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1 The physiological attributes of Gram-negativebacteria associated with spoilage of meat andmeat productsM.L. GARCIA-LOPEZ, M. PRIETO AND A. OTERO

1.1 IntroductionT h e slaughtering an d butchering of food animals provide bacteria with a nopportunity to colonize meat surfaces. A wide range of micro-organismscoming from different sources are introduced to surfaces which containabundant nutrients and which have a high water availability. Only a few ofth e contam inan ts will be able to initiate growth, and only som e of the se willeventually spoil the m ea t by mean s of their biochemical attributes. M an hassearched (until recently in an em pirical m an ne r), for ways to ke ep spoilageorganisms away from meat, to reduce their growth rate, or to select thosewith low spoilage potential. Predominance of different groups of micro-organisms o n m eat dep end s o n th e characteristics of the m eat, the environ-ment in which meat is stored as well as the processing that meat mayundergo.Gram-negative bacteria constitute the greatest spoilage potential formeat and meat products. When fresh meat is chill-stored aerobically,members of the genera Pseudo mon as, Acinetobacter, Psychrobacter andMoraxella display th e fastest growth rates an d hence the greatest spoilagepotential. Species of Shewanella and Enterobacteriaceae need conditionsm ore favourable than those of t he above genera in orde r to develop andproduce spoilage metabolites. Depending upon conditions, the shelf-life offresh m ea t is in th e range of d ays before signs of spoilage (off-odours a ndslime) are evident. An extension of shelf-life is achieved by hindering thegrowth of Gram -negative organisms relative to that of G ram-positive ones(Micrococcaceae and lactic acid bacteria). To achieve this, environmentaland or product conditions (atmosphere, a,, salt and n itrite concentrations,tem per atur e, etc.) tha t favour growth of Gram-positive bacteria in me at ar eselected.The number of micro-organisms on fresh meat surfaces change duringchill storage following a typical microbial growth pattern. Counts of bac-teria in me at ar e in the range 102-105cfu/cm2,but only around 10% are ableto initiate growth (Nychas et al., 1988).T he initial lag phase is attributed tomicrobial adaptation to changing conditions (chill temp eratures and surface

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2 T H E MICROBIOLOGY OF MEAT A N D POULTRYdesiccation). E nsu ing logarithmic grow th ta kes place after cells have accom-mo dated to th e new en vironmental sett ing an d ada pted their metabolism.W hen n um bers exceed lo7cells per cm 2, he first spoilage signs ar e dete cted ,as off-odours. A no th er typical spoilage sign, bacterial slime, is noticeablewith cell density around los cells pe r cm2 (Gill, 1982). Shortly a fter, grow thdeclines and t he stationary phase is reache d. Do min ance of a single micro-bial group is due to its higher growth rates under specific conditions, andthis highe r rat e is eit he r because of metabolism adv anta ges (sub strate trans-form ation and affinity) or toleranc e to factors (psychrotrophism, pH , a, .Th us, it seem s that there are n o interact ions (or they a re indirect) betweenmicroorganisms until one of the genera present reaches its maximum celldensity (Gill an d M olin, 1991).A t the beginning of chill storag e, the role played by the composition ofpost-mortem meat is selective more than limiting. High water contentfavours microbial growth and, although there exist plenty of growth-sus-tainable n utrients, psychrotro phs prefer to use low-molecular-weight com -po un ds rat her tha n complex proteins an d lipids (Gill, 1982).During development of post-mortem rigor, muscular fibres degradeglycogen (first aerobically and then anaerobically) in order to obtain thenecessary ATP to maintain cellular structures and osmotic balance. As aconsequence, proport ions of many of low-molecular-mass substancescha ng e during conversion of muscle into me at (Table 1.1). W hen oxygen isdepleted, anaerobic routes are used and lact ic acid becomes the end-pro du ct of glycolysis, and its accumulation in turn causes th e p H to fall. Innormal circumstances, pH reaches the value of 5.8-5.5, equivalent to0.9-1% of lactic acid in m uscle.

Since nutrient composition does not stop bacteria from growing, otherfactors hav e to be used ei ther to inhibit microbial activity ( E h , a,, temper-atu re, atmos phe re) o r to p rom ote growth of non-spoilage bacteria.Table 1.1 Chemical composition of typical adult mam-malian m uscle post rigor morrisComponent ~~Post-rigor (%)WaterProteinLipidCreatine phosphateCreatineATPIMPGlycogenGlucoseGlucose-6-PLactic acidAmino acidsCarnosine, anserine

751930.70.30.10.10.20.90.40.3

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TH E PHYSIOLOGICAL ATTRIBUTES OF GRAM-NEGATIVE BACTERIA 3Surface desiccation takes place during refrigeration of carcasses. Theevap oration of water fro m the surface causes the a , to dr op during the first

fou r ho urs ( or m or e in connective tissue overlying fat) below limits (0.95)for m eat spoilers such as Pseudomonas and Moraxella (G rau , 1979). Diffu-sion fro m th e inner parts cou nteracts this effect, which can only be used todelay microbial growth in th e first 24 ho urs afte r dressing. Tem per atur e isthe m ain factor used to d ecrease growth of spoilage bacteria on m eat, byincreasing both lag phase and g ene ratio n tim e. Psychrotrophs are especiallyfavoured by the normal practice of refrigerating meat.

1.2 Gram-negative spoilage bacteria in meat and meat products1.2.1 Fresh me atIt is generally recognized that Gram-negative, motile and non-motileaerobic rods and coccobacilli belonging to the genera Pseudomonas,Mora xella, Psychroba cter (formerly Moraxella-like) and Acinetobacter areth e majo r com po nen ts of th e spoilage flora of raw me at sto red aerobicallyun de r refrigeration (Molin an d Te rns trom , 1982, 1986; Shaw an d Latty,1982, 1984, 1988; Pr ieto et al . , 1992a, 1992b; Dro sinos and Bo ard , 1995a).Certain species of psychrotrophic Ente robac teriaceae commonly occur onchilled meat. These organisms, which are able to grow aerobically onadipose tissue and on muscle tissue of high pH (> 6) , appear to be morepreva lent on p ork and lamb (G ra u, 1981; Da inty an d Mackey, 1992). The irgrowth is favou red by tem pe ratu res of 4 C (Blickstad and Molin, 1983).Isolation of Flavobacterium, Alcaligenes, Vibrio, Aeromonas andAlteromonas is reported less frequently (Patterson and Gibbs, 1977;N ottin gh am , 1982; Blickstad and Molin, 1983).T he initial flora of pou ltry skin is partially elimina ted du rin g scalding. Asignificant proportion of the subsequent contaminants are Gram-negativebacteria (Daud et al. , 1979). A ltho ug h th e incidence of psychrotrophs in theinitial flora ( lo3-lo4 pe r cm 2) is variable, th e finished carcasses a re g en er-ally contaminated with large numbers of species capable of survival andeven growth in chilled water. For this reason, it appears that the psy-chrotrop hic flora o n poultry is mor e likely t o b e less variable tha n th at onoth er m eats (Gill, 1986). A t t he time of spoilage, the pre dom inant organ -isms on eviscerated poultry are pseu dom onad s and t o a lesser extent Acine-tobacter and probably Psyc hrobacter. Shewanella putrefaciens may also bepre sen t. This bacterium, which is a poten t spoilage organism, is consideredan important part of the spoilage association even though it may not benumerically dominant (McMeekin, 1977). Other Gram-negative bacteriaFlavobacterium and Enterobacteriaceae) have been recovered on manyoccasions from spoiled chicken an d turkeys (Mc Me ekin, 1975; D au d et al .,

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4 T H E M I C R O B I O LO G Y OF MEAT AND POULTRY1979; Lahellec and Colin, 1979). A relationship has be en observed betweenthe occurrence of the abo ve gene ra and different sites of the poultry carcass.Pseudomonas is do m ina nt on the skin of the breast ( p H 5.7-5.9) an d leg (pH6.4-6.7), bu t the remaining ge ne ra are restricted to tha t of the breast appar -ently because of iLs lower p H value (Ba rnes and Impey, 1968; McM eekin,1975,1977).The spoilage of aerobically stored meat, as with other proteinaceousfoods, is preceded by a phase of variable duration during which bacteriam ake use of c arb oh yd rate s particularly glucose and glucose-6-P as a carb onand energy source (Gill an d N ew ton, 1978).

Glucose is present in post-morrem beef meat at concentrations in therang e 0.1-0.5% (Gill, 1986); it is readily used by microbial cells growing onthe surface of m eat. A t th e ou tset, a diffusion gradient develops from withinthe m uscle. This maintains a n ad eq ua te glucose concentration at the m eatsurface and hence ensures bacteria continue to metabolize carbohydratesthe reb y delaying the utilization of oth er co m pou nds (Gill, 1986). Only w henthe demand from large numbers of bacteria (typically more than l o 7cells/cm2) can no t be m et, d o they attack am ino acids, and cause a rise in th econcentration of ammonia and pH. Gram-negative bacteria appear to beparticularly fitted to use low-molecular-weight com po un ds at refrigerationtempe ratures and in meat of normal p H (5.5-5.8). Th ey have higher growthrates than would-be competitors and thus outnum ber them o n meat sur-faces.Residual glucose values in meat post-mortem can be low as a result ofstress, starvation or fright pr ior t o slaughter of animals. Such circumstancesde ple te the glycogen concentration in live animals. D ue to its organolepticcharacteristics, meat from stressed animals is referred to as DFD (Dark,F i rm, Dry) m ea t . As post-mortem glycolysis is curtailed by low substratecon centra tion, actic acid is not p rodu ced in the no rm al amounts. As glucoselevels are lower than normal, the resulting meat spoils rapidly becauseglucose scarcity prompts bacteria to the early use of amino acids. Theaddition of glucose to D F D me at d oes delay th e onset of spoilage becausebacteria can increase their maximum cell density without attacking aminoacids (Lam bropou lou et al., 1996).

In the first phase of growth on meat, the metabolism of glucose bypseudomonads and other Gram-negative bacteria does not give rise tooffensive off-odo urs. Gr ow th is su ppo rted by carbohydrates, and theircatabolism releases a complex m ixture of substances containing short-chainfa tty acids, ke ton es an d alcohols, th at exhibit a variety of fruity and sweetyodours (Dainty, 1996).The second phase begins when glucose is depleted, and the micro-organisms begin to use am ino acids for energy. This occurs when flora reachnumbers of lo7 bacteria/cm2. Volatiles responsible for the spoilage o do ursin this phase a re well characterized (McMeekin, 1982). A m ino acids

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THE PHYSIOLOGICAL ATTRIBUTES OF GRAM-NEGATIVE BACTERIA 5(cysteine, cystine and methionine) are precursors of hydrogen sulphide,methylsulphide and dimethylsulphide. T he se comp ounds are gen erate d byP seudomonas spp. and En teroba cteriacea e, and produce odo urs describedas putrid an d sulphury. D ea m ina tio n of these amin o acids gives rise to pyru-vate, amm onia, H2S, CH3SH and (CH3)2S. Dimethylsulphide may also bemetabolized from methylation of methylsulphide. It is interesting to notethat P seudomonas spp. degrad e amino acids by d eam ination, while En ter-obacteriaceae also possess the ability to decarboxylate amino acids(McM eekin, 1982).Methylamine, dimethylamine and trimethylamine have also been com-monly detected their formation is associated with the growth of Ps.fluorescens as well as non-fluorescent pseudom onads. A m on g the pseudo -monads, Ps. fragi is considered to be mainly responsible for the productionof ethyl esters having a sw eet, fruity od ou r. Ot he r sulphur-containing com-pounds generated in more advanced stages of spoilage by pseudomonadshave also be en detected. Am ino acids are the source of these products andtheir production occurs when the num bers of bacteria are gre ate r than 10cfu/cm2.

Two main e nd products of amino acid decarboxylation have been identi-fied: cadaverine, from lysine, and putrescine from ornithine or arginine.High correlations between putrescine production and pseudomonadscounts, and between cadaverine and En terobac teriaceae counts have beenob tain ed ( D ain ty and Mackey, 1992). Nevertheless, their use as spoilageindicators is not feasible as the detection occurs when bacterial numbersexceed 107/cm2.Other significant but less studied amines are spermidine,spe rm ine, histamine and tyramine.Experiments done in pure cultures with highly proteolytic strains ofProteus and P seudomonas (Ps. f ragi , Ps. f luorescens) demonstrated theirhigh p rot ea se activity ag ainst myofibrils and sarcoplasmic proteins (Daintyet al., 1983). This is in ag reeme nt with results from taxono mic stud ies (Molinan d T ern stro m , 1982; Shaw and Latty, 1982), which reveal production ofextracellular enzymes by Ps. fragi clusters 1 and 2. Bacteria may use pro-teins if they are th e sole source of carbon and energy, but not w hen easy-to-assimilate compounds are available. Although some species of differentgenera (Pseudom onas , Pro teus, Aerom ona s) are easily shown to be pro teo -lytic or lipolytic on laboratory media, there is clear evidence that thesephen om ena do not contribu te to spoilage as other factors are d etected first.It seems that release of exoproteases from bacteria only occurs in the sta-tionary phase, when cells have attained their maximum density and aminoacids have been depleted. Analysis of myofibrillar and sarcoplasmic pro-teins exhibited no chan ge until num bers exceeded O O cells/cm2 and longafter off-odours an d slime had been d etec ted (McM eekin, 1982).Ev en thoug h m any psychrotrophic bacteria produce lipases, the role o fbacteria in the lipolytic and oxidative changes of meat is generally

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6 THE M I C R O B I O L O G Y OF MEAT A N D POULTRYoverlo oked . So m e expe rimen ts have shown however, th e ability of Ps. fragito increase the level of free fatty acids in meat as well as in a culturemed ium. Pseudom onads and psychrobacters use the same compoun ds onfatty surfaces as they d o on lean m eat, and pro duce the sam e metabolites.N eve rthe less , th e rate of diffusion of low-weight-molecular com po un ds isslower and water activity is lower, thereby reducing their growth rate onfatty surfaces vis-ci-vis that on lean meat. Spoilage is thus first noticed onlean parts of the carcass.

1.2.2 Vacuiimstored mea tThe storage life and keeping quality may be extended by modifying thegaseous atm osph ere surrounding the m eat . Vacuum and modified atmos-ph ere packaging (M A P) are the two metho ds commonly used in wholesalemarketing to modify the gas atm osph ere. Both of these proce dures and con-ventional overwrapped (aerobic) trays are also used in retail marketing(H oo d an d M ead, 1993). Vacuum packaging is the preferred m ethod for thestora ge an d distribution of large pieces of chilled primals or wholesale cuts.Within the vacuum packs, the residual oxygen is rapidly consumed (below1%)by tissue and microbial respiration, and C 0 2 ncreases t o ab out 20%.Co m ple tely anaer ob ic conditions ar e rarely achieved, since all films in com -mercial use have a certain oxygen permeability. Thus, during storage,aerobic Gram-negative bacteria are replaced by the slow growing Gram-positive bacteria (Dainty et al., 1983; Ega n and Ro be rts, 1987; Da inty a ndMackey, 1992). Lactic acid bacteria are the most frequently isolated bac-teria fr om this kind of prod uct since they a re tolera nt to COz and low tem-per atu res. The se bacteria metabolize glucose as they d o in aerob ic me atto p rod uce lactic, isobutanoic. is op enta no ic and acetic acids. This givesm eat a so ur (cheesy, acid) taste and smell. T he accumulation of the se acidsoccurs mainly during the stationary phase and at some point in time themeat is rejected. O th er spoilage phen om en a, such as proteolysis or lipoly-sis ar e very limited o r nonexistent because the G ram positive bacteria havevery limited proteolytic activity.

For a variety of reasons (high initial contamination levels, film perme-ability, storage temp era ture , etc.), Gram -nega tive bacteria (Entero bacteri-aceae and even P seudomonas ) may o n occasions form large populations onvacuum pac ked beef cuts of normal p H (Dain ty et al., 1983; Gill an d Penney,1988). O n vacuum packed po rk, substantial num bers of enterobacteria maybe present throughout storage at -1.5 and 3 C (Gill and Harrison, 1989).These bacteria and Shewanella piitrefaciens have been reported to growreadily on fat and skin tissues of vacuum packed pork, irrespective of thep H of the muscle tissue. G row th of En terobac teriaceae on vacuum packedlam b has also been observed (Gill and Penney, 1985). When p H is 6,

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THE PHYSIOLOGICAL ATTRIBUTES OF GRAM-NEGATIVE BACTERIA 7growth of Shewanella putrefaciens, Alcaligenes. A ero m on as spp. or somespecies of Enterobacteriaceae may cause spoilage.1.2.3 Modified atmosphere packed meatCarb on dioxide is used in th e M A P of m eats because it inhibits microbialgrowth. G enerally, it is used in combination with N2 an d/ or 02 he per-centages used vary from 1 0% to 40 in the case of C 0 2 and 90% to 60%for oxygen. In gene ral, the higher the COzconc entration , the better in termsof inhibition of spoilage organisms. A long shelf-life may be attained in100% C 0 2 . How ever, a product may und ergo chemical changes that a redet rim enta l to mea t quality (Gill and M olin, 1991). Prevalence of slowgrowing lactic acid bacteria is responsible for the extended storage life.However, depending on several factors (pH, storage temperature, initialnumbers, packaging materials, etc.), Enterobacteriaceae and Aeromonasspp. may grow and cause spoilage (Gill and Penney, 198.5, 1988; Gill andHa rriso n, 1989; McM ullen and Stiles, 1993). According to several au tho rs(Asensio et al., 1988; O rdo iiez et al., 1991; D ainty and Mackey, 1992), En ter -obacteriaceae and Pseudomonas are more prevalent on MAP than onvacuum packed meat, especially on pork, their growth being favoured bystorage at ca. 5 C, an d by prio r conditioning in air.T h e growth of som e gene ra of lactic acid bacteria (e.g. Leuconostoc) mayb e favo ure d if oxygen is available. Ev en so spoilage organisms, such aspseudomonads, enterobacteria and Brochothrix can also compete effec-tively, and their numbers are higher than those in vacuum packed meat.Contamination during slaughter and meat conditioning has a large influ-enc e, since if conditions are n ot string ent , spoilage can be caused by severalgroups of bacteria. Lactic acid bacteria and Bvochorhrix are also dom inanton poultry stored in vacuum packs, C02, and nitrogen, sometimesaccompanied by cold-tolerant coliforms, Sh. putrefaciens and Pseudomonas(H oo d and M ead , 1993; Ka kou ri and Nychas, 1994).1.2.4 Meat productsCo m m inu ted mea ts (fresh minced m eat, certain types of fresh sausages andburger-type pro du cts) tend to have a sho rt shelf-life because of the qualityof th e raw ingredients (usually with a high load of micro-o rganism s), as wellas the effect of comminution. The spoilage flora of minced meat stored inair is dominated by Pseudomonas and to a lesser extent by E nterob acteri-aceae (von Holy an d Holzapfel, 1988; Lam brop oulou s et al. , 1996). Differ-en t trea tm ent s (addition of preservatives, vacuum a nd modified atm osp her epackaging) select other organisms such as lactic acid bacteria, Brochothrixand yeasts. Even so bo th groups of Gram -nega tive bacteria ar e normallypre sen t (von Holy a nd Holzapfel, 1988: Nychas an d A rko ud elos , 1990;

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8 THE MICROBIOLOGY OF MEAT AND POULTRYDrosinos and B oard , 1995b; Lam bropoulou et al., 1996). T h e microflora offresh sausages resembles that of minced me at tho ugh type of m eat, preser-vatives and storage temperature influence the selection of the dominanttypes. On some occasions (low sulphite levels, and high storage tempera-tures), Enterob acteriacea e ma y be involved in spoilage of bu rger-typ e prod-ucts.O n e of the major uses of mechanically recovered meat (MRM) is theextension of ground meat products. Significant growth of Pseudomonas,Achromobacter and Flavobacterium may occur in M R M (Swingler, 1982).In mo st cases, spoilage by Gram -ne gat ive bacteria of certain c oo ked bu tuncured meats (whole and restructured joints and poultry, ready meals)results fro m post-processing c ontam ination. Prod ucts stored u npac ked o rpacked in air permeable films tend to develop a spoilage flora dominatedby Pseudomonas (< 5 C) or environmental Enterobacteriaceae (highertemperatures). Occasionally Janthinobacterium lividum has been associatedwith formatio n of slime on roast beef. In vacuum and modified atm osp her epacks stored at high te m per atu res (10 C),Enterobacteriaceae may becom ea significant pro po rtion of the spoilage microflora (Pe nn ey et al., 1993).

Factors regulating microbial grow th underg o grad ual chan ge during thedrying and ripening of meat products. Temperature, a,, pH, and concen-tratio n of additives such as salt, nitrite an d nitr ate, modify th e sensory prop -erties of m ea t an d select particular microorganisms. M icrococcaceae, lacticacid b acteria (Carnobacter ium, Leucon ostoc) , Vibrio , Enterobacteriaceae,and non-motile Gram-negative bacteria (Psychrobacter) are the majorgroups present on cured raw meat p roducts.Bacterial spoilage of cured meats has been reviewed by Gardner (1983)and by Borch et al. (1996). Raw cured m eats include ham and bacon. Theoccurrence of Vibrio spp. as spoilage organisms in bacon is widely recog-nized. Ga rd ne r (1981) demon strated that th ere a re three groups of halophilicvibrios on bacon: Vib. costicola, V ib . costicola subsp. liquefaciens and anunidentified group (probably Vib. costicola) which is the most frequentlyfou nd in the slime on spoiled Wiltshire bacon. O th er Gra m -ne gativ e bacteria(Acinetobacter, A er om on as , Alcaligenes, Janthinobacterium and m embers ofEnte robac teriaceae) have been isolated from the surface of bacon, bu t theirrole in the spoilage has been difficult to prove though Gardner (1982)includes Acinetobacter among the surface spoilage flora of refrigeratedbacon. I nter nal taints such as pocket taint may be caused by Vibrio,Alcali-genes and, on occasions, Proteus inconstans (now Providencia alcalifaciensand Providencia stuartii) (Ga rdne r, 1983). Both Providencia and halophilicspecies of Vibrio have been isolated from bone taints of Wiltshire bacon.Providencia, particularly Prov. rettgeri, appears to be the major cause ofinternal taints in unpumped hams (Gardner, 1983). Spoilage (souring) ofpacked raw c ure d meats is mainly d ue to lactic acid bacteria. H2Sproductioncan result from the growth of Vibrio and members of Enterobacteriaceae on

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THE PHYSIOLOGICAL AlTRIBUTES OF GRAM-NEGATIVE BACTERIA 9vacuum packed bacon held at high temperatures. Providencia (Proteusinconstans) is responsible for cabbage odour due to the production ofmethane thiol from methionine. This type of spoilage is associated withbacon of high pH , and low salt content stored at high tem pe ratu re (Ga rdn er,1982,1983). T h e spoilage microflora of bacon in M A P appears to b e similarto th at of vacuum packed. Proteus and Providencia are also involved in thespoilage of sweet-cured bacon (Varnam and Sutherland, 1995).Spoilage of dry-cured hams by Gram-negative bacteria has also beenrepo rted. Blanco et al. (1994) isolated B urkho l der ia ( P seudom onas )cepaciafrom a spoiled sample (potato defect taint) of Par m a ham. T he strain p ro-duced th e o dou r compound responsible for the p otato defect. Ca nton i etal. (1994) and P apa (1994) fou nd tha t th e putrefactive type spoilage of rawhams was du e to E nterobacteriaceae (mainly to Prot. vulgaris).T her e ar e many types of cooked cured m eats (ha m , luncheon meats ,various sausages, etc.). Most vegetative bacteria are inactivated by heattrea tm en t, but post-process contamination occurs during slicing, portioningor skinning. Although a wide variety of Gram-negative bacteria are oftenrecovered from thes e products, they a re unable t o com pe te with lactic acidbacteria in vacuum or MA packages under refrigerated storage (Borch etal., 1996). O th er processed meats such as ferm ented sausages, dried m eats,canned uncured meats, shelf-stable canned cured meats, etc. are not nor-mally spoiled by Gram-negative bacteria.Since frozen m eat and m eat products (< -12 C d o not allow th e growthof micro-organisms, bacterial spoilage is related to the n um be r and type ofmicro-organisms before freezing as well as to the thawing conditions. It isoften stated that thawed-frozen meat is more perishable than fresh meat,especially because of th e drip exuded fro m thawed m eat . How ever, Lowryan d G ill (1985) concluded tha t w here handling before freezing, storage andthawing have been satisfactory, thawed meat is as microbiologically soundas those that have never been frozen and as such will spoil in exactly thesam e man ner for any given storage conditions.

1.2.5 Offa l sAlthough a great variety of bacteria are isolated from offals or varietymeats (edible offal or glandular meat) after refrigerated storage in air,pseudomonads commonly become dominant with Enterobacteriaceaebeing favoured by storage at 3C rather th an at 0C. Occasionally, Acine to -bacter and A e r o m o n a s are predom inant . Flavobacterium, Moraxella andAlcaligenes have also been found (Hanna et al., 1982a, b). Spoilage ofthaw ed offals is likely to follow the spoilage pat ter n of tha t of th e fresh ones.Thus temperature abuse favours the development of Enterobacteriaceae(Lowry an d Gill, 1985). Lactic acid bacteria and streptococci pre do m ina te

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10 THE M I C R O B I O L O G Y OF M E A T A N D POULTRYon spoiled kidneys and livers (beef and pork) in vacuum packs: Flavo-bacterium, Altero mo nas , Mo raxella , Acinetobacter and Sh. putrefaciens mayalso grow to significant num bers. Entero bacteriac eae app ear to fo rm a sub-stan tial fraction of the flora during t he sto rag e of vacuum packed po rk liversand sweetbreads (Gill and Jeremiah. 1991).1.2.6 Irradiated prod uctsIrradiation of poultry, pork and sausages with doses in the range up to 10kGy is permitted (WHO, 1994) in a number of countries as a means ofextending their refrigerated shelf-life and for inactivation of non-spore-forming pathogenic bacteria and foodborne parasi tes (protozoa andhelm inths). Most G ram -nega tive bacteria (i.e. Pseudomonas and Enter-obacteriaceae) are easily destroyed by low irradiation doses, but Psy-chrobacter imm obil i s , Moraxella and Acinetobacter are m ore resistant. T hespoilage flora of irradiated meat and poultry depends on several factors(initial flora, intrinsic and extrinsic facto rs, radiation do se and at m os ph erein the pack). Moraxellae (most of the strains probably Psychrobacter) aream on g the primary flora of irradiated poultry stored in air at low tem per a-tures (ICM SF, 1980). When the atm osph ere is anaerobic (vacuum and M Apack), the relatively radiation-resistant lactobacilli become dominant inpoultry, pork and beef. A e r o m o n a s spp. were isolated from tem per atu re-abused vacuum pack ed irradiated pork by Lebep e et al. (1990). No nm otileorganisms (probably Psychrobacter imm obi l is ) were dominant on storedirradiated poultry and responsible for the spoilage of irradiated Viennasausages (Shaw an d Latty. 1988).

1.3 Taxonomy and physiology of Gram -negative bacteria associated withspoilage of meat and meat products1.3.1 Gra m-neg ative aerobic moti le rodsG e n u s Pseudomonas. T h e genus Pseudomonas has been subdivided intofive groups on th e basis of nucleic acids similarity studies (Palleroni, 1993).T h e first rR N A similari ty group (G ro up I ) includes bo th fluorescent (Ps . lu-orescens biovars I to IV. Ps. putida biovar A and Ps. lundensis) an d nonflu-oresce nt species (P s . f ragi biovars 1 and 2) which are responsible for lowtem pe ra tur e ae rob ic spoilage of me ats. In practice, these usually account formore than 50% and som etime s up t o 90% of the spoilage flora (Molin andTern strom , 1982,1986: Shaw and Latty. 1982,1984,1988; Prieto etal. , 1992a).Pseudomonas fragi is the most com mon pseud omo nad on spoiled me at w ithan incidence ranging between 56.7 and 79%. Pseudomonas lundensis maybe considered as the second com pon ent of the Pseiidomonas association of

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THE PHYSIOLOGICAL ATTRIBUTES OF GRAM-NEGATIVE BACTERIA 11spoiled m ea t. It is a new species propo sed by M olin et al. (1986) to accomm o-da te strains atta ch ed to o n e of th e ma jor clusters defined by numerical analy-sis in th e studies of M olin and Tern strom (1982) and S haw and Latty (1982).Biovars I and I11 of Ps. fluorescens are also frequently reported onmeat, their incidence being lower than that of Ps. lundensis (5-13%).Pseudomonas pu f ida , which can occur on red meats (less than 5% of thepseudom onad population) does not seem to be of grea t importance in meatspoilage though the species, Ps. sfutze ri (nonfluorescent member of GroupI), Burkholderia (Ps eud om ona s) cepacia (mem ber of G rou p 11) and Ps. flu -orescens have be en associated w ith spoilage of loins pack ed in M A and inpermeable films (Ahmad and Marchello, 1989a, b). Burkholderia cepaciahas also be en associated with th e spoilage of dry cured h am .T h e ab ility of Pseudomonas spp. to grow in refrigerated m ea t is du e partlyt o the metabolism of glucose to 2-0x0-gluconate or glucon ate via theEntn er-Do udoro ff me tabolic pathway (F arber and Idziak, 1982; Nychas etal., 1988). Th ese com pounds ar e not readily assimilable by o ther m icro-organisms, and pseu dom onad s can build up a n extracellular energy reservefor use when glucose is de ple ted . 2-0x0-gluconate o r gluconate have b eenpropo sed recently as good spoilage indicators (Dainty, 1996). Dro sinos andB oar d (1994) have foun d metabolic differences amon g Pseudomonas spp.from m eat. Th ey proposed that dominance of Ps. fragi over Ps. lundensisand Ps. fluorescens is du e to its ability to m etabolize crea tine and crea tinineun de r aerobic conditions. In br oth , pseudom onads use amino acids andlactic acid as the next choice of s ub stra te. Ap parently, lactic acid was usedin broth culture only after glucose had been depleted (Drosinos and B oar d,1994). There are contradictory reports on this issue (Molin, 1985). Inrelation to their spoilage metabolites, pseudomonads produce dimethylsul-phide , but not H2S.This feature distinguishes them from the E nterob acte-riaceae. The inability of Ps. fragi to metabolize creatine and creatinineun de r modified atmospheres in meat broth in the laboratory (Drosinos andB oa rd , 1994) could partly explain the failure of th ese organisms to becom edominant in MAP.

Genus Shew anella. T h e taxonomic status of Shewa nella putrefaciens is notclear. This species has be en assigned to th e ge ne ra Pseudomonas (Ps .putre-faciens) and A l f e r o m o n a s A h .putrefaciens). MacD onell and Colwell(1985)classified this organism in a new genus Shewanella which was estab-lished around this species. It also includes Sh. hanedai (Al teromonashanedai) and Sh. benfhica. Two new species (Sh. alga and Sh. col-welliana-Alt. colw elliana) have be en prop osed also. T h e validity of the newgenus is su pp or ted by the analysis of th e 5s rR N A sequence and by data onpolar lipids, fatty acids and isopreno id qu inones. Ho we ver, the inclusion ofShewanella in the family Vibrionaceae is still controversial.

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THE PHYSIOLOGICAL ATTRIBUTES OF GRAM-NEGATIVE BACTERIA 131.3.2 Gram -negative non mo tile aerobic rodsGe n u s Flavobacterium . T he taxonom y of th e flavobacteria was reviewedin the Prokaryotes (Holmes, 1992). M ore recently, Be rna rde t et al. (1996)provided an amended description of the genus Flavobacterium and pro-posed new combinations for seven described species. In addition, a newspecies, E hydatis was proposed for Cytophaga aquatilis. The emendedgenus Flavobacterium contains bacteria tha t ar e motile by gliding, produ ceyellow colonies and are widely distributed in soil and freshwater habitats.Because of the taxonomic and nomenclatural changes that have been pro-posed, it is difficult to establish the identity of the Flavobacterium spp.associated with meat spoilage.Genera Moraxella, Acinetobacter and Psychrobacter. M otile and non-motile, nonpigmented, nonfermentative Gram-negative saprophytic bac-teria were classified in the first edition of Bergeys Manual of SystematicBacteriology as Achromobacter (Bergey et al., 1923). Thornley (1967)grouped the nonmotile ones in the genus Acinetobacter. La ter, this genuswas modified; the nonmotile, oxidase-negative strains were assigned toAcinetobacter spp., and the nonmotile, oxidase positive bacteria toMoraxella because of their resemblance to Moraxella-like spp. Morerecently, Juni and Heym (1986) described a new species, Psychrobacterimmobi l is , which embraces some of the oxidase-positive strains which ar eunrelated to th e tru e moraxellas, as shown by D N A transformation assay.Since 1996, thr ee new Psychrobacter spp. (Psy . frigidicola, Psy. urativorans,and Psy. glacincola have bee n described. Rossau et al. (1991) propo sed t hecreation of a new family, Moraxellaceae, on the basis of DNA-rRNAhybridization studies, to accommodate the above cited micro-organismswhich were previously included in the family Neisseriaceae. This new familyis divided into two main groups, Acinetobacter and ano the r superclusterwith four subgroups: th e authentic Moraxella spp., M . osloensis, M . atlantaeand a heterogeneous group containing M. phenylpyruvica, Psychrobacterimmobi l is , and allied organisms. Catlin (1991) proposed the new familyBranhamaceae for Moraxella and Branhamella.T he genus Acinetobacter is biochemically an d genetically heterogeneou s.D N A hybridization studies have identified 1 8 phenotypically distincthybridization groups (genospecies) an d species names have b een proposedfor seven of the se group s. Acinetobacter john sonii was th e species found byShaw and Latty (1988) on poultry an d aerobically stored red meats. Acine-tobacter lwofii was the predom inant species isolated from spoiled meat byGennar i et al. (1992).Several auth or s (Shaw and Latty, 1988; G en na ri et al., 1989, 1992; Prie toet al., 1992b) concluded that most of the strains isolated from protein-aceous foods and formerly identified with Moraxella and Moraxella-like

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14 THE M I C R O B I O L O G Y OF M E A T A N D POULTRYmicro-organisms were Psychrobacter immobilis. Prieto et al. (1992b) iso-lated this species and M . phenylpyruvica throug hou t th e storage life of lam bcarcasses. It should be noted that the latter species appears to be closelyrelated to Psy. imm obil is . In fact, Bo wm an et al. (1996) have pro pose d th atM . phenylpyruvica should b e transferred to the genus Psychrobacter as Psy.phenylpyruvicus .It is often assum ed tha t strain s of th e family Moraxellaceae form a sig-nificant po rtion of any spoilage flora on aerobically st ore d me at. It has alsobeen reporte d howev er, that their imp ortance is overstated as they oftenoccur only as a minor p art of the m icroflora and have a low spoiling po ten-tial ( Er ib o and Jay, 1985; G enn ari et al., 1989; Prie to et al., 1992b). Never-theless, it has bee n suggested th at a cinetob acters and Psy. immo bil is couldplay a lipolytic role when they form large po pul ation s or in irrad iated foo ds.This group of bacteria were characterized as poor competitors with alimited enzymatic arsenal (Nychas et al., 1988). They cannot metabolizehexoses but use am ino acids and organic acids as carbon and energ y sources.T h e substrate s used by Psychrobacter are not known (Dainty and Mackey,1992). Acinetobacter use amino acids first , and then lactate. They oftenoccur on m eats toge ther with Pseudomonas , mainly on surfaces of fa t, o r onme ats with interm ediate pH . Thei r incidence declines as storage progresses,when conditions beco me stringen t. Eve n thou gh they use amin o acids, theirmetabolic end products are not offensive. In pure cultures, off-odoursdescribe d as fishy are produ ced. The ir comm ercial imp ortance could com efrom their capacity to restrict (under conditions of maximum cell density)oxygen availability to pseudomonads and Sh. putrefaciens, enhancing theirspoilage potential as they start to attack amino acids an d produ ce H2S.In taxonomic studies, strains identified with Psychrobacter immobiliswere capable of producing acid from a large num be r of carbo hyd rates. Aswith oth er Gram -negative bacteria, the gro up is unable to use many com-pounds as a carbon source. Atypical nonmotile variants of Pseudomonasfragi are no t uncommon on spoiled me at (Shaw an d Latty, 1988; Prie to etal., 1992b). Th es e variants of Ps. fragi ar e closely related to th eir m otile rela-tives, as they sh are many m etabolic properties. Likewise they d o not disap-pear from stored meat as spoilage progresses. In poultry meat, strains ofMoraxellaiAcinetobacter were isolated more frequently from leg (higherpH ) th an breast (M cM eekin, 1975,1977). They also appe ar t o prefer fat sur-faces (Shaw an d Latty, 1988).1.3.3 Facultatively anaerobic Gram -negative rodsFamily Eizterobacteriaceae. A total of 29 genera (14 traditional and 15additional) are included in this family (B ren ne r 1992). Of th ese , Citrobac-ter, Enterobacter, Hafnia, Klebsiella, Kluyvera, Proteus, Providencia andSerratia are associated with meat spoilage (Table 1.2). Escherichia coli and

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THE PHYSIOLOGICAL ATTRIBUTES OF G R A M - N E G A T I V E BACTERIA 15Table 1.2 Ge ner a and species of Enterobacteriaceae found in significant numbers in spoiledmea t and me at products by several authorsGe ner a Species Meat productsCitrobacter

Enter o bacter

HafniaKlebsiella

KluyveraProteus

Pro videncia

Serratia

Citrobacter spp.f reundi ikoseri (C. diversus)aerogenescloacae complexagglomerandErwiniaherbicola complex

Enterobacter spp.

Hafnia spp.alveiKlebsiella spp.pneumoniae subsp.

pneumoniae subsp.pneumoniaeozaenaeKluyvera spp.Proteus spp.vulgarismirabilisProvidencia spp.alcalifacienssruartiirettgeriSerratia spp.liquefaciensmarcescens

Vacuum and M A packed beef lamb a nd poultry,a i r s tored meat an d m eat productsLamb , pork , beef , high p H red m eat ( in a i r , M Aand vacuum), ground meat (a ir and vacuum),poultry , offals (in PV C and vacuum). freshsausages, raw cured m eats (pa cked)Pork, high p H red m eat , ground m eat , poultry,premarinated beef and raw cured meats(in vacuum ). Red meats an d poultry (in air)Beef (in vacuum ), poultry and red meats (in air)

Beef (in air)Bacon (vacuum packed ), raw ham s, cured meats

Internal taints (bacon and h am) , raw cured hamsand bacon (in vacuum a nd M A )

La mb , pork , beef, poultry and high pH meat(in vacuum), high p H meat . poultry, fat , groundpork and fresh sausages (in air)

Yersinia have also been reported. Table 1.2 shows the main genera andspecies of Enterobacteriaceae involved in the spoilage of meat and meatproducts.T h re e species of Citrobacter are curren tly recognized: C. freundii, C. koseri(also called C. diversus and Levinia rnalonatica) and C. arnalonaticus. Theclassification and nomenclature of the genus Enterobacter have undergonemajor changes in the last decade bu t th e situation is still confused. T he re a re14 named species of Enterobacter and probably additional species will beadded f rom the Ent. cloacae and the Erwinia herbicola-Ent. agglomeranscomplexes. T h e latter comprises 12 or more DNA hybridation groups someof which have been assigned to new genera (i.e. Pantoea-Pan. agglornerans).Transfer of Ent. aerogenes to th e genus Klebsiella has also been proposed.Escherichia, which currently contains five species, is a typical mem be r ofthe family. A strong relationship both genetic and phenotypic existsbetw een this genus (mainly E. coli) and Shigella.T he genus Hafnia contains only o ne species (Haf alvei) though two sep-ar ate genospecies ar e recognized. This bacterium has bee n described unde r

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16 T H E MICROBIOLOGY OF M E A T A N D POULTRYseveral names including Enterobacter alvei. T he genus Klebsiella containsfive species, with K. pneumoniae including thr ee subspecies. Two of t he se,K. pneumoniae subsp. pneumoniae and K . pneumoniae subsp. ozaenae, areoften isolated from spoiled meat and meat products. It is difficult to sepa-rate this genus from Enterobacter. Strains of Klebsiella are nonm otile, butnonmotile strains of Enterobacrer also occur. Kluyvera is not commonlyassociated with spoilage of meat. However, Kleeberger et al. (1980)reported that Kluyvera (not identified to species) was dominant on beefs tored a t 7 C and 15 C. T h e species currently recognized ar e Kluy. cryo-crescens and Kluy . ascorbata.

The taxonomy of Proteus and Providencia has undergone markedchanges in the last years. At present, four species are recognized in thegenus Proreus, two of the se being associated with m eat spoilage (P ro t. vzd-garis biogroups and Prot. mirabilis). T he swarming phe nom eno n is charac-teristic of Proteus. Three of the five species of Providencia (Prov.alcalifaciens, Prov. stuartii and Prov. rettgeri) ar e involved in the spoilage ofm eat products. Providencia alcalifaciens and Prov. stuartii were previouslyincluded as two strains A+B) n o ne species of Proteus (Proteus inconstans)and Prov. rettgeri was also formerly listed as a Proteus sp, Ten species ar elisted within the genus Serratia. Serratia liquefaciens is frequently and Serr.marcescens only occasionally involved in meat spoilage. Production ofextracellular enzymes, salt tolerance and relatively low minimal growthtem pe rat ur e ar e characteristics of th ese species. In vacuum packed m eat ,Yersinia spp. and Yer. enterocolitica may form a significant part of themicroflora. T h e latt er species is pathogenic to h um ans , the infection usuallybeing waterborne and food borne.Alth oug h most attentio n is generally paid to th e pathogenic pr oper ties ofparticular g ener a of En terob acteria ceae , som e m em bers of th e family con-stitute an important spoilage group when conditions favour their growthov er that of pseudom onads. Th is gro up includes Serr. liquefaciens, H a t alveiand Ent. agglomerans. Similarly to pseudomonads, they use glucosealthough so m e (e.g. Enterobacter) ap pe ar to have secondary preferences formetabolic intermediates such as glucose-6-P. Amino acids are degradedafte r carbo hyd rates with the release of amines, organic sulphides and H2S.A characteristic of this gr ou p is their ability to p rod uc e H2S, but notdimethylsulphide, a feature that distinguishes them from pseudomonads.D F D m eat is not suitable for vacuum packaging d ue to its high pH , andits tendency to spoil due to H*S, which combines with myoglobin to formsulphmyoglobin (greening). Several species are known to grow on DFDunder anaerobiosis: Serratia liquefac iens, Ha alvei and Yersinia spp.requ ire p H 6 for growth. These organisms spoil mea t d ue t o production ofH2S and greening, the exte nt of which is accentuated in meat rich in myo-globin. Inhibition of these bacteria is achieved by combined use of pH , tem -peratu re and anaerobiosis, but they can grow if these con ditions change.

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THE PHYSIOLOGICAL ATTRIBUTES OF GRAM-NEGATIVE BACTERIA 17Even though Enterobacter has higher affinity for glucose tha n lactic acidbacteria, p H cond itions and metab olic characteristics (ability of lactic acid

bacteria t o get energy from th e use of arginine) preclude Enterobacter frombecoming dominant (McMeekin, 1982). Enterobacter use serine concur-rently w ith glucose. O nc e glucose has b een de plete d, glucose 6-P, lysine,arginine and threon ine ar e used. Serratia liquefaciens produces acetic acid.Aeromonas cause spoilage when traces of o xygen are present (0.1/o . Prov-idencia (P ro v. rettgeri) in cases of tem pe ratu re abuse can sta nd salt concen-tration up to 8 % . Together with Salinivibrio (V ib ri o) costicola, are knownto spoil hams (bone taint) . It is able to metabolize methionine tomethanethiol (cabbage odour).

Family Vibrionaceae. The most recent edition of Bergeys Manual(Bauman and Schubert, 1984) included four genera in the familyVibrionaceae: Vibrio , Ae rom on as, Plesiomonas and Photobacterium. Inrecent years, this family has undergone marked revision and a number ofoth er g en era have be en classified in the family. Aeromonas has bee n placedin a new family, Aeromonadaceae, and Plesiomonas shigelloides includedwithin the genus Proteus as Prot. shigelloides (MacDonell and Colwell,1985). O n the basis of DNA -DN A hybridization stud ies, Janda (1991)recognized 13 species (also called hybridization groups) of Aeromonas ,so m e of which have be en subdivided in to subspecies. Ad ditional hy bridiza-tion groups have also be en proposed.Both Aeromonas and Vibrio have b ee n involved in spoilage of m eat an dm eat products. U nd er aerobic conditions, motile ae rom on ad s, mostly Aer.hydrophila and Aer. caviae, are unable to compete with faster growingorganisms such as pseud om onad s. W hen low levels of oxygen a re available,how ever, they may becom e significant contam inants o n me at of high pH .Ch ang es in the nom enc lature and classification of Vibrio make it difficultto kno w which Vibrio spp. are implicated in the spoilage of cured me ats. Itwould appear that the three groups of Vibrio strains studied by Gardner(1981) can now b e assigned t o Vib . cosricola. Recently, Mellado et al. (1996)proposed tha t this species be transferred to a new gen us, Salinivibrio, as Sal.costicola.Spoilage of products is carried out by bacteria belonging to the genusVibrio , which are favoured by their high salt concentrations. They spoilW iltshire ha ms a nd bacon causing rib taint. T he ir me tabolic contributiont o spoilage and sensory faults of products see m to b e very low, even thoughthey seem to have a strong spoilage potential. Vibrios are psychrotrophswhich can redu ce nitrate and nitr ite, and som e (Vib . costicola subsp. lique-faciens) deg rad e casein and gelatin. S om e strains also produce HzS, andothers can form slime due to d extran production.

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18 THE MICROBIOLOGY OF MEAT AND POULTRY1.4 Origin of Gram -negative bacteria in m eat and meat productsT h e microbiology of red m eat and poultry is determ ined by the conditionsund er which the animals a re reare d, slaughtered and processed. T he mostcritical stages for meat contamination are the slaughter procedures but aconsiderable amount of contamination is also possible during subsequentoperations. With catt le and sheep , the major source of the psychrotrophicspoilage bacteria app ears to be the hides and fleece of animals co ntam inat edby soil and water. Pseudomonas. Acinetobacter and Moraxella were themost com mo n psychrotrophs foun d by Newton et al. (1977,1978) on hidesand fleece, as well as on mea t. Bo th habitats and vegetation are importan treservoirs of the majority of Gram -nega tive bacteria associated with meatspoilage (T able 1.3).In poultry, th e psychrotrophic flora is carried principally on t he fea ther s,but is also found on th e skin. Most of these bacteria are d estroye d duringscalding. Levels of psychrotrophs and Enterobacteriaceae can increase

Table 1.3 Habitats of Gram-negative bacteria associated with meat spoilageAer obic rods Hab itat Facultatively Hab itat OriginanaerobicrodsAcin eto bacter

AlcaligenesAlteromonusFlavobacterium

JanthinobacteriumMoraxella

Pseudomonas

PsychrobacterShewanella

Ubiq uitous , soil ,water and sewage,hum an skinUbiq uitous , soiland waterMarine envi ronmentsWidely distribu ted innatur e. especially inwaterSoil and waterMucosal surfaces

Ubiquitous,fresh and sea water,soil, plants, etc.Aquatic habitats,fish and p oultryAquat ic and marinehabitats

Cirrobacter

EnterobacterHafniaKlebsiella

KluyveraProteus

Providencia

SerratiaAeromonas

VibrioSalinivihrio

Soil , water and sewage N Fma n an d animalsSoil, water, sewage and N FSoil, water and sewage NFIFmam mals and birdsSoil . vegetat ion and NFIFwate r. wild a nddomestic animals,humansSoil, wate r an d sewageIntest ine of humans NFIFand animals.ma nure, soil and

polluted watersSoiled bedding (faeces N Fand ur ine)water and envi ronmentPlants, water and soil , N Fsmall mam malsAquat ic envi ronments ,widely d istribute d inthe environmentAquatic and m arine habitatsHypersal ine

plants

environments~~ ~ ~~~

F, faeca l origin; NF, not of faecal origin; NFIF, both (Mossel et al., 1995).

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THE PHYSIOLOGICAL ATTRIBUTES OF G R A M - N E G A T I V E BACTERIA 19during defeathering. In addition, the water used in washing and chillingcarries a psychrotrophic pop ulation which may rec ontam inate th e poultrycarcasses. Furth er recon tam ination occurs at su bsequ ent stages of process-ing (B rem ner a nd Jo hnston , 1996). Lahellec an d C olin (1979) showed th atpseudomonads form a very small proportion of the psychrotrophs on theoutside of live chicken and turkeys and that Acinetobacter and to a lesserextent Flavobacterium were dominan t. Contam ination with pseudom onadsoccurred during processing from water, hands and materials, and theybeca me dom inant am ong th e psychrotrophic flora at the en d of chilling.T he scalding trea tme nts applied to pigs largely destroys the Gra m-n ega-tive organisms, but the carcasses are then recontaminated from the pro-cessing equipment. Gill and Bryant (1992) demonstrated that spoilagebacteria (Pseudomonas, Acinetobacter and Moraxel la) grew to highnu m be rs in th e accumulated detritus of the dehairing e quipm ent and con t-am inated th e circulating waters. Furth erm ore , they observed that th e com-position of the flora was largely unaltered after the singeing operations.A e r o m o n a s spp. (Aer . hydrophi la and Aer. caviae) also grew well in thisniche, the organisms being then s pread throug hou t the dressing and bre ak-ing lines wh ere they grew furt he r (Gill an d Jon es, 1995). Th es e auth ors iso-lated both species from most of the samples obtained from the eq uipm entin pig slaughtering plants.Patterson and Gibbs (1978) reported that Gram -negative bacteria (nonEnterobacteriaceae) were widely distributed in abattoirs (lairage, slaugh-ter hall , chill room and boning room ), P seudomonas being p resent a t mostsites. M em ber s of the family Enter ob acte riace ae involved in m eat spoilagew ere isolated fro m all sites except carcass wash wa ter an d air samples in thelairages an d boning ro om . B ot h gro up s of bacteria ar e successful coloniz-ers of wet e nviron m ents in the structural and work surfaces within the aba t-toir (Newton er al., 1978). Nortje er al. (1990) investigated the particularcon tribu tion of e ach link in the production chain t o the m icro biai profile ofthe final products (carcasses and minced meat). In the abattoir, Enter-obacteriaceae and P seudomonas were the domin ant psychrotrophs, E nt er -obacteriaceae and micrococci at the wholesaler, and micrococci andpseudomonads at the retai lers . They concluded that Enterobacteriaceaeare com mon psychrotrophs in the m eat production chain possibly originat-ing from the abatto ir and wholesale environmen ts. Using the contami-nation index, Gustavsson an d B orch (1993) studied the con tam ination ofbeef carcasses by psychrotrophic P seudomonas and Enterobacteriaceaedu ring sla ugh ter, chilling an d cutting. Ra pid chilling was identified as a criti-cal processing step . De hid ing an d chilling in cold sto rage roo m s were alsoimplicated as critical processing steps, with respect to aerosol contami-nation and surface cross contamination. Pse udom ona s JIuorescens wasdom inant in atm osph ere samples, as well as those obtained from meat andthe processing environment. Other Gram-negative spoilage bacteria

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20 THE MICROBIOLOGY OF MEAT AND POULTRY(P s. frag i, Ps. lundensis, Acinetobacter and Psychrobacter) were detectedon me at a nd/or in atm osph eric and env ironm ental samples. Carcass washwater was identified by Sierra (1991) as the origin of the fluorescentpse ud om on ads fo un d on freshly dressed lam b carcasses. T h e association ofthes e bacteria with fre e water on surfaces has also been repo rted by D rosi-nos and Board (1995a). A wide spectrum of Gram-negative bacteria(P seu do m on as , Acinetoba cter, Serratia, Enterobacter, Proteus and Vibr io)were recovered by von Holy et al. (1992) from en viro nm en tal samples in am eat processing plant which ma nu fac ture d vacuum-packed , Viennasausages. Th ey concluded that the psychrotrophic nature and simple nutri-t ional requ irem ents of the gene ra enab led them to persist a nd/o r multiplyin/on water, c on den sate , soil, eq uip m ent surfaces, brine solutions and moistfloors. Although Pseudomonas do not grow in brines, they survive in thisenvironment (Gardner, 1982). The probable source of Vibr io in curedmeats is curing brines. Gardner (1981) discussed the origins of these bac-ter ia and identified t he following as possible sou rces of con tamina tion: saltused in brine m anu fac ture , fish meals included in th e diet of pigs and ev enagonal bacteraemia.

1.5 Methods of isolation and identificationT h e preliminary differentiation of th e main groups of Gram -negative m eat-spoiling bacteria is based on a simplified key (Tab le 1.4).

1.5.1Isolat ion. Pseudomonas spp . grow well on non-selective me dia (i.e., platecount agar and blood agar) and on routine primary isolation media (i.e.M acC onk ey and E osine Methylene Blue A gar) when incubated a t a tem-pe ra tur e suitable for their growth (Jep pe sen , 1995).When me at and m eat products a re analyzed, strains of Pseudomonas arecommonly isolated from plates of Plate C oun t Aga r (PC A ), Try ptoneGlucose Yeast Ex tract Ag ar, or Stand ard O ne Nutrient Agar, after incu-bation a t 4-30 C for 2-10 days (Mo lin an d Tern strom, 1982 ,1986; G en na riand D ragotto, 1992; Prieto et al., 1992a; von Ho ly et al., 1992; Ham ilton andA hm ad , 1994). P C A plus 1 % (w /v) NaC l has also been used for their iso-lation (Shaw and Latty, 1982; 1984).Specific media fo r the isolation of Pseudomonas spp. ar e available. Th emedium B of King et al. (1954) is frequently used for the isolation of fluor-escent Pseudomonas spp. Pyoverdin production is enh anc ed and the charac-teristic colonies can be identified un de r U V light.A selective medium, with cephaloridine, fucidin and cetrimide as selec-tive agents, (C FC agar) was described and tested with poultry me at (M ead

Isolation and identification of species of Pseudomonas

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Table 1.4 Simplified key for the differentiation o f the main groups of Gram-negative bacteria which are commonly related with the spoilage of meatand meat uroducts

hactcriaceac

Cell shapeFlagellarOx1dase testMoti l i ty1Jtiliiation o f

arrangement

glucose in theHugh & Leifsonmedium

C + C content ofDNA (rnol%)

Argininedihydrolase

DNAscPigmentation ol

co onichSensitivity t u the

vihriostaticcompound 0 129

Na required for(or htimulates)growthcommon antigenEnterohacterial

Straight or Straight or Straight or Kod or Straight orPolar Polar Polar Pcritrichouh N o n e

curved rod curved rod curved rod cocci curved rodShort rod Rod orN o n e N o n e

cocciRod o r

cocciN o n e

+Oxidative Oxidative Oxidative Oxidative Oxidative

55-64 4447 37-50 52-68 31w2

+ +/-None Pink N o n e None Yellow

+/

4 w 4 7 3 x 4 7

Oxidalivc

4 1 4 7

N o n c

Straight rodIcritrichouslN o n ctiFerriientiltive

3X-001 /-+I-NonelOther\

V111rio/ Arrorrioriir.~Siil ir ir vrhrio

(urved or Short rodPolar Polar

htriiight rod

+Fcrnientativc Fermentative

3x 51 57-63- I t +/-+/Non e None

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22 T H E M I C R O B I O L O G Y OF M E A T A N D P O U LT R Yand A dam s, 1977). It allows the enu me ration of both pigmented and no n-pigmented P se u d o m o n a s spp. Mead and Adams (1977) found that themedium effectively suppressed Gram-positive bacteria and supportedgrowth of Pse i idomonas spp. (including Ps. aeruginosa) , whilst inhibitingmost othe r G ram-negative bacteria. Shewanella putrefaciens was only partlyinhibited. CF C medium can be used for enu meration of P se u d o m o n a s spp.in various types of food (M ea d , 1985). T h e med ium is inoculated by surfaceplating a nd incub ated for 48 h at 25 C. T he re ar e two types of colonies: pig-mented and non-pigmented. 2-5 mm in diameter. Confirmatory tests arenot usually required but in cases of doubt flooding the plates with oxidasereag ent c an be used to distinguish Pse i idomonas spp. from oth er organismswhich m ay be p rese nt (i.e. in som e cases Ser. liquefaciens and yeasts).M em bers of Enterobacteriaceae, present on meat packed und er vacuumor in modified atmo sphe res grow on C FC m edium thereb y giving inflatedcou nts of P se u d o m o n a s . In or de r to differentiate between these two groupsof organisms, S tanbridg e and B oa rd (1994) modified the C FC me dium byaddin g arginine (1Yo wiv) and ph enol red (0.002% wiv). T he pseud om on-ads produce am mo nia from arginine (a pink colour develops in and aro undthe co lonies), wh ereas En terobac teriaceae generally do not use this sub-strat e and pro duce yellow colonies. How ever, with high Entero bacteriac eaecounts and with a spiral plating machine as inoculator, the acid producedby the E nte rob acte riac eae caused yellowing of th e medium and neutralizedthe alkaline drift of pseu dom onad s (Stanb ridge and B oar d, 1994).Purification of strains is commonly achieved by streaking onto NutrientAg ar. Strains are easi ly maintained for 1-3 mon ths on N utrient Agar slopesa t 5 C. Lyophilization or liquid nitrogen techniques have to be used forlong term storage.Preliminary characterization of isolates. Ea ch isolate is tested for oxidasereaction , Gr am reaction an d cell morphology, as well as for production ofacid from glucose (modified H ugh and Leifson me dium ). Isolates which ar eoxidase-positive, G ram -nega tive rods an d pro duce acid from glucose oxida-tively are de em ed to be P se u d o m o n a s spp.Identification of isolates. T h e identification of P se u d o m o n a s spp. is prob -lematic. Simple dicho tom ou s det erm inat ive keys ar e of limited use. Takinginto account the description of species in the Bergey's M a n u a l of System-atic Bacteriology (Pa llero ni, 1984) an d t he later description of Ps. lundensis(Molin et al., 1986), different system s employing tables including m ultiplecharac ters have b een suggested. Ho we ver, many of the strains of m eatorigin show phenotypic properties that do not fit the description of recog-nized species. From various studies of numerical taxonomy ofP se u d o m o n a s strains of meat origin (Molin and Ternstrom, 1982, 1986;

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THE PHYSIOLOGICAL ATTRIBUTES OF G R A M - N E G A T I V E B A C T E R I A 23Shaw and Latty, 1982, 1984), several simplified schemes for the differen-tiation of clusters and species have been described.

The scheme of Molin and Ternstrom (1982) includes eight characters(fluorescent pigments, gelatinase, acid from cellobiose and maltose,utilization of sac ch arate , treh alo se, meso-inositol and benzylamine). F romthe data of Molin and Ternstrom (1986) and Molin et al . (1986), asimplified sch em e with 16 biochemical tests (acid from m alt os e, assimi-lation of D-a rabino se, DL-ca rnit ine, creatine, deoxycholate, D -glucu r-on ate , 4-hydroxybenzonate, hydroxy-L-proline, inosine, meso-inositol,malonate , D -mann itol , muc ate , D-quinate , D-saccharate and D-xylose)has been devised (D rosino s and B oa rd, 1995a).Shaw and Latty (1982) employed 12 characters (fluorescent pigments,and utilization as carbon sources for growth of: benzylamine, butylamine,creatine, malonate, hippurate, mannitol, mucate, saccharate, pimelate,rham nose , and mesaconate). T hey described a computer-assisted prob a-bilistic identification techn ique with 18carbon source utilization tests, to becomplemented with extra carbon source tests for strains not correctlyidentified.W ith da tab as es from th e pheno typic description of species and taxa ofPseudomonas , software for the probabilistic identification of field isolateshas been described (P rieto , 1994).The different species and biovars of fluorescent Pseudomonas can bereasonably identified with a scheme of 15 tests (de nitrification, levan pr o-duction, phena zine pigm ent, and assimilation of L-arab inose, D-xylose, tre -halose, mucate, erythritol, myo-inositol, mesaconate, etha no l, an thra nila te,histamine, trigonelline and quinate) and a computer assisted probabilisticm etho d (G enn ari and D rag otto , 1992). So m e additional tests (assimilationof nicotinate, mannitol, L-tryptophan and D-galactose) are necessary forth e differentiation of biovars A and B of Ps. putida .T h e oxidative capacity of 95 organic substrates included in the Biolog G Nmicroplates (Biolog, Hayward, USA) is less discriminating between thedifferent species of Pseudomonas than the conventional tests of carbonsource utilization (Ternstrom et al., 1993).A semi-automated system of identification based on electrophoresis ofintracellular proteins ( A M B IS ) is useful for the differentiation of referencestrains belonging to biovars I and I11 of Pseudom onas f luorescens (Roweand Fin n, 1991), although som e strains of the oth er th ree biovars of thisspecies cluster a t values of relatively high similarity.A fte r standardization of cultural and chem ical techniqu es, th e fatty acidprofiles (mainly 2- and 3-hydroxy fatty acids) can be used for assigningstrains of Pseudomonas to o ne of th e six major groups described by Ste ad(1992).R ap id detection of add ed reference strains of Ps. fluorescens, Ps. f ragi andPs. aeruginosa (o n m ea t surfaces at levels higher than 104-105 cfu/cm2) is

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24 THE MICROBIOLOGY OF MEAT A N D POULTRYpossible with different enzyme-linked immunosorbent assays using poly-clonal antibodies against live cells, or against protein F of the cell envelopeof Ps. Jluorescens AH-70 (Gonzalez et al., 199 4,1996 ). A similar assay, tha tem ploy s antibod ies against hea t-killed cells of Ps. Jluorescens (and adsorbedwith Ps. aeruginosa) has been used for the specific detection of Ps. Puo-rescens at levels over 3 X lo5 bacteria per ml of an homogenate of meat(Eriksson et al., 1995).Several rRNA targeted probes for the detection of Pseudomonas spp.(mainly those belonging to G ro u p I) have been described (Braun-H ow landet al., 1993; Ludwig et al., 1994). A variety of hybridization methods areavailable.A polymerase chain reaction (PCR) for the detection of Pseudomonasand other Gram-negative and Gram-positive bacteria has been designed(Venkitanarayanan et al., 1996). It em ploys two primers (23 bases and 20bases, respectively) selected from the 2 3s r D N A seq ue nce of Ps. aerugi-nosa. PC R p roducts ar e dete cted by gel electrophoresis. It seems that levelsof spoilage bacteria over 104-105 cfu/cm2 on me at surface can be detec ted,without interference from m eat D N A (V enkitanarayanan et al., 1996).Also, a P C R protoco l, with genu s specific primers, has be en designed forthe assessment of the initial contam ination-level of Pseudomonas (van de rVossen a nd H ofstra, 1996).Finally, in or de r to establish the phylogenetic relationships between newisolates and previously defined taxa, the degree of relatedness of theirgenomes may be used. Several methods are available: ( i) DNA-rRNAhybridization (Palleroni et al. , 1973: D e Vos et al., 1989); (ii) D NA -DN Ahybridization (U rsin g, 1986); (iii) d irect com parison of rRNA sequences(Ludwig et al., 1994); (iv) comparison of macrorestriction patterns bypulsed-field gel electrophoresis (G ro thu es and T um m ler, 1991); an d (v) gelelectrophoresis of stable low molecular weight com po nen ts of the rR N Apool (Hofle, 1992).1.5.2 Isolation and identification of Shewanella, A lterom ona s andAlcaligenesT he old hydrogen sulphide-producing pse udo mo nad s are now assigned toth e species Sh. putrefaciens. W ith strains of m ea t origin, oth er useful char-acters for their differentiation from oth er pseu do m on ads are pink pigmen-tation, inability to produce arginine dihydrolase, and ability to produceD N A se (Molin and Te rnstr om , 1982). Phenotypically, strains of Sh. putre-faciens from fish are quite sep arate from t h e type strain, and differ frompoultry iso lates in their ability to reduce trimethylamineoxide and to assimi-late succinate (Stenstrom and Mo lin, 1990).Strains of G ram -ne gativ e, he tero trop hic , aero bic bacteria w ith singlepolar flagellum, which differ from members of the genus Pseudomonas

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THE PHYSIOLOGICALATTRIBUTES OF G R A M - N E G A T I V E B A C T E R I A 25mainly in D N A G + C content (37-50 mol %, compared with 55-64 molfor P seudomonas spp.) ar e commonly assigned to th e genus Alteromonas.

Gram-negative, oxidase-positive, strictly aerobic rods motile with peritri-chous flagella or cocci which a re unable to attack carbohydrates aerobicallyhave been commonly assigned to the genus Alcaligenes (Busse and Auling,1992). For a reliable identification of isolates with Alcaligenes, chemotaxo-nomic or phylogenetic methods have to be used. The presence of bothubiquinone with eight isoprenoid units in its side chain (Q-8) and hydroxy-putrescine as the characteristic polyamine are considered the minimumrequirements for a reliable allocation to the genus Alcaligenes (Busse andAuling, 1992). Assignm ent to th e species is do ne mainly after analysis of fattyacid profiles, although a simplified scheme with 36 cultural and biochemicalattrib utes may be used (Busse and Auling, 1992). DN A-DN A hybridizationstudies allow a definitive allocation to a defined species (S ne ath , 1989).

1.5.3 Isolation and identification of FlavobacteriumIn g eneral, non -ferm entative, Gra m-n egative , nonmotile, rod-sh aped bac-teria which produce yellow-pigmented colonies are placed in the genusFlavobacterium (Holmes, 1992). These organisms are easily isolatedwithout the need for enr ichment , f rom nutr ient agar- type media (af ter 3 4days at 20-25 C). One of such media for the isolation of organisms of thisgenus from food was propo sed by M cMeekin er al. (1971). Fu rth er identifi-cation is rarely done with strains of meat origin. The genus includes onlystrains with low G+ C content of the D N A (3 0 4 2 % ). Th e last emendeddescription (based on DN A-D NA hybridization, DNA -rRNA hybridiza-tion, Fatty Acid M ethyl E ste r profiles, and PAGE of whole-cell prot eins ) ofthe genus Flavobacterium includes Gram -ne gativ e rods that exhibit glidingmotility (Bernardet et al., 1996). A scheme based on 27 phenotypic charac-ters for the differentiation of 10 species is included in this description(Bernardet et a/. , 1996).

1.5.4 Isolation and ident cation of Ac inetob acter, Moraxella andPsychrobacterMembers of the three genera are easily isolated on standard laboratorym edia, such as Trypticase Soy Agar or B rain H ea rt Infusion Ag ar. The y areisolated also on Plate C ount A gar used fo r total viable counts. Media m adeselective with crystal violet and bile salts (Medium M and Medium B,Gennar i et al., 1992) improved th e recovery of strains when th ese con stitut ea minority of th e microflora. O n both media B an d M, colonies of Acineto-bacter and Psychrobacter ar e convex, opa qu e and light blue (colony colourincreases progressively after prolonged incubation at 5 C).

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26 THE M I C R O B I O L O G Y OF M E A T A N D POULTRYT h e oxidase test and fou r oth er biochemical tests (us e of am inova lerate,2-keto-D-gluconate, glycerol and fructose as carbon sources for growth:

Shaw an d Latty, 1988) and ex am ination of cell morphology (G en nar i et al.,1992) are used for the differentiation of Acinetobacter, Moraxella--Psychrobacter and nonmoti le Pseudomonos . T h e identification of Acineto-bacter and Moraxella-Psychrobacter strains of meat origin is reliablyachieved with the API 20E system (Eribo et al., 1985).T h e identification of mea t isolates with (geno )species of Acinetobacter onthe basis of biochemical characters alone is difficult. Two matrices consist-ing of 22 and 10 diagnostic characters respectively, are available for theidentification of the majority of genomic species (Kampfer et al., 1993).Even so definitive identification can only be don e with D NA -ba sed m etho ds(D N A -D N A hybridization, ribotyping, or restriction frag m ent length poly-m orphism analysis of PC R amplified D N A ; Ge rner-Sm idt, 1992; Novak andKu r, 1995; Va neec houtte et al., 1995).T he D N A transformation assay with Psychrobacter im mobil is hyx7 (Juniand H eym . 1980) is th e best m eth od fo r the differentiation of Psychrobac-ter and Moraxel la phenylpyruvica strains from the true Moraxella. On thebasis of biochemical tests (acid production from m elibiose, L-ara bin ose , cel-lobiose, m altose; oxidative utilization of glucose: pheny lalanine deam inase:urease production: ability to utilize carbon sources), different phenotypicgroups are found among the Psychrobacter and allied organisms isolatedfrom meat and meat products (Gennar i etal . , 1992: Prieto etal. , 1992b). Dif-fere ntia tion of th e two species of Psychrobacter, imm obil is and phenylpyru-vicus associated with meat and meat products is based on variousphen otypic, genotypic an d 1 6 s ribosomal D N A phylogenetic analysis(Bowman er al., 1996).Useful methods for the identification of species of the true moraxellaeare: th e D N A transform ation assay of Juni (1978) for Moraxella osloensis,determination of the 1 6 s r ibosomal D N A sequence (Enr ight et ol., 1994)and DN A-rR NA hybridization analysis (Ro ssau et al., 1991).1.5.5Of th e many selective media devised for the isolation of Ente rob acte riac eae(Bloo d an d Curtis, 1995), Violet R ed Bile G lucose Aga r is the most com-monly used. It includes bile salts and crystal violet as selective agents, aswell as glucose and a p H indicator as a differential system. Ino cula ted andsolidified medium in petri dishes is overlayed with 10 ml of the sameme dium before incubation (at 32 2 C for 2 4 4 8 h). For specific purposes,incubation at 4 C for 10 d , 37 - 1 C or 4 2 4 4 C is used. The recoveryof injured cells can be assisted by shaking dilutions of a food homogenatefor 1 h at room temperature before plat ing. Stressed populat ions fromseveral sources can be resuscitated by being spread on the surface of

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THE PHY SIOL OG ICAL ATTRIBUTES OF G R A M - N E G A T I V E B A C T E R IA 27Tryptone Soya Pepton e Glucose Yeast Extract Agar. After 6 h at room tem-peratu re, this med ium is overlayed with V R B G A a nd the plates are incu-bate d as usual (Mossel et al. , 1995). Confirmatory tests for isolates from suchcolonies are: G ra m stain (-ve), oxidase test (-ve), and ferm enta tion ofglucose in a min eral salts medium covered with sterile mineral oil and incu-bated at 37 C for 24 h (ICM SF , 1978).The majority of strains of Enterobacteriaceae isolated from meat andm eat pro du cts ar e easily identified at th e species level by m ean s of severalrapid systems incorp ora ting multiple biochemical tests. Results of the A P I20 E (24 h of inc ubation ), and A PI Rap id E (4 h of incubation), whichinclude 20 tests (13 com mo n to both systems) can b e interpreted with a com-put er assisted pro gram (C ox and Bailey, 1986). T h e Minitek system, of 35tests, needs to be complemented with supplementary ones (motility, andseveral additional biochemical reactions) (Stiles and Ng, 1981; Ho lm es andHu mp hry, 1988). T he AutoMicrobic System (AM S) an autom ated systemis helpful for identification purposes. With the Enterobacteriaceae-plus(EBC+) 'biochemical card' (29 biochemical tests and one positive-controlbr ot h) an d the A M S, 98-99% of strains of m eat origin can be identified forspecies (Bailey et al., 1985). T h e G ram -nega tive identification (G N I) data-base, to be used for the interpretat ion of the GNI card (which has nowreplaced the EBC+), includes information for the identification of 99species of E nterob acteriace ae a nd o the r Gram -nega tive bacteria.Several numerical identification software p ackages a re available for com-parison of the phen otypic fea tur es of isolates with strains included in severaldata bas es. O ne of these is described by Miller an d A lachi (1996).

1.5.6More than one plating medium is recommended for the isolation ofAeromonas. With samples of meat and meat products, the best results areob taine d with: (i) Bile Salts-Irgasan-Brilliant G re en Aga r, (ii) Am picillinShe ep Blood Ag ar with 30 mgil of Ampicillin (ASBA 30), and /or (iii) StarchAmpicillin A ga r (SA A ). T he media are incub ated at 25-30 C for up to 72h. Apart from the selective agents (bile salts or ampicillin) these mediainclude differential systems (xylose, blood, starch). Other selective mediaar e also available (Pin et al. , 1994; G ob at and Jem m i, 1995; Jepp esen, 1995).Fo r the recovery of injured cells, enrichm ent in alkaline p ept on e water (p H8.7 r 0.1) at 28 C is reco m m end ed be fore plating. Presumptive identifi-cation of typical colonies (showing haemolytic activity, starch hydrolysisand /or non ferm en ting xylose) is simple. A dd ition al tests for the assignmentof isolates to genus are: oxidase (it can be carried out directly on someme dia), ferm entation of glucose and o the r carbohy drates, and resistance tothe vibriostatic agent 0/ 12 9.

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28 T H E M I C R O B I O L O G Y OF MEAT AND POULTRYThe identification to genospecies level may be done with DNA-DNAhybridization studies, random amplified polymorphic DNA polymerase

chain reaction (RA PD -PC R) (Oa key et al., 1996), or comparison of pro-files of cellular fatty acid methyl esters FAME) with those of the es tab-lished hybridization groups (Huys et al., 1995). PC R primers andhybridization pro bes for the rapid d etec tion of som e species of Aerornonashave also been described (Dorsch et al., 1994).1.5.7 Isolation and identification of VibrioM odera tely halophilic Vibrio are isolated from cured meats by plating onmedia with 4-6% NaCI. Enrichment for 48 h at 22 C in brain heart infu-sion broth containing 6 % NaCl and 10 ppm crystal violet is necessary forisolation from certain un cured me ats. Presu m ptive tests are G ram reaction(+v e), catalase, oxidase ( +v e) and utilization of glucose ( H ug h an d Leifsonme dium ). Ma intenan ce m edium is complex and includes 1 0% (w/v) salts. Itis possible to diffe ren tiate Salinivibrio Vibrio)costicola from o the r speciesof Vibr io on the basis of the resp onse to different conc entrations of NaCI,arginine decarboxylase a nd G + C conten t (Mellad o et al., 1996).ReferencesAhmad, H.A. and Marchello, J.A. (1989a) Effect of gas atmosphere packaging on psy-chrotrophic growth and succession on steak surfaces. J. Food Sci. , 54 274-6.Ahmad, H.A.and Marchello. J.A. (1989b) Microbial growth and successions on steaks as influ-enced by packaging procedures. J Food Protect., 52.23&9.Asensio, M.A., Ordoiiez, J.A. and Sanz. B. (1988) Effect of carbon dioxide and oxygen

enriched atmospheres on the shelf-life of refrigerated pork packed in plastic bags. J FoodProtect.. 51, 356-60.Bailey, J.S., Cox, N.A. and Fung. D.Y.C. (1985) Identification of Enterobacteriaceae in foodswith the AutoMicrobic System. J. Food Prorecr., 48 147-9.Barnes, E.M. and Impey, C.S. (1968) Psychrophilic spoilage bacteria of poultry. J. A p p l . Bac-teriol., 31 97-107.Baumann, P. and Schubert, R.H.W. (1984) Family 11. Vibrionaceae Veron 1965, 4245AL, nBergey's Manual of System atic Bacteriolog-y,Vol. I, (eds N.R. Krieg and J.G. Holt), Williamsand Wilkins, Baltimore, pp. 516-17.Bergey, D.H., Harrison, F.C.. Breed, R.S. ,Hammer, B.W. and Huntoon, F.M. (1923).Bergey'sM a n u a l of Determinative Bacteriology, 1st edn. Williams and Wilkins. Baltimore.Bernardet, J.F., Segers, P., Vancanneyt. M. et al. (1996) Cutting a gordian knot: emendedclassification and dtscription of the genus Flavohacterium, emended description of thefamily Flavobacteriaceae, and proposal of Flavohacreriicm hydatis nom. nov. (Basonym,Cytophaga aquatilis Strohl and Tait 1978). Inr. J System. Bacteriol.. 46, 2848.Blanco, D., Barbieri, G., Mambriani, P. et 01 (1994) Study of the 'potato defect' of raw dry-cured ham. Industria Conserve, 69,230-6.Blickstad, E. and Molin, G. (1983) Carbon dioxide as a controller of the spoilage flora of pork,with special reference to temperature and sodium chloride. J Food Protect., 46. 56-63.Blood, R.M. and Curtis, G.D.W. (1995) Media for 'total' Enterobacteriaceae, coliforms andEscherichia coli. Inr. J. Food Microhiol . . 26 3-116.Borch, E., Kant-Muermans, M.L. and Blixt, Y. (1996) Bacterial spoilage of meat and curedmeat products. Int. J Food Microbio l ,33. 103-20.

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THE PHYSIOLOGICALATTRIBUTES OF G R A M - N E G A T I V E BACTERIA 29Bow man , J.P., Cavanagh , J., Austin, J.J. and Sanderson , K. (1996) Novel Psychrobacter speciesfrom A ntarctic ornithogenic soils. Int. J System. Bacteriol. , 46,841-8.Br aun -H ow land , H ., Vescio, P.A. an d Nierzwicki-Bauer, B . (1993) Use of a simplified cell blottechnique a nd 16 s rRNA -directed probes for identification of com mon env ironmental iso-lates. Appl. Environ. Microbiol . , 59, 3219-24.Brem ner, A. and John ston, M. (1996) Poultry Meat Hygiene and Inspection, WB Saunders,London.Br enn er, D.J. (1992) Introduction to the family Enterobacteriaceae, in Th e Prokaryotes. AH an db oo k o n Habitats, Isolation and Identification of Bacteria, 2 edn, (eds A . Balows, H.G.Truper, M. Dworkin, W. Harder and K.H. Schleifer), Springer Verlag, New York, pp.2673-95.Busse, H.-J. and Auling, G. (1992) The genera Alcaligenes and Achromobacter , in T heProkaryotes, 2nd edn, (eds A. Balows, H.G. Truper, M. Dworkin, W. Harder and K.-H.

Sch leifer), Springer V erlag, New York, pp . 2544-55.Cantoni, C. , Bersani, C. and Ronca glia, P.L. (1994) Putrefactive-type spoilage of raw ham.Ingegneria Alimentare le Conserve Animal i , 1 0 , 4 0 4 .Catlin, B.W. (1991) Branham aceae fam. nov., a proposed family to accommo date the generaBranhamella and Moraxella. Int. J System . B acteriol., 41 320-3.Cox, N.A . and Bailey, J.S. (1986) Ente robac teriaceae identification from stock cultures andhigh moisture foods with a four-hour system (A PI R apid E ). J Food Protect., 49 605-7.Da inty, R. H . (1996) Chem icalibiochem ical detection of spoilage. Int. J Food Microbiol. . 33,Dainty, R.H . an d Mackey, B.M. (1992) Th e relationship betw een the pheno typic properties ofbacteria from chill-stored meat and spoilage processes. J Ap pl . Bacteriol., 73, Symp. Suppl.21,103s-114s.Dainty, R .H ., Shaw, B.G . and Ro ber ts, T.A. (1983) Microbial and chemical changes in chill-stored red meats, in Food Microbiology: Adv ance s and Prospects , (eds T.A. Ro berts andF. A . Skinner), A cadem ic Press, London, p p. 151-78.Da ud , H.B., McM eekin, T.A. an d Thom as, C.J . (1979) Spoilage association of chicken skin.Ap pl . Environ . Microbio l ., 37, 39940 1.D e Ley, J., Segers, P., Kersters, K. et al. (1986) Intra - an d intergeneric similarities of the Bor -detella ribosomal ribonucleic acid cistrons. Proposal for a new family, Alcaligenaceae. Int.

J System. Bacteriol., 35, 405-11.D e Vos, P., Van La ndschoot, A ,, Segers, P. et al. (1989) Geno typic relationships and taxon omiclocalization of unclassified Pseudomonas and Pseudomonas- l ike strains by deoxyribonucleicacid:ribosom al ribonucleic acid hybridizations. Int. J System . Bacteriol., 39. 35-49.Dorsch, M., Ashbolt, N.J., Cox, P.T. and Goodman, A.E. (1994) Rapid identification ofA e r o m o n a s species using 16s rDN A targeted oligonucleotide prim ers: a molecular approachbased on screening of environmental isolates. J Ap pl . Bacter iol . , 77,722-6.Drosinos, E .H . and Bo ard, R.G . (1994) Metabolic activities of pseudom onads in batch culturesin extract of minced lamb. J Appl. Bacteriol., 77, 613-20.Drosinos, E. H . and B oar d, R.G . (1995a) Microbial and physicochemical attrib utes of mincedlamb: sources of contamination with pseudomonads. Food Microbiol. , U 189-97.Drosinos, E .H . and B oar d, R.G . (1995b) A survey of minced lamb packaged in modified atmos-pheres. Fleischwirtschaft, 75 327-30.Egan, A.F. and Roberts, T.A. (1987) Microbiology of meat and meat products, in Essays inAgricultural and Food M icrobiology , (eds J.R. Norris and G.L. Pettipher ), Wiley, New York,

Enright, M.C., Carter, P.E., MacLean, I.A. and McKenzie, H. (1994) Phylogenetic relation-ships between some members of the genera Neisseria, Acinetobacter, Moraxella, andKingella based o n partial 1 6 s ribosomal D NA sequence analysis. Int. J System. Bacteriol. ,Eribo, B.E. and Jay, J.M. (1985) Incidence of Acinetobacrer spp. and other Gram-negative,oxidase-negative bacteria in fresh and spoiled groun d beef. Appl. Environ. Microbiol . , 49256-7.Er ibo, B .E., Lall, S.D . and Jay, J.M. (1985) Incidence of Moraxella and o ther Gram -negativeoxidase-positive bacteria in fresh a nd spoiled ground beef. Food Microbiol., 2 , 2 3 7 4 0 .

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30 T H E MICROBIOLOGY OF MEA T AND POULTRYEriksson, P.V., Paola, G.N., Pasetti, M.F. and Manghi, M.A. (1995) Inhibition enzyme-linkedimmunosorbent assay for detection of Pseudomonas juorescei is on meat surfaces. Appl .Environ. Microbiol , , 61, 397-8.Farber, J.M. and Idziak. E.S. (1982) Detection of glucose oxidation products in chilled freshbeef undergoing spoilage. Appl. Environ. Microbiol , ,44 214.Gardner, G.A. (1981) Identification and ecology of salt-requiring Vibrio associated with curedmeats. Meat Sci., 5 71-81.Gardner, G.A. (1982) Microbiology of processing: bacon and ham, in Mear Microbiology, (edM.H. Brown), Applied Science, London, pp. 129-78.Gardner, G.A. (1983) Microbial spoilage of cured meats, in Food Microbiology; Adv ance s andProspects, (eds T.A. Roberts and F.A. Skinner), Academic Press, London. pp. 179-202.Gauthier. G.. Gauthier, M . and Christen, R. (1995) Phylogenetic analysis of the generaAlteromonas, Shewanella and Moritella using genes coding for small-subunit rRNA

sequences and division of the genus Al teromonas into two genera, Al teromonas (emended)and Pseudoalteromonas gen. nov., and proposal of twelve new species combination. Znt. J.System. Bacteriol., 45, 755-61.Gennari, M., Alacqua. G. , Ferri, F. and Serio, M. (1989) Characterization by conventionalmethods and genetic transformation of Neisseriaceae (genera Psychrobacter and Acineto-bacter) isolated from fresh and spoiled sardines. Food Microbiol., 6, 199-210.Gennari, M. and Dragotto. F. (1992) A study of the incidence of different fluorescentPseudomonas species and biovars in the microflora of fresh and spoiled meat and fish, rawmilk, cheese, soil and water. J. Ap pl . Bacteriol . , 72, 281-8.Gennari, M., Parini, M., Volpon, D. and Serio, M. (1992) Isolation and characterization by con-ventional methods and genetic transformation of Psychrobacter and Acinetobacter fromfresh and spoiled meat, milk and cheese. Int. J. Food Microbiol., 15, 61-75.Gerner-Smidt, P. (1992) Ribotyping of the Acinetobacter calcoaceticus-Acinetobacter bau-manii complex. J Clinical Microbiol.. 30, 2680-5.Gill, C.O. (1982) Microbial Interaction with Meats, in Meat Microbiology, (ed M.H. Brown),Applied Science, London, pp. 225-64.Gill, C.O. (1986) The control of microbial spoilage in fresh meats, in Meat and Poultry M icro-biology, (eds A.M. Pearson and T.R. Dutson). Macmillan, Basingstone, pp. 49-88.Gill, C.O. and Bryant, J. (1992) The contamination of pork with spoilage bacteria during com-mercial dressing, chilling and cutting of pig carcasses. Znt. J Food Microbiol., 16, 51-62.Gill, C.O. and Harrison, J.C.L. (1989) The storage life of chilled pork packaged under carbondioxide. Mear Sci. , 26,313-24.Gill, C.O. and Jeremiah. L.E. (1991) The storage life of non-muscle offals packaged undervacuum or carbon dioxide. Food Microbiol., 8,339-53.Gill, 2.0. nd Jones, T. (1995) The presence of Aero rnon as, Listeria and Yersinia in carcassprocessing equipment at two pig slaughtering plants. Food Microbiol , , 12. 13541.Gill, C.O. and Molin, G. (1991) Modified atmospheres and vacuum packaging, in Food Preser-vatives, (eds N.J. Russell and G.W. Gould), Blackie. Glasgow, pp. 172-99.Gill, C.O. and Newton, K.G. (1978) The ecology of bacterial spoilage of fresh meat at chilltemperatures. Meat Sci., 2, 207-17.Gill, C.O. and Penney. N. (1985) Modification of in-pack conditions to extend the storage lifeof vacuum packaged lamb. Mear Sci., 14,43450.

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