Provisional epidemiological cutoff values for standard broth microdilution susceptibility testing of Flavobacterium columnare

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.     

Epidemiological cut‐off values for Flavobacterium psychrophilum MIC data generated by a standard test protocol

Epidemiological cut‐off values were developed for application to antibiotic susceptibility data for Flavobacterium psychrophilum generated by standard CLSI test protocols. The MIC values for ten antibiotic agents against Flavobacterium psychrophilum were determined in two laboratories. For five antibiotics, the data sets were of sufficient quality and quantity to allow the setting of valid epidemiological cut‐off values. For these agents, the cut‐off values, calculated by the application of the statistically based normalized resistance interpretation method, were ≤16 mg L^?1 for erythromycin, ≤2 mg L^?1 for florfenicol, ≤0.025 mg L^?1 for oxolinic acid (OXO), ≤0.125 mg L^?1 for oxytetracycline and ≤20 (1/19) mg L^?1 for trimethoprim/sulphamethoxazole. For ampicillin and amoxicillin, the majority of putative wild‐type observations were ‘off scale’, and therefore, statistically valid cut‐off values could not be calculated. For ormetoprim/sulphadimethoxine, the data were excessively diverse and a valid cut‐off could not be determined. For flumequine, the putative wild‐type data were extremely skewed, and for enrofloxacin, there was inadequate separation in the MIC values for putative wild‐type and non‐wild‐type strains. It is argued that the adoption of OXO as a class representative for the quinolone group would be a valid method of determining susceptibilities to these agents.     

Erythromycin and florfenicol treatment of rainbow trout Oncorhynchus mykiss (Walbaum) experimentally infected with Flavobacterium psychrophilum

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.     

Genetic diversity and antimicrobial resistance of Flavobacterium psychrophilum isolated from cultured rainbow trout,Onchorynchus mykiss (Walbaum), in Spain

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 μg 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.     

Antimicrobial susceptibility pattern of Flavobacterium columnare isolates collected worldwide from 17 fish species

Flavobacterium columnare is the causative agent of columnaris disease in diverse fish species worldwide. Although columnaris is an important disease, the antimicrobial susceptibility pattern of F. columnare is not well studied. Thus, the purpose of this study was to test the in vitro antimicrobial susceptibility of 97 F. columnare isolates collected worldwide between 1987 and 2011 from 17 fish species. The broth microdilution technique was utilized for reliable testing of these fastidious organisms. None of the isolates displayed acquired resistance to florfenicol, gentamicin, ormetoprim‐sulfadimethoxine and trimethoprim‐sulfamethoxazole. Acquired resistance to chloramphenicol was detected in 1%, to nitrofuran in 5%, to oxytetracycline in 11% and to enrofloxacin, flumequine and oxolinic acid in 10%, 16% and 16% of the isolates, respectively, as reflected by a bimodal or trimodal distribution of their minimum inhibitory concentrations (MICs). One isolate showed acquired resistance towards several antimicrobial agents including erythromycin. Another isolate revealed acquired resistance towards – amongst others – ampicillin. The isolates displaying acquired resistance originated from ornamental fish species or Vietnamese catfish, except for two isolates coming from wild channel catfish in which acquired resistance was encountered towards oxytetracycline only. Fifty per cent of the resistant isolates from ornamental fish were shown to have acquired resistance against three classes of antimicrobial agents, assigning these isolates as multiple resistant. These data might indicate less prudent use of antimicrobials especially in ornamental fish species.     

Pharmacokinetic/pharmacodynamic properties and P‐glycoprotein expression in pacific white shrimp,Litopenaeus vannamei after oral administration at three dosages of oxolinic acid

The experiments explored the pharmacokinetics (PK) properties of oxolinic acid (OXA) after oral administration at three dosages (10, 30 and 80 mg/kg) via medicated feed in the shrimp. The results showed that the C_max values of 4.31, 14.93 and 16.62 mg/L and AUC_0–∞ values of 92.61, 252.30 and 364.27 mg hr^?1 L^?1 were observed at three OXA dosage groups in the haemolymph respectively. In the hepatopancreas, C_max values of 7.90, 27.23 and 60.51 mg/kg and AUC_0–∞ values of 42.01, 133.06 and 219.06 mg hr^?1 L^?1 were observed at 0.5 hr post administration respectively. In the muscle, C_max values of 1.62, 5.80 and 7.36 mg/kg and the AUC_0–∞ values of 25.64, 98.10 and 134.24 mg hr^?1 L^?1 were observed at 2 hr post administration respectively. In the gills, C_max values of 2.87, 8.08 and 12.12 mg/kg and the AUC_0–∞ values of 51.38, 118.65 and 206.48 mg hr^?1 L^?1 were observed at 4 hr post administration respectively. In addition, the in vitro MIC values of OXA at three dosages against 132 strains of Vibrio were examined and showed that the minimum inhibitory concentration (MIC) values for OXA primarily ranged from 0.15–1.25 µg/ml, including eight strains of Vibrio showing MIC values ≥5 µg/ml. The MIC₅₀ and MIC₉₀ values of 132 strains were 0.62 and 1.25 μg/ml respectively. The AUC_0–24/MIC₉₀ ratios of Vibrio were 140.4 in 30 mg/kg group. Furthermore, the P‐glycoprotein (P‐gp) expression was determined in shrimp tissues after administration to three dosage groups (10, 30 and 80 mg/kg). The results showed that P‐gp expression was up‐regulated in the hepatopancreas (5.36‐, 13.68‐ and 31.06‐fold respectively) compared with the control group.     

Evaluation of the immune response in rainbow trout fry,Oncorhynchus mykiss (Walbaum), after waterborne exposure to Flavobacterium psychrophilum and/or hydrogen peroxide

The immune response in rainbow trout fry against Flavobacterium psychrophilum was elucidated using an immersion‐based challenge with or without prior exposure to hydrogen peroxide (H₂O₂). Samples were taken from the head kidney 4, 48, 125 and 192 h after immersion, and the regulation of several genes was examined. Bacterial load was assessed based on the presence of 16S rRNA and correlated with gene expression, and the levels of specific antibodies in the blood were measured 50 days post‐infection. Separately, both H₂O₂ and F. psychrophilum influenced gene expression, and pre‐treatment with H₂O₂ influenced the response to infection with F. psychrophilum. Pre‐treatment with H₂O₂ also affected correlation between gene regulation and pathogen load for several genes. A delay in antibody production in H₂O₂‐treated fish in the early phase of infection was indicated, but H₂O₂ exposure did not affect antibody levels 50 days post‐infection. An increasing amount of F. psychrophilum 16S rRNA was found in the head kidneys of infected fish pre‐treated with H₂O₂ relative to the F. psychrophilum group. The results show that a single pre‐treatment with H₂O₂ impairs the response against F. psychrophilum and may intensify infection.     

Immunization of rainbow trout Oncorhynchus mykiss (Walbaum) with a crude lipopolysaccharide extract from Flavobacterium psychrophilum

Control methods for Flavobacterium psychrophilum are limited and oftentimes ineffective; hence, research efforts have focused on vaccine development. This study tested the hypothesis that a crude lipopolysaccharide (LPS) extract from F. psychrophilum will elicit a protective immune response in rainbow trout Oncorhynchus mykiss (Walbaum) against F. psychrophilum challenge. Rainbow trout (mean weight, 3 g) were immunized intraperitoneally with the following treatment and control preparations: 10 μg of crude LPS with or without Freund's complete adjuvant (FCA), 25 μg of crude LPS with or without FCA and saline with or without FCA. Immunization of fish with 10 or 25 μg of crude LPS/FCA resulted in significant antibody responses against F. psychrophilum using ELISA with a whole‐cell lysate as the coating antigen, but only minimal levels of protection were conferred following F. psychrophilum challenge at 14 weeks post immunization. Western blot analyses demonstrated that fish exhibited antibodies specific for low‐molecular mass proteins present in the crude LPS extract, but did not exhibit antibodies specific for F. psychrophilum LPS. The results indicate that higher immunization doses and/or the use of an alternative extraction method that yields larger LPS molecules (23–70 kDa) may be necessary to elicit specific antibody responses against F. psychrophilum LPS.     

Comprehensive antibiotic susceptibility profiling of Chilean Piscirickettsia salmonis field isolates

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.     

Multilocus sequence typing (MLST) analysis of California Flavobacterium psychrophilum reveals novel genotypes and predominance of CC‐ST10 in California salmonid hatcheries

The Gram‐negative bacterium, Flavobacterium psychrophilum, is endemic to California, USA, where it is an important pathogen in salmonid aquaculture, especially in rainbow trout (Oncorhynchus mykiss). Disease outbreaks caused by F. psychrophilum in rainbow trout fingerlings can approach 90% mortality, resulting in millions of dollars of economic losses annually. The focus of this study was to investigate the genetic diversity of 49 F. psychrophilum isolates collected from disease outbreaks in 17 salmonid hatcheries in California, USA, from 2015 to 2018 using multilocus sequence typing. Results suggest California F. psychrophilum isolates are diverse, representing 11 distinct sequence types (STs), three of which were previously undescribed. Still, the majority of genotyped isolates (n = 41) belonged to a single clonal complex (CC), CC‐ST10, which is the largest CC worldwide and has been linked to disease outbreaks on several continents. Results of this study provide evidence of marked intraspecific genetic diversity of F. psychrophilum from California. The biological significance of this genetic variability is unclear but could have implications for future vaccine development and treatments. Further studies investigating the virulence, antigenic, and antimicrobial susceptibility profiles of F. psychrophilum are warranted to better understand the epizootiology of this pathogen in the Western United States.     

Isolation of bacterial probiotic candidates from the gastrointestinal tract of rainbow trout,Oncorhynchus mykiss (Walbaum), and screening for inhibitory activity against Flavobacterium psychrophilum

In this study, 318 bacterial strains were isolated from the gastrointestinal (GI) tracts of 29 rainbow trout, Oncorhynchus mykiss (Walbaum). These bacteria were screened in vitro for their ability to inhibit growth of Flavobacterium psychrophilum, the causative agent of coldwater disease. Bacteria observed to inhibit F. psychrophilum growth were further screened against rainbow trout bile, as an indicator of their ability to survive in the GI tract. This screening resulted in narrowing the pool to 24 bacterial isolates. Those 24 isolates were then tested for pathogenicity in rainbow trout by intraperitoneal injection. Following a 28‐day challenge, eight isolates were shown to cause direct mortality and were eliminated from further study. As a result, 16 bacterial isolates were identified as probiotic candidates with the potential to control or reduce disease caused by F. psychrophilum.     

The effects of elevated pCO2 on growth,shell production and metabolism of cultured juvenile abalone,Haliotis iris

Reduced seawater pH and elevated pCO₂ are important considerations in tank‐based abalone aquaculture, while sea‐based farms may be at risk to ocean acidification reductions in pH. Juvenile Haliotis iris (5–13 and 30–40 mm shell length) were reared in two, 100‐day experiments at ambient pH_nbs (~ 8.1, 450 μatm CO₂), pH 7.8 (~1000 μatm CO₂) and pH 7.6 (~1600 μatm CO₂). Seawater pH was measured and adjusted automatically by bubbling CO₂ into water in replicated flow through tanks. Two separate trials were run, in winter (8.8°C) and summer (16.5°C). Survival and growth were monitored every 30 days, and post experiment measurements of morphometrics and respiration rate undertaken. Growth of shell length and wet weight were negatively affected by reduced pH, with a 2 to 3‐fold reduction in growth of both size classes between ambient and pH 7.6 treatments in the summer experiment. For small juveniles, growth reductions were in conjunction with decreases to shell weight, while large juveniles showed greater resilience in shell production. No changes to respiration rate occurred, suggesting that juveniles may maintain physiological functioning while tolerating dissolution pressure or that they are unable to upregulate metabolism to compensate for pH effects. These data show that CO₂ driven reductions in pH can impact growth, metabolism and biomineralization of abalone, and indicate that water quality and ocean acidification are of importance in aquaculture of the species.     

Assessment of cross‐protection to heterologous strains of Flavobacterium psychrophilum following vaccination with a live‐attenuated coldwater disease immersion vaccine

Bacterial coldwater disease, caused by Flavobacterium psychrophilum, remains one of the most significant bacterial diseases of salmonids worldwide. A previously developed and reported live‐attenuated immersion vaccine (F. psychrophilum; B.17‐ILM) has been shown to confer significant protection to salmonids. To further characterize this vaccine, a series of experiments were carried out to determine the cross‐protective efficacy of this B.17‐ILM vaccine against 9 F. psychrophilum isolates (representing seven sequence types/three clonal complexes as determined by multilocus sequence typing) in comparison with a wild‐type virulent strain, CSF‐259‐93. To assess protection, 28‐day experimental challenges of rainbow trout (Oncorhynchus mykiss) fry were conducted following immersion vaccinations with the B.17‐ILM vaccine. F. psychrophilum strains used in challenge trials were isolated from several fish species across the globe; however, all were found to be virulent in rainbow trout. The B.17‐ILM vaccine provided significant protection against all strains, with relative percent survival values ranging from 51% to 72%. All vaccinated fish developed an adaptive immune response (as measured by F. psychrophilum‐specific antibodies) that increased out to the time of challenge (8 weeks postimmunization). Previous studies have confirmed that antibody plays an important role in protection against F. psychrophilum challenge; therefore, specific antibodies to the B.17‐ILM vaccine strain appear to contribute to the cross‐protection observed to heterologous strain. The ability of such antibodies to bind to similar antigenic regions for all strains was confirmed by western blot analyses. Results presented here support the practical application of this live‐attenuated vaccine, and suggest that it will be efficacious even in aquaculture operations affected by diverse strains of F. psychrophilum.     

The in vitro efficacy of oxytetracycline against re‐isolated pathogenic Aeromonas hydrophila carrying the cytolytic enterotoxin gene through hybrid catfish,Clarias macrocephalus (Günther, 1864) × Clarias gariepinus (Burchell, 1822) in Thailand

Aeromonas hydrophila is a pathogen infecting farmed hybrid catfish, Clarias macrocephalus (Günther, 1864) × Clarias gariepinus (Burchell, 1822) which incurs substantial economic losses in Thailand. The study aimed at a genetic tracking of A. hydrophila infection and the in vitro assessment of the efficacy of antibiotics against its virulent strains. Five clinical strains from catfishes and Nile tilapia were employed. They were 3‐passage re‐isolated through healthy hybrid catfish and the cytolytic enterotoxin gene (AHCYTOEN) of individuals was traced. Each of the re‐isolates at a dose of ~6.67 × 10⁵ CFU/g was intraperitoneally injected into ~15 g‐healthy hybrid catfish and their pathogenicity were observed for 7 days. It was found that AHCYTOEN was carried over whereas typical signs of motile aeromonas septicaemia were found in the specimens. The bacterial strains of Nile tilapia origin did not induce mortality but those of catfish origins (80%–100% rate of mortality). The strains were susceptible to the tetracycline antibiotics, and oxytetracycline produced MIC₅₀ and MBC as low as 0.007–0.031 μg/ml and 1–8 μg/ml respectively. As oxytetracycline specifically inhibited pathogenic A. hydrophila in vitro, it is recommended that an appropriate dosage regimen of the drug should be established.     

Efficacy of a polyvalent injectable vaccine against Flavobacterium psychrophilum administered to rainbow trout (Oncorhynchus mykiss L.)

Flavobacterium psychrophilum is one of the most important pathogens affecting cultured rainbow trout (Oncorhynchus mykiss). Recent information from UK salmonid farms showed country‐wide distribution of genetically and serologically divergent clones, which has hampered the development of a vaccine for rainbow trout fry syndrome. The current study assessed the efficacy of an injectable polyvalent vaccine containing formalin‐inactivated F. psychrophilum in rainbow trout. The vaccine was formulated with an oil adjuvant (Montanide ISA 760VG) or formalin‐killed cells alone. Duplicate groups of trout (60 ± 13 g) were given phosphate‐buffered saline or vaccine formulated with Montanide by intra‐peritoneal (i.p.) injection and challenged by intra‐muscular (i.m.) injection with a homologous and a heterologous isolate of F. psychrophilum at 525 degree days post‐vaccination (dd pv). Significant protection was achieved in vaccinated fish (p = 0.0001, RPS 76% homologous, 88% heterologous). Efficacy of the adjuvanted vaccine was also demonstrated by heterologous challenge at 1155 dd pv resulting in 100% protection, whereas survival in the un‐adjuvanted group was not significantly different from control fish. Levels of specific antibody at 1155 dd pv, as measured by ELISA, were significantly higher in the fish vaccinated with adjuvant when compared with unvaccinated fish.     

Virulence of Flavobacterium psychrophilum isolates in rainbow trout Oncorhynchus mykiss (Walbaum)

Flavobacterium psychrophilum, the causative agent of bacterial cold‐water disease (BCWD) in freshwater‐reared salmonids, is also a common commensal organism of healthy fish. The virulence potential of F. psychrophilum isolates obtained from BCWD cases in Ontario between 1994 and 2009 was evaluated. In preliminary infection trials of rainbow trout juveniles, significant differences (0% to 63% mortality) in the virulence of the 22 isolates tested were noted following intraperitoneal injection with 10⁸ cfu/fish. A highly virulent strain, FPG 101, was selected for further study. When fish were injected intraperitoneally with a 10⁶, 10⁷ or 10⁸ cfu/fish of F. psychrophilum FPG 101, the 10⁸ cfu/fish dose produced significantly greater mortality (p < 0.05). The bacterial load in spleen samples collected from fish every 3 days after infection was determined using rpoC quantitative polymerase chain reaction amplification and by plate counting. Bacterial culture and rpoC qPCR were highly correlated (R² = 0.92); however, culture was more sensitive than the qPCR assay for the detection of F. psychrophilum in spleen tissue. Ninety‐seven per cent of the asymptomatic and the morbid fish had splenic bacterial loads of <2.8 log₁₀ gene/copies and >3.0 log₁₀ gene copies/reaction, respectively, following infection with 10⁸ cfu/fish.     

Pharmacodynamics of amoxicillin against field isolates of Streptococcus parauberis from olive flounder (Paralichthys olivaceus)

Olive flounder is the most important species for the Northeast Asian fish farming industry. However, this species is substantially affected by multiple infectious agents, including Streptococcus parauberis. Evaluation of antibiotics before their application is critical to treat infections and prevent drug resistance. Therefore, in this study, the pharmacodynamics of amoxicillin (AMX) and other antimicrobials against the planktonic‐ and biofilm‐forming bacteria were assessed. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and Time–kill curve assay were analysed using micro‐dilution method. The minimum biofilm eradicating concentration (MBEC) was determined using the Calgary Biofilm device. The effects of temperature, pH, hardness and salinity were detected for both planktonic‐ and biofilm‐forming bacteria. The MIC of AMX ranged from 0.015 to 2 μg/ml, whereas that of cephalexin (CEP), enrofloxacin (ENR) and oxytetracycline (OTC) ranged from 0.125 to 256, 0.125 to >512 and 0.25 to >512 μg/ml respectively. No bacteria were resistant against AMX, while the percentage of resistance to CEP, OTC and ENR were 68.7%, 52.6% and 11.1% respectively. The IC₅₀ of AMX, CEP, ENR and OTC was 0.03, 0.091, 0.015 and 0.213 μg/ml respectively. The MBEC of amoxicillin against S. parauberis ranged from 0.5 to 16 μg/ml. Higher rates of bacterial growth were obtained at 30°C, pH = 8 and salinity of 7.5–10 ppt. The hardness of the media suppressed the bacterial growth. In conclusion, AMX was found to be effective against both the planktonic and the biofilm forms of the prominent fish pathogen, S. parauberis.     

Treatment of the early life stages of scallop (Pecten maximus) with antimicrobial agents; searching for an alternative to chloramphenicol

High mortality rates are often observed in rearing the early stages of the great scallop, Pecten maximus. The addition of antibacterial agents has been necessary to improve larval survival. However, as one antibacterial agent, chloramphenicol, is banned in Norway and Europe the aim of this study is to investigate alternative antibacterial agents. The therapeutic agents investigated in this study were florfenicol, oxytetracycline, oxolinic acid, neomycin and Pyceze. The mean minimum inhibitory concentration (MIC) values were determined for oxytetracycline, oxolinic acid and Pyceze against bacteria isolated from scallop larvae. Two types of treatment regime were investigated on an intermediate scale (20 L). One regime involved continuous exposure of scallop larvae to the therapeutic agent while the other involved a short exposure lasting two hours. All intermediate scale treatments were performed in parallel to large-scale production (800 L) treatment with chloramphenicol. Of the therapeutants investigated, oxolinic acid was the most effective, although only at high concentrations. The short exposure of two hours was ineffective for all therapeutics.     

Evolution of drug resistance and minimum inhibitory concentration to enrofloxacin in <Emphasis Type="Italic">Tenacibaculum maritimum</Emphasis> strains isolated in fish farms

The in vitro susceptibility of 63 isolates of Tenacibaculum maritimum from four fish farms to eight chemotherapeutic agents used for the treatment of bacterial diseases in fish were assessed. The results indicated that all strains were resistant to oxolinic acid and susceptible to amoxicillin, nitrofurantoin, florfenicol, oxytetracycline and trimethoprim-sulphamethoxazole. However, some isolates presented resistance to enrofloxacin and flumequine, ranging from 10 to 30%, and from 25 to 60%, respectively, depending on the farm sampled. These data were used in an attempt to predict whether the resistance to enrofloxacin was static or evolved during the time of sampling from 2003 to 2004. A relationship between the use of enrofloxacin and levels of resistance was detected in the studied farm, increasing significantly from no resistant isolates in 2003 to 44.8% resistant strains in 2004, the year in which this drug was commonly employed. This result was accompanied by a marked decline of about 29.2% of the inhibition zone sizes for the T. maritimum strains in comparison to the initial values (average 21.5 mm). Minimum inhibitory concentration (MIC) of enrofloxacin for 100 T. maritimum strains was determined by the microdilution method. Twenty isolates were resistant to enrofloxacin (> 256 μg ml⁻¹), while the remaining strains showed a bimodal distribution, which ranged from 0.5 to 32 μg ml⁻¹. Our interpretation of the enrofloxacin MIC data suggests that the breakpoint for T. maritimum should be 4 μg ml⁻¹. However, similar studies in other laboratories are necessary to validate this breakpoint value.     

Depletion study and estimation of withdrawal periods for florfenicol and florfenicol amine in pacu (Piaractus mesopotamicus)

The intensive production of farmed fish is at a global all‐time high, and the control of bacteria proliferation in fish farms requires the frequent use of antimicrobials. This practice raises important environmental concerns related to the emergence of antimicrobial‐resistant bacteria strains. Only a few antimicrobial drugs have been approved for use in aquaculture, one of which is florfenicol. This work studies the depletion and withdrawal period of florfenicol and its main metabolite, florfenicol amine, in pacu (Piaractus mesopotamicus), a neotropical characin widely farmed in the southern hemisphere. Juvenile pacu (average weight of 724 g) were stocked in a closed‐loop laboratory system with controlled water temperature (25.8°C), and were fed for 10 consecutive days with a diet containing an intended dose of 10 mg/florfenicol per kg bw. Muscle and skin tissues were collected at 1, 3, 6, 8, 10, 12 and 16 days post‐treatment, and florfenicol and florfenicol amine were quantified using a validated ultra‐high performance liquid chromatography‐tandem mass spectrometry (UPLC‐MS/MS) method. The limits of quantitation for florfenicol and florfenicol amine were 10 ng/g in muscle and 50 ng/g in skin. Considering a maximum residue limit of 1000 ng/g for the sum of florfenicol and florfenicol amine in muscle with skin in natural proportions a withdrawal period of 5 days (water temperature 25.8°C) or 129 degree days was calculated on the basis of the upper limit of the one‐sided 95% confidence interval for the 99^th percentile derived from the residue depletion study.