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Journalof Food Protection, Vol. 65, No. 3, 2002, Pages 575– 582 Copyright Q,InternationalAssociation for FoodProtection

Review Aeromonas Species inFoods

JAMIEH. ISONHOOD AND MARYANNE DRAKE *

Departmentof Food Science, North Carolina State University, Raleigh,North Carolina 27695, USA

MS01-318:Received 28August 2001/ Accepted 16October 2001

ABSTRACT Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/3/575/1674790/0362-028x-65_3_575.pdf by guest on 01 October 2021

Aeromonas specieshave been recognized as potential or emerging foodborne pathogens for more than 20 years. Aero- monadsare estuarine bacteria and are ubiquitous in freshwater ,Žshand shellŽ sh, meats, and fresh vegetables. Actual sourced foodborneoutbreaks are few, but epidemiological evidence suggests that the bacterium can cause self-limiting diarrhea, with childrenbeing the most susceptible population. Most aeromonads are psychrotrophic and can grow in foods during cold storage.Aeromonads are not resistant to foodprocessing regimes and are readily killed by heattreatment. A hostof virulence factorsare present, but the exact role of each in human disease has not been fully elucidated.

Thebacterial genus Aeromonas isnow ofŽ cially clas- cently,pectinolytic activity has been reported in a newly siŽed within the family Aeromonadaceae andconsists of identiŽed strain (80). Most Aeromonas spp.can grow at a 14different conŽ rmed species (51) (anasterisk indicates pHof 5.5 to 9.0. Colonies formed after 24 h onnutrient thata specieshas been isolated from clinicalspecimens): agarare 1 to3 mm indiameter ,convex,smooth, whitish, A.allosaccharophila,A.hydrophila, * A.bestiarum, * A. andtranslucent. Colonies turn a lightbeige color after pro- caviae,* A.encheleia,A. eucrinophila,A. jandaei, * A. po- longedincubation (55). pofŽi, A.media, * A.salmonicida,A. schubertii, * A. sobria, Similar to Vibrio species,aeromonads have been re- A. veronii,* and A. trota.*Thegenus was formerlyclassi- portedto havea viablebut nonculturable state (92). Certain Ž ed within Vibrionaceae, andmisidentiŽ cation of Aero- strainsdisplay a rapiddecline following 12 to 18 h of monas spp. as Vibrio spp.still poses a problemin clinical growthin media containing glucose. This phenomenon is settings (2, 8). Originally,four Aeromonas specieswere referred toas the suicide phenomenon (70). The optimal identiŽ ed: A.hydrophila,A. sobria,A. caviae, and the Ž sh growthtemperature is generally believed to be 28 8C (58, pathogen A.salmonicida(82). Subsequentwork with these 82), buta widegrowth range and variability in the optimum fourspecies indicated distinct biochemical groupings (des- temperaturehave been observed (54,57, 58, 65, 80). Many ignatedphenospecies) and distinct DNA hybridization strainscan grow at ,58C,makingaeromonads particularly groupings(designated genospecies, sometimes referred to signiŽcant in refrigerated foods (24,27, 77, 78). A. hydro- ashybridization groups [HGs]), leadingto variability in phila hasbeen reported to have an optimal growth temper- speciesdesignation and classiŽ cation (1,8, 13, 18, 38, 54, ature of 288C,althoughit cangrow at temperatures ranging 58). Thesegram-negative facultative organisms are ubiq- from 1 to 428C (65, 74). uitousin water and in many foods. Their role in foodborne Aeromonadsare considered opportunistic pathogens of illnessis still not Ž rmlyestablished. The purpose of this bothaquatic and terrestrial animals (25, 67). Since1960, it reviewis to provide a generaloverview of Aeromonas spp. hasbeen speculated that these bacteria may be pathogenic infoods and the role they may play in foodborne illness. to humans (67, 72). In1984, the Food and Drug Admin- GENERAL CHARACTERISTICS istrationintroduced A.hydrophila asa ‘‘new’’ foodborne pathogen (89). Aeromonadshave been considered contro- Aeromonadsare gram-negative, oxidase-positive, glu- versialhuman pathogens, since results from humanvolun- cose-fermenting,facultatively anaerobic rods, and most are teerstudies have been inconclusive (57);however,epide- motileby polar  agella (82). The word Aeromonas was miologicalevidence continues to indicate that these organ- derivedfrom theGreek words aer, meaningair or gas,and (7,26, 62, 69) monas, meaningunit or monad. Cells are typically 1.0 to ismsare capable of causing gastroenteritis 4.4 mminlength and occasionally form Žlamentsof upto andother complications, including wound infections, sep- 8.0 mm.Aeromonads can ferment the sugars glucose, fruc- ticemia,and endocarditis (2, 14, 48). Gastroenteritisin- tose,maltose, and trehalose to acid or toacid and gas. They volvesin ammation of the gastrointestinal tract due to in- canalso hydrolyze starch, dextrin, and glycerol (55). Re- vasionof bacteriainto the intestinal mucosa. Septicemia or bloodpoisoning involves the growth of bacteria in the *Authorfor correspondence. Tel: 919-513-4598; Fax: 919-515-7124; blood (9). Studiesindicate that Aeromonas spp.can act as E-mail: [email protected]. bothinfectious and enterotoxigenic pathogens (56). Non- 576 ISONHOOD AND DRAKE J.FoodProt., Vol. 65, No. 3 motileaeromonads, namely, A.salmonicida, arethought to thermalsurvivor curves. Palumbo et al. (76, 79) reported beobligate Ž shparasites (25). Themotile mesophilic aero- tailingor two-phase thermal survivor curves with three monads,consisting of A.hydrophila,A. sobria, and A. cav- clinicaland two food isolates of A.hydrophila. Isonhood iae, areconsidered causative agents of human gastroenter- (45) alsoreported biphasic thermal survivor curves for two itis,wound infections, and septicemia (34). strains of A.hydrophila. Thetaxonomy of the genus Aeromonas isstill devel- FOODBORNE OUTBREAKS oping.Molecular genetic evidence now indicates that Aero- monas spp.,formerly members of thefamily Vibrionaceae, Currently,reports of foodborne Aeromonas outbreaks arequite different from eachother and from thevibrios lackhard evidence that indisputably establishes the organ- (58). Earlystudies of aeromonadsclassiŽ ed themesophilic, ismas a causativeagent of foodborne illness. Kirov (57) pathogenic,and nonpathogenic strains as A.hydrophila and reviewedincidences of Aeromonas-relatedfoodborne gas- thepsychrophilic strains that were Žshpathogens as A. sal- troenteritisthat occurred between 1977 and 1991. Most out-

monicida(58, 72). Inthe early 1980s, Popoff (82) used breaksinvolved seafood, such as oysters, sashimi, prawns, Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/3/575/1674790/0362-028x-65_3_575.pdf by guest on 01 October 2021 DNA-DNA hybridizationto show that at leastnine distinct andshrimp. Although Aeromonas was implicated,viruses HGs existedamong the mesophilic species A.hydrophila. were notruled out as a possiblecause of the illness. The Amongthe HGs, threeseparate phenotypic groups were onlyreport presenting good evidence leading to Aeromonas foundby using 8 to18 biochemical tests. These three asthe cause of gastroenteritis involved a 38-year-oldman ‘‘phenospecies’’ were named A.hydrophila,A. sobriae, and whoconsumed a ready-to-eatshrimp cocktail. Through ri- A. caviae. Mostclinical laboratories have accepted pheno- botyping,the patient’ s stooland the shrimp were foundto typicclassiŽ cation of the mesophilic aeromonads. A. hy- containidentical Aeromonas spp.This report offers thebest drophila isnow limited to HG1, HG2, andHG3 (58). A evidenceknown from astudyin which molecular tech- decadeago, investigators used DNA-DNA hybridization niqueswere usedto identify Aeromonas spp.as the caus- studiesto apply the classiŽ cation schemes of genomospe- ativeagent in foodborne gastrointestinal infection (47). cies,genospecies, or hybridization groups, which were de- Saad et al. (84) reportedan increased rate of Aeromonas Žnedas having at least 70% DNA homologywith the des- spp.isolated from humanstools during the summer months ignatedtype strain (72). Thesehybridization groups are andhypothesized an association with the incidence of mo- geographicallydispersed. Moreover ,noevidence has sug- tileaeromonads in fresh vegetables.The increased frequen- gesteda signiŽcant correlation between these hybridization cyof human stool– associated aeromonad isolation in the groupsand virulence (67). WhileDNA homologyanalysis summermonths was notlinked with microorganisms iso- isthe traditional tool for speciesconŽ rmation, 16S rRNA latedfrom nearbyriver and tap water ,butit did correlate sequencing,ampliŽ ed fragment length polymorphism, and withlarge numbers of Aeromonas from foodsamples. fattyacid methyl ester analysis have all been proposed to VIRULENCE FACTORS characterizenew isolates (51). Aeromonas spp.have properties that are shared by bac- Therole of aeromonads and the toxins they produce teriain the families Enterobacteriaceae and Vibrionaceae andtheir relationship to virulence in cases of human gas- (55).Aeromonas spp.are not particularly fastidious and can troenteritishave not been fully elucidated. There is no an- growwell on many common laboratory media, such as imalmodel for Aeromonas gastroenteritis.Most cases of MacConkeyagar ,Hektoenenteric agar ,andxylose lysine gastroenteritisassociated with Aeromonas occurin chil- desoxycholateagar (47). Inefforts toisolate Aeromonas dren,elderly people, and immunocompromised people. A spp.,selective media are used that take advantage of unique numberof putative virulence factors have been identiŽ ed, characteristicsof amylaseactivity and ampicillin resistance. includinghemolysins, invasins, adhesins, endotoxin (lipo- Selectivemedia such as glutamate starch penicillin agar polysaccharide[LPS]), proteases,Ž mbriae,pili, capsules, (52), ampicillindextrin agar (40), andstarch ampicillin agar S-layers,siderophores, and a varietyof extracellular en- (77) aretypically used for theisolation of aeromonadsfrom zymes (47,58, 66). Cytotoxicactivity on V erocells has foods.Gavriel and Lamb (33) studiedthe abilities of all occasionallybeen reported and has been associated with threemedia to grow different species of Aeromonas and hemolysinactivity. Several hemolysins may be produced, reportedwide disparity in the recovery of certain Aero- butthe most well characterized of them is aerolysin.Aero- monas species,namely, A.veronii,A. schubertii, and A. lysinis a lyticchannel-forming exotoxin encoded by aerA sobria, indicatingthat not all species are equally recovered (11, 42) thatwas Žrst characterized .25years ago. Other withthese selective media. cytotoxicenterotoxins similar to aerolysin and loosely Aeromonadsdo not exhibit unusual resistance to food groupedas aerolysins have since been isolated and char- processingprocedures. These organisms are not heat- or acterized (15, 30). Heat-labileand heat-stable cytotoxic en- acid-resistantand do not grow at a pHbelow 5 orat a terotoxinshave also been reported (6, 20, 58). Inaddition, sodiumchloride concentration above 3.5% (58,78, 79). two A.hydrophila phospholipaseswere recentlydemon- Combinationsof sodium chloride and polyphosphates are stratedto play a rolein virulence in Ž shand mice (66). alsoinhibitory (76), asare sorbates and smoke (37). The Theformation of S-layershas been considered by some chlorineresistance of aeromonads is also similar to that of tobe an indicator of pathogenicity in gram-negative and othergram-negative bacteria (58). Oneinteresting phenom- gram-positivemicroorganisms. An S-layer is an arrange- enon of Aeromonas speciesis their tendency for biphasic mentof protein or glycoprotein subunits on the cell wall J.FoodProt., Vol. 65, No. 3 AEROMONAS SPECIESIN FOODS 577

(95). Other than A.salmonicida, themesophilic Aeromonas rialstrains, its effects on enteropathogenici tyare still un- spp.of serotype O:11, A.hydrophila and A. sobria, are the clear (68). onlyones thus far toshow the presence of S-layers (58, Abeytaet al. (4) studied A.hydrophila involvedin 472 68). S-layersincrease the ability of the cells to adhere to casesof gastroenteritis occurring in November 1982 asso- andcolonize the intestinal epithelium. Presumably, S-layers ciatedwith the consumption of oysters harvested from Lou- of Aeromonas spp.of serotype O:11 contribute directly to isiana.Samples obtained from theoysters all tested nega- diarrhealsymptoms. The S-layer also allows the bacteria to tive for Salmonella, pathogenic Vibrioparahaemolyticus, beless susceptible to opsonophagocyto sis.Opsonophago- anddiarrhetic shellŽ sh poison. Twenty-three of 28 A. hy- cytosisis phagocytosis of a cellfacilitated by an opsonin, drophila strainstested positive in at least one of the viru- whichis a substancethat binds to bacteria, erythrocytes, or lenceassays, which included the suckling mouse test, the otherparticles, making them more susceptible to phago- adrenalY -1mouse cell test, and hemolysin assays. While cytosis (23).Aeromonas spp.of serotypeO:11 are also fre- thesetests did not prove that A.hydrophila was the sole

quentlyisolated in cases of septicemia (68). causeof the outbreak, they did indicate that A.hydrophila Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/3/575/1674790/0362-028x-65_3_575.pdf by guest on 01 October 2021 Theendotoxin LPS producedby Aeromonas is similar shouldbe considered when foodborne outbreaks involving tothose produced by other gram-negative bacteria. Simi- oystersare investigated (4). Krovaceket al. (61) found few laritiesin the LPS arerelated to a T-independentantigen differencesin virulence factors between human diarrheal thatactivates polyclonal B-cell activation and produces a andmarine environmental isolates of A.hydrophila. Kuhn predominantlyimmunoglobulin M response.The injection et al. (62) reportedthat hemolysin and cytotoxin production ofLPS intoanimals tends to causepyrogenicity, leukopenia were morefrequent for Aeromonas spp.isolated from in- leadingto leukocytosis, shock, hemorrhagic necrosis of tu- dividualswith diarrhea than for thoseisolated from healthy mors,diarrhea, and death (68). Thesereactions are caused individuals.However ,thenumber of environmentalisolates bylipid A, acommonLPS. Excretionof some exotoxins thatexhibited hemolysin and cytotoxin production was hasbeen shown to be dependent on the presence of theO- largerthan the number of human isolates that did. Albert antigenLPS. ThesigniŽ cance of this Ž ndingis that those et al. (6) compared A.hydrophila isolatedfrom children strainslacking the O-antigen LPS (roughstrains) excrete withdiarrhea and with those isolated from healthychildren lesstoxin than those strains that have ample O-antigen LPS andfrom surfacewater inBangladesh. The three toxins (smoothstrains). It is importantto note that O-antigen LPS studiedwere aheat-labilecytotonic enterotoxin (AL T), a isone of the key factors involved in bacterial colonization heat-stabilecytotonic enterotoxin (AST), anda cytotoxic ofthegut mucosa. Research has revealed that these strains, enterotoxin(ACT). Thequantities of Aeromonas isolates whichare characterized by O-antigen LPS, aremore viru- thattested positive for thepresence of the alt gene were lentwhen grown at low temperatures. Some reports of En- similarfor allthree sources. The ast genewas signiŽcantly terobacteriaceae arerelated to these Ž ndings (68). morecommonly encountered in isolates from environmen- Fimbriae(Ž lamentous and nonŽ lamentous) adhesins talsamples than in those from childrenwith diarrhea. Iso- havebeen described in studies of mesophilic Aeromonas latespositive for boththe alt and the ast geneswere sig- spp.These Ž mbriaeenable Aeromonas toadhere to differ- niŽcantly more numerous for childrenwith diarrhea than entcell lines. T wotypes of Ž mbriaehave been character- for theother sources. This led to theconclusion that alt and ized.Fimbriae of theŽ rst typeare short and rigid (S/ R)and ast mayhave a synergisticeffect in inducing diarrhea. For occurin large numbers in bacteria cells. A.hydrophila and astudyin which Aeromonas spp.were theonly pathogens A. sobria arethe most adherent to various surfaces, a Žnd- isolatedfrom thestools of 26 patients, clinical data for 11 ingthat has been attributed to the nonŽ lamentous (outer ofthese patients also suggested that watery diarrhea oc- membrane)adhesins (68). Fimbriaeof the second type are curredwhen both the alt and the ast geneswere present, longand  exible(L/ W)andoccur in smaller numbers in whileloose stools occurred when only the alt gene was bacterialcells. These two types of Žmbriaehave been found present (6). Schiavanoet al. (87) alsoreported mixed re- onboth clinical and environmental isolates of mesophilic sultsfrom astudyof Aeromonas isolatesfrom healthyin- Aeromonas spp.Isolates from different Aeromonas spp. dividualsand from thosesuffering from diarrhea.Cytotoxic sharea highdegree of homologywith the N-terminal amino activityas well as adhesin and invasin production was acidsequences with respect to L/WŽmbriae.Growth at low foundin four of eightdiarrheal isolates but was alsofound temperaturesin a liquidmedium helps to promote Ž mbriae inisolates from healthyindividuals. Of 141food isolates, expressionamong most isolates (68). 66%tested positive for thepresence of a cytolyticentero- A.salmonicida, aŽshpathogen, has been shown to toxingene, as did 67% of clinical isolates and 58% of en- producea capsularpolysaccharide when grown in vivo or vironmentalisolates (56). Ninety-fourpercent of 767shell- inaglucose-richmedium. This capsular polysaccharide has Žshisolates were hemolysinpositive (1). Of thehemolysin- beenan importantsurface structure and a pathogenicityde- positiveisolates, 59% exhibited cytotoxic activity. W ang terminantin virulent strains. A.hydrophila and A. sobria and Silva (93) alsodetected hemolytic activity in 86% of were shownto have the same ability to produce a capsular catŽ sh Aeromonas isolates. polysaccharide.Studies have shown that the ability to pro- VIRULENCE INFOOD ducea capsularpolysaccharide improved the adhesion of thebacteria to varioussurfaces (68). Whilea capsularpoly- Theinfectious dose for Aeromonas spp.in foods is saccharidecertainly enhances the virulence of these bacte- unknown,as is the exact mechanism of how they cause 578 ISONHOOD AND DRAKE J.FoodProt., Vol. 65, No. 3 gastroenteritis. A.hydrophila and A. caviae arethe species illnesswith Aeromonas spp.as the cause could increase in mostcommonly associated with diarrhea. Mateos et al. (65) thefuture because of a higherconsumer demand for less studiedthe expression of selected virulence factors in A. processed,more ‘ ‘natural’’ foods. Aeromonas spp.are com- hydrophila isolatesas a functionof temperature. Environ- monlyisolated from aquaticenvironments such as rivers, mentaland human isolates were studied;environmental iso- lakes,sewage ef uents, marine waters, and chlorinated lateswere inhibitedat 37 8C,whilehuman isolates were not. drinkingwater (4,6, 34, 39, 60, 62, 80) andfrom retail Thehemolytic and cytotoxic activity of environmentaliso- fresh vegetables (84) andfoods of animal origin such as latesdecreased at 37 8C,whilethis activity was stimulated seafood (4, 5, 39), red meat (27,51, 77), groundpork, for somehuman isolates (4 of 9). All environmental isolates poultry (77), raw milk (47, 77), andcheese made from raw were pathogenicfor trout(100% mortality), while only milk (86). Recovery of Aeromonas spp.from estuarineen- 44%(4 of 9)ofthehuman isolates were. Okrendet al. (73) vironmentshas been reported to be seasonal (34, 83). Aero- andTsai and Chen (89) determinedthe toxigenicity of aero- monadsappear to be ubiquitous on freshwater Žsh(both

monadsisolated from meatsand seafood, respectively. Cy- wildand pond-raised) (35,36, 71, 93). Refrigerationof Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/3/575/1674790/0362-028x-65_3_575.pdf by guest on 01 October 2021 totoxinswere detectedin more than 90% of A.hydrophila thesefoods has not proven adequate for control,as re- isolates.In a studyinvolving an A.hydrophila strain iso- searchershave reported growth at 4 and5 8C (16, 24, 27, latedfrom theoyster ,Tsaiet al. (90) investigatedthe pro- 31). Estimatesshow that Aeromonas maycause up to 13% ductionof hemolysin and cytotoxin under different envi- ofthe gastroenteritis cases in the United States (56). ronmentalconditions. A.hydrophila was foundto produce As waterborneand environmental bacteria, Aeromonas bothhemolysin and cytotoxin at 37, 28, and 5 8C. Toxins spp.encounter a rangeof environmental changes that in- were morestable and were producedfaster at 28 8C than at cludeacid and temperature stresses. The ability to adaptto 378C.Inthe presence of 1 to5% (wt/ vol)NaCl in brain awidevariety of environments accounts for itsthreat as a heartinfusion broth, the production of hemolysin and cy- humanpathogen and its frequent isolation from various totoxinwas decreased.T oxinproduction also decreased habitats (53).Aeromonas spp.are considered a potential whenthe pH was raisedor lowered from 7.2.T oxinpro- causeof human gastroenteritis, and water supplies, partic- ductionincreased with an increase in dissolved oxygen dur- ularlydrinking water ,havebeen investigated as sourcesof ingthe stationary growth phase (90).Aeromonas spp. gen- thesebacteria. A recentstudy in northern Scotland focused erallygrow at refrigeration temperatures, and toxin produc- ona drinkingwater distribution system (34). The various tionis not necessarily inhibited (28, 64). Enterotoxinand factorswithin the system, such as chlorine concentration, hemolysinwere producedin meat extracts stored at 5 or pH,temperature, and rainfall, were evaluated.The results 128Cinan experiment that revealed not only that motile ofthe study revealed that although some reservoirs with Aeromonas spp.grew wellin food products at refrigeration lowchlorine concentrations were shownto contain low temperatures,but also that production of enterotoxin and numbers of Aeromonas, theorganism was frequentlyiso- hemolysinby thesebacteria was notlimited by refrigeration latedfrom highlychlorinated reservoirs in signiŽ cant num- of the food (64). bers.Gavriel et al. (34) suggesteda relationshipbetween Astudyby Kirov et al. (59) addressedthe virulence rainfalland the densities of Aeromonas isolated.Extended ofstrains isolated from milk.Isolates recovered from raw periodsof rainfall increased the incidence of the recovery andpasteurized milk were testedfor exotoxinproduction. of Aeromonas from thereservoirs. This increase is thought Moststrains were notenterotoxigenic, and some produced tohave occurred because an increased organic load caused smallamounts of hemolysin. Piliation was observedin theformation of chloramines, which are less effective in moststrains. A. sobria from pasteurizedmilk produced all killingmicroorganisms. ofthe exotoxins (hemolysins, cytotoxin, and enterotoxin) Cottonand Marshall (22) evaluatedmicro ora on pro- measured.This strain grew wellat 43 8C.Almosthalf of cessingequipment in two different catŽ sh processing thestrains showed some ability to bind to Hep-2 cells ( $5 plants. Aeromonas spp.(37.5%) and Pseudomonas spp. bacteriaper cell). This test was runwith control adherent (37.5%)were themost common gram-negative bacteria iso- strainsisolated from humanand chicken feces (12 to 20 lated.The deheaders, conveyors, and cutting boards were bacteriaper cell). Strains grown at lowtemperatures exhib- theprincipal areas in theplant from which Aeromonas spp. itedincreased adhesive ability and piliation. These strains were isolated.Automated Ž lletingmachines were not were ableto grow to large numbers in refrigerated milk shownto harbor aeromonads. An interesting observation withoutspoilage. Evaluation of thisstudy suggests that con- was that Aeromonas spp.were isolatedat ahigherfrequen- sumptionof pasteurized milk with preformed toxins may cyfrom thesmaller ,lessautomated plant than they were beof little concern, since exotoxin production was signif- from thelarger ,automatedplant. The researchers concluded icantlylower in milk than in bacteriological medium. In thatthese differences may have resulted from thediffer- addition,bacteria with the capability to form suchtoxins encesin the frequencies with which equipment was sani- appearrarely in milk isolates (59). tizedin the two plants. The smaller plant cleaned and san- itizedequipment at the end of theprocessing day, while at SOURCES OFCONTAMINATION thelarger ,automatedplant, sanitizer was sprayedon equip- Theubiquity of aeromonads in foods suggests that mentsurfaces during employee break periods, and cleaning foodsare vehicles for illnesscaused by Aeromonas spp. andsanitation operations were carriedout after processing Merinoet al. (68) speculatedthat occurrence of foodborne runs.Furthermore, the results of this study may implicate J.FoodProt., Vol. 65, No. 3 AEROMONAS SPECIESIN FOODS 579

Aeromonas spp.as possibleindicators of processingequip- from raw sheep’s milkcheese (86), thiswas high-pH( .6.4) mentsanitation (22). cheesewithout an added lactic starter .Thus,although Aero- Thecontrol of bioŽ lms on food processing equipment monas isfound in raw milk,it is unlikely to be found in isessentialto minimize the risk of contamination of aprod- fermenteddairy products or pasteurized milk. uctwith pathogens. Aeromonas canform bioŽlms, which Dohertyet al. (27) evaluatedthe growth of A. hydro- contributeto increased resistance to normal bactericidal phila onrefrigeratedlamb meat stored in vacuumand mod- treatments.Bal’ a etal. (10) examinedthe effects of heat iŽed-atmosphere packages at high and neutral pHs. These andchlorine on the inactivation of Aeromonas bioŽ lms on researchersfound that numbers of aeromonadsdecreased in stainlesssteel surfaces. These researchers found that older alltypes of packages tested at 5 8C.However,inhigh-pH bioŽlms were moreheat resistant than were lessestablished lambmeat, aeromonads signiŽ cantly increased or main- bioŽlms. An 8-h bioŽ lm was destroyedwithin 1 minat tainedtheir numbers in all packages except those containing 508Corby exposure to 25 ppm chlorine for 1min.Eight- 100% CO2,inwhich the bacteria slowly died. At 0 8C, A.

day-oldbioŽ lms were effectivelydestroyed by either 60 8C hydrophila was recoveredfrom allsamples with high pHs. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/3/575/1674790/0362-028x-65_3_575.pdf by guest on 01 October 2021 or75 ppm chlorine for 1min. Inhigh-pH minced lamb meat stored at 5 8Cinair or vac- Somestudies have shown that fresh vegetablescan also uumpackages, there was asigniŽcant increase in cell num- harbor Aeromonas spp. (84). Sincevegetables are normally bers (27).A. hydrophila grew slowlyat 2 8C on cooked consumedraw, thepresence of aeromonadson these foods crayŽsh tails with air or vacuum storage, but modiŽ ed-at- canpresent a signiŽcant health risk. A studyconducted in mospherestorage inhibited growth (43). Theability of Brazil (84) isolatedmotile aeromonads from 43of 90veg- Aeromonas spp.to survive and grow at refrigeration tem- etablesamples tested. Most isolates were foundon water- peraturesis a causefor concernand results in a needfor cress,with lettuce having fewer positivesamples and es- othermeans to control this psychrotrophic foodborne patho- caroleshaving slightly fewer positivesamples than lettuce. gen (64). Positivesamples were reportedto range from ,102 to STRESS RESPONSE OFMICROORGANISMS .106 CFU/g.Sixteenpositive samples had counts of $104 CFU/g (84). Jacxsenset al. (46) reportedthat the growth Theuse of mild processing techniques in the produc- of Aeromonas spp. at 78Confresh-cut vegetables was in- tionof food products to meet consumer demand for higher uencedmore by vegetable type than by atmosphere. qualityproducts is increasing in popularity. Foods are pro- Growthoccurred on shredded lettuce and chicory but not cessedwith mild heat treatments, are formulated with less oncarrots or brussels sprouts. V elazquezet al. (91) dem- acid,salt, and sugar ,andare not as dependent on preser- onstratedthat A.hydrophila survivedand grew onfresh vativessuch as sulŽ te and nitrite. For theseminimally pro- tomatosurfaces and in fresh choppedtomatoes at refrig- cessedfoods, psychrotrophic and mesophilic microorgan- erationtemperatures. ismsare a concernbecause of their ability to grow in a Pin et al. (81) conducteda surveyof the frequencies widerange of temperaturesand their tolerance to lowlevels ofmesophilic Aeromonas spp.isolated from variousfoods. of heat (3). Inthe environment and in foods, microorgan- Atotalof 87different foods (meats, water ,dairyproducts) ismsencounter changing conditions and stresses that they were sampledfor thepresence of A.hydrophila,A. sobria, mustsurvive and overcome in order to reproduce (12). and A.caviae.A. hydrophila was isolatedfrom 33of the Someof these stresses include starvation, cold shock, heat 87samples, while A. sobria was isolatedfrom 17samples shock,weak acids, high osmolarity, and high hydrostatic and A. caviae was isolatedfrom 6samples.Isolation fre- pressure (3). Thephysiological, biochemical, and genetic quencieswere highestfor meatproducts such as Ž sh,sea- mechanismsthat different bacteria undergo during certain food,and poultry, with 82.7% of the strains being isolated stresseshave not been elucidated for manymicroorganisms. from theseproducts. The other 17.3% were isolatedfrom However,itis well documented that when microorganisms water,cheese,and milk (81). Ina differentstudy (97), poul- areexposed to a stress,they will adapt and may become trymeat and eggs were investigatedas potential sources of resistantto stronger doses of the stress (homologous stress enterotoxigenic Aeromonas spp.All eggs sampled were free hardening). of Aeromonas. From 130broiler meat samples, 17 Aero- Anotheradaptive response that has been observed is monas strainswere isolated.Of thesestrains, 8 were posi- thecross-protective effect of stress (heterologous stress tiveby the vasopermeability test and 7 were positiveby hardening).Cross-protection occurs when the adaptation of themouse foot pad test (97). Milkhas also been investi- amicroorganismto one stress carries over to the increased gatedas a potentialvehicle of Aeromonas-relatedgastro- survivalof another stress. As anexample of cross-protec- enteritis.In a studyby Kirov et al. (59), 72samplesof raw tion, Listeriamonocytogenes grownunder low-nutrient milkand 183 samples of pasteurized milk were collected conditionswas shownto have an increased tolerance to inAustralia. Aeromonas spp.were recoveredfrom 60%of chlorinesanitizers (12). Thewell-characterized foodborne raw samplesand from only3.8% of pasteurized samples. pathogens L.monocytogenes,Escherichia coli, and Sal- Of thespecies recovered from raw milk,74% were A. hy- monellaenterica allhave documented stress responses that drophila, while A. sobria was thespecies most recovered canlead to homologous and heterologous stress hardening, from pasteurizedmilk (59). Santoset al. (85) reportedthe andthis Ž ndinghas implications for foodprocessing tech- inhibition ofA. hydrophila inthe presence of Lactococcus niques (16,17, 29, 41, 49, 50, 63, 88). lactis subsp. lactis. Althoughaeromonads were isolated Adaptationto host defenses within the gastrointestinal 580 ISONHOOD AND DRAKE J.FoodProt., Vol. 65, No. 3 tractis crucialto enablea gastrointestinalpathogen to reach stress.Following stress, bacteria were notmore heat or thesite of the infection. The acid barrier in the stomach, freeze-thawresistant. Exposure to moderate acidic condi- thephysical barrier of the epithelial cells lining the intes- tionsdid enhance subsequent acid resistance to more ex- tinalwalls, and nonspeciŽ c immuneresponses of thebody tremepH conditions, Ž ndingsthat are similar to resultsre- suchas macrophages are the main barriers that the micro- portedby Karem etal. (53). organismencounters on its path to the gastrointestinal tract Thealternative sigma factor RpoS orchestrates the inhealthy individuals (32). Responseto certain stress con- stressresponses of many of the Enterobacteriaceae, in- ditionssuch as the ones implicated in foods can induce cluding E.coli(19), Salmonella (96), and Shigella(94). stresstolerance and thereby enhance virulence (3, 12). Strains of E. coli or Shigella exneri withoutfunctional From afoodsafety standpoint, it is importantto understand rpoS showa decreasedstress response to acid and starva- theadaptation, survival, and growth of microorganisms in tion,and the cross-protective effects of these stresses are foodsunder such stresses. For acidstress, a low-pHenvi- alsodecreased. Salmonella virulenceis decreased, (96), as

ronmentmay be used in a foodto inhibit the growth of is that of Yersiniaenterocolitica (44), inthe absence of Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/3/575/1674790/0362-028x-65_3_575.pdf by guest on 01 October 2021 microorganisms.In effect, what may actually happen is that RpoS.Yildiz and Schoolnik (98) isolatedand characterized themicroorganism may adapt to this imposed stress in the an rpoS homolog in Vibriocholerae. Mutantsof the Vibrio storedfood, allowing it toadapt and survive the acid barrier rpoS exhibiteda decreasedability to survive environmental ofthe stomach better . stress. rpoS didnot enhance acid resistance for V. cholerae STRESS IN AEROMONAS as it did for Enterobactericeae. An rpoS genehas not been reportedfor Aeromonas. Furtherstudies on the genotypic Acidstress, also called acid tolerance response, in- andphenotypic characterization of the stress response of volvesa complexprocess in which many changes in the Aeromonas willfurther elucidate its role in foodborne dis- levelsof different proteins and other events occur through easeand its pathogenicity. generegulation (32).Aeromonas spp.are not acid-resistant organisms,but they do demonstrate an adaptive stress re- CONCLUSIONS sponseto acid. Karem etal. (53) studiedthe effects of sub- Aeromonas isanestuarine bacterium implicated in oc- lethalacid adaptation followed by a severeacid environ- casionalgastroenteritis outbreaks. Aeromonas isnot unusu- ment.The death of A.hydrophila was rapidat apHof3.5, allyresistant to traditional food processing techniques but butwhen the bacteria were adaptedat a pHof 5.0 for 20 isubiquitous on seafoods, meats, and fresh produce.Ad- minbefore they were challengedat a pHof 3.5, a .5-log ditionalresearch is necessary to elucidate the stress re- increasein survival relative to that with no acid adaptation sponseof this organism and to expand knowledge of vir- was observedwithin 1.5 h afterthe acid challenge. Karem ulenceand pathogenicity and their relation to foods and et al. (53) hypothesizedthat A.hydrophila was ableto adapt foodprocessing. andsurvive in the acid environments by producing ‘ ‘pro- tective’’ proteins.Chloramphenicol-tre atedcells (no protein ACKNOWLEDGMENTS synthesis)did not show an acid tolerance response. On the basisof this research, it seemed clear that proteins were Appreciationis extended to Drs. 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