RESEARCH ARTICLE Open Access
S. pseudin e medius and S. au eus lineages
wi h ansmission abili y ci cula e as
causa i e agen s o in ec ions in pe s o
yea s
Lau a Ruiz-Ripa
1†
, Ca men Simón
2
, Sa a Ceballos
1
, Ca melo O ega
2
, My iam Za azaga
1
, Ca men To es
1
and
Elena Gómez-Sanz
3,4,1*†
Abs ac
Backg ound: S aphylococcus pseudin e medius (SP) and S aphylococcus au eus (SA) a e common colonize s o
companion animals, bu hey a e also conside ed oppo unis ic pa hogens, causing diseases o di e se se e i y. This
s udy ocused on he iden i ica ion and cha ac e iza ion o 33 coagulase-posi i e s aphylococci isola ed om
diseased pe s (28 dogs and i e ca s) du ing 2009–2011 in a e e ina y hospi al in Spain in o de o s ablish he
ci cula ing lineages and hei an imic obial esis ance p o ile.
Resul s: Twen y-eigh isola es we e iden i ied as SP and i e as SA. Nine me hicillin- esis an (MR) isola es (27%)
ca ying he mecA gene we e de ec ed (eigh MRSP and one MRSA). The 55% o SP and SA isola es we e mul id ug-
esis an (MDR). MRSP s ains we e yped as ST71-ag III-SCCmecII/III-(PFGE) A (n=5), ST68-ag IV-SCCmecV-B1/B2
(n=2), and ST258-ag II-SCCmecIV-C (n=1). SP isola es showed esis ance o he ollowing an imic obials [pe cen age
o esis an isola es/ esis ance genes]: penicillin [82/blaZ], oxacillin [29/mecA] e y h omycin/clindamycin [43/e m(B)],
aminoglycosides [18–46/aacA-aphD, aphA3, aadE], e acycline [71/ e (M), e (K)], cip o loxacin [29], chlo amphenicol
[29/ca
pC221
], and ime hop im-sul ame hoxazole [50/d G,d K]. The d K gene was e ealed as pa o he adC-
in eg a ed Tn559 in wo SP isola es. Vi ulence genes de ec ed among SP isola es we e as ollow [pe cen age o
isola es]: sie [100], se-in [100], lukS/F-I [100], sec
canine
[7], and expB [7]. The single MRSA-mecA de ec ed was yped as
011-ST398/CC398-ag I-SCCmecV and was MDR. The me hicillin-suscep ible SA isola es we e yped as 045-ST5/CC5
(n=2), 10576-ST1660 (n=1), and 005-ST22/CC22 (n=1); he 005-ST22 eline isola e was PVL-posi i e and he wo
045-ST45 isola es we e asc ibed o Immune E asion Clus e (IEC) ype F. Mo eo e , he 10576-ST1660 isola e, o
po en ial equine o igin, ha bo ed he lukPQ and scneq genes. Acco ding o animal clinical his o y and da a eco ds,
se e al s ains seem o ha e been acqui ed om di e en sou ces o he hospi al en i onmen , while some SA
s ains appea ed o ha e a human o igin.
(Con inued on nex page)
© The Au ho (s). 2021 Open Access This a icle is licensed unde a C ea i e Commons A ibu ion 4.0 In e na ional License,
which pe mi s use, sha ing, adap a ion, dis ibu ion and ep oduc ion in any medium o o ma , as long as you gi e
app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons licence, and indica e i
changes we e made. The images o o he hi d pa y ma e ial in his a icle a e included in he a icle's C ea i e Commons
licence, unless indica ed o he wise in a c edi line o he ma e ial. I ma e ial is no included in he a icle's C ea i e Commons
licence and you in ended use is no pe mi ed by s a u o y egula ion o exceeds he pe mi ed use, you will need o ob ain
pe mission di ec ly om he copy igh holde . To iew a copy o his licence, isi h p://c ea i ecommons.o g/licenses/by/4.0/.
The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i ecommons.o g/publicdomain/ze o/1.0/) applies o he
da a made a ailable in his a icle, unless o he wise s a ed in a c edi line o he da a.
* Co espondence: [email p o ec ed]
†
Lau a Ruiz-Ripa and Elena Gómez-Sanz con ibu ed equally o his wo k.
3
Ins i u e o Food, Nu i ion and Heal h, ETH Zü ich, Schmelzbe gs asse 7,
LFV B36, 8092 Zu ich, Swi ze land
4
Á ea de Mic obiología Molecula , Cen o de In es igación Biomédica de La
Rioja (CIBIR), Log oño 26006, Spain
Full lis o au ho in o ma ion is a ailable a he end o he a icle
Ruiz-Ripa e al. BMC Ve e ina y Resea ch (2021) 17:42
h ps://doi.o g/10.1186/s12917-020-02726-4
(Con inued om p e ious page)
Conclusions: The equen de ec ion o MR and MDR isola es among clinical SP and SA s ains wi h no iceable i ulence
ai s is o e e ina y conce n, implying limi ed ea men op ions a ailable. This is he i s desc ip ion o MRSA-ST398 and
MRSP-ST68 in pe s in Spain, as well he i s epo o he d K-ca ying Tn559 in SP. This e idences ha cu en ansmissible
lineages wi h mobilizable esis omes ha e been ci cula ing as causa i e agen s o in ec ions among pe s o yea s.
Keywo ds: MRSP, MRSA, MRSP-ST71, MRSA-CC398, Pe s, In ec ion, Tn559
Backg ound
S aphylococcus pseudin e medius (SP) and S aphylococ-
cus au eus (SA) a e ha mless colonize s o he skin and
mucosa o humans and animals [1], bu hey a e also e-
quen ly implica ed in oppo unis ic in ec ions. In pe s,
especially in dogs, SP is mos equen ly de ec ed han
SA, bo h as colonize and as causa i e agen o in ec ion.
I is ecognized as he mos common e iological agen
implica ed in skin and so issue in ec ions, o i is, and
u ina y ac in ec ions in dogs [2–4]. Mo eo e , SP can
also cause diseases in humans, specially associa ed wi h
dog exposu e, which sugges s zoono ic ansmission [4].
SA is also ound in heal hy pe s in a es be ween 8 and
12% [5–7].
Simila o me hicillin- esis an SA (MRSA) in he clin-
ical se ing, me hicillin- esis an SP (MRSP) has become
a wo ldwide p oblem in animal heal h. I is equen ly
associa ed wi h a mul id ug esis ance pheno ype, which
limi s he he apeu ic op ions o e e ina ians. Mo e-
o e , in ecen yea s, se e al epo s ha e e idenced an
inc ease in he esis ance a es o some impo an an i-
mic obials, such as luo oquinolones, in SP isola es e-
co e ed om companion animals in Eu opean coun ies
[8,9]. In SP isola es eco e ed om diseased dogs, p e i-
ous s udies ha e epo ed me hicillin esis ance a es
om 10 o 20% [8,10,11], al hough i a ies no ably de-
pending on he geog aphic egion. Howe e , he me hi-
cillin esis ance a e inc eases up o 60% in isola es
eco e ed om canine pyode ma [12]. The sp ead o
MRSP be ween coun ies is due o he dissemina ion o
well-known speci ic gene ic lineages such as he clone
ST71 in Eu ope, ST68 in he USA, and ST45/ST112 in
Asia [2–4,10,13], al hough all hese clones ha e sp ead
wo ldwide [4].
Rega ding SA, molecula cha ac e iza ion has e ealed ha
companion animals a e colonized o in ec ed by hospi al-
associa ed (HA) and communi y-associa ed (CA) MRSA
clones om humans in close con ac , which sugges s an
an h opozoono ic o igin [14,15]. Li es ock-associa ed (LA)
MRSA-CC398, which is mainly ela ed wi h li es ock and
people wi h li es ock con ac , has also been epo ed causing
in ec ions in pe s in ew occasions [15,16].
In his s udy, we iden i ied and pe o med he molecu-
la cha ac e iza ion o a collec ion o coagulase-posi i e
s aphylococci (CoPS) ob ained om diseased pe s du ing
a 3-yea -sampling pe iod (2009–2011) in he e e ina y
labo a o y a he Uni e si y o Za agoza, Spain.
Resul s
Isola es eco e ed and species iden i ica ion
O he 33 CoPS included in his s udy, 28 we e iden i ied
as SP and i e as SA. The in ec ion si e o he animals
om which samples we e eco e ed is indica ed in
Table 1and Supplemen a y Table 1. Nine me hicillin-
esis an isola es ca ying he mecA gene we e de ec ed
(eigh MRSP and one MRSA), ep esen ing 27% o he
s udied isola es; all o hem, excep one MRSP isola e,
we e eco e ed om dogs. The 55% o SP and SA iso-
la es we e mul id ug- esis an (MDR) ( esis an o a
leas h ee amilies o an imic obial agen s) (Table 1).
Rema kably, pe sis en MRSP o MRSA ca iage, based
on he molecula cha ac e is ics o eco e ed isola es,
was de ec ed in h ee animals e alua ed in subsequen
samplings. S ains in ol ed we e MRSP ST68 clone
C3870 (2 mon hs la e ), MRSP ST71 clone C3871 (1 yea
la e ) and MRSA ST398 clone C3883 (a e 3 mon hs).
None heless, only he ini ial isola e pe animal was in-
cluded in his s udy.
Cha ac e iza ion o SP isola es
Fi e o he eigh MRSP, eco e ed om di e se in ec ion
ypes, we e yped as ST71, ag -III, ha bo ed he S aphylo-
coccal Casse e Ch omosome mec (SCCmec) II-III, and
belonged o he same clone (A). Two MRSP eco e ed
om u ina y- ep oduc i e in ec ions we e yped as ST68,
ag -IV, SCCmec V, and ep esen ed wo di e en sub-
clones (B1 and B2). The emaining MRSP isola e, also
om a u ina y in ec ion, was yped as ST258, ag -II,
SCCmec IV, and displayed pulsed- ield gel elec opho esis
(PFGE) pa e n C.
The an imic obial esis ance a es o he me hicillin-
suscep ible SP (MSSP) and MRSP isola es, a e shown in
Fig. 1. Resis ance o e y h omycin, clindamycin, kanamy-
cin, s ep omycin, cip o loxacin, and ime hop im-
sul ame hoxazole we e signi ican ly highe among MRSP
isola es. Mo eo e , esis ance o gen amicin and ob a-
mycin we e solely de ec ed in MRSP isola es (Fig. 1).
Eigh ou o 28 SP isola es we e MRSP (29%). Nine
MSSP and all MRSP isola es we e MDR, while wo
MSSP we e suscep ible o all an imic obial agen s
Ruiz-Ripa e al. BMC Ve e ina y Resea ch (2021) 17:42 Page 2 o 10
Table 1 Cha ac e iza ion o he 28 S. pseudin e medius and i e S. au eus isola es eco e ed om clinical samples o dogs and ca s in
his s udy
S ain Bac e ial
species
Yea Animal Type o
in ec ion
a
spa-MLST/CC-
ag -SCCmec
An imic obial esis ance
pheno ype
b
An imic obial esis ance
geno ype
Vi ulence
genes
de ec ed
C3871
c
MRSP 2009 Dog B-J ST71-ag III-
SCCmecII-III
PEN-OXA-ERY-CLI-GEN-TOB-
KAN-STR-TET-CIP
-CHL-SXT
blaZ,mecA,e m(B), aacA/aphD,
aphA3,aadE, e (K), ca
pC221
,d G
lukS/F-I, sie , se-
in
C3880 MRSP 2010 Dog S ST71-ag III-
SCCmecII-III
PEN-OXA-ERY-CLI-GEN-TOB-
STR-TET-CIP
-CHL-SXT
blaZ,e m(B), aacA/aphD,aphA3,
aadE, e (K), ca
pC221
, d G
lukS/F-I, sie , se-
in
C3885 MRSP 2010 Dog I ST71-ag III-
SCCmecII-III
PEN-OXA-ERY-CLI-GEN-TOB-
KAN-STR-TET-CIP
-CHL-SXT
blaZ,e m(B), aacA/aphD,aphA3,
aadE, e (K), ca
pC221
,d G
lukS/F-I, sie , se-
in
C5355 MRSP 2010 Dog B-J ST71-ag III-
SCCmecII-III
PEN-OXA-ERY-CLI-GEN-TOB-
KAN-STR-CIP
-SXT
blaZ,mecA,e m(B), aacA/aphD,
aphA3,aadE,d G
lukS/F-I, sie , se-
in
C5613 MRSP 2011 Dog U-R ST71-ag III-
SCCmecII-III
PEN-OXA-ERY-CLI-GEN-TOB-
KAN-STR-TET-CIP
-CHL-SXT
blaZ,mecA,e m(B), aacA/aphD,
aphA3,aadE, e (K), ca
pC221
,d G
lukS/F-I, sie , se-
in
C3866 MRSP 2009 Ca U-R ST68-ag IV-
SCCmecV
PEN-OXA-ERY-CLI-KAN-STR-
TET-CIP
-SXT
blaZ,mecA, e (M), e m(B), aphA3,
aadE,d G
lukS/F-I, sie , se-
in
C3870
d
MRSP 2009 Dog U-R ST68-ag IV-
SCCmecV
PEN-OXA-ERY-CLI-KAN-STR-
TET-CIP
-SXT
blaZ,mecA,e m(B), aphA3,aadE,
e (M), d G
lukS/F-I, sie , se-
in
C3869 MRSP 2009 Dog U-R ST258-ag II-
SCCmecIV
PEN-OXA-ERY-CLI-KAN-STR-
TET-SXT
blaZ,mecA,e m(B), aphA3,aadE,
e (M), d G
lukS/F-I, sie , se-
in
C5344 MSSP 2009 Dog U-R PEN-ERY-CLI-KAN-STR-TET-
CHL-SXT
blaZ,e m(B), aphA3,aadE, e (M),
ca
pC221
,d G
lukS/F-I, sie , se-
in
C5345 MSSP 2009 Ca SI PEN-ERY-CLI-KAN-STR-TET-
CHL-SXT
blaZ,e m(B), aphA3,aadE, e (M),
ca
pC221
,d G
lukS/F-I, sie , se-
in
C5360 MSSP 2011 Dog U-R PEN-ERY-CLI-KAN-STR-TET-
CHL-SXT
blaZ,e m(B), aphA3,aadE, e (M),
e (K), d G,ca
pC221
lukS/F-I, sie , se-
in
C5347 MSSP 2009 Dog U-R PEN-ERY-CLI-TET-CHL-SXT blaZ,e m(B), e (M), ca
pC221
,d G,
d K
h
lukS/F-I, sie , se-
in
C3877 MSSP 2010 Dog U-R PEN-KAN-STR-TET blaZ,aphA3,aadE, e (M) lukS/F-I, sie , se-
in , expB
C5351 MSSP 2010 Ca U-R PEN-KAN-TET blaZ,aphA3, e (M) lukS/F-I, sie , se-
in
C3881 MSSP 2010 Dog U-R STR-TET-CIP
s , e (K), e (M) lukS/F-I, sie , se-
in
C5354 MSSP 2010 Dog U-R PEN-TET-SXT blaZ, e (M), d G lukS/F-I, sie , se-
in
C5358 MSSP 2011 Dog U-R PEN-TET-SXT blaZ, e (M), d G,d K
h
lukS/F-I, sie , se-
in
C3875 MSSP 2010 Dog I PEN-TET blaZ, e (M) lukS/F-I, sie , se-
in
C3876 MSSP 2010 Dog U-R PEN-TET blaZ, e (M) lukS/F-I, sie , se-
in , expB
C5356 MSSP 2011 Dog U-R PEN-TET blaZ, e (M) lukS/F-I, sie , se-
in
C5362 MSSP 2011 Dog I PEN-TET blaZ, e (M) lukS/F-I, sie , se-
in
C5353 MSSP 2010 Dog U-R TET e (M) lukS/F-I, sie , se-
in , sec
canine
C3873 MSSP 2010 Dog U-R PEN blaZ lukS/F-I, sie , se-
in
C3878 MSSP 2010 Dog U-R PEN blaZ lukS/F-I, sie , se-
in
C3879 MSSP 2010 Dog I PEN blaZ lukS/F-I, sie , se-
in , sec
canine
Ruiz-Ripa e al. BMC Ve e ina y Resea ch (2021) 17:42 Page 3 o 10
Table 1 Cha ac e iza ion o he 28 S. pseudin e medius and i e S. au eus isola es eco e ed om clinical samples o dogs and ca s in
his s udy (Con inued)
S ain Bac e ial
species
Yea Animal Type o
in ec ion
a
spa-MLST/CC-
ag -SCCmec
An imic obial esis ance
pheno ype
b
An imic obial esis ance
geno ype
Vi ulence
genes
de ec ed
C5357 MSSP 2011 Dog U-R PEN blaZ lukS/F-I, sie , se-
in
C3874 MSSP 2010 Dog I Suscep ible –lukS/F-I, sie , se-
in
C5359 MSSP 2011 Dog U-R Suscep ible –lukS/F-I, sie , se-
in
C3883
e
MRSA 2010 Dog S 011-ST398/
CC398-ag I-
SCCmecV
PEN-FOX-ERY-CLI-GEN-TOB-
KAN-TET-CIP
g
-SXT
blaZ,mecA,e m(B), e m(C), aacA/
aphD, e (M), e (K), d A,d G
hla,hlb,hld,hlg
C5612 MSSA 2009 Dog U-R 10576-ST1660-
ag II
PEN blaZ lukPQ,scneq,
hla,hlb,hld,
hlg
C5650 MSSA 2009 Ca R 005-ST22/CC22-
ag I
PEN blaZ lukS/F-PV, hla,
hlb,hld,hlg
C5610 MSSA 2011 Dog U-R 045-ST5/CC5-
ag II
PEN blaZ IEC ype F,
lukED,hla,hld,
hlg
C5609 MSSA 2011 Ca I 045-ST5/CC5-
ag II
Suscep ible –IEC ype F,
lukED,hla,hld,
hlg
a
B-J, bones-join s in ec ion; U-R, u ina y- ep oduc i e in ec ion; R, espi a o y in ec ion; I, in egumen a y in ec ion; S, su gical in ec ion; SI, sep ic in ec ion
b
PEN, penicillin; OXA, oxacillin; FOX, ce oxi in; ERY, e y h omycin; CLI, clindamycin; GEN, gen amicin; TOB, ob amycin; KAN, kanamycin; STR, s ep omycin; TET,
e acycline; CIP, cip o loxacin; CHL, chlo amphenicol; SXT, ime hop im-sul ame hoxazole
c
Isola ed again one yea la e o he i s eco e y
d
Isola ed again wo mon hs la e o he i s eco e y
e
Isola ed again h ee mon hs la e o he i s eco e y
Amino acid change S84L and S80I in Gy A and G lA p o eins, espec i ely
g
Amino acid change S84L and S80F in Gy A and G lA p o eins, espec i ely
h
The d K gene was loca ed wi hin he adC-in eg a ed Tn559
Fig. 1 An imic obial esis ance a e o MSSP and MRSP in es iga ed in his s udy. PEN, penicillin; ERY, e y h omycin; CLI, clindamycin; GEN,
gen amicin; TOB, ob amycin; KAN, kanamycin; STR, s ep omycin; TET, e acycline; CIP, cip o loxacin; CHL, chlo amphenicol; SXT, ime hop im-
sul ame hoxazole. The p- alue (Fishe ’s Exac es ) is shown below he igu e. As e isks indica e he an imic obial agen s o which s a is ical
di e ences we e ound be ween he an imic obial esis ance a es o MSSP and MRSP (P < 0.05)
Ruiz-Ripa e al. BMC Ve e ina y Resea ch (2021) 17:42 Page 4 o 10
e alua ed. The blaZ and/o mecA esis ance genes we e
esponsible o β-lac am esis ance in SP isola es. Mac o-
lide and lincosamide esis ance was media ed by he
e m(B) gene in all cases, and aminoglycoside esis ance
by di e en combina ions o aacA/aphD,aphA3, and
aadE esis ance genes. The e (M) and/o e (K) genes
media ed e acycline esis ance. The eigh isola es exhi-
bi ing chlo amphenicol esis ance ha bo ed he ca
pC221
gene, while he d G gene was de ec ed in he 14 isola es
ha displayed esis ance o ime hop im–sul ame hoxa-
zole, in combina ion wi h d K in wo s ains (C5358,
C5347) (Table 1). The amino acid changes S84L and
S80I in he genes encoding he Gy A and G lA p o eins,
espec i ely, we e de ec ed in he eigh cip o loxacin-
esis an SP isola es. None o he SP isola es showed e-
sis ance o ancomycin, linezolid, o usidic acid.
The i ulence genes de ec ed among SP isola es we e
as ollows: sie (100%), se-in (100%), lukS/F-I (100%),
sec
canine
(7%), and expB (7%).
Cha ac e iza ion o SA isola es
The single MRSA isola e de ec ed was yped as 011-
ST398/CC398-ag I-SCCmecV and was eco e ed om a
su gical in ec ion o a dog. This ST398 s ain was esis -
an o β-lac ams, mac olides and lincosamides, amino-
glycosides, e acycline, cip o loxacin, and ime hop im-
sul ame hoxazole, and ca ied he blaZ,mecA,e m(B),
e m(C), aacA/aphD, e (K), e (M), d A, and d G esis -
ance genes. Mo eo e , he amino acid changes S84L and
S80F in he deduced sequences o Gy A and G lA p o-
eins, espec i ely, we e de ec ed. The MRSA isola e ha -
bo ed he haemolysins hla,hlb,hld,andhlg (Table 1).
The me hicillin-suscep ible SA (MSSA) isola es we e
assigned o 045-ST5/CC5 (n=2), 10576-ST1660 (n=1),
and 005-ST22/CC22 (n=1). One MSSA isola e was sus-
cep ible o all an imic obial agen s e alua ed, and he
emaining h ee only showed penicillin esis ance and
ca ied he blaZ gene. The MSSA 005-ST22 eline iso-
la e ha bo ed he genes enconding he Pan on-Valen ine
leukocidin (PVL) and he wo MSSA 045-ST45 ca ied
he scn,chp,sak, and sep genes and, he e o e, we e as-
c ibed o Immune E asion Clus e (IEC) ype F. In e es -
ingly, he 10576-ST1660 isola e ca ied he equid-
adap ed leukocidin lukPQ and he equine a ian o
S aphylococcal Complemen Inhibi o (SCIN). Di e en
combina ions o haemolysins, encoded by hla,hlb,hld,
hlg, and hlg
, we e de ec ed among MSSA isola es
(Table 1).
Gene ic en i onmen o he d K gene
The Tn559-speci ic PCRs e ealed ha he MSSP C5358
ha bo ed a comple e Tn559, which was in eg a ed in he
ch omosomal adC gene. De ec ion o he adC gene
and he Tn559- adC linkage was no possible o s ain
MSSP C5347. The whole genome sequencing (WGS)
analysis o his s ain enabled he iden i ica ion o he
comple e Tn559 and i s in eg a ion posi ion (Fig. 2)
(GenBank accession numbe MT252966). Nucleo ide
and amino acid sequence alignmen o Tn559 C5347
wi h e e ence S aphylococcus au eus ansposon Tn559
(GenBank accession numbe FN677369) e ealed he in-
se ion o one nucleo ide and h ee nucleo ide subs i u-
ions in he non-coding egion downs eam he d K
gene (Fig. 2). Rema kably, a single poin mu a ion in he
hyb idiza ion sequence o he adC o wa d p ime was
de ec ed ( adC- w: 5′-GTC/AGGAATAGGGCGTA-3′),
which esul ed esponsible o he absence o PCR amp-
li ica ion. Mo eo e , nucleo ide and amino acid align-
men wi h he adC gene o S. pseudin e medius s ain
C2719, ha bo ing ansposon Tn558 (GenBank accession
numbe HF679552), e ealed he p esence o se en syn-
onymous poin mu a ions (C27T, G114C, C219A,
C301T, G444C, T516C, G540T) plus wo addi ional
non-synonymous subs i u ions in he deduced RadC se-
quence (K63N, A103D) (Fig. 2). Tn559 ci cula in e me-
dia es we e de ec ed.
Discussion
This is he i s desc ip ion o MRSA-ST398 and MRSP-
ST68 in pe s in Spain, as well he i s epo o he
d K-ca ying Tn559 in SP. This e idences ha cu en
ansmissible lineages wi h mobilizable esis omes ha e
ci cula ed unno iceably in Spain as causa i e agen s o
in ec ions in pe s o yea s.
In his s udy, 28 o he 33 CoPS om diseased pe s
we e iden i ied as SP, which was expec ed since his is
he mos common s aphylococcal species de ec ed bo h
as colonize and cause o in ec ion in companion ani-
mals, specially dogs [4,5]. In his wo k, SP and SA we e
de ec ed causing in ec ion in h ee and wo ca s, espec -
i ely. These species ha e been o me ly eco e ed om
diseased ca s [1,17], howe e , a ecen s udy has e-
po ed p edominance o o he s aphylococcal species,
such as S. elis and S. haemoly icus, among eline in ec-
ions [1].
Fi e o he eigh MRSP isola es de ec ed in his s udy
we e asc ibed o he gene ic lineage ST71 and showed
he same PFGE pa e n. They we e isola ed om i e di -
e en dogs in di e en yea s and he e was no appa en
ela ion among hese animals; howe e , all o hem
unde wen su ge y in he e e ina y hospi al. Rega dless
hey we e assis ed by di e en e e ina ians, and he op-
e a ing oom and/o su ge y ma e ial a e ecu en ly dis-
in ec ed, none o hem can be excluded as po en ial
in ec ion sou ces. MRSP-ST71 is he majo clone in Eu -
ope [2,3], and he e a e al eady ew desc ip ions o his
gene ic lineage in heal hy dogs in Spain [18]. Howe e ,
ecen s udies ha e epo ed a downwa d end in he
Ruiz-Ripa e al. BMC Ve e ina y Resea ch (2021) 17:42 Page 5 o 10

Fig. 2 aG aphical ep esen a ion o he Tn559 s uc u e con aining he d K gene in eg a ed in he ch omosomal adC gene o he isola e S aphylococcus
pseudin e medius C5347 (GenBank accession numbe MT252966) and nucleo ide subs i u ions de ec ed in he non-coding egion downs eam he d K gene
compa ed o S aphylococcus au eus ansposon Tn559 (GenBank accession numbe FN677369). The nucleo ide posi ions a e se based on he whole Tn559.
Nucleo ide inse ions and subs i u ions a e colo ed in yellow. bAmino acid sequence alignmen o adC gene and esul an RadC o S aphylococcus
pseudin e medius s ain C2719 (GenBank accession numbe HF679552), whe e ansposon Tn558 was in eg a ed, ha o S. pseudin e medius C5347 (GenBank
accession numbe MT252966). The posi ion o he p ime pai employed is indica ed in g ey. Nucleo ide subs i u ions a e colo ed in yellow and amino acid
subs i u ions in blue
Ruiz-Ripa e al. BMC Ve e ina y Resea ch (2021) 17:42 Page 6 o 10
p e alence o he MRSP-ST71 lineage among compan-
ion animals in no he n Eu ope [11,19] and F ance [3].
The MRSP-ST258 lineage, which was de ec ed in his
s udy in 2009, seems o be eplacing MRSP-ST71 in
hese coun ies [11,19]. The emaining wo MRSP de-
ec ed in his s udy belonged o he ST68, a gene ic
lineage known o be p edominan in USA, al hough i
has been de ec ed ou side his coun y [4]. Howe e , o
he bes o ou knowledge, his is he i s desc ip ion o
MRSP-ST68 isola ed om companion animals in Spain.
Fo me s udies epo ed MRSA belonging o he HA-
MRSA and CA-MRSA lineages among isola es eco e ed
om companion animals [9,15]; howe e , in his wo k,
he only MRSA isola e de ec ed belonged o he LA-
MRSA CC398 gene ic lineage. Mo eo e , his isola e
lacked he genes o he IEC sys em, which sugges s an
animal o igin. LA-MRSA CC398 eco e ed om dis-
eased dogs has been epo ed in ew occasions [9,15,
16], howe e , o ou knowledge, his is he i s desc ip-
ion in Spain. Rega ding MSSA, isola es belonging o
CC22 and CC5 a e clonal g oups widesp ead among
companion animals [9,15,20]. The emaining MSSA
isola e was yped as 10576-ST1660, and, in e es ingly,
he single desc ip ion o his gene ic lineage is in a con-
empo a y isola e (ob ained in he same week) eco e ed
om one hospi alized equine in he same e e ina y hos-
pi al, which displayed he same an imic obial esis ance
pheno/geno ype and i ulence gene con en [21]. Mo e-
o e , he SA 10576-ST1660 isola e eco e ed in his
s udy ha bo ed he leukocidin lukPQ and he scneq,
which a e equid-adap ed i ulence ac o s [22,23]. This
suppo s he hypo hesis o a plausible equine o igin and
sugges s he ansmission o he MSSA s ain be ween
animal species in he e e ina y hospi al.
The equency o me hicillin esis ance and MDR iso-
la es among CoPS eco e ed om pe s in his s udy ep-
esen s a g ea conce n o e e ina y medicine. This is
also a public heal h p oblem due o he exposu e and in-
e species ansmission o SA/SP among pe s and
owne s [6,24], as well as po en ial ans e ence o esis -
ance genes o human-adap ed s aphylococcal s ains.
The a e o me hicillin esis ance obse ed in his s udy
(27%) is highe han he one de e mined among clinical
isola es om companion animals in se e al coun ies,
such as Aus alia (12%) [10,17], Finland (14%) [19], he
Ne he lands [11], and F ance [8]. As in his epo ,
MRSP-ST71 a e o en MDR [2,11], and hey a e mo e
likely o display luo oquinolone esis ance han o he
STs [10,11]. Fo una ely, in line wi h o me epo s, all
isola es eco e ed he e, bo h SA and SP, we e suscep-
ible o impo an o las eso an imic obials in human
medicine, such as ancomycin and linezolid [2,9].
All SP isola es, bo h MSSP and MRSP, ca ied he
i ulence de e minan s lukS/F-I, sie , and se-in , which
ha e been p e iously de ec ed in bo h commensal and
clinical SP isola es, sugges ing hey may be ubiqui ous in
his s aphylococcal species [5,18,20]. One MSSA 005-
ST22/CC2 eco e ed om a espi a o y in ec ion o a
ca was PVL-posi i e. The PVL is one o he mos im-
po an i ulence de e minan s p oduced by SA, which
has a c i ical ole in he pa hogenesis o skin and so is-
sue in ec ions. CC22 is an impo an gene ic lineage o
PVL-posi i e MSSA also implica ed in hospi al ou -
b eaks [25,26]. Mo eo e , he wo MSSA 045-ST5/CC5
isola es ca ied he human-adap ed scn,chp,sak, and sep
genes o he IEC sys em, which sugges s a human- o-
animal ansmission. To his ega d, o me s udies ha e
de e mined he in e species ansmission abili y o SA
om humans o pe s [6,24], which ep esen s a sou ce
o u he ansmission and a isk o in ec ion.
The d K gene has been a ely de ec ed in SP isola es
[9,27]. Indeed, he d K-ca ying ansposon Tn559 was
i s ly epo ed in a po cine MSSA ST398 isola e [28]
and he ea e in SA belonging o he same gene ic
lineage [21], bu also in En e ococcus aecium [29]. How-
e e , o he bes o ou knowledge, we epo he e he
i s desc ip ion o he Tn559 in SP. This could sugges
an exchange o esis ance genes be ween s aphylococci
and o he G am-posi i e bac e ia, including en e ococci,
o animal bu also om human o igin.
Conclusions
P esen ly ele an MRSP and MRSA gene ic lineages
wi h no iceable i ulence ai s we e de ec ed in isola es
eco e ed du ing 2009–2011, including he i s desc ip-
ion o MRSA ST398 and MRSP ST68 in pe s in Spain.
In addi ion, he appa en dissemina ion o CoPS s ains
in he e e ina y hospi al highligh s he impo ance o
u he in es iga ing SA and SP sou ces and su i al
abili y as con aminan s, hei popula ion s uc u e and
epidemiology, as well as hei an imic obial esis ance
pa e n and ansmission abili y.
Me hods
S udy popula ion
A o al o 33 CoPS isola es ob ained om 28 and i e
diseased dogs and ca s (one isola e/animal), espec i ely,
we e ob ained in he Ve e ina y Labo a o y o he Uni-
e si y o Za agoza (Za agoza, Spain) du ing he yea s
2009 (9 isola es), 2010 (15), and 2011 (9), and we e in-
cluded in his s udy. The samples we e aken om he
in ec ion si e (Supplemen a y Table S1). The isola es
we e s o ed ozen a −80 °C un il hey we e s udied.
Isola ion and iden i ica ion o SP and SA isola es
Iden i ica ion o isola es was pe o med by biochemical as-
says, including colony mo phology, G am s aining, ca a-
lase and DNase ac i i ies, and API20-STAPH. The
Ruiz-Ripa e al. BMC Ve e ina y Resea ch (2021) 17:42 Page 7 o 10
iden i ica ion o SA and SP was de e mined by a mul iplex
PCR ha ampli ies he speci ic nuc gene o SA o S. in e -
medius/SP [30]. Disc imina ion be ween S. in e medius
and SP was ca ied ou by diges ion o he p a gene ampli-
con wi h MboIenzyme[31].
Molecula yping and clonal ela edness
All SA we e subjec ed o spa- yping by PCR and ampli-
con sequencing, and he ob ained sequences we e ana-
lyzed using Ridom S aph-Type so wa e e sion 1.5.21
(Ridom GmbH, Müns e , Ge many) [32]. Mul i Locus
Sequence Typing (MLST) was pe o med in all SA iso-
la es [32], and acco ding o he sequence- ype (ST), he
isola es we e asc ibed o he di e en clonal complexes
(CC). MLST o SP isola es was likewise pe o med as
p e iously desc ibed [33]. All isola es we e cha ac e ized
by ag - yping ollowing s anda d me hodology [32,33].
SCCmec- yping was unde gone in MRSP and MRSA as
p e iously desc ibed [13,34].
PFGE o o al DNA es ic ed wi h SmaIenzymewaspe -
o med on MRSP as p e iously desc ibed [35]. Isola es we e
conside ed di e en clones when hey exhibi ed mo e han
h ee bands o di e ence in PFGE band pa e ns and sub-
clones when PFGE band pa e ns di e ed be ween 1 and 3
bands [36].
An imic obial esis ance pheno ype and geno ype
The suscep ibili y o 17 an imic obials was de e mined
by aga disk-di usion me hod. The an imic obial agen s
es ed we e as ollows (μg/disk): penicillin (10 uni s),
oxacillin (1), ce oxi in (30) e y h omycin (15), clindamy-
cin (2), gen amicin (10), ob amycin (10), kanamycin
(30), s ep omycin (10), e acycline (30), cip o loxacin
(5), mupi ocin (200), ancomycin (30), chlo amphenicol
(30), linezolid (30), usidic acid (10), and ime hop im-
sul ame hoxazole (1.25+ 23.75). The CLSI guidelines
[37] was used o all an imic obials, excep o s ep o-
mycin, mupi ocin, and usidic acid, o which he
me hods and b eakpoin s ecommended by he Socié é
F ançaise de Mic obiologie [38] we e employed.
The p esence o 34 an imic obial esis ance genes was in-
es iga ed by PCR: mecA,blaZ,e m(A), e m(B), e m(C),
e m(T), mph(C), ms (A), ms (B), lnu(A), ga(A), ga(C),
aacA-aphD, aphA3, aadE,aadD,aadA, s , e (K), e (M),
e (L), sa 4 (e en hough s ep o h icin suscep ibili y was no
es ed), exA, exB, c , op A, pox A, ca
ps194
,ca
pC221
,
ca
pC223
,d A,d D,d G,andd K[18,39,40]. Posi i e con-
ols om he collec ion o he Uni e si y o La Rioja we e
included in all PCR assays.
Mu a ions in he genes encoding he Gy A and G lA p o-
eins we e in es iga ed in cip o loxacin- esis an SA and SP
isola es by PCR and sequencing [18,41]. The co esponding
sequences o S. au eus NCTC 8325 (GenBank accession
numbe CP000253) and S. pseudin e medius KM1381
(GenBank accession numbe AM262969 and AM262972)
we e used as e e ences.
De ec ion o i ulence genes
The p esence o he leukocidin genes lukSF-PV, lukM,
lukED,andlukPQ was in es iga ed in all SA isola es [21,
23]. They we e also sc eened o he p esence o haemoly-
sin genes (hla,hlb,hld,hlg,andhlg
), ex olia i e genes
(e a,e b,ande d), and he oxic shock synd ome oxin-1
( s )[21]. PCR-based de e mina ion o he i e genes (scn,
chp,sak,sea, and sep) ha comp ises he IEC sys em as
well he equine a ian o SCIN encoded by scneq we e
likewise in es iga ed [32,42]. In addi ion, SP isola es we e
sc eened o he p esence o he leukocidin gene lukS/F-I,
he ex olia i e genes sie ,expA,andexpB, and he en e o-
oxin genes si-en and sec
canine
by PCR [20].
S a is ical analysis
Po en ial s a is ical di e ences be ween he an imic obial
esis ance a es in MRSP and MSSP isola es we e com-
pa ed using he Fishe ’s Exac es wi h he R Com-
mande p og am. P< 0.05 was conside ed a s a is ically
signi ican esul .
Gene ic en i onmen o he d K gene in S.
pseudin e medius isola es
The possibili y ha he d K gene was loca ed wi hin he
Tn559 and in eg a ed wi hin he ch omosomal adC
gene was in es iga ed by speci ic PCRs a ge ing he di -
e en cons i uen s o Tn559 and hei physical linkage
o adC, as p e iously desc ibed [21,43]. S ains nega-
i e o a leas one p ime combina ion we e submi ed
o WGS.
Whole genome sequencing o Tn559 ca ying s ain
nega i e o adC in eg a ion
WGS was pe o med on SP s ain C5347 using PacBio Se-
quel and Illumina Miseq 2 × 300 bp pla o ms p io phenol-
chlo o o m DNA ex ac ion, as p e iously desc ibed [44].
Raw PacBio eads we e assembled using Canu [45]wi hde-
aul pa ame e s and se ing an es ima ed genome size o 3
Mb. The esul ing assembled con igs we e hen polished as
ollows: Fi s , Illumina aw eads we e quali y- immed using
T immoma ic [46] and aligned agains he assembled PacBio
con igs using Bow ie2 [47]. Then, he esul ing bam iles
we e used o ix indi idual base e o s, indels and local mis-
sassemblies using Pilon [48].
Resul ing genes on he assembled con igs we e p edic ed
using P odigal [49]. RNA and RNA genes we e p edic ed
using RNAscan-SE [50], ssu-align [51] and me a- na [52].
P edic ed p o ein sequences we e compa ed agains he
NCBI n da abase using DIAMOND [53], and agains COG
[54]andTIGFRAM[55] using HMMscan [56] o axo-
nomic and unc ional anno a ion.
Ruiz-Ripa e al. BMC Ve e ina y Resea ch (2021) 17:42 Page 8 o 10
Supplemen a y In o ma ion
The online e sion con ains supplemen a y ma e ial a ailable a h ps://doi.
o g/10.1186/s12917-020-02726-4.
Addi ional ile 1: Table S1. Type o in ec ion, sample and sampling
me hod used o ob ain he hi y- h ee coagulase-posi i e s aphylococci
o his s udy.
Abb e ia ions
SP: S aphylococcus pseudin e medius; SA: S aphylococcus au eus;
MR: me hicillin- esis an ; MDR: mul id ug- esis an ; SCCmec: S aphylococcal
casse e ch omosome; ST: sequence ype; CC: clonal complex; PVL: Pan on-
Valen ine leukocidin; IEC: immune e asion clus e ; MRSA: me hicillin- esis an
S aphylococcus au eus; MRSP: me hicillin- esis an S aphylococcus
pseudin e medius; HA: hospi al-associa ed; CA: communi y-associa ed;
LA: li es ock-associa ed; CoPS: coagulase-posi i e s aphylococci;
PFGE: pulsed- ield gel elec opho esis; MSSP: me hicillin-suscep ible S.
pseudin e medius; MSSA: me hicillin-suscep ible S. au eus;
SCIN: s aphylococcal complemen inhibi o ; MLST: mul ilocus sequence
yping; CLSI: clinical labo a o y s anda ds ins i u e; PCR: polyme ase chain
eac ion; WGS: whole genome sequencing
Acknowledgemen s
The au ho s would like o hank D . Jose Manuel Ha o-Mo eno (Miguel He -
nandez Uni e si y, Spain), o his di ec suppo in WGS assembly and anno-
a ion o S. pseudin e medius C5347.
Au ho s’con ibu ions
LRR, SC and EGS ca ied ou he expe imen s and analyzed he da a. CS and
CO isola ed he s ains used in his wo k. MZ, CT and EGS designed he
p ojec and supe ised he esea ch. All au ho s d a ed he manusc ip and
app o ed i .
Funding
The de elopmen o his s udy was suppo ed by p ojec s SAF2012–35474
and SAF2016–76571-R om he Agencia Es a al de In es igación (AEI) o
Spain and he Fondo Eu opeo de Desa ollo Regional (FEDER) o EU and by
he Ins i u o A agonés de Ciencias de la Salud ICS o Spain. E. Gómez-Sanz
was ini ially unded by a ellowship om he Gobie no de La Rioja, Spain
(sample collec ion and expe imen al wo k) and la e on (da a analysis and
manusc ip e ision) by he Eu opean Union’s F amewo k P og am o
Resea ch and Inno a ion Ho izon 2020 (2014–2020) unde he Ma ie
Sklodowska-Cu ie G an Ag eemen No. 659314 and by in e nal unds o
ETHZ. Lau a Ruiz-Ripa has a p e-doc o al ellowship om he Uni e sidad de
La Rioja (Spain). The unding o ganisms nei he a ec he design o he e-
sea ch no he in e p e a ion o he da a.
A ailabili y o da a and ma e ials
The en i e ansposon Tn559 plus he unca ed adC gene o S.
pseudin e medius C5347, comp ising 5′069 bps, ha e been deposi ed in he
Genbank da abase wi h accession numbe MT252966.
E hics app o al and consen o pa icipa e
No applicable.
Consen o publica ion
No applicable.
Compe ing in e es s
The au ho s decla e ha hey ha e no compe ing in e es s.
Au ho de ails
1
Á ea Bioquímica y Biología Molecula , Uni e sidad de La Rioja, Log oño
26006, Spain.
2
Depa amen o de Pa ología Animal, Uni e sidad de Za agoza,
Za agoza 50013, Spain.
3
Ins i u e o Food, Nu i ion and Heal h, ETH Zü ich,
Schmelzbe gs asse 7, LFV B36, 8092 Zu ich, Swi ze land.
4
Á ea de
Mic obiología Molecula , Cen o de In es igación Biomédica de La Rioja
(CIBIR), Log oño 26006, Spain.
Recei ed: 23 Ap il 2020 Accep ed: 21 Decembe 2020
Re e ences
1. Bie owiec K, Ko zeniowska-Kowal A, Wzo ek A, Rypuła K, Gamian A.
P e alence o S aphylococcus species coloniza ion in heal hy and sick ca s.
Biomed Res In . 2019;2019:4360525.
2. Somayaji R, Rubin JE, P iyan ha MA, Chu ch D. Explo ing S aphylococcus
pseudin e medius: an eme ging zoono ic pa hogen? Fu u e Mic obiol. 2016;
11:1371–4.
3. Be go M, Ma ins-Simoes P, Kilian H, Châ e P, Wo hing KA, No is JM, e al.
E olu ion o he popula ion s uc u e o S aphylococcus pseudin e medius in
F ance. F on Mic obiol. 2018;9:3055.
4. Pi es Dos San os T, Dambo g P, Moodley A, Gua dabassi L. Sys ema ic
e iew on global epidemiology o me hicillin- esis an S aphylococcus
pseudin e medius: in e ence o popula ion s uc u e om Mul ilocus
Sequence Typing Da a. F on Mic obiol. 2016;7:1599.
5. an Duijke en E, Ca y B, G eko C, Mo eno MA, Pomba MC, Pyö älä S, e al.
Re iew on me hicillin esis an S aphylococcus pseudin e medius.J
An imic ob Chemo he . 2011;66(12):2705–14.
6. Gómez-Sanz E, To es C, Lozano C, Za azaga M. High di e si y o
S aphylococcus au eus and S aphylococcus pseudin e medius lineages and
oxigenic ai s in heal hy pe -owning household membe s.
Unde es ima ing no mal household con ac ? Comp Immunol Mic obiol
In ec Dis. 2013;36(1):83–94.
7. Kaspa U, on Lü zau A, Schla mann A, Roesle U, Köck R, Becke K.
Zoono ic mul id ug- esis an mic oo ganisms among small companion
animals in Ge many. Plos One. 2018;13(12):e0208364.
8. Haenni M, de Mo aes NA, Châ e P, Médaille C, Moodley A, Madec JY.
Cha ac e isa ion o clinical canine me icillin- esis an and me icillin-
suscep ible S aphylococcus pseudin e medius in F ance. J Glob An imic ob
Resis . 2014;2(2):119–23.
9. Cou o N, Monchique C, Belas A, Ma ques C, Gama LT, Pomba C. T ends and
molecula mechanisms o an imic obial esis ance in clinical s aphylococci
isola ed om companion animals o e a 16 yea pe iod. J An imic ob
Chemo he . 2016;71(6):1479–87.
10. Wo hing KA, Ab aham S, Coombs GW, Pang S, Sapu a S, Jo dan D, e al.
Clonal di e si y and geog aphic dis ibu ion o me hicillin- esis an
S aphylococcus pseudin e medius om Aus alian animals: disco e y o no el
sequence ypes. Ve Mic obiol. 2018;213:58–65.
11. Duim B, Ve s appen KM, B oens EM, Laa ho en LM, an Duijke en E, Ho dijk
J, e al. Changes in he popula ion o me hicillin- esis an S aphylococcus
pseudin e medius and dissemina ion o an imic obial- esis an pheno ypes in
he Ne he lands. J Clin Mic obiol. 2016;54(2):283–8.
12. Kawakami T, Shiba a S, Mu ayama N, Naga a M, Nishi uji K, Iwasaki T, e al.
An imic obial suscep ibili y and me hicillin esis ance in S aphylococcus
pseudin e medius and S aphylococcus schlei e i subsp. coagulans isola ed
om dogs wi h pyode ma in Japan. J Ve Med Sci. 2010;72(12):1615–9.
13. Pe e en V, Kadlec K, Schwa z S, Ande sson UG, Finn M, G eko C, e al.
Clonal sp ead o me hicillin- esis an S aphylococcus pseudin e medius in
Eu ope and No h Ame ica: an in e na ional mul icen e s udy. J An imic ob
Chemo he . 2010;65(6):1145–54.
14. Loe le A, P ei e DU, Lindsay JA, Soa es Magalhães RJ, Lloyd DH.
P e alence o and isk ac o s o MRSA ca iage in companion animals: a
su ey o dogs, ca s and ho ses. Epidemiol In ec . 2011;139(7):1019–28.
15. Wip JR, Pe e en V. Me hicillin- esis an S aphylococcus au eus isola ed om
dogs and ca s in Swi ze land. Schweiz A ch Tie heilkd. 2016;158(6):443–50.
16. Wi e W, S ommenge B, S anek C, Cuny C. Me hicillin- esis an
S aphylococcus au eus ST398 in humans and animals, Cen al Eu ope. Eme g
In ec Dis. 2007;13(2):255–8.
17. Sapu a S, Jo dan D, Wo hing KA, No is JM, Wong HS, Ab aham R, e al.
An imic obial esis ance in coagulase-posi i e s aphylococci isola ed om
companion animals in Aus alia: a one yea s udy. Plos One. 2017;12(4):
e0176379.
18. Gómez-Sanz E, To es C, Lozano C, Sáenz Y, Za azaga M. De ec ion and
cha ac e iza ion o me hicillin- esis an S aphylococcus pseudin e medius in
heal hy dogs in La Rioja, Spain. Comp Immunol Mic obiol In ec Dis. 2011;
34(5):447–53.
19. G ön hal T, Eklund M, Thomson K, Piipa inen H, Si onen T, Ran ala M.
An imic obial esis ance in S aphylococcus pseudin e medius and he
Ruiz-Ripa e al. BMC Ve e ina y Resea ch (2021) 17:42 Page 9 o 10