The aim of this work is to investigate the degree of variation in the tetracycline resistance encoding plasmid population in strains isolated in the aquatic environment and those isolated in the human compartment. From this data a one or two compartment model will be constructed which best describes the distribution of tetR genes and movement of R+plasmids between compartments of the total environment. This model will then be used to provide an estimate of the possible extent to which the use of anti-microbial agents in aquaculture will have an impact on the use of these agents in human medicine. The choice of tetracycline resistance for construction of this model is based on two considerations:
- the agent is used in both human and veterinary medicine
- the mechanisms of resistance and the genetic determinants of resistance towards this agent have been well characterised
Model construction will be based on analysis of a master strain set of mesophilic Aeromonas and general heterotrophic bacteria. The choice of two separate bacterial groups to include in the master strain set will provide two levels of analysis
- data from the general heterotrophs will provide information of the general plasmid population structure of each environmental compartment
- data from the mesophilic Aeromonads will allow comparison of specific plasmid population structures in related bacterial present in both compartments
In the first year validation was carried out on a limited strain panel of general heterotrophic organisms
and mesophilic Aeromonads from both environments. This decision was informed by the desire to maximise resources while producing protocols of relevance to the programme. The validity of the project is totally dependent on the application of identical methods by each of the three partners.
Through this work a number of problems were identified that required the implementation of some minor chances to the original work plan. These were primarily concerned with the most appropriate media to use for isolation of the master strain set and the most appropriate pre-screening technique for identification of isolates containing transferable oxytetracycline resistance. While significant progress has been achieved on the isolation of general heterotrophs, the problems encountered with the isolation media for mesophilic aeromonads has impeded progress in their isolation. The data for the resolution of this problem has been accumulated by partners #2 and #3 and will be finally resolved at the partner meeting scheduled for March 1998. Filter mating has been adopted as the primary method for pre-screening due to it's greater reliability over the other methods examined.
Analysis of the master strain set will be of three types
- Identification of tetR determinant type through DNA probe hybridisation with specific PCR primers and probes for each determinant.
Partners #1, and to a lesser extent #2, has begun analysis of the master strain set for specific tetR genes using primer sequences obtained from the Primer 3 web-based program. A complete set of bacterial strains containing all of the known tetracycline resistance genes (with the exception of the recently discovered Tet V determinant) has been collected by partner #1. Primer sequences have been obtained for all of the determinants from Primer 3 and are currently awaiting delivery.
- Identification of R+plasmid types (incompatibility) on which tetR determinants are encoded.
Due to the problems encountered with the isolation of mesophilic Aeromonads partner #2 has delayed implementation of R+ plasm-id analysis until year 2.
- Identification and classification of both mesophilic Aeromonads and general heterotrophic bacteria containing transferable tetracycline resistance.
Partner #3 has commenced the preliminary identification and classification of the master strain set as outlined in the original proposal. Preliminary biochemical classification of the field isolates has been determined during year 1 with more detailed identification due to commence in year 2
In year 1 of the programme the majority of the scheduled work in the original work programme has been completed. The major exception to this completion of the methods phase has been the isolation of tetracycline resistant mesophilic Acromonads but mechanisms to resolve this problem are in train. At the end of year 1 a total of 650 or the 1200 strains that will comprise the master strain set have been collected and stored. Preliminary analysis of these strains has begun and is ahead of schedule with regards to their antibiotic susceptibility profiles. Thus, progress in this area is highly satisfactory. In agreement with the change of emphasis outlined, progress made in collecting strains is actually in advance of that planned originally. As a consequence the analysis of these strains are slightly behind the original schedule.
It is considered that this project is proceeding at a pace that will allow its completion within the three-year time scale allotted to it. During this year minor modifications have been made to the original work programme, which, it is believed, have facilitated a more efficient use of the available manpower and resources.
WORK AND ACHIEVEMENTS
Standard operating procedures for the sampling of fish farm effluent/hospital effluent, processing of field samples and pre-screening of isolates for transferable tetracycline resistance have been developed, validated and distributed among the partners. The validated protocols essentially represent the backbone of any future analysis. Thus, the work of the first year was primarily concentrated on ensuring that the data Generated by these protocols would be comparable across the three countries and two environmental compartments and the master strain sets would be suitable for, and generate meaningful date from, the analysis to be undertaken in the second and third year of the project.
Funding SchemeCSC - Cost-sharing contracts
LA22 0LP Ambleside