Chapter21: Hog Heaven
Section: Chapter Questions
Problem 1.2CS
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I need help finding this in the arictle
Why would you look for ARGs in a farming context?
Transcribed Image Text:in Chinese swine farms
Yong-Guan Zhua,b,1,2, Timothy A. Johnsond,1, Jian-Qiang Suª, Min Qiao, Guang-Xia Guob, Robert D. Stedtfelde,
Syed A. Hashshame, and James M. Tiedjed,2
*Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China; Research Center for Eco-
Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China; and "Center for Microbial Ecology, Departments of "Plant, Soil and Microbial
Sciences, and "Civil and Environmental Engineering, Michigan State University, East Lansing, MI 48824
Contributed by James M. Tiedje, December 31, 2012 (sent for review October 31, 2012)
Antibiotic resistance genes (ARGS) are emerging contaminants
posing a potential worldwide human health risk. Intensive animal
husbandry is believed to be a major contributor to the increased
environmental burden of ARGs. Despite the volume of antibiotics
used in China, little information is available regarding the cor-
responding ARGs associated with animal farms. We assessed type
and concentrations of ARGS at three stages of manure processing to
land disposal at three large-scale (10,000 animals per year) commer-
cial swine farms in China. In-feed or therapeutic antibiotics used on
these farms include all major classes of antibiotics except vancomy-
cins. High-capacity quantitative PCR arrays detected 149 unique
resistance genes among all of the farm samples, the top 63 ARGS
being enriched 192-fold (median) up to 28,000-fold (maximum)
compared with their respective antibiotic-free manure or soil con-
trols. Antibiotics and heavy metals used as feed supplements were
elevated in the manures, suggesting the potential for coselection
of resistance traits. The potential for horizontal transfer
because of transposon-specific ARGS is implicated by the enrichment
of transposases-the top six alleles being enriched 189-fold (median)
up to 90,000-fold in manure as well as the high correlation (r2 =
0.96) between ARG and transposase abundance. In addition, abun-
dance of ARGS correlated directly with antibiotic and metal concen-
trations, indicating their importance in selection of resistance genes.
Diverse, abundant, and potentially mobile ARGs in farm samples
suggest that unmonitored use of antibiotics and metals is causing
the emergence and release of ARGs to the environment.
ARGS
concentrated animal feeding operations | horizontal gene transfer |
growth-promoting antibiotics | tetracycline
The
"he spread and aggregation of antibiotic-resistant genes into
multidrug-resistant pathogens is challenging life-saving anti-
biotic therapies (1, 2). Indeed, the expansion of the antibiotic
resistance gene reservoir in the environment has been caused by
antibiotic use in humans and animals (3). Furthermore, a grow-
ing body of direct and indirect evidence from the past 35 y (4)
establishes that farm antibiotic use correlates repeatedly with the
rise and spread of associated resistance genes in human patho-
gens, as well as the direct transfer of antibiotic-resistant bac-
teria from animals to humans (5-8). Antibiotic use has increased
the frequency of horizontal gene transfer and resistance gene
fixation in genomes, leading to the development of diverse re-
sistance genes in genomic islands (9). Acinetobacter baumannii
is a case in point. In 30 y, it evolved from being completely an-
tibiotic-susceptible to being multidrug-resistant by expanding a
genomic island by 66 kb, including 45 resistance genes, which
were horizontally transferred from various genera of bacteria,
some of which likely originated from the environment (10).
Antibiotic-resistant strains can then be distributed worldwide,
aided by a number of human factors, but especially international
travel for commerce, immigration, and recreation (11). Antibi-
otic resistance genes (ARGS) are becoming recognized as envi-
ronmental pollutants, and action is being sought to preserve
the efficacy of antibiotics. The World Organization for Animal
www.pnas.org/cgi/doi/10.1073/pnas.1222743110
Health, together with the US Food and Drug Administration
and the World Health Organization, urge improved regulation of
veterinary antibiotic use in over 100 developing countries (12).
China is the largest antibiotics producer and consumer in the
world. In a 2007 survey, the estimated annual antibiotics pro-
duction in China was 210 million kg, and 46.1% were used in
livestock industries (13), at least four times the amount used in
the US livestock industry in 1999 (14). In China, the use of
antibiotics both for animal disease treatment and growth pro-
motion is unmonitored, which often leads to high use, reflected
by the high concentrations of antibiotic residues (hundreds of
milligrams of tetracycline per kilogram) that are commonly
detected in animal manures (15, 16). Manure is a major source
of antibiotic pollution in the environment, and China produces
an estimated 618 billion kg of swine manure annually (17). Most
veterinary antibiotics are poorly absorbed by the animal and
hence are excreted (18) and dispersed to soil when the manure is
spread as fertilizer, the desired practice for recycling nutrients.
Furthermore, the use of subtherapeutic levels of antibiotics in
animal feeds causes an increase in antibiotic resistance traits in
manure (19, 20), manure-amended soils (21), and downstream
river waters and sediments (22). In addition, metals are added to
swine feed for growth promotion and disease control and may
provide a long-term coselective pressure for antibiotic resistance
(23). The scale of the livestock industry in China and the volume
of antibiotics use provide an opportunity to assess the impact of
large-scale animal farm practices on antibiotic resistance genes
in the environment. Previously, tetracycline resistance (tet) genes
in soils adjacent to representative Chinese swine feedlots were
positively correlated to concentrations of tetracycline residues
(24), raising the question of whether the diversity and abundance
of the antibiotic resistance reservoir extends beyond tetracycline
resistance genes due to the use of additional antibiotics, possible
coselection for other resistance genes, and/or recruitment of
multidrug efflux pump genes.
Although antibiotic-resistant bacteria have been isolated and
characterized from farm soils (21, 25), this method only samples
microbes that are culturable and express their ARGs under those
conditions. ARGs of noncultured populations, as well as "silent" or
unexpressed ARGS (26), are sources of risk because they contrib-
ute to the resistance reservoir and could be horizontally transferred
or expressed under other conditions. We used high-capacity
Author contributions: Y.-G.Z. and J.M.T. designed research; T.A.J., J.-Q.S., M.Q., G.-X.G.,
and R.D.S. performed research; T.A.J., J.-Q.S., R.D.S., and S.A.H. contributed new reagents/
analytic tools; T.A.J., J.-Q.S., and M.Q. analyzed data; and Y.-G.Z., T.A.J., J.-Q.S., and J.M.T.
wrote the paper.
The authors declare no conflict
interest.
Freely available online through the PNAS open access option.
¹Y.-G.Z. and T.A.J. contributed equally to this work.
²To whom correspondence may be addressed. E-mail: tiedjej@msu.edu or ygzhu@rcees.ac.
cn.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1222743110/-/DCSupplemental.
PNAS | February 26, 2013 | vol. 110 | no. 9 | 3435-3440
ENVIRONMENTAL
SCIENCES
![quantitative PCR (qPCR) (19) with 313 validated primer sets,
which target 244 ARGS (Table S1) from all major classes of ARGs,
to extensively sample the antibiotic resistance reservoir. We sam-
pled three large-scale commercial swine farms, each from a differ-
ent region of China, at three stages of manure management:
manure, manure compost, and soil receiving manure compost.
Manure from pigs never fed antibiotics and soil from a pristine
forest in Putian, China were used as experimental controls.
Results
Antibiotics and Metal Concentrations. Antibiotics and their use as
reported by the farmers are listed in Table S2. Total tetracycline
concentrations in these manure and soil samples were as high as
15.2 mg kg and 0.78 mg kg, respectively, as was determined
previously (15). Of the sulfonamides analyzed in this study, sul-
famethoxazole had the highest concentrations for all samples,
ranging from 1.08 to
was
detected in all samples in a range of 0.50-4.81 µg-kg-¹. Of the
fluoroquinolones analyzed in this study, the highest mean
only ofloxacin and
enrofloxacin
amono
concentration of
were
most samples.
(335
tions
of ofloxacin
μg-kg -¹) and enrofloxacin (96.0
ug-kg) were
were observed in Putian compost and soil samples, re-
spectively (Fig. S1). Zinc, copper, and arsenic, used as feed
additives, were also elevated above background concent
concentrations.
The highest mean concentrations of copper, zinc, and arsenic
were detected in Putian manure, Jiaxing compost, and Beijing
manure, respectively, with copper up to 1,700 mg kg-¹
manure
(Fig. S2). The concentration of copper, zinc, and arsenic were
much higher in manure than in compost and soil samples, with
the exception of the Jiaxing compost, in which copper and zinc
had the highest concentrations of all of the samples.
Diversity of Antibiotic Resistance Genes. We detected 149 unique
ARGS among all of the samples, which is three times more types
of ARGS than were found in the control samples (Fig. 14). The
ARGs detected in these farms encompass the three major re-
sistance mechanisms efflux pumps, antibiotic deactivation, and
cellular protection (Fig. 1B) and 1 potentially confer resistance to
most major classes of antibiotics (Fig. 1C). Resistance gene pro-
files indicate the patterns and degrees of enrichment of ARGs for
each site (Fig. 2) and that manure samples cluster separately from
the other samples with the exception of the Putian compost. The
compost and soil samples also cluster separately with the excep-
tion of one of the Beijing compost replicates, which grouped with
the soil samples. Furthermore, Shannon diversity (indicating
richness and abundance) of ARGs from farm samples was sig-
nificantly higher than that of the control samples (Fig. S3).
Abundance of Antibiotic Resistance Genes. ARGs were highly
enriched in the farm samples. We used the sum of the enrichment
of all unique ARGs in a sample to approximate total enrichment
in the farms. Maximum enrichment occurred in the manure
samples at Beijing (121,000-fold) and Jiaxing (39,000-fold) farms,
and in the compost
farm (57,000-fold enrichment),
demonstrating the larg
large expansion of the antibiotic resistance
reservoir in these farms, including the enrichment of up to 19
unique tet genes in a single site (Table S3 gives s enrichment details
for all genes). A total of 63 unique ARGs were significantly
enriched in at least one sample compared with controls at
overall median enrichment of 192-fold for all samples. The max-
imum enrichment of a single ARG was over 28,000-fold in the
Beijing manure (Fig. 34). In terms of absolute abundance, an
aminoglycoside phosphorylation gene aph43 is found 43% as
frequently as the 16S rRNA gene in the manure samples, based on
a 0.58 average value of the delta threshold cycle (ACT) values
(Table S4), meaning this single gene would be found in nearly one
in every second bacterium, assuming a single copy of each gene
in single genomes. In general, enrichment of individual ARGS
an
3436 www.pnas.org/cgi/doi/10.1073/pnas.1222743110
A
No. Resistance >
B
C
السياسي ال
Antibiotic deactivation
□Efflux pump
Cellular protection
Other/ unknown
Aminoglycoside
Beta Lactam
OFCA
OMLSB
Tetracycline
Vancomycin
Other/ efflux
Fig. 1. Antibiotic resistance gene detection statistics. Sample names are
abbreviated with two letters representing location and sample type: first
C, B, J, and P (control, Beijing, Jiaxing, and Putian, respectively) and second
M, C, and S [manure, compost, and soil (with compost amendment), re-
spectively]. Because many resistance genes were targeted with multiple
primers, if multiple primer sets detected the same gene, this was only
counted as detection of a single unique resistance gene. (A) Average number
of unique resistance genes detected in each sample. Error bars represent
SEM of four field replicates. The resistance genes detected in all samples
were classified based on (B) the mechanism of resistance, and (C) the anti-
biotic to which they confer resistance. FCA, fluoroquinolone, quinolone,
florfenicol, chloramphenicol, and amphenicol resistance genes; MLSB,
Macrolide-Lincosamide-Streptogramin B resistance.
decreases in soil samples but is still elevated, with average en-
richment of nearly 100-fold, and some genes were enriched over
1,000-fold compared with the soil control. The Putian soil had
more unique resistance genes enriched at a higher level than the
other two farm soils. When combining the data from all farms, 56,
44, and 17 unique ARGs were statistically elevated in the manure,
compost, and soil samples, respectively.
Transposase Enrichment. Transposases, in parallel to ARGs, were
highly enriched (Fig. 3B). Transposases were found in all sam-
ples (Fig. 2, subgroups A and B) and were enriched up to 90,000-
fold in the manure samples and up to 1,000-fold in the soil
samples. The abundance of ARGS is highly correlated to the
levels of transposases in these farm samples (Fig. 3C) (e.g., as
high as 0.970 for correlation between the abundance of tetra-
cycline resistance genes and transposase genes) (Table S5).
Discussion
Feed Additive Use. These swine farms use a complex mixture of
growth-promoting chemicals, including antibiotics and metals.
However, the individual dosage of each chemical, when considered
alone, on these farms s not excessive compared with other farms
Zhu et al.](/v2/_next/image?url=https%3A%2F%2Fcontent.bartleby.com%2Fqna-images%2Fquestion%2Fa535256e-0857-4f61-a16c-a16363561f95%2Fd275b4d3-d1f6-42f6-a6d8-dcd379d29418%2Fp3pyqar_processed.png&w=3840&q=75)
Transcribed Image Text:quantitative PCR (qPCR) (19) with 313 validated primer sets,
which target 244 ARGS (Table S1) from all major classes of ARGs,
to extensively sample the antibiotic resistance reservoir. We sam-
pled three large-scale commercial swine farms, each from a differ-
ent region of China, at three stages of manure management:
manure, manure compost, and soil receiving manure compost.
Manure from pigs never fed antibiotics and soil from a pristine
forest in Putian, China were used as experimental controls.
Results
Antibiotics and Metal Concentrations. Antibiotics and their use as
reported by the farmers are listed in Table S2. Total tetracycline
concentrations in these manure and soil samples were as high as
15.2 mg kg and 0.78 mg kg, respectively, as was determined
previously (15). Of the sulfonamides analyzed in this study, sul-
famethoxazole had the highest concentrations for all samples,
ranging from 1.08 to
was
detected in all samples in a range of 0.50-4.81 µg-kg-¹. Of the
fluoroquinolones analyzed in this study, the highest mean
only ofloxacin and
enrofloxacin
amono
concentration of
were
most samples.
(335
tions
of ofloxacin
μg-kg -¹) and enrofloxacin (96.0
ug-kg) were
were observed in Putian compost and soil samples, re-
spectively (Fig. S1). Zinc, copper, and arsenic, used as feed
additives, were also elevated above background concent
concentrations.
The highest mean concentrations of copper, zinc, and arsenic
were detected in Putian manure, Jiaxing compost, and Beijing
manure, respectively, with copper up to 1,700 mg kg-¹
manure
(Fig. S2). The concentration of copper, zinc, and arsenic were
much higher in manure than in compost and soil samples, with
the exception of the Jiaxing compost, in which copper and zinc
had the highest concentrations of all of the samples.
Diversity of Antibiotic Resistance Genes. We detected 149 unique
ARGS among all of the samples, which is three times more types
of ARGS than were found in the control samples (Fig. 14). The
ARGs detected in these farms encompass the three major re-
sistance mechanisms efflux pumps, antibiotic deactivation, and
cellular protection (Fig. 1B) and 1 potentially confer resistance to
most major classes of antibiotics (Fig. 1C). Resistance gene pro-
files indicate the patterns and degrees of enrichment of ARGs for
each site (Fig. 2) and that manure samples cluster separately from
the other samples with the exception of the Putian compost. The
compost and soil samples also cluster separately with the excep-
tion of one of the Beijing compost replicates, which grouped with
the soil samples. Furthermore, Shannon diversity (indicating
richness and abundance) of ARGs from farm samples was sig-
nificantly higher than that of the control samples (Fig. S3).
Abundance of Antibiotic Resistance Genes. ARGs were highly
enriched in the farm samples. We used the sum of the enrichment
of all unique ARGs in a sample to approximate total enrichment
in the farms. Maximum enrichment occurred in the manure
samples at Beijing (121,000-fold) and Jiaxing (39,000-fold) farms,
and in the compost
farm (57,000-fold enrichment),
demonstrating the larg
large expansion of the antibiotic resistance
reservoir in these farms, including the enrichment of up to 19
unique tet genes in a single site (Table S3 gives s enrichment details
for all genes). A total of 63 unique ARGs were significantly
enriched in at least one sample compared with controls at
overall median enrichment of 192-fold for all samples. The max-
imum enrichment of a single ARG was over 28,000-fold in the
Beijing manure (Fig. 34). In terms of absolute abundance, an
aminoglycoside phosphorylation gene aph43 is found 43% as
frequently as the 16S rRNA gene in the manure samples, based on
a 0.58 average value of the delta threshold cycle (ACT) values
(Table S4), meaning this single gene would be found in nearly one
in every second bacterium, assuming a single copy of each gene
in single genomes. In general, enrichment of individual ARGS
an
3436 www.pnas.org/cgi/doi/10.1073/pnas.1222743110
A
No. Resistance >
B
C
السياسي ال
Antibiotic deactivation
□Efflux pump
Cellular protection
Other/ unknown
Aminoglycoside
Beta Lactam
OFCA
OMLSB
Tetracycline
Vancomycin
Other/ efflux
Fig. 1. Antibiotic resistance gene detection statistics. Sample names are
abbreviated with two letters representing location and sample type: first
C, B, J, and P (control, Beijing, Jiaxing, and Putian, respectively) and second
M, C, and S [manure, compost, and soil (with compost amendment), re-
spectively]. Because many resistance genes were targeted with multiple
primers, if multiple primer sets detected the same gene, this was only
counted as detection of a single unique resistance gene. (A) Average number
of unique resistance genes detected in each sample. Error bars represent
SEM of four field replicates. The resistance genes detected in all samples
were classified based on (B) the mechanism of resistance, and (C) the anti-
biotic to which they confer resistance. FCA, fluoroquinolone, quinolone,
florfenicol, chloramphenicol, and amphenicol resistance genes; MLSB,
Macrolide-Lincosamide-Streptogramin B resistance.
decreases in soil samples but is still elevated, with average en-
richment of nearly 100-fold, and some genes were enriched over
1,000-fold compared with the soil control. The Putian soil had
more unique resistance genes enriched at a higher level than the
other two farm soils. When combining the data from all farms, 56,
44, and 17 unique ARGs were statistically elevated in the manure,
compost, and soil samples, respectively.
Transposase Enrichment. Transposases, in parallel to ARGs, were
highly enriched (Fig. 3B). Transposases were found in all sam-
ples (Fig. 2, subgroups A and B) and were enriched up to 90,000-
fold in the manure samples and up to 1,000-fold in the soil
samples. The abundance of ARGS is highly correlated to the
levels of transposases in these farm samples (Fig. 3C) (e.g., as
high as 0.970 for correlation between the abundance of tetra-
cycline resistance genes and transposase genes) (Table S5).
Discussion
Feed Additive Use. These swine farms use a complex mixture of
growth-promoting chemicals, including antibiotics and metals.
However, the individual dosage of each chemical, when considered
alone, on these farms s not excessive compared with other farms
Zhu et al.
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