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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image Antibiotic resistance genes in the environment: a comprehensive, multi-phasic survey of prevalence and transfer

Background and objectives

Little data is available concerning antibiotic resistance gene dissemination in bacteria in terrestrial and aquatic environments. Resistance mechanisms vary considerably between strains and inactivating enzymes are readily acquired by horizontal transfer of genes. Whilst the reservoir for such resistance genes within populations of clinical isolates has and is being documented due to their importance in the treatment of infections, no such database exists for populations in soil and water. Few studies have addressed the distribution and diversity of resistance genes in nature in a systematic attempt to determine their prevalence in differing environmental samples and distribution with respect to environmental selection pressure. Nor indeed has any survey been done to quantify the distribution of antibiotic-producing bacteria and their resistance gene pool for comparison with resistance mechanisms in non-producing groups. Important considerations relate to the use of antibiotic resistance marker genes for genetically modified organisms (GMOs) in the environment and the continued use of antibiotics in agriculture, as well as concerns about cross-resistance to clinical antibiotics. The aim of this research work is to provide evidence for the existence of reservoirs of antibiotic resistance genes in the environment and determine the impact of human activities on the dissemination of such resistance within bacterial populations.

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Approach and methodology

The major objective of this proposal was to make a systematic survey of the prevalence, incidence and mobility of genes conferring a resistance to gentamicin, streptomycin and tetracycline antibiotics in different environmental habitats such as soils, rhizosphere, manure, sewage, coastal waters and sediments. The environments analysed comprised sites with heavy and minimal selective pressure. Both culturable and non-culturable populations were surveyed.

The antibiotics which we used were selected due to their very different application profiles. Gentamicin is still a reserve antibiotic in human and animal therapy, tetracycline has been applied in both therapy and agriculture, and streptomycin has been used for decades, mainly in agriculture. Based on existing sequence data for gene families, degenerate PCR primers were designed to allow recovery of potentially novel sequences coding for resistance and production genes.

A survey was made of the natural reservoirs of antibiotic resistance genes in cultured and non-cultured bacterial populations. Using gentamicin, streptomycin and tetracycline as targeted antibiotics, the distribution and abundance of resistance and production genes were determined in samples from a range of environments.

Main findings and outcome

Streptomycin-resistance phenotypes were prevalent in soil, and were markedly affected by streptomycin treatments. Gentamicin resistance was more common in bacteria that were isolated from sewage. This trend was surveyed at the molecular level by evaluation of the diversity of resistance genes present in DNA extracted from the total community at the different sites. The widespread occurrence of streptomycin resistance genes in soil correlated with the presence in all soils of streptomycin-producing bacteria. Gentamicin-producing bacterial genes were rare in soil total community DNA, as were the resistance genotypes. In contrast, sewage and manure contained a diverse range of resistance genes, reflecting human use of tetracycline and gentamicin.



We will extend this work to assess resistance gene mobility by isolating resistance genes located on self-transferable or mobilisable plasmids. The genetic location of these resistance genes on plasmids or transposons will be further characterised. The impact of selective pressure (man-made or by antibiotic-producing strains) on the prevalence of resistance genes, as well as their abundance on mobile genetic elements, will be determined. Furthermore, the impact of selection pressure in specific environmental locations will be assessed to establish bacterial responses to selection in non-clinical environments.

Major publications

Guillaume G., Vebrugge D, Chasseur-Libotte M-J, Moens W & Collard J-M., “PCR typing of tetracycline resistance determinants (tet A to E) in Salmonella enterica serotype Hadar and in the microbial community of activated sludge from hospital and urban wastewater treatment facilities in Belgium”.
FEMS Microbiol Ecol., 32, 2000, pp. 77-85.

Egan S., Wiener P., Kallifidas D. and Wellington E.M.H., “Phylogeny of streptomyces species and evidence for horizontal transfer of entire and partial antibiotic gene clusters”, Antonie van Leeuwonhoek, 2000 (Submitted).

Smalla K., Gebhard F. and Heuer H., “Antibiotic resistance genes as markers in transgenic plants – risk of horizontal gene transfer?”.
Nachrichtenbl. Deut. Pflanzenschutzd., 52, 2000, pp. 62-68.

Egan S. and Wellington E.M.H., “Current use and future prospects for antibiotic resistance gene markers”, in Genetically Engineered Micro-organisms: Method Development from Microcosms to Field, J.K. Jansson, M. Bailey and J.D. van Elsas (eds.), R.G. Landes Company, Austin, Texas, 2000 (in press).
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Contract number

October 1998 – December 2000

E.M.H. Wellington
University of Warwick
Coventry (UK)



J-M. Collard
Federal Ministry of Health and Environment
Brussels (BE)

J.D. van Elsas
Plant Research International (formerly IPO-DLO)
Wageningen (NL)

H. Gürtler
Novo Nordisk
Bagsvaerd (DK)

A. Karagouni
University of Athens (GR)

K. Smalla
Federal Biological Research Centre for Agriculture and Forestry
Braunschweig (DE)

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