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Applied and Environmental Microbiology, April 2001, p. 1494-1502, Vol. 67, No. 4
Department of Animal Sciences, University of
Illinois at Urbana-Champaign, Urbana, Illinois
61801,1 and Illinois State
Geological Survey, Champaign, Illinois 618202
Received 30 November 2000/Accepted 9 January 2001
In this study, we used PCR typing methods to assess the presence of
tetracycline resistance determinants conferring ribosomal protection in
waste lagoons and in groundwater underlying two swine farms. All eight
classes of genes encoding this mechanism of resistance
[tet(O), tet(Q), tet(W),
tet(M), tetB(P), tet(S), tet(T), and otrA] were found in total DNA
extracted from water of two lagoons. These determinants were found to
be seeping into the underlying groundwater and could be detected as far
as 250 m downstream from the lagoons. The identities and origin of
these genes in groundwater were confirmed by PCR-denaturing gradient gel electrophoresis and sequence analyses. Tetracycline-resistant bacterial isolates from groundwater harbored the tet(M)
gene, which was not predominant in the environmental samples and was identical to tet(M) from the lagoons. The presence of this
gene in some typical soil inhabitants suggests that the vector of
antibiotic resistance gene dissemination is not limited to strains of
gastrointestinal origin carrying the gene but can be mobilized into the
indigenous soil microbiota. This study demonstrated that
tet genes occur in the environment as a direct result of
agriculture and suggested that groundwater may be a potential source of
antibiotic resistance in the food chain.
The widespread use of antibiotics in
humans and animals has raised several concerns related to human and
animal health. The principal area of concern has been the increasing
emergence of antibiotic resistance phenotypes in both clinically
relevant strains and normal commensal microbiota. In two recent
studies, a link between the agricultural use of antibiotics and
antibiotic-resistant human infections has been suggested (24,
33). Because consumption of tainted food is considered the main
route of transmission of drug resistance, other possible means of
antibiotic resistance dissemination have received relatively little
attention. One of these possible means could be natural water and soil
environments impacted by antibiotics and antibiotic-resistant bacteria
from agriculture, where antibiotics are used for disease treatment, prophylaxis, and growth promotion. The concern over the use of antibiotics in agriculture, especially for prophylactic and
growth-promoting purposes, has not been limited to the presumed role of
antibiotics in selection of antibiotic-resistant bacteria (pathogenic
or nonpathogenic) in the animal gut. The more debatable issue arising
from chronic low-level exposure to antibiotics is whether this practice
contributes significantly to increased gene frequencies and
dissemination of resistance genes into other ecosystems.
Furthermore, many antibiotics used in animal agriculture are poorly
absorbed in the animal gut. It is estimated that 25% to as much as
75% of the antibiotics administered to feedlot animals could be
excreted unaltered in feces (6, 7) and can persist in soil
after land application (4, 11). There is little
information available concerning the fate of antibiotics in the
environment and their link to the emergence of resistant genotypes
found there. The annual production of livestock and poultry waste in
the United States is nearly 180 million tons (dry weight basis)
(13, 34), and coupled with antibiotic usage, this waste is
a potentially large source of both antibiotics and antibiotic-resistant
bacteria released into the environment.
Lagoons and pit systems are typically used for waste disposal in animal
agriculture. Seepage and runoff into watershed systems are of
particular concern due to potential mobilization of constituents and
exposure of contaminants to humans and other animals. Groundwater, in
particular, constitutes about 40% of the water used for public water
supplies and provides drinking water for more than 97% of the rural
population in the United States
(http://water.usgs.gov/wid/html/GW.html). Recent monitoring studies
have demonstrated the vulnerability of groundwater to seepage from
waste lagoons (19). Over a period of several years, Krapac
and coworkers found indicators such as ammonia and fecal enterococci at
elevated levels in groundwater samples obtained up to 100 m
downstream of from swine waste lagoons, indicating that both long-term
impact and environmental migration of contaminants occur (19,
20).
Recent studies have attempted to evaluate the impact of antibiotic use
on populations of bacteria in natural waters (12, 23, 39).
Antibiotic resistance analysis has been used to identify sources of
fecal pollution (14, 18, 27, 38, 39). The traditional
method involving cultivation and phenotypic testing is still relied
upon but has a clear bias when it is used to determine the
representative phenotypes and genotypes in this environment. The use of
techniques such as PCR and molecular gene probe analysis has allowed
sensitive detection of specific genes in the environment in the absence
of cultivation. PCR amplification of small-subunit ribosomal DNA (rDNA)
genes coupled with denaturing gradient gel electrophoresis (DGGE) has
been used primarily to determine the genetic fingerprints of microbial
communities. The repertoire of genes used for such analyses has been
extended to specific metabolic genes (2, 8, 17, 31, 37)
and, recently, to antibiotic resistance genes (1). In the
latter work, we used a phylogenetic approach to design a set of primers
to target tetracycline resistance genes encoding ribosomal protection
proteins (RPPs). These primers were used to detect the corresponding
tetracycline resistance genes in ruminal fluid, swine feed and feces,
and pig intestinal fecal streptococci. In the present study, we used
these techniques to determine the occurrence and migration of RPP
tetracycline resistance genes in lagoons and in groundwater underlying
two large swine production facilities known to be impacted by waste seepage (19). The use of tetracycline resistance as the
key determinant to monitor resistance genes is relevant due to the common use of this antibiotic in the swine industry. To our knowledge, this is the first study in which the genes for one major class of
antibiotic resistance were characterized in natural groundwater that is directly impacted by animal agriculture.
Description of the sampling sites.
Site A, which began
operation in February 1995, is a finishing operation that houses 4,000 pigs (Fig. 1). Antibiotics are used in
this facility, but the relevant information concerning such use could
not be obtained from the producer. The facility includes a two-stage
waste-handling system in which a concrete settling basin collects most
of the solids before the supernatant liquid passively enters an earthen
lagoon. The area of the lagoon is approximately 1.2 ha, and the lagoon
is unlined. No special construction techniques were used to compress
the soil during lagoon construction. The average depth of liquid in the
lagoon during a study conducted by Krapac et al. (19) was
about 1.5 m. The concrete settling basin is periodically pumped,
and the manure is applied to crop fields both on-site and off-site.
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.4.1494-1502.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Occurrence and Diversity of Tetracycline Resistance Genes in
Lagoons and Groundwater Underlying Two Swine Production
Facilities
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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FIG. 1.
Maps of sites A and C and corresponding stratigraphic
columns indicating the locations and characteristics of sand layers.
The direction of groundwater flow is indicated by large open arrows,
and the locations of monitoring wells are indicated by circles; open
circles represent nested wells screened in deeper sand layers. Well
depths (in meters) are indicated in parentheses. The solid rectangles
represent confinement buildings.
4 m/s. Below the silty sand and gravel is 1.6 to 3 m of silt loam diamicton, which may be colluvial. Below the silt loam
diamicton is a 1- to 2-m-thick lower sand layer composed of sand and
gravel outwash that is being used locally as an aquifer. Four
monitoring wells were installed in this lower sand layer (Fig. 1). The
saturated hydraulic conductivity of this deeper sand was estimated to
be 8.2 × 106 m/s based on slug tests. Below this
sand and gravel is more silt loam diamicton. Logs from water wells
drilled in the vicinity show the presence of discontinuous sand and
gravel outwash units below the diamicton that are used locally as
aquifers. The multiple sand layers make this site particularly
susceptible to leachate migration from the lagoon.
Site C is a farrowing and nursery operation that houses 1,200 sows
(Fig. 1). The facility uses chlortetracycline, which is added once per
quarter for about 2 weeks to the feed (400 g per ton). The feed
consumption rate is about 3 kg/animal per day, and thus, each animal
consumes about 17 g of chlortetracycline over the 2-week period.
The facility began operation in the fall of 1992 and every year
produces approximately 24,000 pigs. The facility uses a single-stage
lagoon. Lagoon water is recycled to partially fill and flush the
shallow pits below the confinement buildings. The area of the lagoon is
approximately 0.8 ha, and the lagoon is unlined. The average depth of
waste in the lagoon was about 6 m. Waste has never been applied to
the crop fields surrounding the lagoon.
Site C is located on a glacial till plain formed during the Illinois
Episode of glaciation. It is underlain by a silt loam glacial diamicton
that is 3 to 15 m thick and overlies shale bedrock (Fig. 1). Thin
(less than 30-cm-thick) glacial gravelly loam layers were found in two
of the seven borings at the site. An intermittent stream borders the
site on the west, and a small pond is located about 100 m south of
the lagoon. Large-diameter bored and dug wells and ponds are the
predominant sources of drinking water in the area.
Groundwater and lagoon sampling. Groundwater was collected from 14 monitoring wells at site A and from six monitoring wells at site C (Fig. 1). Water samples from the waste lagoons were also taken from each site. A polyethylene bailer dedicated to each sample was sterilized with alcohol and rinsed with sterile water prior to use for sample collection. Between 1.5 and 3 well volumes of groundwater was removed from each well before collection as described previously (19). Samples were stored in clean, sterile plastic bottles and kept on ice in the field. Samples were refrigerated at 4°C in the laboratory until they were analyzed.
Bacterial identification and isolation. Tetracycline-resistant bacteria from lagoon and groundwater samples were grown aerobically on Enterococcosel agar (BBL, Cockeysville, Md.) and MR2A agar (9); 20 µg of tetracycline (Sigma, St. Louis, Mo.) per ml was added to each medium. Undiluted groundwater samples (100 µl) were directly plated onto agar media, while lagoon samples were plated by using 10-fold serial dilutions up to 1,000-fold. Media lacking tetracycline were inoculated similarly. Cultures grown on Enterococcosel agar were incubated at 37°C for 48 h. Cultures grown on MR2A agar were incubated at room temperature for up to 14 days. Tetracycline-resistant colonies were further purified by restreaking on the same media and were used for PCR screening as described previously (1). Gram staining was performed with a Protocol kit (Biochemical Sciences, Swedesboro, N.J.).
DNA extraction.
Groundwater samples (250 ml) were
centrifuged at 17,700 × g for 20 min at 4°C. The
supernatants were discarded, and the pellets were washed three times
with 0.1 volume of phosphate-buffered saline (120 mM
NaH2PO4 [pH 8.0], 0.85% NaCl) before
extraction of total DNA by the method of Tsai and Olsen
(36). Lagoon samples (50 ml) were centrifuged at
10,000 × g for 10 min at 4°C before DNA extraction
as described above. DNA (final concentration, 125 ng/µl) was stored
in TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]) at 
20°C.
PCR amplification. The primers and PCR protocol used to amplify tetracycline resistance genes in this study were described previously (1). Briefly, PCR was performed with 25 pmol of each primer and an ExTaq PCR kit (purchased from PanVera Corporation, Madison, Wis.) by using a final reaction mixture volume of 25 µl. Purified DNA (125 ng) or one-half of a 1- to 2-mm-diameter individual colony that was resuspended in sterile water was used as the PCR template. PCR amplification was performed with a GeneAmp PCR system 2400 thermocycler (Perkin-Elmer, Norwalk, Conn.). The temperature program consisted of denaturation at 94°C for 5 min, followed by 30 cycles consisting of 94°C for 30 s, annealing for 30 s, and extension at 72°C for 30 s and a final extension at 72°C for 7 min. The annealing temperatures used for amplification of different RPP genes were as follows: tet(O), 60°C; tet(Q), 63°C; tet(W), 64°C; tet(M), 55°C; tetB(P), 47°C; tet(S), 50°C; otrA, 66°C; and tet(T), 46°C. The PCR protocol was modified for DNA obtained from groundwater samples due to the presence of unidentified PCR-inhibiting substances. For these samples, a second, nested PCR was performed by using 1 µl of the first PCR mixture as the template and amplifying the template for 25 or 30 cycles as described above. Nested PCR was also performed with DNA from lagoon samples when necessary. The control reactions included PCR amplification with bacterial 16S rDNA primers 27F and 1525R (21) and sterile water or DNA of tetracycline-sensitive Escherichia coli EPEC as the negative control template for all primer sets and the positive control strains for each primer set as described previously (1). PCR product aliquots (5 µl) were analyzed by electrophoresis on a 2.5% (wt/vol) agarose gel (NuSieve; FMC Bioproducts, Rockland, Maine) and were stained with ethidium bromide. The sizes of the PCR products obtained from amplification were 167 bp for tet(O), tet(Q), tet(W), tet(M), tetB(P), tet(S), and tet(T) and 212 bp for otrA.
DGGE analysis. DNA from groundwater, lagoons, and bacterial isolates was PCR amplified as described above, except that in the nested PCR the forward primer included a GC clamp at the 5' end. Primers that targeted tetracycline resistance genes and the V3 variable region of 16S rDNA (25) were used. DGGE analyses of the amplified tetracycline resistance genes were performed as described previously (1). Briefly, electrophoretic separation of the PCR products was accomplished by using a polyacrylamide gel with a gradient containing urea and formamide (8% acrylamide, 30 to 60% urea-formamide, 0.5× TAE buffer [pH 7.4]). Electrophoresis was performed at 60°C for 2 h at 150 V and then for 1 h at 200 V, using a D-Code system (Bio-Rad, Hercules, Calif.). The gels were silver stained, and images were captured by using the Bio-Rad Diversity Database fingerprinting software on a G3 Macintosh computer equipped with a Bio-Rad GS-710 calibrated imaging densitometer. The gel standard markers consisted of known mixtures of PCR-amplified RPP tetracycline resistance genes or the V3 variable region of 16S rDNA.
Cloning and sequencing.
DNA bands were excised from DGGE
gels, crushed, equilibrated in 50 µl of sterile water, and then
subjected to three cycles of freezing and thawing (at 20°C and room
temperature). Eluted DNA (1 µl) was reamplified with the
corresponding primers and reelectrophoresed on a DGGE gel as described
above. PCR products were cloned by using a TOPO-TA Cloning kit
(Invitrogen, Carlsbad, Calif.) according to the manufacturer's
instructions. White colonies harboring the corresponding tet
fragments were identified by PCR by using the small amounts of colony
biomass and the corresponding primer sets. Representative bacterial
isolates from the groundwater and lagoons were identified by sequencing
of the cloned 16S rRNA genes, which were amplified with the bacterial
primer set (21). Sequence analysis of the cloned
tet and small-subunit rDNA fragments was performed by the
University of Illinois Keck Center for Functional and Comparative
Genomics. Sequences were analyzed on-line by using the BLAST (Basic
Local Alignment Search Tool) family of programs of GenBank
(22).
Nucleotide sequence accession number. The 16S rDNA sequence data reported in this paper have been deposited in the GenBank database under accession no. AY017049 to AY017063.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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PCR amplification of tet genes from lagoon and
groundwater samples.
All of the known ribosomal protection
tetracycline resistance determinants were detected in the lagoon
samples (Table 1). Most of them were
detected in total DNA from the lagoons after the initial 30 cycles of
PCR; the only exception was otrA, which was detected only
after a second, nested PCR. The most frequently detected determinants
in groundwater samples from both sites were Tet Q, Tet W, Tet M, Tet T,
and Tet O. Groundwater from well A8 contained Tet S, and well C6 water
contained detectable otrA. None of the groundwater samples
contained detectable tetB(P). Background well A7 at site A
contained tet(T), which was also detected in more than
one-half of the wells located downstream of the site lagoon in the
direction of groundwater flow. Site C background well C1 did not
contain any of the ribosomal protection tetracycline resistance
determinants. Two of the shallow wells at site A, wells A10 and A4,
also did not contain any detectable levels of these determinants. Well
C3, located close to the lagoon but perpendicular to the general
direction of groundwater flow away from the lagoon, did not contain any
determinant. Sample A14 from the well located farthest away from the
site A lagoon contained Tet Q, which was the most frequently detected
determinant.
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Phenotypic characterization and tetracycline resistance genotypes
of bacterial isolates.
Approximately 4 × 103 and
3 × 104 CFU per ml were obtained on Enterococcosel
agar containing 20 µg of tetracycline per ml after direct plating of
100 µl of slurry from the lagoons at sites A and C, respectively.
Several presumptive enterococci (gram-positive, catalase-negative
cocci) were randomly selected and isolated (Table 2). All of the enterococcal isolates
contained tet(M) as determined by PCR, and one isolate
(CLE3) also carried tet(S). Another randomly selected
isolate obtained from the Enterococcosel medium was a gram-positive rod
(CLE4) which also harbored the tet(M) gene. Few
tetracycline-resistant colonies were obtained after direct plating of
groundwater on Enterococcosel agar.
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DGGE analysis.
DGGE analysis of the tet(M)
determinant was used to assess conservation of the gene in both
environmental samples and individual isolates. One major band
corresponding to tet(M) was present in all of the
groundwater and lagoon samples (Fig. 2).
Multiple minor bands were present in several samples (e.g., well C7);
however, when these bands were extracted from the gel, reamplified, and reelectrophoresed on a DGGE gel, the migration was identical to the
migration of the major band and the positive control for
tet(M) (data not shown). DGGE analysis for the
tet(M) gene in isolates also demonstrated that there was one
principal band corresponding to the tet(M) control band. The
isolates were also analyzed by DGGE by using primers specific for the
V3 region of the 16S rDNA (Fig. 3). The
results indicated that isolates harboring the same tet(M)
gene were representatives of at least 10 different phylotypes at sites
A and C.
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Identification of tetracycline-resistant bacteria.
Several
tetracycline-resistant isolates were selected for further
identification by 16S rDNA sequencing. The strains that were selected
on Enterococcosel medium belonged to the low-G+C-content gram-positive
bacterial phylum, and the closest relatives were Enterococcus sp., Staphylococcus sp., and
Lactobacillus reuteri (Table
3). Growth on MR2A medium resulted in a
wider range of taxonomic affiliations, including members of the alpha
and gamma subdivisions of the class Proteobacteria and the
Actinobacteridae subclass of high-G+C-content gram-positive
bacteria (Table 3). The only tetracycline-resistant isolate obtained
from the background well at site A was identified as a member of the
genus Bosea, which includes bacteria mostly of soil origin.
With the exception of L. reuteri, all of the isolates had
levels of similarity with database entries between 91 and 96%, which
allowed only approximate taxonomic identification at the genus level
and higher taxa levels.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Tetracycline is commonly used in animal agriculture, particularly in the swine industry (16). Because of this use, selection for tetracycline resistance occurs in the normal swine gut microflora, and in our previous study we demonstrated that the reservoir of tetracycline resistance determinants in the swine intestinal microbiota is substantial and diverse (1). However, the details of what happens with this pool of antibiotic resistance genes further downstream remain largely unknown. Since animal production systems are not closed ecosystems, this pool may be released into the environment. In this study, we demonstrated that a broad range of tetracycline resistance genes occur in two swine waste lagoons and that upon release into the environment these genes can potentially mobilize and persist.
Detection of all of the RPP genes in the waste lagoons at both sites clearly indicated that selection for this drug resistance trait occurs and that a number of resistance genes can be maintained in the microbial populations present. A wide range of RPP genes were detected in groundwater downstream of the waste lagoons. Greater occurrence of RPP genes was detected in wells proximal to both lagoons in the direction of groundwater flow, and the detection of tet(Q) in well A14, located more than 250 m downstream of the lagoon at site A (Fig. 1), suggested that the mobility of resistance genes in the environment can be substantial. Two of the locations at site A had nested wells (Fig. 1, wells A4 and A6), and second wells were screened in deeper sand layers (wells A3 and A5). The occurrence of RPP tetracycline resistance genes was greater in the deep wells than in the corresponding shallow wells, demonstrating that contaminants may be vertically mobile depending on the hydrogeology of the location. Although this study was based on a single sampling event, the data suggest that the presence of the tetracycline resistance genes is due to seepage and movement of groundwater underlying the lagoons. Wells A10 and C3 did not contain any of the tetracycline resistance determinants, as expected due to the locations of the wells relative to the lagoons and the direction of groundwater flow. Thus, there were clear gradients of relative frequency and diversity of tetracycline resistance genes, with the maximal values occurring at waste lagoons and a gradual decline in the direction of groundwater flow; however, the genes were still detectable 250 m downstream. These observations may have important implications for understanding the circulation and acquisition of antibiotic resistance genes. Groundwater constitutes a substantial part of the public water supply in the United States (http://water.usgs.gov/wid/html/GW.html), and at both study sites it is used as the predominant source of untreated drinking water. Thus, along with other ways of acquiring antibiotic resistance, such as consumption of tainted food, the occurrence of antibiotic resistance genes in drinking water provides a possible way for antibiotic resistance to enter the animal and human food chain.
Several tet genes, particularly tet(M),
tet(O), tet(Q), and tet(W), were
dominant in the total communities in the lagoons and groundwater. These
genes were found to be predominant in the gastrointestinal tracts of
pigs and steers (1), and in this study the elevated
frequencies of these genes in the environment surrounding the farms
were consistent with the hypothesis that this occurrence was the result
of gene flow from the animals. Tetracycline-resistant bacteria obtained
from the lagoon and groundwater samples primarily harbored the
tet(M) gene, probably because the cultivation technique was
biased towards aerobic growth and the group of bacteria containing the
known tet(M) genes includes members of a broad range of
genera, including genera of gastrointestinal origin (Fig.
4). Furthermore, it is known that alleles
of tet(M) are present in a broad range of bacteria, and
their transfer is presumably mediated by conjugative transposons
Tn916, Tn1545, and Tn5251 and large
conjugative plasmids. It is notable, however, that the use of two
different media and random selection of tetracycline-resistant colonies
resulted primarily (75%) in bacteria harboring tet(M). No
groundwater isolates were correlated with a high occurrence of Tet T
and Tet O, the two determinants which occur in aerotolerant Streptococcus spp. (Fig. 4). None of the other RPP
tetracycline resistance genes were detected in any of the
tet(M)-harboring isolates except CLE3, which contained both
tet(M) and tet(S). In some tetracycline-resistant
isolates (A7-2, AL-1, AL-5, CL-2, A3-1, and C7-1) no genes conferring
ribosomal protection were detected, and these isolates most likely have
an alternative mechanism of tetracycline resistance, such as
energy-dependent efflux of tetracycline. Anaerobic genera such as
Prevotella, Bacteroides, and Butyrivibrio were
not targeted for cultivation in our study, and consequently, isolates
containing the two most prominent lagoon and groundwater markers
[tet(Q) and tet(W)] were not represented. Few
groundwater samples contained tet(S), otrA, and
tetB(P); however, these determinants were detected in the
lagoons, which suggested that they were present at significantly lower
levels than the other RPP tetracycline resistance genes.
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Enterococci were the dominant fecal bacteria detected in lagoons and in several groundwater samples. Tetracycline-resistant Enterococcus spp. were isolated along with an organism known to be of gastrointestinal origin, L. reuteri. The presence of identical tet(M) alleles in these bacteria suggests that lateral transfer of the gene occurred; however, it cannot be determined if this event occurred in the gut of an animal, in the lagoon environment, or in the groundwater. This gene and tet(O) were found to be circulating among the fecal streptococci in the pig gut (1). In this work, more significantly, the potential for transfer of the tet(M) allele involving groundwater and commensal soil bacteria was evident. Taxonomic analysis of isolates carrying the same tet(M) gene and belonging to the genera Afipia and Microbacterium confirmed that they probably originated from soil. Very little is known about the occurrence of tet(M) and other tetracycline resistance genes in phenotypically tetracycline-resistant bacteria in soil. Most of the tet(M) genotyping work has been done with clinical isolates, and interestingly, the genes are genetically quite diverse (92 to 96.8% identity) and display a mosaic structure consistent with homologous recombination between different lineages (3, 10, 15, 26). In our experiments, we observed extreme homogeneity of these genes in taxonomically (and ecologically) diverse bacteria, including intestinal lactobacilli, enterococci, and staphylococci, as well as soil species of Microbacterium and Afipia (Fig. 4). Consistent with this observation, we hypothesize that in our case we found a point source of genetic contamination originating on farms with the selective pressure of tetracycline. It is likely that transfer events occur in part because of the high density of bacteria in the lagoons, but it is not known to what extent such events occur in less populated groundwater environments. The observed horizontal spread of tet(M) genes to soil bacteria may have several notable implications. First, such spread suggests that antibiotic resistance gene dissemination is not restricted to the release of antibiotic-resistant gastrointestinal microbiota, which may have limited viability outside the gastrointestinal tract. The dissemination vector is not interrupted even if the original antibiotic resistance-harboring bacteria are not viable anymore. Second, when the resistance gene pool is mobilized into the indigenous bacteria in the environment, it has a much better chance of survival, persistence, and mobility, effectively increasing the gene frequency in local populations and having increased potential for reaching other ecosystems.
Another explanation for the elevated occurrence of tet(M) in soil bacteria may be the selective pressure of tetracycline, which may move through the soil layers to the groundwater and select for tetracycline resistance genes residing in soil microbiota. Few previous studies have addressed the fate of antibiotics in the environment and, if they are present, whether they exert any selective pressure for resistance. Tetracycline is not known to be readily degradable and is reportedly strongly absorbed in various soil types (28). This, along with the constant input of tetracycline via animal waste, can potentially provide a concentrated environment in which selection can occur. However, because of the strong sorption of tetracycline in soil (28), this antibiotic may have limited ability to reach distant groundwater layers and is concentrated primarily in soil layers close to lagoons. Despite the possibility of accumulation of tetracycline in soil environments, even low levels can substantially increase the transfer frequency of large conjugative transposons, such as Tn916 (32) and Tn1545 (5), Tn916-like elements (29, 35), or Bacteroides transposons (30) on which tetracycline resistance genes reside. Other factors conducive to resistance persistence and acquisition in the environment are not known, and groundwater is a unique environment for study. In particular, these factors could be naturally occurring compounds with structural similarity to tetracyclines, such as certain plant flavonoids.
In this study, we estimated the pool, diversity, and possible transmission of one group of tetracycline resistance genes in two animal production systems and their surrounding environments. Although previous studies have addressed the occurrence of specific antibiotic resistance characteristics in the environment, none of these studies included a more thorough genotypic characterization of a specific antibiotic resistance trait. While the set of genes which we targeted in our study includes only a limited portion of the bacterial resistance genes that may be present, it provides the basis for using a similar approach in order to combine molecularly and phenotypically based methods to characterize the occurrence, diversity, and routes of transmission of antibiotic genes in nature. The data obtained can be used to address the question of whether environments such as groundwater and soil are significant reservoirs for dissemination of resistance genes. In addition, long-term surveillance can provide an indication of the mobility, persistence, and transfer of resistance genes and begin to address the issue of agriculture as a source of resistance genes for entry into the environment.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801. Phone: (217) 333-8809. Fax: (217) 333-8804. E-mail: aminov{at}uiuc.edu.
Present address: United States Department of Agriculture
Agricultural Research Service, Urbana, IL 61801.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, April 2001, p. 1494-1502, Vol. 67, No. 4
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.4.1494-1502.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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