effects of selection on gene stability and transfer in populations of bacteria
and objectives (1)
The mobility of drug resistance genes in the environment is an important
biosafety issue. Such genes have been used as markers during the construction
of genetically modified crop plants and industrial strains of bacteria.
These markers can be introduced into the environment in GMOs such as pest-resistant
crop plants and in effluents from fermentations of bacterial strains which
produce enzymes and other useful biological and medical compounds.
The conditions affecting gene transfer in the environment have been studied
extensively. However, little is known about the effect of selection on
gene transfer in situ. In highly selective environments, gene transfer
events are readily detected due to the reduced survival of other members
of the biotic community. The aim of this research was to establish whether
selective conditions for antibiotic resistance could be established in
soil either by addition of exogenous antibiotic or by production in
situ by antibiotic-producing streptomycetes. We also wished
to monitor the frequency of such gene transfer events in environments
such as soil, where growth rates are likely to be much lower than those
occurring in vitro or in animal hosts.
(1) This project was a direct follow-on from EC project:
Risk assessment of releasing recombinant streptomycetes into the environment
Approach and methodology
The antibiotics neomycin and thiostrepton were applied to soil microcosms
which had been inoculated with both antibiotic-sensitive (TK23) and resistant,
plasmid-bearing (TK23 pIJ680), strains of Streptomyces lividans.
The effects of antibiotic additions to soil on survival and plasmid transfer
frequency were investigated. The antibiotic, thiostrepton, was applied
to both sterile and non-sterile Warwick soils inoculated with sensitive
and resistant strains of Streptomyces lividans. The loss in activity
of the antibiotic over time, and the effects of the antibiotic on the
survival of these strains was investigated.
The fate of the two antibiotics was studied to determine sensitivity of
detection, extraction efficiencies and inactivation in soil. Neomycin,
a water soluble antibiotic, and thiostrepton, a water insoluble antibiotic,
were chosen because of their use in agriculture and very different solubilities.
Methods for antibiotic extraction and detection were developed to achieve
sensitive assays for monitoring the rate of decay or immobilisation of
antibiotics added to soil.
Main findings and outcome
Neither antibiotic had any effect on survival of sensitive strains in
different soils containing relatively high amounts of clay. However, in
a sandy podzol environment, both thiostrepton and neomycin killed sensitive
streptomycetes. This resulted in a reduction of detected plasmid transfers
through the killing of recipients. In the Warwick and Athens soils tested
(high clay content), neomycin addition to the soil did not affect survival
or plasmid transfer. However, in the Wageningen soil (sandy), in the presence
of neomycin, the sensitive strain, TK23, fell below detection limits (<102
cfu. g-1) after only 30 days of incubation, while without neomycin
the strain reached levels of up to 106 cfu. g-1.
This suggests that neomycin has a detrimental effect on the survival of
TK23. TK24 (pIJ680) grew in the presence of neomycin even at 1000 µg
g-1, although in its absence the strain grew up to 1 log higher.
In contrast to the data obtained in both Warwick and Athens (high clay
content), antibiotic present in Wageningen soil (sandy) appears to have
a killing effect on a sensitive strain.
Thiostrepton lost up to 90% of its activity within 10 days when added
to either sterile or non-sterile soil, while between 10 and 70 days, little
further inactivation occurred. The antibiotic at 1.0 mg g-1
soil had no observable effect on the survival of the sensitive strain
TK23 in sterile soil, but a selective effect was seen for the resistant
auxotrophic strain KT2054. In non-sterile soil, in the absence of thiostrepton,
the KT2054 population fell below the detection limit by day 60. However,
at thiostrepton concentrations of 100 µg g-1 and 1 mg
g-1 soil, the KT2054 population was maintained or increased
Evidence for antibiotic production in soil was observed using streptomycetes
that produce thiostrepton, where over 50 ng g-1 soil was detected
with Streptomyces azureus bacteria used as the inoculant. For soil
microcosms, studies with thiostrepton proved that this antibiotic could
be readily detected in soil and extracted, but problems were encountered
with the water soluble neomycin. The latter antibiotic could not be detected
in soil nor extracted. Using soil microcosms, extraction of thiostrepton
was possible from three different soil types and a sensitive bioassay
was also used to monitor production in situ. Using a range of methods
it was not possible to extract neomycin from soil, except in the case
of Wageningen soil where the minimum detection limit was 500 µg
The results of this study show that the presence of thiostrepton in either
sterile or non-sterile soil offers a selective advantage to a resistant
S. lividans strain, even when that strain is metabolically disabled.
This selective advantage can allow the strain to be maintained in an environment
where it may otherwise die out. Both antibiotics can exert a selective
effect in soils with low clay content but only thiostrepton had a measurable
effect in soils of high clay content where a disabled, unfit strain survived
in non-sterile conditions under selection by thiostrepton addition. Thiostrepton
was also produced naturally in soil, indicating that resistance to this
antibiotic could experience a selective pressure in soil.
Herron P.R., Toth I.K., Heilig G.H.J., Akkermans A.D.L., Karagouni
A. and Wellington E.M.H., Selective effect of antibiotics
on survival and gene transfer of streptomycetes in soil.
Soil Biol. Biochem., 30, 1998, pp. 673-677.
Marsh P., Toth I., Meijer M., Schilhabel M.B. and Wellington E.M.H.,
Survival of the temperate actinophage ØC31 and Streptomyces
lividans in soil and the effects of competition and selection
on the spread of lysogens.
FEMS Microbiol. Ecol., 13, 1993, pp. 13-21.
Karagouni A.D., Vionis A.P., Baker P.W. and Wellington E.M.H., The
effect of soil moisture content on spore germination, mycelium development
and survival of a seeded streptomycete in soil.
Microbial Releases, 2, 1993, pp. 47-51.
October 1991 September 1993
University of Warwick
University of Athens (GR)
Agricultural University of Wageningen (NL)