APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1981, p. 867-8720099-2240/81/040867-06$02.00/0

Vol. 41, No. 4

Enterobacteriaceae Associated with Meats and MeatHandling

MICHAEL E. STILES* AND LAI-KING NGFaculty ofHome Economics and Department ofMicrobiology, The University ofAlberta, Edmonton,

Alberta, Canada T6G 2M8

Received 21 November 1980/Accepted 5 January 1981

The source of Enterobacteriaceae on meats was shown to be associated withthe meat-handling work surfaces in the two packing plants studied. A total of2,343 Enterobacteriaceae were isolated and identified from meat samples andwork surfaces at the packing plants and at the retail facilities. Escherichia colibiotype I and Serratia liquefaciens were detected at all stages of meat handling,indicating that they may be present in meats throughout the meat-handlingsystem. Enterobacter agglomerans and S. liquefaciens were the predominantEnterobacteriaceae at the retail level, but they had limited indicator potentialfor sanitation and hygiene. Klebsiella pneumoniae was a frequent isolate amongEnterobacteriaceae from meats and meat-handling surfaces in the packing plantsbut not at the retail level, indicating that this organism might signal unhygienichandling of meats at the retail level.

Escherichia coli biotype I is one of the pre-dominant Enterobacteriaceae in ground beef(11, 28). With good manufacturing practices, E.coli I contamination on meat is generally consid-ered to come from the skin or hide of animalsduring processing (1, 14, 30) and possibly repre-sents both fecal and nonfecal contamination(27). Furthermore, E. coli I in the stationarygrowth phase survives well in frozen and non-frozen meat (18, 32) and grows in meats atimproper storage temperatures. This fact ledmany workers to criticize the use of E. coli as anindicator of sanitation and hygiene in raw meats(13, 15, 20). In contrast, Mossel and co-workers(5, 21, 24, 25) criticized the use of E. coli as anindicator of food safety for dehydrated, frozen,and refrigerated foods, because they found thatE. coli does not survive well under such condi-tions. As a result, they recommended the use ofEnterobacteriaceae as indicators of food safety(22, 23, 26). Greater concem for non-E. colicoliforms has also been proposed because oftheir increasing involvement in diarrheal dis-eases (34).

E. coli biotype I (IMViC [indole, methyl red,Voges-Proskauer, citrate] ++-- and gas posi-tive in EC broth at 450C) is generally recognizedas an indicator of direct or indirect fecal contam-ination of foods, and E. coli or colifonn bacteriain pasteurized milk and cooked foods generallyindicate postprocessing contamination (16).Klebsiella pneumoniae is also a frequent butnot exclusive inhabitant of the intestines of ani-mals and humans (4, 5, 9, 19), has been impli-

cated in human infections (3, 12), and has asimilar fecal origin, and hence public healthsignificance, as typical E. coli (4, 17). OtherEnterobacteriaceae are of less specific origin;for example, Citrobacter freundii is found infeces but also in soil (4, 29), and Enterobacterspp. are reported to occur only rarely in thehuman intestine (5).

In an earlier study (28), the principal Entero-bacteriaceae in ground beef were found to be E.coli type I, Enterobacter agglomerans, and Ser-ratia liquefaciens. Other Enterobacteriaceaewere also identified on selective media, includingC. freundii, Enterobacter aerogenes, E. cloacae,E. hafniae, and K. pneumoniae. The signifi-cance of these organisms in retail meats couldnot be determined. Hence, our object was tostudy Enterobacteriaceae at different stages ofthe meat-processing chain to determine whetherany associations could be made between bacte-rial isolates and stage in the meat-handling proc-ess.

MATERIALS AND METHODSSample collection. Samples were collected at

packing plants and retail points of meat handling forsubsequent isolation and identification of Enterobac-teriaceae. In the packing plant study, samples werecollected on 18 occasions (7 times at plant A and 11times at plant B) from two federally inspected Cana-dian packing plants, each operating beef and hog killfloors and meat-processing departments. Sampleswere taken to include (i) meat and nonmeat contactsurfaces and (ii) the beef, hog, and processed-meatareas of the plants. For sampling, we used Rodac

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contact plates and violet red bile agar (VRBA). At theretail level, three stores that received supplies directlyor indirectly from these packing plants were used toobtain samples that included: (i) vacuum-packagedbeef primal cuts before they were opened at the retailstore; (ii) vacuum-packaged beef trimnmings for use inground beef manufacture; (iii) packaged pork loins(not vacuum packaged) before they were opened atthe retail store; (iv) frozen and thawed pork sausagepackaged by manufacturers; and (v) meats handled atthe retail level, including beef steaks, pork chops, andground beef.Sample preparation. Samples of integral meats

were collected by a spray gun technique (7) with a100-ml sterile 0.1% peptone water wash. Samples werecollected aseptically in a graduated Erlenmeyer flask(approximately 20 ml per sampling point). Commi-nuted meat samples were refrigerated and taken tothe laboratory for immediate analysis. Comminutedmeats were sampled by blending 11 g of meat with 99ml of sterile 0.1% peptone water for 5 min in a Col-worth stomacher (model 400).

Bacteriology. Appropriate dilutions were platedonto VRBA by the pour plate technique. All VRBAplates were overlaid with 5 ml of VRBA and incubatedat 35°C for 18 to 24 h. After incubation, representativenumbers of bile-precipitating and non-bile-precipitat-ing colony types were randomly picked from theVRBA plates and purified by streaking first on

MacConkey agar and then on nutrient agar. Biochem-ical tests were done on an isolated colony from thenutrient agar plates. Enterobacteriaceae werescreened by Gram stain and oxidase test. The bio-chemical tests were an expanded Minitek (BBL, Bec-ton-Dickinson and Co., Mississauga, Canada) identi-fication system, described previously (31), involving:arabinose, citrate, dulcitol, H2S-indole, inositol, lysine,malonate, o-nitrophenyl-,f-D-galactopyranoside, orni-thine, phenylalanine, raffinose, rhamnose, and ureadisks. Isolates were also inoculated onto phenol redglucose and lactose (1%) broths, nitrate-motility agar,

triple sugar iron agar, and methyl red-Voges-Pros-kauer medium. Supplementary tests were carried outas required.

RESULTS

Table 1 shows the incidence of Enterobacte-riaceae on various packing-plant surfaces. Theraw meat-handling surfaces, when in use, were

the areas most heavily contaminated with En-terobacteriaceae. Rodac plates with >1,000 totoo numerous to count Enterobacteriaceae per

plate frequently involved the meat saw tablesand stainless-steel work surfaces. In addition,the carcass-splitting saw and beef carcass

shrouds were implicated on two occasions each.Cooked-meat contact surfaces were less contam-inated with Enterobacteriaceae, and surfaceswith no meat contact (walls, doors, etc.) were

not apparent reservoirs of Enterobacteriaceae.A total of 2,343 Enterobacteriaceae isolates

were identified, 1,372 from packing-plant sam-

ples and 971 from meats sampled at the retail

level. The distribution of Enterobacteriaceaetypes isolated at the two packing plants (Table2) indicated that K. pneumoniae, E. coli I, andS. liquefaciens were the principal Enterobacte-riaceae isolated, each accounting for 10 togreater than 20% of the isolates. C. freundii,Enterobacter agglomerans, and E. cloacae wereof lesser importance, accounting for 5 to 12% ofthe isolates. Each of the other types accountedfor less than 5% of the isolates. Some differenceswere observed in the order of the frequency withwhich different Enterobacteriaceae were iso-lated at each packing plant. E. coli I was themost common isolate in plant A, whereas K.pneumoniae was the most common at plant B.

In general, the frequency of isolating Entero-bacteriaceae at plant A was lower than that atplant B. A total of 86% of the plates with con-fluent growth were obtained at plant B, com-pared with only 14% at plant A. The sanitationpractices in both plants virtually eliminated allEnterobacteriaceae from inanimate meat-han-dling surfaces (stainless steel, polyethylene cut-ting boards, and PVK or Neoprene rubber foodbelting). Occasional survivors included: E. aer-ogenes, K. ozaenae, K. pneumoniae, and S. Ii-quefaciens. No E. coli I were isolated on equip-ment after cleanup.The data could also be examined on the basis

of the Enterobacteriaceae associated with dif-ferent areas and surfaces within the packingplants (Table 3). The number of isolates for eachsurface type was proportional to the number oforganisms growing on the Rodac samplingplates. This finding confirmed the earlier indi-cation that the raw-meat-handling surfaces werethe most heavily contaminated with Enterobac-teriaceae, cooked-meat contact surfaces wereless contaminated, and surfaces with no meatcontact were not apparent reservoirs of Entero-bacteriaceae in the plants. On raw-meat contactsurfaces, E. coli I, K. pneumoniae, and S. lique-faciens predominated, whereas E. agglomeransand K. pneumoniae were the main types iden-tified on cooked-meat contact surfaces.

Similar frequency distributions (Table 4) werecalculated on the basis of the Enterobacteria-ceae associated with the different product types(beef, pork, and cooked meat). K. pneumoniae,E. coli I, and C. freundii were the principalisolates from beef work surfaces; E. coli I, S.liquefaciens, and K. pneumoniae (in that order)were the principal isolates from pork; and S.liquefaciens, E. cloacae, K. pneumoniae, and E.agglomerans were the principal isolates fromcooked-meat-handling surfaces. On cooked-meat work surfaces, E. coli I and C. freundiiwere relatively minor isolates (<10%).The incidence of E. coli I on equipment and

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TABLE 1. Levels of Enterobacteriaceae detected on VRBA Rodac plates from different surfaces in twopacking plants

Enterobacteriaceae colonies/Rodac plateSurface

None detectable c50 51-100 101-500 >500-TNTCa

In contact with meatsBeef area 16 43 7 14 24

(15.4)b (41.4) (6.7) (13.5) (23.1)Hog area 81 76 18 33 15

(36.3) (34.1) (8.1) (14.8) (6.7)Processed meatsRaw 40 28 8 10 10

(41.7) (29.2) (8.3) (10.4) (10.4)Cooked 20 11 1 0 0

(62.5) (34.4) (3.1) (0.0) (0.0)With no product contactBeef area 38 14 0 0 1

(71.7) (26.4) (0.0) (0.0) (1.9)Hog area 61 7 0 1 1

(87.1) (10.0) (0.0) (1.4) (1.4)Processed meatsRaw 34 1 1 0 0

(94.4) (2.8) (2.8) (0.0) (0.0)Cooked 18 1 0 0 0

(94.7) (5.3 (0.0) (0.0) (0.0)a Too numerous to count; very heavy to confluent growth.b Numbers in parentheses are percentages.

TABLE 2. Frequency of isolation and distribution ofEnterobacteriaceae types obtained from VRBA

contact swabs from two packing plantsNo. of isolates

OrganismPlant A Plant B

E. coli I 73(20.0)a 149(14.8)Other E. coli 6(1.6) 6(0.6)C. freundii 29(7.9) 127(12.6)E. aerogenes 9(2.5) 26(2.6)E. agglomerans 43(11.8) 65(6.5)E. cloacae 30(8.2) 88(8.8)E. hafniae 14(3.8) 39(3.9)K. ozaenae 12(3.3) 24(2.4)K. pneumoniae 57(15.6) 227(22.6)S. liquefaciens 51(13.9) 148(14.7)S. marcescens 3(0.8) 35(3.5)S. rubidaea 5(1.4) 8(0.8)Y. enterocolitica 6(1.6) 9(0.9)Other Enterobacteriaceae 28(7.6) 55(5.5)

a Numbers in parentheses are percentages.

meat-handling surfaces in use was determinedfor each meat type. In the beef-cutting areas ofboth plants, E. coli I was detected on meat-handling surfaces in 14 of 40 samples. E. coli Iwas also detected on a hand wash sink on thebeef kill floor of plant B in four of nine samples.In contrast, E. coli I was found on only 1 of 14tests on walls in the beef kill area. In the beef-cutting area for vacuum-packaged primal cuts,E. coli I was detected on meat-handling surfacesin 26 of 102 samplings.

Similar tests for E. coli I in the pork prepa-

TABLE 3. Distribution of Enterobacteriaceaeamong different surfaces, excluding meats, within

the packing plantsNo. of isolates on surfaces in con-

tact with:Organism

Raw meat Cooked No meatmeat

E. coli I 153(20.0)a 4(3.0) 5(19.2)Other E. coli 7(0.9) 0(0.0) 1(3.9)C. freundii 71(9.3) 8(7.1) 4(15.4)E. aerogenes 8(1.0) 8(7.1) 0(0.0)E. agglomerans 54(7.0) 26(23.2) 3(11.5)E. cloacae 62(8.1) 10(8.9) 1(3.8)E. hafniae 17(2.2) 5(4.5) 0(0.0)K. ozaenae 21(2.7) 9(8.0) 0(0.0)K. pneumoniae 144(18.8) 17(15.2) 3(11.5)S. liquefaciens 138(18.0) 11(9.8) 2(7.7)S. marcescens 23(3.0) 0(0.0) 3(11.5)S. rubidaea 8(1.0) 0(0.0) 2(7.7)Y. enterocolitica 7(0.9) 5(4.5) 0(0.0)Other 54(7.0) 9(8.0) 2(7.7)Enterobacteriaceaea Numbers in parentheses are percentages.

ration areas resulted in fewer E. coli I isolateson the kill floor equipment (5 positive of 34 tests)than on the beef kill floor. However, there wasa marked increase in the frequency of E. coli Idetected in the pork-cutting areas (51 positive of130 tests). For example, stainless-steel chutes forpork cuts had 8 of 18 samplings that were E. coliI positive; plastic cutting boards, 7 of 20; rubberbelting, 7 of 19; and stainless-steel work tables,13 of 22.The emulsion preparation area for cooked-

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TABLE 4. Distribution of Enterobacteriaceae isolated from surfaces and meats in packing plants associatedwith different meat types

No. of isolates

Organism Processed meatBeef area Hog area

Raw Cooked

E. coli I 86(14.4)a 100(24.9) 31(13.2) 5(3.6)Other E. coli 3(0.5) 7(1.8) 2(0.8) 0(0.0)C. freundii 87(14.6) 37(9.2) 22(9.4) 10(7.3)E. aerogenes 19(3.2) 3(0.8) 4(1.7) 9(6.5)E. agglomerans 43(7.2) 18(4.5) 17(7.3) 30(21.7)E. cloacae 33(5.6) 27(6.7) 46(19.7) 12(8.7)E. hafniae 32(5.5) 4(1.0) 12(5.1) 5(3.6)K. ozaenae 9(1.5) 15(3.7) 3(1.3) 9(6.5)K. pneumoniae 174(29.1) 54(13.4) 35(15.0) 21(15.2)S. liquefaciens 56(9.4) 83(20.6) 45(19.2) 15(10.9)S. marcescens 15(2.5) 20(5.0) 3(1.3) 0(0.0)S. rubidaea 5(0.8) 5(1.2) 3(1.3) 0(0.0)Y. enterocolitica 4(0.7) 2(0.5) 2(0.9) 7(5.1)Other Enterobacteriaceae 32(5.4) 27(6.7) 9(3.8) 15(10.9)

a Numbers in parentheses are percentages.

meat products gave many E. coli I-positive sam-plings (18 of 86 tests). The incidence of E. coliI in this area of plant B was higher than at plantA. However, in the cooked-product handlingareas of the two plants, the incidence of E. coliI was markedly reduced (only 4 positive of 123samplings), and all of these positive sampleswere from the wiener-handling equipment atplant A (4 of 28 tests).Throughout the two plants, surfaces with no

meat contact (walls, doors, etc.) were tested on102 occasions. Only one swab gave a positive E.coli I test. The incidence of Enterobacteriaceaein general on these surfaces was low (Table 1).The distribution of different types of Entero-

bacteriaceae (Table 5) on vacuum-packagedbeefrevealed S. liquefaciens as the predominantisolate (29.7%) and E. coli I as the next mostimportant isolate. In contrast, for ground beefprepared from supplies that had been centrallyprepared but not vacuum packaged, the predom-inating Enterobacteriaceae were E. agglomer-ans, S. liquefaciens, and E. coli I. Frozen porksausage, packaged by the manufacturer, re-vealed that S. liquefaciens was the predominantEnterobacteriaceae; E. agglomerans and E. coliI were also major isolates. In contrast to thepacking-plant samples, K. pneumoniae was anisolate of minor importance in samples collectedat the retail level.Enterobacteriaceae were also isolated from

retail store trim. Of 115 isolates, the principalorganisms were E. agglomerans (43.5%), E. coliI (20.9%), and S. liquefaciens (13.9%). Samplesfrom beef steaks and pork chops contained farfewer Enterobacteriaceae, and as a result only56 and 45 isolates were identified from these

samples, respectively. E. coli I was not detectedamong the beef steak isolates and representedonly 2.2% of the isolates from pork chops. Thepredominant Enterobacteriaceae on both meattypes were S. liquefaciens, which accounted for32.1% of beef steak and 46.7% of pork chopisolates; E. agglomerans accounted for 30.4 and20.0%, respectively. The only other isolate ofanysignificance was E. hafniae, which accounted for16.1% of the isolates from beef steak and 8.9% ofthose from pork chops.Whereas E. coli isolates were principally bio-

type I, with IMViC reaction ++--, the K. pneu-moniae isolates had heterogeneous IMViC bio-types. The 284 K. pneumoniae of packing-plantorigin included 59 (20.8%) IMViC type --++,112 (39.4%) +-++, and 39 (13.7%) ++++. Incomparison, the 118 K. pneumoniae isolatesfrom meats at the retail level included 43 (36.4%)IMViC type --++, 47 (39.8%) +-++, and 10(8.5%) ++++. This represents a high percentageof indole-positive strains.

DISCUSSIONThe distribution of Enterobacteriaceae in the

packing plants was primarily on meat-handlingsurfaces. Surfaces that did not normally come incontact with meats were not implicated as res-ervoirs of Enterobacteriaceae, and any buildupof Enterobacteriaceae during the day's opera-tions appeared to be controlled by sanitationpractices. Nonetheless, some work surfaces de-veloped excessively high contaminating loads ofEnterobacteriaceae during the work day, whichwould act as continual sources of contaminationfor products coming in contact with them. Fromthese results, we conclude that the Enterobac-

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TABLE 5. Distribution ofEnterobacteriaceae isolated from VRBA plates from different meats sampledbefore handling at the retail level

Vacuum-packaged Ground beef from packaged supplies Packaged porkOrganism beef trim and pri- sausage

mal cuts High fat Low fat sausage

E. coli I 22(15.2)a 48(20.9) 28(14.8) 31(15.0)Other E. coli 3(2.1) 2(0.9) 4(2.1) 2(1.0)C. freundii 9(6.2) 15(6.5) 10(5.3) 4(1.9)E. aerogenes 8(5.5) 10(4.3) 5(2.6) 6(2.9)E. agglomerans 14(9.6) 59(25.6) 59(31.2) 32(15.5)E. cloacae 9(6.2) 10(4.3) 15(7.9) 11(5.3)E. h.afniae 17(11.7) 12(5.2) 8(4.2) 4(1.9)K. ozaenae 5(3.5) 3(1.3) 8(4.2) 11(5.3)K. pneumoniae 1(0.7) 6(2.6) 9(4.8) 13(6.7)S. liquefaciens 43(29.7) 53(23.0) 37(19.6) 74(35.7)S. rubidaea 6(5.1) 3(1.3) 1(0.5) 1(0.5)Y. enterocolitica 5(3.5) 1(0.4) 0(0.0) 1(0.5)Other Enterobacteriaceae 3(2.1) 8(3.5) 5(2.6) 1(0.9)

a Numbers in parentheses are percentages.

teriaceae on the meat contact surfaces are abuildup from meats and their ongoing handling.As such, the stage at which the surfaces can beconsidered unsanitary is difficult to decide, es-pecially since buildup can be attributed, in part,to growth of the bacteria on these surfaces.

E. coli I, K. pneumoniae, and S. liquefacienswere generally the dominant Enterobacteria-ceae in the raw-meat-handling areas ofthe pack-ing plants. In contrast, the incidence of Entero-bacteriaceae on cooked (processed)-meat worksurfaces and products was dramatically reduced,as would be expected with the heat treatment.However, E. cloacae, S. liquefaciens, K. pneu-moniae, and E. coli I were the predominantisolates from surfaces before processed productswere cooked, whereas E. agglomerans, K. pneu-moniae, and S. liquefaciens were the most com-mon isolates from cooked-meat work surfacesused after the products had been cooked. Thesedata indicated that K. pneumoniae and E. coli,both of which could be of fecal origin (4, 9), werepredominant Enterobacteriaceae on meat-han-dling surfaces in the packing plants.At the retail level, even on meats before han-

dling by the butchers, the predominating Enter-obacteriaceae had changed to S. liquefaciens,E. agglomerans, and E. coli I. K. pneumoniaehad become a relatively infrequent and minorisolate. This suggested a change in the Entero-bacteriaceae surviving on meats, growth of pay-chrotrophic Enterobacteriaceae (5) on meatsduring the marketing process, or both. E. coli Iwas a frequent Enterobacteriaceae isolate on allsamples except processed meats sampled aftercooking. As a result, E. coli I in a product suchas raw ground beef could have no meaning forsanitation at the retail level. S. liquefaciens wasalso present as a major Enterobacteriaceae iso-

late throughout the meat-handling process.However, E. agglomerans was a minor isolateat the packing plants, but it frequently becamea major isolate, with S. liquefaciens, in retailsamples.The Enterobacteriaceae with greatest indi-

cator potential in these samples was K. pneu-moniae. This organism was frequently isolatedas a dominant Enterobacteriaceae in meats andon meat-handling surfaces in the packing plant,but it was only infrequently found on meats atthe retail level. This observation was confirmedby data of Cox and Mercuri (11) in their studyof Enterobacteriaceae on retail meats, whereasNewton et al. (27) found that 21.5% of the En-terobacteriaceae from hides and meat at pack-ing plants were K. pneumoniae. These resultssuggest that this organism has a short survivaltime in meats, so that its detection at the retaillevel could indicate recent contamination, whichin turn could indicate unsanitary and unhygienichandling.

Studies on the distribution of Klebsiella spp.in a hospital kitchen indicated that 6 of 13 rawmeats contained Klebsiella, but that heaviestcontamination (>103/g) was observed on salads(8). Evidence of contamination in the kitchenwas presented. The use of E. coli I as an indi-cator organism has always been predicated onits restricted occurrence in nature comparedwith the ubiquitous nature of E. aerogenes (5).Differentiation of K. pneumoniae and E. aero-genes poses even greater difficulties because ofthe similarity in their biochemical characteris-tics (2, 10, 11, 22). K. pneumoniae can be selec-tively enumerated, based on morphology onmethyl violet agar medium (6); if contaminantson vegetables can be related to fecal origin (3),this organism might indeed be a more meaning-

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ful Enterobacteriaceae to use as an indicator forsanitation and hygiene of meats at the retaillevel than E. coli I.

ACKNOWLEDGMENTS

This work was supported by a research contract withAgriculture Canada. In addition, we acknowledge the cooper-ation of the meat-packing plants and retail stores that madethis study possible.

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