Sludge Watch ==> Antibiotic Resistance Genes as Emerging Contaminants: Studies in Colorado
Maureen Reilly
maureen.reilly at sympatico.ca
Sun Nov 5 15:04:41 EST 2006
Sludgewatch Admin:
Sludgewatch hates to nag...but the EPA should really be doing its homework
on this antibiotic resistance issue. Look...antibiotic resistance is
showing up in the treated wastewater. Remember the EPA and the FDA and the
local organic certifier are allowing treated wastewater to be sprayed on
that spinach and lettuce in the Salinas Valley...
And lets not forget the poor dairy cows fed on sludged crops... ingesting
antibiotic resistant bacteria, too.
If you want to read the tables at the end of the research study ... go to
the online copy.
.......................................................................................................
http://pubs.acs.org/cgi-bin/sample.cgi/esthag/asap/html/es060413l.html
Environ. Sci. Technol., ASAP Article 10.1021/es060413l S0013-936X(06)00413-5
Web Release Date: August 15, 2006
Copyright © 2006 American Chemical Society
Antibiotic Resistance Genes as Emerging Contaminants: Studies in Northern
Colorado
Amy Pruden,* Ruoting Pei, Heather Storteboom, and Kenneth H. Carlson
Department of Civil and Environmental Engineering, Colorado State
University, Fort Collins, Colorado 80523
Received for review February 20, 2006
Revised manuscript received July 10, 2006
Accepted July 17, 2006
Abstract:
This study explores antibiotic resistance genes (ARGs) as emerging
environmental contaminants. The purpose of this study was to investigate the
occurrence of ARGs in various environmental compartments in northern
Colorado, including Cache La Poudre (Poudre) River sediments, irrigation
ditches, dairy lagoons, and the effluents of wastewater recycling and
drinking water treatment plants. Additionally, ARG concentrations in the
Poudre River sediments were analyzed at three time points at five sites with
varying levels of urban/agricultural impact and compared with two previously
published time points. It was expected that ARG concentrations would be
significantly higher in environments directly impacted by urban/agricultural
activity than in pristine and lesser-impacted environments. Polymerase chain
reaction (PCR) detection assays were applied to detect the presence/absence
of several tetracycline and sulfonamide ARGs. Quantitative real-time PCR was
used to further quantify two tetracycline ARGs (tet(W) and tet(O)) and two
sulfonamide ARGs (sul(I) and sul(II)). The following trend was observed with
respect to ARG concentrations (normalized to eubacterial 16S rRNA genes):
dairy lagoon water > irrigation ditch water > urban/agriculturally impacted
river sediments (p < 0.0001), except for sul(II), which was absent in ditch
water. It was noted that tet(W) and tet(O) were also present in treated
drinking water and recycled wastewater, suggesting that these are potential
pathways for the spread of ARGs to and from humans. On the basis of this
study, there is a need for environmental scientists and engineers to help
address the issue of the spread of ARGs in the environment.
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Introduction
The spread of antibiotic-resistant pathogens is a growing problem in the U.
S. and around the world. Recently a 2000 World Health Organization (WHO)
report (1) focused on antibiotic resistance as one of the most critical
human health challenges of the next century and heralded the need for "a
global strategy to contain resistance". According to the report, more than
two million Americans are infected each year with resistant pathogens and 14
000 die as a result. The rapid growth of the problem emphasizes the need for
intervention. For example, vancomycin is currently considered to be the most
powerful antibiotic of "last resort", yet within 10 years the incidence of
vancomycin-resistant enterococci (VRE) increased in the United States from
0% to 25% (2, 3). Resistance to penicillin, the antibiotic that originally
revolutionized human health 50 years ago, is now as high as 79% in
Staphylococcus pneumoniae isolates in South Africa (4, 5). Alarmingly,
diseases that were once considered to be eradicated, such as tuberculosis,
are now beginning to make a comeback because of antimicrobial resistance (1,
6, 7). As with other dangerous pollutants that spread in the environ ment
and threaten human health, there is a need for environmental scientists and
engineers to help address the critical problem of microbial resistance to
antibiotics.
The rise of antibiotic resistance is considered to be closely linked with
the widespread use of antibiotic pharmaceuticals in humans and animals. In
particular, more than one-half of the antibiotics used in the U. S. are
administered to livestock for purposes of growth promotion or infection
treatment (8, 9). In both animals and humans, up to 95% of antibiotics can
be excreted in an unaltered state (10, 11). Some removal has been observed
in wastewater treatment plants (WWTPs); however, as is true with the larger
problem of pharmaceutical compounds, WWTPs are not designed for the removal
of micropollutants (12-14). Residual antibiotics thus are released into the
environment where they may exert selection pressure on microorganisms. While
overprescribing or other improper use/disposal of antibiotics in humans is
generally considered to contribute to the problem, several studies have also
linked agricultural antibiotic use with antibiotic-resistant infections in
humans (15-23). For example, avoparcin, an antibiotic growth-promoter used
in poultry, was recently banned in Europe because of its association with
the development of vancomycin-resistant enterococci (24).
Because of the direct selection pressure that antibiotics exert on organisms
carrying antibiotic resistance genes (ARGs), the transport pathways of
antibiotic-resistant microorganisms and the ARGs that they carry are
expected to be similar to the pathways of antibiotic pharmaceuticals. In
fact, it is likely that ARGs persist further in the pathway, considering
that in many cases they are maintained in the microbial populations even
after the antibiotic selection pressure has been removed (25-28). Also,
horizontal gene transfer (HGT) is a major mechanism for sharing ARGs between
microbes and has been documented to occur between nonpathogens, pathogens,
and even distantly related organisms, such as Gram-positive and
Gram-negative bacteria (25, 29-31). In many cases, ARGs have been discovered
to occur as part of multiple antibiotic resistant (MAR) superintegrons,
which may contain over 100 ARG cassettes (32). These MAR superintegrons
cause multiple-drug resistance in organisms, meaning that even when very
different antibiotics are used, one antibiotic may coselect for resistance
to other antibiotics (5, 33). MAR gene cassettes and ARGs are notorious for
being associated with plasmids and/or transposons that facilitate HGT.
Finally, even if cells carrying ARGs have been killed, DNA released to the
environment has been observed to persist, to be protected from DNAse,
especially by certain soil/clay compositions, and to be eventually
transformed into other cells (34-36). For all of these reasons, ARGs in and
of themselves can be considered to be emerging "contaminants" for which
mitigation strategies are needed to prevent their widespread dissemination.
The purpose of this study was to document the occurrence of tetracycline and
sulfonamide ARGs in various environmental compartments in northern Colorado.
These two ARG groups were chosen because sulfonamide and tetracycline
antibiotics have been previously characterized in Poudre River sediments and
shown to relate to urban/agricultural activity (37). The breadth of the
study included Cache La Poudre (Poudre) River sediments, dairy lagoon water,
ir rigation ditch water, a wastewater recycling plant (WRP), and two
drinking water treatment plants (DWTPs). The hypothesis was that
environmental compartments most directly impacted by urban/agricultural
activity would have significantly higher concentrations of ARGs than less
impacted and pristine environments. Irrigation ditch waters, which were
directly adjacent to farms, were investigated as a potential pathway of ARGs
from farms to the Poudre River, while the WRP and the DWTPs were explored as
potential routes of human environmental input and consumption. The
presence/absence of several ribosomal protection factor tetracycline ARGs
and folic acid pathway sulfonamide ARGs was determined using a polymerase
chain reaction (PCR) detection assay, and four commonly occurring ARGs were
further quantified by quantitative real-time PCR (Q-PCR). Documenting the
baseline occurrence of ARGs in a cross-section of environmental compartments
will take a step toward understanding and modeling the fate and transport
phenomena associated with these emerging contaminants.
Experimental Section
Poudre River Sediment Sampling. Because of its pristine origins and zonation
corresponding to land use, the Poudre River has served as a good model for
relating human and agricultural activities with the occurrence of antibiotic
pharmaceuticals (37) and ARGs (38). Five sampling sites were the focus of
this study, numbered sequentially in the direction of flow from west to
east, with the following characteristics: site 1, pristine location at the
river origin in the Rocky Mountains; site 2, light-agriculture-influenced
area; site 3, urban-influenced area at the outlet of the Fort Collins Drake
WWTP; site 4, heavy-agriculture-influenced area between Fort Collins and
Greeley; and site 5, heavy-agriculture- and urban-influenced area just east
of Greeley, which is a major center for the meat-packing industry. Over 90
confined animal feeding operations (CAFOs), dairies, and ranches are located
between sites 3 and 5. Further attributes of the Poudre River watershed that
contribute to its suitability for investigating the impacts of urban and
agricultural activity on antibiotics and ARGs have been described previously
(37, 38).
Sediment samples were collected along the Poudre River at the five sites on
August 18, 2005, October 27, 2005, and February 17, 2006. The flow rates on
these three dates were 1.04, 14.19, and 0.14 m3 s-1, respectively (U. S.
Geological Survey station number 06752260, Fort Collins, CO). Sampling at
three points in time provided insight into potential temporal variations in
ARG concentrations, and the February 17th date is exactly 1 year later than
a previously published sampling date (38). The upper sediments (about 5 cm)
from the middle and two sides of a cross-section at each site were sampled
and composited. Samples were collected using a shovel and mixed well in
sterilized centrifuge tubes. Fifty-five grams of mixed sample at each site
were stored at -80 C for subsequent molecular analysis.
Bulk Water Sampling. Irrigation ditch waters were investigated as a
potential pathway of ARGs from farms to the Poudre River. Grab samples of
bulk water were collected in sterile containers from irrigation ditches on
August 18, 2005, corresponding to the August sampling date of the Poudre
River sediments. All irrigation ditches were located between site 4 and site
5 on the Poudre River within a 3.5 km × 2 km zone north of the river, and a
total of ten locations were sampled. To investigate a potential source of
ARGs within this zone, a microaerophillic dairy lagoon (~1 mg/L dissolved
oxygen in the upper 1 m) and an anaerobic dairy lagoon (0 mg/L dissolved
oxygen) from an anonymous farm located 8 km from site 5 were sampled on
October 20, 2005. Finally, source water, and pre-chlorinated, and
post-chlorinated bulk water were collected from two anonymous DWTPs and an
anonymous WRP in northern Colorado in February, 2005. The DWTP was studied
as a potential direct route of ARGs to consumers, and the WRP was considered
a potential human input into the environment. To collect fine particulates
from the dilute ditch water, DWTP, and WRP samples for subsequent analysis,
500 mL of well-mixed sample was filtered using a 0.45 m glass fiber filter
(Whatman). This concentration step was not required for dairy lagoon
samples.
DNA Extraction. DNA was extracted from 0.5 g of composited sediment using
the FastDNA Spin Kit for Soil (MP Biomedicals) and from 1.8 mL of dairy
lagoon water using the Ultraclean Microbial DNA Kit (MoBio Laboratories,
Inc.) according to manufacturer protocol. Both approaches employ a
bead-beating procedure. For fine particulates collected on filters from bulk
water, the filters were cut into small pieces and added directly to the
extraction tubes. Extraction yield and the quality of the DNA were verified
by agarose gel electrophoresis and spectrophotometry.
Detection and Quantification of ARGs. Polymerase chain reaction detection
assays were used for broad-scale screening of the presence/absence of five
ribosomal protection factor tetracycline ARGs (tet(BP), tet(O), tet(S),
tet(T), and tet(W)) (39) and four folic acid pathway sulfonamide ARGs
(sul(I), sul(II), sul(III), and sul(A)). Development and validation of sul
primers was described in Pei et al. (38). Positive controls consisted of
cloned and sequenced PCR amplicons obtained from Poudre River sediments.
Both positive and negative controls were included in every run, and negative
signals were confirmed by spiking positive control template into the sample
to verify a signal. Forty cycles were used to improve chances of product
formation from low initial template concentrations. Further details on
reaction mixes and temperature programs are available in Pei et al. (38);
note that annealing temperatures for tet primers vary from Aminov et al.
(39). Two tetracycline ARGs (tet(W) and tet(O)) and two sulfonamide ARGs
(sul(I) and sul(II)) that were commonly occurring according to the PCR
presence/absence assays were further quantified by Q-PCR using a SybrGreen
approach. For further details on Q-PCR methods, see Pei et al. (38).
Eubacterial 16S rRNA genes were quantified according to the TaqMan Q-PCR
method described by Suzuki et al. (40) so that ARGs could be normalized to
the total bacterial community. This provided a means to correct for
potential variations in extraction efficiencies. By quantification of 16S
rRNA genes, it was also possible to compare ARGs proportionally between
samples of different overall population sizes. Matrix effects associated
with extraction of DNA from environmental samples were corrected for by
performing spiked matrix control tests and determining template sup pression
factors as described in Pei et al. (38). All Q-PCR analyses were performed
using a Cepheid SmartCycler (Sunnyvale, CA).
Statistics. The influences of the environment (sites, ditch water, and dairy
lagoons) on the normalized and non-normalized copies of ARGs were analyzed
using the Mixed Procedure, which fits a variety of mixed linear models to
data. This provides the flexibility of simultaneously modeling means,
variances, and covariances (41-44). Through the use of this test, it was
thus possible to comprehensively compare overall differences between
different environmental compartments with respect to ARG concentrations. For
comparison of the five Poudre River sites, multiple sampling time points
were treated as replicates. Mixed Procedures were conducted using SAS 9.0
(SAS Institute Inc., Cary, NC). A p-value <0.05 was considered to indicate
significance. Averages and standard deviations of all data were determined
using Microsoft Excel, 2003.
Results and Discussion
Occurrence of ARGs in Northern Colorado. Figure 1 summarizes the Q-PCR data
obtained for the four ARGs at the five Poudre River sites, while Figure 2
summarizes the same analyses for the ditch waters and dairy lagoon water.
When August 2005 data for the Poudre River sediments are compared with the
dairy lagoon and ditch water, the following trend is observed with respect
to ARG concentrations: dairy lagoon water > ditch water > river sediments (p
< 0.0001), for all ARGs except sul(II), which was absent from the ditch
waters. This is based on pooling of all 10 ditch water sites, the two dairy
lagoons, and sites 4 and 5, which were directly adjacent to the ditch water
sampling locations. Within each of these three pools, there was no
statistical difference observed among the samples. Therefore, it was
observed as expected that environmental compartments most directly impacted
by human/agricultural activity showed higher concentrations of ARGs. This
trend is even stronger in considering absolute quantities of ARGs (not
normalized to 16S rRNA genes), because the concentration of cells in the
dairy lagoon water was orders of magnitude higher than that of the ditch
water or the sediments.
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Figure 1 Distribution of four ARGs (sul(I), sul(II), tet(O), and tet(W)) in
Poudre River sediments on three sampling dates, compared to two previously
published sampling dates (April 13, 2004, and February 17, 2005 (38)), as
determined by Q-PCR: site 1, pristine site; site 2, light agricultural
activity; site 3, heavy urban activity; site 4, heavy agricultural activity;
site 5, heavy urban and agricultural activity. Error bars represent the
standard deviation of six measure ments from three independent Q-PCR runs
analyzing DNA extract from composite samples.
Figure 2 Distribution of four ARGs (sul(I), sul(II), tet(O), and tet(W)) at
10 sampling points of irrigation ditch water (DW-1-DW-10) located between
site 4 and site 5 compared with that of a microaerophillic dairy lagoon
(LW-AE) and an anaerobic dairy lagoon (LW-AN). DW samples were concentrated
from 500 mL, and LW samples were extracted directly from 1.8 mL. All samples
were normalized to the total 16S rRNA genes. Error bars represent three
independent Q-PCR runs in duplicate. The labels a and b indicate that the
data sets fell into two statistically different groups, according to the
Mixed Procedure.
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In developing a hypothetical pathway for ARGs, a trend is not as clear. The
overall trend in terms of ARG concentra tions of dairy lagoon water > ditch
water > river sediments suggests that on-farm compartments, such as lagoons
may be the source of ARGs, which are subsequently attenuated in ditch water
before reaching Poudre River sediments. However, this trend is not supported
in terms of sul(II), which is entirely absent from the ditch water and
therefore cannot be the source of what is observed in the Poudre River
sediments. An alternative source of the sul(II) that appears at sites 4 and
5 could instead be human inputs. This is supported by the data presented in
Figure 1, in which it is observed that sul(II) is consistently present at
high levels on average at site 3, which is at the point of discharge of the
Drake WWTP, while consistently lower (comparing each date sampled) at site 4
(entirely absent for the October event) and equivalent or lower at site 5,
which has mixed human/agricultural inputs. Because sul(II) is present in the
dairy lagoon waters, it must also have agricultural sources, but it may
attenuate too quickly to be transported to the ditches and subsequently to
the river sediments. On the basis of this study and a previous study (38),
it is appears that of the four ARGs quantified sul(II) is the most sensitive
indicator of human/agricultural impact, and thus it is suggested that it
attenuates quickly in the absence of direct inputs. The other ARGs in the
Poudre River sediments at sites 4 and 5 may be of either/both human and
agricultural origin, since they followed a decreasing trend from the dairy
lagoon through the ditch water but were also present at site 3.
In addition to having higher concentrations of three out of four of the
ARGs, the dairy lagoon water was also observed to have more different kinds
of ARGs present than the irrigation ditch water according to the PCR assay
(Table 1). Together with the Q-PCR results, these data further support the
concept that there is some attenuation of ARGs between any linkages that may
connect dairy lagoon water and irrigation ditch water. Future work should
implement ARG fingerprinting/source tracking to fully characterize the
potential pathways.
Temporal Variations of ARG in Poudre River Sediments. As observed in a
previous study that compared a high-flow sampling point (6.8 m3 s-1, April
2004) with a low-flow sampling point (0.6 m3 s-1, February 2005), the ARG
concentrations in the Poudre River sediments are variable with time (38). To
better understand temporal variations in ARG concentrations, the Poudre
River sediments were sampled at three additional time points and compared
with the two previously published time points. The February sampling point
in this study took place exactly 1 year after the previous February event.
In support of the relationship between ARG concentration and relative
environment impact observed above, the pristine site (site 1) consistently
had the lowest average concentrations of ARGs with time, with sul(II)
completely absent and no individual ARG consistently present at all five
sampling times (Figure 1). When presence/absence of ARGs are compared, site
2 appears to be the next lowest in terms of overall impacts. For example,
sul(II) is consistently absent at site 2, and tet(O) was absent in one of
the five sampling events, whereas these genes were consistently present at
sites 3, 4, and 5. In terms of ARG concentrations, tet(W) and tet(O) at site
2 were equal or less than site 3; however, these two genes were sometimes
higher and sometimes lower than at sites 4 and 5. On the basis of ARG
averages and presence/absence of ARGs, sites 1 and 2 were the least
impacted, as expected.
When the Mixed Procedure was applied to the data, in which the time points
were pooled as replicates, it was found that there was no statistical
difference between the five sites for the 16S normalized data, except in the
case of sul(II) (p = 0.0117). However, when the same test was performed with
non-normalized data, it was found that sites 1 and 2 were statistically
lower than sites 3, 4, and 5 in terms of sul(I) (p = 0.00296), sul(II) (p =
0.0199), and tet(O) (p = 0.0102). Though normalizing to 16S genes provides a
comparison of ARGs as a proportion of the total population, arguably it may
be the absolute quantities of ARGs that are more critical.
While spatial variations in ARGs could be fairly well-characterized, it is
difficult to identify clear temporal patterns. Comparison of the two
February sampling dates that were exactly a year apart provides some
insight. All four genes were either the same on average for both events
(tet(O) for sites 1 and 4 and sul(II) for sites 4 and 5) or higher in the
2006 event (all other genes, except sul(II) at sites 1 and 2, where it was
not present) (Figure 1). This suggests the possibility that all ARGs are
increasing in concentration with time. However, the trends in between these
two dates do not support this. Only tet(W) and tet(O) at site 3 increase
consistently with time. All remaining ARGs at the five sites either decrease
before increasing (e.g., tet(W) at site 2 and sul(II) at site 3), are
constant and then increase (e.g., tet(O) at site 2 and tet(W) at site 1), or
increase and then decrease (e.g., tet(W) at sites 4 and 5) (Figure 1).
Therefore, no clear trend was identified with time.
It was also attempted to analyze trends in the data with respect to river
flow rate. This was of interest because flow rate directly relates to runoff
and nonpoint source inputs, which were hypothesized in the previous study to
play a role in the observed increase in the number of kinds of ARGs detected
in Poudre River sediments (38). The October 2005 sampling date provided a
second sampling date at high flow (14.9 m3 s-1), compared to the previously
published April 2004 high-flow sampling date (6.8 m3 s-1). (All other dates
were at or below 1.0 m3 s-1.) Interestingly, all four ARGs increased on
average at site 5 in comparing the high-flow October event with the
immediately previous low-flow event in August (Figure 1). At site 4, tet(W)
and tet(O) increased, but sul(II) stayed the same, and sul(I) decreased.
There was no effect at all at site 3, which is affected primarily by point
discharge rather than runoff, site 2, or site 1. However, attempts to plot
ARG concentrations versus flow rate did not reveal any clear trend. Thus, it
is still not possible to make a conclusive judgment on the effect of flow
rate on ARG concentrations, though the role of nonpoint source inputs merits
further investigation. To accomplish this, it would be necessary to gather
more data with time/flow or monitor a much more controlled and smaller-scale
system.
Wastewater Recycling Plant and Drinking Water Treat ment Plants. A PCR
presence/absence assay was conducted on the influent, intermediate effluent,
and final effluent of two drinking water treatment plants (DWTP "a" and DWTP
"b") and the pre-chlorinated and chlorinated effluent of a WRP. It was
observed that both tet(W) and tet(O) were present at detectable levels in
all samples except the source water for DWTP "a" (Figure 3). This indicates
that the same two genes that were common in various environmental
compartments in northern Colorado are also present in treated recycled
wastewater and bulk drinking water. These two genes also showed a response
to the level of impact; e.g., they were highest in dairy lagoon water and
ditch water and lowest on average at the pristine site. On the basis of the
intensity of the signal, they were also higher in the recycled wastewater
than in the drinking water, as would be expected. Though these two ARGs are
not directly associated with any known human pathogens, they may be
indicators of linksbetween human/agricultural activity and ARGs in drinking
water. Considering that drinking water is a direct route to human consumers,
this emphasizes the need to better understand the pathways by which ARGs are
spread in the environment and potential ways that the spread of ARGs may be
reduced. For example, vancomycin resistance genes were found in drinking
water biofilms in a recent study (45). Considering that vancomycin is
typically the antibiotic of last resort when all else fails, this
underscores the need to address this issue before it is too late. One
possibility may be to make simple modifications to wastewater and drinking
water treatment plants to reduce the spread of ARGs.
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Figure 3 Agarose gel analysis of PCR presence/absence (in duplicate) of two
ARG families, tet(W) and tet(O): + = positive control; - = negative control.
The presence of a band at the same molecular weight as + indicates the
presence of an ARG: 1 = WRP effluent; 2 = WRP chlorinated effluent; 3 = DWTP
a influent; 4 = DWTP a treated water pre-chlorination; 5 = DWTP a treated
water post-chlorination; 6 = DWTP b influent water; 7 = DWTP b treated water
pre-chlorination; 8 = DWTP b treated water post-chlorination. The band
appearing below 200 bp is consistent with a primer dimer.
--------------------------------------------------------------------------------
ARGs as Emerging Contaminants. On the basis of this study it is clear that
ARGs are present in various environmental compartments, including river
sediments, irrigation ditch water, dairy lagoon water, DWTPs, and a WRP.
Furthermore, quantitative techniques incorporating Q-PCR provide a means to
compare the concentrations of ARGs associated with the known urban and
agricultural impacts, which provides a more direct measure than previous
culture-based methods. On the basis of this occurrence survey, it is argued
that ARGs are emerging contaminants that need to be further studied in the
paradigm of environmental science and engineering. The concept of ARGs as
"pollutants" has also been suggested by Rysz and Alvarez (46).
It should be noted that besides the tetracycline and sulfonamide ARGs that
were the focus of this study, there are numerous other ARGs that have been
described in the literature and likely even more that have not yet been
discovered, each potentially with its own unique properties. Thus, each ARG
may have different behaviors with respect to fate and transport and response
to physical, chemical, and/or biological treatment. In terms of defining
fate and transport characteristics of ARGs in general, it is expected that
their behavior will be distinct in comparison to "typical" contaminants. For
example, ARGs may be sequestered with bacteria, which are themselves
transported, or they may be present as naked DNA bound to clay particles
(47). Furthermore, ARGs may actually amplify in the environment under some
conditions. This is indeed a unique contaminant property. Considering the
significance of the problem of the spread of antibiotic resistance, further
effort by environmental researchers to better understand these emerging
contaminants is well-warranted. This is especially true as the rate of
discovery and development of new antibiotics is continually declining (48),
while the corresponding develop ment and spread of resistance is occurring
at a rapid pace. On the basis of this study, understanding ARGs as emerging
contaminants can add a new and important angle to helping to approach this
important problem.
Acknowledgment
Funding for this study was provided by U. S. Department of Agriculture
(USDA) NRI Watersheds program, the USDA Agricultural Experiment Station at
Colorado State University, and the National Science Foundation CAREER
program. The authors thank Jessica Davis, Kathy Doesken, and Sung-Chul Kim
for coordinating collection of ditch and dairy lagoon water samples as well
as the anonymous providers of the drinking water and recycling plant
samples.
This article is part of the Emerging Contaminants Special Issue.
* Corresponding author phone: (970)491-8814; fax: (970)491-8671; e-mail:
apruden at engr.colostate.edu.
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Table 1. PCR Presence/Absence Assay of Various ARGs in Ditch (DW)a and Dairy
Lagoon (LW) Waterb
ARG
DW-1
DW-2
DW-3
DW-4
DW-5
DW-6
DW-7
DW-8
DW-9
DW-10
LW-AE
LW-AN
+ control
tet(BP)
-
-
-
-
-
-
-
-
-
-
-
-
+
tet(O)
+
+
+
+
+
+
-
-
+
+
+
+
+
tet(S)
-
-
-
-
-
-
-
-
-
-
-
-
+
tet(T)
-
-
-
-
-
-
-
-
-
-
+
+
+
tet(W)
+
+
+
+
+
+
+
+
+
+
+
+
+
sul(I)
+
+
+
+
+
+
+
+
+
+
+
+
+
sul(II)
-
-
-
-
-
-
-
-
-
-
+
+
+
sul(III)
-
-
+
+
+
-
-
-
-
-
+
+
+
sul(A)
-
-
-
-
-
-
-
-
-
-
-
-
+
a Collected August 18, 2005.b Collected October 20, 2005.
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