Veterinary Microbiology 170 (2014) 157161

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Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic

Short Communication

Clonal diversity, virulence-associated genes and antimicrobial

resistance prole of Staphylococcus aureus isolates from nasal
cavities and soft tissue infections in wild ruminants in
Italian Alps
Camilla Luzzago a,*, Clara Locatelli b, Alessia Franco c, Licia Scaccabarozzi b,
Valentina Gualdi d, Roberto Vigano` e, Giuseppe Sironi a, Martina Besozzi a,
Bianca Castiglioni f, Paolo Lanfranchi a, Paola Cremonesi f, Antonio Battisti c
a

Department of Veterinary Science and Public Health, University of Milan, Milan, Italy
Department of Health, Animal Science and Food Safety, University of Milan, Milan, Italy
c
Istituto Zooprolattico Sperimentale delle Regioni Lazio e Toscana, Rome, Italy
d
Parco Tecnologico Padano, Lodi, Italy
e
AlpVet, Crodo (VB), Italy
f
Institute of Agricultural Biology and Biotechnology, Milan, Italy
b

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 22 August 2013
Received in revised form 22 January 2014
Accepted 24 January 2014

Staphylococcus aureus is a commensal and a pathogenic bacterium that causes a wide

variety of diseases in humans and animals with a high impact on public health and the
livestock industry. S. aureus virulence pattern, antimicrobial resistance prole and host
specialization are of great concern both in livestock and in companion animals. Concerning
wild animals, S. aureus carriage and antimicrobial resistance prole has been recently
investigated in free-ranging species both in aquatic and terrestrial environment. Here we
report genotyping (spa typing, Multilocus Sequence Typing and SCCmec typing), virulence
and antimicrobial resistance prole of four S. aureus isolated in Alpine chamois (Rupicapra
r. rupicapra) and roe deer (Capreolus capreolus), euthanized due to walking impairment and
signs of disorientation. S. aureus was isolated from nasal cavities in both wild ruminant
species and in soft tissue infections in chamois. A marked S. aureus genetic heterogeneity
was detected: spa type t1523, sequence type 45 (Clonal Complex 45), and spa type t1328,
ST22 (CC22) from the nasal cavities and the liver of a chamois kid respectively, t1773,
ST700 (CC130) from an adult chamois abscess, and a new sequence type, ST2712,
belonging to CC97 from the roe deer nasal cavities.
One of the main ndings was the conrmation that the t1328, ST22 isolate, obtained
from the liver of the chamois kid, was a methicillin-resistant S. aureus (MRSA) harbouring a
SCCmec cassette type IV. The set of virulence marker and toxin genes investigated showed
proles characteristic of the S. aureus lineages detected, including those of the human
adapted ST (CC) 22 and ST (CC) 45 isolates.
2014 Elsevier B.V. All rights reserved.

Keywords:
Staphylococcus aureus
Virulence factor
MRSA
Genotyping
Chamois
Wild ruminants

* Corresponding author at: Department of Veterinary Science and Public Health, University of Milan, Via Celoria 10, 20133 Milan, Italy.
Tel.: +39 2 50318068; fax: +39 2 50318079.
E-mail address: camilla.luzzago@unimi.it (C. Luzzago).
0378-1135/$ see front matter 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.vetmic.2014.01.016

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C. Luzzago et al. / Veterinary Microbiology 170 (2014) 157161

1. Introduction

2.2. PCR of virulence factors

Staphylococcus aureus is a commensal and a pathogenic

bacterium that causes a wide variety of diseases in humans
and animals with a high impact on public health and the
livestock industry. S. aureus virulence pattern, antimicrobial resistance prole and host specialization are of great
concern both in livestock and in companion animals that
can act as transient carriers or as a reservoir of zoonotic
(Huijsdens et al., 2006) and human strains (Sung et al.,
2008). Concerning free-living wild ruminants, a low
prevalence of methicillin-resistant S. aureus (MRSA)
carriage was recently reported in healthy Iberian Ibex
(Capra pyrenaica hispanica) and red deer (Cervus elaphus)
(Porrero et al., 2013). Here we report genotyping (spa
typing, Multilocus Sequence Typing and SCCmec typing),
virulence and antimicrobial resistance proles of S. aureus
isolated from nasal cavities and infection cases in Alpine
chamois (Rupicapra r. rupicapra) and roe deer (Capreolus
capreolus).

The DNA was extracted from the isolates with a

commercial kit (QIAamp DNA Mini Kit; Qiagen GmbH,
Hilden, Germany) and PCR reactions were performed
targeting virulence-associated genes using primers and
protocols described previously [nuc, sea, sec, sed, seg, seh,
sei, sej, sek, and sel (Cremonesi et al., 2005); coa, clfA, spa, tst,
seb, see, eta, and etb (Akineden et al., 2001); lukE (Fournier
et al., 2008); LukS-PV-lukF-PV (PVL) and mecA (McClure
et al., 2006); blaZ (Martineau et al., 2000), sak, fmtB, scn,
and chp (Sung et al., 2008); LukELukD and LukM
LukFPV(P83) (Jarraud et al., 2002; Kaneko et al., 1997);
cna (Zecconi et al., 2006)].

2. Materials and methods

2.1. Sampling
S. aureus isolates were obtained from a convenience
samples consisting of three animals that had been
euthanized due to walking impairment and disorientation
symptoms by gamekeepers, in compliance with the local
ethical guidelines, in the north-western Italian Alps
(Verbano Cusio Ossola province), in autumn 2011. In
the area, a population of 3500 chamois and 2500 roe deer
were estimated to be on an overall suitable area of
65.756 ha at the 2011 census. Moreover, wild ruminants
are in close contact with goat and sheep ocks and cattle
herds during summer in overlapping pastures. Afterwards
and to date, no other wild ruminant with similar clinical
signs has been culled by the gamekeepers. The three
animals comprised two chamois, a three year old male and
a kid, and a 12 year old female roe deer. The whole
animals were stored at
20 8C and a post-mortem
examination was performed within a range of 721 days.
Samples of selected organs (spleen, lymph node, rumen,
abomasum, liver, kidney, lung and heart in adult chamois
and roe deer; spleen, liver and kidney in the kid chamois)
were collected in 10% buffered formalin, embedded in
parafn-wax and stained with haematoxylin and eosin
(HE) for histopathological examination. Samples for
bacteriological analysis were (i) swabs collected from
nasal cavities, (ii) organs (brain, lung, liver, spleen and
kidney) and (iii) an abscess in an enlarged submandibular
lymph node of the three year old chamois. The samples
from nasal cavities were incubated at 37 8C for 1820 h in
MuellerHinton broth added with to 6.5% NaCl to enhance
staphylococcal growth. Samples from the organs were
cultured onto 5% sheep blood agar plates both with and
without previous 24 h brain heart infusion enrichment.
Bacterial colonies were identied as S. aureus by colony
morphology, gram-stain, haemolytic patterns, catalase
and coagulase reaction. Conrmation was obtained by
PCR (Baron et al., 2004).

2.3. Genotyping
Spa typing was performed by DNA sequencing of the Xregion of the Staphylococcus Protein A (spa typing, Harmsen
et al., 2003), with repeats and spa types determined by
Ridom StaphType software (Ridom GmbH, Wurzburg,
Germany). Seven housekeeping genes were used for MLST
typing, according to standard protocol (http://saureus.mlst.net) as described by Enright et al. (2000). All
PCR were performed in singleplex on previously extracted
DNA and PCR products were analyzed by agarose gel
electrophoresis. Unique single bands of the expected size
were puried with a commercial kit (Wizard1 SV Gel and
PCR Clean-up System, Promega, Italy) and sequenced on
both strands using an ABI 3730 automated DNA sequencer
with the ABI PRISM1 BigDyeTM Terminator Sequencing
Kits v 3.1 following manufacturers protocol. The DNA
sequences were analyzed and manipulated with BioEdit
(http://www.mbio.ncsu.edu/BioEdit/bioedit.html)
and
consensus sequence (CAPcontig assembly program)
was veried by comparison to the sequence databases at
http://saureus.mlst.net/databases in order to obtain the
allelic prole and the sequence type (ST) for each tested
strain. The e-BURST algorithm was instead used to assign
the MLST clonal complex (CC) (http://eburst.mlst.net).
Typing of the Staphylococcal Cassette Chromosome mec
(SCCmec) on one mecA-positive isolate was obtained by
means of a multiple-PCR approach, as previously described
(Kondo et al., 2007).
2.4. Antimicrobial susceptibility testing
S. aureus antimicrobial susceptibility was tested by the
disk diffusion method according to Clinical Laboratory
Standards Institute recommendations (CLSI, 2008). Antimicrobial activities towards a wide spectrum of antimicrobials used in human and animal therapy were tested:
penicillin G (10 IU), ampicillin (10 mg), amoxicillin/
clavulanic acid (20/10 mg), cefoxitin (30 mg), ceftiofur
(30 mg), and ceftriaxone (30 mg) to represent b-lactams;
enrooxacin (5 mg), ciprooxacin (5 mg), tetracycline
(30 mg), doxycycline (30 mg), trimethoprimsulfamethoxazole (1.25/23.75 mg), erythromycin (15 mg), streptomycin
(10 mg), kanamycin (30 mg), gentamicin (10 mg), tobramycin (10 mg). All the antibiotic disks were purchased
from Oxoid (Basingstoke, UK). CLSI clinical breakpoints

C. Luzzago et al. / Veterinary Microbiology 170 (2014) 157161

were used for interpretation of the results (CLSI, 2008). As a

quality control strain, S. aureus ATCC 25923 was employed.
Fluoroquinolone (ciprooxacin) and glycopeptide (vancomycin) susceptibility was further evaluated by broth
microdilution, and results interpreted according to CLSI
standard M100-S23 (CLSI, 2013).

Table 1
Genotypic proles, virulence and antimicrobial resistance genes of S.
aureus isolates from nasal cavities and soft tissue infections in wild
ruminants.
Chamois kid

3. Results
S. aureus was isolated from the nasal mucosa of the roe
deer, from the nasal mucosa and liver of the kid chamois
and from the abscess of the submandibular lymph node of
the adult chamois. No bacterial growth other than
staphylococci has been detected. At the post-mortem
examination, the animals showed good kidney fat deposits
and regular contents of the stomach compartments. An
acute brinous pericarditis and a liver steatosis were
observed in the adult chamois while no macroscopic
lesions were observed in the kid and roe deer. By
histopathological examination, pulmonary hyperaemia
and focal myocardic hemorrhages and necrosis were
detected in the adult chamois. Sarcocystis spp. were rarely
observed in cardiomyocytes. No other microscopic lesions
were observed. No microscopic changes were noted in the
kid and roe deer. Cocci were not observed by histopathological examination.
The isolate from the chamois liver (ID2775) was
resistant by the disk diffusion method to penicillin,
ampicillin, amoxicillin and clavulanic acid, cefoxitin,
ceftiofur, ceftriaxone, ciprooxacin and enrooxacin.
Fluoroquinolone resistance was further conrmed by
broth microdilution (MIC > 8 mg/L). Methicillin-resistance
was conrmed by the presence of the mecA gene. All other
isolates proved to be methicillin-susceptible S. aureus
(MSSA) and showed susceptibility to all the drugs tested.
The only exception was penicillin resistance observed in
the isolate from the nose of the kid chamois (ID 2774), and
was conrmed by the presence of the blaZ gene (Table 1).
The isolates of the chamois kid from nasal mucosa (ID
2774) and liver (ID2775) were MSSA spa type t1523, ST45
(CC45) and MRSA t1328, ST22 (CC22,) SCCmec type IV,
respectively, while the isolate from the adult chamois
resulted a MSSA t1773, ST700, a single locus variant (SLV)
of ST130, within the CC130 MSSA. The MSSA detected from
the roe deer mucosa was a new sequence type ST2712, a
SLV of ST97, within the CC97, and was classied as spa type
t1773. The genotypic proles, including virulence and
antimicrobial resistance genes of S. aureus isolates are
summarized in Table 1. All S. aureus isolates were positive
for spa, coa, lukE, clfA, nuc, fmtB genes while the two ST22
and ST45 isolates only for the cna gene. The sak, scn and chp
genes were detected in the ST22 and ST45 isolates of
chamois kid only, while the staphylococcal enterotoxin
genes were present in all the three lineages detected in the
chamois isolates, with ST22 and ST45 also harbouring seg
and sei genes, within the enterotoxin gene cluster. The
chamois abscess isolate t1773 ST700 (CC130) harboured
the leukotoxin LukE-LukD, LukM-LukF-PV(P83) and tst1
genes. None of the isolates harboured genes encoding for
exfoliative toxins A and B (eta, etb) and Panton-Valentine
Leukocidin (LukS-PV-lukF-PV).

159

MRSA/MSSAc
Spa type
Sequence type
Clonal complex
Gene abbreviation
mecA
blaZ
spa
coa
lukE
clfA
nuc
fmtB
cna
sak
scn
chp
LukELukD
LukM
lukF-PV(P83)
LukS-PV-lukF-PV (PVL)
sea
seb
sec
sed
see
seg
seh
sei
sej
sel
tst1

Chamois
adulta

Roe deer

Nasal
mucosa
id 2774b

Liver
id 2775

Abscess
id 2701

Nasal
mucosa
id 2773

MSSA
t1523
45
CC45

MRSA
t1328
22
CC22

MSSA
t1773
700
CC130

MSSA
t1773
2712
CC97

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+

+
+
+

+
+

+
+

No other specimens have been submitted to laboratory.

Isolate identication number.
c
MSSA: Methicillin-susceptible S. aureus; MRSA: Methicillin-resistant
S. aureus.
b

4. Discussion
Despite the limited number of animals and S. aureus
isolates tested, this report raises interesting issues
regarding clonal diversity, virulence and antimicrobial
resistance proles detectable in wild ruminant species that
are widespread in Europe. Recently MLST was applied to
MRSA detected on the skin and nasal swabs of apparently
healthy free-living ruminants, namely Iberian ibex and red
deer (Porrero et al., 2013). In particular, three MRSA
isolates were found in the group of 485 tested animals,
with these isolates belonging to the ST398 genotype,
which is the most frequent ST detected in livestock in
Europe, including Italy, despite a more evident heterogeneity of lineages observed in Italian primary productions
(Battisti et al., 2010).
In our study, S. aureus soft tissue infections were
observed in chamois, and a contamination or colonization
of nasal mucosa was also detected in both wild ruminant
species. One of the main ndings was that the chamois
liver isolate was a ST (CC) 22 MRSAIV. This is a

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C. Luzzago et al. / Veterinary Microbiology 170 (2014) 157161

widespread human epidemic clone, also known as UKEMRSA-15, occurring both in hospitals and in outpatients,
also in Italy (Campanile et al., 2009). The isolate has a
virulence gene prole fully compatible with human
EMRSA-15, including uoroquinolone resistance which is
commonly observed in this clonal complex (Qi et al., 2005).
A plausible explanation for this rare occurrence of a MRSA
in a free-living chamois kid could be the exposure to a
natural environment contaminated by human outdoor
activities or by other accidental carrier species. The other
isolate from a chamois kid also showed a genotypic prole
typical of human-adapted S. aureus lineages. The ST (CC) 45
isolate, proved to be spa type t1328, with a repeat
succession very similar to other spa types prevalent within
the CC45 and associated with human colonization and
infection. Interestingly, a MRSA isolate within this clonal
complex was detected from wild boar meat in Spain,
although the Authors did not exclude a possible contamination of the positive sample by food-handlers (Lozano
et al., 2009). Unexpectedly, spa type t1773 in addition to
chamois abscess was found also in the isolate from the roe
deer nasal mucosa, a ST2712 within the CC97, a clonal
complex which in Italian livestock is usually associated
with other spa types (Battisti et al., 2010; Feltrin et al.,
2013). This nding might be explained with the occurrence
of a horizontal transfer of the X-region, between two
ruminant-associated lineages (possibly from a CC130
donor), similarly to what has been demonstrated for spa
type t899, acquired by ST398 from the ST9 lineage in a pigrearing environment (Price et al., 2012). Concerning the
virulence prole of the S. aureus isolates, the immune
evasion gene cluster, which is rare in animal species and
highly prevalent in human strains (Sung et al., 2008), was
detected in the two human-adapted clones only, as
expected. Conversely, the isolate detected from lesions
(submandibular lymph-node abscess) in the adult chamois
had an overall genotypic prole typical of ruminant
(bovine)-associated lineages, including variants of leukotoxins and tst genes. Spa type t1773, CC130 is a clone
already detected in Italian domestic ruminants (Battisti,
unpublished). Further evidences of its domestic ruminant
origin were obtained by the positive results for Luk-MLukF-PV(P83) and tst1 (RF122 bovine allele) genes. The
latter is a variant of the gene encoding the toxic shock
syndrome toxin and located with sec and sel genes on
mobile genetic element called bovine staphylococcal
pathogenicity island (Fitzgerald et al., 2001). In this case,
a contamination of pastures by sympatric livestock seems
the most likely source of exposure for wild ruminants.
In summary, though the data from this study are
inadequate to draw any association between the S. aureus
virulence prole and the clinical debility of the animals,
clones associated with human or domestic ruminant hosts
where detected in free ranging wild ruminants living in
areas where human activities and free-range livestock
farming also occur.
It would be interesting to characterize a wider number of
isolates from wild and domestic species that are in potential
contact with each other, such as goat and sheep ocks
and cattle herds during summer pasture for an insight
into host-pathogen interactions in sympatric populations.

Acknowledgments
We wish to thank the Corpo di Polizia Provinciale di
Verbania and Ente Parco Nazionale Val Grande for eld
support. Dr. L. Abraham for the English revision of the
manuscript.

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