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 (88 Flavobacterium psychrophilum isolates and 134 Yersinia ruckeri isolates) and 313 motile Aeromonas 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. ruckeri isolates 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.

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 tetA positive (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 and tetA among the OTCresistant aeromonads, tetE and 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.

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.

No abstract

Abshract-Experimental results of a wideband heterodyne second-order optical phase-locked loop with 1.5 p m semiconductor lasers are presented. The loop has a bandwidth of 180 MHz, a gain ol' 181 dBHz, and a propagation delay of only 400 ps. A beat signal of 8 MHz linewidth is phase locked to become a replica of a microwave reference source close to carrier with a noise level of -125 dBc/Hz. The total phase variance of the locked carrier is 0.04 rad' and carriers can be generated in a continuous range from 3 to 18 GHz. The loop reliability is excellent with an average time to cycle slip of 10" s and an acquisition range of 640 MHz.