Differences in Antibiotic Resistance Patterns of Enterococcus faecalis and Enterococcus faecium Strains Isolated from Farm and Pet Animals

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, My 2001, p Vol. 45, No /01/$ DOI: /AAC Copyright 2001, Americn Society for Microbiology. All Rights Reserved. Differences in Antibiotic Resistnce Ptterns of Enterococcus feclis nd Enterococcus fecium Strins Isolted from Frm nd Pet Animls PATRICK BUTAYE,* LUC A. DEVRIESE, AND FREDDY HAESEBROUCK Lbortory of Veterinry Bcteriology nd Mycology, Deprtment of Pthology, Bcteriology, nd Poultry Diseses, Fculty of Veterinry Medicine, University of Ghent, B-9820 Merelbeke, Belgium Received 14 June 2000/Returned for modifiction 28 September 2000/Accepted 7 Februry 2001 The prevlence of cquired resistnce in 146 Enterococcus fecium nd 166 Enterococcus feclis strins from frm nd pet nimls, isolted in 1998 nd 1999 in Belgium, ginst ntibiotics used for growth promotion nd for therpy ws determined. Acquired resistnce ginst flvomycin nd monensin, two ntibiotics used solely for growth promotion, ws not detected. Avoprcin (glycopeptide) resistnce ws found spordiclly in E. fecium only. Avilmycin resistnce ws lmost exclusively seen in strins from frm nimls. Resistnce rtes were higher in E. fecium strins from broiler chickens thn in strins from other niml groups with tylosin nd virginimycin nd in E. feclis s well s in E. fecium strins with nrsin nd bcitrcin. Resistnce ginst mpicillin ws minly found mong E. fecium strins from pets nd ws bsent in E. feclis. Tetrcycline resistnce occurred most often in strins from frm nimls, while enrofloxcin resistnce, only found in E. feclis, occurred eqully mong strins from ll origins. Resistnce ginst gentmicin ws very rre in broiler strins, wheres resistnce rtes were high in strins from other origins. It cn be concluded tht resistnce ginst ntibiotics used solely for growth promotion ws more prevlent in E. fecium strins thn in E. feclis strins. With few exceptions, resistnce ginst the different ctegories of ntibiotics ws more prevlent in strins from frm nimls thn in those from pets. Following the emergence of vncomycin (glycopeptide) resistnce in enterococci, discussion on the use of growthpromoting ntibcterils hs risen. The fer of possible trnsfer of resistnce genes from niml bcteri to humn bcteri led to the bn on the use in niml feeds of most growthpromoting ntibiotics in the Europen Community (6). Reports of resistnce in enterococci ginst growth-promoting gents re rre (1, 5, 15). Most dt on resistnce ginst growth promoters concern the prevlence of resistnce ginst glycopeptides in enterococci from frm nimls. Only one report hs delt with resistnce rtes in enterococci from pet nimls ginst therpeutic ntibiotics not used in niml feeds (21). However, only limited number of strins nd origins were investigted. Since the prevlence of resistnce ginst ntibiotics in enterococci isolted from pet nimls is lrgely unknown, we compred the resistnce sitution of enterococci from frm nimls to tht from pets. Most growth-promoting ntibcterils re not used in the ltter ctegory of nimls. The dt obtined re discussed in respect to the mjor trends in ntibiotic usge in the different niml groups. Resistnce prevlences of enterococci from nimls re compred to recent dt on resistnce prevlences mong humn enterococci. MATERIALS AND METHODS Bcteril strins. A totl of 146 strins of Enterococcus fecium nd 166 strins of Enterococcus feclis were isolted in Belgium from 1998 to They originted from the feces of different pet nimls (divided into strins originting * Corresponding uthor. Present ddress: CODA-CERVA-VAR, Groeselenberg 99, 1180 Brussels, Belgium. Phone: Fx: E-mil: pbut@vr.fgov.be. from mmmlin pets nd those from vin pets) nd frm nimls (broiler chickens, bovines, nd fttening pigs), s listed in Tble 1. Among mmmlin pet nimls were cts, dogs, hmsters, squirrels, monkeys, rbbits, rts, nd horses. Among the pet birds were pigeons, geese, ducks, prrots, prkeets, swns, cnries, nd some other psseriformes. There ws no connection between the strins from pets nd the strins from frm nimls. Strins from pet nimls were derived from pets ttending the different clinics t the Fculty of Veterinry Medicine, University of Ghent, or were derived from nimls brought in for necropsy t the Deprtment of Pthology of the Fculty of Veterinry Medicine, University of Ghent. Ech strin ws representtive of single origin: single frm or owner originting from the Flemish provinces of Belgium. No dt on previous ntibiotic usge (for therpy or for growth promotion) were collected. In generl, Belgin frm nimls re fed diet contining growth promoter. This study ws performed fter the bnning of voprcin (bnned in 1997), nd some strins were collected shortly fter the bnning of bcitrcin, tylosin, spirmycin, nd virginimycin (bnned in Jnury 1999). Fecl smples were inoculted on Columbi CNA blood gr (Gibco, Pisley, United Kingdom), selective medium contining colistin nd nlidixic cid, nd incubted overnight in ir supplemented with 5% CO 2 or on Slnetz nd Brtley gr (Oxoid, Bsingstoke, United Kingdom). Enterococcus-like colonies were purified nd identified s described erlier (12, 13, 14). Two control strins, E. feclis ATCC nd Stphylococcus ureus ATCC 29213, were included in the tests, s recommended in the Ntionl Committee for Clinicl Lbortory Stndrds (NCCLS) stndrd procedures for testing therpeutic ntibiotics (NC- CLS M31-A) (18). The E. fecium type strin, LMG T, ws included s n dditionl control. Antibcterils. The growth-promoting ntibcterils were obtined s lbortory stndrd powders. The following ntibiotic preprtions were tested: voprcin (Roche, Bsel, Switzerlnd), virginimycin (Pfizer, Rixensrt, Belgium), bcitrcin (67,000 IU/g; Sigm, St. Louis, Mo.), tylosin (Sigm), vilmycin (Eli Lilly, Indinpolis, Ind.), nd nrsin (Eli Lilly). The ntibiotics were dissolved in pproprite solvents to mke stock solutions contining 1,000 g/ml, or 1,000 IU (USP)/ml in the cse of bcitrcin, nd then further diluted in sterile distilled wter ccording to the methods recommended by the NCCLS (18). The ntibiotics not listed in M31-A were voprcin nd bcitrcin dissolved in distilled wter, virginimycin dissolved in ethnol, nd vilmycin nd nrsin dissolved in methnol. Further dilutions were performed s prescribed by the NCCLS. The therpeutic ntibiotics enrofloxcin (Byer AG, Leverkusen, Germny), 1374

2 VOL. 45, 2001 ANTIBIOTIC RESISTANCE IN ENTEROCOCCI FROM ANIMALS 1375 TABLE 1. Number of enterococcl strins tested No. tested Origin E. fecium E. feclis Broiler Swine Ruminnts Pet, vin Pet, mmmlin oxytetrcycline (Sigm), mpicillin (Sigm), gentmicin (Schering-Plough, Kenilworth, N.J.), nd streptomycin (Sigm) were prepred s described bove. Susceptibility tests. MIC tests were crried out on Mueller-Hinton II gr (Becton Dickinson, Cockeysville, Md.) contining doubling dilutions of the ntibiotics (7). Concentrtions from 0.06 to 256 g/ml were tested for ll ntibiotics except streptomycin nd gentmicin, for which only the concentrtions 500, 1,000, nd 2,000 g/ml were tested. Antibiotic-free gr pltes were included s controls. Inocul were prepred by diluting overnight BHI (Oxoid) cultures in buffered sline to density of 0.5 on the McFrlnd turbidity scle nd were diluted 40-fold before inocultion. Pltes were seeded with pproximtely 10 5 CFU. The pltes were incubted in ir t 37 C for 24 h. The strins showing resistnce to voprcin were tested for the presence of vn genes by using multiplex PCR for the detection of the vna, vnb, vnc 1, nd vnc 2 genes s described before (14). Strins for which the mpicillin MICs were 16 g/ml were tested for the production of -lctmse by the nitrocefin test (Oxoid) (19). High-level resistnce ginst streptomycin nd gentmicin ws defined s MIC of 2,000 nd 500 g/ml, respectively, s indicted by the NCCLS guidelines (M31-A) (18). Brekpoints of other therpeuticlly used substnces were derived from the sme source. Sttisticl nlysis. Differences in prevlence of resistnces mong E. fecium nd E. feclis from different origins were nlyzed by the Fischer exct test. A significnce level of 0.05 ws used. RESULTS E. fecium nd growth-promoting ntibiotics. All strins were nturlly resistnt to flvomycin (MIC t which 90% of isoltes re inhibited [MIC 90 ], 256 g/ml), lthough strins from broiler chickens nd swine hd wider MIC distribution rnge (Tble 2) thn strins from other origins. No resistnce ws found ginst monensin (MIC rnge, between 0.12 nd 4 g/ml). Three strins were resistnt to the glycopeptide ntibiotic voprcin. One strin originted from broiler chicken, one from pet rbbit, nd one from bovine. Their resistnce ws medited by the vna gene, s demonstrted by PCR. Lowered susceptibility to nrsin ws minly evident in strins from broiler chickens nd swine. High vilmycin MICs were only found mong the frm niml strins. E. fecium nd ntibiotics used for both growth promotion nd therpy. Tylosin-resistnt strins were isolted from ll origins. Significntly more resistnt strins were found in frm nimls thn in pet nimls (P 0.001). Virginimycin-resistnt strins (MIC, 16 g/ml) were isolted from broilers, swine, nd pet. Strins for which the bcitrcin MICs were 16 IU/ml or higher were found minly in the collections from broilers nd swine. E. fecium nd therpeutic ntibiotics. Ampicillin resistnce ws seen in strins from ll origins except from pet birds. No -lctmse production ws detected in these strins. Resistnt strins were significntly more frequent in the collection from mmmlin pets thn in those from frm nimls nd from vin pets (P 0.001). Resistnce ginst streptomycin (MIC, 2,000 g/ml) ws not detected in strins isolted from ruminnts. No sttisticlly significnt differences were seen mong the different origins for this type of resistnce. Gentmicin-resistnt E. fecium strins (MIC, 500 g/ml) were isolted from ll origins but minly from swine. Almost ll strins from broilers, pigs, nd ruminnts were resistnt to oxytetrcycline, while strins from pet nimls were significntly less often resistnt to this ntibiotic (P 0.001). A monomodl distribution of the enrofloxcin MICs ws obtined, rnging from 0.5 to 8 g/ml. The enrofloxcin MIC for the E. fecium type strin (LMG11423 T [equivlent to ATCC T ]) ws consistently in the higher rnge (8 g/ml). E. feclis nd growth-promoting ntibiotics. No resistnce ws found ginst the ntibiotics flvomycin, voprcin, nd monensin. Resistnce ginst nrsin occurred in strins from ll origins, but minly in poultry strins. Avilmycin resistnce ws found in one strin ech from broiler chicken, pig, nd duck. E. feclis nd ntibiotics used for both growth promotion nd therpy. Resistnce ginst tylosin in E. feclis strins from ll origins ws high. No significnt differences were noted mong the different niml origins. High virginimycin MICs were seen for strins from swine nd broilers only. High bcitrcin MICs occurred for strins from ll origins, but strins from broilers were more often ffected thn strins from other origins. E. feclis nd therpeutic ntibiotics. No resistnce ws found ginst mpicillin. High-level streptomycin resistnce (MIC, 2,000 g/ml) occurred significntly more often mong strins from swine thn in strins from pets nd ruminnts (P 0.01), nd significntly more streptomycin-resistnt strins were isolted from broiler chickens thn from pet birds (P 0.01). In contrst, high-level gentmicin resistnce (MIC, 500 g/ml) ws not found mong broiler strins. There were no significnt differences in gentmicin resistnce prevlence mong strins from other origins. Tetrcycline resistnce ws extremely frequent in strins from ll origins. Not single tetrcycline-susceptible strin ws present in the porcine collection. The enrofloxcin MIC 90 ws 2 g/ml, while few strins were inhibited only by 8 to 64 g/ml (Tble 2), indicting the presence of resistnce. The MICs of the growth promoters for qulity control strins were within the rnges published erlier (4, 5, 7), nd those of other ntibiotics s published in the NCCLS stndrds (M31-A) for the S. ureus nd E. feclis strins (18). DISCUSSION Although ntibiotics used solely for growth promotion re not included in ntionl nd interntionl stndrds for susceptibility testing, nd universlly recognized brekpoints re not vilble, certin indictions re useful. This is notbly the cse with virginimycin, bcitrcin, nd vilmycin. For virginimycin, MICs of 16 g/ml cn be considered s indicting resistnce, becuse resistnce genes hve been demonstrted only in E. fecium strins not inhibited by concentrtions of 16 g/ml nd higher (16). The bcitrcin MICs for E. fecium strins show rther extended rnge. This indictes tht different resistnce mechnisms might be present. However, nothing is known bout the mechnisms or bout the genes encoding this type of resistnce in enterococci. Avilmycin-

3 1376 BUTAYE ET AL. ANTIMICROB. AGENTS CHEMOTHER. Antibiotic nd host origin TABLE 2. Susceptibility of E. fecium nd E. feclis strins from frm nimls nd pets E. fecium E. feclis MIC 90 Rnge % Susceptible MIC 90 Rnge % Susceptible Flvomycin Broilers NA b NA Swine NA NA Ruminnts NA NA Pet, vin NA NA Pet, mmmlin NA NA Avoprcin Broilers NA NA Swine NA NA Ruminnts NA NA Pet, vin NA NA Pet, mmmlin NA NA Monensin Broilers NA NA Swine NA NA Ruminnts NA NA Pet, vin NA NA Pet, mmmlin NA NA Nrsin Broilers NA NA Swine NA NA Ruminnts NA NA Pet, vin NA NA Pet, mmmlin NA NA Avilmycin Broilers NA NA Swine NA NA Ruminnts NA NA Pet, vin NA NA Pet, mmmlin NA NA Virginimycin Broilers NA NA Swine NA NA Ruminnts NA NA Pet, vin NA NA Pet, mmmlin NA NA Bcitrcin Broilers NA NA Swine NA NA Ruminnts NA NA Pet, vin NA NA Pet, mmmlin NA NA Tylosin Broilers Swine Ruminnts Pet, vin Pet, mmmlin Ampicillin Broilers Swine Ruminnts Pet, vin Pet, mmmlin Oxytetrcycline Broilers Swine Ruminnts Pet, vin Pet, mmmlin Continued on following pge

4 VOL. 45, 2001 ANTIBIOTIC RESISTANCE IN ENTEROCOCCI FROM ANIMALS 1377 Antibiotic nd host origin TABLE 2 Continued E. fecium E. feclis MIC 90 Rnge % Susceptible MIC 90 Rnge % Susceptible Enrofloxcin Broilers NA NA Swine NA NA Ruminnts NA NA Pet, vin NA NA Pet, mmmlin NA NA Streptomycin Broilers 500 2, , , , Swine 1,000 2, , ,000 2, , Ruminnts 500 1, ,000 2, , Pet, vin 500 1, , , , Pet, mmmlin 500 2, , , , Gentmicin Broilers , Swine 500 2, , , , Ruminnts , , , Pet, vin , , , Pet, mmmlin , , , Microgrms per milliliter. Bcitrcin MICs re in interntionl units per milliliter. b NA, not pplicble (no NCCLS brekpoint vilble). resistnt strins seemingly hve two types of resistnce: lowlevel type nd high-level type. This ws lso evident in Dnish study (1). Resistnce ginst everninomycin, relted ntibiotic, hs been shown to be medited by muttion in the ribosoml L16 protein in Streptococcus pneumonie (2). If resistnce in enterococci is lso medited by point muttions, double muttion or muttion combined with specific resistnce gene might explin the two levels of resistnce observed in the present study. No cquired resistnce ws found ginst flvomycin nd monensin, two ntibiotics used solely for growth promotion. Monensin results obtined in this study were similr to the results obtined in Dnish investigtion (1). In the ltter study, few E. fecium strins for which the flvomycin MICs were low were reported. However, it must be noted tht identifiction errors my occur when phenotypic identifiction is used (3), nd it hs been shown tht severl enterococcl species cn be clssified s nturlly flvomycin resistnt while others re nturlly susceptible (4). The flvomycin MIC for the E. fecium type strin used s n dditionl control in the present investigtion ws consistently 256 g/ml. Only three E. fecium strins were resistnt to voprcin, glycopeptide ntibiotic with full cross-resistnce with vncomycin (6). This reltively low resistnce rte is due to the fct tht no selective enrichment nd isoltion in medi contining vncomycin ws pplied. This fctor strongly influences the isoltion frequencies of resistnt strins (9). Ionophores re included routinely in broiler feed for the prevention of coccidiosis. In swine, slinomycin is llowed for growth promotion, nd in ruminnts, monensin is used for the sme purpose. These products re not used for pet nimls. This might explin the differences seen between the enterococci from different niml origins. Nrsin resistnce, fully cross-resistnt with slinomycin (8), ws found only in E. fecium from frm nimls, minly broilers. Since the resistnce mechnism nd resistnce genes re unknown, no specific dditionl test is vilble for confirming resistnce. In contrst, nrsin resistnce in E. feclis ws present in strins from ll origins, but resistnt strins were isolted minly from broilers. The prevlences of resistnces to ntibiotics used both therpeuticlly nd for growth promotion were generlly much higher thn those for resistnces to the ntibiotics used solely for growth enhncement. However, it should be noted tht for the ntibiotics virginimycin nd bcitrcin, only topicl preprtions re vilble. These re only spordiclly used in mmmlin pet nimls nd not t ll in pet birds. Bcitrcinresistnt enterococci were found to occur in severl niml species, including pet birds. The reson for this remins uncler. Tylosin nd relted mcrolides re the only ntibiotics in this group tht re commonly pplied in the therpy of pet nd frm nimls. This is reflected by the high resistnce prevlence in pet nimls s well s in frm nimls. Nevertheless, significntly more tylosin-resistnt E. fecium strins were isolted from frm nimls thn from pets, possibly reflecting the more extensive use of this ntibiotic in frm nimls. For the therpeuticlly most importnt ntibiotics in humn enterococcl infections, certin differences were noted between the resistnce sitution in niml nd in humn clinicl strins. The resistnce rtes mong niml strins were lower thn those found mong humn E. fecium strins isolted from hospitl ptients in recent Europen study (20), while the rtes mong ruminnt nd mmmlin pet strins were similr to those found in humn strins in 1993 in Belgium (22). High-level streptomycin resistnce, defined s MIC of 2,000 g/ml, hs been reported to be generlly more frequent thn high-level gentmicin resistnce, defined s MIC of 500 g/ml (17, 20). This ws not evident in our study except for the poultry strins, in which gentmicin resistnce ws rre. The prevlence of gentmicin resistnce in E. fecium strins from swine, ruminnts, nd vin pets ws higher thn tht recently reported in humn strins (20), while the opposite ws true for E. feclis.

5 1378 BUTAYE ET AL. ANTIMICROB. AGENTS CHEMOTHER. Enterococcl infections re rre in nimls nd re not treted s such (10, 11). However, s intestinl inhbitnts, enterocci re under selective pressure from every ntibiotic dministered tht is ctive ginst them. Tetrcyclines re still used frequently in nimls, nd resistnce rtes were very high in both the E. fecium nd E. feclis collections, especilly in strins originting from swine. Only one porcine E. fecium strin ws found to be susceptible, nd ll porcine E. feclis strins were resistnt. Enrofloxcin is only mrginlly ctive ginst E. fecium. Although ll strins were in the intermedite or resistnt ctegory of the NCCLS susceptibility criteri, they cnnot be considered s hving cquired resistnce, since not only the enrofloxcin MICs for the field strins were monomodlly distributed. The brekpoint of this ntibiotic is the medin of the monomodl distribution of the MICs for the E. fecium strins tested here. Moreover, the MIC for the E. fecium type strin used ws 8 g/ml, which is bove the brekpoint. In conclusion, resistnce ginst growth-promoting ntibiotics nd ntibiotics used for both growth promotion nd therpy, s well s resistnce ginst therpeutic ntibiotics, ws present minly in strins from food nimls. -Lctm resistnce, more frequently found in strins from mmmlin pets, ws the only exception in the lst ctegory of gents. ACKNOWLEDGMENTS This work hs been supported by the Reserch Fund of the University of Ghent, Ghent, Belgium, Code BOZF99/N24/030. We thnk A. Vn De Kerckhove for her skilled technicl ssistnce. REFERENCES 1. Arestrup, F. M., F. Bger, N. E. Jensen, M. Mdsen, A. Meyling, nd H. C. Wegener Surveillnce of ntimicrobil resistnce in bcteri isolted from food nimls to ntimicrobil growth promoters nd relted therpeutic gents in Denmrk. APMIS 106: Adrin, P. V., W. Zho, T. A. Blck, K. J. Shw, A. S. Hre, nd K. 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