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Lumpfish is a novel farmed species used as cleaner fish for the removal of lice from farmed salmon. As often with new, farmed species, there are challenges with bacterial infections. The frequency of prescription of antibiotic agents to lumpfish is increasing, despite the lack of knowledge about appropriate doses, duration of treatment and application protocols for the various antibacterial agents. In the current study, we have tested the effect of medicated feed with florfenicol (FFC), oxolinic acid (OA) and flumequine (FLU) on lumpfish experimentally challenged with Vibrio anguillarum, atypical Aeromonas salmonicida and Pasteurella atlantica. We found that all three antibacterial agents efficiently treated lumpfish with vibriosis using 10 and 20 mg kg-1 day-1 of FFC, 25 mg kg-1 day-1 of OA and 25 mg kg-1 day-1 FLU, whereas only FFC (20 mg kg-1 day-1 ) had good effect on lumpfish with pasteurellosis. None of the antibacterial agents were efficient to treat lumpfish with atypical furunculosis. FFC 20 mg kg-1 day-1 showed promising results in the beginning of the experiment, but the mortality increased rapidly 14 days post-medication. Efficient treatment is important for the welfare of lumpfish and for reducing the risk of development of antibiotic-resistant bacteria. To our knowledge, this is the first study to establish protocols for antibacterial treatment of lumpfish.

Surveillance of antibiotic resistance is of paramount importance for animal welfare and production. Despite aquaculture being a main source of animal protein, studies on antibiotic susceptibility in fish pathogens are scarce. Renibacterium salmoninarum, the aetiological agent of bacterial kidney disease (BKD), is one of the most common bacterial pathogens affecting salmon farming. In this work, we present an analysis of susceptibility patterns using determinations of minimum inhibitory concentration (MIC) for 65 field isolates, which were collected over seven years (2013-2019) from Atlantic salmon (Salmo salar) and coho salmon (Oncorhynchus kisutch) farms across southern Chile. The MIC protocol described by the Clinical Laboratory Standards Institute (CLSI) was used, but with microdilution instead of macrodilution and eight instead of four days of incubation. Two laboratories independently conducted analyses to provide data on the epidemiological cut-off values for R. salmoninarum to florfenicol, oxytetracycline and erythromycin. By using two calculation methods, our results provide evidence for an evolving subpopulation of non-wild-type isolates for the macrolide erythromycin, which is consistent with the respective treatment frequencies prescribed against BKD. Contrasting with what was expected, R. salmoninarum isolates were most susceptible to florfenicol and oxytetracycline, both of which are widely used antibiotics currently used in the Chilean salmon industry. The presented findings can serve as a reference for national or international antibiotic surveillance programmes, for both MIC interpretation and to identify emerging resistance to the conventional drugs used in BKD management. Finally, our results indicate that an 8-day incubation period for establishing MIC values of R. salmoninarum should be considered in a future revision of the CLSI guidelines.

Different antibiotic-based drugs are being used for the treatment of Aeromonas hydrophila infection in rainbow trout, and several studies emphasize the use of medicinal plants as immunostimulants for prophylactic measure against Aeromoniasis disease. However, therapeutic effects of aqueous methanolic extracts of tetra (Cotinus coggygria) against A. hydrophila in rainbow trout were not investigated. Four different concentrations of tetra extract (0 [control], 4, 8 and 12 mg/100 µl) and also two different positive control groups (florfenicol and doxycycline antibiotics) were administered orally using feeding needles to individual rainbow trout, Oncorhynchus mykiss of all experimental groups twice a day after intramuscular inoculation of A. hydrophila. The study period was for 10 days. On 0th, 3rd, 7th and 10th day, blood and tissues were collected from the fish and changes in humoral immune responses, haematology and immune-related gene expressions were determined. In the study, superoxide radical production was decreased generally in all experimental groups except in 12 mg tetra and florfenicol treatments compared to control (p .05). Lysozyme activity was generally decreased (p .05), or no differences were observed in all experimental groups compared to the control. Myeloperoxidase activity was significantly increased in florfenicol-treated fish group on 7th day (p .05). Generally, myeloperoxidase activity showed an increase in almost all tetra-treated groups. Haematological parameters increased but were not significantly high enough in treatments. Almost all immune-related gene expressions were significantly enhanced on 3rd and 10th day of the study. Survival rate of 53.33% was found in control group. There were no significant differences in survival between control and 4 mg tetra-treated group (p .05). All the other groups' survival rate was significantly increased compared to control. The highest survival rate was found in florfenicol group (80%). In 12 mg tetra-, doxycycline- and 8 mg tetra-treated groups, survival rate was recorded as 74.44%, 70% and 70%, respectively. Our results suggest that tetra methanolic extract is an effective therapeutic remedy against A. hydrophila infection in rainbow trout at the dose of 24 mg/32.34 g body weight/day.

Optimized dosing regimen is key to the effective use of antibacterials and to minimizing drug-related side effects. The current study established a pharmacokinetic-pharmacodynamic (PK-PD) model for the determination of optimal antibacterial dosing regimen in fish taken into consideration the temperature-dependent PK and the pathogen-dependent antimicrobial susceptibility, using florfenicol (FF) in Nile tilapia as an example. The calculated optimal dosages significantly varied by temperature and target MIC levels, ranging from 2.23 (MIC 1 µg/ml at 24°C) to 34.88 mg kg-1 day-1 (MIC 4 µg/ml at 32°C). The appropriateness of the calculated dosages was successfully verified by the in vivo studies. After 5 days of oral administration of the calculated optimal dosage at 24°C, the predicted plasma drug values were in line with the mean observed Cmin(ss) while at 28 and 32°C underestimation of the Cmin(ss) in a dose-dependent manner was observed and likely due to the occurrence of non-linear PK at high dosages. The averaged serum protein binding of FF was 19.1%. Our results demonstrated the appropriateness and clinical applicability of the developed PK-PD approach for the determination of optimal dosing regimens at given temperatures and MICs. Saturation metabolism and PK non-linearity of FF in tilapia warrant further study.

The gliding aquatic bacterium Flavobacterium columnare causes columnaris disease, a common problem for wild and farmed freshwater fish worldwide. Recently, a broth microdilution method was standardized to test the susceptibility of F. columnare against antimicrobials commonly used in aquaculture. We used this new method to measure the minimal inhibitory concentrations (MICs) of ten antimicrobials against 120 F. columnare isolates. The resulting MIC frequency distributions for each antimicrobial (1 MIC/isolate) were used to estimate epidemiological cut-off values (ECVs) which separate isolates with typical wild-type (WT) susceptibility from isolates with decreased non-wild-type (NWT) susceptibility. We identified 22 NWT isolates with elevated MICs relative to the ECV that covered 99.9% of the MIC distribution against one or more of the antimicrobials: ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, oxolinic acid or oxytetracycline. Ten of the NWT isolates had decreased susceptibility to a single antimicrobial class, six isolates to two antimicrobial classes and six isolates to three or more antimicrobial classes. The MIC frequency distributions and provisional cut-off values provide data needed to set epidemiological cut-off values to monitor for the development of antimicrobial resistance among F. columnare.

The pharmacokinetics of florfenicol (FF) in turbot (Scophthalmus maximus) was studied after single intravenous (10 mg kg-1 ) and oral (100 mg kg-1 ) administration. The plasma concentration-time data of florfenicol were described by an open one-compartment model. The elimination half-life (t1/2 ) was estimated to be 21.0 h, and the total body clearance, Cl, was determined as 0.028 L kg h-1 . The apparent volume distribution (Vd ) was calculated to be 0.86 L kg-1 and the mean residence time (MRTiv ) was 30.2 h. Following oral administration, the maximum plasma concentration (Cmax ) of 55.4 μg mL-1 was reached at 12 h (Tmax ). The absorption constant (ka ) was 0.158 h-1 . The bioavailability was estimated to be 57.1%. The low bioavailability observed at higher doses was explained by the saturation of the mechanisms of absorption. The drug absorption process was limited by its inherent low solubility, which limited the amount of available FF absorbed in the gastrointestinal tract. Based on the pharmacokinetic data, an optimal dosing schedule for FF administration is hereby provided. Based on the minimum inhibitory concentration found for susceptible strains of Aeromonas salmonicida, oral FF administration of first, an initial dose of 30 mg FF kg-1 , followed by 6 maintenance doses at 18 mg kg-1 /daily could be effective against furunculosis in turbot.

This study reports on the characterization of Vagococcus salmoninarum using phenotypic, serological, antigenic, genetic and proteomic methods. All strains of V. salmoninarum were resistant to most of the antimicrobials tested, and only 10% of strains were sensitive to florfenicol. Serological analysis demonstrated a high antigenic homogeneity within the species. No cross-reaction was detected with other fish pathogenic species causing streptococcosis (Lactococcus garvieae, Streptococcus parauberis, Streptococcus iniae, Streptococcus agalactiae, Carnobacterium maltaromaticum) using serum against V. salmoninarum CECT 5810. Electrophoretic analysis of cell surface proteins and immunoblot supported the antigenic homogeneity within V. salmoninarum strains. Moreover, limited diversity was detected using genomic (RAPD, ERIC-PCR and REP-PCR) and MALDI-TOF-MS analyses. The phenotypic, genomic and proteomic methods tested allowed the rapid differentiation of V. salmoninarum from the other species causing streptococcosis. However, MALDI-TOF-MS is the most promising method for typing and characterization of V. salmoninarum.

Flavobacterium psychrophilum is responsible for significant economic losses in rainbow trout aquaculture. Antimicrobial treatment remains the primary means of control; however, there are limited choices available for use. The objectives of the study were therefore to determine the minimum inhibitory concentrations for erythromycin and florfenicol in selected F. psychrophilum isolates and to evaluate their clinical treatment efficacy in experimentally infected rainbow trout. All isolates tested had moderate susceptibility to florfenicol and erythromycin except one isolate, which had low susceptibility to erythromycin. Two isolates (one with moderate and one with low susceptibility to erythromycin) were used in an experimental infection trial. Rainbow trout juveniles were injected intraperitoneally with 108 cfu/fish and after mortality had begun, fish were given erythromycin- and florfenicol-medicated feed at a rate of 75 mg kg- 1 day- 1 and 10 mg kg- 1 day- 1 fish body weight, respectively, for 10 consecutive days. The splenic F. psychrophilum load was determined using an rpoC quantitative PCR throughout the 30-day trial. Relative to antibiotic-free controls, erythromycin treatment significantly (p < 0.05) reduced mortality of rainbow trout juveniles infected with FPG101, even when treatment was initiated after clinical signs developed.

Two replicated controlled trials were conducted to determine the efficacy of florfenicol against Aeromonas salmonicida and Vibrio salmonicida infections in Atlantic salmon, Salmo salar L., smolts kept in 25‰ salt water. Infection with A. salmonicida was treated with florfenicol, oxolinic acid, oxytetracycline, trimethoprim/sulphadiazine or flumequine, whereas the V. salmonicida infection was treated with florfenicol or oxolinic acid only. A. salmonicida infection was induced by the introduction of cohabitant fish previously inoculated intraperitoneally. Medication started simultaneously in all test tanks on the first day of specific mortality among test fish. V. salmonicida infection was induced by intraperitoneal inoculation of all test fish. Medication started 1 day after infection. Medicated feeds were produced by coating the antibacterials on standard feed pellets, and administered twice daily for 10 consecutive days. With the dose used in the present trials, florfenicol was highly effective in reducing specific mortalities due to both infections. It was slightly more effective than oxolinic acid and trimethoprim/sulphadiazine against A. salmonicida infection. There was no significant difference between florfenicol and oxolinic acid in reducing specific mortalities due to V. salmonicida.

Pharmacokinetics and elimination of florfenicol and florfenicol amine in grouper held in sea water at 23.3 ± 0.8 °C were studied using HPLC method after they were given a single peroral dose of florfenicol at 24 mg kg(-1) body weight. Florfenicol was rapidly absorbed from intestine and distributed extensively to all the tissues examined. The maximum concentrations (Cmax , μg g(-1) or μg mL(-1) ) in plasma and tissues were observed at 2-6 h (the time to reach maximum concentration, Tmax ) except for bile (Tmax = 24 h) and were in the order of intestine (52.02 ± 25.07) > bile (49.41 ± 28.16) > gill (45.12 ± 11.10) > plasma (28.28 ± 5.43) > liver (21.97 ± 12.08) > muscle (21.63 ± 6.12) > kidney (20.88 ± 11.28) > skin (19.10 ± 5.88). The drug distribution level was higher in plasma than in extravascular tissues except for bile, based on the ratios of the area under concentration-time curve between tissue and plasma (AUCtissue/plasma ). The elimination of florfenicol was rapid in fish, and the corresponding half-lives (T1/2β ) in the order of magnitude were bile (13.92 h) > muscle or liver (12.31 h) > skin (11.77 h) > plasma (11.57) > gill (11.04 h) > intestine (10.55 h) > kidney (10.05 h). The delayed Tmax , lower Cmax and longer T1/2β for florfenicol amine compared with florfenicol were measured in grouper.

Routine application of antimicrobials is the current treatment of choice for rainbow trout fry syndrome (RTFS) or bacterial coldwater disease (BCWD) caused by Flavobacterium psychrophilum. In this study, the antimicrobial susceptibilities of 133 F. psychrophilum isolates, 118 of which were from the UK, were evaluated by broth microdilution and disc diffusion methods following VET04-A2 and VET03-A guidelines of Clinical and Laboratory Standards Institute (CLSI), respectively. Isolates were categorized as wild type (fully susceptible, WT) or non-wild type (NWT) using normalized resistance interpretation (NRI)-determined cut-off values (COWT ). Broth microdilution testing showed that only 12% of UK isolates were WT to oxolinic acid (MIC COWT ≤ 0.25 mg/L) and 42% were WT for oxytetracycline (MIC COWT ≤ 0.25 mg/L). In contrast, all the isolates tested were WT (MIC COWT ≤ 2 mg/L) for florfenicol, the main antimicrobial for RTFS control in the UK. Disc diffusion-based COWT values were ≥51 mm for 10 μg amoxicillin, ≥44 mm for 30 μg florfenicol, ≥30 mm for 2 μg oxolinic acid and ≥51 mm for 30 μg oxytetracycline. There was a high categorical agreement between the classifications of the isolates by two testing methods for florfenicol (100%), oxytetracycline (93%) and oxolinic acid (99%).

An increase in fish production has consequently brought an increase in infectious diseases in fish farms. The use of chemotherapic drugs is the most effective instrument against common bacterial agents. The number of registered drugs for use in aquaculture is limited and often veterinary practitioners resort to the off-label use of chemotherapic agents authorized for different food-producing animal species. Florfenicol is well known for its outstanding effect against various pathogenic bacteria affecting fish, and therefore, it may be a useful drug for off-label use in aquaculture. The aim of this study was to evaluate the depletion of florfenicol and its major metabolite, florfenicol amine, from the edible tissue of two fish species, rainbow trout and sea bream, following treatment with medicated feed at a dosage of 10 mg kg(-1) of bw day(-1) , for 10 consecutive days. At prefixed time points after the end of administration (0.25, 1, 2, 3, 4, 6, 7, 10, 14 and 21 days after treatment), edible tissues (muscle plus adherent skin) from 15 individuals in each group were collected and analysed by HPLC, to determine concentration of the drug in the tissue. On the basis of the obtained concentrations, withdrawal times of florfenicol in the two species were calculated. The results indicate that a drug withdrawal time of 500 °C-day, as established by Directive 2004/28/EC, for off-label drug use is more than satisfactory to guarantee the healthiness of fish products against the risk of drug residues.

A major concern in aquaculture is the use of chemical therapeutics, such as antibiotics, because of their impact on the environment as well as on the fish product. As a potential tool for reducing antibiotic use, we tested the application of low-frequency ultrasound as a method for enhancing antibiotic uptake. Rainbow trout juveniles (Oncorhynchus mykiss) were exposed to two different concentrations of oxytetracycline (OTC), flumequine (FLU) and florfenicol (FLO), administered by bath after the application of ultrasound. After exposure, concentrations of these substances were measured in the liver and blood of treated fish. Results showed that the ultrasound treatment can significantly increase the uptake for all three antibiotics. The uptake of OTC for example, in fish exposed to an OTC concentration of 20 mg L-1 after prior treatment with ultrasound, was similar to the OTC concentrations in their liver and blood to fish exposed to 100 mg L-1 without sonication. For FLU and FLO, the use of ultrasound caused significant differences of uptake in the liver at high antibiotic concentrations. This suggests that the use of ultrasound as a technique to deliver antibiotics to fish can ultimately reduce the amount of antibiotics discharged into the aquatic environment.

Francisella noatunensis subsp. orientalis (Fno) (syn. F. asiatica) is an emergent Gram-negative facultative intracellular bacterium. Although it is considered one of the most pathogenic bacteria in fish, there are no commercially available treatments or vaccines. The objective of this project was to determine the most efficacious concentration of florfenicol (FFC) [10, 15 or 20 mg FFC kg(-1) body weight (bw) per days for 10 days] administered in feed to control experimentally induced infections of Fno in Nile tilapia, Oreochromis niloticus (L.), reared in a recirculating aquaculture system. The cumulative mortality of fish that received 0, 10, 15 or 20 mg FFC kg(-1) bw per day was 60, 37, 14 and 16%, respectively. Francisella noatunensis subsp. orientalis genome equivalents were detected in water from all challenged groups with slight reduction in the concentration in the florfenicol-treated groups 4 days after treatment. The mean LOG of CFU Fno mg(-1) spleen was 3-5 and was present in all challenged groups at necropsy 11 days after treatment (21 days after challenge). Results show that florfenicol administered at doses of 15 and 20 mg FFC kg(-1) bw per days for 10 days significantly reduced mortality associated with francisellosis in Nile tilapia.

Two experiments were conducted to test the effectiveness of different therapeutants against a mixed infection of Aeromonas hydrophila and Flavobacterium columnare in sunshine bass. Experiment 1 evaluated copper sulphate, florfenicol-medicated feed and potassium permanganate (KMnO4) against a natural mixed infection. Experiment 2 further evaluated copper sulphate as a treatment to control an experimental mixed infection. In experiment 1, naturally infected untreated fish had the lowest final survival per cent, at 71%, while florfenicol-medicated feed at 15 mg kg−1 body weight for 10 days or copper sulphate at 2.1 mg L−1 (1% of the total alkalinity) for 24 h produced the highest final survivals, at 90% and 88%, respectively. The final survival of the naturally infected fish administered florfenicol-medicated feed was significantly different (P

Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease (CWD) and rainbow trout fry syndrome (RTFS) in salmonids. These diseases are a major problem in the aquaculture industry in Spain, and a better understanding of the epidemiology of F. psychrophilum isolates is necessary to improve management strategies. In this study, to investigate genetic variability of this bacterium, pulsed-field gel electrophoresis after DNA digestion with endonuclease StuI, plasmid profiling analysis and antimicrobial susceptibility testing were undertaken with 25 isolates of F. psychrophilum from Spain. These isolates were classified into 17 patterns by PFGE analysis, which were grouped into four clusters and seven independent branches. Twenty isolates (80%) possessed plasmids of 3.5 kb (n = 13) or 5.5 kb (n = 7). No plasmids were associated with antibiotic resistance to oxytetracycline (OTC) or florfenicol (FLO). Twenty isolates (80%) had minimum inhibitory concentrations (MICs) to OTC of between 2.4 and 9.7 microg mL(-1), and all isolates were susceptible to FLO. A relationship between the origin of the isolates and PFGE genotypes was found. Plasmid profile typing correlated with PFGE profile typing, whereas no correlation was found between antimicrobial susceptibility testing and PFGE profiles. These results suggest that the population of F. psychrophilum with pathogenic potential in northern Spain is quite heterogeneous.

Antibiotics have been extensively used against infections produced by Piscirickettsia salmonis, a fish pathogen and causative agent of piscirickettsiosis and one of the major concerns for the Chilean salmon industry. Therefore, the emergence of resistant phenotypes is to be expected. With the aim of obtaining a landscape of the antimicrobial resistance of P. salmonis in Chile, the susceptibility profiles for quinolones, florfenicol and oxytetracycline (OTC) of 292 field isolates derived from main rearing areas, different hosts and collected over 5 years were assessed. The results allowed for the determination of epidemiological cut-off values that were used to characterize the pathogen population. This work represents the first large-scale field study addressing the antimicrobial susceptibility of P. salmonis, providing evidence of the existence of resistant types with a high incidence of resistance to quinolones. Remarkably, despite the amounts and frequency of therapies, our results disclosed that the issue of resistance to florfenicol and OTC is still in the onset.

This study was performed to determine the efficacy of orally administered florfenicol in the treatment of experimentally induced vibriosis (Listonella anguillarum) in cod, Gadus morhua. The L. anguillarum strain HI-610 was used. This strain has a minimal inhibitory concentration value of 0.5 mg L(-1) against florfenicol. Fifteen groups of 40 fish each were challenged by bath with 1.7 x 10(5) CFU mL(-1) for 1 h. Three days following challenge, medication with florfenicol was introduced in 12 of the groups. The dosages used were 10 mg kg(-1) day(-1) for 10 consecutive days in marine or salmonid pellets, 10 mg kg(-1) day(-1) for five consecutive days in marine pellets or administered at days 1, 2, 4, 6 and 8 following initiation of treatment. Among challenged unmedicated fish mortality started at day 3 post-challenge reaching a final cumulative mortality of 77% at day 15. The experiment was terminated at day 26. In the medicated groups, the majority of deaths occurred from days 3-7 post-challenge reaching final cumulative mortalities of 31% and 52%, respectively, for the fish given marine and salmonid pellets for 10 consecutive days. The fish treated with medicated marine pellets for five consecutive days and at days 1, 2, 4, 6 and 8 (sequential feeding) following initiation of treatment had cumulative mortalities of 52% and 38%, respectively. Survival of medicated fish in all groups was significantly (P < 0.005) greater than survival of challenged unmedicated fish. Furthermore, a significant difference (P < 0.001) in survival was found between fish treated for 10 consecutive days using marine pellets and the groups using marine pellets for five consecutive days and salmonid pellets for 10 consecutive days. Twenty four hours following last medication, six fish had mean plasma concentrations of 3.3 +/- 1.7 and 3.5 +/- 2.8 microg mL(-1), respectively, in fish treated for 10 consecutive days using marine and salmonid pellets. Corresponding values for fish treated for five consecutive days and by sequential feeding were 2.2 +/- 2.3 and 1.7 +/- 0.7 microg mL(-1), respectively.

The efficacy of florfenicol in the control of natural outbreaks of furunculosis in Atlantic sallmon, Salmo salar L. was evaluated on five Norwegian farms during the summer of 1992. A total of 115 245 first- or second-generation fish received florfenicol with feed at a dose rate of 10mgkg 1body weight daily for 10 consecutive days. Comparisons were made with similar pens of fish medicated with the preferred treatment for eaeh location. In the 10 days following introduction of therapy, all treatments were associated with a rapid decline in mortality caused by infection with Aeromonas salmonicida. Florfenicol-medicated salmon had a lower specific mortality than those given oxolinie acid, flumequine or a trimethoprim and sulphadiazine eombination. No adverse reactions to treatment with florfenicol were observed.

A sensitive microbioassay for the detection of antibacterials used in the therapy of farmed fishes is described. Three indicator organisms (Bacillus subtilis, Bacillus cereus and Escherichia coli) were evaluated for their sensitivity to seven antibacterial agents (Enrofloxacin, Sarafloxacin, Florfenicol, Cosumix, oxolinic acid, amoxycillin and oxytetracycline). The limits of detection using the microbioassay were compared with those for a conventional radial diffusion assay. The microbioassay was two to four times more sensitive for all antibacterials tested except for amoxycillin, where the end point was the same. The sensitivity of the microbioassay to the presence of oxytetracycline in sea water was evaluated using Bacillus cereus as an indicator organism. The results indicate that the microbioassay can be adapted for use with sea water samples if the samples are filtered before use.

The minimum inhibitory concentrations (MICs) of 29 antimicrobial agents were determined for some representative Aeromonas salmonidda, Yersinia ruckeri and Vibrio isolates. Eleven of them, including three widely used in clinical practice, were further tested against 28–36 recent field isolates of A. salmonidda. The fluoroquinolones were inhibitory at very low concentrations with 50% of A. salmonidda isolates having a MIC of enro-floxacin ≤0·02μg ml-1. The distribution of MICs to the fluoroquinolones and to oxolinic acid were similar indicating possible cross resistance. Amoxycillin, florfenicol, minocycline, rifampicin and the potentiated sulphonamides had a single peak MIC distribution with values of ≤1·25μg ml-1. Minimum inhibitory concentrations of the fluoroquinolones were four or more times greater at 10°C compared with 22°C; this was not so with amoxycillin, florfenicol or the sulphachlorpyridazine-trimethoprim (S-TP) combination. A. salmonicida was rapidly killed at 22°C by the fluoroquinolones and S-TP whereas oxolinic acid and amoxycillin killed within 24h. Florfenicol was bacteriostatic. The therapeutic potential of some of these antibiotics is considered.

The minimum inhibitory concentrations (MICs) of 15 chemotherapeutic agents were tested against 146 Lactococcus garvieae strains isolated from 1999 to 2006 in Japan. The agents used included chloramphenicol, ciprofloxacin, erythromycin (EM), enoxacin, fleroxacin, florfenicol, kanamycin, lincomycin (LCM), norfloxacin, oxolinic acid, orbifloxacin, ofloxacin, benzylpenicillin, streptomycin and tetracycline (TC). Of the tested strains, 46 showed high levels of resistance to EM, LCM and TC. Twelve of these strains were detected to be carrying transferable R-plasmids using a conjugation experiment and, using Southern hybridization, were shown to have the same structure as the R-plasmid. The remaining 34 resistant strains had a similar DNA structure to that of the R-plasmid as confirmed by polymerase chain reaction (PCR) using primers designed from sites in the transferable R-plasmid. The EM and TC resistance genes were classified into the ermB and tetS groups using PCR. We also detected gyrA and/or parC mutants that are highly resistant to old and new generation quinolones. This study revealed that transferable R-plasmids encoding EM, LCM and TC are widely distributed and are conserved regardless of the area and/or time of collection.