burns 40 (2014) 288–294

Available online at www.sciencedirect.com

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Antibiotic susceptibility and resistance of

Staphylococcus aureus isolated from fresh porcine
skin xenografts: Risk to recipients with thermal
injury

Stacey-Ann Busby a, Andrew Robb b, Sue Lang a, Yasu Takeuchi c,

Pavel Vesely d, Linda Scobie a,*
a
Department of Life Sciences, Glasgow Caledonian University, Glasgow G4 0BA, UK
b
Scottish MRSA Reference Laboratory, Stobhill Hospital, 133 Balornock Road, Glasgow, UK
c
MRC/UCL Centre for Medical Molecular Virology and Wohl Virion Centre, Division of Infection & Immunity, UCL,
London, UK
d
CEITEC – Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic

article info abstract

Article history: The previous use of fresh porcine xenografts at the Prague Burn Centre had raised concerns
Accepted 1 June 2013 over the transmission of zoonotic pathogens. This study examines the risk of zoonotic
Staphylococcus aureus colonisation of burn patients from fresh porcine skin xenografts.
Keywords: Samples were collected from the nares, skin and perineum of commercial pigs (n = 101) and
Infection were screened for methicillin sensitive S. aureus (MSSA) and resistant S. aureus (MRSA). The
MRSA efficacy of the antibiotic wash used in decontamination of the pigskin was tested against
MSSA planktonic- and biofilm-grown isolates. The spa type of each isolate was also confirmed.
Porcine skin All pig swabs were negative for MRSA but 86% positive for MSSA. All planktonic-grown
isolates of MSSA were sensitive to chloramphenicol and nitrofurantoin and 44% of isolates were
resistant to streptomycin. Isolates grown as biofilm exhibited higher rates of antimicrobial
resistance. Sequence analysis revealed three distinct spa types of the MRSA ST398 clonal type.
This finding demonstrates the existence of a MSSA reservoir containing spa types
resembling those of well-known MRSA strains. These MSSA exhibit resistance to antibiotics
used for decontamination of the pigskin prior to xenograft. Amended use of procurement
could allow the use of fresh pigskin xenografts to be reinstated.
# 2013 Elsevier Ltd and ISBI. All rights reserved.

1. Introduction
Due to the shortage of human skin for allo-transplantation,
fresh pig skin was frequently used for xenografting at the
Prague Burn Centre (PBC) [1]. However, the use of pigs as a skin
source for xenotransplantation raises concerns over the
potential for zoonotic infection [2,3].
In the treatment for severe burns patients, fresh or frozen
porcine xenografts have proven to be an effective temporary

* Corresponding author at: School of Health and Life Sciences, Department of Life Sciences, Glasgow Caledonian University, Glasgow G4
0BA, UK. Tel.: +44 1413318534.
E-mail address: linda.scobie@gcu.ac.uk (L. Scobie).
Abbreviations: MRSA, methicillin resistant Staphylococcus aureus; MSSA, methicillin sensitive Staphylococcus aureus; CA, community
acquired; HA, hospital acquired; LA, livestock associated; Spa gene, encoding pathogenic factor Protein A.
0305-4179/$36.00 # 2013 Elsevier Ltd and ISBI. All rights reserved.
http://dx.doi.org/10.1016/j.burns.2013.06.006
 burns 40 (2014) 288–294
biological skin substitute [1,4]. Xenografts adhere well to
excised wounds and when removed a clean granulating
wound remains. This promotes rapid epithelial in-growth in
superficial burns and prepares the wound bed for autografting
in deep dermal burns [1]. The PBC in the Czech Republic have
successfully used commercial pigs for over 10,000 skin strip
xenografts annually for severely burned patients and a skin
tissue bank has been established [1,4,5]. The skin xenografts
are in contact with the recipient for up to eight weeks during
treatment. Fresh viable full thickness pig skin strips are
treated with a saline/antibiotic solution prior to application [1].
Indeed, other centres currently use fresh porcine skin with
ongoing success and procurement and preservation techni-
ques are constantly being improved [1,6].
Previous studies on PBC patients receiving fresh porcine
skin xenografts have reported that patients were colonised
with a number of different pathogens with the majority of
these being staphylococci [7]. In addition, methicillin sensitive
Staphylococcus aureus (MSSA) has been shown to be the most
frequently isolated organism from recipients followed by
methicillin resistant S. aureus (MRSA) [5]. S. aureus is a common
opportunistic pathogen associated with a wide range of
diseases from superficial skin and soft tissue infections to
severe life threatening infections including endocarditis and
septicaemia [8]. MRSA is a documented problem in hospital
settings [9] and since the late 1990s there has been a significant
increase in the number of MRSA infections reported outside of
the health care environment categorised as community
acquired MRSA (CA-MRSA) [10]. In general CA-MRSA strains
are more susceptible to antibiotics than hospital acquired
MRSA (HA-MRSA) [11]. Over the past decade a third category of
MRSA has emerged, the livestock associated MRSA (LA-MRSA
[11]).
It has been previously described that MRSA strains evolve
from MSSA strains by acquisition of the mecA gene, which is
carried in a mobile genetic element called the staphylococcal
cassette chromosome mec (SCCmec). This gene confers
resistance to virtually all b-lactam antimicrobials by encoding
an alternative penicillin binding protein (PBP2a), which has a
lowered affinity for b-lactam antibiotics [12]. Epidemiological
information on S. aureus can be obtained through various
methods such as multilocus sequence typing (MLST) and spa
typing, which is based on the sequencing of the polymorphic X
region of the S. aureus protein A gene (spa gene). [11]. It is
suggested that LA-MRSA has been evolved by multiple
introductions of the SCCmec element into MSSA and studies
have shown that strains with identical spa-types can carry
different SCCmec elements [13,14].
To date, two major lineages of LA-MRSA have been
described. MRSA multilocus sequence type (ST) 398 of swine
origin is predominant in Europe and North America and ST9 in
Asia [13,15]. As with humans, colonisation occurs in the nasal
passage, perineum and skin and it has now become an
important global zoonotic pathogen documented in horses,
cats, dogs and cattle. European surveillance studies show that
LA-MRSA ST398 isolates typically belong to 4 major spa types
(t011, t108, t034, and t899) and several minor spa types (e.g.,
t1197, t1451, t1456) [16].
Many studies have also reported the prevalence of MSSA
ST398 in swine [14] and porcine MSSA is known to be present
289
on pigskin and has been described in China, Japan and
Denmark [15–18].
S. aureus, including LA-MRSA, is a significant cause of skin
infections in pigs. Transmission of LA-MRSA between pigs and
pig farmers, pig farmers and their families, veterinarians and
companion animals in small-animal healthcare, and in equine
hospitals has been reported [19,20]. While it has been shown
that LA-MRSA can cause disease in humans, there is currently
no evidence to date, that LA-MRSA has been transmitted
between humans [21].
Finally, it is known that bacteria can exist in two growth
states, planktonic and sessile. Most studies are carried out
using planktonic bacteria, however, in nature, bacterial
populations exist as biofilms. Biofilms exhibit altered pheno-
types with respect to growth, antibiotic resistance and gene
expression and are thought to be the predominant bacterial
phenotype on both healthy and diseased skin [22–24].
Therefore, the carriage of MRSA and/or MSSA on pigskin used
for xenografts could potentially be a risk factor for colonisa-
tion and subsequent infection in burn patients [25].
As a moratorium had been introduced in the Czech
Republic preventing the continuation of this technique, the
purpose of the study was to demonstrate if there was any risk
from the use of fresh viable commercial pig skin. Based on
previous studies demonstrating S. aureus as a common
commensal on pigs skin [7] and the reports on zoonotic S.
aureus infections in individuals [11] the aim of this study was
to; (i) screen a commercial pig herd used for skin xenografts for
the presence of MSSA and MRSA, (ii) assess the clonal
structure of S. aureus isolated from these pigs, (iii) and
determine the antimicrobial susceptibility of planktonic-
and biofilm-associated strains to antimicrobials used to
decontaminate porcine skin prior to xenografting at the PBC.
2. Materials and methods
2.1. Animal sampling
A total of 101 pigs from a commercial herd in Prague used for
skin xenografts were swabbed for the presence of S. aureus.
Three swabs were taken from each pig, one from the
perineum, one from the nasal cavity and an additional swab
from the skin. Each swab was placed into Amies charcoal
medium (Sterilin, UK) for transport and shipped at 4 8C within
24 h of collection.
2.2. Isolation of S. aureus
Each swab was initially cultured on a blood agar plate (BAP)
and in ContrastTM MRSA broth (Oxoid Ltd., Hampshire, UK)
and incubated for 24 h at 37 8C. Positive ContrastTM MRSA
broth cultures were inoculated onto BrillianceTM chromogenic
agar (Oxoid, Hampshire, UK), non-selective 5% BAP (Oxoid
Ltd., Hampshire, UK) [26] and incubated overnight at 37 8C in
air. After incubation, BAP and Brilliance chromogenic agar
were examinded and potential S. aureus were submitted to a
catalase, DNase and StaphaureuxTM latex agglutination test
(Oxoid Ltd., Hampshire, UK) as per manufacturer’s instruction
[27]. The positive control used was the S. aureus NCTC 6571
290 burns 40 (2014) 288–294

Oxford strain. Those confirmed to be S. aureus were stored at
80 8C in brain heart infusion broth (Oxoid Ltd., Hampshire,
UK) supplemented with 10% glycerol for future study. Working
cultures were maintained on brain heart infusion agar (Oxoid
Ltd., Hampshire, UK) stored at 4 8C.
2.3. Antibiotic susceptibility testing
2.3.1. Minimum inhibitory concentration (MIC)
Oxacillin broth microdilution antibiotic susceptibility testing
was performed following the recommendations of the British
Society of Antimicrobial Chemotherapy (BSAC). In brief,
morphologically similar colonies were touched and trans-
ferred to Mueller Hinton broth (Oxoid Ltd., Hampshire, UK) and
incubated for 18 h at 37 8C with aeration at 200 rpm. The
organism suspension was adjusted to McFarland 0.5 turbidity
standard (1  108 colony forming units (cfu/ml)) and working
solution produced by performing a 1:1000 dilution in then
Mueller Hinton broth to a final concentration of 1  105 cfu/
ml. Oxacillin (Sigma, Dorset, UK) was prepared in the range 0–
128 mg/L by two fold serial dilution. 75 ml of working bacterial
suspension was combined with 75 ml of oxacillin in a
microtitre plate (Sterlin, UK) and incubated at 37 8C for 18 h.
The MIC was defined as the lowest concentration of oxacillin
that completely inhibited growth. S. aureus NCTC 6571 was
used for quality control.
2.3.2. Susceptibility of planktonic- and biofilm-grown strains
to antibiotic wash
The susceptibility of planktonic- and biofilm-grown isolates to
the antibiotic wash containing streptomycin (4 g/L), chloram-
phenicol (6 g/L) and nitrofurantoin (2.4 g/L) was determined by
broth dilution. An isolate working concentration of
1  105 cfu/ml was prepared as previously described. Chlor-
amphenicol and nitrofurantoin were initially diluted in
ethanol and dimethyl formamide (DMF).
One millilitre of bacterial suspension and 1 ml of antibiotic
were combined and incubated at 37 8C for 18 h. Prior to the
addition of the antibiotic a control viable cell count was
performed by inoculating 100 ml of bacterial suspension on
brain heart infusion agar (Oxoid, Hampshire, UK). Following
the addition of antibiotic 100 ml of bacterial suspension, taken
at 0, 5, 10, 15 and 20 min, was inoculated on to brain heart
infusion agar (Oxoid, Hampshire, UK). The plates were
incubated at 37 8C for 18 h and the bacterial cell survival
calculated as a percentage of the antibiotic free control. S.
aureus NCTC 6571 was included for quality control [30].
Biofilm production was performed following the method
described by Smith et al. [28] for each test isolate, 150 ml of the
working bacterial suspension (1  105 cfu/ml) was dispensed
to 9 wells of a 96-well microtitre plate (Sterilin, UK) in duplicate
and incubated at 37 8C on a rocking platform at 20 oscillations
per minute for 24 h. Planktonic cells were removed by washing
in phosphate buffered saline (PBS) (Oxoid Ltd., Hampshire, UK)
three times. Biofilms was challenged with 150 ml of antibiotic.
The nine duplicate wells were untreated with antibiotic to
serve as a control. To assess bacterial cell death, 150 ml of
AlamarBlue1 (Invitrogen, UK), diluted 1 in10 with PBS, was
added to each well and the plate incubated in the dark for 1 h
at 37 8C on a rocking platform at 20 oscillations per minute.
Results were read at Abs580 nm and the percentage cell
survival was calculated by averaging the results for each strain
at each time point/antibiotic treatment, then expressing as a
percentage of the control.
2.3.3. Biofilm formation protocol
Samples were grown in BHI broth to a final cell density of
5  105 cfu/ml. 100 ml was placed into 6 replicate wells of a 96
well microtitre plate and incubated for 24 h at 37 8C. Super-
natants were removed and cells washed with PBS then air
dried for 15 min and heat fixed in 80 8C oven for 30 min. Plate
was stained for 15 min with 0.5% (w/v) crystal violet. Plate was
washed with distilled water three times and 100 ml of 70%
ethanol added to remove stain. Absorbance was measured at
570 nm.
2.3.4. DNA extraction and Spa typing
Genomic DNA from S. aureus strains was extracted following
the method of Kobayashi et al. and Leonard et al. [29,30].
Briefly, three to five colonies were suspended in NET buffer
(10 mM Tris, 1 mM EDTA, 10 mM NaCl) containing 10 units of
achromopeptidase (Sigma, UK) and incubated at 50 8C for 10–
15 min. The x region of the spa gene was amplified by PCR with
primers 1113(F) 50 -AGACGATCCTTCGGTGA-30 and 1517(R) 50 -
GCTTTTGCAATGTCATTTACTG-30 as previously described by
Harmsen et al. [31]. DNA sequences were obtained using an
ABI 3500xL sequencer (Applied Biosystems, Foster City, CA).
Spa types are assigned through the Ridom web server (http://
spa.ridom.de/spatypes.html). Spa types with similar repeat
profiles are grouped together as part of the same lineage.
3. Results
3.1. S. aureus isolation
Of the 101 pigs screened, no MRSA was isolated from any of the
herds, however, a total of 87 (86% = 87/101 isolates) MSSA were
isolated from the commercial pigs. MSSA isolates from the pig
population were distributed unequally between the nasal
passage, skin and perineum, with the skin resulting in the
Fig. 1 – Distribution of MSSA isolates by sample site.
 burns 40 (2014) 288–294 291

highest number of isolates followed by the nares then the the MSSA isolates were resistant to the decontamination
Table 1 – Planktonic growth of nine MSSA strains isolated from the pigskin used for xenografts. Spa types for each strain
are indicated and are all of the MLST sequence type ST398. Antibiotic resistance and susceptibility are indicated as colony
forming units/ml.
MSSA Spa Growth of strains after 5 min exposure to antibiotic Growth of strains after 20 min exposure to
isolate type wash (cfu/ml) antibiotic wash (cfu/ml)

Streptomycin Chloramphenicol Nitrofurantoin Streptomycin Chloramphenicol Nitrofurantoin

3 t1456 0 0 0 0 0 0
5 t1456 3.6  10 5 0 0 3.6  10 5 0 0
6 t1456 6.2  10 3 0 0 0 0 0
7 t10569 3.3  10 3 0 0 0 0 0
10 t1456 3  10 5 0 0 3  10 5 0 0
11 t034 0 0 0 0 0 0
18 t034 0 0 0 0 0 0
22 t1456 3  10 5 0 0 3  10 5 0 0
23 t1456 1.9  10 5 0 0 1.9  10 5 0 0

perineum at 61%, 50% and 21% respectively (Fig. 1). Five
animals from the commercial herd were found to have MSSA
in all three sites analysed but isolates from either one or two
sites were most common from the rest of the animals tested
(data not shown).
3.2. Sensitivity of MSSA isolates to antibiotic wash in
planktonic and biofilm state
protocol used. Before decontamination an active bioburden
was present for all isolates (data not shown). 14% of the MSSA
isolates from pig skin were selected at random for antibiotic
susceptibility testing. Only three of the nine isolates tested, 3,
11 and 18 showed a reduction in survival after 5 min when
planktonically grown in streptomycin (Table 1). After exposure
to streptomycin for 20 min, only four isolates remained
resistant 5, 10, 22 and 23 (Table 1).

Porcine MSSA is known to be present on pigskin and as no In the presence of chloramphenicol, all isolates were non-
MRSA was isolated, it was deemed necessary to determine if viable after 5 min when directly exposed (Table 1). No survival

Fig. 2 – Percentage bacterial cell survival of biofilm producing isolates when exposed to individual antibiotics. Survival was
not reduced in any of the isolates when exposed to streptomycin (A). Cell survival was reduced in isolates 7, 18, 22 and 23
(4/9) after exposure to chloramphenicol (B). Cell survival was reduced in 7, 10, 11, 18 and 23 (5/9) after exposure to
nitrofurantoin (C). Each point represents the mean of results from duplicate experiments, with six wells per experiment.
Ox = positive control NCTC 6571 Oxford strain with and without antibiotic treatment.
292 burns 40 (2014) 288–294
after 5 min was observed for any strains when nitrofurantoin
was added to the broth (Table 1).
We know that S. aureus produce biofilm, a complex matrix
that exhibits resistance to antibiotics not seen in their
planktonic growth state. Of the 9 biofilm producing MSSA
isolates, it was observed that all of the biofilm grown strains
were resistant to streptomycin (Fig. 2A). MSSA isolates 3, 5, 6,
10 and 11 remained viable after 20 min exposure to chloram-
phenicol (Fig. 2B); likewise 3, 5, 6 and 22 exhibited enhanced
cell survival after 20 min exposure to nitrofurantoin (Fig. 2C).
3.3. Spa typing of MSSA strains isolated
Spa gene sequence analysis of the nine isolates tested
demonstrated three separate spa types. Six isolates belonged
to spa type t1456, two to spa type t034 and a single isolate to spa
type t10569 (Table 1). All of these were identified as being
associated with the clonal complex 398 (CC398) to which ST398
belongs.
4. Discussion
Due to the increasingly limited source of human skin
allografts, it has become necessary to develop new wound
dressing materials that can substitute. Porcine skin is readily
available at low cost and is a common substitute for skin
allografts in deep seated burn wounds [1,4]. Although fresh
porcine skin is cheaper and easier to use, it has a more potent
antigenicity and a higher rate of bacterial contamination than
irradiated or gluteraldehyde treated allografts [1,4]. Thus it is
important to quantify the infectious risk in fresh viable skin
compared to other non-vital products in use.
Colonisation and infection caused by S. aureus in pigs is well
documented [32]. Therefore, the use of skin from pigs
colonised with S. aureus could pose a significant risk to
patients with severe burns and reduced immune-competence.
A study by Vrankova et al., undertaken at the Prague Burns
Centre (PBC) in the late 1990s, had shown that S. aureus,
including MRSA, was the most frequently isolated bacterial
species from patient’s pre and post-transplant [5]. Likewise,
another study indicated one incident of S. aureus infection post
graft [33] although the source of infection was not described.
Conversely, the majority of studies to date, using tissue
banked pigskin indicate no graft originated infection [34,35].
In the present study, detection of S. aureus was analysed
from three different anatomical sites on pigs used for
xenografts. Overall the isolation rate of S. aureus, all MSSA,
in this sample groups was 86% (87/101 isolates). No MRSA were
isolated. S. aureus isolation was not evenly distributed across
the three sampling sites with the greatest number of isolates
being detected in the skin (61%). Nasal colonisation was found
to be 38% and the least number of isolates were obtained from
the perineal swabs (24%). Previous reports have indicated that
S. aureus is more frequently isolated from slaughter pig’s nasal
sites when compared to the perineum [36] with other data
showing similar levels of isolates being recovered from skin
and nares [37]. The high degree of MSSA isolates we detected
on the skin surface may be due to the fact that the skin sites
are more readily exposed to the environment, which could
lead to more prominent colonisation. In addition, density of
animals in the farming environment may also contribute to
this. A study by Verhegghe et al. has shown a similar rate of S.
aureus isolation, in commercial pigs, from Belgium [38].
Due to large quantities of antimicrobial agents used in the
production of food animals, it is generally accepted that
animals and humans constitute overlapping reservoirs of
antimicrobial resistance, and consequently the use of anti-
microbials in animals can impact on public health [39]. Our
results show that all MSSA isolates in the planktonic cell assay
were susceptible to chloramphenicol and nitrofurantoin with
only five of the nine isolates showing susceptibility to
streptomycin after 20 min. However, when these same
isolates were grown as biofilm, 100% of the isolates exhibited
resistance to streptomycin, 55% (5/9) exhibited resistance to
chloramphenicol and 44% (4/9) exhibited resistance to
nitrofurantoin. A study by Sakoulas et al. has shown that
strong biofilm producing S. aureus are more inherently
resistant to the effects of antibiotic treatment [40]. Our results
are also consistent with those published by Rose et al. where
biofilm producing S. aureus were more resistant to antibacte-
rial therapy than isolates grown as planktonic cells [41]. Not so
clear, however, is the extent to which this resistance can be
attributed to the structure of biofilms rather than the
physiology and density of bacteria within them. The formation
of biofilms on the pig skin is of concern and alternative
treatments or combination therapy should be explored to
optimise outcomes in biofilm-associated infections. Studies
have been carried out previously looking at microbial resis-
tance in porcine skin/tissue biobanks and what appropriate
combinations are suitable for decontamination [6,42] and this
information should be exploited.
As mentioned, data indicates that MRSA ST398 strains may
evolve from a variety of MSSA strains and recent studies have
reported the prevalence of MSSA ST398 in swine [17,18]. In the
present study, we examined the spa types of MSSA isolates
obtained from the commercial swine in order to evaluate the
possibility of the emergence of MRSA in pigs used for skin
xenografts. Our analysis of the S. aureus isolates identified
three spa types; two were from the major group (t034) and the
remainder in the minor type category (t1456 and t1056). All of
these were associated with the ST398 livestock associated S.
aureus (LA-MSSA) lineage as confirmed using the Ridom
database (http://spaserver.ridom.de). Two specific spa types,
t034 (22.2%) and t1456 (66.6%) clustered within the group
tested supporting previous evidence that specific clones are
predominant within farms [13]. A rare spa type, t10569 (repeat
succession 11-10-21-17-34-24-34-17-25) was identified in one
isolate. While this spa type does not cluster, by BURP analysis,
with the other spa types (t034 and t1456) this isolate was typed
by MLST at the reference laboratory and the sequence type
also belonged to the ST398/CC398 clonal background. Inter-
estingly, the resistant strains of MSSA identified belonged to
spa type t034 (second most common in pigs) and t1456 (less
common in pigs). MSSA identified in the study by Osadebe
et al. belonged to spa types t337 and t034 and they suggested
that swine MRSA, like human, may emerge from pre-existing
MSSA reservoirs which acquire the SCCmec gene cassette [43].
Indeed, as mentioned a number of other studies have
identified MSSA ST 398 in pigs [15–18]. It could be hypothesised
 burns 40 (2014) 288–294
that accquistion of SCCmec leading to antibiotic resistance
and LA-MRSA could be encouraged by the selective pressure of
antibiotic use in commercial pigs. Indeed, these spa types have
been shown to colonise humans and are commonly found in
professionals in contact with pigs, however, the risk of spread
between humans is unknown [13]. Therefore, the presence of
anti-microbial resistant MSSA spa types in commercial swine
used for xenografting should be monitored.
This work and others [16,44] suggests that due to the
evidence of human to animal transmission, biosafety prac-
tices for fresh pigskin xenografts should be unified and animal
screening should complement existing surveillance systems
for MSSA and MRSA zoonoses. With improved procurement
and preservation methods at PBC, there appears to be a limited
risk to burns patients receiving vital pigskin from colonisation
with S. aureus. Indeed, the use of fresh and frozen pigskin is an
economically viable option when compared with other
alternative dressings. With regard to the commercial herd
in this study, alternative antimicrobials such as the combina-
tion of amphotericin, ampicllin and ceftazidime suggested by
Csonge et al. could improve the suitability of the skin for
xenografting [6]. In addition, to ensure that LA-MRSA has not
arisen in this source herd, periodic testing should take place.
European guidelines regarding the procurement and
preservation of xenograft tissue are now available and can
be implemented to ensure the low risk of S. aureus colonisation
associated with this practice. PBC are currently using and have
demonstrated the successful use of biological acellular pig
dermis for burn treatment, reducing the overall risk of
infection [45]. However, these results suggest that re-estab-
lishment using fresh pigskin at PBC would be acceptable as
there appears to be no elevated infectious risk with respect to
S. aureus.
Conflict of interest
This manuscript, including related data, figures and tables has
not been previously published and that the manuscript is not
under consideration elsewhere.
All authors confirm that there are no conflicts of interest.
Acknowledgements
The authors would like to acknowledge the assistance of Dr.
Blahoslava (CZ MVDr) for sample collection at Cesky Brod near
Prague. This work has been supported by a European
Commission FP6 funded project LSHB-CT-2006-037377,
Xenome.
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