resistance genes in the environment: a comprehensive, multi-phasic survey
of prevalence and transfer
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
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
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
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.
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,
Smalla K., Gebhard F. and Heuer H., Antibiotic resistance
genes as markers in transgenic plants risk of horizontal
Nachrichtenbl. Deut. Pflanzenschutzd., 52, 2000, pp.
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).
October 1998 December 2000
University of Warwick
Federal Ministry of Health and Environment
J.D. van Elsas
Plant Research International (formerly IPO-DLO)
University of Athens (GR)
Federal Biological Research Centre for Agriculture and Forestry