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Frequency and distribution of resistance to oxytetracycline in micro-organisms isolated from marine fish farm sediments following therapeutic use of oxytetracycline
Aquaculture Aquaculture 123 (1994) 43-54
Frequency and distribution of resistance to oxytetracycline in micro-organisms isolated from marine fish farm sediments following therapeutic use of oxytetracycline Joe Kerry”, Maura Hiney”, Rosie Coynea, Dave Cazabon”, Saoirse NicGabhainnb, Peter Smith”>* “Fish Disease Group, Department ofMcrobiology, University College Galway, Galway, Ireland ‘Department of Health Promotion, University College Galway, Galway, Ireland
(Accepted 26 January 1994)
Abstract The background level of resistance to oxytetracycline in sediments free of anthropogenic influences was determined on 2216 V agar with 25 ,ug*g-’ oxytetracycline. The mean frequency of resistance in 153 samples taken in Galway Bay was 1.2 & 1.8%. The impact of oxytetracycline therapy on the frequency of resistance in the sediments under a marine fish farm was investigated on two occasions. In the first investigation, oxytetracycline was detected at a concentration of 9.9 ? 2.9 ,ug*g-’ in the sediments under a cage that received 865 g oxytetracycline per day for 10 days, but no significant rise in resistance frequency was detected. In the second investigation, oxytetracycline was detected at a concentration of 10.9 f 6.5 pg*g-’ in the sediments under a cage block that received 175 kg oxytetracycline over 12 days. The frequency of resistance reached 16.0 f 8.9% after the treatment. The frequency declined at an exponential rate ( r2 = 0.89 ) with a half-life of 26 days. At 73 days after the end of therapy the frequency, in under-cage samples, was not significantly higher than the background level. At the end of the therapy elevated frequencies of resistance were detected up to 75 m from the edge of the cage block and in samples where the levels of oxytetracycline were below the limit of detection ( 1.2 ,/.fg*g-’ ). Thirty-three days after the end of the therapy the frequency of resistance in all samples not directly under the cages was not significantly higher than in samples taken from sediments free of anthropogenie influence.
*Corresponding author. 0044~8486/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDZ 0044-8486 (94)00026-K
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J. Kerry et al. /Aquaculture 123 (1994) 43-54
1. Introduction In marine salmon farms the use of oxytetracycline has been demonstrated to coincide with an increased frequency of resistant microflora in the sediments under the fish cages (Husevag et al., 1991; Samuelsen et al., 1992; Torsvik et al., 1988 ), and with the isolation of oxytetracycline-resistant fish pathogens (Bjorklund et al., 1990, 1991; Husevag et al., 1991). Nygaard et al. (1992) demonstrated that the selection of resistant strains may occur as a result of the presence of oxytetracycline in the marine sediments. They added 50 ppm oxytetracycline to boxes of sediment placed on the sea bottom and demonstrated an increase in resistance from 5 rt 1% to 16 & 1.5% over a 12-month period. Hansen et al. ( 1993 ) reported that the addition of 400 ppm of oxytetracycline to sediments in laboratory aquaria resulted in the selection of an increase in frequency of oxytetracycline resistance in the microflora. This frequency rose slowly to a maximum of 35% after 185 days. Samuelsen et al. ( 1992 ) reported frequencies of oxytetracycline resistance of over 100% in the sediment under cages following therapy. The frequencies remained at between 10 and 50% for the subsequent 18 months. Torsvik et al. ( 1988) also reported long-term effects on the frequency of oxytetracycline resistance in sediment flora resulting from the therapeutic use of oxytetracycline. For the 13 months following medication the levels of resistance were between 18% and 26%. Fish pathogens have been detected in farm sediments (Enger et al., 1989; Bjorklund et al., 1991; Husevag et al., 1991) and therefore increases in the frequency of resistance to antimicrobials in this environment may have significance for the success of future antimicrobial therapy of disease in fish. Intra-species transfer of plasmid-encoded resistances has been reported in laboratory simulation of the marine environment (Angles et al., 1993; Goodman et al., 1993). Genetic transfer, mediated by transformation, has also been shown to occur in the marine environment (Stewart and Sinigalliano, 1990). It is possible, therefore, that elevated frequencies of resistance in fish farm sediments may have implications for therapy in terrestrial, and more importantly, human 1983). This investigation was undertaken to determine the impact of oxytetracycline use on the frequency of resistance in the sediment microflora, to determine the persistence of any increase in frequency with respect to time and to determine the area of sediment affected.
2. Materials and methods
Sample sites for determination of background resistance frequencies Galway Bay is a large inlet in the middle of the west coast of Ireland. The bay is approximately 50 km long in an east-west direction, and 16 km north to south. The dominant current circulation is anti-clockwise. The only significant density of human population is Galway city (pop. 50000) situated in the north-east cor-
J. Kerry et al. /Aquaculture 123 (1994) 43-54
45
ner of the bay. Marine salmon farming is concentrated in the north-west of the bay. For the initial survey of the background frequency of resistance a sample site (A) on the north shore of the bay approximately half-way between Galway city and the nearest fish farms was used. For the extended survey, four additional sampling sites were selected, one at the extreme south-east of the bay, and three on the north shore between Galway city and the fish farming sites. Site 2 was close to the main sewage outfall from Galway city. One sample at this site was taken 600 m from the outfall. Sites 1, 3 and 4 were at least 10 km distant from any fish farm or significant sewage discharge. Samples were collected between April and June, at which time the water temperature was between 13 and 15 ‘C. No differential was recorded in the temperature of sediment cores down to 20 cm depth. At each site triplicate core samples of sediment were collected by divers at distances of 300,900 and 1500 m off shore. At site A, additional samples were also taken at 600 and 200 m off shore. Fish farm investigations
The details of the fate of oxytetracycline following the first two therapeutic treatments investigated in this work have been reported elsewhere (Coyne et al., 1994). The layout of the fish cages and sample sites is shown in Fig. 1. In the first investigation 5 samples were collected from under a cage in block 6 at 3, 12 and 19 days after a lo-day treatment with oxytetracycline at 125 mg-kg- ‘. The total amount of oxytetracycline used in the cage was 865 g. In the second investigation, 17 samples were collected under and outside cage block 7 (Fig. 1). The total amount of oxytetracycline used during the 12-day treatment was 175 kg. Samples were collected after 10 days therapy and at 19, 33 and 73 days after the end of therapy. A third investigation was carried out in order to compare various methods of bacteriological enumeration. In this investigation 10 samples were taken under a cage in block 6. The cage received 160 g of oxytetracycline per day for 14 days. Samples were taken 1 day after the end of the therapy. Sample collection
Sediment samples from all sites were collected by divers using plexiglass core tubes of 4.5 cm i.d. and 25 cm length according to the method of Samuelson et al. ( 1992). Samples were transported to the laboratory on ice in an insulated container and bacterial analysis carried out immediately on arrival. Slices corresponding to 2 cm of the sediment core, measured with a ruler, were sequentially pushed from the core tube with a piston. Sediment samples used for drug residue analysis were stored at - 20” C until analysed by the HPLC method of Coyne et al. (1994). Bacteriological analysis
In all investigations 1 g aliquots of samples were diluted in phosphate-buffered saline and plated, in duplicate, on 22 16 V agar (Zobell, 194 1) (ZV) and the same medium with 25 pg*ml-’ oxytetracycline. Plates were counted after 72 h
46
J. Kerry et al. /Aquaculture 123 (1994) 43-54 N
+ BLOCK 6
Predominant
Current
4
Direction
s”2
Fig. 1. Layout of cage blocks and sampling sites. In the first fish farm investigation, samples were taken from under cage marked X in block 6. In the second fish farm investigation, samples were taken at the sites labeled x in block 7.
incubation at 22 oC. Longer incubation at this temperature resulted in fungal overgrowth of bacterial colonies and consequent diffkulties in determining counts. All colonies with an obviously fungal morphology were not included in the counts. The frequency of oxytetracycline resistance was determined by dividing the number of colonies on ZV agar containing oxytetracycline by the number of colonies on ZV agar without oxytetracycline. This figure was then multiplied by 100 to generate the percentage of resistant colony-forming units. The significance of different frequencies of resistance detected in sets of samples was determined using the Student t-test. A comparative media trial was performed where sediment samples (y1= 10 ) were also plated on TSCA (Samuelsen et al., 1992 ) at 15 ‘C for 7 days and on sea water TSA (SWTSA) (Nygaard et al., 1992) at 18°C for 10 days.
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J. Kerry et al. / Aquaculture 123 (1994) 43-54
3. ResuIts Establishment of background resistance levels in Galway Bay
In the initial survey of background resistance at site A the frequency of resistance in the samples from the top 2 cm of the sediment showed a significant inverse correlation with the distance of the sample from the shore ( r2=0.90; p= 0.037). A similar correlation with distance was not seen in samples taken from the lower (2-4 cm and 4-6 cm) depths. A comparison of the frequencies at different depths at each distance from the shore showed significantly higher frequencies in the top 2 cm than in the 2-4 cm sample (p=O.O2) or the 4-6 cm sample (p=O.O2). No significant differences were detected between the frequencies at depths of 2-4 and 4-6 cm. A larger survey of background resistance was then undertaken to determine if these variations in resistance frequency with respect to distance from the shore and depth of sample were general in nature or were site-specific. Analysis of the data from the four additional sites did not confirm the general nature of the observations made from the data collected at site A. A three-way analysis of variance was carried out on resistance frequencies found at the 36 sample sites (3 distances x 3 depths x 4 locations). This analysis identified a main effect of distance and a two-way interaction between location and depth (Table 1). Further analyses of the main effect of distance showed that all differences were in the unpredicted direction, i.e. resistance frequencies increased the further the sample was from the shore. As these differences were unpredicted, post-hoc Scheffe t-tests were applied, but none proved significant. Similarly, after the simple main effects of depth at location and location at depth were carried out and Scheffe t-tests were conducted for those that proved significant, the original preTable 1 Three-way ANOVA on the resistance frequency data from sites 2-4 Source of variance
sum of squares
Location
Degrees of freedom
Variance estimate
22.91
4
5.73
9.76
2
4.88
Locationx depth
73.81
8
9.23
Distance
46.73
2
23.36
40.07
8
5.01
24.23
4
6.06
56.31
16
3.52
263.21
89
2.96
Depth
Location
x
distance
Depthxdistance Location
x
N/Location
depth x
x
distance
depth x distance
J. Kerry et al. /Aquaculture 123 (1994) 43-54
48
diction concerning depth was not confirmed. Thus no reliable differences were identified between any of the samples in this larger survey. The overall mean frequency of oxytetracycline resistance in all 153 cores analysed was 1.2 2 1.8%. The significance of data collected from fish farms was determined by comparison with the distribution of frequencies in these background samples using Student’s t-test. Resistance frequencies in individual samples were considered as meaningfully elevated if they exceeded the mean plus one standard deviation (i.e. 3%). Of the 27 cores taken at site 2,9 were taken from sediments 600 m distant from the main sewage outfall of Galway city. The mean resistance frequency in these 9 cores was 0.52 20.26%. which was not significantly different from the background (g=O.12). Frequency of resistance under$sh farms In the first fish farm investigation the frequency of resistance was determined in 5 samples taken under a single cage in cage block 6 (Fig. 1) . The increase in resistance frequency detected was not significant. Prior to the start of therapy the frequency of resistance was 0.3 + 0.05%. Three days after the end of the lo-day therapy the mean frequency was 2.4 & 3.9%. These data were not significantly higher than the.background frequency (p= 0.1). The frequency fell to 1.3 -t 0.6% 9 days later and to 0.5 + 0.2 28 days later. The concentrations of oxytetracycline detected in the same (top 2 cm) samples were 9.9 2 2.9, 4.9 ? 1.7 and 2.3 2 0.5 ,ug*g-‘, respectively (Coyne et al., 1994). In the second fish farm investigation the layout of sample sites (see Fig. 1) was designed to allow for the determination of both increased frequency of resistance under the cage block and horizontal distribution of increases in samples taken at a distance from cage block 7. With respect to the 7 samples taken directly under the cage block (Fig. 1) the mean frequency of resistance after 10 days of therapy was 16.0 + 8.9% (Fig. 2). Nineteen days after the end of the 12-day therapy, the frequency in these samples was 9.6 t 11.3%. The high variation in this figure was 25 -
I
10
30 Days
after
50
70
therapy
Fig. 2. Concentration of oxytetracycline and percentage of oxytetracycline-resistant microflora in the 7 samples taken from the sediments under cage block 7 during the second investigation. n = oxytetracycline pg-g-‘; 0 =percentage resistance in sediment microflora.
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the result of the detection of 35% resistance in one sample. The frequency had fallen to 3.3 + 1.2% and 1.9 + 0.8% at 33 and 73 days after the end of therapy, respectively. At 73 days, the frequencies of resistance were not significantly different from those in the background samples (p=O.2). The decline in the increased frequency of resistance was exponential ( r2 = 0.89) and the half-life was 26 days. One hundred and forty-one days after the end of therapy the frequency of resistance was 1.5 +_1.1%. The concentrations of oxytetracycline detected in thesesampleson thesame dayswere 10.926.5,3.3? 1.7, 1.6+0.4pg-g-r (Coyne et al., 1994). Seventy-three days after the end of therapy the concentration of oxytetracycline, in all samples, was below the limit of detection. The data from the analysis of samples designed to determine the extent of the horizontal distribution of increases in resistance frequency are shown in Table 2. After 10 days of therapy meaningful rises in resistance frequency ( > 3.0%) were detected in all samples except that taken 100 m to the west of the cage block. In all other directions the limit of the horizontal distribution of the increase was not reached. Oxytetracycline was detected at 1.6 pg*g-’ in the sample 25 m to the west of the cage block and was below the limit of detection ( 1.2 ,ug*g-’ ) in all other samples. In 8 of the 10 samples, therefore, elevated oxytetracycline resistance occurred in the absence of detectable oxytetracycline concentrations. The distribution of increased resistance does not follow the westward bias predicted by the model based on current flow measurements (Coyne et al., 1994). The increases in resistance frequency were greater in samples taken to the north and south of the cages than to the west. Nineteen days after the end of therapy the frequency of resistance in 7 of the 10 samples had decreased, but overall the decreases were not significant (p = 0.12 ) . Thirty-three days after the end of therapy 9 of the 10 samples showed a further Table 2 Frequency of resistance in samples not directly under cage block Site
Wl N2 El E2 Sl s2 Wl w2 w3 W4
Distance from cage
Nearest OTC on day -2*
Percentage resistance
(m)
(m)
Day -2”
2.5 50 25 50 25 50 25 50 75 100
50 15 25 50 25 50 0 25 50 75
“The distance to the nearest sample site that contained days before the end of a 12-day therapy. **Days after the end of a 12&y therapy.
9.6
4.5 4.4 4.9 10 11 10 6.1 6.0 0.7
Day lgb
Day 33b
4.1 4.0 5.1 5.0 5.0 2.8 5.0 3.2 1.1 4.8
1.2 2.2 0.6 1.7 2.0 2.1 2.0 2.3 2.5 1.4
detectable oxytetracycline
( > 1.2 pg.g- *) 2
50
J. Kerry et al. / Aquaculture 123 (1994) 43-54
decrease and the overall frequency was significantly different from that recorded after both 10 days therapy (p=O.O04) and 19 days after therapy (p=O.O03). At this time the frequencies at all of the 10 sample sites were within one standard deviation of the mean background resistance frequency and the overall frequencies were not significantly higher than the background (p= 0.18 ) . Comparison of bacteriological methods On one occasion the frequency of resistance was determined using three culture methods. Following the use of 32.6 kg of oxytetracycline over 14 days at cage block 6, 10 samples were taken 1 day after the end of therapy from under a single cage that received 2.2 kg during the treatment. The frequency of resistance was 12.2 2 16.3% on ZV medium incubated at 22” C for 72 h, 11.7 & 10.4% on TSCA incubated at 15 ‘C for 7 days and 16.2 5 13.6% on SWTSA incubated at 18 ‘C for 10 days. None of these data was significantly different (ZV vs. TSCA, p= 0.97; ZV vs. SWTSA, p=O.37; TSCA vs. SWTSA, p= 0.44).
4. Discussion Solid surface culture methods have been shown to detect a low percentage of the viable bacteria in sea water (Jannasch and Jones, 1959; Buck, 1979; Kogure et al., 1980). The sub-set of the flora cultured will depend on the composition of the medium used (Buck, 1974). Samuelsen et al. ( 199 1) suggested that the fraction of the total viable bacteria that can be cultured from fish farm sediments may be as low as 0.02%. This inefficiency means that the frequencies of resistance determined by culture methods may not accurately reflect the frequency of resistance in the total viable microflora. In fresh water, free of anthropogenic influence, Magee and Quinn ( 199 1) found that oxytetracycline resistance was confined to Pseudomonas, species only and that such oxytetracycline-resistant Pseudomonads comprised 1.8% of the total strains they isolated. They failed to detect evidence that the resistances were plasmid-encoded and suggested that the strains they isolated may have been intrinsically resistant. Intrinsic, and therefore non-transferable, resistance to oxytetracycline has been reported in Pseudomonas aeruginosa (Russell and Chopra, 1990). On the other hand Jones et al. ( 1986) failed to detect any oxytetracyclineresistant strains in the waters of remote upland tarns. It is possible that Pseudomonads may play a dominant role in contributing to the background frequency of oxytetracycline resistance in marine sediments free from anthropogenic influence. If this is true, then it might be predicted that the background frequencies would be higher in aerobic environments, such as the surfaces of sediments, than in the more anaerobic lower levels of such sediments. Further, it is reasonable to postulate that terrestrial Pseudomonads, derived from streams and soil run-off water, might make some contribution to the background levels of resistance in inshore sediments and therefore the background frequency might show an inverse relationship with distance from the land.
J. Kerry et al. /Aquaculture 123 (1994) 43-54
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The results of the initial survey of the background resistance at site A provided some evidence in favour of both these hypotheses. The data from the extended survey, however, demonstrated that even though these hypotheses may be valid in some, particular, situations, they cannot be generalised to all sites in Galway Bay. For this reason, the background resistance frequency used in the analysis of the results from the fish farm sediments was the overall mean of all 153 cores analysed. This background frequency is dependent on the media employed, the selective level of oxytetracycline chosen and on the time and temperature of incubation. It is essentially only valid for samples of sediment taken from Galway Bay and counted on ZV agar containing 25 pgm-’ oxytetracycline and read after 72 h incubation at 22 oC. The background level of resistance to oxytetracycline in marine sediments unassociated with fish farms has variously been re: ported as < 1% by Samuelsen et al. (1992), 0.8% by Torsvik et al. (1988) and 5 + 1% by Nygaard et al. ( 1992). All these authors however, provide little detail as to the source or number of samples used to determine these values. In the first fish farm investigation, when samples were collected from sediments under one cage, no significant increase in resistance frequency was detected in the presence of 9.9 + 2.9 Fg.g- ’ oxytetracycline. In contrast, in the second investigation, when the mean oxytetracycline concentration under the whole cage block was 10.9+6.5 pg-g-l, significant increases were detected. This discrepancy raises questions as to the causal link between sediment oxytetracycline concentrations and the frequency of resistance detected in that environment. The peak mean frequency in samples from under the cage block in the second investigation was similar to that reported by Torsvik et al. ( 1988) but dramatically lower than that reported by Samuelsen et al. ( 1992). The variations in media and incubation conditions used complicates a comparison of our results with these observations. The comparison of the different bacteriological methods presented here would, however, indicate that the use of ZV medium at 22°C for 3 days may not produce frequencies significantly different from the use of TSCA at 15 “C for longer times (6 and 7 days respectively) as employed by Torsvik et al. (1988) and Samuelsen et al. (1992). In the sediments studied by Samuelsen et al. ( 1992) the peak levels of oxytetracycline were 2.5- to 25-fold higher than those detected in this work and this may ‘account for the difference in the frequencies of resistance detected. Both Torsvik et al. ( 1988) and Samuelsen et al. ( 1992) reported long-term effects on resistance frequencies. In contrast, in this work, the half-life of the increased frequencies under the cages was 26 days. As opposed to the numerical value of the frequencies of resistance, this difference in the half-lives of the elevated frequencies cannot be a function of differences in methodology. Samuelsen et al. ( 1992) reported half-lives of 125, 144 and 87 days for oxytetracycline in the sediments that they studied. This contrasts with the 13-day halflife of oxytetracycline in the sediments studied in the second investigation. These differences in the persistence of oxytetracycline may in turn have influenced the difference in the persistence of elevated frequencies of resistance. Coyne et al. ( 1994) have, however, presented a more detailed comparison of the two sites
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J. Kerry et al. / Aquaculture 123 (I 994) 43-54
studied in this work and that studied by Samuelsen et al. ( 1992) and it is possible that other parameters may have an importance. The data from the second sea farm investigation does not allow for the determination of the limit of the area of sediment in which elevated resistance frequencies occur either after 10 days of therapy or 19 days after the end of the therapy. After 10 days of therapy the area where increased resistance could be detected was significantly greater than the area in which oxytetracycline could be detected. For example, in the most southerly sample analysed (50 m from the nearest site at which oxytetracycline was detected) the frequency of resistance was approximately 11% (Table 2). This raises the question as to whether the oxytetracycline-resistant strains were selected for at the sites where they were detected. It is possible that they were selected in sediments containing significant levels of oxytetracycline, i.e. those under the cage block, but that the microflora is more laterally mobile than the oxytetracycline. Oxytetracycline is known to bind to particulate matter in sediments (Sithole and Guy, 1987a,b). The bottom current flows at this site are predominantly westerly (Coyne et al., 1994) and this hypothesis would lead to the prediction that the distribution of elevated resistance frequencies should show a bias to the west of the cage block. This bias was not found (Table 2). Bjiirklund et al. ( 1990, 199 1) have shown that selection for resistance can occur in the fish and it is possible that the resistant strains detected in the sediment were selected in this environment. Even if this was the case, it is not clear as to why this should result in the lateral distribution found. In conclusion, the use of oxytetracycline in a marine salmon farm, with a location and husbandry style typical of the Irish industry, has in one case resulted in no significant rise in the frequency of resistance in the under-cage sediments. In a second case a significant, but transitory, increase in the frequency of resistant microflora was detected over an undetermined area. The extent to which the increased frequency of resistance is contributed to by strains with transferable resistance genes, or by those with non-transferable, intrinsic, resistance, has not been determined. This question must be answered if the implications of these data for future therapy of fish, animals and humans is to be addressed. Acknowledgements
The original stimulus for this work resulted from conversations with Drs. E-M Bernoth and A.E. Ellis. We thank Seamus Bonner, Deirdre Gilroy, Sinead Rudden and Sean Pender for their technical assistance and the staff of the fish farm studied for their co-operation and good will. This work was funded from the internal resources of the Fish Disease Group. References Angles, M-L., Marshall, K.C. and Goodman, A.E., 1993. Plasmid transfer between marine bacteria in the aqueous phase and biofilms in reactor microcosms. Appl. Environ. Microbial., 59: 1843-850.
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Bjorklund, H., Bondestam, J. and Bylund, G., 1990. Residues of oxytetracycline in wild fish and sediments from fish farms. Aquaculture, 86: 359-367. Bj8rklund, H.V., Rabergh, CM. and Bylund, G., 199 1. Residues of oxolinic acid and oxytetracycline in fish and sediments from fish farms. Aquaculture, 97: 85-96. Buck, J.D., 1974. Effects of medium composition on the recovery of bacteria from sea water. J. Exp. Mar. Biol. Ecol., 15: 2534. Buck, J.D., 1979. The plate count in aquatic microbiology. p. 19-28. In: J.W. Costerton and R.R. Colwell (Editors), Native Aquatic Bacteria: Enumeration Activity and Ecology. American Society for Testing Materials, Philadelphia. Coyne, R., Hiney, M., O’Connor, B., Kerry, J., Cazabon, D.J. and Smith P., 1994. Concentration and persistence of oxytetracycline in sediments under a marine salmon farm. Aquaculture, 123: 3142. Enger, O., Husevag, B. and Goksoyr, J., 1989. Presence of the fish pathogen Vibrio salmonicida in fish farm sediments. Appl. Environ. Microbial. 55: 2815-28 18. Goodman, A.E., Hild, E., Marshall, KC. and Hermansseon, M., 1993. Conjugative plasmid transfer between bacteria under simulated marine oligotrophic conditions. Appl. Environ. Microbial., 59: 1035-1041. Hansen, P.K., Lunestad, B.T. and Samuelsen, O.B., 1993. Effects of oxytetracycline, oxolinic acid, and flumequine on bacteria in an artificial marine fish farm sediment. Can. J. Microbial., 39: 1307-1312. Husevag, B., Lunestad, B.T., Johannessen, P.J., Enger, 0. and Samuelsen. O.B., 1991. Simulantaneous occurrence of Vibrio salmonicida and antibiotic resistant bacteria in sediments at abandoned aquaculture sites. J. Fish Dis., 14: 63 l-640 Jannasch, H.W. and Jones, G.E., 1959. Bacterial populations in sea water as determined by different methods of enumeration. Limnol. Oceanogr., 4: 128-140. Jones, J.G., Gardener, S., Simon, B.M. and Pickup, R.W., 1986. Antibiotic resistant bacteria in Windermere and two remote upland tams in the English Lake District. J. Appl. Bacterial., 60: 443453. Kogure, K., Simidu, U. and Taga, N., 1980. Distribution of viable marine bacteria in neritic sea water around Japan. Can. J. Microbial., 26: 318-323. Lunestad, B.T., 1992. Fate and effects of antibacterial agents in aquatic environments. In: C.M. Michel and D.J. Alderman (Editors), Chemotherapy in Aquaculture: From Theory to Reality. Off. Int. Epizoot., Paris 567~~. Magee, A.M. and Quinn, J.P., 1991. Antibiotic resistance in the bacteria of a remote upland river catchment. Lett. Appl. Microbial., 13: 145-149. Nakajima, T., Suzuki, M., Harada, K., Inoue, M. and Mitsuhashi, S., 1983. Transmission of R plasmids in Vibrio anguillarum to Vibrio cholera. Microbial. Immunol., 27: 195-198. Nygaard, K., Lunestad, B.T., Hektoen, H., Berge, J.A. and Hormazabal, V., 1992. Resistance to oxytetracycline, oxolinic acid and furazolidone in bacteria from marine sediments. Aquaculture, 104: 31-36. Russell, A.D. and Chopra, I., 1990. Understanding Antibacterial Action and Resistance. Ellis Horwood Ltd., Chichester, UK Samuelsen, O.B., Solheim, E. and Lunestad, B.T., 1991. Fate and microbiological effects of furazolidone in a marine aquacultural sediment. Sci. Total Environ., 108: 275-283. Samuelsen, O.B., Torsvik, V. and Ervik, A., 1992. Long range changes in oxytetracycline concentration and bacterial resistance towards oxytetracycline in a fish farm sediment after medication. Sci. Total Environ., 114: 25-36. Sithole, B.B. and Guy, R.D., 1987a. Models for tetracycline in aquatic environments. I. Interaction with bentonite clay systems. Water Air Soil Pollut., 32: 303-315. Sithole, B.B. and Guy, R.D., 1987b. Models for tetracycline in aquatic environments. II. Interaction with humic substances. Water Air Soil Pollut., 32: 3 15-32 1 Stewart, G.J. and Sinigalliano, C.D., 1990. Detection of horizontal gene transfer by natural transformation in native and introduced species of bacteria in marine and synthetic sediments. Appl. Environ. Microbial., 65: 1818-1824.
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Torsvik, V.L., Sorheim, R. and Gokwyr, J., 1988. Antibiotic resistance of bacteria from fish farm sediments. ICES Report, C.M. 1 988/F: 10. Zobell, C.E., 1941. Studies on marine bacteria. 1. the cultural requirements of heterotrophic species. J. Mar. Res.. 4: 42-75.
Frequency and distribution of resistance to oxytetracycline in micro-organisms isolated from marine fish farm sediments following therapeutic use of oxytetracycline Joe Kerry”, Maura Hiney”, Rosie Coynea, Dave Cazabon”, Saoirse NicGabhainnb, Peter Smith”>* “Fish Disease Group, Department ofMcrobiology, University College Galway, Galway, Ireland ‘Department of Health Promotion, University College Galway, Galway, Ireland
(Accepted 26 January 1994)
Abstract The background level of resistance to oxytetracycline in sediments free of anthropogenic influences was determined on 2216 V agar with 25 ,ug*g-’ oxytetracycline. The mean frequency of resistance in 153 samples taken in Galway Bay was 1.2 & 1.8%. The impact of oxytetracycline therapy on the frequency of resistance in the sediments under a marine fish farm was investigated on two occasions. In the first investigation, oxytetracycline was detected at a concentration of 9.9 ? 2.9 ,ug*g-’ in the sediments under a cage that received 865 g oxytetracycline per day for 10 days, but no significant rise in resistance frequency was detected. In the second investigation, oxytetracycline was detected at a concentration of 10.9 f 6.5 pg*g-’ in the sediments under a cage block that received 175 kg oxytetracycline over 12 days. The frequency of resistance reached 16.0 f 8.9% after the treatment. The frequency declined at an exponential rate ( r2 = 0.89 ) with a half-life of 26 days. At 73 days after the end of therapy the frequency, in under-cage samples, was not significantly higher than the background level. At the end of the therapy elevated frequencies of resistance were detected up to 75 m from the edge of the cage block and in samples where the levels of oxytetracycline were below the limit of detection ( 1.2 ,/.fg*g-’ ). Thirty-three days after the end of the therapy the frequency of resistance in all samples not directly under the cages was not significantly higher than in samples taken from sediments free of anthropogenie influence.
*Corresponding author. 0044~8486/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDZ 0044-8486 (94)00026-K
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J. Kerry et al. /Aquaculture 123 (1994) 43-54
1. Introduction In marine salmon farms the use of oxytetracycline has been demonstrated to coincide with an increased frequency of resistant microflora in the sediments under the fish cages (Husevag et al., 1991; Samuelsen et al., 1992; Torsvik et al., 1988 ), and with the isolation of oxytetracycline-resistant fish pathogens (Bjorklund et al., 1990, 1991; Husevag et al., 1991). Nygaard et al. (1992) demonstrated that the selection of resistant strains may occur as a result of the presence of oxytetracycline in the marine sediments. They added 50 ppm oxytetracycline to boxes of sediment placed on the sea bottom and demonstrated an increase in resistance from 5 rt 1% to 16 & 1.5% over a 12-month period. Hansen et al. ( 1993 ) reported that the addition of 400 ppm of oxytetracycline to sediments in laboratory aquaria resulted in the selection of an increase in frequency of oxytetracycline resistance in the microflora. This frequency rose slowly to a maximum of 35% after 185 days. Samuelsen et al. ( 1992 ) reported frequencies of oxytetracycline resistance of over 100% in the sediment under cages following therapy. The frequencies remained at between 10 and 50% for the subsequent 18 months. Torsvik et al. ( 1988) also reported long-term effects on the frequency of oxytetracycline resistance in sediment flora resulting from the therapeutic use of oxytetracycline. For the 13 months following medication the levels of resistance were between 18% and 26%. Fish pathogens have been detected in farm sediments (Enger et al., 1989; Bjorklund et al., 1991; Husevag et al., 1991) and therefore increases in the frequency of resistance to antimicrobials in this environment may have significance for the success of future antimicrobial therapy of disease in fish. Intra-species transfer of plasmid-encoded resistances has been reported in laboratory simulation of the marine environment (Angles et al., 1993; Goodman et al., 1993). Genetic transfer, mediated by transformation, has also been shown to occur in the marine environment (Stewart and Sinigalliano, 1990). It is possible, therefore, that elevated frequencies of resistance in fish farm sediments may have implications for therapy in terrestrial, and more importantly, human 1983). This investigation was undertaken to determine the impact of oxytetracycline use on the frequency of resistance in the sediment microflora, to determine the persistence of any increase in frequency with respect to time and to determine the area of sediment affected.
2. Materials and methods
Sample sites for determination of background resistance frequencies Galway Bay is a large inlet in the middle of the west coast of Ireland. The bay is approximately 50 km long in an east-west direction, and 16 km north to south. The dominant current circulation is anti-clockwise. The only significant density of human population is Galway city (pop. 50000) situated in the north-east cor-
J. Kerry et al. /Aquaculture 123 (1994) 43-54
45
ner of the bay. Marine salmon farming is concentrated in the north-west of the bay. For the initial survey of the background frequency of resistance a sample site (A) on the north shore of the bay approximately half-way between Galway city and the nearest fish farms was used. For the extended survey, four additional sampling sites were selected, one at the extreme south-east of the bay, and three on the north shore between Galway city and the fish farming sites. Site 2 was close to the main sewage outfall from Galway city. One sample at this site was taken 600 m from the outfall. Sites 1, 3 and 4 were at least 10 km distant from any fish farm or significant sewage discharge. Samples were collected between April and June, at which time the water temperature was between 13 and 15 ‘C. No differential was recorded in the temperature of sediment cores down to 20 cm depth. At each site triplicate core samples of sediment were collected by divers at distances of 300,900 and 1500 m off shore. At site A, additional samples were also taken at 600 and 200 m off shore. Fish farm investigations
The details of the fate of oxytetracycline following the first two therapeutic treatments investigated in this work have been reported elsewhere (Coyne et al., 1994). The layout of the fish cages and sample sites is shown in Fig. 1. In the first investigation 5 samples were collected from under a cage in block 6 at 3, 12 and 19 days after a lo-day treatment with oxytetracycline at 125 mg-kg- ‘. The total amount of oxytetracycline used in the cage was 865 g. In the second investigation, 17 samples were collected under and outside cage block 7 (Fig. 1). The total amount of oxytetracycline used during the 12-day treatment was 175 kg. Samples were collected after 10 days therapy and at 19, 33 and 73 days after the end of therapy. A third investigation was carried out in order to compare various methods of bacteriological enumeration. In this investigation 10 samples were taken under a cage in block 6. The cage received 160 g of oxytetracycline per day for 14 days. Samples were taken 1 day after the end of the therapy. Sample collection
Sediment samples from all sites were collected by divers using plexiglass core tubes of 4.5 cm i.d. and 25 cm length according to the method of Samuelson et al. ( 1992). Samples were transported to the laboratory on ice in an insulated container and bacterial analysis carried out immediately on arrival. Slices corresponding to 2 cm of the sediment core, measured with a ruler, were sequentially pushed from the core tube with a piston. Sediment samples used for drug residue analysis were stored at - 20” C until analysed by the HPLC method of Coyne et al. (1994). Bacteriological analysis
In all investigations 1 g aliquots of samples were diluted in phosphate-buffered saline and plated, in duplicate, on 22 16 V agar (Zobell, 194 1) (ZV) and the same medium with 25 pg*ml-’ oxytetracycline. Plates were counted after 72 h
46
J. Kerry et al. /Aquaculture 123 (1994) 43-54 N
+ BLOCK 6
Predominant
Current
4
Direction
s”2
Fig. 1. Layout of cage blocks and sampling sites. In the first fish farm investigation, samples were taken from under cage marked X in block 6. In the second fish farm investigation, samples were taken at the sites labeled x in block 7.
incubation at 22 oC. Longer incubation at this temperature resulted in fungal overgrowth of bacterial colonies and consequent diffkulties in determining counts. All colonies with an obviously fungal morphology were not included in the counts. The frequency of oxytetracycline resistance was determined by dividing the number of colonies on ZV agar containing oxytetracycline by the number of colonies on ZV agar without oxytetracycline. This figure was then multiplied by 100 to generate the percentage of resistant colony-forming units. The significance of different frequencies of resistance detected in sets of samples was determined using the Student t-test. A comparative media trial was performed where sediment samples (y1= 10 ) were also plated on TSCA (Samuelsen et al., 1992 ) at 15 ‘C for 7 days and on sea water TSA (SWTSA) (Nygaard et al., 1992) at 18°C for 10 days.
41
J. Kerry et al. / Aquaculture 123 (1994) 43-54
3. ResuIts Establishment of background resistance levels in Galway Bay
In the initial survey of background resistance at site A the frequency of resistance in the samples from the top 2 cm of the sediment showed a significant inverse correlation with the distance of the sample from the shore ( r2=0.90; p= 0.037). A similar correlation with distance was not seen in samples taken from the lower (2-4 cm and 4-6 cm) depths. A comparison of the frequencies at different depths at each distance from the shore showed significantly higher frequencies in the top 2 cm than in the 2-4 cm sample (p=O.O2) or the 4-6 cm sample (p=O.O2). No significant differences were detected between the frequencies at depths of 2-4 and 4-6 cm. A larger survey of background resistance was then undertaken to determine if these variations in resistance frequency with respect to distance from the shore and depth of sample were general in nature or were site-specific. Analysis of the data from the four additional sites did not confirm the general nature of the observations made from the data collected at site A. A three-way analysis of variance was carried out on resistance frequencies found at the 36 sample sites (3 distances x 3 depths x 4 locations). This analysis identified a main effect of distance and a two-way interaction between location and depth (Table 1). Further analyses of the main effect of distance showed that all differences were in the unpredicted direction, i.e. resistance frequencies increased the further the sample was from the shore. As these differences were unpredicted, post-hoc Scheffe t-tests were applied, but none proved significant. Similarly, after the simple main effects of depth at location and location at depth were carried out and Scheffe t-tests were conducted for those that proved significant, the original preTable 1 Three-way ANOVA on the resistance frequency data from sites 2-4 Source of variance
sum of squares
Location
Degrees of freedom
Variance estimate
22.91
4
5.73
9.76
2
4.88
Locationx depth
73.81
8
9.23
Distance
46.73
2
23.36
40.07
8
5.01
24.23
4
6.06
56.31
16
3.52
263.21
89
2.96
Depth
Location
x
distance
Depthxdistance Location
x
N/Location
depth x
x
distance
depth x distance
J. Kerry et al. /Aquaculture 123 (1994) 43-54
48
diction concerning depth was not confirmed. Thus no reliable differences were identified between any of the samples in this larger survey. The overall mean frequency of oxytetracycline resistance in all 153 cores analysed was 1.2 2 1.8%. The significance of data collected from fish farms was determined by comparison with the distribution of frequencies in these background samples using Student’s t-test. Resistance frequencies in individual samples were considered as meaningfully elevated if they exceeded the mean plus one standard deviation (i.e. 3%). Of the 27 cores taken at site 2,9 were taken from sediments 600 m distant from the main sewage outfall of Galway city. The mean resistance frequency in these 9 cores was 0.52 20.26%. which was not significantly different from the background (g=O.12). Frequency of resistance under$sh farms In the first fish farm investigation the frequency of resistance was determined in 5 samples taken under a single cage in cage block 6 (Fig. 1) . The increase in resistance frequency detected was not significant. Prior to the start of therapy the frequency of resistance was 0.3 + 0.05%. Three days after the end of the lo-day therapy the mean frequency was 2.4 & 3.9%. These data were not significantly higher than the.background frequency (p= 0.1). The frequency fell to 1.3 -t 0.6% 9 days later and to 0.5 + 0.2 28 days later. The concentrations of oxytetracycline detected in the same (top 2 cm) samples were 9.9 2 2.9, 4.9 ? 1.7 and 2.3 2 0.5 ,ug*g-‘, respectively (Coyne et al., 1994). In the second fish farm investigation the layout of sample sites (see Fig. 1) was designed to allow for the determination of both increased frequency of resistance under the cage block and horizontal distribution of increases in samples taken at a distance from cage block 7. With respect to the 7 samples taken directly under the cage block (Fig. 1) the mean frequency of resistance after 10 days of therapy was 16.0 + 8.9% (Fig. 2). Nineteen days after the end of the 12-day therapy, the frequency in these samples was 9.6 t 11.3%. The high variation in this figure was 25 -
I
10
30 Days
after
50
70
therapy
Fig. 2. Concentration of oxytetracycline and percentage of oxytetracycline-resistant microflora in the 7 samples taken from the sediments under cage block 7 during the second investigation. n = oxytetracycline pg-g-‘; 0 =percentage resistance in sediment microflora.
J. Kerry et al. /Aquaculture 123 (1994) 43-54
49
the result of the detection of 35% resistance in one sample. The frequency had fallen to 3.3 + 1.2% and 1.9 + 0.8% at 33 and 73 days after the end of therapy, respectively. At 73 days, the frequencies of resistance were not significantly different from those in the background samples (p=O.2). The decline in the increased frequency of resistance was exponential ( r2 = 0.89) and the half-life was 26 days. One hundred and forty-one days after the end of therapy the frequency of resistance was 1.5 +_1.1%. The concentrations of oxytetracycline detected in thesesampleson thesame dayswere 10.926.5,3.3? 1.7, 1.6+0.4pg-g-r (Coyne et al., 1994). Seventy-three days after the end of therapy the concentration of oxytetracycline, in all samples, was below the limit of detection. The data from the analysis of samples designed to determine the extent of the horizontal distribution of increases in resistance frequency are shown in Table 2. After 10 days of therapy meaningful rises in resistance frequency ( > 3.0%) were detected in all samples except that taken 100 m to the west of the cage block. In all other directions the limit of the horizontal distribution of the increase was not reached. Oxytetracycline was detected at 1.6 pg*g-’ in the sample 25 m to the west of the cage block and was below the limit of detection ( 1.2 ,ug*g-’ ) in all other samples. In 8 of the 10 samples, therefore, elevated oxytetracycline resistance occurred in the absence of detectable oxytetracycline concentrations. The distribution of increased resistance does not follow the westward bias predicted by the model based on current flow measurements (Coyne et al., 1994). The increases in resistance frequency were greater in samples taken to the north and south of the cages than to the west. Nineteen days after the end of therapy the frequency of resistance in 7 of the 10 samples had decreased, but overall the decreases were not significant (p = 0.12 ) . Thirty-three days after the end of therapy 9 of the 10 samples showed a further Table 2 Frequency of resistance in samples not directly under cage block Site
Wl N2 El E2 Sl s2 Wl w2 w3 W4
Distance from cage
Nearest OTC on day -2*
Percentage resistance
(m)
(m)
Day -2”
2.5 50 25 50 25 50 25 50 75 100
50 15 25 50 25 50 0 25 50 75
“The distance to the nearest sample site that contained days before the end of a 12-day therapy. **Days after the end of a 12&y therapy.
9.6
4.5 4.4 4.9 10 11 10 6.1 6.0 0.7
Day lgb
Day 33b
4.1 4.0 5.1 5.0 5.0 2.8 5.0 3.2 1.1 4.8
1.2 2.2 0.6 1.7 2.0 2.1 2.0 2.3 2.5 1.4
detectable oxytetracycline
( > 1.2 pg.g- *) 2
50
J. Kerry et al. / Aquaculture 123 (1994) 43-54
decrease and the overall frequency was significantly different from that recorded after both 10 days therapy (p=O.O04) and 19 days after therapy (p=O.O03). At this time the frequencies at all of the 10 sample sites were within one standard deviation of the mean background resistance frequency and the overall frequencies were not significantly higher than the background (p= 0.18 ) . Comparison of bacteriological methods On one occasion the frequency of resistance was determined using three culture methods. Following the use of 32.6 kg of oxytetracycline over 14 days at cage block 6, 10 samples were taken 1 day after the end of therapy from under a single cage that received 2.2 kg during the treatment. The frequency of resistance was 12.2 2 16.3% on ZV medium incubated at 22” C for 72 h, 11.7 & 10.4% on TSCA incubated at 15 ‘C for 7 days and 16.2 5 13.6% on SWTSA incubated at 18 ‘C for 10 days. None of these data was significantly different (ZV vs. TSCA, p= 0.97; ZV vs. SWTSA, p=O.37; TSCA vs. SWTSA, p= 0.44).
4. Discussion Solid surface culture methods have been shown to detect a low percentage of the viable bacteria in sea water (Jannasch and Jones, 1959; Buck, 1979; Kogure et al., 1980). The sub-set of the flora cultured will depend on the composition of the medium used (Buck, 1974). Samuelsen et al. ( 199 1) suggested that the fraction of the total viable bacteria that can be cultured from fish farm sediments may be as low as 0.02%. This inefficiency means that the frequencies of resistance determined by culture methods may not accurately reflect the frequency of resistance in the total viable microflora. In fresh water, free of anthropogenic influence, Magee and Quinn ( 199 1) found that oxytetracycline resistance was confined to Pseudomonas, species only and that such oxytetracycline-resistant Pseudomonads comprised 1.8% of the total strains they isolated. They failed to detect evidence that the resistances were plasmid-encoded and suggested that the strains they isolated may have been intrinsically resistant. Intrinsic, and therefore non-transferable, resistance to oxytetracycline has been reported in Pseudomonas aeruginosa (Russell and Chopra, 1990). On the other hand Jones et al. ( 1986) failed to detect any oxytetracyclineresistant strains in the waters of remote upland tarns. It is possible that Pseudomonads may play a dominant role in contributing to the background frequency of oxytetracycline resistance in marine sediments free from anthropogenic influence. If this is true, then it might be predicted that the background frequencies would be higher in aerobic environments, such as the surfaces of sediments, than in the more anaerobic lower levels of such sediments. Further, it is reasonable to postulate that terrestrial Pseudomonads, derived from streams and soil run-off water, might make some contribution to the background levels of resistance in inshore sediments and therefore the background frequency might show an inverse relationship with distance from the land.
J. Kerry et al. /Aquaculture 123 (1994) 43-54
51
The results of the initial survey of the background resistance at site A provided some evidence in favour of both these hypotheses. The data from the extended survey, however, demonstrated that even though these hypotheses may be valid in some, particular, situations, they cannot be generalised to all sites in Galway Bay. For this reason, the background resistance frequency used in the analysis of the results from the fish farm sediments was the overall mean of all 153 cores analysed. This background frequency is dependent on the media employed, the selective level of oxytetracycline chosen and on the time and temperature of incubation. It is essentially only valid for samples of sediment taken from Galway Bay and counted on ZV agar containing 25 pgm-’ oxytetracycline and read after 72 h incubation at 22 oC. The background level of resistance to oxytetracycline in marine sediments unassociated with fish farms has variously been re: ported as < 1% by Samuelsen et al. (1992), 0.8% by Torsvik et al. (1988) and 5 + 1% by Nygaard et al. ( 1992). All these authors however, provide little detail as to the source or number of samples used to determine these values. In the first fish farm investigation, when samples were collected from sediments under one cage, no significant increase in resistance frequency was detected in the presence of 9.9 + 2.9 Fg.g- ’ oxytetracycline. In contrast, in the second investigation, when the mean oxytetracycline concentration under the whole cage block was 10.9+6.5 pg-g-l, significant increases were detected. This discrepancy raises questions as to the causal link between sediment oxytetracycline concentrations and the frequency of resistance detected in that environment. The peak mean frequency in samples from under the cage block in the second investigation was similar to that reported by Torsvik et al. ( 1988) but dramatically lower than that reported by Samuelsen et al. ( 1992). The variations in media and incubation conditions used complicates a comparison of our results with these observations. The comparison of the different bacteriological methods presented here would, however, indicate that the use of ZV medium at 22°C for 3 days may not produce frequencies significantly different from the use of TSCA at 15 “C for longer times (6 and 7 days respectively) as employed by Torsvik et al. (1988) and Samuelsen et al. (1992). In the sediments studied by Samuelsen et al. ( 1992) the peak levels of oxytetracycline were 2.5- to 25-fold higher than those detected in this work and this may ‘account for the difference in the frequencies of resistance detected. Both Torsvik et al. ( 1988) and Samuelsen et al. ( 1992) reported long-term effects on resistance frequencies. In contrast, in this work, the half-life of the increased frequencies under the cages was 26 days. As opposed to the numerical value of the frequencies of resistance, this difference in the half-lives of the elevated frequencies cannot be a function of differences in methodology. Samuelsen et al. ( 1992) reported half-lives of 125, 144 and 87 days for oxytetracycline in the sediments that they studied. This contrasts with the 13-day halflife of oxytetracycline in the sediments studied in the second investigation. These differences in the persistence of oxytetracycline may in turn have influenced the difference in the persistence of elevated frequencies of resistance. Coyne et al. ( 1994) have, however, presented a more detailed comparison of the two sites
52
J. Kerry et al. / Aquaculture 123 (I 994) 43-54
studied in this work and that studied by Samuelsen et al. ( 1992) and it is possible that other parameters may have an importance. The data from the second sea farm investigation does not allow for the determination of the limit of the area of sediment in which elevated resistance frequencies occur either after 10 days of therapy or 19 days after the end of the therapy. After 10 days of therapy the area where increased resistance could be detected was significantly greater than the area in which oxytetracycline could be detected. For example, in the most southerly sample analysed (50 m from the nearest site at which oxytetracycline was detected) the frequency of resistance was approximately 11% (Table 2). This raises the question as to whether the oxytetracycline-resistant strains were selected for at the sites where they were detected. It is possible that they were selected in sediments containing significant levels of oxytetracycline, i.e. those under the cage block, but that the microflora is more laterally mobile than the oxytetracycline. Oxytetracycline is known to bind to particulate matter in sediments (Sithole and Guy, 1987a,b). The bottom current flows at this site are predominantly westerly (Coyne et al., 1994) and this hypothesis would lead to the prediction that the distribution of elevated resistance frequencies should show a bias to the west of the cage block. This bias was not found (Table 2). Bjiirklund et al. ( 1990, 199 1) have shown that selection for resistance can occur in the fish and it is possible that the resistant strains detected in the sediment were selected in this environment. Even if this was the case, it is not clear as to why this should result in the lateral distribution found. In conclusion, the use of oxytetracycline in a marine salmon farm, with a location and husbandry style typical of the Irish industry, has in one case resulted in no significant rise in the frequency of resistance in the under-cage sediments. In a second case a significant, but transitory, increase in the frequency of resistant microflora was detected over an undetermined area. The extent to which the increased frequency of resistance is contributed to by strains with transferable resistance genes, or by those with non-transferable, intrinsic, resistance, has not been determined. This question must be answered if the implications of these data for future therapy of fish, animals and humans is to be addressed. Acknowledgements
The original stimulus for this work resulted from conversations with Drs. E-M Bernoth and A.E. Ellis. We thank Seamus Bonner, Deirdre Gilroy, Sinead Rudden and Sean Pender for their technical assistance and the staff of the fish farm studied for their co-operation and good will. This work was funded from the internal resources of the Fish Disease Group. References Angles, M-L., Marshall, K.C. and Goodman, A.E., 1993. Plasmid transfer between marine bacteria in the aqueous phase and biofilms in reactor microcosms. Appl. Environ. Microbial., 59: 1843-850.
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Bjorklund, H., Bondestam, J. and Bylund, G., 1990. Residues of oxytetracycline in wild fish and sediments from fish farms. Aquaculture, 86: 359-367. Bj8rklund, H.V., Rabergh, CM. and Bylund, G., 199 1. Residues of oxolinic acid and oxytetracycline in fish and sediments from fish farms. Aquaculture, 97: 85-96. Buck, J.D., 1974. Effects of medium composition on the recovery of bacteria from sea water. J. Exp. Mar. Biol. Ecol., 15: 2534. Buck, J.D., 1979. The plate count in aquatic microbiology. p. 19-28. In: J.W. Costerton and R.R. Colwell (Editors), Native Aquatic Bacteria: Enumeration Activity and Ecology. American Society for Testing Materials, Philadelphia. Coyne, R., Hiney, M., O’Connor, B., Kerry, J., Cazabon, D.J. and Smith P., 1994. Concentration and persistence of oxytetracycline in sediments under a marine salmon farm. Aquaculture, 123: 3142. Enger, O., Husevag, B. and Goksoyr, J., 1989. Presence of the fish pathogen Vibrio salmonicida in fish farm sediments. Appl. Environ. Microbial. 55: 2815-28 18. Goodman, A.E., Hild, E., Marshall, KC. and Hermansseon, M., 1993. Conjugative plasmid transfer between bacteria under simulated marine oligotrophic conditions. Appl. Environ. Microbial., 59: 1035-1041. Hansen, P.K., Lunestad, B.T. and Samuelsen, O.B., 1993. Effects of oxytetracycline, oxolinic acid, and flumequine on bacteria in an artificial marine fish farm sediment. Can. J. Microbial., 39: 1307-1312. Husevag, B., Lunestad, B.T., Johannessen, P.J., Enger, 0. and Samuelsen. O.B., 1991. Simulantaneous occurrence of Vibrio salmonicida and antibiotic resistant bacteria in sediments at abandoned aquaculture sites. J. Fish Dis., 14: 63 l-640 Jannasch, H.W. and Jones, G.E., 1959. Bacterial populations in sea water as determined by different methods of enumeration. Limnol. Oceanogr., 4: 128-140. Jones, J.G., Gardener, S., Simon, B.M. and Pickup, R.W., 1986. Antibiotic resistant bacteria in Windermere and two remote upland tams in the English Lake District. J. Appl. Bacterial., 60: 443453. Kogure, K., Simidu, U. and Taga, N., 1980. Distribution of viable marine bacteria in neritic sea water around Japan. Can. J. Microbial., 26: 318-323. Lunestad, B.T., 1992. Fate and effects of antibacterial agents in aquatic environments. In: C.M. Michel and D.J. Alderman (Editors), Chemotherapy in Aquaculture: From Theory to Reality. Off. Int. Epizoot., Paris 567~~. Magee, A.M. and Quinn, J.P., 1991. Antibiotic resistance in the bacteria of a remote upland river catchment. Lett. Appl. Microbial., 13: 145-149. Nakajima, T., Suzuki, M., Harada, K., Inoue, M. and Mitsuhashi, S., 1983. Transmission of R plasmids in Vibrio anguillarum to Vibrio cholera. Microbial. Immunol., 27: 195-198. Nygaard, K., Lunestad, B.T., Hektoen, H., Berge, J.A. and Hormazabal, V., 1992. Resistance to oxytetracycline, oxolinic acid and furazolidone in bacteria from marine sediments. Aquaculture, 104: 31-36. Russell, A.D. and Chopra, I., 1990. Understanding Antibacterial Action and Resistance. Ellis Horwood Ltd., Chichester, UK Samuelsen, O.B., Solheim, E. and Lunestad, B.T., 1991. Fate and microbiological effects of furazolidone in a marine aquacultural sediment. Sci. Total Environ., 108: 275-283. Samuelsen, O.B., Torsvik, V. and Ervik, A., 1992. Long range changes in oxytetracycline concentration and bacterial resistance towards oxytetracycline in a fish farm sediment after medication. Sci. Total Environ., 114: 25-36. Sithole, B.B. and Guy, R.D., 1987a. Models for tetracycline in aquatic environments. I. Interaction with bentonite clay systems. Water Air Soil Pollut., 32: 303-315. Sithole, B.B. and Guy, R.D., 1987b. Models for tetracycline in aquatic environments. II. Interaction with humic substances. Water Air Soil Pollut., 32: 3 15-32 1 Stewart, G.J. and Sinigalliano, C.D., 1990. Detection of horizontal gene transfer by natural transformation in native and introduced species of bacteria in marine and synthetic sediments. Appl. Environ. Microbial., 65: 1818-1824.
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Torsvik, V.L., Sorheim, R. and Gokwyr, J., 1988. Antibiotic resistance of bacteria from fish farm sediments. ICES Report, C.M. 1 988/F: 10. Zobell, C.E., 1941. Studies on marine bacteria. 1. the cultural requirements of heterotrophic species. J. Mar. Res.. 4: 42-75.