Vol. 39, No. 1INFECTION AND IMMUNITY, Jan. 1983, p. 184-1920019-9567/83/010184-09$02.00/0Copyright © 1983, American Society for Microbiology

Characterization of Plasmids in Bacterial Fish PathogensALICIA E. TORANZO,t JUAN L. BARJA,t RITA R. COLWELL, AND FRANK M. HETRICK*

Department of Microbiology, University of Maryland, College Park, Maryland 20742

Received 7 September 1982/Accepted 14 October 1982

Plasmid profiles of representative fish pathogens, Aeromonas salmonicida,Aeromonas hydrophila, Vibrio anguillarum, Pasteurella piscicida, Yersinia ruck-eri, Edwardsiella tarda, and Renibacterium salmoninarum, were determined byagarose gel electrophoresis with four different plasmid detection methods. Acombination of two methods was required to detect the plasmids present in thesestrains and to calculate precisely the molecular weights of the plasmids. Of 38strains, 28 harbored one or more plasmids, with the majority of strains demon-strating multiplasmid banding. Similarity in plasmid banding between strains wasnoted and related to geographic source. Five strains of A. salmonicida possessedsix plasmid bands having molecular weights of 8.6 x 106, 8.4 x 106, 8.1 x 106, 3.6x 106, 3.5 x 106, and 3.4 x 106. Four P. piscicida isolates shared three plasmidbands having molecular weights of 37 x 106, 15 x 106, and 5 x 106, and five A.hydrophila strains harbored a common plasmid having a molecular weight of ca.

20 x 106 to 30 x 106. The highest-molecular-weight plasmids (145 x 106 and 130 x

106) were detected in V. anguillarum. From curing experiments, it was found thatin A. hydrophila strain 79-62, a loss of resistance to tetracycline was associatedwith loss of plasmid content in all susceptible derivatives, suggesting plasmid-mediated tetracycline resistance. Cell surface characteristics and metabolicproperties were also modified in cured derivatives of A. hydrophila strain 79-62.

With the expansion of fish culture in recentyears, problems associated with bacterial fishpathogens have increasingly occurred. Concom-itantly, a variety of important properties ofmicroorganisms have been proven to be plasmidmediated. Plasmids encode virulence determi-nants that could be responsible for specific sur-face antigens permitting attachment of bacteriato fish cells, or alternatively, virulence determi-nants may operate via enterotoxin production aspart of the invasion process (19, 22, 23, 47). Theuse of antibiotics in the treatment of infectiousdiseases of fishes has resulted in the expansionof R plasmids in commercial aquaculture (7, 8,13, 45), owing to the selective pressure exer-cised by chemotherapeutic agents when usedover an extended period of time (2, 3, 9).The presence of plasmids in bacterial fish

pathogens may pose a potential public healthhazard, since plasmids from animals may betransferred to humans either directly, by infec-tion with pathogens such as Aeromonas hy-drophila or Edwardsiella tarda (28, 29), or indi-rectly, if they are transferred to humanpathogens such as Vibrio cholerae or Escherich-ia coli by way of pathogenic fish bacteria (1, 2).

t Present address: Departamento de Microbiologfa, Facul-tad de Biologia, Universidad de Santiago de Compostela,Spain.

Crosa et al. (16, 17) have demonstrated thatvirulence in Vibrio anguillarum is related to thepresence of a plasmid having a molecular weightof 47 x 106. However, there are no reportsshowing physical evidence of plasmids in fishpathogens other than V. anguillarum or associa-tion of plasmids with specific phenotypic traits.The objective of the study reported here was

to determine the plasmid profiles of repre-sentative bacterial fish pathogens received fromseveral culture collections and geographic areas.Several plasmid detection procedures were em-ployed, and the results were compared.

MATERIALS AND METHODS

Bacterial strains. Bacteria used in this study andtheir sources are listed in Table 1. The taxonomicposition of these strains was confirmed with morpho-logical and biochemical tests by methods described byShotts and Bullock (40). A. hydrophila, Aeromonassalmonicida, Yersinia ruckeri, and Edwardsiella tardastrains were maintained in tryptic soy agar (DifcoLaboratories, Detroit, Mich.), and V. anguillarum andPasteurella piscicida strains were maintained in nutri-ent agar (Difco) supplemented with 0.5% yeast extractand 2% NaCl. All strains were routinely cultured forplasmid screening at 25°C in nutrient broth with 0.5%yeast extract and appropriate salt concentration (0.5 or2% NaCI). Renibacterium salmoniarum was main-tained at 15°C in Mueller-Hinton agar or broth (Oxoid,Columbia, Md.) supplemented with 0.1% (wt/vol) L-

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PLASMIDS IN FISH PATHOGENS 185

TABLE 1. Strains of bacterial fish pathogens used in this study

Species Strain Donor" Source Place oforigin

A. salmonicida

A. hydrophila

V. anguillarum

P. piscicida

Y. ruckeri (ERM)

Edwardsiella tarda

R. salmoninarum (BKD)

ATCC 14174ATCC 270133.93123/76H22/77V5/80E8101G8103

ATCC 7966ATCC 9071ATCC 154671.251.5481-8379-6280-Al80-A280-A3Y6267-P-24

775ATCC 19105ATCC 19106ATCC 19109ATCC 14181ATCC 19264

ATCC 17911SB2-KKKB 7703MP 7801NGA 7901MZS 8001

11.411.29

81-48EDK-1KGE 7901

Lea-1-74

ATCCATCCD. P. AndersonB. Austin and C. RodgersB. Austin and C. RodgersB. Austin and C. Rodgersr. Aokir. Aoki

ATCCATCCATCCD. P. AndersonD. P. AndersonA. E. Toranzo and F. M. Hetrickr. WellbornA. E. Toranzo and J. L. BarjaA. E. Toranzo and J. L. BarjaA. E. Toranzo and J. L. BarjaT. Aokir. Aoki

. H. CrosaATCCATCCATCCATCCATCC

ATCCR. RobohmT. KitaoT. KitaoT. KitaoT. Kitao

D. P. AndersonD. P. Anderson

T. WellbornT. AokiT. Aoki

B. Austin and C. Rodgers

Salvelinus fontinalisOncorhynchus masouSalmo salarUnknown fish-speciesSalmo truttaSalmo gairdneriOncorhynchus rhodurusOncorhynchus rhodurus

Frog, "red-leg"Used oil emulsionsHumanSalmo truttaLepomis macrochirusIctalurus puntactusSalmo gairdneriSalmo gairdneriSalmo gairdneriAnguilla japonicaPlecoglossus altivelis

Oncorhynchus kisutchMercenaria mercenariaCrassostrea virginicaMercenaria mercenariaSalmo truttaGadus callaris

Roccus americanusMorone saxatilisSeriola quinqueradiataSeriola quinqueradiataSeriola quinqueradiataSeriola quinqueradiata

Salmo gairdneriOncorhynchus tshawytscha

Ictalurus puntactusAnguilla japonicaTilapia nilotica

Oncorhynchus tshawytscha

U.S.A.JapanU.S.A.IrelandEnglandEnglandJapanJapan

U.S.A.U.S.A.U.S.A.U.S.A.U.S.A.U.S.A.U.S.A.SpainSpainSpainJapanJapan

U.S.A.U.S.A.U.S.A.U.S.A.ScotlandDenmark

U.S.A.U.S.A.JapanJapanJapanJapan

U.S.A.U.S.A.

U.S.A.JapanJapan

U.S.A.

a ATCC, American Type Culture Collection, Rockville, Md.; J. H. Crosa, Department of Microbiology andImmunology, Oregon University Medical School, Portland, Oreg.; B. Austin, Fish Disease Laboratory,Weymouth, England; D. A. Anderson, National Fisheries Health Laboratory, Leetown, W. Va.; A. E. Toranzoand J. L. Barja, Department of Microbiology, University of Santiago de Compostela, Spain; T. Kitao,Department of Fish Faculty Agriculture, Miyazaki University, Japan; T. Wellborn, Mississippi CooperativeExtension Service, Mississippi State University, Jackson, Miss.; T. Aoki, Faculty of Agriculture, MiyazakiUniversity; R. Robohm, National Marine Fisheries Service, Milford, Conn.

cystein hydrochloride and 20% (vol/vol) inactivatedfetal bovine serum (GIBCO Laboratories, Grand Is-land, N.Y.).Drug sensitivity patterns of all strains were deter-

mined by the method of Bauer et al. (11) on Mueller-Hinton agar. The following antibiotic concentrationswere used (in micrograms per milliliter): ampicillin, 10;chloramphenicol, 30; kanamycin, 30; erythromycin,15; streptomycin, 10; tetracycline, 30; gentamicin, 10;

nalidixic acid, 30 (all from BBL Microbiology Sys-tems, Cockeysville, Md.); sulfadiazine, 300; and nitro-furantoin, 300 (both from Difco). Susceptibility toantibiotics in strains failing to grow on Mueller-Hintonmedium was tested with nutrient agar with 0.5% yeastextract and 2% NaCl.

Plasmid detection. Bacteria were grown in 3 to 4 mlof the appropriate medium until late exponentialphase. Cultures were centrifuged for 3 min at 12,000 x

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186 TORANZO ET AL.

g in 1.5-ml Eppendorf vials, using a microcentrifuge(Eppendorf model 5412). Four plasmid DNA isolationprocedures were employed. In a first screening, amodification (37) of the Eckhardt method (18) wasused. The pellets obtained after centrifugation weresuspended in Tris borate buffer (89 mM Tris-12.5 mMNa2 EDTA-89 mM boric acid [pH 8.0]) with 10%glycerol. Fifteen ,ul of resuspended cells were loadedin the agarose gel, and 15 p.1 of RLT mixture (20 ,ug ofRNase A [type III A; Sigma Chemical Co., St. Louis,Mo.] per ml-2 mg of lysozyme [Sigma] per ml-0.05%bromophenol blue in Tris borate buffer) was added.After 20 min, 15 p.1 of sodium dodecyl sulfate (SDS)mixture (Tris borate buffer-10% glycerol-2% SDS)was added to each sample in the wells. When lysis wasobserved, the samples were electrophoresed in TBEbuffer (89 mM Tris-2.5 mM Na2 EDTA-89 mM boricacid [pH 8.2]).

Following the procedure of Holmes and Quigley(26), cells from overnight cultures or several coloniespicked from plates were resuspended in 50 p.1 of lysissolution (8% sucrose-5% Triton X-100 in 50 mM Tris-50 mM Na2 EDTA [pH 8.0]), to which was added 4 p.1of lysozyme (10 ,ug/ml). The vials were placed inboiling water for 40 s and centrifuged for 10 min.Supernatants were recovered, and plasmid DNA wasprecipitated with an equal volume of isopropanol at-20°C for 10 min. Pellets obtained by centrifugationfor 5 min were resuspended in TES buffer (30 mMTris-5 mM Na2 EDTA-50 mM NaCl [pH 8.0]), and thesamples were analyzed by electrophoresis in TBEbuffer.For improved characterization of plasmid content of

the strains, two methods were employed. Followingthe method of Portnoy et al. (38), cells were washed inTS buffer (50 mM Tris-0.1 M NaCl [pH 8.0]) andresuspended in 40 p.1 of the same buffer. Cells weretransferred to a clean 1.5-ml microcentrifuge tubecontaining 600 p.1 of lysis buffer (TS buffer plus 4%SDS [pH 12.3]). After 20 min at 37°C, neutralizationwas accomplished by the addition of 40 p.1 of 2 M Trisbuffer (pH 7.0). Chromosomal DNA was precipitatedby adding 160 p.l of 5 M NaCl. The samples werechilled in an ice bath overnight and centrifuged for 5min, and the supernatants were decanted into othertubes. Plasmid DNA was precipitated with 550 p.1 ofcold isopropanol and placed at -20°C for 3 h. Theprecipitate was collected by centrifugation for 3 minand resuspended in 40 p.l of TES buffer and 10 p.1 oftracking dye mixture (0.07% bromophenol blue-7%SDS-20% Ficoll).The second method used was that of Kado and Liu

(30) with some modification. Cell pellets were resus-pended directly in 50 to 75 p.1 of lysis solution (3% SDSin 50 mM Tris acetate buffer [pH 12.4]) and incubatedat 55 to 60°C for 45 min in a water bath, after which theplasmid DNA was extracted with an equal volume ofphenolchloroform solution (1:1, vol/vol). The emul-sion was separated by centrifuging for 10 min at 12,000x g. The aqueous phase was transferred by capillarypipette to a plastic microculture plate and mixed withtracking dye solution (0.25% bromocresol purple-509oglycerol in Tris acetate buffer).

Gel electrophoresis. DNA samples (20 to 25 p.1) wereelectrophoresed through 0.5 to 0.7% agarose (type 1;Sigma) in either TBE buffer at 150 V, 60 mA for 5.5 hby the method of Portnoy et al. (38) or Tris-acetate

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buffer (40 mM Tris-acetate-2 mM Na2 EDTA [pH 7.9])at 150 V, 110 mA for 4 h by the method of Kado andLiu (30).

Electrophoresis was performed on a horizontal ap-paratus fitted with a water-cooling plate. The gels (5mm thick) were stained for 2 h in 0.5 p.g of ethidiumbromide solution per ml, destained in water, andphotographed with a Polaroid MP4 camera, using type55 P/N film and 23A, 9, and 2B filters.

Bacterial reference strains. The following referenceplasmids were included in the agarose gel electropho-resis (molecular weights are given in parentheses):TP116 (143.7 x 106), R40a (96 x 106), R27 (112 x 106),Ri (60 x 106), RP4 (36 x 106), Sa (25 x 106), RSF1030(5.6 x 106), and plasmids from Escherichia coli V517(ranging between 36 x 106 and 1.4 x 106).

Molecular weights of the plasmids in the bacterialfish pathogen DNA preparations were calculated byplotting the relative mobility of the reference plasmidsagainst log molecular weight.Curing experiments. Curing experiments were car-

ried out with mitomycin C (Calbiochem, La Jolla,Calif.) and ethidium bromide (Sigma). Cells weregrown for 24 h in broth containing 0.5 and 0.05 p.g ofmitomycin C per ml. Concentrations of ethidium bro-mide used were 0.4 and 0.04 mg/ml. After treatment,cells were plated on a solid medium, and when growthwas observed, the plates were replicated onto freshmedium either containing appropriate antibiotics orwithout antibiotics. Colonies which grew on mediumwith antibiotics, as well as those failing to grow, werepicked randomly from master plates to test for plasmidcontent and antibiotic sensitivity.

RESULTSPlasmid detection. Comparison of the four

methods used to detect plasmids in this studyyielded evidence that optimal results wereachieved with the methods of Kado and Liu (30)and Portnoy et al. (38). These methods yieldedthe best resolution of plasmid bands and made itpossible to estimate accurately their molecularweights. In general, the Kado and Liu method(30) was more effective in detecting high-molec-ular-weight plasmids (>70 x 106), whereas themethod of Portnoy et al. (38) proved useful inresolving low-molecular-weight plasmids, nota-bly those in multiplasmid-bearing strains such asA. salmonicida; however, plasmids having mo-lecular weights higher than 90 x 106 were usual-ly lost.The method of Newland et al. (37) did not

resolve the plasmid bands in the multiplasmidicstrains, and the method of Holmes and Quigley(26) was not as repeatable as the other methodsemployed; consequently, these procedures werenot used for molecular-weight determinations.

Plasmid content of bacterial fish pathogens. Atotal of 38 representative bacterial fish patho-gens were examined by four plasmid detectionprocedures, as described above. Results showedthat 28 strains (73.7%) harbored one or moreplasmids. Of the strains bearing plasmids, 23(82.1%) were multiplasmidic, and almost all car-

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PLASMIDS IN FISH PATHOGENS 187

TABLE 2. Number and molecular weights of plasmid bands detected in fish pathogens by two differentplasmid detection methods

No. of plasmidbands detected

Plasmids by method of:Strain present Mol wt (x106)a Antibiotic resistance'

(no.) Portnoy Kadoet al. Liu

A. salmonicidaATCC 14174ATCC 270133.93123/76H22/77V5/80E8101G8103

A. hydrophilaATCC 7966ATCC 9071ATCC 154671.251.5481-8379-6280-Al80-A280-A3Y-6267-9-24

V. anguillarum775ATCC 19105ATCC 19106ATCC 19109ATCC 14181ATCC 19264

P. piscicidaATCC 17911SB2-KKKB 7703MP 7801NGA 7901MZS 8001

+ (9)+ (4)+ (8)+ (7)+ (7)+ (9)+ (5)+ (3)

+ (2)+ (2)+ (1)+ (3)+ (2)+ (3)+ (2)

+ (1)+ (1)+ (1)+ (4)

+ (2)

+ (2)+ (5)+ (4)+ (5)+ (6)+ (8)

9 94 NDC8 86 76 79 94 52 3

55, 9.3, 8.6, 8.4, 8.1, 4.2, 3.6, 3.5, 3.455, 17, 6.7, 6.59.3, 8.6, 8.4, 8.1, 4.2, 3.6, 3.5, 3.472, 8.6, 8.4, 8.1, 3.6, 3.5, 3.455, 8.6, 8.4, 8.1, 3.6, 3.5, 3.455, 9.3, 8.6, 8.4, 8.1, 4.2, 3.6, 3.5, 3.4110, 9.3, 8.6, 4.2, 3.6110, 8.6, 3.6

2 ND 2.5, 1.62 ND 9.5, 4

1 283 2 25, 9.5, 2.52 1 25,7.23 3 28, 7.5, 3.42 1 25,7.2

1113

2

Nb, SdSdNb, SdNb, SdNb, SdSdSm, SdNb, Sm, Na, Sd

Am, Nb, Em, SdAm, Nb, Em, Te, Sd, NfAm, Nb, SdAm, Nb, Em, SdAm, Nb, Em, SdAm, Nb, SdAm, Nb, Em, Sm, Te, SdAm, Nb, Em, Sm, SdAm, Nb, Em, Sm, Cm, SdAm, Nb, Em, Sm, SdAm, SdAm, Nb, Em, Sm, Sd

1 471 501 1454 130, 80, 39, 35

2 5.5, 2.6

2 1 20,73 3 65, 25, 18, 6.5, 34 3 37,30,15,55 3 37, 18, 15, 5.5, 56 4 37,30,15,6.5,5,37 6 80, 55, 37, 30, 15, 6.5, 5, 3

Am, Km, Em, Sm, SdAm, SdKm, Em, Sm, SdKm, Sm, SdAm, SdAm, Sd

Km, Em, Sm, SdKm, Em, Sm, SdKm, Em, Sm, SdKm, Em, Sm, SdKm, Em, Sm, SdKm, Em, Sm, Sd

3 2 72, 32, 25, 15

Edwardsiella tarda81-48EDK-1KGE 7901

+ (1) 1 1 78 Nb, Em, Te, SdNb, Em, Sm, SdNb, Em, Sm, Sd

R. salmoninarumLea 1-74d

Y. ruckeri11.411.29

+ (4) Nb, Em, SdNb, Em, Sd

a Determined by agarose gel electrophoresis.b Abbreviations: Am, ampicillin; Km, kanamycin; Em, erythromicin; Sm, streptomycin; Te, tetracycline; Gm,

gentamicin; Cm, chloramphenicol; Na, nalidixic acid; Nb, novobiocin; Sd, sulfadiazine; Nf, nitrofurantoin.C ND, Not determined.d Resistant to terramycin, aureomycin, and polymyxin (20).

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ried one or more small plasmids (molecularweight, 1 x 106 to 10 x 106). Plasmids withmolecular weights of -20 x 106 were also de-tected in 24 strains (85.7%). Five strains har-bored only a single plasmid with a high molecu-lar weight. Table 2 shows the molecular weightsof plasmids detected by the methods of Kadoand Liu (30) and Portnoy et al. (38).A. salmonicida and P. piscicida most fre-

quently carried more than one plasmid. Five A.salmonicida strains revealed six plasmid bandsin common, with molecular weights of 8.6 x 106,8.4 x 106, 8.1 x 106, 3.6 x 106, 3.5 x 106, and 3.4x 106. Four of these strains harbored a plasmidhaving a molecular weight of 55 x 106. TheJapanese isolates revealed a plasmid having amolecular weight of 110 x 106 that was visibleonly by the Kado and Liu method (30); however,their low-molecular-weight plasmids were com-mon to other American and European isolates.The plasmid banding patterns found in A. sal-monicida strains are shown in Fig. 1, lanes Bthrough G, and Fig. 2, lanes E through I. Most ofthese strains contained two series of plasmidbands in which the upper bands, close to thechromosomal DNA with molecular weights of9.3 x 106, 8.6 x 106, 8.4 x 106, and 8.1 x 106,are the open circular forms, corresponding tothe circular covalently closed forms with molec-ular weights of 4.2 x 106, 3.6 x 106, 3.5 x 106,and 3.4 x 106, respectively (Table 2).

Pasteurella strains isolated from Yellowtail(Seriola quinqueradiata) in Japan shared at leastthree plasmids with molecular weights of 37 x106, 15 x 106, and 5 x 106 (Fig. 3, lanes Bthrough E, and Fig. 4b, lanes F through I).American isolates showed a different pattern(Fig. 3, lane F, and Fig. 4b, lane E), but alldemonstrated the same drug resistance pattern.The highest-molecular-weight plasmids (145 x

106 and 130 x 106; Fig. 4, lane D) were detectedin strains of V. anguillarum by the Kado and Liumethod (30). The presence of plasmids with veryhigh molecular weights appears to be character-istic of V. anguillarum isolated from striped bass(Morone saxatilis) (A. E. Toranzo, J. L. Barja,S. A. Potter, R. R. Colwell, F. M. Hetrick, andJ. H. Crasa, manuscript in preparation). Sevenof twelve A. hydrophila strains harbored plas-mids, five of which, although from differentorigins, carried at least one plasmid with amolecular weight between 20 x 106 and 30 x 106(Fig. 2, lanes B and C). Plasmids were notobserved to be present in the strains receivedfrom the American Type Culture Collection(Fig. 3, lanes L and M).

Y. ruckeri (enteric red mouth bacteria) strain11.4 yielded four different plasmid bands (Fig. 3,lane I, and Fig. 4b, lane B). This strain belongsto the serotype which contains the most virulent

isolates of Y. riuckeri (T. M. Cook, personalcommunication). Only the Edwardsiella tardastrain isolated from channel catfish (Ictaluruspunctatius) carried a plasmid, which had a mo-lecular weight of 78 x 106 (Fig. 4c, lane A). R.salmoninarum was tested for plasmid contentdirectly from agar plates because of its slow,relatively poor growth in liquid medium. Noplasmids were detected.

Antibiotic resistance patterns determined forthe fish pathogens included in this study (Table2) showed that all strains were resistant tosulfadiazine. One strain of A. hydrophila (80-A2)was found to be resistant to chloramphenicol,another A. hydrophila strain (ATCC 9071) wasresistant to nitrofurantoin and tetracycline, andA. hydrophila strain 79-62 and Edwardsiellatarda strain 81-48 were also resistant to tetracy-cline. Sulfadiazine, chloramphenicol, tetracy-cline, and nitrofurantoin have been or are beingused for the control of fish diseases.Curing experiments. Curing experiments were

attempted with five representative fish bacteria,including V. anguiillarulm ATCC 19106, A. sal-monicida ATCC 14174, A. hydrophila 80-A2, A.hydrophila 79-62, and Y. ruckeri 11.4, to deter-mine changes in plasmid content associated withantibiotic resistance patterns. The number ofcolonies tested from each strain ranged between500 to 700.A. hydrophila 79-62 and A. salmonicida

ATCC 14174 produced derivatives demonstrat-ing a change in drug sensitivity. The frequencyof isolation of tetracycline-susceptible deriva-tives of A. hydrophila 79-62 was 85%, aftergrowth in either ethidium bromide or mitomycinC, and the loss of tetracycline resistance wasconcomitant with the loss of plasmid content.Sulfadiazine-susceptible derivatives of A. sal-monicida ATCC 14174 were obtained aftergrowth in ethidium bromide with a frequency of40%. However, loss of plasmids or changes inplasmid mobilities in the sulfadiazine-sensitivederivatives of this strain were not observed.

Neither antibiotic-susceptible nor plasmidlessderivatives were isolated from A. hydrophila 80-A2, V. anguillarlum ATCC 19106, or Y. ruckeri11.4.To evaluate alterations in cell surface charac-

teristics arising in tetracycline-sensitive isolatesof A. hydrophila 79-62, three different tests,agglutination in acriflavine (0.2%), stability ofcells after boiling, and sensitivity to bactericidalaction of normal fresh serum, were done (36).Although parental and cured isolates of A. hy-drophila 79-62 were resistant to normal guineapig serum and did not agglutinate in acriflavine,the virulence characteristic of precipitation afterboiling was observed only in the parental strain.Cured isolates lost this property.

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PLASMIDS IN FISH PATHOGENS 189

FIG. 1. Detection of plasmids in six strains of A.salmonicida using the method of Portnoy et al. (38).Lane A, Sa plasmid; lane B, A. salmonicida V5/80;lane C, A. salmonicida H22n7; lane D, A. salmonicida123/6; lane E, A. salmonicida ATCC 27013; lane F,A. salmonicida ATCC 14174; lane G, A. salmonicida3.93; lane H, Escherichia coli V517. Arrow indicateschromosomal band.

Morphological and biochemical characteris-tics tested for both parental and tetracycline-sensitive derivatives of A. hydrophila 79-62showed metabolic properties of indole and gelat-inase production to be lost in cured A. hydrophi-la derivatives.

DISCUSSIONIn this study, several plasmid detection proce-

dures were evaluated, and the plasmid contentof representative fish pathogens was character-ized. Although the in-the-well lysis method ofNewland et al. (37) was rapid and useful forprimary screening and plasmids with very highmolecular weights could be visualized, it had thedisadvantage of not resolving clearly the plasmid

A B C D E F G H J K

FIG. 2. Detection of plasmids in bacterial fishpathogens using the method of Portnoy et al. (38).Lane A, R27 plasmid; lane B, A. hydrophila 79-62;lane C, A. hydrophila 80-A2; lane D, Escherichia coliV517; lane E, A. salmonicida G8103; lane F, A.salmonicida E8101; lane G, A. salmonicida ATCC14174; lane H, A. salmonicida 123/76; lane I, A.salmonicida ATCC 27013; lane J, P. piscicida ATCC17911; lane K, Rl plasmid. Arrow indicates chromo-somal band.

bands of multiplasmid-bearing strains, e.g., A.salmonicida and P. piscicida.Only with the methods of Kado and Liu (30)

and Portnoy et al. (38) could the molecularweights of the plasmids in the bacterial strainstested be calculated with reasonable accuracy.The different buffers used in these two methodsmay account for the observed different migra-tion rates of the plasmid bands, as plasmidsmasked in the chromosomal band by one proce-dure became visible by the other. However,samples extracted by the Portnoy method andrun in Tris-acetate buffer used in the Kado andLiu method did not allow visualization of theplasmids previously lost in the extraction steps.Thus, a combination of both methods is neces-sary to determine the total plasmid content of agiven strain.

Plasmids associated with properties such asenterotoxigenicity, production of hemolysin, orspecific surface antigens permitting attachmentof the bacteria to host cells have been reportedin human pathogens such as Escherichia coli(19, 24, 34, 47), Yersinia enterocolitica (22, 48),and Yersinia pseudotuberculosis (23). However,

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FIG. 3. Detection of plasmids in 13 bacterial fishpathogens using the procedure of Portnoy et al. (38).Lane A, Escherichia coli V517; lane B, P. piscicidaKB 7703; lane C, P. piscicida MP 7801; lane D, P.piscicida NGA 7901; lane E, P. piscicida MZS 8001;lane F, P. piscicida ATCC 17911; lane G, A. salmoni-cida 3.93; lane H, A. salmonicida ATCC 14174; lane I,

Y. ruckeri 11.4; lane J, V. anguillarum 775; lane K, V.anguillarum ATCC 19109; lane L, A. hydrophilaATCC 7901; lane M, A. hydrophila ATCC 15467; laneN, A. hydrophila 80-A2. Arrows indicate chromo-somal bands.

to date, correlation of virulence and plasmidcarriage in fish pathogens has been reportedonly for V. anguillarium (14, 16, 17), in whichthe virulence plasmid specifies an iron-seques-tering system, enabling bacteria to survive inconditions of iron limitation. Under these condi-tions, at least two new outer membrane proteinsare formed, one of which is inducible only whenthe virulence plasmid is present (15).With the continued use of antibiotics in the

treatment of fish diseases, plasmid-mediatedantibiotic resistance is increasing. Japaneseworkers have detected by conjugation the Rplasmids of drug-resistant strains of fish patho-gens, e.g., A. hydrophila (5, 7), A. salmonicida(4), V. anguillarum (2, 7, 9), Vibrio spp. (6), andEdwardsiella tarda (1). However, at present, thephysical evidence of plasmids in these strainsand correlation with specific characteristics,such as antibiotic resistance, have not beendemonstrated.The use of several agarose gel electrophoresis

techniques in this study has permitted the deter-mination of plasmid profiles in bacterial fishpathogens. Furthermore, similarities betweenstrains isolated from different geographical areas

have been detected. The majority of the strainsexamined (85%) harbored at least one plasmidwith a high molecular weight, indicating thepossibility of direct transfer within the bacterialpopulation. These plasmids may be able to medi-ate mobilization of smaller plasmids.

Finding conjugative plasmids encoding eithervirulence or antibiotic resistance in bacteriafrom fish culture systems is recognized as indic-ative of a potential human health hazard. Thus,A. hydrophila, a ubiquitous component of theaquatic environment (25, 31) and a normal in-habitant of the intestinal tract of fish (42), is alsoa significant bacterial pathogen for fish (12),other cold-blooded animals (41), and humans(29). Similarly, Edwardsiella tarda is known tobe an inhabitant of the intestinal flora of hu-mans, snakes, and aquatic animals (39, 46), apathogen for humans (28), and the causativeagent of diseases in eels cultured in Japan (44)and channel catfish in the United States (35).

Plasmids can be transferred indirectly to hu-man pathogens via pathogenic fish bacteria. Araiet al. (10) reported that R factors from V.anguillarum were stable in V. cholerae as wellas in Escherichia coli, and the transfer of Rplasmids from A. hydrophila to Edwardsiellatarda in fish-culturing ponds has been demon-strated (1). These facts suggest that plasmidsmay spread among bacterial populations inaquaculture systems, with the potential develop-ment of serious public health problems.From curing experiments, it was observed

that A. hydrophila 79-62 lost its resistance totetracycline, which was accompanied by loss ofplasmids in all susceptible derivatives. Thus, itis concluded that tetracycline resistance is plas-mid mediated in this strain of Aeromonas. Cellsurface characteristics were modified to somedegree in cured strains ofA. hydrophila 79-62, aswas demonstrated by the loss of characteristicprecipitation after boiling, a property present inthe parental strain. The relationship betweencell surface characteristics and virulence in mo-tile Aeromonas strains has been reported byMittal et al. (36). Instability after boiling andresistance to fresh serum appear to be associat-ed with the most virulent of the strains of A.hydrophila isolated from moribund fish. Howev-er, since virulent strains were also found to besensitive to serum, according to these authors,the significance of an A. hydrophila isolate couldbe assessed by stability after boiling, followedby application of the acriflavine test.

Metabolic properties, such as indole and ge-latinase production, were lost along with theplasmids in the derivatives of A. hydrophila 79-62. Metabolic properties that are plasmid medi-ated, such as citrate utilization, urease and pro-tease production, raffinose, and lactose and

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PLASMIDS IN FISH PATHOGENS 191

A B C D E F G H A B C

FIG. 4. Detection of plasmids in bacterial fish pathogens using the method of Kado and Liu (30). Arrowsindicate chromosomal band. (a) Lane A, Sa plus RSF1030; lane B, RP4 plus RSF1030; lane C, Escherichia coliV517; lane D, V. anguillarum ATCC 19109; lane E, V. anguillarum 775; lane F, V. anguillarum ATCC 14181. (b)Lane A, V. anguillarum ATCC 19105; lane B, Y. ruckeri 11.4; lane C, A. salmonicida ATCC 14174; lane D, A.salmonicida 3.93; lane E, P. piscicida ATCC 17911; lane F, P. piscicida MZS 8001; lane G, P. piscicida NGA7901; lane H, P. piscicida MP 7801; lane I, P. piscicida KB 7703. (c) Lane A, Edwardsiella tarda 81-48; lane B,A. hydrophila 79-62; lane C, Escherichia coli V517.

sucrose utilization, have been reported for Esch-erichia coli (21, 27, 43) as well as for severalspecies of Streptococcus (32, 33). Thus, in addi-tion to antibiotic resistance, other properties areplasmid mediated in A. hydrophila 79-62.Because neither antibiotic-sensitive nor plas-

midless derivatives of Y. ruckeri 11.4, A. hydro-phila 80-A2, or V. anguillarum ATCC 19106were isolated, it is not clear whether antibioticresistance is plasmid mediated in these strains.

In summary, the major finding of this study isthe demonstration of plasmid profiles for select-ed species of known bacterial fish pathogens,and the first step in determining plasmid func-tion in these species has been achieved. Since anextensive variety of properties are plasmid me-diated, further studies are necessary if a particu-lar plasmid is to be identified unequivocablywith a specific phenotypic property in multiplas-mid-bearing strains.

ACKNOWLEDGMENTSWe gratefully thank the donors who kindly supplied bacteri-

al strains used in this study.Alicia E. Toranzo and Juan L. Baria acknowledge the Juan

March Foundation and Ministry for Education of Spain forfellowships.

This work was supported by National Science Foundation

grant DEB-77-14646, National Oceanic and Atmospheric Ad-ministration grant NA 80 RA D00042, and Maryland PowerPlant Siting Program contract P84-81-04.

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