当前位置：文档库 › 粪肥有助于滋生耐药微生物Bloom of resident antibiotic-resistant bacteria in soil

粪肥有助于滋生耐药微生物Bloom of resident antibiotic-resistant bacteria in soil

Bloom of resident antibiotic-resistant bacteria in soil following manure fertilization

Nikolina Udikovic-Kolic a,b,1,Fabienne Wichmann a,c,1,Nichole A.Broderick a,and Jo Handelsman a,2

a Department of Molecular,Cellular,and Developmental Biology,Yale University,New Haven,CT06511;

b Division for Marine and Environmental Research, Rudjer Boskovi

c Institute,Zagreb10000,Croatia;an

d c Biosafety Research,Stat

e Laboratory Basel,4012Basel,Switzerland

Edited by W.Ford Doolittle,Dalhousie University,Halifax,Canada,and approved September8,2014(received for review May28,2014)

The increasing prevalence of antibiotic-resistant bacteria is a global threat to public health.Agricultural use of antibiotics is believed to contribute to the spread of antibiotic resistance,but the mecha-nisms by which many agricultural practices influence resistance remain obscure.Although manure from dairy farms is a common soil amendment in crop production,its impact on the soil micro-biome and resistome is not known.To gain insight into this im-pact,we cultured bacteria from soil before and at10time points after application of manure from cows that had not received an-tibiotic treatment.Soil treated with manure contained a higher abundance ofβ-lactam–resistant bacteria than soil treated with inorganic fertilizer.Functional metagenomics identifiedβ-lactam–resistance genes in treated and untreated soil,and indicated that the higher frequency of resistant bacteria in manure-amended soil was attributable to enrichment of resident soil bacteria that har-borβ-lactamases.Quantitative PCR indicated that manure treat-ment enriched the bla CEP-04gene,which is highly similar(96%)to a gene found previously in a Pseudomonas sp.Analysis of16S rRNA genes indicated that the abundance of Pseudomonas spp. increased in manure-amended soil.Populations of other soil bac-teria that commonly harborβ-lactamases,including Janthinobac-terium sp.and Psychrobacter pulmonis,also increased in response to manure treatment.These results indicate that manure amend-ment induced a bloom of certain antibiotic-resistant bacteria in soil that was independent of antibiotic exposure of the cows from which the manure was derived.Our data illustrate the unintended consequences that can result from agricultural practices,and dem-onstrate the need for empirical analysis of the agroecosystem. dairy cow manure|β-lactam antibiotics

A griculture affects human health through both the con-

sumption and production of food for the human diet.Ma-nure from pig and cattle farms is commonly used as a substitute for inorganic nitrogen and phosphorus fertilizers for agricultural crops worldwide,especially in organic farming practices(1–6). With the increasing consumer demand for organically produced food,the use of animal manure,which conforms to organic conventions,will likely increase in the future.According to the National Organic Program,raw manure may be used up to90–120d before harvest,depending on the crop,and composted manure may be applied at any time.There are no restrictions on the source of manure(1).

Animal manure is an important reservoir of antibiotic-resistant bacteria,antibiotic-resistance genes(collectively known as the“resistome”),and pathogens(2,7–12).Although antibiotic use increases antibiotic-resistance genes and resistant bacteria in manure(13–16),antibiotic-resistant bacteria are also abundant in manure from animals with no history of antibiotic treatment, indicating the natural presence of bacteria intrinsically resistant to antibiotics in animal gastrointestinal tracts(2,17,18). There is increasing concern about the use of manure as an agricultural amendment because of its possible contribution to the pool of resistance genes to resident soil bacteria and pathogens(2,19).Antibiotic-resistance genes from the soil resistome can enter the food chain via contaminated crops or groundwater(5,20),and have potential consequences for human health if transferred to human pathogens.Studies assessing the impact of fertilization with pig manure on the soil resistome have shown that excessive application of manure from farms with in-tensive sulfonamide use can lead to an increase of antibiotic-resistance genes in soil(2,3);however,most studies have found that such increases are transient when the manure is applied at recommended rates(2,21,22).Cow manure from dairy farms, which useβ-lactam antibiotics predominantly to prevent and treat diseases(23),is commonly used in crop production,but its impact on the soil resistome has yet to be investigated.

Along with its impact on the soil resistome,the application of manure can affect the composition and functional properties of soil microbial communities,as has been demonstrated by com-munity fingerprinting(21,24).Recent advances in DNA-based analysis,such as metagenomics and quantitative PCR(qPCR), offer greater precision in such studies,enabling identification of affected community members(25)and their resistance genes(4). In the present study,we assessed the impact of cow manure on the composition and resistance profiles of bacterial communities in soil.Our results show that manure from cows that had not been treated with antibiotics increased the populations of resi-dent soil bacteria harboring genes for resistance toβ-lactam antibiotics,whereas inorganic fertilizers did not.These results demonstrate the complexity,and at times nonintuitive conse-quences,of agricultural

practices.

Author contributions:N.U.-K.,F.W.,and J.H.designed research;N.U.-K.and F.W.per-formed research;N.U.-K.,F.W.,N.A.B.,and J.H.analyzed data;and N.U.-K.,F.W.,N.A.B., and J.H.wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition:The sequences reported in this paper have been deposited in MG-RAST, https://www.wendangku.net/doc/5c10113967.html,/(project ID8945,“YaleFarmSoil”;metagenomes4562248.3–4562264.3,4562266.3–4562295.3,and4570842.3)and GenBank(accession nos. KM113767–KM113773).

1N.U.-K.and F.W.contributed equally to this work.

2To whom correspondence should be addressed.Email:jo.handelsman@https://www.wendangku.net/doc/5c10113967.html,.

This article contains supporting information online at https://www.wendangku.net/doc/5c10113967.html,/lookup/suppl/doi:10. 1073/pnas.1409836111/-/DCSupplemental.

https://www.wendangku.net/doc/5c10113967.html,/cgi/doi/10.1073/pnas.1409836111PNAS Early Edition|1of6M I C R O B I O L O G Y

获取更多语音讲解，关注订阅号钢钢发声

Results

Manure Fertilization Increases Total and β-Lactam –Resistant Culturable Bacteria in Soil.We compared the effect of application of either

cow manure or inorganic fertilizer [nitrogen,phosphorus,and potassium (NPK)]on the populations of total and β-lactam –resistant culturable bacteria in soil.We cultured soil before fertilization and at 10time points after application of manure or NPK.Culturing indicated that manure directly added 107colony-forming units (CFU)per gram of soil,which approximately dou-bled the culturable bacteria in the soil.Soil populations of culturable bacteria remained significantly higher (P <0.05)in samples treated with manure compared with those treated with inorganic fertilizer from the time of application until 94d after treatment (Fig.1A ).

We focused on resistance to β-lactam antibiotics,such as cephalothin,because this class of drugs is commonly used to treat mastitis in dairy cows.The baseline level of culturable bacteria resistant to cephalothin was substantially higher in un-treated soil than in manure (7.4%vs.0.67%).Consequently,the culturable population of β-lactam –resistant bacteria from day 1was not significantly different in manure-treated soils compared with NPK-treated soils (Fig.1B ).At later time points (days 12–94),significantly higher populations of resistant bacteria were isolated from manure-treated soil samples than from the NPK-treated soil samples,indicating that manure treatment of soil induced the growth of cephalothin-resistant bacteria originating from either the soil or the manure.

Identification of Genes Conferring β-Lactam Resistance.To de-

termine the origin of the culturable antibiotic-resistant bacteria,we constructed five metagenomic fosmid libraries,including four libraries from cultured β-lactam –resistant bacteria isolated from soil after manure or NPK treatment and one library from the manure that had been used for fertilization (Table S1).We used samples obtained at 52d after treatment,because this is when the greatest differences between treatments were detected.From our metagenomic libraries covering a total of 143Gb of DNA,we identified seven unique clones conferring resistance to cepha-lothin (Table 1).Two genes originated from the manure library and five originated from the cultured β-lactam –resistant bacterial community.All seven genes closely matched β-lactamase sequences in GenBank with high sequence identities (68–99%).β-lactamase genes (bla )are grouped into four Ambler classes based on their primary structure (26,27).Phylogenetic analyses assigned the seven unique β-lactamase sequences to three of the four Ambler classes (A,B,and C;Fig.S1),thereby demonstrating that the approach of applying functional metagenomics provides access to a broad range of β-lactamases from two very different environments.

Manure Treatment Induces a Bloom of Cephalothin-Resistant Culturable Soil Bacteria.We assessed the presence and abundance of the

newly identified resistance genes in manure and in soil fertil-ized with manure or NPK over time.PCR with primers specific for each of the seven genes amplified five of them (bla CEP-01,bla CEP-02,bla CEP-03,bla CEP-04,and bla CEP-05)from DNA ex-tracted from soil or manure.Primers directed toward two of the genes,bla CEP-06and bla CEP-07,yielded nonspecific products and were not analyzed further.End-point PCR and qPCR in-dicated that the β-lactamases that originated from manure (bla CEP-01and bla CEP-03)were detected in soil only at early time points after manure treatment (Fig.2A and B ).By 38d after treatment,manure-derived resistance genes were no longer amplified from soil,and these genes were not detected in samples from the NPK-treated beds.Similarly,the genes found in soil bacteria (bla CEP-02,bla CEP-04,and bla CEP-05)were not amplified from manure.Two genes,bla CEP-02and bla CEP-05,were detected in both manure-and NPK-treated soils,but there was no effect of treatment on their relative abundance (Fig.2C ).In contrast,bla CEP-04,which was found in soil before treatment,was significantly enriched in manure-treated soil at all but one time point (day 38)through 94d after treatment.The sequence of bla CEP-04closely matched (96%)a β-lacta-mase from a Pseudomonas sp.,a genus commonly found in soil (Fig.2C ).Taken together,these data demonstrate that the increased β-lactam resistance in manure-treated soil was re-lated not to the persistence of resistant manure bacteria,but rather to an increase in abundance of soil resistance genes,particularly a gene closely related to a β-lactamase from a Pseudomonas sp.

Effects of Manure and NPK Treatments on Microbial Communities.

Culturing and qPCR suggested that the increase in cephalothin-resistant bacteria after manure treatment was attributed to growth of a Pseudomonas sp.native to soil,prompting us to explore the effect of manure treatment on the composition of the soil bacterial community.Quality-filtered sequences of 117,65716S rRNA genes amplified from metagenomic DNA from a total of 49soil and manure samples were analyzed.This corresponded to an average of 2,401sequences per sample,with an average read length of 477bp.Sequence clustering yielded a total of 7,018operational taxonomic units (OTUs),each containing sequences that shared at least 97%identity.Manure and NPK treatments resulted in distinct soil community structures (Fig.3;adonis:R 2=16%,P <0.001).Manure-treated soil had less phylogenetic di-versity than NPK-treated soil (P <0.001,Welch ’s t test).Com-pared with manure treatment,NPK did not significantly (P =0.08)affect the number of taxa in soil (species richness),although nine OTUs that affiliated with the Rhizobiales,Xanthomonadales,or Acidobacteria were significantly enriched by NPK treatment (Fig.4A ).Bacterial communities in manure were distinct from those in both treated and untreated soil communities (Fig.S2)and were excluded from β-diversity analyses.Bacterial communities in replicate samples from manure-treated soils formed temporally distinct clusters,whereas the communities in samples from NPK-treated soils clustered more randomly,with no obvious concor-dance with time of sampling (Fig.4and Fig.S2).The time effect was significant (adonis:R 2=18%,P <0.001),but by 130d after treatment,the manure-treated communities were similar to NPK-treated communities (Fig.3).

Manure Treatment Enriches Taxa That Commonly Carry Resistance to β-Lactam Antibiotics.The addition of manure affected the soil

community structure differently than the addition of NPK fertil-izer.The abundance of 10taxa that originated from soil increased in soil after manure treatment (Fig.4A ).Conversely,the abun-dance of eight taxa present in manure but not in untreated soil were found in manure-treated soil and decreased over time,

Days after treatment Days after treatment

Soil + NPK

L o g (t o t a l C F U /g )

L o g (r e s i s t a n t C F U /g )

Fig.1.Effects of manure on the abundances of culturable soil bacteria.Dynamics of total (A )and cephalothin-resistant (B )culturable bacteria in soil after treatment with manure or inorganic fertilizer (NPK).Each value is the mean ±SD of three replicates.All time points,except day 38,revealed sig-nificantly more culturable CFU in manure-treated soil until day 94after treatment (*P <0.05,multiple t test).Dotted line indicates the average populations of total and resistant soil bacteria before treatment.

2of 6|https://www.wendangku.net/doc/5c10113967.html,/cgi/doi/10.1073/pnas.1409836111

Udikovic-Kolic et al.

获取更多语音讲解，关注订阅号钢钢发声

following a trajectory similar to that of manure-derived β-lacta-mases (Fig.4B ).Two soil OTUs that affiliated with the Pseu-domonadaceae family (OTUs 726and 1500),one of which affiliated with the genus Pseudomonas (OTU 726),and a group of OTUs affiliated with the Janthinobacterium genus (OTUs 8555and 2161;Fig.4E and F )were highly enriched in manure-treated soils,but were present at low abundance in NPK-treated soils and were not found in manure (Fig.4C and D ).This is especially in-teresting given that the metallo-β-lactamase –encoding gene bla CEP-07identified in the functional metagenomic screen is highly similar (95%)to a gene from Janthinobacterium lividum .A third group of OTUs that were highly enriched by manure treatment but not de-tected in NPK-treated soils affiliated with Psychrobacter pulmonis (OTUs 9776,4110,and 9413;Fig.4G ).OTUs affiliated with Pseudomonas and Janthinobacterium genera were abundant at later dates (between days 38and 66after treatment),indicating that their dynamics differed from those of Psychrobacter in re-sponse to manure.The community analyses demonstrate that manure treatment had a greater effect than NPK on soil com-munity richness and structure.We identified significant shifts of certain phylotypes and confirmed a higher abundance of Pseu-domonas spp.at the total community level.Other β-lactamase –harboring bacteria (Janthinobacterium sp.and P.pulmonis )were also enriched in the manure-treated soils,likely explaining the altered antibiotic-resistance gene profile of manure-treated soils.Discussion

The increasing prevalence of antibiotic-resistance genes among both clinically important pathogens and environmental bacteria is a global threat to human health in the 21st century.It is im-perative to understand the sources and behaviors of resistance genes to enable the development of strategies to reduce their abundance and dissemination.Current knowledge does not pro-vide a sufficiently detailed portrait of the evolution and movement of antibiotic-resistance genes to enable reliable predictions about their behavior or the design of precise strategies to manage them.Livestock operations that use antibiotics are closely associated with increases in antibiotic-resistant bacteria in animal caretakers (28,29),meat processors (30),and others who live in the vicinity of livestock facilities (31).Manure from antibiotic-treated animals provides a direct source of antibiotics and antibiotic-resistant bac-teria,and thus application of manure to soil as a fertilizer frequently

increases levels of antibiotic-resistant bacteria and their genes in the soil (2,32).These increases have been traced to introduction of manure containing resistance genes and resistant organisms (3),residual antibiotics that select for resistant bacteria already resident in the soil (32),or a combination of these contributors.Here we present evidence of an additional mechanism by which manure increases antibiotic-resistant bacteria.Manure from ani-mals that had not been treated with antibiotics induced a bloom of Pseudomonas and Janthinobacterium spp.and increased the abundance of a gene encoding AmpC,a β-lactamase commonly found in these genera along with metallo-β-lactamases.

We identified two β-lactamases in manure that share signifi-cant similarity with β-lactamases from members of the Gram-negative anaerobic genera Selenomonas (99%)and

Bacteroides

Soil + Manure

Soil + NPK

101010101010

101010101

1010101010Days after treatment

r e

u r e Days after treatment

b e

f o

r e

412

94130m a n u r e

1010101010101010Days after treatment

L o g (b l a /16S )

bla bla Fig.2.Dynamics of manure-derived and soil-derived β-lactamases in soil after treatment with manure or NPK.(A and B )End-point PCR amplification (A )and qPCR amplification (B )of β-lactamases bla CEP-01and bla CEP-03iden-tified from manure in soil.(C )Dynamics of the relative abundance of the β-lactamases bla CEP-02,bla CEP-05,and bla CEP-04in soil after treatment with manure or inorganic fertilizer (NPK)measured by qPCR.Each value is the mean ±SD of three biological replicates calculated from three technical replicates of each.The dotted line indicates the average number of gene copies before treatment.Significance (P <0.05)indicated by one-way ANOVA is indicated with asterisks.None of the genes presented here were detected in manure samples.

Table 1.Cephalothin-resistance genes identified in clones of functional metagenomic libraries built in this study Gene

designation GenBank accession no.Library Cephalothin MIC,μg/mL

Length,aa e-value %ID Accession no.of closest

match

Closest match (BLASTX)

bla CEP-01KM113767MAN_uncultured 64284099WP_019542800β-lactamase

(Selenomonas bovis )

bla CEP-02KM113768B04_cultured 2563160100WP_016087836β-lactamase 3

(Bacillus cereus )

bla CEP-03KM113769MAN_uncultured 64455068WP_005841913β-N -acetyl-glucosaminidase

(Bacteroides vulgatus )

bla CEP-04

KM113770

B06_cultured

512

385

WP_020798140β-lactamase class

C [Pseudomonas sp.G5(2012)]

bla CEP-05KM113771

B04_cultured 256377091

ACH58999LRA-10

(uncultured bacterium BLR10)

B06_cultured B07_cultured bla CEP-06KM113772B09_cultured 2562807E-17382BAL14456Metallo-β-lactamase

(Serratia marcescens )

bla CEP-07

KM113773

B09_cultured

256

288

3E-158

ABK64020Metallo-β-lactamase

(Janthinobacterium lividum )

The results of the alignment to the best BLASTX hits,their identity (%),and accession numbers in the nonredundant GenBank database are provided.MIC,minimal inhibitory concentration.

Udikovic-Kolic et al.

PNAS Early Edition |3of 6

M I C R O B I O L O G Y

获取更多语音讲解，关注订阅号钢钢发声

(68%).PCR and qPCR results revealed that neither the β-lac-tamase –encoding genes nor bacteria found in manure persisted in the soil over time.These results suggest that the antibiotic-resistant organisms and the genes responsible for their re-sistance were enriched from among resident soil bacteria,rather than introduced in the manure.

Our analysis identified five β-lactamases from cultured soil bacteria.Soil is a reservoir of divergent β-lactamases,irrespective

of anthropogenic influences (33,34).Elevated abundance of a gene encoding an AmpC-family β-lactamase persisted for up to 130d after manure treatment,as did elevated populations of Pseudomonas spp.,which are common sources of β-lactamases in nosocomial infections (35,36).In addition,metallo-β-lactamases similar to those that we found in soil are a serious threat to human health.Metallo-β-lactamases hydrolyze a broad spectrum of target β-lactams,and there are no metallo-β-lactamase inhibitors approved for clinical use.Other highly abundant taxa in manure-treated soils were Psychrobacter spp.,composing up to 10%of all sequences at day 12.Psychrobacter spp.are halotolerant and psy-chrotolerant and are abundant in ornithogenic soil in Antarctica (37).They also have been found to harbor AmpC-β-lactamases (38).Given that we conducted our sampling during winter,and that manure is typically very saline,our identification of a Psychrobacter sp.is not surprising.

A concern raised by our findings is the possibility that these resistance genes might spread from residents of farm soil to human pathogens.This is especially important given the use of manure fertilization in cropping systems.Genes encoding AmpC-type and metallo-β-lactamases are typically located on the chromosome (39,40),however,making the risk of transfer low.Furthermore,a re-cent study demonstrated that although novel antibiotic-resistance genes were commonly isolated from soil,with β-lactamases among the most abundant,mobility elements were rarely associated with these resistance genes (41).The lack of mobile elements in the regions flanking the resistance genes that we found in soil rein-forces previous work suggesting that despite their abundance and rapid response to selection pressure,many antibiotic-resistance genes from soil-dwelling bacteria may face barriers that prevent their spread to clinical settings.

Even in the absence of transferability,elevated abundance of antibiotic-resistant bacteria is a threat when the resistance

NMDS1

N M D S 2

Fig.3.Temporal changes in soil community structures before treatment and after treatment with manure or NPK.Bray –Curtis similarity coefficients were calculated from relative OTU abundances of bacterial soil communities across three biological replicates of soil before treatment,treated with manure or treated with inorganic fertilizer (NPK),and plotted on a non-metric multidimensional scaling (NMDS)graph.The 2D stress was 0.12.In-creasing symbol size indicates time since manure treatment.Treatment effects were significant in all adonis combinations (P <0.001;soil and manure-treated communities,R 2=13%;soil before treatment and manure-treated communities,R 2=15%;soil before and NPK-treated communities,R 2=6%).

121201119494

66665252665238383894

13013038669466130949452386638152112121301300001201305201

01356913b e f o r m a n u r n =1n =61356913

b e

f o r m a n u r n =1Janthinobacterium sp. OTUs Psychrobacter pulmonis OTUs days after treatment

1356913

b e f o

r m a n u r n =1Pseudomonas sp. OTUs 1356913

b e f o r

m a n u r n =1Janthinobacterium sp. OTUs r e l a t i v e a b u n d a n c e o f t a x a

1356913b e f o r m a n u r n =1n =61356913b e f

o r m a n u r n =1Manure OTUs

Pseudomonas sp. OTUs r e l a t i v e a b u n d a n c e o f t a x a

Heatmap most abundant OTUs

day

relative abundance

most abundant taxa

all taxa O T U I D

T a x o n o m y

r e l a t i v e a b u n d a n c e o f t a x a

days after treatment

A F

Fig.4.Dynamics of the most abundant OTUs and β-lactamase harboring OTUs in response to treatment with manure or NPK.(A )Heat map of the relative abundance of the 59most abundant OTUs composing more than 10%across all 49samples.OTUs with similar occurrence patterns are highlighted with the same colors.The OTUs in bold type were inspected more closely.(B )Relative abundance of manure OTUs found in soil.(C )Relative abundance of three P.pulmonis OTUs enriched in manure-treated soil.(D )Relative abundance of two Pseudomonadaceae OTUs enriched in soil after manure treatment.(E )Relative abundance of two Pseudomonadaceae OTUs in soil treated with inorganic fertilizer (NPK).(F )Relative abundance of two Janthinobacterium OTUs enriched in soil after manure treatment.(G )Relative abundance of two Janthinobacterium OTUs in soil treated with NPK.P.pulmonis OTUs were not detected in NPK-treated soils,nor were they found in manure.Unless stated otherwise,all means and SDs were calculated from three biological replicates.

4of 6|https://www.wendangku.net/doc/5c10113967.html,/cgi/doi/10.1073/pnas.1409836111

Udikovic-Kolic et al.

获取更多语音讲解，关注订阅号钢钢发声

determinants reside in organisms such as Pseudomonas and Janthinobacterium spp.,which are opportunistic human patho-gens(42,43).The enrichment of these antibiotic-resistant strains could increase the likelihood of their entry into the food chain on crops grown in manured soil.Pseudomonas spp.are of particular concern because they are used in agricultural settings as disease-suppressive and plant growth-promoting agents(44), and are also responsible for nosocomial infections(45).In this regard,we queried the Pseudomonas genome database(46)to assess the presence and abundance ofβ-lactamase genes.Of the 49fully sequenced Pseudomonas strains,38contain AmpC-type β-lactamases,including most of those used in agriculture(47), and all but three strains contain at least one metallo-β-lactamase. Moreover,a recent study reported an increase in the abundance of Pseudomonas spp.in response to soil amendment with pig manure (48),suggesting that Pseudomonas spp.may be particularly re-sponsive to manure amendment and important environmental reservoirs ofβ-lactam antibiotic-resistance genes.

A focus of future research will be on identifying the compo-nent of manure that leads to the proliferation of bacteria that carryβ-lactamases.The nutrients in manure may favor the growth of Pseudomonas and Janthinobacterium spp.,which are fast-growing and well adapted to many environmental stresses (44,49).If nutrients are the key driver,then there may be a similar enhancement of antibiotic-resistant bacteria around plant roots,which secrete vast amounts of carbon into the soil.In addition to nutrients,heavy metals,which are commonly used as additives in animal feed,are alternative candidates that might impose a selection pressure.Heavy metals have been shown to coselect for metal resistance and antibiotic resistance(50–52). Interestingly,even low concentrations of metal in soils select for resistance to various antibiotic classes,includingβ-lactams(53), owing to shared genetic or physiological mechanisms of re-sistance to antibiotics and metals(50).Another future direction is to determine whether other types of antibiotic-resistance genes respond similarly to manure treatment and whether manures from other animals enrich the same genera.If the mechanism depends on the fact that genera such as Pseudomonas are highly adaptable and resistant to stress,then we would expect a similar response to other manure types and cows fed a different diet, which is consistent with previous work(48).

Our study highlights the role of unexpected selection pressures that increase the abundance of resident soil bacteria that are resistant to antibiotics.This finding indicates the importance of understanding the behavior of antibiotic-resistance genes in the environment,including their response to agricultural practices and movement into the food supply.These observations invite further study of the abundance of antibiotic-resistant bacteria on vegeta-bles,which are often raised in manure-fertilized soil and eaten raw,thereby providing a potential route for antibiotic-resistance genes to migrate from the environment to human ecosystems. Materials and Methods

Field Experiment,Soil Fertilization,and Soil Sampling.The field experiment was conducted at the Yale Farm at West Campus(West Haven,CT).Three replicate vegetable beds with no previous history of manure application were used for each treatment.The soil consisted of loamy sand soil(79%sand,16% silt,and5%clay;pH7.4)with an organic matter content of87g kg?1and a total nitrogen content of3.7g kg?1.

The manure used in this study was collected from the pens of dairy cows that had not been treated with antibiotics(University of Connecticut)and tested for nutrient(NPK)and heavy metal content(Table S2).The level of Zn detected in the manure was considerably higher than its maximum recom-mended limit in animal manure for land application(54).The manure was incorporated into soil beds(depth,15cm)at a level of20kg per bed.Beds treated with NPK were amended with the same amount of each nutrient present in the manure.On each sampling day,10soil cores(1.5cm in diameter and 12cm deep)were collected at random and pooled.Bacteria were cultured from beds at1d before(day0)manure or NPK treatment and then at10time points after treatment:days1,12,19,38,52,66,80,94,108,and130.At day4after treatment,samples were collected for qPCR analyses,but not cultured(Fig.S3). Culture-Based Isolation of Bacteria from Soil and Manure and Construction of Metagenomic Libraries.To quantify populations of culturable bacteria in soil and manure,samples from serial10-fold dilutions in PBS were cultured on R2A agar plates(Remel)with and without cephalothin(50mg/mL).CFU were counted after a5-d incubation at28°C.Cephalothin-resistant bacteria were scraped from the plates(10?3dilutions),pooled,and stored at?80°C.Fro-zen samples obtained at52d after manure or NPK application were used to build metagenomic libraries.

Fosmid libraries were constructed from two pools of cultured soil bacteria from each treatment and the manure sample.DNA for libraries was extracted using the Aurora technology(https://www.wendangku.net/doc/5c10113967.html,)and subjected to end-repair and ligation with the pCC2Fos vector(Epicentre),following the manufacturer’s recommendations.Library storage and size estimation were performed according to previously published protocols(55).

Identification of Clones Resistant to Cephalothin.The pooled clones from each metagenomic library were grown in50mL of LB supplemented with chlor-amphenicol(20μg/mL)for2h at37°C and200rpm.Cultures were plated on LB containing chloramphenicol(20μg/mL)and cephalothin(50μg/mL).As-sessment of the diversity of resistant fosmid clones,minimum inhibitory concentration assays,and subcloning of genes conferring resistance to ceph-alothin were conducted according to previously published protocols(55). Plasmid DNA from resistant subclones was purified using the Qiaprep Kit (Qiagen)and sequenced at the DNA Analysis Facility at Yale University(http:// https://www.wendangku.net/doc/5c10113967.html,).Geneious version6.0.5was used for protein sequence comparison,and phylogenetic analyses were conducted with CLUSTALW(56).

DNA Extraction,qPCR,and End-Point PCR.DNA was extracted from soil and manure for qPCR and16S rRNA gene sequencing with the ZR Fecal DNA MiniPrep Kit according to the manufacturer’s recommendations and further purified using the nucleic Aurora acid purification instrument(Boreal Genomics).Amplification was done using10ng of DNA with iQ Supermix (Bio-Rad)in a total volume of20μL.Primers used for qPCR are listed in Table S3.Thermal cycling conditions for all but one primer pair(i.e.,PrNU.K.017/ PrNU.K.018)were as follows:an initial denaturation step of3min at95°C, 45cycles of15s at95°C,and1min at60°C.For PrNU.K.017/PrNU.K.018,an annealing/extension temperature of56°C was used.Specificity of primer pairs was verified by melting curve analysis.PCR efficiency was tested with serial dilutions of DNA samples,and all ranged between89%and103%.The relative abundance of antibiotic-resistance genes was calculated by dividing the respective bla gene abundance by16S rRNA gene copy number.End-point PCR was performed using Hotstar Taq DNA Polymerase(Qiagen) according to the manufacturer’s recommendations,and PCR products were separated on a3%agarose gel.

Pyrosequencing and Sequence Analyses of16S rRNA Genes.Multiplexed pyrosequencing(Roche454FLX with Titanium reagents)from the V1-V3 regions of bacterial16S rRNA genes was used to assess changes in bacterial soil community structure before and after manure or NPK treatment.Aurora-purified DNA was amplified and sequenced using standard protocols at the Research and Testing Laboratories,Lubbock,TX(www.researchandtesting. com).Primers28forward and519reverse were chosen(57).The default workflow in QIIME v1.7was used for sequence processing,quality control, and OTU selection.Each sample was rarefied to904sequences.Alpha di-versity was described for each sample using Faith’s phylogenetic diversity (58)and the number of OTUs(defined at97%sequence identity).Differ-ences were assessed using Welch’s t test.Permuted ANOVA was performed to test for differences in treatment and time using the“adonis”function in the R environment for statistical computing(59)with the“vegan”commu-nity ecology package(60).

A heat map visualization of OTUs with relative abundances was performed with the heatmap.2function of the“gplots”package.Only the OTUs that each composed more than0.1of the relative abundance across all samples were included(top59OTUs).A Mantel test with Pearson’s correlation co-efficient between the full and reduced datasets of Bray–Curtis matrices revealed that they were highly correlated(r=0.91,P<0.001).Additional information on is provided in SI Materials and Methods.

Sequences were deposited in MG-RAST,https://www.wendangku.net/doc/5c10113967.html,(pro-ject ID8945,“YaleFarmSoil”;metagenomes4562248.3–4562264.3,4562266.3–4562295.3,and4570842.3;Table S4)and the GenBank database(accession nos. KM113767–KM113773)(Table1).

Udikovic-Kolic et al.PNAS Early Edition|5of6M I C R O B I O L O G Y

获取更多语音讲解，关注订阅号钢钢发声

ACKNOWLEDGMENTS.We thank Prof.Sheila Andrew and Mary Margaret Cole(University of Connecticut)for coordinating access to the Kellogg Dairy Unit and Justin Freiberg for providing plots at the Yale Farm at West Campus. We thank Ashley Ferguson and Nicole Price for technical assistance,Ashley Shade for invaluable support in bacterial community analyses,and Philipp Engel for a critical review of an earlier version of the manuscript.This research was funded by a grant from the Fulbright Foundation,Swiss National Science Foundation Grant PBZHP3-138800,US National Science Foundation Grant MCB-1243671,and US National Institutes of Health Grant1R13GM090574and Kirschstein National Research Service Award T32Training Grant for Genomics and Proteomics5T32HG003198(to Yale University).

1.65.Federal Register246(2000),pp80548–80596.

2.Heuer H,Smalla K(2007)Manure and sulfadiazine synergistically increased bacterial

antibiotic resistance in soil over at least two months.Environ Microbiol9(3):657–666.

3.Heuer H,et al.(2011)Accumulation of sulfonamide resistance genes in arable soils

due to repeated application of manure containing sulfadiazine.Appl Environ Mi-crobiol77(7):2527–2530.

4.Jechalke S,et al.(2013)Increased abundance and transferability of resistance genes

after field application of manure from sulfadiazine-treated pigs.Appl Environ Mi-crobiol79(5):1704–1711.

5.Marti R,et al.(2013)Impact of manure fertilization on the abundance of antibiotic-

resistant bacteria and frequency of detection of antibiotic resistance genes in soil and on vegetables at harvest.Appl Environ Microbiol79(18):5701–5709.

6.Zhou Z,Raskin L,Zilles JL(2010)Effects of swine manure on macrolide,lincosamide,

and streptogramin B antimicrobial resistance in soils.Appl Environ Microbiol76(7): 2218–2224.

7.Ferens WA,Hovde CJ(2011)Escherichia coli O157:H7:Animal reservoir and sources of

human infection.Foodborne Pathog Dis8(4):465–487.

8.Zhou W,Wang Y,Lin J(2012)Functional cloning and characterization of antibiotic

resistance genes from the chicken gut microbiome.Appl Environ Microbiol78(8): 3028–3032.

9.Zhu YG,et al.(2013)Diverse and abundant antibiotic resistance genes in Chinese

swine farms.Proc Natl Acad Sci USA110(9):3435–3440.

10.Binh CTT,Heuer H,Kaupenjohann M,Smalla K(2008)Piggery manure used for soil

fertilization is a reservoir for transferable antibiotic resistance plasmids.FEMS Mi-crobiol Ecol66(1):25–37.

11.Smith KE,et al.(2004)Outbreaks of enteric infections caused by multiple pathogens

associated with calves at a farm day camp.Pediatr Infect Dis J23(12):1098–1104. 12.Durso LM,Harhay GP,Bono JL,Smith TPL(2011)Virulence-associated and antibiotic

resistance genes of microbial populations in cattle feces analyzed using a meta-genomic approach.J Microbiol Methods84(2):278–282.

13.Schwaiger K,et al.(2009)Tetracycline in liquid manure selects for co-occurrence of

the resistance genes tet(M)and tet(L)in Enterococcus faecalis.Vet Microbiol139(3-4): 386–392.

14.Bibbal D,et al.(2007)Impact of three ampicillin dosage regimens on selection of

ampicillin resistance in Enterobacteriaceae and excretion of blaTEM genes in swine feces.Appl Environ Microbiol73(15):4785–4790.

15.Blake DP,et al.(2003)Influence of tetracycline exposure on tetracycline resistance

and the carriage of tetracycline resistance genes within commensal Escherichia coli populations.J Appl Microbiol94(6):1087–1097.

16.Looft T,et al.(2012)In-feed antibiotic effects on the swine intestinal microbiome.

Proc Natl Acad Sci USA109(5):1691–1696.

17.D’Costa VM,et al.(2011)Antibiotic resistance is ancient.Nature477(7365):457–461.

18.Stanton TB,Humphrey SB,Stoffregen WC(2011)Chlortetracycline-resistant intestinal

bacteria in organically raised and feral swine.Appl Environ Microbiol77(20): 7167–7170.

19.Andrews RE,Jr,Johnson WS,Guard AR,Marvin JD(2004)Survival of enterococci and

Tn916-like conjugative transposons in soil.Can J Microbiol50(11):957–966.

20.Chee-Sanford JC,Aminov RI,Krapac IJ,Garrigues-Jeanjean N,Mackie RI(2001)Oc-

currence and diversity of tetracycline resistance genes in lagoons and groundwater underlying two swine production facilities.Appl Environ Microbiol67(4):1494–1502.

21.Binh CTT,et al.(2007)Short-term effects of amoxicillin on bacterial communities in

manured soil.FEMS Microbiol Ecol62(3):290–302.

22.Sengel?v G,et al.(2003)Bacterial antibiotic resistance levels in Danish farmland as

a result of treatment with pig manure slurry.Environ Int28(7):587–595.

23.Oliver SP,Murinda SE,Jayarao BM(2011)Impact of antibiotic use in adult dairy cows

on antimicrobial resistance of veterinary and human pathogens:A comprehensive review.Foodborne Pathog Dis8(3):337–355.

24.Jechalke S,et al.(2014)Structural and functional response of the soil bacterial

community to application of manure from difloxacin-treated pigs.FEMS Microbiol Ecol87(1):78–88.

25.Nacke H,et al.(2011)Pyrosequencing-based assessment of bacterial community

structure along different management types in German forest and grassland soils.

PLoS ONE6(2):e17000.

26.Ambler RP(1980)The structure of beta-lactamases.Philos Trans R Soc Lond B Biol Sci

289(1036):321–331.

27.Jacoby GA,Munoz-Price LS(2005)The new beta-lactamases.N Engl J Med352(4):

380–391.

28.Nijsten R,London N,van den Bogaard A,Stobberingh E(1996)Antibiotic resistance

among Escherichia coli isolated from faecal samples of pig farmers and pigs.

J Antimicrob Chemother37(6):1131–1140.

29.Nijsten R,London N,van den Bogaard A,Stobberingh E(1996)In vitro transfer of

antibiotic resistance between faecal Escherichia coli strains isolated from pig farmers and pigs.J Antimicrob Chemother37(6):1141–1154.30.Boost M,Ho J,Guardabassi L,O’Donoghue M(2013)Colonization of butchers with

livestock-associated methicillin-resistant Staphylococcus aureus.Zoonoses Public Health60(8):572–576.

31.Kozak GK,Boerlin P,Janecko N,Reid-Smith RJ,Jardine C(2009)Antimicrobial re-

sistance in Escherichia coli isolates from swine and wild small mammals in the prox-imity of swine farms and in natural environments in Ontario,Canada.Appl Environ Microbiol75(3):559–566.

32.Ghosh S,LaPara TM(2007)The effects of subtherapeutic antibiotic use in farm ani-

mals on the proliferation and persistence of antibiotic resistance among soil bacteria.

ISME J1(3):191–203.

33.Allen HK,Moe LA,Rodbumrer J,Gaarder A,Handelsman J(2009)Functional meta-

genomics reveals diverse beta-lactamases in a remote Alaskan soil.ISME J3(2): 243–251.

34.Demanèche S,et al.(2008)Antibiotic-resistant soil bacteria in transgenic plant fields.

Proc Natl Acad Sci USA105(10):3957–3962.

35.D’Costa VM,McGrann KM,Hughes DW,Wright GD(2006)Sampling the antibiotic

resistome.Science311(5759):374–377.

36.Gupta V,Sidhu S,Chander J(2012)Metallo-β-lactamase–producing nonfermentative

gram-negative bacteria:An increasing clinical threat among hospitalized patients.

Asian Pac J Trop Med5(9):718–721.

37.Bowman JP,Cavanagh J,Austin JJ,Sanderson K(1996)Novel Psychrobacter species

from Antarctic ornithogenic soils.Int J Syst Bacteriol46(4):841–848.

38.Feller G,Zekhnini Z,Lamotte-Brasseur J,Gerday C(1997)Enzymes from cold-adapted

microorganisms:The class C beta-lactamase from the antarctic psychrophile Psy-chrobacter immobilis A5.Eur J Biochem244(1):186–191.

39.Rossolini GM,et al.(2001)Metallo-beta-lactamase producers in environmental mi-

crobiota:New molecular class B enzyme in Janthinobacterium lividum.Antimicrob Agents Chemother45(3):837–844.

40.Jacoby GA(2009)AmpCβ-lactamases.Clin Microbiol Rev22(1):161–182.

41.Forsberg KJ,et al.(2014)Bacterial phylogeny structures soil resistomes across hab-

itats.Nature509(7502):612–616.

42.Patijanasoontorn B,et al.(1992)Hospital-acquired Janthinobacterium lividum septi-

cemia in Srinagarind Hospital.J Med Assoc Thai75(2,Suppl2):6–10.

43.Yoshino Y,et al.(2011)Pseudomonas putida bacteremia in adult patients:Five case

reports and a review of the literature.J Infect Chemother17(2):278–282.

44.Weller DM(2007)Pseudomonas biocontrol agents of soilborne pathogens:Looking

back over30years.Phytopathology97(2):250–256.

45.Berg G,Eberl L,Hartmann A(2005)The rhizosphere as a reservoir for opportunistic

human pathogenic bacteria.Environ Microbiol7(11):1673–1685.

46.Winsor GL,et al.(2011)Pseudomonas Genome Database:Improved comparative

analysis and population genomics capability for Pseudomonas genomes.Nucleic Acids Res39(Database issue):D596–D600.

47.Shen X,Hu H,Peng H,Wang W,Zhang X(2013)Comparative genomic analysis of four

representative plant growth-promoting rhizobacteria in Pseudomonas.BMC Ge-nomics14:271.

48.Ding GC,et al.(2014)Dynamics of soil bacterial communities in response to repeated

application of manure containing sulfadiazine.PLoS ONE9(3):e92958.

49.Pantanella F,et al.(2007)Violacein and biofilm production in Janthinobacterium

lividum.J Appl Microbiol102(4):992–999.

50.Seiler C,Berendonk TU(2012)Heavy metal-driven co-selection of antibiotic resistance

in soil and water bodies impacted by agriculture and aquaculture.Front Microbiol3: 399.

51.Baker-Austin C,Wright MS,Stepanauskas R,McArthur JV(2006)Co-selection of an-

tibiotic and metal resistance.Trends Microbiol14(4):176–182.

52.Ji X,et al.(2012)Antibiotic resistance gene abundances associated with antibiotics

and heavy metals in animal manures and agricultural soils adjacent to feedlots in Shanghai,China.J Hazard Mater235-236:178–185.

53.Knapp CW,et al.(2011)Antibiotic resistance gene abundances correlate with metal

and geochemical conditions in archived Scottish soils.PLoS ONE6(11):e27300.

54.Birchall S,Dillon C,Wrigley R(2008)Effluent and manure management database for

the Australian dairy industry.Available at https://www.wendangku.net/doc/5c10113967.html,/index.php?

id=48.Accessed January16,2014.

55.Wichmann F,Udikovic-Kolic N,Andrew S,Handelsman J(2014)Diverse antibiotic

resistance genes in dairy cow manure.MBio5(2):e01017.

https://www.wendangku.net/doc/5c10113967.html,rkin MA,et al.(2007)ClustalW and ClustalX version 2.Bioinformatics23(21):

2947–2948.

57.Hartmann M,et al.(2012)Significant and persistent impact of timber harvesting on

soil microbial communities in Northern coniferous forests.ISME J6(12):2199–2218.

58.Faith DP(1992)Conservation,evaluation and phylogenetic diversity.Biol Conserv

61(1):1–10.

59.R Development Core Team(2011)R:A language and environment for statistical

computing.Available at https://www.wendangku.net/doc/5c10113967.html,.Accessed December15,2013.

60.Oksanen JF,Blanchet G,Kindt R,Legendre P,Minchin PR,et al.(2011)Vegan:Com-

munity ecology package.R package version2.0.Available at https://www.wendangku.net/doc/5c10113967.html,.Accessed December6,2013.

6of6|https://www.wendangku.net/doc/5c10113967.html,/cgi/doi/10.1073/pnas.1409836111Udikovic-Kolic et al.

获取更多语音讲解，关注订阅号钢钢发声