Maura Hiney

Distribution of Oxytetracycline Resistance Plasmids between Aeromonads in Hospital and Aquaculture Environments: Implication of Tn1721 in Dissemination of the Tetracycline Resistance Determinant Tet A

ABSTRACT Oxytetracycline-resistant (OTr) 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 OTr 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 OTr 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-kbEcoRI restriction fragment. Hybridization withinc-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 OTr 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 differentAeromonas 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.

Bacterial resistance to antimicrobial agents used in fish farming: A critical evaluation of method and meaning

Abstract The use of antimicrobial agents in aquaculture has resulted in the increase in the frequency of strains resistant to these agents. Potentially these resistant strains can have an impact on the therapy of fish diseases, the therapy of human diseases, or the environment of the fish farms. Analysis of the extent of these impacts is hindered by the limited information available and the variation in methods that have been used. There is, for example, considerable variation in the methods used to measure the sensitivity of strains and in the criteria used to determine the clinical significance of these laboratory data. It is important that some standardisation of sensitivity testing methods is attempted. The available data on the frequency of resistance in fish pathogens suggest that the use of antimicrobial agents in aquaculture has significantly reduced the therapeutic options for treating fish diseases. The data available to assess the impact of the use of these agents in aquaculture on the therapeutic options for the treatment of human infections are incomplete. At present, no quantitative assessment of this risk can be attempted. Considerations of the data on the impact of the veterinary use of these agents on the therapy of human diseases would suggest that the extent of the risk represented by their use in aquaculture is small. The epidemiology of the human pathogens that have been associated with fish would tend to confirm this assessment. There is little data pertaining to the ecology of R plasmids in the natural environment. The significance of these plasmids in transferring resistance determinants from the aquatic compartment to the human compartment can, at present, only be assessed at a theoretical level. However, such a theoretical analysis suggests that the contribution of R plasmids, selected in the aquatic environment, to the frequency of resistance in human pathogens is probably very small. Fish farmers will have to develop methods of husbandry that limit the rate at which resistant strains emerge. Without these changes in husbandry, fish farming will rapidly enter the preantibiotic era. It is probable that these changes will also have the effect of reducing any impact of antimicrobial agents used in aquaculture on the environment outside the fish farm.

Concentration and persistence of oxytetracycline in sediments under a marine salmon farm

Abstract The concentration of oxytetracycline in the sediment under two adjacent cage blocks in a marine salmon farm was determined following the therapeutic use of the drug. The sediment cores were grey, indicating some build-up of organic material. Infaunal polychaetes were present as were mobile fauna including crabs, starfish and flat fish. There were no significant accumulations of undigested feed pellets under the cages. At one block where 186 kg oxytetracycline was used over 10 days the oxytetracycline concentrations were determined under a single cage that received 8.65 kg oxytetracycline during the treatment. Peak concentrations in the top 2 cm of the sediment were 9.9±2.9 μg·g −1 . This declined at an exponential rate ( r 2 =0.99) with a half-life of 16 days. At the second block the oxytetracycline concentration was measured with a sampling programme designed to determine the horizontal and vertical distribution of oxytetracycline under the whole cage block during and after a treatment where 175 kg of oxytetracycline were used over 12 days. Peak concentrations, in the top 2 cm of the sediments under the cage block, were 10.9±6.5 μg·g −1 and this declined at an exponential rate ( r 2 =0.99) with a half-life of 13 days. Nineteen days after the end of the therapy oxytetracycline was detected at depths of up to 8 cm in the sediment, but the concentration of the antibacterial agent had decreased at all levels in the sediment 14 days later. At the end of the treatment oxytetracycline was detected in an area of the sediment less than twice the area of the cages themselves. Data on current flow and sedimentation rate were used to generate a predictive model of the area of sediment that would be subject to the deposition of both pelleted fish feed and fish faeces. Oxytetracycline was confined to the area of sediment predicted to be subject to feed deposition that was directly under and slightly to the west of the cage block. Oxytetracycline was not detected in the area predicted to be subject to faecal deposition only.

Fish feed as a source of oxytetracycline-resistant bacteria in the sediments under fish farms

Abstract Concentrations of oxytetracycline and the frequency of oxytetracycline resistance in the environmental microflora were monitored following the therapeutic use of this agent at a marine fish farm. 529 kg of oxytetracycline were administered over a 24 day period at an average of 1.4 kg per cage per day. Three days after the end of the therapy 4.6 ± 3.7 μg/g oxytetracycline were detected in the sediments and the frequency of resistance in the sediment microflora was 9.0 ± 5.3%. A rise in the frequency of resistance in this flora to 26 ± 8.7% occurred 24 days after the therapy. This rise was not associated with any increase in the concentrations of oxytetracycline in the sediment. At this time the frequency of resistance in the flora isolated from mussels suspended above the sediments (36 ± 8.5%) was significantly ( P = 0.005) higher than that present in the sediment flora. The feed used on the farm 24 days after the end of therapy was shown to contain 4.6 × 10 4 oxytetracycline-resistant cfu/g. The distribution of phenotypic groups in the oxytetracycline-resistant flora in this feed and in the sediments during the peak in resistance were compared with those from other marine environments. These data demonstrated that resistant flora in feed can, under certain circumstances, significantly contribute to the resistant flora detected in sediments under fish cages.

Spatial distribution of oxytetracycline and elevated frequencies of oxytetracycline resistance in sediments beneath a marine salmon farm following oxytetracycline therapy

Abstract The concentrations of oxytetracycline and the frequencies of oxytetracycline resistant microorganisms were determined in 11 samples taken from the sediments in the vicinity of a block of fish cages at a marine salmon farm. The cage block contained 10 tonnes of Atlantic salmon smolts and a total of 20 kg of oxytetracycline were administered during the 12 day treatment. Samples cores were collected by divers 5 days after the end of the period of therapy and the top 2 cm of each core was analysed. HPLC analysis was able to quantify the oxytetracycline concentrations in three of the six samples taken directly under the cage block. The mean concentration under the cage was between 0.65 and 1.2 μg g −1 ( n = 6) depending on the values attributed to samples where the concentrations were below the level of quantitation (1.2 μg g −1 ). In the five samples taken from locations not directly under the cage block oxytetracycline was only detected in the sample taken adjacent to, and down current from, the cage block. This sample was collected 10 m to the west of the cage block and contained 4.2 μg g −1 oxytetracycline. These data indicate that oxytetracycline was confined to an area of the sediment which was smaller in extent than the area of the cage block itself. The frequencies of resistance to oxytetracycline in the microflora cultured from the samples were determined by differential plating on 2216V media, containing 25 and 100 μg ml −1 oxytetracycline. Analysis of eighty three samples from sites free of overt human influence demonstrated that the background levels of resistance at these two selection concentrations were 1.3 ± 1.3% and 0.4 ± 0.6%, respectively. Elevated frequencies of resistance were detected in samples from a wider area than the cage block. The median frequency of resistance in the samples ( n = 6) taken from directly under the cage block was 1.4% at 100 μg ml −1 and 5.3% at 25 μg ml −1 . In the samples ( n = 5) taken from outside the cage block the frequencies were 5.3% at 100 μg ml −1 and 8.5% at 25 μg ml −1 . There was no correlation between the concentration of oxytetracycline in a sample and the frequency of resistance that was determined in the culturable microflora in that sample.

Frequency and distribution of resistance to oxytetracycline in micro-organisms isolated from marine fish farm sediments following therapeutic use of oxytetracycline

Abstract the background level of resistance to oxytetracycline in sediments free of anthrogogenic influences was determined on 2216 V agar with 25 μg·g −1 oxytetracycline. The mean frequency of resistance in 153 samples taken in Galway Bay was 1.2 ±1.8%. The impact of oxytetracycline therapy on the frequency of resistance in the sediments under a marine fish farm was investigated on two occasions. In the first investigation, oxytetracycline was detected at a concentration of 9.9 ± 2.9 μg·g −1 in the sediments under a cage that received 865 g oxytetracycline per day for 10 days, but no significant rise in resistance frequency was detected. In the second investigation, oxytetracycline was detected at a concentration of 10.9 ± 6.5 μg·g −1 in the sediments under a cage block that received 175 kg oxytetracycline over 12 days. The frequency of resistance reached 16.0 ± 8.9% after the treatment. The frequency declined at an exponential rate ( r 2 =0.89) with a half-life of 26 days. At 73 days after the end of therapy the frequency, in under-cage samples, was not significantly higher than the background level. At the end of the therapy elevated frequencies of resistance were detected up to 75 m from the edge of the cage block and in samples where the levels of oxytetracycline were below the limit of detection (1.2 μg·g −1 ). Thirty-three days after the end of the therapy the frequency of resistance in all samples not directly under the cages was not significantly higher than in samples taken from sediments free of anthropogenic influence.

Validation of Polymerase Chain Reaction-based techniques for proxy detection of bacterial fish pathogens: Framework, problems and possible solutions for environmental applications

Validation is a process that establishes the extent to which a technique generates meaningful data when it is applied, for a specific purpose, in a specific experimental context. This review develops a framework for the performance of validation studies of the application of PCR-based techniques to the detection of fish pathogenic bacteria in the environment. It is argued that the potential for the application of these techniques to environmental microbiology will only be realised if adequate attention is paid to their validation. The framework developed here advocates the evaluation of the quantitative, qualitative and reliability criteria of a technique at four levels of experimental complexity. The problems encountered in assessing each of these criteria of validity at each level of complexity are discussed. Three of the levels of experimental complexity, in vitro studies, studies in seeded matrices and studies in incurred matrices can be performed within the laboratory. Consideration of the problems inherent in these laboratory-based validation studies demonstrates that they cannot provide evidence of adequate validity for field applications but it is suggested that their performance will be cost-effective. Their major role is in providing rational grounds for rejecting techniques whose application to environmentally derived matrices has been shown to be invalid. It is argued that, at the fourth level of experimental complexity represented by field studies, comparative and predictive validation provide powerful tools for establishing the value of any application of a PCR-based technique. Such studies are, however, time consuming and their performance cannot be envisaged with techniques that could have been rejected during laboratory-based studies.

3 – Covert Aeromonas salmonicida Infections

Publisher Summary The existence of clinically inapparent Aeromonas salmonicida infections has been recognized almost as long as the disease itself. Clinically inapparent infections were common and could persist in populations of fish for periods of months. These infections could be latent. If infected fish populations were stressed, overt clinical furunculosis could be precipitated. Fish with these infections were capable of acting as carriers. They could shed sufficient bacteria to transmit the infection to other fish. Fish with these clinically inapparent infections played a major role in the epizootiology of furunculosis. A variety of methods, of differing efficiency, that have been employed to detect these infections are discussed in this chapter. The covert infections are classified into three categories: incubatory clinical infections, subclinical infections, and commensal infections. These infections are persistent in nature and play a central role in the spread of the disease. Covert infections are the key to understanding the epizootiology of overt furunculosis.

Transient presence of oxytetracycline in blue mussels (Mytilus edulis) following its therapeutic use at a marine Atlantic salmon farm

On 15 occasions, during and after a therapeutic treatment of fish in an Atlantic salmon farm, oxytetracycline concentrations were determined in samples of blue mussels collected at two sites in the vicinity of the farm. Oxytetracycline was not detected in any samples of mussels collected 20 m from the cage block at a depth of 1 m. Those mussels sampled from immediately under one cage (Cage 65) at the farm (10–11 m depth) contained a concentration of 10.2 μg oxytetracycline g−1 of soft tissue on the last day of treatment. After the end of oxytetracycline administration the concentration detected in these samples declined exponentially (r2 = 0.94) with a half-life of approximately 2 days. At the time of the treatment the farm was stocked with 144 tonnes of fish and 186 kg of oxytetracycline were administered during a ten day treatment. In muscle samples taken from fish (n = 5) in Cage 65, after 8 days of therapy the mean oxytetracycline concentration was 1.3 ± 0.9 μg g−1. Three days after the end of the therapy the top 2 cm of the sediments beneath Cage 65 contained 9.9 ± 2.9 μg g−1 (n = 5). These tissue and sediment concentrations lie within the ranges of values that have previously been reported for such treatments. It is argued that residues present in filter feeding bivalves that occur as a consequence of the therapeutic use of oxytetracycline in marine fish farms are unlikely to present a significant human health hazard.

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.