The objectives are to determine the effects of selection on the stability of antibiotic resistance genes amongst populations of soil bacteria. Resistant phenotypes will be selected for in soil by addition of antibiotics and by their production in situ. Subsequent effects on gene stability and transfer will be determined for chromosomal, plasmid and phage borne genes.
The role of natural or artificial selection in soil must be measured to accurately assess the fate of genes in the natural environment and evaluate any risks associated with new gene introductions. Enabling technology is being developed to study the process of selection in the soil within a realistic time scale. In order to carry out the study it was first necessary to obtain genetically marked Streptomycetes. Genes for antibiotic resistance to thiostrepton and neomycin were sought as these antibiotics had been selected for subsequent soil enrichments. The second objective was the development of a soil microcosm system which required the selection and characterization of soil at specified sites, and use of batch, fed-batch and continuous microcosms with methods for the extraction of bacteria and antibiotics from the soil. The characterization of selected soils, involved physical, chemical and biological study. The latter required assessment of indigenous resistance levels to thiostrepton and neomycin in streptomycete populations. Antibiotic extraction techniques were required for detection and identification of antibiotics produced in soil and added to soil. As the former was known to be very low, a high level of sensitivity was required. Cell extractions were required for monitoring both populations and deoxyribonucleic acid (DNA) in soil. The aim here was to enable monitoring of cell replication during sporulation. Thus methods allowing differentiation of spores and vegetative cells were needed to determine if the fed-batch and continuous microcosms were allowing rounds of replication. Molecular methods were intended as a secondary source of information concerning the fate of introduced genes. In this case differential extraction of DNA from spores and vegetative cells was needed together with exploitation of polymerase chain reaction (PCR) to improve sensitivity for detection of genetic markers.
Transposons were found to be extremely unstable and an alternativ e strategy forchromsomal marking was developed. This involved the use of the plasmid series pGM (Muth et al 1989) which allow insertion of markers into the streptomycete chromosome as these plasmids act suicide vectors, unable to replicate at 35 C.
The development of a new soil model microcosm, the fed-batch system has been accomplished which allow continuous rounds of germination. This will provide a more accurate and realistic assessment of antibiotic resistance gene transfer and stability in soil. New methods for sensitive extraction and identification of antibiotics produced in, or added to soil have been developed.
The project is continuing.
The survival and dissemination of genes in soil microbial populations may be influenced by their selective advantage. The project aims to investigate the ways in which selection might act for antibiotic resistance in soil bacteria using the genes for thiostrepton resistance (tsr) and aminoglycoside resistance (aph I, II, V). A number of aspects will be considered to determine how these genes might spread in soil populations and if indigenous resistance is affected by the selection pressure imposed. The three participating laboratories will investigate gene stability by introducing resistance genes on plasmids, transposons and within amplified sections of the chromosome. Parallel experiments involving continuous soil columns will be run to allow comparison between the different sources of the genes.
The mobility of tsr within populations of introduced and indigenous streptomycetes will be studied at all three laboratories while Tn5 (aph II) will be studied in marked plasmids and transposons. The aph II gene being selected for work with amplified sequences on the chromosome. The stability of genes aph I and II will also be investigated in pseudomonad populations introduced into soil. Selection for the genes will be simulated by the enrichment of soil with antibiotic solutions and by introduction of antibiotic-producing streptomycetes (in nutrient-enriched soil). Genes will be tracked by direct probing and PCR of soil DNA and by analysis of phenotype and genotype of soil isolates.
Our aim is to determine the following:
Can selection for antibiotic resistance genes occur in soil?
Genes thought to be lost, following die-off through death of unfit inoculants, can reappear with selection?
The location of the gene is important for stability and mobility, but to what extent does this matter after several generations in soil?
Funding SchemeCSC - Cost-sharing contracts
6703 CT Wageningen