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The susceptibility to oxolinic acid of 48 stains of Aeromonas salmonicida was established using the disc diffusion and agar dilution protocols of Alderman and Smith (2001) [Alderman, D., Smith, P., 2001. Development of draft protocols of standard reference methods for antimicrobial agent susceptibility testing of bacteria associated with fish disease. Aquaculture 196, 211–243.]. Both methods generated data that had a bimodal distribution. With respect to the agar dilution method an unambiguous breakpoint MIC of 0.25 mg/l allowed all strains to be classified as susceptible or tolerant. The equivalent figure for the disc diffusion data was 30 mm. Spearman's rank correlation analysis demonstrated a high degree of correlation ( r = 0.89) between the zone diameters and the log of the MIC values for members of the strain set. The breakpoints estimated in this work are compared to those estimated by other workers from data generated from different protocols. The issue of the clinical relevance of the breakpoints generated from the distribution of susceptibility data is discussed.

Two protocols designed to detect Aeromonas salmonicida, one based on an enzyme linked immunosorbent assay (ELISA) and one on a polymerase chain reaction (PCR), had previously performed well in extensive laboratory-based validation studies. These proxy methods were used together with direct culture to examine 223 kidneys of wild salmonid fish and 35 sediment samples collected from their habitat. Direct culture failed to detect the organism in any of the kidney samples. ELISA generated positive results from 23.3% of the kidneys and PCR from 20.6% but the degree of agreement between the two proxy methods was not greater than would have been predicted by chance (p>0.05). A total of 60% of the samples generating a positive response in ELISA were negative by PCR and 56% of PCR-positive samples were negative by ELISA. The overall concordance was, therefore, only 27%. With respect to the sediment samples, ELISA generated positive results from 53% and PCR from 33%. Again, the degree of agreement between the two proxy methods was not significantly greater than chance and the concordance was only 24%. The data generated by the two proxy methods investigated in this work was employed in a comparative validation of their application to the detection of A. salmonicida in these sample matrices. The hypothesis that both the PCR and ELISA protocols used in this work can validly be used in this way is clearly inconsistent with the data obtained. This analysis could not, however, discriminate between the hypotheses that neither of the methods possessed validity for these applications or that one of them possesses considerably greater validity than the other. The extensive laboratory-scale validation studies to which the two proxy methods used in this work have been submitted are discussed. It is argued that these laboratory studies provided no grounds for predicting the lack of validity that was detected when these proxy methods were applied to field samples. The results presented in this work provide strong evidence that laboratory-scale studies are not adequate to validate the application of proxy methods to field samples.

The survival of Aeromonas salmonicida was determined in sediment–water containing laboratory microcosms. Survival was estimated by calculating the decimal reduction times (DRT) of colony forming ability (cDRT) on Coomassie brilliant blue tryptone soya agar (TSA) (CBBA). Two sources of sediment and water were used in these experiments. One source, an outflow of a freshwater lake, produced sediments that were acidic and rich in humic acids. The other, a local river, gave sediments that were neutral and low in humic acids. The decimal reduction times in microcosms constructed from the humic acid-rich materials were of the order of 0.3 days, whereas those determined in microcosm, composed of river-derived materials, were approximately 40 days. This 100-fold difference in the decimal reduction times was demonstrated to be a function of the sediment source rather than the source of the water. The estimates of survival of A. salmonicida outside its host represent a critical element in any assessment of the risk of furunculosis transmission. The demonstration here, that such survival is highly matrix dependent, demonstrates that it is unlikely that a single figure could be used as an estimate of such survival in any formal quantitative risk assessment process.

Two laboratory microcosms, constructed of sterilised sediment and water taken from different locations, were seeded with Aeromonas salmonicida. A non-quantitative PCR, a heterogeneous, non-competitive, sandwich ELISA and colony formation were employed to generate survival data. In one microcosm, all three analytical methods generated positive results over the full 279 days of the incubation of the system at 18 °C. In the second microcosm, constructed with humic acid-rich material, colony formation was undetectable after 1 day but the PCR and ELISA methods generated positive results for up to 269 days. Direct injection into fish of material from the two microcosms both seeded with a passaged strain of A. salmonicida was used to determine the disease-producing potential of the bacteria in these systems. A direct correlation was observed between the presence of colony-forming units and the ability to produce furunculosis in test fish. None of the samples that generated positive PCR and ELISA signals but in which colony formation was not detected proved capable of inducing disease in fish. The significance of these data for the application of proxy methods in epizootiological studies is discussed.

The aim of this study was to investigate the relationship between antimicrobial tolerance and taxonomic diversity among the culturable oxytetracycline-resistant (Ot(r)) heterotrophic bacterial population in two Belgian aquatic sites receiving wastewater either from human medicine or from aquaculture. The study of Ot(r) heterotrophs and mesophilic Aeromonas spp. allowed comparison of tolerance data at the intergenus as well as at the intragenus level. In total, 354 independently obtained Ot(r) isolates were subjected to antimicrobial tolerance testing and identified by GLC analysis of their cellular fatty acid methyl esters (FAMEs), by API 20E profiling and/or by Fluorescent Amplified Fragment Length Polymorphism (FAFLP) DNA fingerprinting. In general, Ot(r) hospital heterotrophs displayed a higher frequency (84%) of ampicillin (Amp) tolerance compared to the Ot(r) heterotrophs from the freshwater fishfarm site (22%). FAME results indicated that this effect was linked to the predominance of intrinsically ampicillin-resistant Ot(r) Aeromonas strains over representatives of Acinetobacter and Escherichia coli within the hospital strain set. Among the Ot(r) mesophilic Aeromonas strain set, the global tolerance profiles of the two sites only differed in a higher number of kanamycin (Kan) -tolerant strains (43%) for hospital aeromonads in comparison with the fishfarm aeromonads (8%). To some extent, this finding was correlated with the specific presence of Aeromonas caviae DNA hybridisation group (HG) 4. Collectively, these results suggest that the profiles for Amp and Kan tolerance observed in both sites arose from taxonomic differences in the culturable Ot(r) bacterial population at the generic or subgeneric level. In addition, our identification data also revealed that Enterobacter sp., Stenotrophomonas maltophilia, and A. veronii biovar sobria HG8 may be considered potential indicator organisms to assess microbial tolerance in various compartments of the aquatic environment.

Tanks (39 cm × 30 cm × 27 cm) containing marine sediment overlaid with various amounts of sterilised commercial fish food pellets and seawater were used to study the emergence of microorganisms capable of forming colonies on TSCA media in both the presence and absence of 25 μg/ml oxytetracycline. All experimental systems were free of oxytetracycline. Initial levels of culturable organisms in the sediments used in these experiments were 7.1 · 10 4 cfu/g of which 0.2% were capable of growth on the oxytetracycline-containing agar. In tanks containing no feed, the number of resistant cfu/g increased slightly, from 1.8 · 10 4 to 5.1 · 10 4 cfu/g, over 70 days incubation at 8.5–12 °C. In the tank containing low levels (1–2 cm depth) of feed, the increase in resistant organisms was from 4.9 · 10 3 to 2.3 · 10 4 cfu/g. There was no significant increase in the relative size of the resistant sub-population in either tank. In the tank containing high levels (16–17 cm depth) of fish feed, the levels of oxytetracycline-resistant cfu' s/g rose from below the limit of detection ( 1 ) to 1.9 · 10 8 and their relative abundance rose to 34% by the end of the experiment ( t = 70 days). The resistant flora isolated from the tanks with high levels of feed were characteristically slowgrowing and resistant to high levels (> 512 μg/ml) of oxytetracycline. Over 50% of these strains were insensitive to oxolinic acid, cotrimoxazole and furazolidone, but the frequency of sensitivity to ampicillin and chloramphenicol was high. In contrast, the flora isolated from the sediment used in all tanks exhibited more rapid growth and lower levels of resistance to oxytetracycline. These strains were more frequently sensitive to the other antimicrobial agents.

The fish pathogen, Aeromonas salmonicida, could be detected only by bacteriological culture from the kidney of dead or moribund fish in one tank in a hatchery rearing Atlantic salmon (Salmo salar L.) smolts. However, by using a DNA probe specific for this species, allied to a PCR assay, the pathogen could be detected in water, feces and effluent samples taken from this fish tank. Also, the presence of the pathogen was found in effluent samples from two fish tanks containing apparently healthy fish. Subsequently, the presence of pathogen in these tanks was confirmed by an increase in the daily mortality rate and by a plate culture from moribund fish.

A DNA fragment that is specific to Aeromonas salmonicida has been isolated from a genomic DNA library by differential hybridization. The specificity of this fragment as a DNA probe for A. salmonicida was shown by hybridization against reference strains and clinical isolates of A. salmonicida, related aeromonads, and species from several other bacterial genera. The sensitivity of detection by a polymerase chain reaction test, based on this fragment, was approximately two A. salmonicida cells.

Summary This ecotaxonomic study compared the antibiotic tolerance among culturable oxytetracyline-resistant (Ot r ) heterotrophic strains isolated from two aquatic environments representing human activities in health care and aquaculture, namely hospital effluents and freshwater fishfarms. Using a standardized methodology, samples taken in England and Ireland were analyzed to determine the antibiotic tolerance profiles of two groups of culturable Ot r bacterial isolates at the intergeneric and intrageneric level comprising heterotrophs (189 strains) and mesophilic Aeromonas spp. (153 strains), respectively. Antibiogram data of heterotrophic isolates revealed that Irish hospital strains comprised higher frequencies of multi-tolerance than those originating from fishfarm environments whereas a reverse correlation was found among the English heterotrophs. Polyphasic identification of the isolates using fatty acid analysis and API 20E profiling showed that this difference arose from the unique taxonomic diversity within each heterotrophic strain set. Acinetobacter (27%) and Brevundimonas (22%) were predominant among the Irish Ot r fishfarm isolates, whereas isolates originating from the English aquaculture site almost entirely consisted of Stenotrophomonas maltophilia (86%) exhibiting high frequencies of tolerance to ampicillin and streptomycin. Within both the English and the Irish Ot r Aeromonas strain sets, on the other hand, the hospital strain sets displayed higher numbers of multi-tolerant isolates than to fishfarm isolates although country-specific differences were observed for individual antimicrobial agents. The typical occurrence of kanamycin-tolerant aeromonads in the Irish hospital site could to some extent be linked to the typical presence of A. hydrophila DNA hybridization group (HG) 3 strains as determined by fatty acid analysis and fluorescent amplified fragment length polymorphism (FAFLP) fingerprinting. Essentially, these data indicate that tolerance profiles in a specific environment of one country do not necessarily reflect the corresponding tolerance profiles of the same type of environment in another country, and this mainly as a result of the unique taxonomic composition of each site. Ot r representatives of Acinetobacter, S. maltophilia , and A. veronii biovar sobria HG8 were common to most if not all of the four sites under study, indicating that these three taxa may serve as potential indicator organisms for monitoring antibiotic tolerance among indigenous bacterial populations in various aquatic environments.

Oxytetracycline-resistant (OT r ) mesophilic aeromonads were recovered from untreated hospital effluent (72 isolates) and from fish farm hatchery tanks (91 isolates) at sites within the English Lake District, Cumbria, England. The transfer of OT r plasmids from these isolates was investigated. Using Escherichia coli J53-1 as a recipient, 11 isolates from the hospital site and 6 isolates from the fish farm site transferred OT r plasmids (designated pFBAOT1 to 17). Original isolates were identified using fatty acid methyl ester and fluorescent amplified fragment length polymorphism comparisons as either Aeromonas hydrophila HG3 (eight isolates), A. veronii b.v. sobria HG8 (six isolates), and A. caviae HGB5 (one isolate). One isolate remained unidentified, and one could not be assigned a taxonomic designation beyond the genus level. Plasmids pFBAOT1 to -17 were screened for the presence of the tetracycline resistance determinants Tet A to E and Tet G. Only determinant Tet A (10 plasmids) was detected in these plasmids, with 7 tet gene determinants remaining unclassified. In all cases, Tet A was located on a 5.5-kb Eco RI restriction fragment. Hybridization with inc-rep probes N, P, Q, W, and U showed pFBAOT3, -4, -5, -6, -7, -9, and -11, from the hospital environment, to be IncU plasmids. Further, restriction fragment length polymorphism (RFLP) analyses and DNA probing demonstrated that pFBAOT plasmids were closely related to IncU OT r plasmids pASOT, pASOT2, pASOT3, pRAS1 (originally isolated from A. salmonicida strains from fish farms in Scotland and Norway, respectively), and pIE420 (isolated from a German hospital E. coli strain). In addition, DNA analyses demonstrated that plasmids pRAS1 and pIE420 had identical RFLP profiles and that all fragments hybridized to each other. The presence of tetracycline resistance transposon Tn 1721 in its entirety or in a truncated form in these plasmids was demonstrated. These results provided direct evidence that related tetracycline resistance-encoding plasmids have disseminated between different Aeromonas species and E. coli and between the human and aquaculture environments in distinct geographical locations. Collectively, these findings provide evidence to support the hypothesis that the aquaculture and human compartments of the environment behave as a single interactive compartment.

Standard bacteriology and enzyme-linked imrnunosorbent assay (ELISA) were employed to detect Aeromonas salmonicida in samples of hdney, intestine and mucus from pre-smolt Atlantic salmon Salmo salar with stress-inducible furunculosis infections. No A. salrnonicida was isolated by bacteriological culture of samples from 30 unstressed salmon. In the same samples. ELISA detected antigen in 15 intestinal samples, but none in any of the kidney or mucus samples. The frequency of stress-inducible infections in a paired group of Atlantic salmon was 7/29 when the kidneys of the stressed fish were examined bacteriologically and 13/29 when ELISA results from kidney, intestinal content and mucus were combined. If ELISA results from kidneys alone were considered the frequency of detection was 8/20. In 2 separate experiments brown trout enrichment was used to detect A. salmonicida in pre-smolt Atlantic salmon with stress-inducible infections and compared to the standard stress test. The pathogen was detected in the combined mucus, fins and gills of 7/20 and 14/20 fish and in the blood of 3/20 and 2/20 fish respectively. The frequency of stress-inducible infections in paired groups of fish from the same populations was 10/20 and 16/20 respectively. These data suggest that the intestine may be the primary location of A. salmonjcida in salmon with stress-inducible infections and that colonisation of the mucus, fins and gills may also occur. The implications of these findings for the design of methods to eliminate such infections are discussed.