Bloom of resident antibiotic-resistant bacteria in soil

following manure fertilization

Nikolina Udikovic-Kolica,b,1, Fabienne Wichmanna,c,1, Nichole A. Brodericka, and Jo Handelsmana,2
a
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06511; bDivision for Marine and Environmental Research,
Rudjer Boskovic Institute, Zagreb 10000, Croatia; and cBiosafety Research, State Laboratory Basel, 4012 Basel, Switzerland

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 mechanisms 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 microbiome and resistome is not known. To gain insight into this impact, we cultured bacteria from soil before and at 10 time points
after application of manure from cows that had not received antibiotic treatment. Soil treated with manure contained a higher
abundance of -lactamresistant bacteria than soil treated with
inorganic fertilizer. Functional metagenomics identified -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 harbor -lactamases. Quantitative PCR indicated that manure treatment enriched the blaCEP-04 gene, which is highly similar (96%) to
a gene found previously in a Pseudomonas sp. Analysis of 16S
rRNA genes indicated that the abundance of Pseudomonas spp.
increased in manure-amended soil. Populations of other soil bacteria that commonly harbor -lactamases, including Janthinobacterium sp. and Psychrobacter pulmonis, also increased in response
to manure treatment. These results indicate that manure amendment 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 demonstrate the need for empirical analysis of the agroecosystem.
dairy cow manure

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 intensive sulfonamide use can lead to an increase of antibioticresistance 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 use -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 community 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 resident soil bacteria harboring genes for resistance to -lactam
antibiotics, whereas inorganic fertilizers did not. These results
demonstrate the complexity, and at times nonintuitive consequences, of agricultural practices.
Significance

| -lactam antibiotics

The increasing prevalence of antibiotic-resistant bacteria is one

of the most serious threats to public health in the 21st century.
One route by which resistance genes enter the food system
is through amendment of soils with manure from antibiotictreated animals, which are considered a reservoir of such
genes. Previous studies have associated application of pig
manure with the dispersal of sulfonamide-resistance genes to
soil bacteria. In this study, we found that dairy cow manure
amendment enhanced the proliferation of resident antibioticresistant bacteria and genes encoding -lactamases in soil even
though the cows from which the manure was derived had not
been treated with antibiotics. Our findings provide previously
unidentified insight into the mechanism by which amendment
with manure enriches antibiotic-resistant bacteria in soil.

griculture affects human health through both the consumption and production of food for the human diet. Manure 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 (16).
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 to 90
120 d 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 antibioticresistant bacteria, antibiotic-resistance genes (collectively known
as the resistome), and pathogens (2, 712). Although antibiotic
use increases antibiotic-resistance genes and resistant bacteria in
manure (1316), 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
www.pnas.org/cgi/doi/10.1073/pnas.1409836111

Author contributions: N.U.-K., F.W., and J.H. designed research; N.U.-K. and F.W. performed 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,
http://metagenomics.anl.gov/ (project ID 8945, YaleFarmSoil; metagenomes 4562248.3
4562264.3, 4562266.34562295.3, and 4570842.3) and GenBank (accession nos.
KM113767KM113773).
1

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

To whom correspondence should be addressed. Email: jo.handelsman@yale.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.

1073/pnas.1409836111/-/DCSupplemental.

PNAS Early Edition | 1 of 6

MICROBIOLOGY

Edited by W. Ford Doolittle, Dalhousie University, Halifax, Canada, and approved September 8, 2014 (received for review May 28, 2014)

Results
Manure Fertilization Increases Total and -LactamResistant 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 -lactam
resistant culturable bacteria in soil. We cultured soil before
fertilization and at 10 time points after application of manure or
NPK. Culturing indicated that manure directly added 107 colonyforming units (CFU) per gram of soil, which approximately doubled 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 94 d after
treatment (Fig. 1A).
We focused on resistance to -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 untreated soil than in manure (7.4% vs. 0.67%). Consequently, the
culturable population of -lactamresistant bacteria from day 1
was 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 NPKtreated 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 -Lactam Resistance. To determine the origin of the culturable antibiotic-resistant bacteria,
we constructed five metagenomic fosmid libraries, including four
libraries from cultured -lactamresistant 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 52 d after treatment, because this is when
the greatest differences between treatments were detected. From
our metagenomic libraries covering a total of 143 Gb of DNA,
we identified seven unique clones conferring resistance to cephalothin (Table 1). Two genes originated from the manure library and
five originated from the cultured -lactamresistant bacterial
community. All seven genes closely matched -lactamase sequences
in GenBank with high sequence identities (6899%). -lactamase
genes (bla) are grouped into four Ambler classes based on their
primary structure (26, 27). Phylogenetic analyses assigned the seven
unique -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 -lactamases from two very different environments.

Soil + Manure

Apr

May

Jan

Feb

Mar

Apr

May

9.5

7.5
7.0
6.5

8.5
8.0
7.5

94

8.0

9.0

80

66

38

52

7.0
6.5

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 significantly more culturable CFU in manure-treated soil until day 94 after
treatment (*P < 0.05, multiple t test). Dotted line indicates the average
populations of total and resistant soil bacteria before treatment.

2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1409836111

newly identified resistance genes in manure and in soil fertilized with manure or NPK over time. PCR with primers specific
for each of the seven genes amplified five of them (blaCEP-01,
blaCEP-02, blaCEP-03, blaCEP-04, and blaCEP-05) from DNA extracted from soil or manure. Primers directed toward two of
the genes, blaCEP-06 and blaCEP-07, yielded nonspecific products
and were not analyzed further. End-point PCR and qPCR indicated that the -lactamases that originated from manure
(blaCEP-01 and blaCEP-03) were detected in soil only at early time
points after manure treatment (Fig. 2 A and B). By 38 d 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 (blaCEP-02, blaCEP-04, and blaCEP-05) were not
amplified from manure. Two genes, blaCEP-02 and blaCEP-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, blaCEP-04, which was found in soil before
treatment, was significantly enriched in manure-treated soil at
all but one time point (day 38) through 94 d after treatment.
The sequence of bla CEP-04 closely matched (96%) a -lactamase from a Pseudomonas sp., a genus commonly found in soil
(Fig. 2C). Taken together, these data demonstrate that the
increased -lactam resistance in manure-treated soil was related 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 -lactamase from a
Pseudomonas sp.
Effects of Manure and NPK Treatments on Microbial Communities.

Culturing and qPCR suggested that the increase in cephalothinresistant 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 117,657 16S rRNA
genes amplified from metagenomic DNA from a total of 49 soil
and manure samples were analyzed. This corresponded to an
average of 2,401 sequences per sample, with an average read
length of 477 bp. Sequence clustering yielded a total of 7,018
operational 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: R2 =
16%, P < 0.001). Manure-treated soil had less phylogenetic diversity than NPK-treated soil (P < 0.001, Welchs t test). Compared 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 -diversity analyses. Bacterial communities in
replicate samples from manure-treated soils formed temporally
distinct clusters, whereas the communities in samples from NPKtreated soils clustered more randomly, with no obvious concordance with time of sampling (Fig. 4 and Fig. S2). The time effect
was significant (adonis: R2 = 18%, P < 0.001), but by 130 d after
treatment, the manure-treated communities were similar to NPKtreated communities (Fig. 3).

13

Days after treatment

10

0
13

10

80

94

52

66

38

19

12

Days after treatment

6.0

6.0

8.5

Mar
*

12

Log (total CFU/g)

9.0

Soil + NPK

Feb
*

19

Jan

9.5

Log (resistant CFU/g)

Manure Treatment Induces a Bloom of Cephalothin-Resistant Culturable

Soil Bacteria. We assessed the presence and abundance of the

Manure Treatment Enriches Taxa That Commonly Carry Resistance to

-Lactam Antibiotics. The addition of manure affected the soil

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

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

blaCEP-01

Library

Cephalothin
MIC, g/mL

Length,
aa

e-value

% ID

KM113767

MAN_uncultured

64

284

99

blaCEP-02

KM113768

B04_cultured

256

316

100

blaCEP-03

KM113769

MAN_uncultured

64

455

68

blaCEP-04

KM113770

B06_cultured

512

385

96

blaCEP-05

KM113771

256

377

91

blaCEP-06

KM113772

B04_cultured
B06_cultured
B07_cultured
B09_cultured

256

280

7E-173

82

blaCEP-07

KM113773

B09_cultured

256

288

3E-158

95

Accession
no. of closest
match

Closest match
(BLASTX)

WP_019542800 -lactamase
(Selenomonas bovis)
WP_016087836 -lactamase 3
(Bacillus cereus)
WP_005841913 -N-acetyl-glucosaminidase
(Bacteroides vulgatus)
WP_020798140 -lactamase class
C [Pseudomonas sp.
G5(2012)]
ACH58999
LRA-10
(uncultured bacterium
BLR10)
BAL14456
Metallo--lactamase
(Serratia marcescens)
ABK64020
Metallo--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.

80

94
13
0
m
an
u
N re
TC

52
66

38

b+
be
fo
1 re

1k

Soil + Manure

10-3

300
200

10-4

100

10-5

blaCEP-03

300
200

10-6

16S rRNA

100
300
200

10-7

10-6
10-7

**

**

**

94

80

Days after treatment

10-7

13

Days after treatment

blaCEP-04

94
m 130
an
ur
e

10-6

80

10-7

66

10-5

52

10-6

38

10-4

4
12

10-3

10-5

10-4

66

10-3

10-5

38

10-2

re

Log (bla/16S)

10-1

10-4

52

blaCEP-01
blaCEP-03

10 0

blaCEP-05

10-3

100

Log (bla/16S)

blaCEP-01

Soil + NPK

blaCEP-02

12

bp

12

Days after treatment

fo

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 imperative 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 provide 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 bacteria, 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 animals 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 -lactamase commonly
found in these genera along with metallo--lactamases.
We identified two -lactamases in manure that share significant similarity with -lactamases from members of the Gramnegative anaerobic genera Selenomonas (99%) and Bacteroides

be

following a trajectory similar to that of manure-derived -lactamases (Fig. 4B). Two soil OTUs that affiliated with the Pseudomonadaceae family (OTUs 726 and 1500), one of which
affiliated with the genus Pseudomonas (OTU 726), and a group of
OTUs affiliated with the Janthinobacterium genus (OTUs 8555
and 2161; Fig. 4 E 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. 4 C and D). This is especially interesting given that the metallo--lactamaseencoding gene blaCEP-07
identified 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 detected 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 38 and 66 after treatment), indicating
that their dynamics differed from those of Psychrobacter in response to manure. The community analyses demonstrate that
manure treatment had a greater effect than NPK on soil community richness and structure. We identified significant shifts of
certain phylotypes and confirmed a higher abundance of Pseudomonas spp. at the total community level. Other -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.

Fig. 2. Dynamics of manure-derived and soil-derived -lactamases in soil

after treatment with manure or NPK. (A and B) End-point PCR amplification
(A) and qPCR amplification (B) of -lactamases blaCEP-01 and blaCEP-03 identified from manure in soil. (C) Dynamics of the relative abundance of the
-lactamases blaCEP-02, blaCEP-05, and blaCEP-04 in 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.

PNAS Early Edition | 3 of 6

MICROBIOLOGY

GenBank
accession
no.

Gene
designation

895
2323
3332

0.06

3783
4211
9299

0.015

Pseudomonas sp. OTUs

manure-treated soil
726
1500

0.010

0.04

0.005

0.02

0.08

re
fo

94

Janthinobacterium sp. OTUs

manure-treated soil
8555
2161

0.06

0.010

13

be

m
a
n= nu
1 re

Pseudomonas sp. OTUs

NPK-treated soil
726
1500

1
12

be
n=fore
m 6
a
n= nu
1 re

D
0.015

38
52
66

0.000

0.00

0.04

0.005

0.02

0.000

Janthinobacterium sp. OTUs

NPK-treated soil

0.08

8555
2161

0.06

0.10

m
a
n= nu
1 re

re
fo

be

94

13

52
66

1
12

38

be
fo
re
m
a
n= nu
1 re

0.00

Psychrobacter pulmonis OTUs

manure-treated soil
9776
4110
9413

0.08
0.06

0.04

0.04

0.02

0.02

0
be
n=fore
m 6
a
n= nu
1 re

94

13

52
66

38

days after treatment

1
12

0.00

0.00
be
fo
re
m
a
n= nu
1 re

8555
5225
9776
3332
895
2323
9299
9968
7390
4877
5404
5731
5338
8386
9601
7771
3241
4170
58
7744
4110
9413
2228
4211
2047
3783
2161
726
1500
2327
9662
7705
7077
3582
2570
8149
8049
694
8932
7567
7039
9144
3837
2525
8923
5079
7891
4951
2518
523
3137
4679
1503
4019
11
6125
9661
5217
Janthinobacterium sp.
Micrococcaceae sp.
Psychrobacter pulmonis
Trichococcus sp.
Aerococcaceae sp.
Peptostreptoccaceae sp.
Peptostreptoccaceae sp.
Acidobacteria-6 sp.
Bradyrhizobium sp.
Xanthomonadales sp.
Hyphomicrobiaceae sp.
Rhodoplanes sp.
Sinobacteraceae sp.
Acidobacteria-6 sp.
Rhodoplanes sp.
Sinobacteraceae sp.
Micrococcaceae sp.
Flavobacterium sp.
Sejongia sp.
Flavobacterium sp.
Psychrobacter pulmonis
Psychrobacter pulmonis
Aerococcaceae sp.
Erysipelothrix sp.
Turicibacter sp.
Clostridiaeae sp.
Janthinobacterium sp.
Pseudomonas sp.
Pseudomonadaceae sp.
Hyphomicrobiaceae sp.
Roseiflexales sp.
Anaerolineae sp.
Rhodobiaceae sp.
Rhodobiaceae sp.
Rhodospirillales sp.
Devosia sp.
Balneimonas sp.
Rhodospirillales sp.
Acidobacteria-6 sp.
Rhodoplanes sp.
Flammeovirgaceae sp.
Pirellulaceae sp.
Acidobacteria-6 sp.
Hyphomicrobiaceae sp.
Rhodoplanes sp.
Rhizobiales sp.
Hyphomicrobiaceae sp.
Hyphomicrobiaceae sp.
Gaiellaceae sp.
Anaerolineae sp.
Actinomycetales sp.
Rhodoplanes sp.
Sinobacteraceae sp.
Betaproteobacteria sp.
Gaiellaceae sp.
Sinobacteraceae sp.
Acidobacteria-6 sp.
Methylocaldum sp.

Manure OTUs
OTUs enriched by manure
OTUs enriched by NPK

Taxonomy

OTU ID

relative abundance of taxa

0
1
0
52
130
0
12
0
0
0
130
130
12
12
1
52
1
38
66
38
52
94
94
130
66
94
66
38
130
130
94
38
38
38
52
66
52
52
66
66
94
94
1
1
1
0
12
12
12

Manure OTUs
manure-treated soil

94

day

0.08

13

0.1 0.2 0.3

relative abundance

94

soil + manure
manure

13

soil before
soil + NPK

0.08

94

0.04

13

most abundant taxa

relative abundance of taxa

all taxa

38
52
66

A Heatmap most abundant OTUs

52
66

(68%). PCR and qPCR results revealed that neither the -lactamaseencoding genes nor bacteria found in manure persisted
in the soil over time. These results suggest that the antibioticresistant organisms and the genes responsible for their resistance were enriched from among resident soil bacteria,
rather than introduced in the manure.
Our analysis identified five -lactamases from cultured soil
bacteria. Soil is a reservoir of divergent -lactamases, irrespective

52
66

Fig. 3. Temporal changes in soil community structures before treatment

and after treatment with manure or NPK. BrayCurtis 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 nonmetric multidimensional scaling (NMDS) graph. The 2D stress was 0.12. Increasing symbol size indicates time since manure treatment. Treatment
effects were significant in all adonis combinations (P < 0.001; soil and manuretreated communities, R2 = 13%; soil before treatment and manure-treated
communities, R2 = 15%; soil before and NPK-treated communities, R2 = 6%).

12

0.4

38

0.2

38

0.0
NMDS1

relative abundance of taxa

0.2

1
12

0.4

0.4

of anthropogenic influences (33, 34). Elevated abundance of a

gene encoding an AmpC-family -lactamase persisted for up to
130 d after manure treatment, as did elevated populations of
Pseudomonas spp., which are common sources of -lactamases in
nosocomial infections (35, 36). In addition, metallo--lactamases
similar to those that we found in soil are a serious threat to
human health. Metallo--lactamases hydrolyze a broad spectrum
of target -lactams, and there are no metallo--lactamase inhibitors
approved for clinical use. Other highly abundant taxa in manuretreated soils were Psychrobacter spp., composing up to 10% of all
sequences at day 12. Psychrobacter spp. are halotolerant and psychrotolerant and are abundant in ornithogenic soil in Antarctica
(37). They also have been found to harbor AmpC--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--lactamases are typically located on the chromosome (39,
40), however, making the risk of transfer low. Furthermore, a recent study demonstrated that although novel antibiotic-resistance
genes were commonly isolated from soil, with -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 reinforces 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

0.0

0
1
12
38
52
66
94
130

1
12

days

0.2

NMDS2

0.2

soil before
soil + manure
soil + NPK

days after treatment

Fig. 4. Dynamics of the most abundant OTUs and -lactamase harboring OTUs in response to treatment with manure or NPK. (A) Heat map of the relative
abundance of the 59 most abundant OTUs composing more than 10% across all 49 samples. 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.

4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1409836111

Udikovic-Kolic et al.

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, and 5% clay; pH 7.4) with an organic matter content of 87 g kg1 and a
total nitrogen content of 3.7 g kg1.
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 recommended limit in animal manure for land application (54). The manure was
incorporated into soil beds (depth, 15 cm) at a level of 20 kg per bed. Beds
treated with NPK were amended with the same amount of each nutrient
present in the manure. On each sampling day, 10 soil cores (1.5 cm in diameter and
12 cm deep) were collected at random and pooled. Bacteria were cultured from
beds at 1 d before (day 0) manure or NPK treatment and then at 10 time points

Udikovic-Kolic et al.

after treatment: days 1, 12, 19, 38, 52, 66, 80, 94, 108, and 130. At day 4 after
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 serial 10-fold dilutions in PBS were cultured on
R2A agar plates (Remel) with and without cephalothin (50 mg/mL). CFU were
counted after a 5-d incubation at 28 C. Cephalothin-resistant bacteria were
scraped from the plates (103 dilutions), pooled, and stored at 80 C. Frozen samples obtained at 52 d 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 (www.borealgenomics.com) and subjected to
end-repair and ligation with the pCC2Fos vector (Epicentre), following the
manufacturers 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 in 50 mL of LB supplemented with chloramphenicol (20 g/mL) for 2 h at 37 C and 200 rpm. Cultures were plated on
LB containing chloramphenicol (20 g/mL) and cephalothin (50 g/mL). Assessment of the diversity of resistant fosmid clones, minimum inhibitory
concentration assays, and subcloning of genes conferring resistance to cephalothin 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://
dna-analysis.research.yale.edu). Geneious version 6.0.5 was 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 and 16S rRNA gene sequencing with the ZR Fecal DNA
MiniPrep Kit according to the manufacturers recommendations and further
purified using the nucleic Aurora acid purification instrument (Boreal
Genomics). Amplification was done using 10 ng of DNA with iQ Supermix
(Bio-Rad) in a total volume of 20 L. 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 of 3 min at 95 C,
45 cycles of 15 s at 95 C, and 1 min at 60 C. For PrNU.K.017/PrNU.K.018, an
annealing/extension temperature of 56 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 between 89% and 103%. The
relative abundance of antibiotic-resistance genes was calculated by dividing
the respective bla gene abundance by 16S rRNA gene copy number. Endpoint PCR was performed using Hotstar Taq DNA Polymerase (Qiagen)
according to the manufacturers recommendations, and PCR products were
separated on a 3% agarose gel.
Pyrosequencing and Sequence Analyses of 16S rRNA Genes. Multiplexed
pyrosequencing (Roche 454 FLX with Titanium reagents) from the V1-V3
regions of bacterial 16S rRNA genes was used to assess changes in bacterial
soil community structure before and after manure or NPK treatment. Aurorapurified DNA was amplified and sequenced using standard protocols at the
Research and Testing Laboratories, Lubbock, TX (www.researchandtesting.
com). Primers 28 forward and 519 reverse were chosen (57). The default
workflow in QIIME v1.7 was used for sequence processing, quality control,
and OTU selection. Each sample was rarefied to 904 sequences. Alpha diversity was described for each sample using Faiths phylogenetic diversity
(58) and the number of OTUs (defined at 97% sequence identity). Differences were assessed using Welchs 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 community ecology package (60).
A heat map visualization of OTUs with relative abundances was performed
with the heatmap.2 function of the gplots package. Only the OTUs that
each composed more than 0.1 of the relative abundance across all samples
were included (top 59 OTUs). A Mantel test with Pearsons correlation coefficient between the full and reduced datasets of BrayCurtis 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, http://metagenomics.anl.gov (project ID 8945, YaleFarmSoil; metagenomes 4562248.34562264.3, 4562266.3
4562295.3, and 4570842.3; Table S4) and the GenBank database (accession nos.
KM113767KM113773) (Table 1).

PNAS Early Edition | 5 of 6

MICROBIOLOGY

determinants reside in organisms such as Pseudomonas and

Janthinobacterium spp., which are opportunistic human pathogens (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 of -lactamase genes. Of the
49 fully sequenced Pseudomonas strains, 38 contain AmpC-type
-lactamases, including most of those used in agriculture (47),
and all but three strains contain at least one metallo--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 responsive to manure amendment and important environmental
reservoirs of -lactam antibiotic-resistance genes.
A focus of future research will be on identifying the component of manure that leads to the proliferation of bacteria that
carry -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 (5052).
Interestingly, even low concentrations of metal in soils select for
resistance to various antibiotic classes, including -lactams (53),
owing to shared genetic or physiological mechanisms of resistance 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 vegetables, 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.

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
Grant 1R13GM090574 and Kirschstein National Research Service Award
T32 Training Grant for Genomics and Proteomics 5T32HG003198 (to
Yale University).

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Udikovic-Kolic et al.

Supporting Information
Udikovic-Kolic et al. 10.1073/pnas.1409836111
SI 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) between January 18 and May 28, 2013, to
simulate winter manure application for spring vegetable planting.
Maximum and minimum daily temperatures for this experimental
period are shown in Fig. S3. The experimental units were vegetable beds (above ground structures containing soil) with lumber
frames covering a surface area of 122 244 cm. Three replicate
beds per treatment were used. To prevent freezing, the beds
were covered with a textile hoop during the first five weeks.
Loamy sand soil with no previous history of manure application
was obtained from the Yale Farm and placed in the beds. Particle composition of the soil was 79% sand, 16% silt, and 5% clay
(University of Wisconsin-Verona Soil and Plant Analysis Laboratory). The organic matter content was 87 g kg1, total nitrogen
content was 3.7 g kg1, and pH was 7.4. The abundance of the
heavy metals Cu and Zn was low, 0.9 mg kg1 and 6.1 mg kg1,
respectively.
The manure used in this study was obtained from a dairy farm
(University of Connecticut) that uses -lactam antibiotics when
necessary to treat and prevent mastitis. After treatment, cows are
housed separately for at least 2 wk. The manure used in this
study was collected from the pens of approximately 80 untreated
dairy cows. Manure was tested for nutrient [nitrogen, phosphorus, and potassium (NPK)] and heavy metal content at the University of Wisconsin-Marshfield Soil and Forage Analysis Lab
(Table S2). The level of Zn detected in the manure was higher
than the maximum recommended limit in animal manure for land
application (1). The manure was mechanically incorporated into
soil beds (depth, 15 cm) at a level of 20 kg per bed to simulate a
typical manure application rate for Wisconsin farming (67.2
metric tons per hectare). Beds treated with NPK were amended
with the same amount of each nutrient present in the manure
(added as ammonium nitrate, superphosphate, and potassium
sulfate). On each sampling day, 10 soil cores (1.5 cm in diameter
and 12 cm deep) were randomly collected and pooled by hand
mixing. Bacteria were cultured from beds at 1 d before (day 0)
manure or NPK treatment, and at 10 time points after treatment
(days 1, 12, 19, 38, 52, 66, 80, 94, 108 and 130). At day 4 after
treatment, soil cores were collected and used for quantitative
PCR (qPCR) analyses, but not cultured (Fig. S3).
Culturing Bacteria from Soil and Manure Samples and Construction of
Fosmid Libraries. To determine total CFU from soil and manure,

a 1-g sample (wet weight) was vortexed in 9 mL of PBS (10 mM,

pH 7) to disperse the bacteria. Serial 10-fold dilutions in PBS were
cultured on three replicate R2A agar plates (Remel) with and
without cephalothin (50 mg/mL) to enumerate resistant and total
bacteria. Cephalothin, a -lactam antibiotic, was chosen for this
study because its relatives are used to treat and prevent diseases
in dairy cows. The culture medium was supplemented with cycloheximide (100 mg/mL) to inhibit growth of fungi. To standardize log CFU per mass, soil and manure dry weights were
determined by weighing 1-g portions before and after drying at
105 C; samples were dried until their mass no longer decreased
on heating. CFU of solid media were determined after incubation
for 5 d at 28 C. Cephalothin-resistant bacteria were scraped from
the plates (103 dilutions), pooled, and stored at 80 C in R2A
broth supplemented with 30% glycerol. Frozen samples obtained
at 52 d after manure or NPK application were used to build
metagenomic libraries.
Udikovic-Kolic et al. www.pnas.org/cgi/content/short/1409836111

Fosmid libraries were constructed from two pools of cultured

soil bacteria of each treatment and the manure sample.
DNA was extracted from the pools using the Small Volume
Bacterial DNA Extraction Protocol (BG-1060006-AA-D;
www.borealgenomics.com). DNA was extracted from manure
using the Aurora High-Capacity 2050 kb DNA from Soil Protocol
(106-0021-BB-D). DNA was subjected to end-repair and ligation
with the pCC2Fos vector (Epicentre) following the manufacturers
recommendations. Clones from fosmid libraries were scraped from
plates, pooled, and stored at 80 C in LB broth supplemented
with 30% glycerol and chloramphenicol (20 g/mL). Library size
was estimated using previously published protocols (2).
Identification of Clones Resistant to Cephalothin. The clones from
each metagenomic library were pooled and grown in 50 mL of LB
supplemented with chloramphenicol (20 g/mL) for 2 h at 37 C
and 200 rpm. Serial dilutions were plated on LB plates containing
chloramphenicol (20 g/mL) and cephalothin (50 g/mL). The
diversity of resistant fosmid clones was assessed with PstI digests
of total fosmid DNA visualized after agarose gel electrophoresis.
Minimum inhibitory concentration assays of resistant clones were
conducted in MuellerHinton broth according to previously published protocols (2). To subclone genes conferring resistance, we
used the pET28b vector and followed previously published protocols (3 46). Plasmid DNA from resistant subclones was purified
using the Qiaprep kit (Qiagen) and sequenced at the DNA Analysis
Facility at Yale University (http://dna-analysis.research.yale.edu)
using the T7 promoter and terminator primers. Geneious version
6.0.5 was used for protein sequence comparison and phylogenetic analyses. Sequence alignments were performed using
CLUSTALW (4), and the phylogenetic trees were inferred using
maximum likelihood. Bootstrap values were calculated based on
100 replications.
DNA Extraction, qPCR, and End-Point PCR. DNA was extracted from
soil and manure for qPCR and 16S rRNA gene sequencing with
the ZR Fecal DNA MiniPrep Kit according to the manufacturers
recommendations and then further purified using an Aurora
nucleic acid purification instrument, Aurora (Boreal Genomics).
Ten ng of DNA was used for amplification with iQ Supermix
(Bio-Rad) in a total volume of 20 L. The 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 for 3 min at 95 C, 45 cycles of
15 s at 95 C, and 1 min at 60 C. For PrNU.K.017/PrNU.K.018,
an annealing/extension temperature of 56 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 between 89% and 103%. The relative AR gene abundance
was calculated by dividing the respective bla gene abundance by
16S rRNA gene copy number to eliminate variation contributed
by differential DNA extraction and amplification efficiency between samples. End-point PCR was performed using Hotstar Taq
DNA Polymerase (Qiagen) according to the manufacturers recommendations, and PCR products were separated on a 3% agarose gel.
Pyrosequencing and Sequence Analyses of 16S rRNA Genes. Multiplexed pyrosequencing (Roche 454 FLX with titanium reagents)
from the V1V3 regions of bacterial 16S rRNA genes was used to
assess changes in bacterial soil community structure before and
after manure or NPK treatment. Aurora-purified DNA was
1 of 5

quantified using Qubit (Life Technologies), and then amplified

and sequenced using standard protocols at the Research and
Testing Laboratories, Lubbock, TX (www.researchandtesting.
com). Primers 28 forward (bacteria-specific; 5-GAGTTTGATCNTGGCTCAG 3) and 519 reverse (universal; 5-GTATTACCGCGGCTGCTGG 3) were chosen because they amplified
well and have previously been applied successfully for soil DNA
(5). The default workflow in QIIME v1.7 was used for sequence
processing, quality control, and operational taxonomic unit (out)
selection. A more stringent window size of 25 (instead of the default window size of 0) was used to filter sequences. After PyNAST
alignment, chimeric sequences, singletons, and chloroplast sequences were removed. Each sample was rarefied to 904 sequences
to match the sequence numbers across samples. Alpha diversity
was described for each sample using Faiths phylogenetic diversity

(6) and the number of OTUs (defined at 97% sequence identity).

Differences were assessed using Welchs t test. Permuted
ANOVAs was used to test for differences in treatment and time
after excluding the manure sample using the adonis function
according to methods described by Shade et al. (7) in the R environment for statistical computing (8) with the vegan community
ecology package (9).
A heat map visualization of OTUs with relative abundances was
performed with the heatmap.2 function of the gplots package.
Only the OTUs that each composed more than 0.1 of the relative
abundance across all samples (top 59 OTUs) were included. A
Mantel test (vegan package) with Pearsons correlation coefficient
between the full and reduced datasets of BrayCurtis matrices
revealed that they were highly correlated (r = 0.91, P < 0.001).

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

for the Australian dairy industry. Available at www.dairyingfortomorrow.com/
index.php?id=48. Accessed January 16, 2014.
2. Wichmann F, Udikovic-Kolic N, Andrew S, Handelsman J (2014) Diverse antibiotic resistance genes in dairy cow manure. MBio 5(2):e01017.
3. Winsor GL, et al. (2011) Pseudomonas Genome Database: Improved comparative
analysis and population genomics capability for Pseudomonas genomes. Nucleic Acids
Res 39(Database issue):D596D600.
4. Larkin MA, et al. (2007) ClustalW and ClustalX version 2. Bioinformatics 23(21):
29472948.

5. Hartmann M, et al. (2012) Significant and persistent impact of timber harvesting on soil
microbial communities in northern coniferous forests. ISME J 6(12):21992218.
6. Faith DP (1992) Conservation, evaluation and phylogenetic diversity. Biol Conserv
61(1):110.
7. Shade A, McManus PS, Handelsman J (2013) Unexpected diversity during community
succession in the apple flower microbiome. MBio 4(2):0060200612.
8. DCosta VM, et al. (2011) Antibiotic resistance is ancient. Nature 477(7365):457461.
9. Oksanen JF, Blanchet G, Kindt R, Legendre P, Minchin PR, et al. (2011) Vegan: Community ecology package. R package version 2.0. Available at http://vegan.r-forge.rproject.org. Accessed December 6, 2013.

substitutions/site

WP_019542800| Selenomonas bovis

89
1 0 0 YP_004896674| Acidaminococcus intestini

bla(CEP-01)

100

0.3

100

WP_016087836| Bacillus cereus

class A
TEM

identified from soil in this study

identified from manure in this study
clinical isolates
other soil metagenomic libraries

bla(CEP-02)
CDB98800| Bacteroides sp. CAG:443

100

WP_005841913| Bacteroides vulgatus

99

bla(CEP-03)
WP_020798140| Pseudomonas sp. G5(2012)
100

bla(CEP-04)
WP_008054664| Pseudomonas sp. GM78

99

CBZ41773| Pseudomonas aeruginosa

100

100

ACX31161| Pseudomonas aeruginosa

class C
AmpC

100

CBZ41772| Pseudomonas aeruginosa

WP_020654670| Massilia niastensis

100
100

ACH58999| LRA-10 uncultured bacterium

bla(CEP-05)

ACS83721| Amo1 uncultured bacterium

bla(CEP-06)

100

100

BAL14456| Serratia marcescens

WP_008450411| Janthinobacterium sp. HH01

100

class B
metallo-bla

ACS83724| Crb11 uncultured bacterium

100

ABK64020| Janthinobacterium lividum

bla(CEP-07)

Fig. S1. Phylogenetic relationship of -lactamase sequences identified in this study. -lactamase genes were identified by subjecting metagenomic libraries to
selection for cephalothin resistance. Metagenomic libraries were constructed with DNA from bacteria cultured from soil or dairy cow manure. Cephalothinresistant clones were sequenced, and the gene responsible for resistance was identified. Related deduced protein sequences, including those from clinical
isolates retrieved from GenBank, were used to construct the phylogenetic tree. Proteins marked with boxes were detected in soil samples using qPCR. Numbers
represent bootstrap values for 100 replications. Bootstrap values >80 are shown.

Udikovic-Kolic et al. www.pnas.org/cgi/content/short/1409836111

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soil + manure
manure

days
0
1
12
38
52
66
94
130

0.4

Bray-Curtis distance
0.7
1.0

soil before
soil + NPK

Fig. S2. Hierarchical cluster of bacterial soil communities based on BrayCurtis similarities. BrayCurtis similarity coefficients were calculated from relative OTU
abundances in bacterial soil communities across three biological replicates of soil treated with manure or inorganic fertilizer (NPK).

Temperature ( C)

30

Jan

Feb

Mar

Apr

May

Minimum
Maximum

20
10
0
-10

38 52 66 80 94 108
Days after treatment

1 12 19

0
13

culturing
qPCR
pyrosequencing
Fig. S3. Daily temperatures during the field experiment. Maxima and minima daily temperatures recorded over the course of the study in 2013 at the Yale
Farm at West Campus. Different colored arrows (green, red, and blue) indicate the time points at which the corresponding analyses were performed.

Table S1. Features of the metagenomic libraries constructed in this study from cultured oil bacteria at 52 d after
manure or inorganic fertilizer (NPK) application and from dairy cow manure
Library name
B04_cultured
B06_cultured
B07_cultured
B09_cultured
MAN_uncultured

Origin

Treatment

No. of clones

Average insert size, kb

Amount of cloned DNA, Gb

Soil_bed 4
Soil_bed 6
Soil_bed 7
Soil_bed 9
Manure

Soil + manure
Soil + manure
Soil + NPK
Soil + NPK
Manure

810,980
450,000
464,800
1,960,000
84,142

36.2
35
38.1
41.3
34.3

29.4
15.8
17.7
80.9
2.9

Table S2. Chemical composition of the dairy cow manure

g kg1 (dry matter)
% moisture Total N P2O5 K2O
85.40

4.97

Udikovic-Kolic et al. www.pnas.org/cgi/content/short/1409836111

2.61

mg kg1 (dry matter)

Zn

Cu

Cd

Pb C/N pH

2.53 428.37 40.41 <0.4 <2

13

8.2

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Table S3. PCR primer sequences, targets, annealing temperatures, and amplicon lengths
Primer

Gene targeted

Pr_NU.K.03
Pr_NU.K.06
Pr_NU.K.04
Pr_NU.K.05
Pr_NU.K.17
Pr_NU.K.18
Pr_FW039
Pr_FW046
Pr_FW062
Pr_FW063
Pr_FW047
Pr_FW048

blaCEP-05
blaCEP-04
blaCEP-02
blaCEP-01
blaCEP-03
16S rRNA

Primer sequence (53)

Amplicon size, bp

Primer annealing
temperature, C

Ref.

CATGCCGCCCACCTTGAAGTA
CCATGGTCAATTATGCGTGGGG
CGAAGCCTACGGGGTGAAAAC
GCCAGCAGACCATCCAGTGTGA
CCCCAAAGGCATTAGCTACTAGTC
TTGCGATATCATTTCTGGTTCC
GCTACAAACCTGATACGCGT
CGAACAAATTTCCGCCAGCG
GTTGTTGGATACGCGGACG
CAGCCGGATAAAGGACCATG
CCTACGGGAGGCAGCAG
ATTACCGCGGCTGCTGG

212

59

This study

198

59

This study

198

57

This study

191

59

(1)

193

59

This study

194

59

(2)

1. Oksanen JF, Blanchet G, Kindt R, Legendre P, Minchin PR et al. (2011). Vegan: Community ecology package. R package version 2.0. Available at http://vegan.r-forge.r-project.org.
Accessed December 6, 2013.
2. Muyzer G, de Waal EC, Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified
genes coding for 16S rRNA. Appl Environ Microbiol 59(3):695700.

Udikovic-Kolic et al. www.pnas.org/cgi/content/short/1409836111

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Table S4. Accession numbers of 16S rRNA datasets

Treatment

Days

Replicate

MG-RAST ID

Untreated soil
Untreated soil
Untreated soil
Untreated soil
Untreated soil
Untreated soil
Manure
Soil + manure
Soil + manure
Soil + manure
Soil + manure
Soil + manure
Soil + manure
Soil + manure
Soil + manure
Soil + manure
Soil + manure
Soil + manure
Soil + manure
Soil + manure
Soil + manure
Soil + manure
Soil + manure
Soil + manure
Soil + manure
Soil + manure
Soil + manure
Soil + manure
Soil + NPK
Soil + NPK
Soil + NPK
Soil + NPK
Soil + NPK
Soil + NPK
Soil + NPK
Soil + NPK
Soil + NPK
Soil + NPK
Soil + NPK
Soil + NPK
Soil + NPK
Soil + NPK
Soil + NPK
Soil + NPK
Soil + NPK
Soil + NPK
Soil + NPK
Soil + NPK
Soil + NPK

0
0
0
0
0
0

1
1
1
12
12
12
38
38
38
52
52
52
66
66
66
94
94
94
130
130
130
1
1
1
12
12
12
38
38
38
52
52
52
66
66
66
94
94
94
130
130
130

A
B
C
D
E
F

A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C

4562248.3
4562249.3
4562250.3
4562251.3
4562252.3
4562253.3
4562296.3
4562254.3
4562255.3
4562256.3
4562260.3
4562261.3
4562262.3
4562266.3
4562267.3
4562268.3
4562272.3
4562273.3
4562274.3
4562278.3
4562279.3
4562280.3
4562284.3
4562285.3
4562286.3
4562290.3
4562291.3
4562292.3
4562257.3
4562258.3
4562259.3
4562263.3
4562264.3
4570842.3
4562269.3
4562270.3
4562271.3
4562275.3
4562276.3
4562277.3
4562281.3
4562282.3
4562283.3
4562287.3
4562288.3
4562289.3
4562293.3
4562294.3
4562295.3

Udikovic-Kolic et al. www.pnas.org/cgi/content/short/1409836111

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