Transferable drug resistance in bacteria from fish-farm sediments

RUTH-ANNE SANDAA, VIGDIS LID TORSVIK, AND JOSTEIN GOKSQ~YR University of Bergen, Department of Microbiology and Plant Physiology, Jahnebakken 5, N-5007 Bergen, Norway

Received March 3 1, 1992

Revision received May 22, 1992

Accepted May 26, 1992

SANDAA. R.-A., TORSVIK, V. L., and GOKS@YR, J. 1992. Transferable drug resistance in bacteria from fish-farm sediments. Can. J . Microbiol. 38: 1061-1065.

Antibiotic-resistant bacteria were isolated from sediment samples collected beneath two fish farms west of Bergen (Norway). The samples were collected just after the fish had been treated with oxytetracycline. Eighty-four bacterial isolates were tested for susceptibility to antibacterial agents. Most of the isolates were resistant to oxytetracycline, kanamycin, and sulfamethoxazole. Transferable plasmid-related resistance was shown by direct cell transfer and agarose gel electrophoresis. Among 34 multiple-resistant isolates, 7 isolates were able to transfer resistance to Escherichia coli HB101. Phenotypical characterization indicated that these seven isolates belonged to the genera Vibrio and Pseudomonas. The results indicate that sediments beneath fish farms may serve as a reservoir for transferable antimicrobial resistance genes.

Key words: drug resistance, gene transfer, marine sediment bacteria.

SANDAA. R.-A., TORSVIK, V. L., et GOKS@YR, J. 1992. Transferable drug resistance in bacteria from fish-farm sediments. Can. J . Microbiol. 38 : 1061-1065.

Des batteries rksistantes aux antibiotiques ont ktk isolkes de prklevements de sediments rkcoltes au-dessous d'installa- tions d'klevage de poissons a l'ouest de Bergen (Norvkge). Les Cchantillons ont ktk prelevks juste aprks un traitement des poissons avec de l'oxytetracycline. La sensibilitk aux antibiotiques a ktk verifike chez 84 souches ainsi isolkes. La majo- rite des souches ktaient resistantes a l'oxytCtracycline, a la kanamycine et au sulfamkthoxazole. Une rksistance plasmi- dique transfkrable a ktk confirmke par transfert cellulaire direct et electrophorkse sur gel d'agarose. Parmi 34 souches demontrant une rksistance multiple, 7 ktaient capables de transfkrer la resistance a Escherichia coli HB101. La caracte- risation phknotypique indique que ces sept souches appartiennent aux genres Vibrio et Peudomonas. Ces resultats con- firment que les skdiments prksents sous les installations d'klevage de poissons peuvent constituer un reservoir de gknes de rksistance transfkrable aux antibiotiques.

Mots clks : rksistance aux antibiotiques, transfert de gknes, bactkries de skdiments marins. [Traduit par la redaction]

Introduction The incidence of antibiotic-resistant bacteria in the envi-

ronment has increased dramatically as a consequence of the widespread use of antibacterial agents (Baya et al. 1986). Bacteria with the highest level of resistance are generally isolated from environments with the greatest potential for contamination by antibacterial agents, e.g., hospitals and hospital sewage effluent, fish farms, and waste waters (Datta 1969; Fontaine and Hoadley 1976; Toranzo et al. 1984; McPherson and Gealt 1986).

Spread of R plasmids in bacteria from polluted environ- ments may cause transfer of resistance genes to bacteria that are pathogenic to either fish or homoiothermic animals. Aoki et al. (1974) demonstrated transferable R factors in Vibrio anguillarum strains from diseased fish. Recent stud- ies have shown that resistance can be transferred from Escherichia coli to the fish pathogen V. anguillarum (R.-A. Sandaa, unpublished).

Most of the studies involving gene transfer in natural envi- ronments have been performed in rivers, lakes, soil, and waste water. Very little is known about the transfer of resis- tant genes in marine environments.

Transferable antibiotic resistance has not previously been reported in Norwegian aquaculture. In this study we have examined a highly polluted marine environment, i.e., a fish- farm sediment, to see whether it may be a reservoir of resis-

' ~ u t h o r to whom all correspondence should be addressed. Printed in Canada / lmprime au Canada

tant bacteria, with the capability of transferring resistant genes.

Materials and methods Sampling

Sediment samples from two fish farms west of Bergen, Norway, were collected in April 1988. The samples were collected just after the fish had received a standard treatment with oxytetracycline, as a result of an outbreak of cold-water vibriosis. The dosage was 75 mg/kg fish per day for 10 days. The antibiotic was administrated orally through oxytetracycline-coated pellets. The samples were col- lected below the feeding place of three cages. Sterile Perspex cylinders with rubber stoppers were used, and the samples were collected by divers. The samples were kept cold during transport to the laboratory and were analyzed immediately after arrival at the laboratory. Subsamples for analysis were taken from the top 2 cm of the sediment cores.

Bacterial counts and isolation Portions of 10 g of wet sediment were homogenized in a Waring

blender at low speed with 90 mL of cold Rila marine solution (Rila Products, Teaneck, N. J.) for 1 min. Dilutions were made in Rila marine solution, and samples were plated on tryptone soya citrate agar (TSCA) (20 g tryptone soya broth (Oxoid Ltd., London, United Kingdom), 10 g trisodium citrate dihydrate, 15 g Difco agar (Difco Laboratories, Detroit, Mich.), 1000 mL sterile 70% sea water) with and without oxytetracycline (25 pg/mL). Lunestad and Goksqyr (1990) have recently shown that only 5% of the oxytetracycline in seawater exists in free form as a result of com- plex formation with magnesium and calcium. Thus the concen- tration of free oxytetracycline in the medium with 70% seawater

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1062 CAN. J . MICROBIOL. VOL. 38, 1992

m .-

60 4-

- m so 4-

4- .t

0 40

8 30

20

10

0 OT DO S K RL NA W N OX0 FL C NV FR AMP

Antibacterial Substance

FIG. 1. Percentage of total isolated bacteria susceptible to antibacterial substances. The bacteria are classified into three groups according to the diameter of the inhibition zone. The diam- eter of the discs was 6 mm. o, inhibition zone 6-10 mm; D, inhibi- tion zone 10-26 mm; m, inhibition zone >26 mm. OT, oxytetra- cycline; DO, doxycycline; S, streptomycin; K, kanamycin; RL, sulphamethoxazole; NA, nalidixic acid; W, trimethoprim; N, neomycin; 0 x 0 , oxolinic acid; FL, flumequine; C, chloram- phenicol; NV, novobiocin; FR, furazolidone; AMP, ampicillin.

was approximately 5 pg/mL. The plates were incubated for 10 days at 15 "C. A total of 84 colonies were randomly picked and isolated from TSCA with 25 pg oxytetracycline/mL (19 strains) and TSCA (65 strains).

Disc diffusion susceptibility testing The antibacterial susceptibility was determined for all the isolates

by using susceptibility test discs (Oxoid Ltd.). The tests were per- formed as recommended by the manufacturers. The isolates were spread on the surface of tryptone soya broth agar (TSA) sup- plemented with 70% seawater, and incubated for 24 h at 15°C before measuring the inhibition zones. Susceptibility to the antibiotics was measured as the diameter of the inhibition zone. Escherichia coli B6 was used as a control organism for verifica- tion of the antibacterial effect of the discs on TSA plates. The drug content per disc was 30 pg oxytetracycline (OT), 30 pg nalidixic acid (NA), 30 pg chloramphenicol (C), 25 pg novobiocin (NV), 25 pg streptomycin (S), 50 pg furazolidone (FR), 30 pg kanamycin (K), 5 pg trimethoprim (W), 10 pg ampicillin (AMP), 30 pg neomycin (N), 5 pg oxolinic acid (OXO), 5 pg flumequine (FL), 25 pg sulphamethoxazole (RL), and 30 pg doxycycline (DO). To determine whether the medium contained end products of folate metabolism Streptococcus faecalis ATCC 29212 was used as a con- trol (Lennette 1985). Such products could permit organisms to avoid effects of inhibitors that interfere with either the biosynthesis of dihydrofolate (e.g., sulfonamides) or its reduction products (e.g., trimethoprim) (Ferone et al. 1975). Resistance patterns of E. coli recipients and transconjugants were also determined by use of susceptibility test discs. The E. coli strains were spread on the surface of Luria Bertani agar (LB) (10 g Bacto tryptone (Difco), 5 g Bacto yeast extract (Difco), 10 g NaCl, 15 g Difco agar, 100 mL distilled water; pH adjusted to 7.5 with NaOH) and incubated at 37°C.

Mating procedure Thirty-four of the isolates with multiple antibiotic resistance were

chosen as donors in the conjugation experiments. Escherichia coli HBlOl, with streptomycin resistance, was used as the recipient strain. Escherichia coli 7 103, with the helper plasmid pRK2013 (48 kb with kanamycin resistance), was used in triparental matings. The donors were grown on TSA with 70% seawater and 25 pg

oxytetracycline/mL. The isolates were incubated at 15°C. The recipient E. coli HBlOl was grown on LB plates with 25 pg streptomycin/mL. Escherichia coli 7103 was grown on LB plates with 25 pg kanamycin/mL. Escherichia coli strains were incubated at 37°C. Tryptone soya broth, supplemented with NaCl to a final concentration of 1.5070, was used for growth of all strains in liquid medium. The membrane filter mating, with some modifications, was carried out as described by Genthner et al. (1988). Aliquots (1 mL) of log-phase cultures of the recipient and donor were mixed in a tube. Cell densities of recipient and donor cells were 2 x 10' and 1 x 10' cells/mL, respectively. After mixing, the cells were filtered on a 0.2-pm Unipore membrane filter, type 11307 (Sartorius GmbH, Goettingen, Germany). The filters were transferred to a Petri dish containing TSA with 1.5% NaCl and incubated at 22°C. After 24 h incubation, the filters were removed and stirred in a Vortex mixer in 2 mL A-14 buffer (39.33 mM Na2HP04.2H20, 22.04 mM KH2P04, 68.45 mM NaC1, 0.81 mM MgS04.7H20, pH 7.2) to remove the bacteria from the filter. One hundred microlitres of appropriate dilutions was then plated on media selec- tive for the exconjugants (LB plates with 25 pg oxytetracycline/mL and 200 pg steptomycin/mL). Plates were incubated at 40°C for 48 h. Transfer frequency was defined as the number of excon- jugants per recipient cell. Both the recipients and donors were tested for growth on the selective media at 40°C as a control. None showed any growth.

Plasm id screening Potential donors, recipients, and transconjugants were screened

for plasmids by using the alkaline lysis procedure of Kado and Liu (1981), modified by L. Q)vre& and R. Sgrheim (University of Bergen, unpublished). The isolated sediment bacteria from a 1- to 2-cm2 area of TSA plates were removed and diluted in 100 pL Tris-EDTA (TE) buffer (40 mM Tris-acetate, 1 mM EDTA, adjusted to pH 8.0) supplemented with 1.5% NaCl. A 100-pL volume of freshly made lysozyme solution (5 mg lysozyme (Sigma Chemical Co., St. Louis, Mo.) per millilitre TE buffer) was added, and the mixture was incubated at 37°C for 10 min. Then 200 pL lysis buffer (70 mM Na,P04. 12H20, 30 mM Na2HP04. 2H20, 3 % sodium dodecyl sulfate, pH 12.14) was added. Escherichia coli cells were scraped off LB plates, suspended in TE buffer, and treated with 300 pL lysis solution. The lysates were deproteinized, first with phenol-chloroform (1: l), then with chloroform - isoamyl alcohol (24:l). An aliquot of DNA was electrophoresed in 0.7% agarose, stained with ethidium bromide, and photographed. Plasmids from E. coli V517 (Macrina et al. 1978) were run as molecular weight standards and used to estimate the size of the plasmids in the sediment bacteria as described by Rochelle et al. (1985).

Characterization of bacterial strains The isolates showing conjugative transfer of resistance genes were

characterized phenotypically, by the following tests: aerobic pro- duction of acid from mannitol, ribose, starch, sucrose, glycerol, mannose, n-acetylglucosamine; metabolism of glucose; proteolysis of gelatin; nitrate reductase; chitinase activity; Voges-Proskauer; production of indole from tryptophane; action of 6-galactosidase and caseinase; and Simmon's citrate. All tests were carried out as described by Hansen and Sgrheim (1991). Presence of cytochrome oxidase and catalase were tested on fresh cultures. Gram reaction was tested on fresh cultured colonies, using 3% KOH (Buck 1982). Morphology and motility were recorded using a phase-contrast microscope. Pigmented colonies were assayed on TSCA plates. The sensitivity to 2,4-diamino-6,7-diisopropylpteridine (0/129) was tested by using susceptibility discs with 10 and 150 pg 0/129 (Oxoid) per millilitre.

Results The antibacterial susceptibility of 84 bacterial isolates,

determined by the disc diffusion test, are shown in Fig. 1. The isolates were classified into three groups according to

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TABLE 1 . Transfer of resistance from sediment bacteria to E. coli HBlOl by using oxytetracycline as a selective transfer marker

Resistance Resistance patterns Transfer Transfer frequencies Strain pattern being transferred frequencies with helper plasmida

OT, DO, S, W, RL OT 1 . 2 ~ 6.5 x l o p 4 OT, DO, NA, S, K, OT, K 5.8 x l o p 5

W, N, RL, 0x0, FL OT, DO, NA, S, K, OT, K 5.8 x l o p 5 1 . 2 ~

N, RL, 0x0, FL OT, K, W, KL, OT, K 5.8 x lo- '

0x0, FL OT, K, W OT, NV 2.9 x 10 - 4

OT, NA, S, K, AMP, OT, NV, AMP N, RL, 0x0, FL

OT, S, W, RL OT, NV, AMP 5.9 x N, S, W

'Plasmid pRK2013 in E. coli 7103 for transfer of mobilizable plasmids. 'Recipient E. coli HBlOl .

the size of the inhibition zone. A high percentage of the isolates were resistant to oxytetracycline, doxycycline, streptomycin, sulphame.thoxazole, and kanamycin. Of the isolates, 33-44% were resistant to the quinone group of antibiotics: nalidixic acid, oxolinic acid, and flumequine. Similar resistance patterns were found for isolates that were resistant to trimethoprim and neomycin. Few isolates were resistant to chloramphenicol, novobiocin, furazolidone, and ampicillin.

The results of the conjugation experiment are given in Table 1. Of the 34 multiple-resistant isolates tested, 7 were able to transfer resistance to E. coli HB101. Escherichia coli 7103 with the broad-range plasmid pRK2013 was used to mediate conjugation of mobilizable but nonconjugative plasmids. The plasmid did improve the frequency of transfer for some isolates (A9, A1 1, K25).

Plasmid bands exhibiting similar electrophoretic mobilities were observed in agarose gels of all the transconjugants and their respective donors. Estimation of the molecular weights of the bands resulted in values from 1.4 to 35.5 MDa (Table 2).

The results of the phenotypic characterization of the isolates being able to transfer resistance genes are presented in Table 3. All isolates were Gram-negative rods. The Voges- Proskauer test and the action of gelatinase, caseinase, and chitinase gave negative results for all strains. All isolates were resistant to 0/129. On the basis of the results given in Table 3, strains A9, A10, and A1 1 probably belong to the genus Vibrio and strains K14, K18, K25, and 27B are classified as Pseudornonas species.

Discussion It was shown that the percentage of oxytetracycline-

resistant bacteria was very high (2 100%) immediately after the medication in April (R.-A. Sandaa, unpublished results). The fish farm had a disease outbreak with subsequent medication with oxytetracycline some months prior to this investigation. Diseased fish have a reduced appetite; therefore a large fraction (approximately 80%) of the drug- coated pellets will not be consumed but will reach the sedi- ment beneath the fish farm (Samuelsen 1988). Most of the oxytetracycline will, according to Samuelsen (1988), be washed out of the sediment during the first weeks, but

TABLE 2. Content of plasmids in donors and transconjugants E. coli HBlOl

Donor Plasmid content of Plasmid strains donor strains (MDa) transferred (MDa)

A9, A10, A l l 1.8, 1.4 K14 21.6, 1.8, 1.4 K18 35.5,26.8,20.0, 11.7,

1 1 .o, 9.4, 7.4, 3.7, 2.7, 1.4

K25 32.2, 26.8, 24.7, 17.3, 15.1, 13.7, 13.3, 8.3, 6.3, 5.3

27 B 2.5, 1.8, 1.4

detectable concentrations are still present 3-6 months after medication. Low oxytetracycline concentrations will not kill the bacteria, but offer favorable conditions for resistant strains to evolve. After a new medication, the resistant bac- teria will have a selective advantage. This may explain why the samples collected immediately after medication con- tained such a high percentage of oxytetracycline-resistant bacteria. The fact that most of the isolated strains showed multiple drug resistance indicates that a potential for transferring resistance genes was present.

Seven of the drugs used in the antibiotic susceptibility test (Fig. 1) are of special interest since they are used for medica- tion in Norwegian fish farms, these are oxytetracycline, furazolidone, oxolinic acid, streptomycin, flumequine, and combinations of trimethoprim and sulfadiazine ('T'ribrissenR). The results of the disc diffusion test show that many bacte- ria were resistant to some of these drugs. The isolates exhib- ited resistance against antibacterial agents that they had presumably never been exposed to. This may be due to accu- mulation of transposable resistant elements or R plasmids with resistance to other antibacterial agents than those selected for (Levin 1986). Oxytetracycline was chosen as the selective marker in the conjugation experiments for two reasons: (i) it has been the most used antibacterial agent in Norwegian fish farms and (ii) the genes that code for oxytetracycline resistance are located near the transfer genes (Freifelder 1985).

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TABLE 3. Phenotypic characterization of strains able to transfer resistance genes

Test A9 A10 A l l K14 K18 K25 27B

Morphology (rods) Gram Motility Pigmentationa Nitrate reduction Chitinase Voges-Proskauer Indole Simmon's citrate Catalase Cytochrome oxidase Gelatinase P-galactosidase Caseinase Carbohydrate utilization , Mannitol

Ribose Starch Sucrose Glycerol Mannose n-Acetylglucosamine

Glucose oxidative Glucose fermentative Susceptibility to

0/129, 150 pg

"Positive test indicates strains that are yellow. b ~ , resistant.

Transferable resistance to oxytetracycline, kanamycin, and ampicillin have previously been reported in fish culture (Aoki et al. 1987), although these R factors were isolated from bacteria in diseased fish. Table 1 shows that not all of the resistance markers were transferred. Resistance to oxolinic acid and nalidixic acid are probably chromosomal and thus not directly transferable. Resistance to novobiocin is found in the exconjugants, but not in the donor and recip- ient. This can be due to low expression of novobiocin resis- tance in the donor but expression in the recipient. It has been reported (Belas et al. 1984) that the ability to express antibiotic resistance, encoded by transposons, may vary in different strains.

Plasmids with the same molecular weights were found in both the transconjugants and their donors, except for K18 and the corresponding E. coli transconjugant (Table 2). This isolate contained a plasmid with lower molecular size than the donor, indicating that only a part of a plasmid had been transferred. Some of the transconjugants had plasmids with low molecular sizes only. Such plasmids are too small to carry genes for conjugal transfer (Saunders 1984). This indicates that some large plasmids, not detected by gel elec- trophoresis, have acted as helper plasmids in mobilization of the smaller plasmids. McClure et al. (1989) have recently shown that a nonconjugative plasmid can be mobilized in situ by a conjugative plasmid occurring in the same cell. Plasmids with large molecular sizes often occur in low copy numbers and may, therefore, be difficult to detect by gel electrophoresis (Grinsted and Bennett 1988). It seemed that a number of different plasmids were transferred. It was possible to link resistance directly to particular plasmids for

two of the seven strains (K18, 27B). In one case resistances to tetracycline and kanamycin were transferred (K18), in the other resistances to tetracycline, novobiocin, and ampicillin were transferred (27B).

This study was carried out under conditions that we believe are favorable for conjugation with respect to nutrient availability and temperature. By removing microorganisms from their native ecological niches, the selective pressures exerted by the environment are changed or eliminated. In the natural environment, temperature, nutrient availability, pH, and bacterial density are some of .the parameters affect- ing gene transfer (Stotzky et al. 1991). Trevors and Oddie (1986) found that mating pair formation by E. coli did occur at temperatures as low as 15°C in soil and water. Steward and Koditschek (1980) found that transfer of resistance genes in sediment could occur at 10°C. The number of colony- forming units (cfu) and the total number of bacteria mea- sured in the sediment were approximately 10' and lo9 bac- teria per gram sediment, respectively (R.-A. Sandaa, unpublished). Considering that bacteria often occur in microenvironments, contact between cells could easily occur. The sediments beneath fish farms consist mainly of waste fodder and fecal material from the fish, and are high in available nutrients. Achtman (1975) observed that mating mixtures of E. coli consist of aggregates of cells (2 to 20) instead of mating pairs of two cells only. It is possible that aggregates of mating cells might consist of a number of dif- ferent types of bacteria. In an environment such as sedi- ments, aggregate formation would probably increase and be more stable on sediment particles. These factors would actually increase the transfer frequency. Naturally occur-

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ring R plasmids in sediments have been observed by Steward and Koditschek (1980), Goyal et al. (1979), and Fredrickson et al. (1988).

We conclude that conjugative transfer of resistance genes may occur even in a marine environment, and the pool of resistance genes in the marine environment is amplified by the use of antibiotic and (or) chemolherapeutic agents in aquaculture. Many fish pathogens can survive in marine environments (Hoff 1989; Husevig et al. 1991). The occur- rence of bacteria with transferable resistant plasmids and fish pathogenic bacteria in sediments creates situations where transfer of resistance to the pathogenic bacteria is possible.

Acknowledgments The authors are thankful to R. Sgrheim and B.T. Lunestad

for valuable help during this study. Part of this work was supported by the Council of Nordic Ministers.

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