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Applied and Environmental Microbiology, November 1999, p. 5139-5141, Vol. 65, No. 11
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Gene Transfer in the Gastrointestinal
Tract
Trudy
Netherwood,1,2,*
R.
Bowden,1,2
P.
Harrison,1,2
A. G.
O'Donnell,2
D. S.
Parker,1 and
H. J.
Gilbert1
Department of Biological and Nutritional
Sciences1 and Department of Agriculture
and Environmental Science,2 University of
Newcastle-upon-Tyne, Newcastle-upon-Tyne NE1 7RU, Great Britain
Received 26 April 1999/Accepted 9 August 1999
 |
ABSTRACT |
The maximum in vivo transfer rate of plasmid pAM
1 in the gut was
0.03 transconjugant per recipient cell, and this rate could be
simulated in vitro only by forced filter mating. Transfer was not
detected in liquid culture matings. Our findings demonstrate that in
vitro methods, such as forced filter mating and liquid mating,
underestimate the in vivo rates of gene transfer.
| |
TEXT |
Genetically modified organisms
(GMOs) are being developed and approved for use in many areas of food
production; these organisms include microbial GMOs that are used in
cheese and bread making (1, 7) and a variety of GMO food
crops (11). Safety studies have indicated that the use of
GMOs presents no greater risk than the use of the original unmodified
products (10). However, there are still public and
scientific concerns about the safety of foods produced by gene
manipulation technology. One of the main concerns is the possibility of
gene transfer from GMOs to organisms in the environment, particularly
transfer of genes to pathogenic bacteria that could increase their
virulence. Gene transfer has been observed in the gastrointestinal (GI)
tracts of mammals (2, 6). However, several studies have
showed that gene transfer in the environment, such as in the GI tract
of rats (14) and in wastewater treatment plants (4,
5), occurs at rates that are 101- to
105-fold lower than the rates measured under laboratory
conditions. In this study we investigated the persistence of GMO
probiotics and transgene transfer in the avian GI tract.
Persistence of a GMO in the GI tract.
For a GMO to have an
impact in the gut, the probiotic strain must be stable and be able to
transiently colonize the gut; however, ideally, it should not persist
following removal of the probiotic from the diet. The GMO probiotic
used in this study was a genetically manipulated Enterococcus
faecium strain (NCIMB 11508) with chromosomally encoded rifampin
resistance (Rifr), in which the Ruminococcus
flavefaciens -1,4-glucanase was present on a
non-self-transmissible plasmid (pVACMC1, conferring erythromycin
resistance [Eryr]) and was maintained as an
extrachromosomal element (15). The construct was 98% stable
for five generations in vitro and produced 0.125 U of cellulase/ml of
culture, which was equivalent to 1.4 U of cellulase/mg of total protein.
The in vivo studies were carried out by using broiler chicks and
probiotic added to feed at a concentration of 105 CFU/g;
the method used is described in the accompanying paper (8).
Probiotic bacteria were recovered from the GI tract by selecting for
Rifr colonies on MRS medium (Oxoid Ltd.). This selection
strategy was appropriate as the background level of Rifr
bacteria in the GI microflora is <101 CFU/ml, 100,000-fold
lower than the level of the probiotic in the GI tract. The number of
generations of the GMO probiotic that occurred during transit of the GI
tract is not known, but the plasmid was detected in 69% (percentage of
Rifr enterococci which were also Eryr) of GMO
probiotic isolates obtained from the GI tract following 4 weeks of
probiotic treatment. Thus, while the GMO probiotic was maintained in
the gut, a major proportion retained the recombinant genotype.
The carriage rate of the probiotics was initially determined to be
10
5 CFU/g of gut contents. After 28 days of probiotic
feeding, the
GMO carriage rate had increased to 10
7 CFU/g
of gut contents, while the level of unmodified probiotic
remained
10
5 CFU/g (Fig.
1). This may
indicate that the GMO had a selective
advantage over the unmodified
strain, which may have been due
to the production of cellulase which
provided an accessible energy
source for the microorganism and the
host.
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FIG. 1.
Persistence of probiotics in the cecum following removal
from the feed: numbers of CFU (log10) of probiotics
isolated on selective media containing rifampin/g of cecum contents, as
determined at 2-day intervals after the probiotics were removed from
the diet. The probiotics were removed from the diet following 28 days
of treatment. The error bars indicate standard errors of the means.
Three replicates were used in each experiment.
|
|
Neither the GMO nor unmodified probiotic strains persisted in the GI
tract of chickens for more than 5 days following removal
from the diet
(Fig.
1). Therefore, the probiotic strains do not
permanently colonize
the avian GI tract when the rate of application
of the probiotic in the
feed is 10
5 CFU/g, the rate used in this
study.
To investigate whether pVACMC1 was transferred to the endogenous avian
microflora, 5,000 Ery
r cellulase-producing bacteria
recovered from the GI tracts of
birds inoculated with the probiotic
were found to be Rif
r, suggesting that these organisms were
probiotic strains. In addition,
after the probiotic strain was washed
out following removal of
the strain from the diet, no Ery
r
cellulase-expressing bacteria were detected in the GI tract.
Although
it is not possible to conclusively prove that pVACMC1
was not
transferred to the endogenous avian microflora, we detected
no transfer
events, indicating that if the plasmid was transferred,
the transfer
occurred at a very low
rate.
Gene transfer in the avian GI tract.
One of the major concerns
in using genetically modified organisms in food production is the risk
of gene transfer to other organisms in the environment. Naturally
occurring gene transfer in the environment takes place relatively
infrequently (3) and is therefore difficult to measure
experimentally with currently available techniques. To estimate the
rate of transfer of transgenes from the GMO probiotic, in vitro and in
vivo techniques were compared by using broad-host-range conjugative
mobilizing plasmid pAM1 (26 kb, Eryr) to determine the
relative sensitivity of each of the methods. The in vitro method used
included the filter mating technique of Sasaki et al. (12)
and liquid mating, which consisted of the same basic method involving
mixing optimum concentrations of log-phase donors and recipients with
and without a filter step.
The transfer rate of recombinant plasmid pVACMC1 (Ery
r) was
investigated initially in vitro under forced filter mating conditions
by using 10
8 CFU of the donor strain (Rif
r
Ery
r encoded by pVACMC1) per ml and 10
7 CFU of
the Rif
r recipient strain per ml and in the presence of the
mobilizing
Ery
r plasmid pAM
1 at a concentration of
10
8 CFU/ml. This was considered the worst-case scenario for
detection
of gene transfer from the recombinant probiotic. Gene
transfer,
as shown by the appearance of Rif
r
Ery
r cellulose-expressing colonies, was not detected under
these conditions
but may have occurred at a rate of less than
10
8, which was the lowest level of
detection.
The gene transfer rate of conjugative plasmid pAM
1 from a
Rif
s probiotic strain to a Rif
r probiotic
strain in the GI tract was found to be 0.03 transconjuant
per donor
organism at probiotic concentrations of 10
5 CFU of donor
(Rif
s probiotic strain)/ml of gut contents and
10
4 CFU of recipient (Rif
r probiotic strain)/ml
of gut contents (Fig.
2). This rate can
be considered greater than the rate predicted by the in vitro
study
(also 0.03 transconjugant per donor), but it was obtained
under optimal
forced filter mating conditions at maximal probiotic
concentrations
(
12).
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FIG. 2.
Rates of mobilization of the probiotic in the chicken GI
tract: distribution of gene transfer events in the crop, duodenum, and
cecum on four different sampling dates. The rate of mobilization rate
was the mean number of transconjugants per recipient cell. The error
bars indicate standard errors. Three replicates were used in each
experiment.
|
|
The gene transfer rate is most commonly expressed as the number of
transconjugants per recipient cell, but this method of
expression does
not take into account the volume in which the
organisms are placed and
therefore the potential for intimate
contact that is required for
transfer to take place. In vitro,
the volume in which mating takes
place is infinitely small, and
the donors and recipients are in
intimate contact on a filter;
thus, the potential for conjugal transfer
should be expected to
be much greater than the potential for conjugal
transfer in the
GI tract. Therefore, we also should consider the
density of the
bacteria in the mating mixture. Our in vitro transfer
rate is
similar to the maximal rates reported previously
(
13), and therefore
the discrepancy between the in vitro and
in vivo rates is not
due to suboptimal application of in vitro
techniques but truly
reflects the inadequacy of the techniques used to
represent in
vivo
conditions.
Gene transfer in vitro was not detected without filter mating. Liquid
mating at a donor concentration of 10
8 CFU/ml and a
recipient concentration of 10
7 CFU/ml was carried out, both
with and without gut contents, but
no transfer was
observed.
Neither the filter mating technique nor the liquid mating technique
accurately represent the natural conditions in the gut.
The effects of
bacterial binding to the gut wall membrane and
solid matter in the
lumen create conditions for gene transfer
at estimated bacterial
concentrations that are 1,000 fold lower
than the concentrations used
in vitro. Therefore, the in vivo
conditions actually represent the
worst-case scenario for gene
transfer rather than the optimized in
vitro conditions. However,
this presents a problem in assessing the
risk of the GMO probiotic,
as the methods used to detect transfer in
vivo have severe sensitivity
limitations and tend to underestimate the
true rate of transfer
in
vivo.
The in vivo measurements obtained for sample sites and individuals were
not consistent (Fig.
2). Most of the transfer occurred
in the cecum,
but transfer was detected in only 5 of the 72 chicks
tested (the
detection rates were represented by counts of between
30 and 200 CFU/plate at rates of dilution of gut contents of 10
0.5
to 10
1). Transfer may have occurred at rates below the
level of detection
in the other individuals, but techniques with
greater sensitivity
are necessary to improve the accuracy of the
estimated in vivo
transfer rates. The current method produced large
numbers of CFU
when transfer did occur, but this seemed to occur in a
minority
of the chicks tested. Transfer was not detected in some
chicks,
which may have been due to individual variability of the
chicks.
For example, gene transfer may have occurred with a different
bacterial species, unidentified factors in the GI tract may have
inhibited transfer, or
E. faecium may have entered an
unculturable
state. The relatively low concentrations of the donor and
recipient
probiotics in the gut (10
5 and 10
4
CFU/ml) could have contributed to the sporadic detection of gene
transfer, as these concentrations are near the limit of sensitivity
of
the culture
techniques.
Within the gut, a probiotic organism is not present as a synchronized
culture, and only a small proportion of the cells may
be at a stage of
growth capable of conjugation. Thus, the in vivo
culture technique
probably underestimated the measurable rate
of transfer in the avian GI
tract, as only the recipient probiotic
strain was detected and this
strain represented a very minor population
of the enterococci in the GI
tract, all of which were potential
recipients but were difficult to
detect in the presence of a large
number of background Ery
r
donor
organisms.
The presence of other bacteria and solid matter may also reduce the
likelihood that a donor and a recipient meet and therefore
the
likelihood of gene transfer. As the intestinal tract contains
many
naturally competent bacteria at concentrations greater than
probiotic
application rates, we cannot predict that transfer to
other bacteria
does not occur in the GI tract via transformation.
The resulting
transformants would not have been detected on the
selective media which
we used, and therefore the true rate of
transfer would have been
underestimated. The presence of DNases
in the GI tract, however,
reduces the likelihood of transfer by
this
mechanism.
In conclusion, our findings indicate that the true gene transfer rates
in vivo are higher than the rates determined in vitro,
which means that
there are indigenous effects in the GI tract
that promote a higher rate
of transfer than the rate observed
in vitro. Similar "hot spots"
for gene transfer have been identified
in the environment (for example,
the bean phylloplane [
9])
and are characterized by the
availability of nutrients and high
bacterial
densities.
| |
ACKNOWLEDGMENTS |
This work was supported by a grant from the United Kingdom Ministry
of Agriculture, Fisheries and Food as part of the Novel Foods Programme.
We thank Harry Flint, Rowett Research Institute, Aberdeen, United
Kingdom for donation of the pVACMC1 plasmid.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Agriculture and Environmental Science, King George VI Building,
University of Newcastle-upon-Tyne, Newcastle-upon-Tyne NE1 7RU,
Great Britain. Phone: 0191 222 5044. Fax: 0191 222 5228. E-mail:
trudy.netherwood{at}newcastle.ac.uk.
| |
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Applied and Environmental Microbiology, November 1999, p. 5139-5141, Vol. 65, No. 11
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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