Why did the researchers discover enrichment for ARGs that confer resistance to antibiotics that were not used at the sites tested?

prephase and find answer in article
Transcribed Image Text:quantitative PCR (qPCR) (19) with 313 validated primer sets, which target 244 ARGS (Table $1) 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-¹ and 0.78 mg kg-¹, 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: 3.02 µg-kg-¹ (Fig. S1). Sulfadiazine was also detected: all samples in a range of 0.50-4.81 ug.kg Of the fluoroquinolones analyzed in this study, only ofloxacin and enrofloxacin were observed in most samples. The highest mean concentration of ofloxacin (335 µg-kg-¹) and enrofloxacin (96.0 ug.kg ¹) 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 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 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. 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). The tet Abundance of Antibiotic Resistance Genes. ARGs were highly enriched in the farm samples. We used the of the enrichment of all unique ARGs in a sample to approximate total enrichment in the farms. Maximum enrichment occurred in the manure samples sat Beijing (121,000-fold) and Jiaxing (39,000-fold) farms, and in the compost at the Putian farm (57,000-fold enrichment), demonstrating the large expansion of the antibiotic resistance reservoir in these farms, including the enrichment of up to 19 unique t genes in a single site (Table S3 gives enrichment details for all genes). A total of 63 unique ARGs were significantly enriched in at least one sample compared with controls at an 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 3436 www.pnas.org/cgi/doi/10.1073/pnas.1222743110 B 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 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 is not excessive compared with other farms Zhu et al.
Transcribed Image Text:A B C E D D Zhu et al. Fold Change D D WN ANN-AW-AWA SunSSSSSSSMMMW 1PBBBBBB00 | Soil+Compost Control Manure Manure Compost 10-³0 10³ Fig. 2. Resistance gene profile from the farm sites. Each column is labeled with the sample name (same abbreviation scheme as in Fig. 1, with numbers representing field replicates), and each row is the results from a single primer set. Values plotted are the AAC, with the control soil being the reference sample for all samples. The legend denotes corresponding fold change values, which is a log scale. All primer sets (223) that showed amplification in at least one sample are shown. Columns were clustered based on Bray-Curtis diversity measures. Black boxes delineate resistance profiles: (A) enriched in all sam- ples, including control manure (CM); (B) enriched in all farm samples, but not the CM; (C) widely enriched in most of the farm samples but not the CM; (D) genes that were enriched in the Putian compost but not the Putian manure; and (E) strongly enriched in CM and farm manures. globally. Total tetracyclines in manure and soil samples were as high as 15.2 mg kg-¹ and 0.78 mg kg, respectively (15), which is within the range reported for some European manures between 2002 and 2005 (14). However, other farms in China use higher concentrations of antibiotics; for example, tetracycline and sul- fonamide concentrations in manure reported previously (16) were as high as 764 mg kg-¹ and 20 mg kg-¹, respectively, whereas in this study their maximum concentrations were only 15 mg kg-¹ and 5 µg-kg manure, respectively. However, the Jiaxing and Putian farms used 13 types of antibiotics, which is close to the estimate of the number of antibiotics used in fisheries along the entire Thai coastline (27). In addition to antibiotics, metals used as feed ad- ditives contributed to the complex mixture of selective pressures 5 in these farms. The metal feed additives zinc, copper, and arsenic were elevated above background concentrations at levels typical in Chinese swine farms (28) and only slightly higher than concen- trations reported in the United States and Europe [maximum values reported as 1,300-1,550 mg copper-kg-¹ manure (29)]. Al- though no single antibiotic or metal concentration is excessive in these farms, it is the number of additives used that is striking. The effect of mixtures of resistance selecting agents is unknown but presumably increases the likelihood of coresistance in genetic elements (9). Enlarged Diversity and Abundance of the Environmental Resistance Reservoir. This study documents the breadth and extent of the antibiotic resistance reservoir in large-scale animal production facilities. Furthermore, we provide measures to estimate the field- scale response to composting and I subsequent soil application representing typical manure manure management practices in China as 400 van operons is possible under general, genes potentially conferring a case study. The diverse set of resistance genes detected (Fig. 1) potentially confer resistance to all major classes of antibiotics, including antibiotics critically y important for human medicine (30), such as macrolides (mphA and erm genes), cephalosporins (bla- TEM and blaCTX-M), aminoglycosides (aph and aad genes), and tetracycline (tet genes). Although a number of vancomycin re- sistance genes were detected in these farm samples (Fig. 1C), we do not expect significant phenotypic resistance to vancomycin because detection levels were low and resistance is s dependent on multigene van operons (1, 25), which we did not detect. However, our detection of individual van genes may be an indication that enrichment for van alternative conditions. In resistance to aminoglycosides, tetracyclines, sulfonamide, flor- fenicol, and quaternary ammonium compounds were enriched most broadly in all farm samples. Beta lactam and macrolide re- sistance genes were enriched primarily in manure samples, al- though they may still be present but at levels below detection in the downstream samples. A previous study using a similar qPCR method, sampling only a few individual pigs, detected 57 re- sistance genes, but only 8 were enriched (19). D'Costa et al. (25) found resistance to a broad of antimicrobials but only considered cultured actinomycete strains. One specific type of resistance studied broadly is that for tetracyclines. In a survey of 14 tet genes among hundreds of tetracycline-resistant soil isolates, Ghosh and LaPara (21) found that the most common genes were tetL, tetA, tetM, and tetG (tetW was not included in their survey). We detected 22 of the 28 tetracycline resistance genes targeted on our array. The most abundant tet genes (based on ACT values, Table S4) in the manure were tetQ, tetW, tetX, tet (32), teto, tetM, tetL, and tetG, whereas in the soil they were tetG, tetL, tetA, and tetW, the latter set being similar to those found in soil by Ghosh and LaPara (21). The increased number of re- sistance genes we detected compared with previous studies reflects our sampling at the herd and field levels and the use of a high-throughput qPCR method of detection. range The resistance genes found in our samples were not limited to the antibiotics administered. Aminoglycosides were not used in the Putian farm, but more than 10 aminoglycoside resistance genes were enriched in that farm up to more than 10,000-fold. Similarly, flor was enriched 500-fold in the Jiaxing compost but amphenicols were not known to be used at that farm. Coen- richment of these genes is most likely due to aggregation of re- sistance genes on mobile genetic elements (19, 31-34), as has been observed directly (35). In addition, the abundance of ARGS in these samples is correlated with the concentrations of anti- biotics, as well as with 1 copper, zinc, and arsenic (Fig. 3 and Table S5). The presence of heavy metals provides another coselective pressure for antibiotic resistance (23) and may aid in long-term persistence of ARGs during manure management and disposal (36). Only a few multidrug efflux pumps (qacEA1 and dfrA1) PNAS February 26, 2013 vol. 110| no. 9 | 3437 ENVIRONMENTAL

Question

Why did the researchers discover enrichment for ARGs that confer resistance to antibiotics that were not used at the sites tested?

prephase and find answer in article

quantitative PCR (qPCR) (19) with 313 validated primer sets,
which target 244 ARGS (Table $1) 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-¹ and 0.78 mg kg-¹, 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: 3.02 µg-kg-¹ (Fig. S1). Sulfadiazine was also
detected: all samples in a range of 0.50-4.81 ug.kg Of the
fluoroquinolones analyzed in this study, only ofloxacin and
enrofloxacin were observed in most samples. The highest mean
concentration of ofloxacin (335 µg-kg-¹) and enrofloxacin (96.0
ug.kg ¹) 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 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 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.
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).
The
tet
Abundance of Antibiotic Resistance Genes. ARGs were highly
enriched in the farm samples. We used the of the enrichment
of all unique ARGs in a sample to approximate total enrichment
in the farms. Maximum enrichment occurred in the manure
samples
sat Beijing (121,000-fold) and Jiaxing (39,000-fold) farms,
and in the compost at the Putian farm (57,000-fold enrichment),
demonstrating the large expansion of the antibiotic resistance
reservoir in these farms, including the enrichment of up to 19
unique
t genes in a single site (Table S3 gives enrichment details
for all genes). A total of 63 unique ARGs were significantly
enriched in at least one sample compared with controls at an
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
3436 www.pnas.org/cgi/doi/10.1073/pnas.1222743110
B
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 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 is not excessive compared with other farms
Zhu et al.

Transcribed Image Text:A B C E D D Zhu et al. Fold Change D D WN ANN-AW-AWA SunSSSSSSSMMMW 1PBBBBBB00 | Soil+Compost Control Manure Manure Compost 10-³0 10³ Fig. 2. Resistance gene profile from the farm sites. Each column is labeled with the sample name (same abbreviation scheme as in Fig. 1, with numbers representing field replicates), and each row is the results from a single primer set. Values plotted are the AAC, with the control soil being the reference sample for all samples. The legend denotes corresponding fold change values, which is a log scale. All primer sets (223) that showed amplification in at least one sample are shown. Columns were clustered based on Bray-Curtis diversity measures. Black boxes delineate resistance profiles: (A) enriched in all sam- ples, including control manure (CM); (B) enriched in all farm samples, but not the CM; (C) widely enriched in most of the farm samples but not the CM; (D) genes that were enriched in the Putian compost but not the Putian manure; and (E) strongly enriched in CM and farm manures. globally. Total tetracyclines in manure and soil samples were as high as 15.2 mg kg-¹ and 0.78 mg kg, respectively (15), which is within the range reported for some European manures between 2002 and 2005 (14). However, other farms in China use higher concentrations of antibiotics; for example, tetracycline and sul- fonamide concentrations in manure reported previously (16) were as high as 764 mg kg-¹ and 20 mg kg-¹, respectively, whereas in this study their maximum concentrations were only 15 mg kg-¹ and 5 µg-kg manure, respectively. However, the Jiaxing and Putian farms used 13 types of antibiotics, which is close to the estimate of the number of antibiotics used in fisheries along the entire Thai coastline (27). In addition to antibiotics, metals used as feed ad- ditives contributed to the complex mixture of selective pressures 5 in these farms. The metal feed additives zinc, copper, and arsenic were elevated above background concentrations at levels typical in Chinese swine farms (28) and only slightly higher than concen- trations reported in the United States and Europe [maximum values reported as 1,300-1,550 mg copper-kg-¹ manure (29)]. Al- though no single antibiotic or metal concentration is excessive in these farms, it is the number of additives used that is striking. The effect of mixtures of resistance selecting agents is unknown but presumably increases the likelihood of coresistance in genetic elements (9). Enlarged Diversity and Abundance of the Environmental Resistance Reservoir. This study documents the breadth and extent of the antibiotic resistance reservoir in large-scale animal production facilities. Furthermore, we provide measures to estimate the field- scale response to composting and I subsequent soil application representing typical manure manure management practices in China as 400 van operons is possible under general, genes potentially conferring a case study. The diverse set of resistance genes detected (Fig. 1) potentially confer resistance to all major classes of antibiotics, including antibiotics critically y important for human medicine (30), such as macrolides (mphA and erm genes), cephalosporins (bla- TEM and blaCTX-M), aminoglycosides (aph and aad genes), and tetracycline (tet genes). Although a number of vancomycin re- sistance genes were detected in these farm samples (Fig. 1C), we do not expect significant phenotypic resistance to vancomycin because detection levels were low and resistance is s dependent on multigene van operons (1, 25), which we did not detect. However, our detection of individual van genes may be an indication that enrichment for van alternative conditions. In resistance to aminoglycosides, tetracyclines, sulfonamide, flor- fenicol, and quaternary ammonium compounds were enriched most broadly in all farm samples. Beta lactam and macrolide re- sistance genes were enriched primarily in manure samples, al- though they may still be present but at levels below detection in the downstream samples. A previous study using a similar qPCR method, sampling only a few individual pigs, detected 57 re- sistance genes, but only 8 were enriched (19). D'Costa et al. (25) found resistance to a broad of antimicrobials but only considered cultured actinomycete strains. One specific type of resistance studied broadly is that for tetracyclines. In a survey of 14 tet genes among hundreds of tetracycline-resistant soil isolates, Ghosh and LaPara (21) found that the most common genes were tetL, tetA, tetM, and tetG (tetW was not included in their survey). We detected 22 of the 28 tetracycline resistance genes targeted on our array. The most abundant tet genes (based on ACT values, Table S4) in the manure were tetQ, tetW, tetX, tet (32), teto, tetM, tetL, and tetG, whereas in the soil they were tetG, tetL, tetA, and tetW, the latter set being similar to those found in soil by Ghosh and LaPara (21). The increased number of re- sistance genes we detected compared with previous studies reflects our sampling at the herd and field levels and the use of a high-throughput qPCR method of detection. range The resistance genes found in our samples were not limited to the antibiotics administered. Aminoglycosides were not used in the Putian farm, but more than 10 aminoglycoside resistance genes were enriched in that farm up to more than 10,000-fold. Similarly, flor was enriched 500-fold in the Jiaxing compost but amphenicols were not known to be used at that farm. Coen- richment of these genes is most likely due to aggregation of re- sistance genes on mobile genetic elements (19, 31-34), as has been observed directly (35). In addition, the abundance of ARGS in these samples is correlated with the concentrations of anti- biotics, as well as with 1 copper, zinc, and arsenic (Fig. 3 and Table S5). The presence of heavy metals provides another coselective pressure for antibiotic resistance (23) and may aid in long-term persistence of ARGs during manure management and disposal (36). Only a few multidrug efflux pumps (qacEA1 and dfrA1) PNAS February 26, 2013 vol. 110| no. 9 | 3437 ENVIRONMENTAL

Accepted Answer

The discovery of enrichment for antibiotic resistance genes (ARGs) that confer resistance to…