Morten Sichlau Bruun

Occurrence of antimicrobial resistance in fish-pathogenic and environmental bacteria associated with four danish rainbow trout farms.

ABSTRACT Surveillance of bacterial susceptibility to five antimicrobial agents was performed during a 1-year period in and around four freshwater fish farms situated along a stream in western Denmark. Besides assessing the levels of antibiotic resistance among the culturable fraction of microorganisms in fish, water, and sediment samples, two major fish pathogens (88Flavobacterium psychrophilum isolates and 134Yersinia ruckeri isolates) and 313 motileAeromonas isolates, representing a group of ubiquitous aquatic bacteria, were isolated from the same samples. MICs were obtained applying a standardized agar dilution method. A markedly decreased susceptibility of F. psychrophilum isolates to most antimicrobial agents presently available for use in Danish aquaculture was detected, while the collected Y. ruckeriisolates remained largely sensitive to all therapeutic substances. Comparing the inlet and outlet samples, the increase of the antibiotic-resistant proportions observed among the culturable microflora was more pronounced and statistically significant among the motile aeromonads. High levels of individual and multiple antimicrobial resistances were demonstrated within the collected flavobacteria and aeromonads, thus indicating a substantial impact of fish farming on several groups of bacteria associated with aquacultural environments.

Incidence, Distribution, and Spread of Tetracycline Resistance Determinants and Integron-Associated Antibiotic Resistance Genes among Motile Aeromonads from a Fish Farming Environment

ABSTRACT A collection of 313 motile aeromonads isolated at Danish rainbow trout farms was analyzed to identify some of the genes involved in high levels of antimicrobial resistance found in a previous field trial (A. S. Schmidt, M. S. Bruun, I. Dalsgaard, K. Pedersen, and J. L. Larsen, Appl. Environ. Microbiol. 66:4908–4915, 2000), the predominant resistance phenotype (37%) being a combined oxytetracycline (OTC) and sulphadiazine/trimethoprim resistance. Combined sulphonamide/trimethoprim resistance (135 isolates) appeared closely related to the presence of a class 1 integron (141 strains). Among the isolates containing integrons, four different combinations of integrated resistance gene cassettes occurred, in all cases including a dihydrofolate reductase gene and a downstream aminoglycoside resistance insert (87 isolates) and occasionally an additional chloramphenicol resistance gene cassette (31 isolates). In addition, 23 isolates had “empty” integrons without inserted gene cassettes. As far as OTC resistance was concerned, only 66 (30%) out of 216 resistant aeromonads could be assigned to resistance determinant class A (19 isolates), D (n = 6), or E (n = 39); three isolates contained two tetracycline resistance determinants (AD, AE, and DE). Forty OTC-resistant isolates containing large plasmids were selected as donors in a conjugation assay, 27 of which also contained a class 1 integron. Out of 17 successful R-plasmid transfers to Escherichia coli recipients, the respective integrons were cotransferred along with the tetracycline resistance determinants in 15 matings. Transconjugants were predominantly tetApositive (10 of 17) and contained class 1 integrons with two or more inserted antibiotic resistance genes. While there appeared to be a positive correlation between conjugative R-plasmids andtetA among the OTC-resistant aeromonads, tetEand the unclassified OTC resistance genes as well as class 1 integrons were equally distributed among isolates with and without plasmids. These findings indicate the implication of other mechanisms of gene transfer besides plasmid transfer in the dissemination of antibiotic resistance among environmental motile aeromonads.

Antimicrobial resistance patterns in Danish isolates of Flavobacterium psychrophilum

The resistance pattern of Flavobacterium psychrophilum to the antimicrobial agents used in fish farming in Denmark was assessed in vitro using an agar dilution method. After identification of 387 isolates from clinical outbreaks of rainbow trout fry syndrome (RTFS) and the environment, the isolates were tested and the resulting antibiograms were used to predict the theoretical therapeutic efficacy and to evaluate if resistance had changed as a course of time. Antimicrobial agents included in this investigation were oxolinic acid (OXA), amoxicillin (AMX), potentiated sulfadiazine, oxytetracycline (OTC) and florfenicol (FLO). We found that F. psychrophilum isolates divided in susceptible and resistant clusters reflecting the reduced efficacy in practice when using OTC and AMX. The most recent isolates were less susceptible to AMX and OXA, whereas resistance to OTC seemed stable over the last 5 years. Apparently, F. psychrophilum carries intrinsic resistance towards the potentiated sulfonamides, and in correlation with this, we found very few susceptible isolates. All isolates were susceptible to FLO. The method used for determining minimum inhibitory concentration (MIC) follows the American guideline from The National Committee for Clinical Laboratory Standards (NCCLS) with some modifications to comply with the growth demand of this fastidious bacterium. We suggest future investigations use these guidelines as a benchmark.

Detection of Flavobacterium psychrophilum from fish tissue and water samples by PCR amplification

Rainbow trout fry syndrome and cold‐water disease, caused by Flavobacterium psychrophilum, are important diseases in farmed salmonids. Some of the presently available techniques for the detection of Fl. psychrophilum are either time consuming or lack sufficient sensitivity. In the present investigation, the possible detection of Fl. psychrophilum from fish tissue and water samples was examined using nested PCR with DNA probes against a sequence of the 16S rRNA genes. The DNA was extracted using Chelex® 100 chelating resin. The primers, which were tested against strains isolated from diseased fish, healthy fish, fish farm environments and reference strains, proved to be specific for Fl. psychrophilum. The obtained detection limit of Fl. psychrophilum seeded into rainbow trout brain tissue was 0·4 cfu in the PCR tube, corresponding to 17 cfu mg−1 brain tissue. The PCR‐assay proved to be more sensitive than agar cultivation of tissue samples from the brain of rainbow trout injected with Fl. psychrophilum. In non‐sterile fresh water seeded with Fl. psychrophilum the detection limit of the PCR‐assay was 1·7 cfu in the PCR tube, corresponding to 110 cfu ml−1 water. The PCR‐assay detected Fl. psychrophilum in water samples taken from a rainbow trout farm, but Fl. psychrophilum could not be isolated using inoculation on selective agar. The method presented here has the potential to detect low levels of Fl. psychrophilum in fish tissue and in water samples, and the technique can be a useful tool for understanding the epidemiology of Fl. psychrophilum.

Efficiency of oxytetracycline treatment in rainbow trout experimentally infected with Flavobacterium psychrophilum strains having different in vitro antibiotic susceptibilities

Abstract The medication effect of oxytetracycline on groups of rainbow trout fry experimentally infected with three strains of Flavobacterium psychrophilum was investigated. The infection model was based on intraperitoneal injection of the pathogen and treatment was done using medicated feed resulting in 100 mg oxytetracycline/kg fish for 10 days. The three F. psychrophilum strains had different antimicrobial susceptibilities and successful treatment was only obtained in the trial using a strain with a MIC OTC of 0.25 μg/ml. No effect of treatment was seen in the group infected with a strain having MIC OTC of 8.0 μg/ml and only little effect was seen when the strain MIC OTC was 4.0 μg/ml. This shows that it is valid to predict the treatment efficiency of OTC from in vitro data facing an outbreak of rainbow trout fry syndrome. The importance of doing susceptibility testing is emphasized, and as shown the selection of media for antimicrobial susceptibility testing of F. psychrophilum is important.

Conjugal Transfer of Large Plasmids Conferring Oxytetracycline (OTC) Resistance: Transfer between Environmental Aeromonads, Fish-Pathogenic Bacteria, and Escherichia coli

Abstract The ability of environmental, motile Aeromonas species and fish-pathogenic bacteria to transfer oxytetracycline resistance to various bacterial species was assessed in vitro and in situ. Oxytetracycline-resistant motile aeromonads and A. salmonicida with large plasmids were used as donors; the recipients were a rifampicin-resistant Escherichia coli, the fish pathogens Yersinia ruckeri and Flavobacterium psychrophilum, a Pseudomonas putida strain, and motile aeromonads originating from aquatic environments. Transfers of oxytetracycline resistance were seen in both laboratory and environmental settings. In vitro transfers from motile Aeromonas spp. to E. coli using filter mating yielded transfer frequencies ranging from 1.4 × 10−9 to 6.8 × 10−5 transconjugants per recipient. Tetracycline resistance determinants A and D and unidentified tetracycline resistance determinants were transferred, usually on plasmids of approximately 150 kilo-base-pairs in size. In vitro transfer from motile Aeromonas spp....

Chemical surface disinfection of eggs of Baltic cod, Gadus morhua L.

The effect of two disinfectants on eggs and larvae of Baltic cod, Gadus morhua, was investigated. The eggs were disinfected for 10 min using various concentrations of either glutaraldehyde (100, 200, 400, 600 and 800 mg L(-1)) or iodophor (10, 50, 100 and 150 mg L(-1)), 1-4-days post-fertilization. Bactericidal effect of disinfection, survival to hatching, hatching success and larval abnormalities were assessed. Larval survival was recorded at 5-, 10- and 15-days post-hatch (dph). Although Baltic cod eggs have an unusually thin chorion, they could tolerate surface disinfection. A reduction in bacterial growth was observed with increased concentrations of disinfectant (3.0 x 10(7)-1.6 x 10(1) CFU mL(-1)). Abnormalities in newly hatched larvae were not related to disinfection. Survival of the yolk sac larvae was significantly better for eggs treated with 400 mg L(-1) glutaraldehyde for 10 min at 10 and 15 dph. Effective disinfection was also recorded using 100 mg L(-1) Actomar K30. Egg batch effect rather than initial bacterial concentration, disinfectant type or incubation method determined the survival of the eggs to hatching and survival of larvae. Because of the carcinogenic effect of glutaraldehyde, iodophor is recommended for routine disinfection of cod eggs.