In your opinion, is antibiotic use at farm sites promote the development and spread of antibiotic resistant genes in bacteria? Why or why not please explain based on the article

Transcribed Image Text:as to define soil and landscape features that would minimize dis- persal to the human food chain. Resistance gene diversity and abundance patterns specific to each management type indicate the influence of the antibiotics as a selective pressure. These profiles show that generally samples of the same management type clustered together (Fig. 4). The relationships between the structure of detected ARGs and an- tibiotic and heavy metal concentrations were assessed with ca- nonical correspondence analysis. Manure samples grouped separately by the first axis and were strongly affected by arsenic, copper, and tetracycline concentrations, which are likely among the dominant factors driving the changes in structures of ARGS on these farms (Fig. 4). Although only three farms are included in this study, regardless of their location (a separation of over 2,000 km), composting technique, or antibiotic dosage, the ARGs' resistance profiles are similar, indicating that similar reservoirs of ARGs are likely common across China and in other countries where management practices are similar. The diversity and abundance of ARGS reported in this study is alarming and clearly indicates that unmonitored use antibiotics and metals on swine farms has expanded the diversity and abun- dance of the antibiotic resistance reservoir in the farm environ- ment. The coenrichment of ARGs and transposases further exacerbates the risks of transfer of ARGs from livestock animals to human-associated bacteria, and then spread among human pop- ulations (4, 6). Policies and management tools to facilitate prudent use of antibiotics and heavy metals, including their combined use, in animal industries and animal waste management are needed. Decreased resistance levels have been observed in Europe after the disuse of agricultural antibiotics (51). Pig manure, with its abundant and diverse ARGs and sheer volume, is a major source of resistance genes and as such a public health hazard. Microbes from manure, compost, or soil containing the ARGs are subject to dispersal via runoff into rivers (22), leaching to subsurface waters, air dispersal via dust, human travel, and distribution of agricultural products, including compost for gardening, which could expand a local contamination to regional and even global scales (6, 11). CCA2 (4.56%) 2- 0 CS BM JM PM BC JC A PC BS ● JS ▲ PS Sulfonamide Soil Control Soil -2 Quinolone 0 Compost Zn Cu As Tetracycline Manure 2 CCA1 (10.06%) Fig. 4. Canonical correspondence analysis (CCA) compares the abundance of detected resistance genes (symbols) and the concentration of heavy metals and antibiotics (arrows). The results showed that pig manure samples were positively correlated to the concentrations of copper, zinc, arsenic, and Materials and Methods Sampling. A total of 36 samples were collected in 2010 from three Chinese provinces including (from north to south) Beijing (Beijing farm), Zhejiang (Jiaxing farm), and Fujian (Putian farm). The manure and compost samples were obtained from representative swine farms with an animal intensity of 10,000 market hogs or more per year. Soil samples were collected from a nearby agronomic field to which manure-based compost had been applied. Four replicates were taken from each sample type and farm, and all of the samples were kept on dry ice during transportation and stored at -80 °C before DNA extraction and chemical analysis. These are typical large-scale swine farms. Pigs are continuously housed on concrete. The manure was sampled within 1 d after excretion in all cases. In Beijing, compost was managed in outdoor windrows with aeration for 2 wk. In Jiaxing, pile composting was used with regular stirring (one or two times per day) for about 10 d. In Putian they used pile composting with limited aeration for 2-4 wk. In Jiaxing and Putian, compost products are packed and sold as commercial organic fertilizer for local farmers. For soil amendment, the composted manure spreading rate varies but is ~10 tons/hectare, applied once per year. At the Beijing and Jiaxing farms, the soil had been receiving manure compost for more than 2 y, and the most recent application was 2 mo before sampling. At the Putian farm, the soil had been receiving manure compost for more than 3 y, and the most recent application was 1 wk before sampling. Control samples received no known antibiotic input. The control soil is from a pristine forest in Putian, China. This soil has had no anthropogenic antibiotic input and has an abundance of ARGs and diversity profile similar to another temperate-region, antibiotic-free grassland soil we studied. The control pig manure samples were mixtures of DNA extracted from feces from pigs birthed from a mother with no antibiotic exposure and grown in facilities with no antibiotic exposure but fed a normal grower diet (ref. 19 gives further details). Sample CM1 was taken from six 84-d-old pigs not fed antibiotics. Samples CM2-4 were each taken from a single animal at three time points between 86 and 104 d of age. The control manure was used as a comparison against the farm manures, and the control soil was used as a comparison against both the farm compost and farm soil. Antibiotic and Metal Quantitation. Concentrations of sulfonamides and qui- nolones were analyzed in this study, including sulfadiazine, sulfamerazine, sulfamethoxydiazine, sulfamethazine, sulfamethoxazole, norfloxacin, ofloxacin, enrofloxacin, and ciprofloxacin. Previously, 5 target tetracyclines and 10 deg- radation products were analyzed (15). Metals were analyzed in air-dried, milled samples after oxidative digestion in sealed tubes by inductively coupled plasma-mass spectrometry (7500cx; Agilent). Quantities were determined relative to reference standards. Sample extraction and analysis procedures for antibiotics and metals are described in SI Materials and Methods. DNA Extraction. High-molecular-weight community DNA was extracted by the freeze-grinding, SDS-based method (52) and was purified using a low- melting agarose gel followed by phenol extraction. DNA concentration and quality were determined with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc.). Primer Design. A majority of the primer sets (247) were designed, used, and validated in a previous study (19). For this study, 89 new primer sets were designed for categories of resistance genes not previously targeted as thoroughly. The same design parameters were used as before (19). Refer- ence sequences were harvested from the Antibiotic Resistance Genes Data- base (http://ardb.cbcb.umd.edu/). Additional validation of the primer sets was performed and is described in SI Materials and Methods. Quantitative PCR. All quantitative PCR reactions were performed using the Applied Biosystems OpenArray platform, as described previously (19), except that a threshold cycle (CT) of 27 was used as the detection limit. Generally the technical triplicates were tested during separate testing occasions (plate and day of testing) as a method of quality control. The AAC, method of comparison (53) was used to compare relative abundance between samples: ACT=CT. (ARG) - CT,(165) [1]
Transcribed Image Text:A B Transposase Fold Increase C Total Transposases 105 104 10³ 10² 10¹ 10⁰ 105 104 10³ 10² 10¹ 10⁰ 10-1 300 250 200 150 100 50 0 0 ■ 58 0 ■ Manure · O ■ Copper O Transposase Oxytetracycline 54 0 O -- so 43 0 13 D ■ - 35 D Compost Soil+Compost 8 56 0 0 104 3438| www.pnas.org/cgi/doi/10.1073/pnas.1222743110 103 10² 10-1 200 400 600 800 1000 1200 1400 Total Antibiotic Resistance 101 10⁰ 17 Oxytetracycline (µg kg-¹) 10³ 10² 10¹ 10⁰ 10-1 Copper (mg kg-¹) Fig. 3. Abundance of resistance genes and transposases. In the box plots, the symbols indicate the following: box, 25th to 75th percentile; horizontal line, median; whiskers, 10th and 90th percentile; and square, maximum value. The y axis is a log scale of fold increase: farm manure compared with the control manure, and farm compost or soil compared with the control soil. (A) Only statistically enriched resistance genes are represented. The number above each site indicates the number of primer sets that yielded statistically signifi- cant results. (B) Summary of all nine primer sets used to target different transposase alleles (in B, top whisker represents the maximum value). (C) Correlation of total resistance and transposase abundances, oxytetracycline concentration, and copper concentration. Total antibiotic resistance and total transposases values are the sum of AAC+ values of all assays of that type in each sample. The sample identifiers below B apply to both A and B. were broadly enriched (Table S6). It seems that genes with specific mechanisms of resistance were preferably enriched in these high- selection-pressure environments. The diversity of resistance genes enriched and coenriched at the farm level is concerning because this broad set of ARGS or a subset thereof could be (co)enriched or transferred to pathogens under future selection conditions. Swine farms are known hotspots for pervasive and abundant antibiotic resistance both in antibiotic-free animals (19, 37) and, especially, in antibiotic-treated animals (26, 38). The level of enrichment of individual resistance genes is on par with previous field-scale studies. Tetracycline resistance genes were enriched 10² to 10-fold in cattle waste lagoons (39), and the median enrichment of ARGs in this manure was about 10-fold. Of bac- terial isolates from swine and chicken manure, 92% and >80%, respectively, were found resistant to at least one antibiotic (26, 31). We estimate about 43% of bacteria possess at least the aphA3 gene; hence, it is feasible that upward of 90% of the entire community would carry one of the other 148 resistance genes detected. Considering all antibiotic resistance genes com- bined in the manure or compost samples, we estimate a total of 50,000-fold enrichment (Table S3). Although enrichment of in- dividual resistance genes is similar to that found in previous studies, we were able to capture a more complete picture of the total level of the antibiotic resistance reservoir. Potential for Horizontal Gene Transfer of ARGs. This study high- lights that ARGs in swine farms are not only diverse but are also remarkably abundant, which together offers a higher sta- tistical probability of dispersal, further selection, and/or hori- zontal transfer in the environment. The emergence and spread of ARGS are closely associated with mobile genetic elements such as plasmids, integrases, and transposases (20, 40, 41). The high de- gree of transposase enrichment and correlation with abundance of ARGs suggests that horizontal gene transfer may have aided the enrichment of ARGs. The transposases detected most fre- quently belong to the IS6 family of insertion sequences, which are typically found flanking an array of genes, often resistance genes (42). The most abundant member of the IS6 family in these samples, IS26, has been isolated, along with integrons in multi- drug-resistant plasmids in enterobacteria (43). Integrons most commonly contain resistance cassettes encoding aadA genes (44), as well as qacEAI and sul2, which were among the most enriched genes in this study. The Putian farm ARGs that are more enriched in the compost than in the manure (Fig. 2, D boxes and Table S6) are predominately aadA and other aminoglycoside resistance genes and their enrichment may be due to their pres- ence in integrons that also hold a resistance gene cassette relevant to the drugs used on the farm (34, 45). Additionally, the combi- nation of antibiotics and metals may provide a stronger selection for realized horizontal gene transfer within the microbial com- munity than either alone (9, 23, 34, 46). It appears that a number of factors in swine farms could contribute to elevated rates of horizontal gene transfer, including elevated concentrations of antibiotics, metals, ARGs, and mobile genetic elements, making subsequent dispersal, (co)enrichment, or horizontal transfer, in- cluding to human-associated bacteria, more probable. Role of Manure Management in Controlling ARGS. The long-term goal of manure management is to remove environmental con- taminants while disposing of this high-volume waste product and capturing its value to improve soil fertility. The goal in the case of ARGS is to identify practices that decrease their concentration to a greater degree than by simple dilution (47). Manure composting decreased the abundance of ARGs at Beijing, but abundance remained nearly the same in the Jiaxing manure, while at Putian composting actually increased the abundance of ARGs. Com- posting concentrated sulfonamides (Fig. S1), sulfonamide resistance genes, and some metals (Fig. S2), consistent with the observation that sulfonamide resistance genes are more recalcitrant than tet- racycline genes (22, 48, 49). The common practice of spreading compost on soil was not sufficient to reduce abundance of ARGS to background levels, and the Putian soil showed up to 3,000-fold enrichment. However, the practice decreased concentrations of ARGs substantially below compost levels. The relatively high en- richment of ARGs in Putian soil may be due to a higher manure/ soil ratio and/or shorter time before sampling after amendment compared with other farms. These observations highlight the need to determine adequate composting time to reduce resistance levels before release to the more uncontrolled environment (50), as well Zhu et al.

Question

In your opinion, is antibiotic use at farm sites promote the development and spread of antibiotic resistant genes in bacteria? Why or why not please explain based on the article

Transcribed Image Text:as to define soil and landscape features that would minimize dis- persal to the human food chain. Resistance gene diversity and abundance patterns specific to each management type indicate the influence of the antibiotics as a selective pressure. These profiles show that generally samples of the same management type clustered together (Fig. 4). The relationships between the structure of detected ARGs and an- tibiotic and heavy metal concentrations were assessed with ca- nonical correspondence analysis. Manure samples grouped separately by the first axis and were strongly affected by arsenic, copper, and tetracycline concentrations, which are likely among the dominant factors driving the changes in structures of ARGS on these farms (Fig. 4). Although only three farms are included in this study, regardless of their location (a separation of over 2,000 km), composting technique, or antibiotic dosage, the ARGs' resistance profiles are similar, indicating that similar reservoirs of ARGs are likely common across China and in other countries where management practices are similar. The diversity and abundance of ARGS reported in this study is alarming and clearly indicates that unmonitored use antibiotics and metals on swine farms has expanded the diversity and abun- dance of the antibiotic resistance reservoir in the farm environ- ment. The coenrichment of ARGs and transposases further exacerbates the risks of transfer of ARGs from livestock animals to human-associated bacteria, and then spread among human pop- ulations (4, 6). Policies and management tools to facilitate prudent use of antibiotics and heavy metals, including their combined use, in animal industries and animal waste management are needed. Decreased resistance levels have been observed in Europe after the disuse of agricultural antibiotics (51). Pig manure, with its abundant and diverse ARGs and sheer volume, is a major source of resistance genes and as such a public health hazard. Microbes from manure, compost, or soil containing the ARGs are subject to dispersal via runoff into rivers (22), leaching to subsurface waters, air dispersal via dust, human travel, and distribution of agricultural products, including compost for gardening, which could expand a local contamination to regional and even global scales (6, 11). CCA2 (4.56%) 2- 0 CS BM JM PM BC JC A PC BS ● JS ▲ PS Sulfonamide Soil Control Soil -2 Quinolone 0 Compost Zn Cu As Tetracycline Manure 2 CCA1 (10.06%) Fig. 4. Canonical correspondence analysis (CCA) compares the abundance of detected resistance genes (symbols) and the concentration of heavy metals and antibiotics (arrows). The results showed that pig manure samples were positively correlated to the concentrations of copper, zinc, arsenic, and Materials and Methods Sampling. A total of 36 samples were collected in 2010 from three Chinese provinces including (from north to south) Beijing (Beijing farm), Zhejiang (Jiaxing farm), and Fujian (Putian farm). The manure and compost samples were obtained from representative swine farms with an animal intensity of 10,000 market hogs or more per year. Soil samples were collected from a nearby agronomic field to which manure-based compost had been applied. Four replicates were taken from each sample type and farm, and all of the samples were kept on dry ice during transportation and stored at -80 °C before DNA extraction and chemical analysis. These are typical large-scale swine farms. Pigs are continuously housed on concrete. The manure was sampled within 1 d after excretion in all cases. In Beijing, compost was managed in outdoor windrows with aeration for 2 wk. In Jiaxing, pile composting was used with regular stirring (one or two times per day) for about 10 d. In Putian they used pile composting with limited aeration for 2-4 wk. In Jiaxing and Putian, compost products are packed and sold as commercial organic fertilizer for local farmers. For soil amendment, the composted manure spreading rate varies but is ~10 tons/hectare, applied once per year. At the Beijing and Jiaxing farms, the soil had been receiving manure compost for more than 2 y, and the most recent application was 2 mo before sampling. At the Putian farm, the soil had been receiving manure compost for more than 3 y, and the most recent application was 1 wk before sampling. Control samples received no known antibiotic input. The control soil is from a pristine forest in Putian, China. This soil has had no anthropogenic antibiotic input and has an abundance of ARGs and diversity profile similar to another temperate-region, antibiotic-free grassland soil we studied. The control pig manure samples were mixtures of DNA extracted from feces from pigs birthed from a mother with no antibiotic exposure and grown in facilities with no antibiotic exposure but fed a normal grower diet (ref. 19 gives further details). Sample CM1 was taken from six 84-d-old pigs not fed antibiotics. Samples CM2-4 were each taken from a single animal at three time points between 86 and 104 d of age. The control manure was used as a comparison against the farm manures, and the control soil was used as a comparison against both the farm compost and farm soil. Antibiotic and Metal Quantitation. Concentrations of sulfonamides and qui- nolones were analyzed in this study, including sulfadiazine, sulfamerazine, sulfamethoxydiazine, sulfamethazine, sulfamethoxazole, norfloxacin, ofloxacin, enrofloxacin, and ciprofloxacin. Previously, 5 target tetracyclines and 10 deg- radation products were analyzed (15). Metals were analyzed in air-dried, milled samples after oxidative digestion in sealed tubes by inductively coupled plasma-mass spectrometry (7500cx; Agilent). Quantities were determined relative to reference standards. Sample extraction and analysis procedures for antibiotics and metals are described in SI Materials and Methods. DNA Extraction. High-molecular-weight community DNA was extracted by the freeze-grinding, SDS-based method (52) and was purified using a low- melting agarose gel followed by phenol extraction. DNA concentration and quality were determined with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc.). Primer Design. A majority of the primer sets (247) were designed, used, and validated in a previous study (19). For this study, 89 new primer sets were designed for categories of resistance genes not previously targeted as thoroughly. The same design parameters were used as before (19). Refer- ence sequences were harvested from the Antibiotic Resistance Genes Data- base (http://ardb.cbcb.umd.edu/). Additional validation of the primer sets was performed and is described in SI Materials and Methods. Quantitative PCR. All quantitative PCR reactions were performed using the Applied Biosystems OpenArray platform, as described previously (19), except that a threshold cycle (CT) of 27 was used as the detection limit. Generally the technical triplicates were tested during separate testing occasions (plate and day of testing) as a method of quality control. The AAC, method of comparison (53) was used to compare relative abundance between samples: ACT=CT. (ARG) - CT,(165) [1]

A
B
Transposase
Fold Increase
C
Total Transposases
105
104
10³
10²
10¹
10⁰
105
104
10³
10²
10¹
10⁰
10-1
300
250
200
150
100
50
0
0
■
58
0
■
Manure
·
O
■ Copper
O
Transposase
Oxytetracycline
54
0
O
--
so
43
0
13
D
■
-
35
D
Compost Soil+Compost
8
56
0
0
104
3438| www.pnas.org/cgi/doi/10.1073/pnas.1222743110
103
10²
10-1
200 400 600 800 1000 1200 1400
Total Antibiotic Resistance
101
10⁰
17
Oxytetracycline (µg kg-¹)
10³
10²
10¹
10⁰
10-1
Copper (mg kg-¹)
Fig. 3. Abundance of resistance genes and transposases. In the box plots, the
symbols indicate the following: box, 25th to 75th percentile; horizontal line,
median; whiskers, 10th and 90th percentile; and square, maximum value. The
y axis is a log scale of fold increase: farm manure compared with the control
manure, and farm compost or soil compared with the control soil. (A) Only
statistically enriched resistance genes are represented. The number above
each site indicates the number of primer sets that yielded statistically signifi-
cant results. (B) Summary of all nine primer sets used to target different
transposase alleles (in B, top whisker represents the maximum value). (C)
Correlation of total resistance and transposase abundances, oxytetracycline
concentration, and copper concentration. Total antibiotic resistance and total
transposases values are the sum of AAC+ values of all assays of that type in
each sample. The sample identifiers below B apply to both A and B.
were broadly enriched (Table S6). It seems that genes with specific
mechanisms of resistance were preferably enriched in these high-
selection-pressure environments. The diversity of resistance genes
enriched and coenriched at the farm level is concerning because
this broad set of ARGS or a subset thereof could be (co)enriched
or transferred to pathogens under future selection conditions.
Swine farms are known hotspots for pervasive and abundant
antibiotic resistance both in antibiotic-free animals (19, 37) and,
especially, in antibiotic-treated animals (26, 38). The level of
enrichment of individual resistance genes is on par with previous
field-scale studies. Tetracycline resistance genes were enriched
10² to 10-fold in cattle waste lagoons (39), and the median
enrichment of ARGs in this manure was about 10-fold. Of bac-
terial isolates from swine and chicken manure, 92% and >80%,
respectively, were found resistant to at least one antibiotic (26,
31). We estimate about 43% of bacteria possess at least the
aphA3 gene; hence, it is feasible that upward of 90% of the
entire community would carry one of the other 148 resistance
genes detected. Considering all antibiotic resistance genes com-
bined in the manure or compost samples, we estimate a total of
50,000-fold enrichment (Table S3). Although enrichment of in-
dividual resistance genes is similar to that found in previous
studies, we were able to capture a more complete picture of the
total level of the antibiotic resistance reservoir.
Potential for Horizontal Gene Transfer of ARGs. This study high-
lights that ARGs in swine farms are not only diverse but are
also remarkably abundant, which together offers a higher sta-
tistical probability of dispersal, further selection, and/or hori-
zontal transfer in the environment. The emergence and spread of
ARGS are closely associated with mobile genetic elements such as
plasmids, integrases, and transposases (20, 40, 41). The high de-
gree of transposase enrichment and correlation with abundance
of ARGs suggests that horizontal gene transfer may have aided
the enrichment of ARGs. The transposases detected most fre-
quently belong to the IS6 family of insertion sequences, which
are typically found flanking an array of genes, often resistance
genes (42). The most abundant member of the IS6 family in these
samples, IS26, has been isolated, along with integrons in multi-
drug-resistant plasmids in enterobacteria (43). Integrons most
commonly contain resistance cassettes encoding aadA genes (44),
as well as qacEAI and sul2, which were among the most enriched
genes in this study. The Putian farm ARGs that are more
enriched in the compost than in the manure (Fig. 2, D boxes and
Table S6) are predominately aadA and other aminoglycoside
resistance genes and their enrichment may be due to their pres-
ence in integrons that also hold a resistance gene cassette relevant
to the drugs used on the farm (34, 45). Additionally, the combi-
nation of antibiotics and metals may provide a stronger selection
for realized horizontal gene transfer within the microbial com-
munity than either alone (9, 23, 34, 46). It appears that a number
of factors in swine farms could contribute to elevated rates of
horizontal gene transfer, including elevated concentrations of
antibiotics, metals, ARGs, and mobile genetic elements, making
subsequent dispersal, (co)enrichment, or horizontal transfer, in-
cluding to human-associated bacteria, more probable.
Role of Manure Management in Controlling ARGS. The long-term
goal of manure management is to remove environmental con-
taminants while disposing of this high-volume waste product and
capturing its value to improve soil fertility. The goal in the case of
ARGS is to identify practices that decrease their concentration to
a greater degree than by simple dilution (47). Manure composting
decreased the abundance of ARGs at Beijing, but abundance
remained nearly the same in the Jiaxing manure, while at Putian
composting actually increased the abundance of ARGs. Com-
posting concentrated sulfonamides (Fig. S1), sulfonamide resistance
genes, and some metals (Fig. S2), consistent with the observation
that sulfonamide resistance genes are more recalcitrant than tet-
racycline genes (22, 48, 49). The common practice of spreading
compost on soil was not sufficient to reduce abundance of ARGS
to background levels, and the Putian soil showed up to 3,000-fold
enrichment. However, the practice decreased concentrations of
ARGs substantially below compost levels. The relatively high en-
richment of ARGs in Putian soil may be due to a higher manure/
soil ratio and/or shorter time before sampling after amendment
compared with other farms. These observations highlight the need
to determine adequate composting time to reduce resistance levels
before release to the more uncontrolled environment (50), as well
Zhu et al.

Accepted Answer

ARGs stands for antibiotic resistance genes, which are genes that encode proteins or enzymes that…