayconstitute relatively low risks for humanlisteriosis. portedstudies cited in this review clearly point to thefood Participationin this database should be expected for aca- processingenvironment as the most relevant target for in- demicand government investigators, analogous to the entry terventionsthat will reduce the incidence of Listeria in ofnucleotide sequence data in an accredited database, foods.A clearneed currently exists for theoreticaltools that J.FoodProt., Vol. 65, No. 11 L.MONOCYTOGENES VIRULENCE AND PATHOGENICITY 1823 willanalyze the available data on Listeria prevalencein throughoutthe country and would also allow the incorpo- foodprocessing facilities and in foods with enhanced ana- rationof comparative data from scientistsin other coun- lyticand predictive potential. There is especially a needfor tries.The importance of such an endeavor for enhancing modelingthe niche complexity of Listeria inthe food pro- foodsafety and public health cannot be overemphasized. cessingfacilities. T obeeffective, such modeling would Wealsoneed the active participation of industry in haveto bespeciŽc totheprocessing facility in termsof the establishinga collectionof strains speciŽ c toprocessing productthat is being processed (type, volume, and other environments,so that strains and species from processing keyvariables). ‘ ‘Complexitymeasures,’ ’ asboth a concept microhabitatscan be adequately represented. Such a plan anda formalism,are already common in certain branches shouldinclude protocols to safeguardparticipating industry ofthe new mathematics (17, 201), andsuch measures as membersfrom anyputative actions arising from strainsor thesecould be adapted to a moreformal description of the speciessubmitted to the collection. manybacterial niches within the food processing environ- ment.Such studies will enhance our ability to understand 4.4.Genomics and proteomics: expected impact. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/11/1811/1672430/0362-028x-65_11_1811.pdf by guest on 26 September 2021 andsubsequently control the incidence of Listeria in food Thecurrently available typing tools, including serotyping processingenvironments. andmolecular subtyping, largely fail to provide adequate Suchmodels would serve as good ‘ ‘costingtools’ ’: insightinto strain virulence and the likelihood that any giv- theywould help deŽ ne the facilities’ speciŽ c maintenance enstrain will cause illness. The reasons are primarily that needsand critical control points, thereby facilitating the es- theresolution of these tools is relatively limited and rele- timationof the Ž nancialresources that would be required vantonly to selected small regions of the genome, which for anycorrective action. areexamined without reference to the rest of theorganism’ s Importantcontributions could be made by theoretical geneticendowment and adaptive potential. With the com- studiesthat address outcomes on the basis of interactive pletionof the complete genome sequence determination of processes(e.g., between microorganisms in abioŽlm or be- L.monocytogenes (strainEGD) (74), anewgeneration of tweenmicrobial cells and a surface),as well as by any genome-leveltools is becoming available for straintyping, studiesthat address linked and repetitive processes. The including,but not limited to, DNA chips(microarrays). conclusionsfrom suchstudies should be formulatedso that Genemicroarrays will permit whole-genome comparisons theyare meaningful to both the industrial user and the mi- amongdiverse strains and the identiŽ cation of strain- or crobiologist.Effective model construction can only be de- lineage-speciŽc sequences.The hybridization of DNA mi- liveredby teams that include applied mathematicians as croarrayswith genomic DNA from otherstrains (genomo- wellas microbiologists. A regular,periodic(e.g., every 5 typing)will identify genomic regions of divergence in dif- years)literature review of models such as that proposed ferentstrains and will provide rigorous estimates of genetic shouldbe undertaken, again by individuals with combined variationin distinct lineages, as is beingalready done with expertisein applied mathematics and microbiology, and otherpathogens (55, 173). Furthermore,expression mi- shouldfocus on theindustrial user and the processing plant croarrayswill be utilized to determine the expression pat- microbiologist. ternof the organism’ s genes(representing all or aselected fractionof the genome) in response to speciŽ c conditions. 4.2.Additional microbiologic and epidemiologic For typingpurposes, differences in such multigene or ge- studies. Additionalsurveys are needed to understand the nome-levelexpression proŽ les among different strains may contributionof other segments of the farm-to-fork contin- beespecially useful. Expression proŽ les speciŽ c for strains uumto the burden of foodborne listeriosis. Examples in- thatare of specialepidemiologic relevance (e.g., strains im- cludefood service settings such as restaurants, salad bars, plicatedin epidemics)may be determined.With the support anddelicatessens as wellas consumerpreparation and han- ofadequate epidemiologic data, virulence-related genomic dlingin the home. Household pets can reportedly shed and signatures(at the DNA sequenceor geneexpression level) carry L.monocytogenes withoutshowing symptoms, as can areexpected to emerge from analysisof thegenomes. Such householdmembers who are asymptomatic carriers ofthe tools,along with other DNA sequence–based typing tools organism.W eshouldassess the contribution, if any, of (e.g.,multiple-locus sequence typing), will allow the rapid thesesources to the burden of human illness. In addition, screeningof isolates relative to the constellation of genetic specialattention should be givento the ecology of lineages markers characteristicof virulent strains of diverse sero- thatare highly relevant epidemiologically, such as epidemic typesas well as those characteristic of especially trouble- clonesand serotype 4b strains, for theidentiŽ cation of cur- somelineages, such as those implicated in outbreaks. rentlyunknown reservoirs and ampliŽ cation niches. Contributionsare also expected from proteomicanal- 4.3.Construction ofa strainlibrary for an aug- ysisof the organism’ s completeprotein signatures. Current mented typingdatabase. Weneedan augmented PulseNet methodologiessuch as matrix-assisted laser desorption-ion- (or equivalent)database for foodbornepathogens that izationmass spectrometryhold promise not only for typing wouldhave the infrastructure and resources needed to make purposesbut for theidentiŽ cation of expression patterns ittruly accessible to all accredited, properly trained, and uniqueto selected strains or lineages. Strain- or lineage- properlyequipped microbiologists in government, industry, speciŽc proteinsare likely to be identiŽ ed, and additional andacademic sectors. This would greatly facilitate the insightwill be obtained from theidentiŽ cation of proteins monitoringof food and environmental contamination thathave unique expression patterns (e.g., timing of ex- 1824 KATHARIOU J.FoodProt., Vol. 65, No. 11 pression,stability, and amount). It will be essential that lowingintragastric and intravenous inoculation in mice. Microb. strainsof special epidemiologic and food safety relevance Pathog.20:247– 253. 15.Bayles, D. O.,B. A.Annous,and B. J.Wilkinson. 1996. Cold stress areadequately represented in such genomic and proteomic proteinsinduced in Listeriamonocytogenes inresponse to temper- studies. ature downshockand growth at lowtemperatures. Appl.Environ. Microbiol.62:1116– 1119. ACKNOWLEDGMENTS 16.Bayles, D. O.,and B. J.Wilkinson.2000. Osmoprotectants and cryoprotectantsfor Listeriamonocytogenes. Lett.Appl. Microbiol. Theauthor thanks ILSI-North America forsupport for the writing 30:23–27. ofthis review andespecially thanksMs. Catherine Nnoka and Drs. Don 17.Bazykin, A. 1998.Non-linear dynamics of interactingpopulations. Zinkand Alex Malaspina for their encouragement. The areas ofresearch WorldScientiŽ c Publishing,River Edge, N.J. fromthe author’ s laboratorythat are includedin thisreview were partially 18.Ben Embarek, P .K.1994.Presence, detectionand growth of Lis- supportedby ILSI-North America andby USDA grant99-35201-8183. teriamonocytogenes inseafoods:a review.Int. J. FoodMicrobiol. Theauthor thanks all themembers ofherlaboratory for their involvement 23:17–34. inthese research areas andis gratefulto Drs. FranzFiedler and Doug

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