Ole Bent Samuelsen

Bacterial resistance to antimicrobial agents used in fish farming: A critical evaluation of method and meaning

Abstract The use of antimicrobial agents in aquaculture has resulted in the increase in the frequency of strains resistant to these agents. Potentially these resistant strains can have an impact on the therapy of fish diseases, the therapy of human diseases, or the environment of the fish farms. Analysis of the extent of these impacts is hindered by the limited information available and the variation in methods that have been used. There is, for example, considerable variation in the methods used to measure the sensitivity of strains and in the criteria used to determine the clinical significance of these laboratory data. It is important that some standardisation of sensitivity testing methods is attempted. The available data on the frequency of resistance in fish pathogens suggest that the use of antimicrobial agents in aquaculture has significantly reduced the therapeutic options for treating fish diseases. The data available to assess the impact of the use of these agents in aquaculture on the therapeutic options for the treatment of human infections are incomplete. At present, no quantitative assessment of this risk can be attempted. Considerations of the data on the impact of the veterinary use of these agents on the therapy of human diseases would suggest that the extent of the risk represented by their use in aquaculture is small. The epidemiology of the human pathogens that have been associated with fish would tend to confirm this assessment. There is little data pertaining to the ecology of R plasmids in the natural environment. The significance of these plasmids in transferring resistance determinants from the aquatic compartment to the human compartment can, at present, only be assessed at a theoretical level. However, such a theoretical analysis suggests that the contribution of R plasmids, selected in the aquatic environment, to the frequency of resistance in human pathogens is probably very small. Fish farmers will have to develop methods of husbandry that limit the rate at which resistant strains emerge. Without these changes in husbandry, fish farming will rapidly enter the preantibiotic era. It is probable that these changes will also have the effect of reducing any impact of antimicrobial agents used in aquaculture on the environment outside the fish farm.

Long-range changes in oxytetracycline concentration and bacterial resistance towards oxytetracycline in a fish farm sediment after medication

Following 10 days medication with oxytetracycline, marine sediment was sampled beneath three selected cages (cages 1, 2 and 3) at a fish farm over a period of 18 mnd., in order to detect any change in the sediment oxytetracycline concentration, bacterial number and bacterial resistance towards the drug. The bulk of oxytetracycline disappeared during the first weeks, but it persisted in the sediment at lower concentrations for quite some time after the medication. Half-life (t(1/2)) of oxytetracycline in the sediment was measured as: 125, 144 and 87 days under cages 1, 2 and 3, respectively. At the end of the medication, all three sediments had greater than 100% oxytetracycline-resistant bacteria. This value dropped to 20% after 72 days and stabilised at levels of between 10 and 50%. The change in bacterial numbers, described as total and plate counts, was due to seasonal variations rather than to the medication.

Degradation of oxytetracycline in seawater at two different temperatures and light intensities, and the persistence of oxytetracycline in the sediment from a fish farm

A rapid procedure for the quantitative determination of the antibiotic oxytetracycline in sediments and in seawater using high performance liquid chromatography is presented. The mean recoveries from the sediment and from seawater were 98.4% and 99.5%, respectively. The limit of detection was 0.1 ppm for sediment and 0.01 ppm for seawater when injecting 150 μgl of sample. The persistence of oxytetracycline in the sediment was highly dependent on the rate of sedimentation. Based on measurements during the first 12 weeks and with a very small sedimentation rate, the half-life (t12) of the antibiotic was 32 days. When the oxytetracycline layer was immediately covered with an additional 4 cm of sediment, the degradation was much slower (t12=64 days). The degradation of oxytetracycline in seawater was rapid. The half-lives were 128 and 168 h at 5 and 15°C, respectively, under illumination for 24 h a day with a 40-W fluorescent tube, and 390 and 234 h, respectively, in aquaria maintained in darkness.

Anaesthesia of farmed fish: implications for welfare

During their life cycle as farmed animals, there are several situations in which fish are subjected to handling and confinement. Netting, weighing, sorting, vaccination, transport and, at the end, slaughter are frequent events under farming conditions. As research subjects, fish may also undergo surgical procedures that range from tagging, sampling and small incisions to invasive procedures. In these situations, treatment with anaesthetic agents may be necessary in order to ensure the welfare of the fish. The main objective of this paper is to review our knowledge of the effects of anaesthetic agents in farmed fish and their possible implications for welfare. As wide variations in response to anaesthesia have been observed both between and within species, special attention has been paid to the importance of secondary factors such as body weight, water temperature and acute stress. In this review, we have limited ourselves to the anaesthetic agents such as benzocaine, metacaine (MS-222), metomidate hydrochloride, isoeugenol, 2-phenoxyethanol and quinaldine. Anaesthetic protocols of fish usually refer to one single agent, whereas protocols of human and veterinary medicine cover combinations of several drugs, each contributing to the effects needed in the anaesthesia. As stress prior to anaesthesia may result in abnormal reactions, pre-anaesthetic sedation is regularly used in order to reduce or avoid stress and is an integral part of the veterinary protocols of higher vertebrates. Furthermore, the anaesthetic agents that are used in order to obtain general anaesthesia are combined with analgesic agents that target nociception. The increased use of such combinations in fish is therefore included as a special section. Anaesthetic agents are widely used to avoid stress during various farming procedures. While several studies report that anaesthetics are effective in reducing the stress associated with confinement and handling, there are indications that anaesthesia may in itself induce a stress response, measured by elevated levels of cortisol. MS-222 has been reported to elicit high cortisol release rates immediately following exposure, while benzocaine causes a bimodal response. Metomidate has an inhibitory effect on cortisol in fish and seems to induce the lowest release of cortisol of the agents reported in the literature. Compared to what is observed following severe stressors such as handling and confinement, the amount of cortisol released in response to anaesthesia appears to be low but may represent an extra load under otherwise stressful circumstances. Furthermore, anaesthetics may cause secondary adverse reactions such as acidosis and osmotic stress due to respiratory arrest and insufficient exchange of gas and ions between the blood and the water. All in all, anaesthetics may reduce stress and thereby improve welfare but can also have unwanted side effects that reduce the welfare of the fish and should therefore always be used with caution. Finally, on the basis of the data reported in the literature and our own experience, we recommend that anaesthetic protocols should always be tested on a few fish under prevailing conditions in order to ensure an adequate depth of anaesthesia. This recommendation applies whether a single agent or a combination of agents is used, although it appears that protocols comprising combinations of agents provide wider safety margins. The analgesic effects of currently used agents, in spite of their proven local effects, are currently being debated as the agents are administrated to fish via inhalation rather than locally at the target site. We therefore recommend that all protocols of procedures requiring general anaesthesia should be complemented by administration of agents with analgesic effect at the site of tissue trauma.

Stability of antibacterial agents in an artificial marine aquaculture sediment studied under laboratory conditions

Abstract Applying high-performance liquid chromatography (HPLC) and microbiological assays, various antibacterial agents were tested for their stability in an artificial marine aquaculture sediment under laboratory conditions. HPLC analysis revealed that oxolinic acid (OXA), flumequine (FLU), oxytetracycline (OTC) and sulfadiazine (SUL) were stable, while sulfadimethoxine (SMX) decreased by ca. 20% during the experimental period of 180 days. Ormethoprim (OMP) and trimethoprim (TMP) were unstable and could not be detected after 1 and 2 months, respectively. Microbiological test-systems measured no decrease in the antimicrobial activity of sediments to which OXA, FLU, SUL and SMX were added. In the OTC- and OMP-treated sediments, no antibacterial activity was found 1 month after addition of the antibacterial agents. Two months following administration the antibacterial activity in the TMP-treated sediment was considerably reduced and after 3 months no antibacterial activity could be observed.

Photostability of eight antibacterial agents in seawater

The stability and activity of eight antibacterial agents dissolved in seawater were determined using UV-spectroscopy, high-performance liquid chromatography and microbiological assays. The samples were illuminated by daylight at sea level and submerged to a depth of 1 meter in the sea. Oxytetracycline, furazolidone, oxolinic acid and flumequine were degraded and lost their antibacterial activity during the experimental period of 21 days at sea level. Some degradation was found for sulfadiazine and sulfadimethoxine, while trimethoprim and ormethoprim were found to be stable. However, no significant decrease in antibacterial activity was observed for these substances. When exposed to underwater light intensities, only oxytetracycline, furazolidone and to a certain extent flumequine were decomposed. All other substances were found to be stable. Only oxytetracycline and furazolidone showed reduced antimicrobial activity when exposed to underwater light intensities.

Efficacy of orally administered florfenicol and oxolinic acid for the treatment of vibriosis in cod (Gadus morhua)

Abstract This study was performed to determine the efficacy of orally administered oxolinic acid and florfenicol in the treatment of experimentally induced vibriosis in cod Gadus morhua . The Vibrio anguillarum strain HI-610 was used. This strain has minimum inhibitory concentration (MIC) values of 0.016 mg/l against oxolinic acid and 0.5 mg/l against florfenicol. Ten groups of 40 fish each were challenged by bath, 8.5×10 6 cells/ml for 1 h. Three days following challenge, medication with oxolinic acid or florfenicol was introduced in eight of the groups. The dosages used were 10 or 20 mg/kg day for both antibacterials and administered at days 1, 2, 4, 6, 8 and 10 following initiation of treatment for oxolinic acid and daily for 10 consecutive days for florfenicol. Among challenged unmedicated fish, the mortality started at day 3 post-challenge reaching a final cumulative mortality of 87.5% at day 22 when the experiment was terminated. In the medicated groups, the majority of deaths occurred from days 3 to 5 post-challenge reaching final cumulative mortalities of 34% and 28%, respectively, for the fish treated with 10 and 20 mg/kg of oxolinic acid and 31% and 20%, respectively, for the fish treated with 10 and 20 mg/kg of florfenicol. Survival of medicated fish in all groups were significant ( p p >0.1) in survival was however found between groups with parallel treatment or between groups given different drugs, dosages or medication regimens. Twenty-four hours following last medication, fish ( n =5) given a daily dosage of 10 mg/kg of florfenicol had mean plasma and muscle concentrations of 5.0±1.6 mg/l and 4.6±0.9 mg/kg, respectively. Corresponding values for fish given 20 mg/kg day of florfenicol were 6.5±1.3 mg/l (plasma) and 7.0±2.7 mg/kg (muscle). The plasma and muscle concentrations for fish treated with oxolinic acid were 0.8±0.2 mg/l and 1.9±0.4 mg/kg, respectively, when administered a dosage of 10 mg/kg day and 1.4±0.6 mg/l and 3.4±1.1 mg/kg, respectively, for the fish given a dosage of 20 mg/kg day.

Efficacy of Orally Administered Florfenicol in the Treatment of Furunculosis in Atlantic Salmon

Abstract This study was performed to determine the efficacy of orally administered florfenicol to treat experimentally induced furunculosis in Atlantic salmon Salmo salar held in seawater. A strain of the causative bacterium, Aeromonas salmonicida subsp. salmonicida, sensitive to florfenicol was used. In two trials, cohabitation challenges were performed by introducing six (trial 1) or eight (trial 2) fish—challenged in advance by an intraperitoneal injection of 2.2 × 104 colony-forming units—to eight aquaria, each containing 40 healthy fish. The treatment groups in both trials consisted of three groups receiving medication at 10, 13, and 16 d, respectively, postchallenge and one control group. An unchallenged, unmedicated group was used to determine the natural mortality in the population. The recommended therapeutic dose of 10 mg florfenicol/kg fish daily for 10 d was used. A higher mortality rate was observed in both trials for challenged unmedicated control fish than for unchallenged fish. In both tri...

Minimum inhibitory concentrations of chloramphenicol, florfenicol, trimethoprim/sulfadiazine and flumequine in seawater of bacteria associated with scallops (Pecten maximus) larvae

Abstract The purpose of this study was to find the minimum inhibitory concentrations (MIC) for the following antibacterial agents: chloramphenicol, florfenicol, flumequine and the combination trimethoprim/sulfadiazine to bacteria associated with scallop ( Pecten maximus ) larvae. To evaluate possible effects of components in seawater to the antimicrobial activity of these agents, MIC values were established on Mueller Hinton agar dissolved in either distilled water added 2% NaCl or 25‰ seawater. For flumequine and trimethoprim/sulfadiazine, the MIC values increased significantly using 25‰ seawater compared to 2% NaCl. Chloramphenicol and florfenicol did not show any significant increase in MIC values using 25‰ seawater compared to 2% NaCl. A significant increase in MIC values was found for chloramphenicol in the second egg group, using 25‰ seawater compared to 2% NaCl. It is concluded that flumequine, trimethoprim/sulfadiazine and chloramphenicol are, to a varying degree, antagonised by components in seawater.

Fate and microbiological effects of furazolidone in a marine aquaculture sediment

Furazolidone is used in the treatment of bacterial diseases in farmed fish. During application a large proportion of the administered drug reaches the environment directly or via feces. The persistence and metabolism of furazolidone in sediment from a Norwegian salmon farm is described. Furazolidone, in contrast to oxytetracycline and oxolinic acid, is actively metabolized by microorganisms in the sediment. The main metabolite is 3-(4-cyano-2-oxobutylidene-amino)-2-oxazolidone. This is a well known metabolite of the degradation of furazolidone in mammals, fish and Escherichia coli. 3-(4-Cyano-2-oxobutylideneamino)-2- oxazolidone had no detectable antibacterial activity. The half-life of furazolidone in the sediment at 4 degrees C was calculated to be 18 h.