Methicillin-resistant Staphylococcus aureus in horses and horse personnel: an investigation of several outbreaks.

Abstract

At the Veterinary Microbiological Diagnostic Center, the Netherlands, the percentage of methicillin-resistant Staphylococcus aureus (MRSA) isolates found in equine clinical samples increased from 0% in 2002 to 37% in 2008. MRSA of spa-type t064, belonging to MLST ST8 and spa-types t011 and t2123, both belonging to the livestock-associated MLST ST398, predominated. During an outbreak of post-surgical MRSA infections in horses at a veterinary teaching hospital in 2006/2007, MRSA isolates of spa-type t2123 were cultured from 7 horses and 4/61 personnel which indicated zoonotic transmission. After intervention the outbreak stopped. However, another outbreak occurred in 2008, where 17 equine MRSA isolates of spa-type t011 (n=12), t2123 (n=4), and t064 (n=1) were found. This time, 16/170 personnel were positive for MRSA with spa-type t011 (n=11) and t2123 (n=5). Personnel in close contact with horses were more often MRSA-positive (15/106) than those without (1/64). Screening of horses upon admission showed that 9.3% were MRSA-positive predominantly with spa-type t011. Weekly cross-sectional sampling of all hospitalized horses for 5 weeks showed that 42% of the horses were MRSA-positive at least once, again predominantly with spa-type t011, which suggests that nosocomial transmission took place. Fifty-three percent of the environmental samples were MRSA-positive, including samples from students' and staff members' rooms, and all were spa-type t011. This indicates that humans contribute to spreading the organism. Culturing of samples employing high-salt pre-enrichment performed better than a comparable method without pre-enrichment. Our results show that nosocomial transmission occurs in equine clinics and suggests that personnel play a role in the transmission.

Full text

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Methicillin-resistant Staphylococcus aureus in horses and horse
personnel: An investigation of several outbreaks
E. van Duijkerena,*, M. Molemanb, M.M. Sloet van Oldruitenborgh-Oosterbaanb, J. Multema,
A. Troelstrac, A.C. Fluitc, W.J.B. van Wameld, D.J. Houwersa, A.J. de Neelinge, J.A. Wagenaara,f
aDepartment of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, PO Box 80165, 3508 TD Utrecht, Utrecht University, The Netherlands
bDepartment of Equine Medicine, Faculty of Veterinary Medicine, Utrecht University, The Netherlands
cDepartment of Medical Microbiology, University Medical Center Utrecht, Utrecht University, The Netherlands
dDepartment of Medical Microbiology and Infectious Diseases, Erasmus Medical Center, Rotterdam, The Netherlands
eNational Institute of Public Health and the Environment, Laboratory for Infectious Diseases and Screening, Bilthoven, The Netherlands
fCentral Veterinary Institute of Wageningen UR, Lelystad, The Netherlands
1. Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) is
an emerging equine pathogen. Initial reports of MRSA
infections in horses largely concerned sporadic infections
associated with veterinary hospitals (Hartmann et al.,
1997; Seguin et al., 1999). However, the number of reports
onMRSAcolonizationandinfectionsinhorsesisincreasing
and they are now also associated with private practices
and the community (Weese and van Duijkeren, 2009). The
prevalence of MRSA colonization published so far varied
between 0% and 4.7%onhorse farms inEurope, Canadaand
Veterinary Microbiology xxx (2009) xxx–xxx
A R T I C L E I N F O
Article history:
Received 29 May 2009
Received in revised form 9 July 2009
Accepted 3 August 2009
Keywords:
Horse
MRSA
Staphylococcus aureus
Transmission
Environment
A B S T R A C T
At the Veterinary Microbiological Diagnostic Center, the Netherlands, the percentage of
methicillin-resistant Staphylococcus aureus (MRSA) isolates found in equine clinical
samples increased from 0% in 2002 to 37% in 2008. MRSA of spa-type t064, belonging to
MLST ST8 and spa-types t011 and t2123, both belonging to the livestock-associated MLST
ST398, predominated.
During an outbreak of post-surgical MRSA infections in horses at a veterinary teaching
hospitalin2006/2007,MRSAisolatesofspa-typet2123wereculturedfrom7horsesand4/61
personnel which indicated zoonotic transmission. After intervention the outbreak stopped.
However, another outbreak occurred in 2008, where 17 equine MRSA isolates of spa-type
t011 (n=12), t2123 (n=4), and t064 (n=1) were found. This time, 16/170 personnel were
positive for MRSA with spa-type t011 (n=11) and t2123 (n=5). Personnel in close contact
with horses were more often MRSA-positive (15/106) than those without (1/64).
Screening of horses upon admission showed that 9.3% were MRSA-positive
predominantly with spa-type t011. Weekly cross-sectional sampling of all hospitalized
horses for 5 weeks showed that 42% of the horses were MRSA-positive at least once, again
predominantlywithspa-typet011,whichsuggeststhatnosocomialtransmissiontookplace.
Fifty-three percent of the environmental samples were MRSA-positive, including samples
from students’ and staff members’ rooms, and all were spa-type t011. This indicates that
humans contribute to spreading the organism. Culturing of samples employing high-salt
pre-enrichment performed better than a comparable method without pre-enrichment.
Our results show that nosocomial transmission occurs in equine clinics and suggests
that personnel play a role in the transmission.
? 2009 Elsevier B.V. All rights reserved.
* Corresponding author. Tel.: +31 30 2533 603; fax: +31 30 2533 199.
E-mail address: e.vanduijkeren@uu.nl (E. van Duijkeren).
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North America (Busscher et al., 2006; Baptiste et al., 2005;
Vengust et al., 2006; Weese et al., 2005), between 2.9% and
10.9% in horses admitted to equine clinics (Weese et al.,
2006; Van den Eede et al., 2009) and 16% in horses in an
equine hospital (Baptiste et al., 2005). In hospitalized
horses, surgical site infections predominate, whereas joint,
incision and skin/soft tissue infections are most common
in community-onset infections (Weese and van Duijkeren,
2009). In Canada and North America most equine MRSA
infections are caused by a human epidemic clone of
Multilocus Sequence Type (MLST) ST8 (Canadian MRSA-5
or USA500) (Weese and van Duijkeren, 2009). It has been
suggested that this MRSA is horse-adapted because of its
predominance in horses and horse personnel (Weese and
van Duijkeren, 2009). Recently, there have been reports on
colonizationandinfectionofhorseswithMRSAST398(Van
den Eede et al., 2009; Witte et al., 2007), a MRSA clone
associated with livestock (de Neeling et al., 2007; Gravel-
and et al., 2008). The emergence of two clusters of MRSA
infections at an equine veterinary teaching hospital in
2006/2007 and 2008, respectively, and the subsequent
demand for outbreak management and MRSA control
measures, prompted us to perform a study on colonization
rates of horses and horse personnel, the possible occur-
rence of nosocomial transmission and the degree of
contamination of the hospital’s interior.
2. Materials and methods
2.1. First outbreak investigation
2.1.1. Horses
In December 2006 and early 2007, 7 horses originating
from different provinces in the Netherlands developed
MRSA infections shortly after colic surgery at the
veterinary teaching hospital. Six patients had surgical
wound infections and one had thrombophlebitis. All
isolates were genotyped by Pulsed-Field Gel Electrophor-
esis (PFGE), spa-typing, SCCmec-typing and Multiple Locus
Variable-number tandem repeat Analysis (MLVA) as
described below. One isolate was typed by MLST.
2.1.2. Personnel
In February 2007, 61 persons who had been in contact
with the above patients at the hospital were screened on a
voluntary basis. Two persons refused to participate. All
human samples were taken from both nares and from the
throat and were cultured using the culturing method 2
described below.
This outbreak initially stopped after thorough cleaning
of the hospital and strict isolation of the patients.
2.2. Second outbreak investigation
Almost a year later a second outbreak occurred from
January to October 2008.
2.2.1. Horses
A total of 12 horses (hospitalized and outpatients)
developed MRSA infections and 5 horses housed in the
same stables were found to be MRSA-positive in nasal
samples. Isolates were further indentified by spa-typing
and 7 of these were analyzed by MLVA.
2.2.2. Personnel
From May to July 2008, a second screening of the
personnel at the clinic was performed and 106 persons in
close contact with the horses (veterinarians, animal
attendants and students) were sampled. Persons who
did not want to participate were asked to note all animal
contacts on a daily basis. This time, no one refused to
participate. Samples were taken from both nares and from
the throat. In addition, samples were taken from 64
personnel without close contact with the horses but
working at the radiology department, in technical main-
tenanceandforthecleaningcompany.Samplesweretaken
and cultured as described.
2.3. Screening of horses at admission to the hospital
DuringNovemberandDecember2008,samplesfromthe
naresofall horses(n=259)justbeforeenteringthe hospital
were taken in duplicate and cultured for MRSA using two
culture methods (below). All isolates were spa-typed.
2.4. Screening of the hospitalized horses
During the same period, cross-sectional sampling of all
hospitalized horses was performed 5 times with a weekly
interval leading to a total of 319 samples from 149 horses.
On Mondays, all horses present at the clinic were sampled.
The number of patients sampled varied between 79 on the
firstsamplingdayto31onthelast.Onaverage,atotalof2.1
samples were taken per horse. The first 79 samples were
cultured by the two methods; the other 240 samples were
cultured using pre-enrichment with Muller Hinton broth
and 6.5% NaCl (method 1) only. All isolates were spa-typed.
2.4.1. Sampling of the horses
Per horse, one cotton-tipped swab was inserted into
bothnares(?10 cm)andretractedwhilerotatingtheswabin
contact with the nasal mucosa. For screening of horses at
admission and on the first sampling of the hospitalized
horses, this was done twice in order to obtain duplicate
samples. Samples were processed within 24 h.
2.4.2. Environmental sampling
To assess the environmental contamination in Decem-
ber 2008, surface wipes of the environment (n = 36) were
taken using sterile gauzes (Cutisoft1, BSN Medical BV,
Almere, the Netherlands). A surface of approximately
10 cm ?30 cm was gently rubbed using both sides of the
gauze. Wipes were taken in duplicate at the adjacent sites.
Each wipe was taken wearing new sterile gloves to prevent
cross-contamination, put into sterile containers and
immediately transported to the laboratory. The duplicate
samples were assigned to both culture techniques at
convenience. The sampling sites were seven stable units
(A, B, D, F, H, O and R), six isolation units, two surgical
theatres, two preparation rooms, two examination rooms,
the equine reproduction clinic, the farriery, the intensive
care supply room, the anaesthesia unit, the surgical
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sterilizing unit (both sending side and receiving side),
three locker rooms of the staff, animal attendants and
students, two students’ rooms, two staff rooms, animal
attendants canteen, students canteen, pantry in the staff
building and lunch room of the reproduction clinic. All
MRSA isolates were spa-typed.
2.5. Culturing technique
Two culture techniques using a broth with aztreonam
and ceftizoxime were compared, one with (method 1) and
another without (method 2) pre-enrichment in Mueller
Hinton with 6.5% NaCl. Method 1 had been used
successfully to detect MRSA, including ST398, in meat
samples (van Loo et al., 2007) and method 2 is the standard
procedure at the University Medical Center Utrecht for
clinical as well as screening cultures of humans. From a
previous study we knew that method 1 performed better
for culturing MRSA of ST398 from samples from pigs and
calves (Graveland et al., 2009), but we did not know if this
was also true for samples from horses.
2.5.1. Method 1
Swabs were put into tubes containing 5 ml Mueller
HintonBroth(MHB),containing6.5%NaCl.Containerswith
the wipes were filled with 50 ml of this broth. After
overnightincubationat37 8Conemlofthepre-enrichment
broth was transferred to 9 ml phenolred mannitol broth
(PHMB) (BioMe ´rieux, Marcy l’Etoile, France) with 5mg/ml
ceftizoxime and 75mg/ml aztreonam. This enrichment
broth was also incubated overnight at 37 8C and 10ml of
the PHMBbrothwas subsequentlyplatedontosheepblood
agar (Biotrading, Mijdrecht, The Netherlands) and bril-
liance MRSA agar (Oxoid, Basingstoke, United Kingdom).
2.5.2. Method 2
Swabs were put into a tube with 5 ml tryptone soya
broth (TSB) containing 4% NaCl, 1% mannitol, 16mg/ml
phenol red, 50mg/ml aztreonam and 5mg/ml ceftizoxime.
The containers with wipes were filled with 50 ml of this
broth. After incubation for 48h at 378C, 10ml was plated
onto sheep blood agar (Biotrading, Mijdrecht, The Nether-
lands) and MRSA brilliance agar (Oxoid, Basingstoke,
United Kingdom).
2.6. Identification of bacteria
Suspect colonies were identified as Staphylococcus
aureus (S. aureus) using standard techniques: colony
morphology, Gram stain, catalase and coagulase and by
a latex agglutination test (Pasteurex Staph Plus, Bio-Rad
Laboratories, Hercules, USA). Colonies suspect of being
MRSA were tested by PCR for the S. aureus specific DNA
fragment (Martineau et al., 1998), the mecA gene (De
Neeling et al., 1998), and the Panton-Valentine leucocidin
toxin genes (Lina et al., 1999).
2.7. Genotyping
All isolates from the first outbreak and a selection of
isolates from different spa-types were genotyped by PFGE
using SmaI as restriction enzyme according to the
Harmony protocol (Murchan et al., 2003), MLVA (Schouls
et al., 2009) and SCCmec-typing (Ito et al., 2001; Ito et al.,
2004; Okuma et al., 2002). At least one isolate of each spa-
type was analyzed by MLST (Enright et al., 2000).
All human, equine and environmental MRSA isolates
were spa-typed by sequencing of the repetitive region of
the protein A gene spa (Harmsen et al., 2003). Data were
analyzed using the Ridom Staphtype software version 1.4
(www.ridom.de/staphtype).
2.8. Retrospective study of S. aureus isolates from clinical
samples from horses
VMDC’s database was searched for all equine S. aureus
isolates from 2002 to 2008. As from January 1st, 2002, all S.
aureus isolates with unusual antimicrobial resistance
patterns had routinely been tested for the presence of mecA
byPCRandmecA-positiveisolatesweresubjectedtofurther
genotyping and stored as part of VMDC’s strain collection.
During this period the culture techniques for S. aureus did
not change and all isolates were identified as described
above. Only first isolates from a patient and only isolates
fromclinicalinfectionswereincludedinthesetofdata.Four
isolates of spa-type t064 and one isolate of spa-type t011
were chosen at random for SCCmec-typing and MLST as
described above. In addition, eight isolates of spa-type t64
and one isolate of spa-type t011 were analyzed by MLVA.
3. Results
3.1. First outbreak investigation
3.1.1. Horses
The 7 clinical isolates from 2006 and 2007 were all non-
typeable by PFGE, MLVA-type MT0568, SCCmec-type IVa
and spa-type t2123 and were PVL-negative. The isolate
analyzed by MLST was ST398.
3.1.2. Personnel
4/61 personnel were found positive, all with the same
MRSA strain with MLVA-type MT0568 and spa-type t2123.
3.2. Second outbreak investigation
3.2.1. Horses
TheclinicalMRSAisolates(n=12)in2008werespa-type
t2123 (n=2), t011 (n=9) and t064 (n=1), respectively.
Seven isolates were analyzed by MLVA: the isolates of spa-
type t2123 were both MLVA-type MT0568 and 5 isolates of
spa-type t011 were MLVA MT0398 (Tables 1 and 2). The
horse which had a surgical wound infection with t064 also
carried an MRSA of spa-type t2123 in its nose. Of the five
positivecontacthorsesonehadaMRSAwithspa-typet2123,
three type t011, whereas one isolate was not available for
typing (Table 1). All isolates were PVL-negative.
3.2.2. Personnel
16/170 personnel at the clinic were found to be MRSA-
positive. From the 106 persons in close contact with
horses, 15 (14. 2%) were found to be MRSA-positive. From
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the remaining 64 persons without direct contact with
horses, only one person (1.6%) working for the cleaning
company in the stables was found MRSA-positive. Persons
only cleaning offices and persons working at the radiology
department or in technical maintenance were negative.
The spa-types found were t011 (n =11) and t2123 (n =5).
All isolates were PVL-negative.
3.3. Screening of horses at admission to the veterinary
teaching hospital
Screening of the horses before entering the hospital
showed that 24/259 horses (9.3%) tested MRSA-positive.
Twenty-one of these MRSA (87.5%) were spa-type t011,
one t064, one t2123 and one was lost before typing. All
typed isolates were PVL-negative.
3.4. Screening of the hospitalized horses
Screening of the hospitalized horses revealed that 88 of
324 (27%) samples wereMRSA-positiveand 62of149 (42%)
horses. Four of these horses were already MRSA-positive at
admissionwhile21werenegativeand37werenotsampled
at admission, because they were already hospitalized when
the investigation started. Most horses were MRSA-positive
only once, but one horse was MRSA-positive 5 times
(Table 3). All MRSA isolates except one were spa-type t011.
One horse had spa-type t2123, but this horse was already
positive with the same spa-type at admission and had been
hospitalizedatthisclinicbeforein2004and2007.AllMRSA
were negative for the PVL toxin genes.
3.5. Screening of the environment
From the environmental wipes, 19/36 (53%) tested
positive for MRSA: 4/7 stable units, 3/6 isolation units, 2/2
preparation rooms, 2/2 examination rooms, the farriery,
the anaesthesia unit, the intensive care supply room, 2/2
staff rooms, the animal attendants’canteen, the students’
room and the students’ canteen. All environmental isolates
had spa-type t011 and were PVL-negative.
3.6. Comparing two culture methods
Culturing method 1 using MHB with 6.5% NaCl as pre-
enrichment yielded more MRSA-positive samples than
method2 usingTSBbrothwith4%NaClandaztreonamand
ceftizoxime as enrichment. Of the 338 equine samples
tested by both methods, 53 (15.7%) were MRSA-positive
using pre-enrichment with MHB and NaCl (method 1),
whereas only 12 (3.5%) samples were MRSA-positive using
the TSB broth (method 2). All MRSA-positive samples
found by method 2 were also detected using method 1. Of
the 36 environmental wipes, 18 (50%) samples were found
positive by method 1 whereas only 6 (16.7%) samples were
positive by method 2. One sample was found positive by
method 2 and not by method 1.
3.7. Retrospectivestudyof clinical MRSAisolatesatthe VMDC
The percentage of clinical MRSA isolates on the total
number of clinical equine S. aureus isolates found at the
VMDC increased from 0% in 2002 to 37% in 2008 (Fig. 1).
Table 1
Spa-types and repeat succession of the equine MRSA isolates found in the
present study.
Spa-typeTandem repeat
t011
t2123
t588
t064
t451
008-16-02-25-34-24-25
008- - -- - - -- - - -- - -25
008-16-02- - - - - -24-25
11-19-12-05-17-34-24-34-22-25
11- - - -12-05-17-34-24-34-22-25
Table 3
Cross-sectional sampling of the hospitalized horses.
Number of
samples/horse
Number of positive
samples/horse
Number of horses
with this result
1
1
2
2
2
3
3
3
4
4
4
4
4
5
5
5
0
1
0
1
2
0
1
3
0
1
2
3
4
0
1
5
47
25
13
7
6
3
2
3
22
12
2
1
2
2
1
1
Table 2
Distribution of spa-types of clinical equine MRSA isolates among the equine clinics.
Clinic
Spa-types found at this clinic Number of isolates
with this spa-type
Clinical condition
Veterinary teaching hospitalt011, t064, t2123
n = 9, n = 1, n = 9 Post-operative wound infections,
wound infections, thrombophlebitis
Arthritis, endometritis, conjunctivitis,
peritonitis, post-operative wound infections
Post-operative wound infections, abscess,
pneumonia
Arthritis
Abscess, endometritis
Endometritis
Post-operative wound infection
Referral clinic At064, t011
n = 8, n = 4
Referral clinic Bt064, t011, t588
n = 6, n = 3, n = 1
Clinic C
Clinic D
Clinic E
Clinic F
t064
t011
t451
t064
n = 1
n = 2
n = 1
n = 1
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Most MRSA originated from the veterinary teaching
hospital described in the first part of this study and two
large equine referral clinics, but these 3 clinics together
account for about 50% of all equine samples submitted to
the VMDC. Only 5 incidental cases were found at 4 other
equine clinics (Table 2). The most prevalent MRSA at both
referral clinics had spa-type t064, which is typeable by
PFGE using SmaI (RIVM-type 6b), MLST ST 8, MLVA-type
MT0265 and SCCmec-type IVa. MRSA with spa-type t011
(non-typeable by PFGE, MLVA-type MT0398, SCCmec-type
IVa and known to be ST398) was the second most common
MRSAinthose tworeferral clinics(Table2, Fig.2). Spa-type
t588 was only found once. Spa-type t451, which differs
fromt064 byonlyonerepeat,was alsofoundonceinoneof
the 4 clinics with only incidental cases of MRSA. Notably,
spa-typet2123wasnot foundinhorsesat anyequineclinic
except at the veterinary teaching hospital (Table 2) nor in
any other animal species (data not shown).
4. Discussion
Earlier studies among equine clinical isolates in the
Netherlands (van Duijkeren et al., 2004) and a survey
among 200 healthy horses (Busscher et al., 2006) did not
reveal MRSA. The reason for the sharp increase in the
number of equine MRSA infections since 2006 is currently
unknown. The high prevalence of ST398 in pigs and veal
calves in the Netherlands (de Neeling et al., 2007;
Graveland et al., 2008) and the fact that horses are often
kept on farms with other livestock and are sometimes
treated by veterinarians who are also involved in the
treatment of livestock are likely explanations for the
increase of ST 398 in horses. MRSA ST8 of spa-type t064
have also been found in horses and staff at a veterinary
hospital in Ireland (Moodley et al., 2006) and MRSA ST398
spa-type t011 have been found in horses and staff in a
teaching hospital in Vienna (Cuny et al., 2008). The
international travel and trade of horses may favour the
dissemination of the MRSA. Whether changes in the use of
certain antimicrobials are responsible for the increase
remains to be determined.
All equine and human MRSA isolates from the first
outbreakatthehospitalwere ofthe rarespa-typet2123.As
the horses had had no direct contact with each other it is
likely that nosocomial transmission took place, which is
supported by the fact that MRSA with this spa-type had
and has not been found in animals outside this clinic with
one exception: a horse which was positive with spa-type
t2123 during our pre-admission study in 2008. This horse,
however, had been admitted to this hospital in November
2004 and in October 2007. Unfortunately, no cultures were
performed at that time, but it is most likely that this horse
became infected at an earlier hospitalization. Spa-type
t2123 is also very rare in humans in the Netherlands: in
2007 and 2008 only 4 isolates had this spa-type, while it
was not found in 2006, except for the isolates related to
this outbreak (https://mrsa.rivm.nl/flash/flash.aspx). In
this view there is a strong case that zoonotic transmission
did occur at the hospital.
The index case of this outbreak was not identified, but
we postulate that spa-type t2123 (repeat succession 08-
25) has emerged from the more common and closely
related spa-type t011 (08-16-02-25-34-24-25) by the
loss of five repeats. Notably, the isolates of spa-type t011
and t2123 found at the hospital shared the same
resistance pattern (data not shown) and had the same
SCCmec-type. A similar observation was made during an
MRSA outbreak in a permanent care facility for people
with visual and intellectual disabilities, where two
different spa-types were discovered: the rare spa-type
t2383 (08–16) and spa-type t011 (Fanoy et al., 2009).
Another indication that new spa-types emerge through
the loss of repeats is that spa-type t451 (11-12-05-17-34-
24-34-22-25) was found once and this spa-type seems to
be related to the more common spa-type t064 (11-19-12-
05-17-34-24-34-22-25) from which it differs only by the
absence of repeat 19. spa-type t064 is also closely related
to
spa-typet008(11-19-12-21-17-34-24-34-22-25),
which has been isolated from horse personnel in Vienna
(Cuny et al., 2008).
Fig. 1. The number of clinical equine S. aureus and MRSA isolates found at
the VMDC from 2002 to 2008.
Fig.2. Thedistribution ofthe 3predominantspa-types foundat theVMDC
in clinical equine samples from 2002 to 2008.
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Pleasecitethisarticleinpressas:vanDuijkeren,E.,etal.,Methicillin-resistantStaphylococcusaureusinhorsesandhorse
personnel: An investigation of several outbreaks. Vet. Microbiol. (2009), doi:10.1016/j.vetmic.2009.08.009