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Genome-wide association study reveals a locus

for nasal carriage of Staphylococcus aureus in

Danish crossbred pigs

Article in BMC Veterinary Research · December 2015

DOI: 10.1186/s12917-015-0599-y

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et al. BMC Veterinary Research

(2015) 11:290

DOI 10.1186/s12917-015-0599-y

RESEARCH ARTICLE

Open Access

Genome-wide association study reveals a

locus for nasal carriage of

Staphylococcus

aureus

in Danish crossbred pigs

Per Skallerup

, Carmen Espinosa-Gongora

, Claus B. Jørgensen

, Luca Guardabassi

and Merete Fredholm

Abstract

Background:

Staphylococcus aureus

is an important human opportunistic pathogen residing on skin and mucosae

of healthy people. Pigs have been identified as a source of human colonization and infection with methicillin-

resistant

Staphylococcus aureus

(MRSA) and novel measures are needed to control zoonotic transmission. A recent

longitudinal study indicated that a minority of pigs characterized by high nasal load and stable carriage may be

responsible for the maintenance of

S. aureus

within farms. The primary objective of the present study was to detect

genetic loci associated with nasal carriage of

S. aureus

in Danish crossbred pigs (Danish Landrace/Yorkshire/Duroc).

Results:

Fifty-six persistent carriers and 65 non-carriers selected from 15 farms surveyed in the previous longitudinal

study were genotyped using Illumina’s Porcine SNP60 beadchip. In addition,

spa

typing was performed on 126

aureus

isolates from 37 pigs to investigate possible relationships between host and

S. aureus

genotypes. A single

SNP (MARC0099960) on chromosome 12 was found to be associated with nasal carriage of

S. aureus

at a genome-wide

level after permutation testing (p = 0.0497) whereas the association of a neighboring SNP was found to be borderline

(p = 0.114). Typing of

S. aureus

isolates led to detection of 11

spa

types belonging to the three main

S. aureus

clonal

complexes (CC) previously described in pigs (CC9, CC30 and CC398). Individual carriers often harbored multiple

aureus

genotypes and the host-pathogen interaction seems to be independent of

S. aureus

genotype.

Conclusion:

Our results suggest it may be possible to select pigs genetically resistant to

S. aureus

nasal colonization as

a tool to control transmission of livestock-associated MRSA to humans.

Keywords:

Pigs, Staphylococcus aureus colonization, MRSA control

Background

Staphylococcus aureus

is a significant human pathogen

causing wound and skin infections, endocarditis and

bacteremia [1, 2]. It has long been recognized that

healthy individuals may be colonized on skin and mu-

cosae, the most frequent carriage site in humans being

the anterior nares [3, 4]. Longitudinal studies have dem-

onstrated the existence of three

S. aureus

nasal carriage

patterns, i.e., persistent carriers (~20 % of the popula-

tion), non-carriers (~50 %) and intermittent carriers

(~30 %) [3, 4]. There is evidence showing that nasal car-

riage is associated with a higher risk of

S. aureus

infec-

tion [5, 6]. Colonization of the host is a complex process

* Correspondence:

[email protected]

Department of Veterinary Clinical and Animal Sciences, University of

Copenhagen, Frederiksberg, Denmark

Full list of author information is available at the end of the article

which is influenced by host factors, bacterial factors, and

environmental factors [6–8]. Although the heritability of

S. aureus

carriage was not significant in two human

studies [9, 10], single nucleotide polymorphisms (SNPs)

associated with nasal carriage have been found for several

candidate genes, e.g., C-reactive protein (CRP), interleukin

4 (IL-4), and glucocorticoid receptor (NR3C1) [11–15]. In

addition, genetic loci associated with susceptibility to S.

aureus infection have been reported in murine studies [16,

17]. Collectively, these studies suggest that host gene vari-

ants underlie differences in susceptibility to

S. aureus

colonization. However, candidate gene studies suffer from

the limitation that they only assess the effects of specific

genes picked by the researcher based on hypothesized in-

volvement in disease etiology. In contrast, genome-wide

association studies (GWAS) survey the entire genome,

Open Access

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International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and

reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to

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(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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et al. BMC Veterinary Research

(2015) 11:290

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and many of the associations found in GWAS identify

novel candidate genes [18, 19].

Pigs and other livestock species carrying methicillin-

resistant

S. aureus

(MRSA) clonal complex (CC) 398

may act as a source of human colonization and infection

[20, 21]. Spread of this livestock-associated MRSA clone

is presently regarded as a threat to public health, and ef-

fective control measures preventing transmission of

MRSA to farmers and other people exposed to livestock

are urgently needed [22, 23]. A recent longitudinal study

revealed that a minority of pigs characterized by high

nasal load and stable carriage may be responsible for the

maintenance of

S. aureus

within farms [24]. The object-

ive of the present study was to detect loci associated

with nasal carriage of

S. aureus.

Danish crossbreds clas-

sified as persistent carriers (n = 56) and non-carriers (n

= 65) were genotyped by GWAS using Illumina’s Porcine

SNP60 beadchip [25]. A single SNP on chromosome 12

was found to be genome-wide significant after permuta-

tion testing. The region of interest was inspected and we

identified four candidate genes which may control

aureus

colonization in pigs.

were part of routine examinations and diagnosis of ani-

mals normally used at production farms. All handling of

animals was performed by trained personnel and

veterinarians.

Genotyping of pigs

To detect QTLs associated with

S. aureus

carrier status,

we genotyped all pigs using diagnostic blood samples

collected in EDTA tubes (VWR, USA). DNA was ex-

tracted using either a salting out procedure with minor

modifications [26] or MasterPure™ Complete DNA and

RNA Purification Kit (Epicentre Biotechnologies, USA)

according to the manufacturer’s instructions. The concen-

tration and purity of DNA was measured on a NanoDrop

1000 spectrophotometer (Thermo Fisher Scientific, USA).

2500 ng of each sample was submitted for genotyping to

GeneSeek, Inc. (http://www.neogeneurope.com). Samples

were genotyped for 61,565 SNPs using Illumina’s Porcine

SNP60 beadchip [25].

Isolation and genotyping of

S. aureus

Methods

Phenotypic characterization of pigs

Our study population comprised 56 persistent carriers

and 65 non-carriers from 15 farms located in the central

part of Jutland, Denmark (three specific-pathogen-free

(SPF) and 12 non-SPF farms). Most pigs were pheno-

typed in the previous longitudinal study [24] and 21 add-

itional pigs were recruited for this study on four of the

farms surveyed in the longitudinal study. Nasal swabs

(Dryswab™, MWE, UK) were collected from all pigs

three times on a weekly basis. Pigs that were

S. aureus-

positive on all three sampling points were classified as

persistent carriers whereas non-carriers were negative

on at least two sampling points and with no more than

100 CFU/swab in the remaining sample. In order to en-

sure exposure to a minimum colonization pressure, non-

carriers were included only if they originated from farms

where at least one persistent carrier was detected. The

distribution of persistent carriers and non-carriers

among farms is shown in Additional file 1: Table S1. All

pigs were crossbreeds (Danish Landrace/Yorkshire/

Duroc) of approximately 70 kg. Pedigree details were

not available but since the farmers used mixed semen to

produce the offspring, the sample was expected to com-

prise a mixture of half-sibs (by sow and boar) and more

distantly related pigs.

According to Danish laws (Danish Animal Experimen-

tation Act, Chapter 1, Paragraph 1, point 3) no ethical

approval was required for this study since the blood

samples collected from the animals were taken for diag-

nostic purposes. All procedures concerning the animals

To study possible relationships between host and

S. aur-

eus

genotypes,

S. aureus

was isolated from 37 persistent

carriers whose nasal swabs had been stored at−80 °C in

the previous study [24]. Swabs were directly plated onto

SaSelect agar (Biorad, USA) and enriched in Müller-Hin-

ton broth containing 2.5 % of NaCl to enhance

S. aureus

detection. After overnight incubation one presumptive

aureus

colony was randomly selected for each sampling

point. Additional colonies were isolated if they had clear

morphological features suggesting the presence of differ-

ent strains on the same plate. If

S. aureus

was not de-

tected by direct plating, the enrichments were further

processed as described above. All isolates were charac-

terized by

spa

typing [27, 28] and

spa

types were

assigned using Ridom Staphtype software, version 2.2.1

(Ridom GmbH, Würzburg, Germany). Associations be-

tween

spa

types and clonal complexes (CC) were deter-

mined according to information available in the scientific

literature. For

spa

types not previously associated to a

clonal complex, BURP cluster analysis (Ridom StaphType

software, version 2.2.1) was used to infer association [27].

Statistical analyses

Data were analyzed in R version 3.1.0 [29]. Genotype

data were analyzed and visualized using the GenABEL

package [30, 31] except for Manhattan plots which were

made using the qqman package [32]. SNPs were ex-

cluded prior to analysis if genome position was not pro-

vided (n = 12,627) or if they were located on sex

chromosomes (n = 1381). SNP genotype data were sub-

jected to quality control (QC) measures. GenABEL ap-

plies QC filters using an iterative process; for individuals

we used the following criteria, call rates > 0.95, false

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discovery rate (FDR) for unacceptably high heterozygos-

ity < 0.01 and identity-by-state (IBS) < 0.95 (based on

2000 markers); for SNPs we used the following criteria

(number of SNPs that did not pass the threshold), call

rate > 0.95 (2500), minor allele frequency (MAF) > 0.05

(7012), and SNPs in Hardy-Weinberg equilibrium with

p-values

> 0.05 (15,073). After QC a total of 23,919 auto-

somal SNPs mapped to build Sscrofa 10.2 and 121 indi-

viduals (56 carriers, 65 non-carriers) were included in

the final analysis.

We estimated the average relatedness between pigs by

computing an

marker-based genomic kinship

matrix for all pairs of pigs. Kinship coefficients between

two individuals (average

identical-by-state

value) were

estimated using 23,919 autosomal SNPs which had

passed QC as described elsewhere [31]. The genomic

kinship matrix was transformed to a distance matrix

which was then subjected to multidimensional scaling

analysis and plotted in two dimensions (principal com-

ponent axes) [31].

The association between SNP genotype and nasal car-

riage of

S. aureus

(binary trait) was tested in GenABEL

using an allelic association test with 1 df. We included

farm as a covariate in the model. To adjust for multiple

testing with a high number of SNPs, we derived the em-

pirical distribution of the chi-square statistic after 10,000

permutations of the whole dataset. Genome-wide signifi-

cance was set to empirical

p-values

< 0.05. We used gen-

omic control [33] to adjust for any inflation of the test

statistic. Calculations of linkage disequilibrium (LD) and

visualization of LD were performed in Haploview ver-

sion 4.2 [34]. Since the annotation of the pig genome se-

quence is incomplete, we also interrogated the human

orthologue of the candidate region (HSA: 17q12) using

builds Sscrofa10.2 and GRCh38 accessed through the

Ensembl genome browser (www.ensembl.org).

The association between total number of

spa

types

colonizing over the three-week period (1, 2, or 3) and

SNP MARC0099960 genotype (GG, GA, AA) was tested

using Fisher’s Exact Test for Count Data. We tested if

colonization by each clonal complex (CC9, CC30, and

CC398) was non-random among SNP MARC0099960

genotypes using Fisher’s Exact Test for Count Data. In

addition to the genotype model, we also tested a domin-

ance/recessive model (GG vs. GA and AA; GG and GA

vs. AA).

Results

The 23,919 SNPs which passed quality testing were used

to compute a genomic kinship matrix for all pairs of pigs

(Fig. 1a). Inspection of the matrix suggested that the pigs

in our sample were only distantly related. We next applied

multidimensional scaling to a distance matrix calculated

from the genomic kinship matrix (Fig. 1b). The plot did

not suggest any stratification of data with respect to

aureus

carriage (persistent carriers vs. non-carriers) or

farm (data not shown). We constructed a quantile-

quantile plot (Fig. 1c) and calculated the genomic inflation

factor (λ

= 1.06) which also confirmed that genetic con-

founding was not an issue in our dataset.

Fig. 1

From left to right:

Distribution of genomic relationships between pairs of pigs (kinship matrix).

Multidimensional scaling plot (n = 121).

0, non-carriers; 1, persistent carriers; PC, principal component.

Quantile-quantile plot. Black line, the expected distribution of association test

statistics under the null hypothesis of no association is plotted against observed values. Any deviation from the X-Y line suggests a consistent

difference between persistent carriers and non-carriers e.g., due to genetic confounding. At the extreme of the distribution, the observed chi-square

values are higher than expected by chance which indicates true association. Red line, fitted slope

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A GWAS was performed on the final dataset (Fig. 2).

One locus (SNP MARC0099960) on porcine chromo-

some 12 demonstrated association with carriage of

aureus.

The effect was genome-wide significant after

permutation testing (p < 0.05; Table 1; Fig. 2). A

neighboring SNP, ALGA0104951, in high LD with

MARC0099960 (r

= 0.806), did not reach genome-

wide significance but was borderline significant after

permutation testing (Table 1). These two SNPs are

both located in an intergenic region.

To define the haplotype structure within the region,

LD blocks were analyzed using Haploview. The mea-

sures of pairwise LD are shown in Fig. 3 where Block 1

indicates a region of 234 kb showing LD with SNP

MARC0099960. The proposed candidate region is flanked

by SNP markers ASGA0093685 and ALGA0123748, both

showing no or weak LD with Block 1. Thus, the two SNPs

(MARC0099960, ALGA0104951) delineate a haplotype

block and since LD to flanking markers is weak we con-

servatively use these flanking markers as coordinates and

delineate our QTL to SSC12: 42,422,021–43,436,573.

With the limitations of the annotation of the porcine gen-

ome assembly, this QTL encompasses four annotated

genes encoding chemokines (CCL1,

CCL2, CCL8, CCL11).

The following 11

spa

types were observed among the

126

S. aureus

isolates from persistent carriers (frequency

in brackets): t011 (11 %), t034 (76 %), t337 (5 %), t1333

(16 %), t1334 (8 %), t1580 (3 %), t2315 (16 %), t2370

(3 %), t2462 (11 %), t3131 (14 %), and t5817 (5 %). Thir-

teen pigs (35 %) were found to carry the same strain

throughout the study, while the remaining 24 pigs car-

ried either two (59 %) or three (5 %) different strains

(Additional file 1: Table S1). The identified

spa

types

have previously been associated with CC9 (t337, t1334,

t2315, t2462, t3131) [35–37], CC30 (t1333) [38] and

CC398 (t011, t034, t1580, t2370) [35, 39]. We did not

find any reported clonal complex association for

spa

type

t5817, which belonged to CC9 according to BURP cluster

analysis. The association between SNP MARC0099960

and host colonization was independent of

S. aureus

genotype.

Discussion

To our knowledge the work presented here is the first

attempt to decipher the host genetic factors involved in

nasal carriage of

S. aureus

in pigs. The GWAS demon-

strated a significant association between a SNP marker

located in a non-coding region (SNP MARC0099960) and

nasal carriage of

S. aureus,

while the association of a

nearby polymorphism in high LD with SNP MAR

C0099960 (SNP ALGA0104951) was borderline signifi-

cant. Regardless, the results presented here are prelimin-

ary and the association should be replicated in other pigs

to confirm the findings.

The frequency of the G allele at the SNP MAR

C0099960 locus was twice as high in carrier pigs

Fig. 2

Manhattan plots for GWAS of nasal

Staphylococcus aureus

carriage in Danish crossbred pigs. The analysis included 65 non-carriers and 56

persistent carriers which were genotyped at 23,919 autosomal SNPs.

p-values

were corrected using lambda statistic to account for genetic

confounding. Upper figure shows raw

p-values;

a suggestive significance threshold (p = 1 ×10

−5

is indicated with a horizontal line. Lower figure

shows permuted dataset after 10,000 permutations; the horizontal line shows the genome-wide significance threshold (p = 0.05)

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Table 1

Statistics for two lead single nucleotide polymorphisms (SNPs) associated with nasal carriage of

Staphylococcus aureus

Danish crossbred pigs (56 persistent carriers and 65 non-carriers)

SNP ID

MARC0099960

ALGA0104951

Chromosome

Position (bp)

43,145,785

43,380,247

A1/A2

G/A

G/T

MAF (persistent carriers)

0.63

0.61

MAF (non-carriers)

0.32

0.33

6.37×10

−6

1.38×10

−5

0.0497

0.1135

MAF

minor allele frequency;

minor allele;

major allele

Allelic association statistic adjusted for genomic control (Pc1df)

Permutation test statistic after 10,000 permutations adjusted for genomic control

compared to non-carriers of

S. aureus

(Table 1). The

majority (31 out of 36) of the 121 genotyped pigs that

were homozygous for the A allele were non-carriers of

aureus

while the majority (20 out of 28) that were

homozygous for the G allele were persistent carriers,

suggesting that the G allele is associated with suscepti-

bility to nasal carriage of

S. aureus.

A total of 31 and 26

pigs were heterozygotes in the group of persistent- and

non-carriers, respectively. Since both SNPs are located

in a non-coding part of the genome, our results indicate

that the haplotype tagged by these two SNPs contains

one or several genes with an effect on

S. aureus

nasal

carriage.

We used the Ensembl genome browser to interrogate

our region of interest (build Sscrofa10.2) and the human

orthologue of the region of interest (build GRCh38). In-

spection of our ~1 Mb QTL region revealed that it en-

compasses a cluster of four chemokine genes (CCL1,

CCL2, CCL8,

and

CCL11).

A causative variant may be a

SNP located in an exon of a protein-coding gene (chan-

ging the amino acid sequence of the protein), a regula-

tory part of a gene (altering the expression level), or a

copy-number variant of a gene [40, 41]. Chemokines are

expressed by a variety of cells to help direct immune

cells of the innate and adaptive branch of the immune

system to the site of foreign antigen [42]. There is evi-

dence suggesting some of our candidate chemokines

may be invoked following bacterial colonization;

S. aur-

eus

antigens have been shown to stimulate expression of

CCL1 by dendritic cells [43] while another study demon-

strated human alveolar epithelial cells produced CCL2

following stimulation by LPS, a component of the gram-

negative cell wall [44]. CCL2 has chemotactic properties

for monocytes [45]; indeed, recruitment of macrophages

required expression of the CCL2-binding chemokine re-

ceptor 2 in a mouse model of

Steptococcus pneumoniae

colonization [46]. CCL1 and CCL11 had direct anti-

microbial activity against

S. aureus

while CCL2 and

CCL8 did not have any effect on this pathogen [47].

The host factors underlying the differences in

S. aur-

eus

carriage are not yet fully understood [6]. Studies in

a murine model have suggested

S. aureus

clearance is

T-cell mediated and happens via an IL-17A-dependent

recruitment of neutrophils [48]. While the adaptive im-

mune response was found to be important, these au-

thors were not able to demonstrate that B-cells were

crucial. In agreement with these findings there is

evidence showing that immunity to pneumococcal

Fig. 3

Linkage disequilibrium (LD) plot showing r

x 100-values (correlation coefficient) with standard Haploview color scheme (ranging from

white when r

= 0 to black when r

= 1). Block 1 indicates a region of 234 kb showing LD with SNP MARC0099960. The proposed candidate

region is flanked by SNP markers ASGA0093685 and ALGA0123748, both showing no or week LD to block 1

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colonization is antibody independent but does require

CD4

T cells [49].

Research in humans and murine models using infec-

tion with

S. aureus

as phenotypic trait have suggested

different positional candidate genes, e.g.,

SEH1L,

TNFAIP8, KLK,

and

CDON

[16, 17, 50, 51]. However,

none of these genes are situated in or close to our QTL

region. Genetic studies in human populations are chal-

lenged by a considerable genetic heterogeneity which

may explain why previous efforts have shown a non-

significant heritability of nasal carriage of

S. aureus

[9,

10]. In contrast, pigs may be used as a convenient model

since they are much less heterogeneous and smaller

sample sizes are needed to detect genetic variants associ-

ated with complex traits [52]. The pig model may be

used to further explore

S. aureus

colonization mecha-

nisms in humans; e.g., by taking advantage of the possi-

bility to control various factors under experimental

settings (e.g., housing conditions, known inoculation

doses, known pedigrees, etc.).

GWAS for host susceptibility to infectious pathogens

should take the genome of the microorganism, i.e., strain

information, into account [53]. This notion is particu-

larly relevant to

S. aureus

colonization, which is the re-

sult of a complex interplay between host and bacterial

factors [54–56]. All

S. aureus spa

types identified in the

present study have previously been isolated from pigs

[35, 36, 38, 39] except t3131 (CC9) which has only been

reported in cattle [37]. CC398-associated

spa

types were

isolated from most (33/37) persistent carriers, suggesting

that CC398 is the most prevalent

S. aureus

lineage in

Danish crossbreed pigs. Even though the study was not

designed to study coexistence of different lineages in the

nasal cavity of pigs (i.e., only one or two isolates were

spa

typed from each sample), our results show that per-

sistent carriers can be colonized by several lineages dur-

ing a period of three weeks and by more than one strain

at the same time.

The association between SNP MARC0099960 and host

colonization was independent of

S. aureus

genotype.

This is valuable information considering that

S. aureus

a highly clonal microorganism and one clonal lineage

(CC398) accounts for most livestock-associated MRSA

infections in Europe [20, 21]. Identification of genetic

markers associated with nasal carriage of

S. aureus

may

be used in breeding to select animals with reduced sus-

ceptibility to colonization by this organism. Such a

breeding program may serve as an unexplored option to

prevent spread of livestock-associated MRSA to humans.

A similar approach was used to detect the locus respon-

sible for enterotoxigenic

E. coli

diarrhea in piglets, and a

genetic marker test which distinguishes between sus-

ceptible and resistant animals has been developed [57].

The findings presented here may also improve our

understanding of the host mechanisms underlying

aureus

colonization in both pigs and humans. The new

locus detected in the present study provides a basis for

further exploration by validation and functional testing

of the markers and candidate genes.

Conclusion

We have identified significant association between a

SNP marker located in a non-coding region (SNP

MARC0099960) and nasal carriage of

S. aureus.

The

QTL region encompasses a cluster of four chemokine

genes (CCL1,

CCL2, CCL8,

and

CCL11)

which are po-

tential candidate genes for nasal carrige. Our results sug-

gest it may be possible to select pigs genetically resistant

S. aureus

nasal colonization as a tool to control trans-

mission of livestock-associated MRSA to humans.

Additional file

Additional file 1: Table S1.

Pig and

spa

type distribution across farms.

(PDF 16 kb)

Abbreviations

S. aureus:

Staphylococcus aureus;

CC: Clonal complex; MRSA: Methicillin-resistant

Staphylococcus aureus.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

PS carried out the genetic studies, statistical analysis and drafted the

manuscript. CEG carried out the bacteriological study. CBJ participated in the

design of the study and discussion of results. MF and LG conceived of the

study, participated in its design, discussion of results and helped to draft the

manuscript. All authors read and approved the final manuscript.

Acknowledgements

The authors wish to thank Tina Neergaard Mahler, Christel Ammitzböll

Halberg and Ditte Lundt Gamborg for excellent technical assistance.

Author details

Department of Veterinary Clinical and Animal Sciences, University of

Copenhagen, Frederiksberg, Denmark.

Department of Veterinary Disease

Biology, University of Copenhagen, Frederiksberg, Denmark.

Received: 7 July 2015 Accepted: 18 November 2015

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