Comparison of the antimicrobial tolerance of oxytetracycline-resistant heterotrophic bacteria isolated from hospital sewage and freshwater fishfarm water in Belgium.

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.

Distribution of oxytetracycline resistance plasmids between aeromonads in hospital and aquaculture environments: implication of Tn1721 in dissemination of the tetracycline resistance determinant tet A.

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 EcoRI 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 Tn1721 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.

Characterization of oxytetracycline-resistant heterotrophic bacteria originating from hospital and freshwater fishfarm environments in England and Ireland.

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.

Acute phase proteins in salmonids: evolutionary analyses and acute phase response.

Inflammation induces dramatic changes in the biosynthetic profile of the liver, leading to increased serum concentrations of positive acute phase (AP) proteins and decreased concentrations of negative AP proteins. Serum amyloid A (SAA) and the pentraxins C-reactive protein (CRP) and serum amyloid P component (SAP) are major AP proteins: their serum levels can rise by 1000-fold, indicating that they play a critical role in defense and/or the restoration of homeostasis. We have cloned SAA and a SAP-like pentraxin from salmonid fish species. The salmonid SAA shares approximately 70% amino acid identity with mammalian AP SAA. When salmonids are challenged with an AP stimulus, i.e., Aeromonas salmonicida, SAA responds dramatically as a major AP reactant. The salmonid pentraxin shows approximately 40% amino acid identity to both mammalian SAP and CRP. Evolutionary analysis suggests the presence of only a single such protein in teleosts and lower animal species. Surprisingly, the salmonid pentraxin behaves as a negative AP reactant, reminiscent of the SAP-like Syrian hamster female protein, in that hepatic mRNA concentrations decline to 50% of prestimulus levels. This study reinforces the hypothesis that SAA induction is an essential and universal feature of the vertebrate AP response and that it represents part of an ancient host defense system. Conversely, the species-dependent heterogeneity of pentraxin expression during the vertebrate AP response supports the possibility that its most important ancestral (and perhaps present) function is not related to its AP behavior.

Detection of Aeromonas salmonicida, causal agent of furunculosis in salmonid fish, from the tank effluent of hatchery-reared Atlantic salmon smolts.

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.

DNA probe for Aeromonas salmonicida.

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.