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Journalof Food Protection, Vol. 66, No. 6, 2003, Pages 931– 936 Copyright q,InternationalAssociation for FoodProtection

Enterococcus faecalis and Enterococcus faecium Isolates from Milk,Beef, andChicken andTheir Antibiotic Resistance

W.CHINGWARU, S.F.MPUCHANE, AND B. A. GASHE*

Departmentof Biological Sciences, University ofBotswana, Gaborone, Private Bag 0022, Gaborone, Botswana

MS02-282:Received 28August 2002/ Accepted 2December 2002

ABSTRACT Downloaded from http://meridian.allenpress.com/jfp/article-pdf/66/6/931/1674782/0362-028x-66_6_931.pdf by guest on 01 October 2021

Theoccurrence and antibiotic resistance of enterococci, especially Enterococcusfaecalis and Enterococcusfaecium, in milk,beef, and chicken in Gaborone, Botswana, were studied. Enterococci were isolated from these sources with the use of bileesculin agar and identiŽ ed with API 20Strep kits. Antibiotic resistance was determined by the disk diffusion method. Theantibiotics tested were vancomycin, teicoplanin, ampicillin, tetracycline, and cephalothin. Among the 1,467 enterococci isolatedfrom the samples, E. faecalis (46.1%) and E. faecium (29.0%)were found to be the predominant species. Other enterococcalspecies made up 25% of the isolates. More than 96 and 97% ofthe E. faecalis and E. faecium isolates,respectively, werefound to be resistant to ampicillin. Almost 34, 27.3, and 22.4% of the E. faecalis isolatesfrom milk, beef, and chicken, respectively,were also resistant to cephalothin. The percentages of E. faecium isolatesthat were found to be resistant to cephalothinwere 32.8, 16.9, and 17.3% for milk, beef, and chicken, respectively. Resistance to vancomycin was widespread. Itwas found that 18.8, 7.8, and 13.1% of the E. faecalis isolatesfrom milk, beef, and chicken samples, respectively, were resistantto vancomycin. In contrast, 32.8, 24.7, and 30.7% of the E. faecium isolatesfrom milk, beef, and chicken samples, respectively,were resistant to vancomycin. Isolates that were resistant to multipledrugs were found in relativelylarge numbers.

Enterococciare found in a varietyof environments. tion (3, 37). Someenterococci are resistant to commonly Theyfrequently occur in large numbers in dairy and food usedantibiotics such as ampicillin, tetracycline, and ceph- products (12, 13). Theyalso occur in humans and animals alothin.However ,suchenterococci can be signiŽ cantly re- (16, 38). Enterococcihave been regarded as harmless com- ducedwith vancomycin and teicoplanin therapy. A problem mensalsand have been used as indicators of fecalcontam- ariseswhen these enterococci are also resistant to vanco- ination (14). Enterococcican survive for longperiods on mycinand teicoplanin. environmentalsurfaces and on the hands of health care Thereis little information available on the prevalence workers (23). Severalinvestigators have found enterococci, ofdrug-resistant enterococci in southern Africa. AsigniŽ- particularlythose that are resistant to multiple drugs, on cantpercentage of peoplewho are immunocompromised as variousobjects in ahospitalenvironment, including on bed aresultof acquired immunodeŽ ciency syndrome (AIDS) rails,night tables, curtains, bathroom sinks, toilet rings, andthose under intensive care in health care centers live in electronicthermometers, and other patient care equipment thisregion. In Botswana, unfortunately, no assessment of (24,25, 29). Enterococcihave recently been identiŽ ed as theprevalence of antibiotic-resistant enterococci among hu- secondaryinvaders in hospital-acquired infections (27). manimmunodeŽ ciency virus– AIDS patientshas been car- Sincethe 1980s, numbers of infections in hospitalized pa- riedout, but there is a generallyhigh level of antibiotic- tientsdue to enterococcihave increased tremendously (17). resistantenterococci among hospitalized patients. When pa- Colonizationwith vancomycin-resistant enterococci tientsare infected with drug-resistant enterococci, there (VRE) innonhospitalized patients has been on the increase maybe a problemwith the availability of or the selection inthe United States and Europe (3). Inthe United King- ofan appropriate drug for treatment.The appearance and dom,VRE were isolatedfrom raw sewageand from farm increasein the incidence of vancomycin elsewhere has be- animals,including pigs, chickens, ducks, and turkeys, but comea causefor greatconcern. VRE couldbe comingpart- noVRE were recoveredfrom cattleand sheep (3). Some lyfrom foodanimals, and this possibility has prompted fermentedtraditional foods in Africa alsoharbor entero- muchresearch on microbial isolates, mainly in the devel- cocci,although their resistance patterns are not known (12). oped world. Thedevelopment of the resistance of enterococci to van- Reservoirsfor antibiotic-resistantenterococci have not comycinin animals is mainly attributed to the use of anti- beencompletely determined. Animals, human food, and the biotics,especially avoparcin (a glycopeptideantibiotic), in inanimateenvironmental components have been suspected animalfeeds as a growthpromoter (9). InEurope, evidence sourcesof some of the resistant clinical isolates (2, 4, 15, suggeststhat foodborne VRE maycause human coloniza- 18, 19). Muchinformation on the antibiotic resistance of enterococciisolated from clinicalsources has accumulated *Authorfor correspondence. Tel: 267-355-2599; Fax: 267-585097; (1), butthe levels of antibiotic resistance of enterococci E-mail: [email protected]. isolatedfrom foodshave not yet been thoroughly docu- 932 CHINGWARU ETAL. J.FoodProt., Vol. 66, No. 6

TABLE 1. Enterococcalspecies from different sources %ofpositivesamples (no.positive/ no.tested) Enterococcus species Milk Beef Chicken

E. faecalis 42.8(411/ 970) 57.2(154/ 269) 46.9(107/ 228) E. faecium 28.2(278/ 970) 28.6(77/ 269) 32.9(75/ 228) E.gallinarum 7.9(77/ 970) 5.9(16/ 269) 7.0(16/ 228) E. avium 6.2(60/ 970) 3.0 (8/269) 5.3(12/ 228) E. durans 2.5(24/ 970) 2.6 (7/269) 0 (0/228) E. hirae 2.4(23/ 970) 2.6 (7/269) 0 (0/228) E.casseliavus 9.6(93/ 970) 0 (0/269) 7.9(18/ 228) E. mundtii 0.4 (4/970) 0 (0/269) 0 (0/228) Downloaded from http://meridian.allenpress.com/jfp/article-pdf/66/6/931/1674782/0362-028x-66_6_931.pdf by guest on 01 October 2021 mented (13). Ithas been realized that there is a needto Chemicalsand media. Allof themedia and antibiotic disks assessthe occurrence of drugresistance in animal products usedin the study were obtained from Oxoid. widelyused for foodin Botswana. This paper reports on Antibioticsusceptibility testing. Eachof the characterized enterococci,especially Enterococcusfaecalis and Entero- Enterococcus specieswas inoculated in Mueller-Hinton broth coccusfaecium, isolatedfrom milk,beef, and chicken and (Oxoid).The  askswere incubated at 25 8Cona Gallenkamp theirpatterns of susceptibilityto Ž veof theantibiotics that shaker(200 rpm) for 24 h orslightly longer depending on the arecommonly directed against them. growthpattern for the Enterococcus speciesinvolved. AmodiŽed version of the method of Steinberg and Lehrer MATERIALS AND METHODS (34) wasused to obtain an absorbance of 0.6 at 600 nm. One Samplingperiod. Thisstudy was conducted between May milliliterof the cell suspension was transferred into 15 to 20 ml ofmoltenMueller-Hinton agar ,mixedthoroughly, and poured into 1999and July 2000. plates.The mixture was then allowed to solidify in the plates. Milk. Milkwas either purchased from supermarkets or ob- Antibioticdisks (ampicillin [10 mg],vancomycin [30 mg], tainedfrom dair yfarmslocated around Gaborone. A totalof 227 teicoplanin[30 mg],tetracycline [10 mg],and cephalothin [30 milksamples (142 raw-milk samples and 84 pasteurized-milk mg])were then placed onto the medium with sterile forceps. Care samples)were obtained. Samples were transported to the labora- wastaken to ensurethat the disks were completely in contactwith toryin acoolbox in 20 to30min. From each milk sample, 1 ml theagar .Theplates were incubated at 37 8Cfor24 h. The diam- wasaseptically transferred into test tubes containing 9 mlof 0.1% etersof the zones of inhibition were measured with a ruleras sterilepeptone water (Mast Diagnostics, Merseyside, UK). Further recommendedby the NCCLS (28) andWilley et al. (40).Staph- dilutionwas carried out as deemed necessary. From the diluted ylococcusaureus ATCC 29213was used as a controlstrain for samples,0.1 ml was spread plated onto bile esculin agar (BEA; the test. CM888).Plates were incubated at 45 8C for 24 h (26). RESULTS AND DISCUSSION Beefand chicken. Fifty-sevenbeef samples and 58 chicken Enterococcaldiversity in the samples. A total of sampleswere purchased from supermarkets around Gaborone. 1,467enterococcal isolates were pickedfrom the342 sam- Portions(25 g each)of minced and unprocessed beef or chicken sampleswere transferred into stomacher bags containing 225 ml ples(227 milk, 57 beef, and 58 chicken samples). These ofsterile peptone water .Thesamples were homogenized with a isolatescomprised 970 isolates from milk,269 from beef, stomacher(Seaward Stomacher 400, T ekmar,Cincinnati,Ohio) for and228 from chicken. E. faecalis (46.1%) and E. faecium 1min.A 0.1-mlportion of the diluted sample was spread plated (29.0%)accounted for mostof theisolates from thediffer- ontoBEA andincubated at 45 8C for 24 h (20). entsamples (T able1). The remaining species identiŽ ed were Enterococcuscasseli avus (7.6%), Enterococcusgal- Isolation. Aftermeat and milk samples had been incubated linarum (7.4%), Enterococcusavium (5.5%), Enterococcus on BEA at 458Cfor24 h, colonies showing brown halos were durans (2.1%), Enterococcushirae (2.0%), and Enterococ- presumedto be enterococcus colonies (26). Aminimumof four colonieswere picked, transferred onto fresh BEA, andincubated cus mundtii (0.3%)(T able1). A similarspectrum of en- at 458Cfor24 h. Gramstains were carried out for each isolate to terococcihad previously been isolated from animaland an- determinepurity and reaction. Gram-positive cocci forming short imalproducts elsewhere (8, 10). Eightspecies were isolated chainsin pairs were subcultured onto nutrient agar (CM3) slants from milk,but only six and Ž vespecieswere isolatedfrom andincubated at 37 8Cfor24 h. The isolates were maintained at beefand chicken samples, respectively (T able1). 48Cwithperiodic subculturing. E. faecalis strainswere isolatedfrom 57.2%of thebeef samples.This percentage was muchlower than that report- IdentiŽcation. Purecultures were streaked onto Columbia edby Kleinet al. (20) (87%).Enterococci occasionally iso- agarbase (Oxoid, Basingstoke, UK) supplementedwith 5% sheep bloodand incubated at 37 8Cfor24 h. These cultures were used latedfrom animalproducts, such as E.casseliavus, are not toinoculate galleries of API 20Strep(bioMe ´rieux,Paris, France) partof the normal  oraof cattle. In this study, E. durans forthe identiŽ cation of enterococci. Readings were taken after 4 and E. hirae, whichare suspected to originate from the handafter 24 h asspeciŽ ed by the manufacturer. Species names environment,were alsoisolated from beef (10). Similar en- wererecorded for sample codes. terococcalspecies diversity in poultry was reportedby De J.FoodProt., Vol. 66, No. 6 ENTEROCOCCI INMILK,BEEF, AND CHICKEN 933

Vriese etal. (11). De Vrieseet al. (8) highlightedthe Ž nd- TABLE 2. Resistancepatterns for Enterococcusfaecalis isolates ing that E. faecium and E. faecalis arecommonly isolated fromdifferent sources from poultryand poultry products, while E.gallinarum and No. of E. avium arerarely isolated from suchproducts (T able1). No. of Resistance resistant Enterococciare known to exist in the feces of animals Source isolates patterna strains% resistance andto persist in the environment for longperiods. Thus, Milk 415 A 400 96.4 thedegree of contamination of milk, beef, or chicken by V 78 18.8 thesebacteria generally depends on the maintenance of Te 78 18.8 propercleanliness and hygiene during milking or slaughter. T 76 18.3 Sinceenterococci can grow at refrigeration temperatures Ce 140 33.7 (58C)andat elevated temperatures (45 8C)andcan with- AV 10 2.4 standpasteurization temperatures it is possible to Ž ndthem ATe 14 3.4 inlarge numbers in milk. AT 43 10.4 ACe 64 15.4 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/66/6/931/1674782/0362-028x-66_6_931.pdf by guest on 01 October 2021 Antibioticresistance of E. faecalis and E. faecium VTe 1 0.2 isolates. Almostall of the E. faecalis and E. faecium iso- TeT 1 0.2 latesfrom themilk, beef, and chicken samples were resis- TeCe 3 0.7 tantto ampicillin (T ables2 and3). Calia (6) and Lerner TCe 3 0.7 (21) havealso reported high levels of ampicillinresistance AVTe 13 3.1 for thesespecies. V ariousresearchers have noted an in- AVT 6 1.4 AVCe 10 2.4 creasein theampicillin resistance of these species since the VTeT 3 0.7 early 1990s (35). However,Thalet al. (36) reportedthat VTeCe 2 0.5 ampicillin-resistantenterococci were notpresent in either AVTeT 1 0.2 chickensor cowsfed antibiotic-free feeds. The proportions AVTeCe 14 3.3 ofenterococci that were resistantto only one of the other AteTCe 5 1.2 antibioticsranged from 18to 49% (T ables2 and3). Such AVTeTCe 5 1.2 largeproportions of ampicillin-resistant enterococci leaves Beef 154 A 153 99.4 onlythe glycopeptides, vancomycin, and teicoplanin as V 12 7.8 drugsof choicefor thetreatment of enterococcalinfections Te 34 22.1 (6). Klein et al. (20) isolatedenterococci that were sensitive T 9 5.8 tomost drugs, including ampicillin, from beefsamples in Ce 42 27.3 Germany.These researchers then recommended the use of ATe 7 4.5 ampicillinfor patientswith enterococcal infections origi- ACe 22 14.3 natingfrom meat.On the basis of our Ž ndings,the use of AVTe 7 4.5 AVCe 1 0.6 ampicillinfor thetreatment of E. faecalis or E. faecium ATCe 13 8.4 infectionsin animals and in patients at healthcare facilities ATeCe 2 1.3 inBotswana may not produce the desired effect. It is nec- AVTeCe 1 0.6 essaryto consider the resistance pattern of the enterococci AVTeT 2 1.3 inquestion before administering any antibiotic (7). In Bo- ATeTCe 1 0.6 tswana,cattle are left in the pasture and in a few casesare AVTeTCe 1 0.6 givenfeeds without antibiotics or growth promoters until Chicken 107 A 100 93.5 theyare slaughtered; hence, they should have harbored en- V 14 13.1 terococcithat were sensitiveto the antibiotics used in the Te 20 18.7 tests.However ,thesecattle might have received antibiotics T 2 1.9 for thetreatment of diseases, e.g., mastitis. Chickens in Bo- Ce 24 22.4 tswanaregularly receive feeds supplemented with growth ATe 7 6.5 promoters.Although we were unableto obtain information ACe 16 15.0 regardingthe extent of the use of antibiotics on dairy and AVCe 12 11.2 ATeCe 3 2.8 poultryfarms, we suspectedtheir use to be common. AVTeT 2 1.9 Thelevel of enterococcal resistance to cephalothin (a cephalosporinwith a b-lactamring like ampicillin), al- a A,ampicillin;V ,vancomycin;T e,teicoplanin; T ,tetracycline; thoughnot comparable to thelevel for ampicillin,was also Ce,cephalothin. high.As muchas 33.7, 27.3, and 22.4% of the E. faecalis isolatesfrom themilk, beef, and chicken samples, respec- tively,were resistantto cephalothin. In addition, 32.8, 16.9, verylow for isolatesfrom beefand chicken samples (T ables and17.9% of the E. faecium isolatesfrom milk,beef, and 2and3). T etracyclineinhibits protein synthesis, while the chicken,respectively, were resistantto cephalothin. fourremaining antibiotics are all cell wall directed. The Levelsof resistance to tetracyclinewas highfor E. fae- modeof action of tetracycline may be a factorin the low calis and E. faecium isolatesfrom milksamples but were levelsof resistance of the enterococcal isolates from the 934 CHINGWARU ETAL. J.FoodProt., Vol. 66, No. 6

TABLE 3. Resistancepatterns for Enterococcusfaecium isolates levelsfor enterococcalisolates in this study were generally fromdifferent sources higher(13.1% of isolates from chickenwere resistantto No. of vancomycinand 18.7% were resistantto teicoplanin, and No. of Resistance resistant 7.8%of isolates from beefwere resistantto vancomycin Source isolates patterna strains% resistance and22.1% were resistantto teicoplanin) than those reported byLevy and Salyers (22) for enterococcalisolates from Milk 274 A 267 97.4 foodin Denmark. These investigators reported that 9% of V 90 32.8 Te 86 31.4 their E. faecium isolatesand none of their E. faecalis iso- T 72 26.3 latesfrom chickenexhibited resistance to vancomycin. Ce 90 32.8 Noneof their isolates from beefshowed such resistance. AV 19 6.9 Almostall of thesamples contained ampicillin-resistant en- ATe 18 6.9 terococci.Generally, fewer samplesshowed resistance to AT 25 9.2 allof the antibiotics except ampicillin in pasteurized-milk Downloaded from http://meridian.allenpress.com/jfp/article-pdf/66/6/931/1674782/0362-028x-66_6_931.pdf by guest on 01 October 2021 ACe 38 13.9 andmeat samples than in raw-milk samples. Enterococcal VTe 5 1.8 resistancelevels in pasteurized-milk and meat samples AVTe 18 6.6 rangedfrom 22.2to 52.7% for vancomycin,from 18to AVT 6 2.2 56.1%for teicoplanin(although the level was 5.9%for AVCe 3 1.1 threeraw milksamples), from 37to 57.1%for tetracycline, ATeT 6 2.2 VTeCe 3 1.1 andfrom 37to 52.6% for cephalothin.Enterococcal resis- AVTeCe 8 2.9 tancelevels in raw-milk samples ranged from 72to 84% AVTCe 6 2.2 for vancomycin,from 68to 75% for teicoplanin,from 68 AVTeTCe 21 7.7 to74% for tetracycline,and from 61to 74% for cephalo- Beef 77 A 74 96.1 thin.Robredo et al. (32) recoveredstrains of VRE from V 19 24.7 27.2%of the chicken samples they obtained from super- Te 15 19.5 marketsin Spain. Descheemaeker et al. (7) foundall of the T 5 6.5 strains of E. faecium theyisolated from fecalsamples of Ce 13 16.9 poultryand other animals in Belgium to be glycopeptide AV 11 14.3 resistant.However ,Qadriet al. (30) foundthat 46% of en- ATe 4 5.2 terococcalisolates from poultrysamples contained VRE. AT 2 2.6 Of theseVRE isolates,33% were E. faecium isolatesand ACe 5 6.5 therest (67%) were E.gallinarum isolates.V anden Braak AVTe 3 3.9 AVCe 1 1.3 et al. (39) reportedthat 79% of the poultry samples they ATCe 1 1.3 collectedin The Netherlands contained VRE, and 59% of AVTeCe 2 2.6 theirVRE isolateswere identiŽed as vancomycin-resistant AVTeT 2 2.6 E. faecium. Thepresent study also revealed a predominance Chicken 75 A 75 100 ofvan comycin-resistant E. faecium inbeef sa mples V 23 30.7 (24.7%)and in chicken samples (30.7%). E. faecalis ac- Te 18 24.0 countedfor 7.8and 13.1% of the beef and chicken isolates, T 7 9.3 respectively.Such a predominanceof vancomycin-resistant Ce 13 17.3 E. faecium and E. faecalis was alsonoted for milksamples. AV 11 14.7 Thepresence of enterococciwith extensive antibiotic resis- ATe 4 5.3 tancein beef and chicken samples could have been due to AT 2 2.7 thecontamination of the carcasses with environmental (es- ACe 4 5.3 peciallyfecal) enterococci during slaughter and processing. AVTe 4 5.3 AVCe 4 5.3 Multiresistancepatterns of E. faecalis and E. fae- AVTeT 5 6.7 cium. The E. faecalis strainsisolated from milk,beef, and AVTeCe 3 4 chickenexhibited 22, 15, and 10 resistance patterns, re- a A,ampicillin;V ,vancomycin;T e,teicoplanin; T ,tetracycline; spectively.Five (1.2%) of the415 E. faecalis isolatesfrom Ce,cephalothin. milkand one of the isolates from beefexhibited resistance toall of the antibiotics used in the test (T able2). The per- centage of E. faecalis isolatesthat were resistantto four samples.It may also indicate less use of this antibiotic in antibioticsin milk was 4.8%.There were few E. faecalis animalhusbandry. isolatesfrom beef(3.2%) or chicken (1.8%) that exhibited Ingeneral, it was E. faecium, rather than E.faecalis, resistanceto four or more antibiotics. The level of resis- thatappeared to be more resistant to vancomycin, teico- tanceincreased as the number of antibiotics decreased. On planin,tetracycline, and cephalothin in all samples (T able theother hand, E. faecium exhibited18, 14, and 13 resis- 3).The trend for resistanceto teicoplanin was similarto tancepatterns in milk, beef, and chicken samples, respec- thatfor resistanceto vancomycin. Glycopeptide resistance tively.There were higherpercentages of multiple-drug re- J.FoodProt., Vol. 66, No. 6 ENTEROCOCCI INMILK,BEEF, AND CHICKEN 935 sistancefor E. faecium isolatesfrom beefand chicken than 8.De Vriese, L.A.,J. Hommez, R.Wijfels,and F .Haesebrouck.1991. for E. faecalis isolatesfrom thosesources. At least7.7% Enterococcaland streptococcal species isolatedfrom intestinal  ora of poultry. J.Appl.Bacteriol. 71:46–50. E. faecium of the isolatesfrom milkwere resistantto all 9.De Vriese, L.,M. Ieven,H. Goossens,P .Vandamme, B.Pot,J. antibiotics.However ,wellover 13.5% were resistantto four Hominez,and F .Haesebrouck.1996. Presence ofvancomycin resis- ormoreantibiotics in milk.None of the E. faecium isolates tantenterococci in farm andpet animals. Antimicrob.Agents Chem- from beefand chicken were resistantto all Ž veantibiotics. other. 40:2285–2287. Of the E. faecium isolatesfrom beefand chicken samples, 10.De Vriese, L.A.,L. Laurier,P .De Herdt,and F .Haesedrouck.1992. 11.7and 16%, respectively, were resistantto three or more Enterococcaland streptococcal species isolatedfrom feces ofcalves, youngcattle anddairy cows. J.Appl.Bacteriol. 72:29–31. antibiotics.Some of the E. faecalis and E. faecium isolates 11.De Vriese, L.A.,A. VanDe Kerckhove,R. Kilpper-Balz,and K. H. from thedifferent samples were resistantto vancomycin Shleifer.1987. Characterization and identiŽ cation of Enterococcus aloneor in combinations. species isolatedfrom intestines of animals. Int.J. Syst.Bacteriol. 37:257–259. CONCLUSIONS 12.Gashe, B.A.1985.Involvement of lactic acid bacteria inthe fer-

mentationof tef ( Eragrostistef ),anEthiopian fermented food. J. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/66/6/931/1674782/0362-028x-66_6_931.pdf by guest on 01 October 2021 Theantibiotics used in this study represented the major Food Sci. 50:800–801. groupsof antibiotics used in health care centers, namely, 13.Giraffa, G.,andF .Sisto.1997. Susceptibility to vancomycin isolated b-lactams(ampicillin), cephalosporins (cephalothin), gly- fromdairy products. Lett.Appl. Microbiol. 25:335–338. copeptides(vancomycin and teicoplanin), and tetracyclines 14.Greenberg, A. E.,A.D.Eaton,and L. S.Clesceri. 1992.Standard (tetracycline).The high levels of multiresistanceof theen- methodsfor the examination of water andwastewater, 18thed. American PublicHealth Association,Washington, D.C. terococcalisolates has caused concern about the antibiotic 15.Jayarao, B. M.,andS. P.Olivers.1992. Aminoglycoside-resistant resistancesituation for enterococcifrom milk,beef, and Streptococcus and Enterococcus species isolatedfrom bovine mam- chicken,the types of foods chie y consumedby people mary secretions. J.Dairy Sci. 75:991–997. worldwide (5, 31, 33). Wefoundvery high levels of en- 16.Jensen, B. B.1998.The impact offeed additiveson the microbial terococcalresistance to the most important antibiotics (sin- ecologyof young pigs. J.Anim.Feed Sci. 7:45–64. glyand in combination) used to controlhuman enterococcal 17.Jensen, L. B.,A.M.Hammerum, R.L.Poulsen,and H. Westh.1999. Vancomycin-resistant Enterococcusfaecium strainswith highly sim- infections,especially glycopeptides (vancomycin and tei- ilar pulse-Želd gel electropho resis patternscontaining similar coplanin).Such resistance is likely to be passed from live- Tn1546-likeelements isolatedfrom a hospitalizedpatient and pigs stockto humans. It would be interesting to assess the an- in Denmark. Antimicrob.Agents Chemother. 43:724–725. tibioticresistance situation for enterococcalisolates from 18.Klare, I.,H.Heier,H.Claus,G. Bohme,S. Marin,G. Seltman,R. healthcare institutions in Botswana. Hakenbeck,V .Antanassova,and W .Witte.1995. Enterococcusfae- cium strainswith vanA-mediated high-levelglycopeptide resistance ACKNOWLEDGMENTS isolatedfrom animal foodstuffsand fecal samples ofhumansin the community. Microbiol.Drug Resist. 1:265–272. We thankMrs. A. Sosomefor her help in the acquisition of all of 19.Klare, I.,H.Heier, H.Claus,and W .Witte.1993. Environmental thematerials usedin this research. Thehelp rendered by members ofthe strains of Enterococcusfaecium withinducible high-level resistance Reservoirsof Antibiotic Resistance Network(ROAR), anInternet-based toglycopeptide. FEMSMicrobiol.Lett. 106:23–30. network,with regard to the current antimicrobial resistance situationin 20.Klein, G., A. Pack,and G. Reuter.1998.Antibiotic resistance of healthcare centers andin food animals is appreciated.Special thanks go enterococciand occurrence ofvancomycin-resistant enterococci in tothe Belgian Government for Ž nancialsupport to W .Chingwaru. raw-minced beef andpork in Germany. Appl.Environ. Microbiol. 65:1830–1835. REFERENCES 21.Lerner ,S.A.1996.Aminoglycosides, p. 1– 12. In V.T.Andriole 1.Aguirre, M., andM. D.Collins.1993. Lactic acid bacteria andhu- (ed.),Current review ofinfectiousdiseases. CurrentMedicine, Phil- man infections. J.Appl.Microbiol. 25:335–338. adelphia. 2.Bates, J.,J. Z.Jordens,and D. T.GrifŽths. 1994. Farm animals as 22.Levy, S. B.,and A. A.Salyers.1998. DANMAP 97report— con- aputativereservoir for vancomycin-resistant enterococcal infection sumptionof antimicrobial agents and occurrence ofantimicrobial in man. J.Antimicrob.Chemother. 34:507–516. resistance inbacteria fromfood animals, foodsand humans. Res- 3.Bogle, B. R.,and G. S.Bogle.1997. 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