Campylobacterjejuni and Campylobacter coli Isolated from Healthy

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1985, p /85/ $02.00/0 Copyright 1985, Americn Society for Microbiology Vol. 22, No. 3 Occurrence of Plsmids nd Antibiotic Resistnce mong Cmpylobcterjejuni nd Cmpylobcter coli Isolted from Helthy nd Dirrheic Animls WAYNE C. BRADBURY* AND DONNA L. G. MUNROE Deprtment of Microbiology, University of Toronto nd Toronto Generl Hospitl, Toronto, Ontrio, Cnd M5G IL7 Received 11 Mrch 1985/Accepted 28 My 1985 Serologiclly defined strins of Cmpylobcter jejuni nd Cmpylobcter coli from helthy nd dirrheic nimls were exmined for the occurrence of plsmid DNA in ssocition with the ntibiotic susceptibility of the bcteril host nd the helth sttus of the niml host. Of ll cmpylobcter orgnisms surveyed, 53% (116 of 200) contined plsmid DNA. A plsmid occurrence rte of 73.8% ws obtined for C. coli from helthy pigs, contrsted by lower plsmid occurrence rtes for C. coli from dirrheic pigs (30%) nd from ll dirrheic nimls (21.4%). For C. jejuni, in contrst, only 13.6% of helthy cttle contined plsmid DNA, contrsted by higher plsmid occurrence rte of 31.2% from dirrheic cttle. A high plsmid occurrence rte of 75.8% ws observed for C. jejuni from helthy chickens. Cmpylobcter plsmids rnged in size from '1 to 86 megdltons. Antibiotic susceptibility for 52 niml isoltes (excluding chickens) indicted tht most isoltes were susceptible to knmycin, erythromycin, gentmicin, tetrcycline, nd compound sulfonmide, wheres few were susceptible to bcitrcin (19.2%); pproximtely hlf were susceptible to mpicillin (55.8%) nd streptomycin (51.9%), nd no isoltes were susceptible to penicillin G. More isoltes contining plsmids were resistnt to mpicillin, tetrcycline, nd gentmicin thn were isoltes not crrying plsmids, there being sttisticlly significnt difference for tetrcycline nd gentmicin, which suggested tht these two ntibiotics were probbly plsmid medited. The ntibiotic susceptibility ptterns of 21 chicken isoltes of C. jejuni, by contrst, were different in tht most were susceptible to mpicillin in ddition to knmycin, erythromycin, nd gentmicin, wheres few were susceptible to compound sulfonmide, streptomycin, nd tetrcycline in ddition to penicillin G nd bcitrcin. A 30- or 39-megdlton plsmid, or both, common to mny of the chicken isoltes ws usully ssocited with tetrcycline resistnce. Cmpylobcter jejuni nd to lesser extent Cmpylobcter coli hve been ssocited with both humn nd niml diseses, most commonly cusing gstroenteritis (4, 5, 9, 17, 19, 25, 34). Cmpylobcter spp. now hve been isolted from wide rnge of hosts (34, 37, 42, 44), nd it TABLE 1. bcteri (29, 31). The common ssocition between Cmpylobcter spp. nd humn disese mkes them potentil source of ntibiotic resistnce genes, lthough informtion on the susceptibility of Cmpylobcter spp. from nimls is lcking. Bcteril ntibiotic resistnce genes POR in niml isoltes of C. coli nd C. jejuni (excluding chicken isoltes) POR (%) in: Animl species Helthy Dirrheic Totl no. psm C. coli C. jejuni C. coli C. jejuni Pig 45/61 (73.8) 1/1 3/10 (30) 3/3 52/75 (69.3) Cttle/diry cow 3/22 (13.6) 0/3 10/32 (31.2) 13/57 (22.8) Dog 1/2 1/2 Fox 1/1 1/1 Got 1/1 1/1 Mink 1/1 1/1 Prtridge 0/1 0/1 Totl percentges of POR in niml isoltes were: helthy C. coli, 73.8%; helthy C. jejuni, 17.4%; dirrheic C. coli, 21.4%; dirrheic C. jejuni, 42.5%. The totl percentge of strins with plsmids ws 50%o. now is ccepted tht the niml reservoir represents mjor source of infection for humns (16, 34). Furthermore, there is concern tht the widespred use of ntibiotics, s therpeutic gents nd s feed dditives, in food-producing nimls, my led to the development of ntibiotic-resistnt * Corresponding uthor. 339 commonly re crried on plsmids, s re virulence properties such s ttchment fctors, toxin production, nd invsiveness (11). Although C. jejuni nd C. coli re known to crry plsmids (2, 7, 40, 41), no plsmid yet hs been correlted to pthogenicity nd only tetrcycline resistnce hs been shown to be crried on trnsferble 38- megdlton (MD) plsmid (40, 42). In this study, C. jejuni

2 340 BRADBURY AND MUNROE J. CLIN. MICROBIOL. Strin TABLE 2. Plsmids nd ntibiotic susceptibility mong niml isoltes of C. jejuni nd C. coli (excluding chicken isoltes) Plsmids (MD) Susceptibility tob: T A P G K E ST S B D44 86, 33, (-) SP20c 25, 4.9, 3.7, 2.6, 2.4, 2.2, Sp39c 86, 80, 32, 26.5, SP51c 86, 29, SP120 86, 50, SP52c 86, 32, 26, 10.5, 10.4, 10.05, 6.5-b3d _ SP60c 86, 25, 8.5, 4.9, 3.7, 2.6, 2.4, 2.3, 1.5, 1.2, < SP97c 86, 26, 10.4, 10.05, 6.5-->3d (-) SP106C 86, 23, 3.6, 2.35, 2.1, P5c 86, 25 - (-) _ - + _ P9C 86, P20C 86, P6c 86, 34, 22, P18C 4.2, 2.3, 2.05, 1.5, (-) (-) P23 86, 55, 32, 27.5, 26, 25, 12, 8.8, d DC20 86, 25, 24, DC26 86, 33, 2.5 (-) D , (-) D42 86, 30, 25 (-) D45 86, 27, 23, 21.5, (-) D70 86, 33, (-) D46 86, 33, D12 86, 33, 25 (-) + (-) P P7 29, 18.5->7.3d P16 86, (-) D80 86, 33, 25 (-) (-) DC, Diry cttle (helthy); D12, D31, D42, D44, D45, D46, nd D70, dirreic cttle; SP, slughterhouse pigs (helthy); P5, P6, P9, P18, P20, nd P23, dirrheic pigs; P2, dirrheic dog; P7, dirrheic got; P16, dirrheic fox; D80, dirrheic mink. b T, Tetrcycline; A, mpicillin; P, penicillin G; G, gentmicin; K, knmycin; E, erythromycin; ST, streptomycin; S, compound sulfonmide; B, bcitrcin; +, sensitive; -, resistnt; (-), intermedite. c Isoltes re C. coli. d Arrows denote the ldder ptterns of negtively supercoiled plsmids described in the text. nd C. coli strins isolted from helthy nd dirrheic nimls (27) were exmined for the occurrence of plsmid DNA in ssocition with the ntibiotic susceptibility of the bcteril host nd the helth sttus of the niml host. MATERIALS AND METHODS Source of isoltes. A totl of 200 C. jejuni nd C. coli isoltes were obtined from J. F. Prescott, Ontrio Veterinry College, Guelph, Ontrio, Cnd. Isoltes included 75 porcine, 62 chicken, 57 bovine, 2 cnine, nd 1 ech of fox, got, mink, nd prtridge. Isoltes from helthy pigs nd chickens were obtined from slughterhouses, nd isoltes from helthy cttle were from different frms in Ontrio. Isoltes from dirrheic nimls were obtined from the dignostic clinic nd Veterinry Services Brnch Lbortory of the Ontrio Veterinry College. The species nd serotype of ech isolte were determined previously by Munroe et l. (27). DNA preprtion. Plsmid DNA ws prepred by the method of Brdbury et l. (7). Cells for the plsmid preprtions were grown on Brucell gr (Brucell broth; BBL Microbiology Systems, Cockeysville, Md.; 1.5% gr; Difco Lbortories, Detroit, Mich.) contining 5% citrted clf blood. Eight Escherichi coli strins crrying stndrd plsmids with the following nine moleculr weights were used to mesure the size (in D) of the cmpylobcter plsmids: 86 x 106, 62 x 106, 47 x 106, 36 x 106, 26 x 106, 7.4 x 106, 4.2 x 106, 5.2 x 106, nd 2.6 x 106. Antibiotic susceptibility testing. The test strins were grown on Brucell gr contining 5% citrted clf blood. Pltes were incubted t 37 C for 36 h in 7.0% C02 incubtor. Colonies were suspended in sline (0.85%) nd djusted to pproximtely 105 CFU/ml. Mueller-Hinton gr pltes were swbbed for confluent growth with ech of the test orgnisms. Antibiotic disks were plced in the center of ech plte; pltes were incubted for 48 h t 37 C under 7.0% C02, with the exception of tetrcycline which required reduced C02 concentrtion. This ws chieved by plcing pltes in Torbl nerobic jr which ws first evcuted, nd then 80 cm3 of ir ws dded. This jr then ws filled with the contents of second jr which hd been set up nerobiclly. The jrs were incubted for 48 h t 37 C. Antibiotics tested. From the mny ntibiotics listed for use s feed supplements nd therpeutics in the Cndin niml husbndry industry (3), the following nine ntibiotics were chosen for testing the susceptibility of the cmpylobcter orgnisms: tetrcycline (5 nd 30 lig) (Becton Dickinson nd Co., Cockeysville, Md.), penicillin G (10,ug), mpicillin (10 zug), gentmicin (10,ug), knmycin (30,ig), erythromycin (15,ug), streptomycin (10,ug), bcitrcin (10,ug), nd compound sulfonmide (300,ug) (Oxoid Ltd., London, Englnd). Susceptibility testing ws by stndrd procedures (23). RESULTS Plsmid nlysis. Serologiclly defined strins (27) of C. jejuni nd C. coli from helthy nd dirrheic nimls were exmined for the occurrence of plsmid DNA in ssocition with the ntibiotic susceptibility of the bcteril host nd the helth sttus of the niml host. Of ll cmpylobcter

3 ;~~~~~~~~~~~~~~~~~~~~~~~~~ L -i2- _ -..: :. ~~~~~~~~~~~~~~~~~~~~~~ LANE }) A_ l l FIG. 1. Agrose (0.7%) gel electrophoresis of plsmid DNA from C. jejuni nd C. coli isolted from dirrheic (D) nd helthy (H) nimls (excluding chickens). Plsmid moleculr weights were determined by comprison with E. coli mrker plsmids of known moleculr weights. Plsmid moleculr weights nd ntibiotic susceptibility dt re presented in Tble 2. Lnes: 1 to 7, pig; 8 to 15, cttle; 16, mink; 17, dog; 18, fox; 19, got. The brckets illustrted in lnes 3, 5, 6, nd 19 denote the ldder ptterns of negtively supercoiled plsmids described in the text. Lnes 1 to 7, 8 to 15, nd 16 to 19 were run in seprte gels. Md, MD. Isolte identifictions re described in Tble 2, footnote. orgnisms surveyed, 53% (116 of 200) contined plsmid DNA, nd ll isoltes were screened t lest twice (Tble 1). With the exception of 62 C. jejuni isoltes from helthy chickens which were nlyzed seprtely, the plsmid occurrence rtes (POR) for 138 cmpylobcter isoltes re presented in Tble 1. A POR of 73.8% (45 of 61) ws obtined for C. coli from helthy pigs, contrsted by lower POR of 30% (3 of 10) nd 21.4% (3 of 14) for dirrheic pigs nd ll other dirrheic nimls, respectively. In contrst, for C. jejuni, POR of 13.6% (3 of 22) ws obtined for helthy cttle, contrsted by 31.2% (10 of 32) for dirrheic cttle. Furthermore, the POR for C. jejuni from ll dirrheic nimls ws higher (42.5% or 17 of 40) thn tht from helthy nimls (17.4% or 4 of 23). Bsiclly, the presence of plsmid DNA vried ccording to the helth sttus of the niml host, there being lower POR for C. coli from dirrheic pigs s compred with higher POR for C. jejuni from dirrheic cttle nd other niml species. This suggested link between the presence of plsmids in C. jejuni nd the occurrence of dirrhe in its niml host; however, similr link could not be estblished for C. coli. Plsmid DNA, for instnce, ws found infrequently mong C. coli isolted from dirrheic pigs nd other nimls. This difference ws striking in pigs, considering not only the high POR for C. coli in helthy pigs but lso the lrge number of plsmids present in ech isolte. Of 69 plsmid-contining isoltes (Tble 1) of C. jejuni nd C. coli from helthy nd dirrheic nimls, 27 were selected for moleculr weight determintions of their plsmids by compring their mobilities with nine plsmids of known moleculr weight rnging from 2.6 x 106 to 82 x 106 D (Tble 2). The plsmid profiles of 19 of the 27 plsmidcontining isoltes (Tble 2) re presented in Fig. 1. Cmpylobcter plsmids rnged in size from <1 x 106 to 86 x 106 D. Among the cttle, pigs, nd other isoltes, some plsmids with identicl moleculr weights were found in more thn one isolte. For instnce, n 86 x 106-D plsmid ws found in 85% (23 of 27) of the cmpylobcter isoltes from both dirrheic nd helthy nimls; similrly, 66.6% (18 of 27) hd common plsmids in the rnge of 25 x 106 to 26 x 106 D, nd 44.7% (11 of 27) hd common plsmids in the rnge of 30 x 106 to 34 x 106 D. Two isoltes shred ldder ptterns rnging in size from 3 x 106 to 6.5 x 106 D (Fig. 1, sp52, sp97, brckets in lnes 5 nd 6). Similr ldder ptterns lso were clerly visulized for two other isoltes which rnged in size from 2.7 x 106 to 5.8 x 106 D (Fig. 1, p23, brckets in lne 3) nd from 7.3 x 106 to 18.5 x 106 D (Fig. 1, p7 in lne 19). Preliminry Si nuclese nlysis indicted (unpublished dt) tht the ldder ptterns re negtively supercoiled plsmid DNA with relxed forms represented by the steps in the ldder ptterns. Ech step differed in size by pproximtely 0.1 x 106 to 0.3 x 106 D. These ldder ptterns were not unlike those recently observed for negtively supercoiled pbr322 in E. coli (15). Similr ldder ptterns in C. coli isolte obtined from sheep nd C. jejuni isolte from mrmoset were observed previously (7). In most cses chicken isoltes from single flock were the sme serotype nd contined the sme plsmids (Fig. 2) nd ntibiotic susceptibility ptterns (Tble 3). This suggegted common source for the dissemintion of C. jejuni mong chickens in flock, nd it ws suspected tht the high POR of 75% (47 of 62) obtined ws due to repet isoltion of identicl orgnisms from different birds of the sme flock. For this reson these isoltes were nlyzed seprtely. Five different plsmid profiles representtive of different flocks were observed mong the chicken isoltes, nd in ~~~~~~~~~~~~~~~~~~~~~~~1 VOL. 22, 1985 POR AND ANTIBIOTIC RESISTANCE IN C. JEJUNI AND C. COLI 341 PIGS CATTLE OTHERS P6 P18 P23 sp1i6 SP52 SP97 sp60 dc2o dc26 d31 d42 d45 d70 d46 P12 d80 P2 P16 P7 D D D H H H H H H D D D D D D D D D xmd i.2-

4 342 BRADBURY AND MUNROE J. CLIN. MICROBIOL. CHICKENS x Mc 86- FIG. 2. Agrose (0.7%) gel electrophoresis of plsmid DNA from C. jejuni isolted from helthy (H) chickens. Plsmid moleculr weights were determined s described in the text. Lnes 1 to 3, 4 to 9, 10 to 14, nd 15 to 18 were run in seprte gels. The time of overnight electrophoresis vried between 14 nd 18 h t 35V. In most cses chicken isoltes of the sme serotype from single flock demonstrted the sme plsmids nd ntibiotic susceptibility ptterns. Lnes: 1 to 3, Penner (27, 32) serotype 5; 5 to 9, serotype 2; 10 to 14, serotype 3. In some cses different serotypes hd identicl plsmids, s ws the cse for serotypes 3 (lnes 15 nd 16) nd 1 (lnes 17 nd 18). Plsmid nd ntibiotic susceptibility dt for isoltes SC3, SC8, SC17, SC28, SC32, SC135, SC136, SC138, SC141, SC146, nd SC154 re presented in Tble 3. severl instnces different flocks shred plsmids of identicl moleculr weight (Tble 3, Fig. 2). Figure 2 illustrtes the five plsmid profiles observed: 54 x 106 (lne 4); 54 x 106, 26 x 10e, nd 25 x 106 (lnes 5 to 9); 30 x 106 nd 47 x 106 (lnes 1 to 3); 30 x 106 nd 39 x 106 (lnes 15 to 18); 39 x 106 (lnes 10 to 14). Antibiotic susceptibility. A totl of 52 cmpylobcter orgnisms with nd without plsmids from dirrheic nd helthy nimls (excluding chickens) were tested for their susceptibility to nine ntibiotics commonly used s feed supplements nd therpeutics in the Cndin niml husbndry industry (3) (Tble 4). Tble 2 presents the results of ntibiotic susceptibility of 27 of these plsmid-contining orgnisms, nd for comprison, Tble 5 presents the susceptibility of 25 isoltes without plsmid DNA. All isoltes listed in Tbles 2 nd 5 were compred to correlte the ntibiotic sensitivity with the presence or bsence of plsmid DNA in the host orgnism (Tble 6). In generl, most isoltes were susceptible to knmycin, erythromycin, nd compound sulfonmide, wheres few isoltes were susceptible to bcitrcin (19.2% or 10 of 52); pproximtely hlf were susceptible to mpicillin (55.8% or 29 of 52) nd streptomycin (51.9% or 27 of 52), nd no isoltes were susceptible to penicillin G (Tble 4). When the susceptibility of isoltes crrying plsmids ws compred with tht of isoltes without plsmids, results were similr for susceptibility to knmnycin, erythromycin, streptomycin, compound sulfonmide, bcitrcin, nd penicillin G (Tble 6); however, more isoltes contining plsmids were resistnt to tetrcycline, gentmicin, nd mpicillin thn were isoltes not crrying plsmids. Sttisticlly, the difference between isoltes hrboring plsmids nd those without plsmids ws not significnt for mpicillin (P > 0.005) but ws significnt for tetrcycline (P = ) nd gentmicin (P = ). This suggested tht ntibiotic resistnce to tetrcycline nd gentmicin ws probbly plsmid medited, lthough there did not pper to be consistent reltionship between prticulr plsmid nd resistnce to n ntibiotic. By comprison, 21 chicken isoltes of C. jejuni tested for their ntibiotic susceptibility showed different ptterns in tht most were susceptible to mpicillin in ddition to knmycin, erythromycin, nd gentmicin, wheres few were susceptible to compound sulfonmide, streptomycin, nd tetrcycline in ddition to penicillin G nd bcitrcin (Tble 3). On compring the ntibiotic susceptibility of 15 isoltes with plsmids nd 6 isoltes without plsmids, tetrcycline ws found to be significntly ssocited with isoltes with plsmids thn with those without plsmids, s ws the cse for the other niml isoltes, but not for gentmicin. Furthermore, 30 x 106- or 39 x 106-D plsmid, or both, ws usully ssocited with tetrcycline resistnce in chicken isoltes of C. jejuni (Tble 7, Fig. 2). DISCUSSION Although the origin of the mny genes coding for ntibiotic resistnce remins specultive, it is well known tht genetic vrition in bcteri hs been rpid, especilly in their resistnce to ntibiotics (36); strins resistnt to mny ntibiotics hve been selected where ntibiotic use is intensive nd multiple (12, 45). For instnce, there is widespred use of ntibiotics in the niml husbndry in Cnd (3), nd it is estimted tht nerly hlf the ntibiotics sold in the United Sttes re given to food-producing nimls (31). Consequently, the spred of ntibiotic-resistnt Slmonell spp. to

5 VOL. 22, 1985 POR AND ANTIBIOTIC RESISTANCE IN C. JEJUNI AND C. COLI 343 TABLE 3. Antibiotic susceptibility mong chicken isoltes of C. jejuni with nd without plsmid DNA Strin Visit no. Serotype Plsmids Susceptibility tob: (MD) T A P G K E ST S B SC (-) SC ,26, SC ,26, SC , SC , SC , SC SC (-) SCS (-) SC , (-) SC , SC , (-) SC SC (-) (-) SC SC (-) SC SC (-) - (-) SC , (-) SC , (-) (-) SC , (-) + - (-) (-) SC, Slughterhouse chickens. b +, Sensitive; -, resistnt; (-), intermedite. For n explntion of ntibiotic bbrevitions, see Tble 2, footnote b. In most cses, isoltes obtined on the sme visit to the slughterhouse demonstrted identicl serotypes, plsmids, nd ntibiotic susceptibility ptterns. A 30- or 39-MD plsmid, or both, ws usully ssocited with tetrcycline resistnce. Downloded from humns, for instnce, by wy of contminted met is becoming incresingly more evident (14, 28, 43). As result of this, there is concern tht there my be link mong ntibiotic use in niml feeds, the development nd presence of ntibiotic resistnce mong bcteri in food-producing nimls, nd ntibiotic-ssocited bcteril infection in humns. Animl feed supplementtion with ntibiotics is known to produce ntibiotic-resistnt bcteri in the norml enteric flor of the nimls (1, 38); however, where ntibiotic use hs been restricted, the incidence of strins resistnt to the gent hs declined (22). There is little doubt tht the veterinry nd medicl use of ntibiotics plys crucil role in selecting resistnce; the outstnding question is the impct of ech on the dissemintion of resistnce genes through lrge popultions of nimls nd humns. Nine ntibiotics were selected becuse of their use s supplements nd growth promoters in feeds in the Cndin niml husbndry industry (3); we found tht the cmpylobcter susceptibilities to these nine ntibiotics re consistent with those from other studies, lthough vrition does generlly occur from study to study (6, 9, 18, 44). In this study most cmpylobcter orgnisms, excluding chicken isoltes, were susceptible to knmycin, compound sulfonmide, streptomycin, erythromycin, gentmicin, nd tetrcycline, lthough resistnce to tetrcycline nd gentmicin ws not uncommon. Similr studies hve shown tht there is high frequency of resistnce to mpicillin nd penicillin G (18); likewise, we hve found ll the cmpylobcter orgnisms to be resistnt to penicillin G nd pproximtely hlf to be resistnt to mpicillin. In ddition, most orgnisms were resistnt to bcitrcin, nd nerly hlf were resistnt to streptomycin. In contrst, chicken isoltes of C. jejuni differed in tht they were susceptible to mpicillin in ddition to knmycin, erythromycin, nd gentmicin, wheres few were susceptible to compound sulfonmide, streptomycin, nd tetrcycline in ddition to penicillin G nd bcitrcin. We lso compred ntibiotic resistnce mong bcteri with nd without plsmids nd found tht higher frequency of tetrcycline nd gentmicin resistnce ws observed mong isoltes with plsmids thn mong isoltes without plsmids. With the exception of chicken isoltes of C. jejuni in which 30- or 39-MD plsmid, or both, ws usully ssocited with tetrcycline resistnce, there did not pper to be consistent reltionship between prticulr plsmid nd resistnce to n ntibiotic. A 38-MD plsmid encoding TABLE 4. Susceptibility of niml isoltes of C. jejuni nd C. coli to nine ntibiotics (excluding chicken isoltes) Antibiotic tested Totl no. (%) of strins susceptible Tetrcycline (75) Ampicillin (55.8) Penicillin G... 0 Gentmicin (71.1) Knmycin (90.4) Erythromycin (88.5) Streptomycin (51.9) Compound sulfonmide (88.5) Bcitrcin (19.2) The totl number of strins tested ws 52. on September 25, 2018 by guest

6 344 BRADBURY AND MUNROE J. CLIN. MICROBIOL. TABLE 5. Antibiotic susceptibility mong niml isoltes of C. jejuni nd C. coli without plsmid DNA (excluding chicken isoltes) Strin' Susceptibility tob: T A P G K E ST S B SP9OC P14C (-) D26C DC (-) D D (-) P19c (-) DC (-) DC DC DC (-) DC (-) D (-) D D D D Pli + (-) (-) P P SP61c (-) SP67c + + (-) SP1i8C D18c + (_) ploc DC, Diry cttle (helthy); Pli, P13, D14, P15, D39, D66, D74, D78, D79, nd D69, dirrheic cttle; SP, slughterhouse pigs (helthy); P10, P14, D18, nd D26, dirrheic pigs; P19, dirrheic prtridge. b +, sensitive; -, resistnt; (-), intermedite. For n explntion of ntibiotic bbrevitions, see Tble 2, footnote b. c Isoltes re C. coli. tetrcycline resistnce ws previously identified in C. jejuni nd C. coli (40-42), but trnsfer of tetrcycline resistnce by conjugtion cn occur with no evidence of extrchromosoml DNA in the tetrcycline-resistnt trnsconjugnts (24). In this study, tetrcycline resistnce lso ws observed mong isoltes tht did not possess 30- or 39-MD plsmid, which suggested tht tetrcycline resistnce my be crried on more thn one size of plsmid. Tetrcycline resistnce hs been ssocited with trnsposble elements (13, 20), s hs gentmicin resistnce (30), thus the potentil exists for movement between replicons TABLE 6. nd subsequent trnsfer between other bcteri in humns. Indeed, it is well recognized tht both C. jejuni nd C. coli re found s pthogens in nimls, food, nd humns nd tht the serotypes commonly found in humn disese re frequently isolted from environmentl nd niml sources, cmpylobcter enteritis in humns being zoonosis (16, 27). Furthermore, in recent surveillnce study of the possible linkges between humn infection with C. jejuni nd its contmintion of food it ws found tht enteritis due to C. jejuni ws more common thn tht due to Slmonell spp., nd the study concluded tht the consumption of poultry products ppered to be the min route of trnsmission in the flow of C. jejuni to humns (44). It ws pprent from our study tht niml isoltes of Cmpylobcter spp. frequently hrbored plsmid DNA of different moleculr weight. The high frequency of plsmid DNA observed mong niml isoltes my result from the overll exposure of the orgnisms to ntibiotics present in niml feeds. Under norml conditions the bcteril cell hs no need for plsmids nd they tend to be lost, but when plsmid function becomes essentil for survivl, s for exmple when ntibiotics re used, cells contining the pproprite plsmid will be selected t the expense of those tht do not (12, 22). These environmentl pressures seem to induce significnt moleculr chnges, resulting in the derivtion of new genetic informtion. Often these new genetic loci re trnsient nd highly unstble nd my be lost s rpidly s they were gined. Besides ntibiotic resistnce, other phenotypic trits such s virulence, toxicity, nd hydrocrbon ctbolism re exmples of this phenomenon (10). Coincidentlly, ll of these trits re plsmid encoded, which ccounts for their instbility nd trnsient nture. A 33% POR for niml nd humn isoltes of Cmpylobcter spp. recently ws demonstrted (7). Plsmid DNA ws observed more frequently in C. coli isoltes thn in C. jejuni isoltes. In the present study, exmintion of C. jejuni nd C. coli confirmed this, in tht higher POR of 64% ws observed for ll C. coli isoltes compred with 33% for ll C. jejuni isoltes (or 54.4% including chicken isoltes). The cmpylobcter orgnisms of the host nimls lso were exmined for the occurrence of plsmids in reltion to their helth sttus, nd it ws found tht there ws lower POR for C. coli from dirrheic pigs, contrsted by higher PORs for C. jejuni from dirrheic cttle nd other niml species, which suggested link between plsmids in C. jejuni nd the occurrence of dirrhe in its niml host. Although no single plsmid could be ssocited with the dirrheic Comprison of ntibiotic susceptibility mong niml isoltes of C. jejuni nd C. coli with nd without plsmids (excluding chicken isoltes) No. susceptible Antibiotic tested Cttle strins Pig strins Totl strinsb P+ (9) P- (16) P+ (14) P- (8) P+ (27) P- (25) Tetrcycline Ampicillin 4 il Penicillin G O O Gentmicin Knmycin 8 16 il Erythromycin 8 16 il Streptomycin Compound sulfonmide Bcitrcin p+, With plsmids; P-, without plsmids. Numbers within prentheses re totl numbers of strins. b In ddition to pig nd cttle isoltes, other isoltes re included.

7 VOL. 22, 1985 POR AND ANTIBIOTIC RESISTANCE IN C. JEJUNI AND C. COLI 345 TABLE 7. Antibiotic susceptibility of chicken isoltes of C. jejuni with nd without plsmid DNA No. of Chicken isoltes (C. jejuni) Antibiotic tested susceptible P+ (15) P- (6) Tetrcycline 3 (20%) 6 (0%) Ampicillin 12 (80%) 6 Penicillin 0 (100%) 0 (100o) Gentmicin 15 (0%) 6 Knmycin 15 6 Erythromycin 15 6 Streptomycin 6 (40%c) 2 (33.3%) Compound Sulfonmide 2 (13.3%) 3 (50%) Bcitrcin 0 0 p +, With plsmids; P-, without plsmids. Numbers within prentheses re totl numbers of isoltes. condition, the most commonly occurring plsmids were n 86-, 25- to 26-, nd 30- to 34-MD species. By contrst, erlier studies indicted tht few humn clinicl isoltes of C. jejuni strins contin plsmids (7, 8, 26, 33) nd tht in one humn outbrek of C.jejuni enteritis, the humn isoltes were devoid of plsmids, wheres identicl isoltes from cttle contined 30- to 34-MD plsmids (8). Although there hs been no firm evidence to dte of plsmids ssocited with cmpylobcter disese (39), recent evidence emerging from two seprte lbortories now suggests two pthogenic mechnisms ssocited with cmpylobcter infections: invsion nd enterotoxin production (secretory) (21, 35). It ws suggested tht some strins of C. jejuni might be nonpthogenic, wheres others my hve seprte plsmid-induced properties of either invsiveness or enterotoxigenicity s is known to occur for E. coli orgnisms (21). Thus, it is possible tht there my be different modes of infection, tht my or my not be medited by plsmids, for different cmpylobcter species nd strins. There is no doubt tht the close ssocition between cmpylobcter nd humn enteric disese mkes it potentil gent for the dissemintion of ntibiotic resistnce genes mong humn bcteril flor. 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