Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 2, February 2007255
Methicillin-resistant Staphylococcus aureus of clonal
lineage ST398 that exhibits related spa types and contains
SCCmec elements of types IVa or V has been isolated from
colonized and infected humans and companion animals
(e.g., dog, pig, horse) in Germany and Austria. Of particu-
lar concern is the association of these cases with cases of
nosocomial ventilator-associated pneumonia.
worldwide, mainly associated with intrahospital and inter-
hospital dissemination of particular epidemic clonal line-
ages of the S. aureus population (hMRSA; ). MRSA
primarily associated with healthcare facilities may also be
disseminated to the community through colonized medical
staff or discharged patients. The emergence and spread of
MRSA in the community during the past 5 years, inde-
pendent of the healthcare setting and in the absence of typ-
ical risk factors for nosocomial MRSA infections, are
matters of further concern. These community-acquired
MRSA infections are less broadly resistant to antimicro-
bial agents than are healthcare-associated MRSAand often
contain the determinants lukS-lukF, which code for
Panton-Valentine leukocidin (2).
Even though MRSA has been known as a nosocomial
pathogen for >30 years, its development in companion ani-
mals and livestock has been rare (3). Recent reports, how-
ever, have documented MRSA infections in animals such
as horses from Canada (4) and Europe (3) and pets (5,6).
Of particular interest is whether MRSAmay be transmitted
ethicillin-resistant Staphylococcus aureus (MRSA)
has become an infection control problem in hospitals
between animals and humans. MRSA of clonal lineage
sequence type (ST) 22 is widely disseminated in human
hospitals in the United Kingdom and Central Europe. The
demonstration of this lineage among MRSA isolates from
staff and from pets in a small animal referral hospital in
United Kingdom suggests transmission between humans
and animals (5). Nasal colonization of veterinary staff with
MRSA(ST8) from infections in horses in a veterinary hos-
pital was frequently observed in Canada (4), and it was
also recorded in an Austrian university veterinary hospital
where horses were affected by MRSA of clonal lineage
We report on molecular characterization of MRSA,
from sporadic infections in humans and in various animal
species, that belong to clonal lineage ST398 according to
multilocus sequence typing (MLST). These isolates were
further characterized by spa-sequence typing (repeat poly-
morphism of the X-region of the spa gene) and by PCR for
grouping of staphylococcal cassette chromosome mec
(SCCmec) elements, which contain the mecA gene and of
which at least 5 basic types have been described.
MRSA isolates from infections in humans and in ani-
mals were sent to the National Reference Center for
Staphylococci at the Robert Koch Institute, Wernigerode
Branch, in Germany, for typing by means of SmaI-
macrorestriction pattern as well as spa typing. Selected
isolates also underwent MLST. Four human isolates were
grown from nasal swabs taken from the staff of a veteri-
nary practice at Veterinary Analytical Center, Geesthacht,
Germany. All isolates were primarily grown on sheep
blood agar and confirmed by standard procedures as
S taphyloc oc c us aureus S T 398
in Humans and Animals,
Wolfgang Witte,* Birgit Strommenger,* Christian Stanek,† and Christiane Cuny†
*Robert Koch Institute, Wernigerode, Germany; and †Veterinary
University, Vienna, Austria
S. aureus. Eleven additional MRSA specimens of lineage
ST398 (1 isolate per patient affected) were found among
4,370 MRSA isolates from patients with recognized infec-
tions. These isolates were identified by indigestibility of
their whole cellular DNA when subjected to SmaI-
macrorestriction analysis. Animal isolates were collected
from 1 dog and 1 foal at the Veterinary Analytical Center,
Geesthacht, Germany; from 1 pig at the diagnostic labora-
tory of the Institute for Microbiology and Infectious
Diseases, School of Veterinary Medicine, Hannover,
Germany; and from 2 horses at the Department of
Orthopaedics, Veterinary University, Vienna, Austria.
Procedures and primers for DNA extraction and PCR
detection of resistance genes were as described previously
(6). Macrorestriction patterns were determined by using
lysis of cells, deproteinization and digestion of DNA (here
by SmaI and ApaI), and pulsed-field gel electrophoresis (7).
The polymorphic X-region of the protein Agene (spa)
was amplified and sequenced according to the Ridom
StaphType standard protocol (www.ridom.org). The result-
ing spa-types were assigned by using the Ridom
StaphType software package (Ridom GmbH, Würzburg,
Germany). The BURP algorithm, implemented in the most
recent Ridom StaphType software version, was used for
cluster analysis of spa types (7).
Primers used for MLST correspond to the protocol as
described previously (8), with the exception of the forward
primer for tpi; we used the sequence tpif 5′-GCAT
TAGCAGATTTAGGCGT-3′. Antimicrobial susceptibility
testing was performed by broth microdilution, performed
according to DIN 58940, Deutsches Institut für Normung
(9). SCCmec elements of types I to IV were characterized
by using a PCR approach, including a combination of dif-
ferent PCRs (6). To demonstrate SCCmec-elements of type
V, we used primers type VF/type VR, as described by
Zhang et al. (10), as well primer pair ccrC9f 5′-CACT-
TAATCCATGTACACAG-3′ and ccrC-R (10).
The following set of primers was used for PCR for
virulence-associated genes: tst, sea, seb, sec, sed, see, as
described by Johnson et al. (11); for lukS-lukF, forward 5′-
3′; for cna, forward 5′-CGGTTCCCCCATAAAAGT
GAAG-3′, reverse 5′-CCCATAGCCTTGTGGATTTG-3′.
Annealing temperature was 55° C; cyclic scheme and fur-
ther conditions were as reported previously (6).
Specimen collection, characterization of the isolates,
data processing, and exchange of data were performed
within the framework of German public health activities
for infection control and prevention of MRSA dissemina-
tion. Ethical approval was obtained within this framework
Characteristics of the 20 MRSA isolates investigated
are shown in the Table. All isolates share MLST ST398
with the allelic profile 3-35-19-2-20-26-39. Three different
spa-types are obviously related (Figure). Types t11 and t34
may have been derived from each other by either deletion
or duplication of 2 repeats; t1197 and t11 differ by a single
nucleotide polymorphism. BURP analysis of these spa-
types groups them as a separate cluster unrelated to other
BURP clusters (7). A peculiarity of S. aureus of clonal lin-
eage ST398 is the indigestibility of whole cellular DNAby
restriction enzyme SmaI. Therefore, SmaI macrorestriction
patterns generate only 1 large fragment because of protec-
tion by a novel DNA methylation enzyme (12). We also
found poor digestion by the isoschizomeric enzyme XmaI.
However, digestion by enzyme ApaI generated similar
fragment patterns that differed at most by 3 fragments
independent of spa types.
The 2 horse isolates from the Vienna veterinary uni-
versity contained SCCmec elements of group IVa. For all
other isolates investigated, PCR indicated SCCmecV.
These findings suggest that MRSA of ST398 from horses
are unrelated to the other isolates and probably have
evolved independently by acquisition of a different
In addition to mecA, all investigated isolates contained
tetM; isolates from animals and humans from Lower
Saxony also contained ermA. The nosocomial human and
horse isolates contained ermC; in the horse isolates, aph2″-
aac6′–mediating aminoglycoside resistance was demon-
strated. PCR was negative for virulence-associated genes
and for lukS-lukF (coding for Panton-Valentine leuko-
cidin), tst, sea, seb, sec, and sed, as well as for cna (colla-
Isolates of clonal lineage ST398 seem not to be fre-
quently represented among the S. aureus population. They
were not recorded by Grundmann et al. (13) among a pop-
ulation sample of nasal colonizers in the Nottingham area
in the United Kingdom and were not found among 108 iso-
lates from carriers in a rural territory in northern Germany
(S. Holtfreter et al., unpub. data). Only 2 notations of
ST398 are found in the S. aureus MLST database
(www.mlst.net), 1 from the Netherlands and 1 from the
Cape Verde Islands.
Among 11,250 isolates of various origin (colonization
and infections in hospitals as well in the community in
humans from all Germany) typed from 1992 through 2003,
no isolates refractory to SmaI macrorestrition analysis
were seen. Therefore, a rather recent emergence of MRSA
ST398 among humans seems likely. However, MRSA of
lineage ST398 had been reported from infections in pigs
256 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 2, February 2007
and from nasal colonization in pig farmers in France (14).
A more recent report from the Netherlands describes
MRSA of ST398 (spa t108, which is in the same BURP
cluster as t11 and t34) in pigs and in humans who had con-
tact with pigs (15). A comparison of the allelic profile of
ST398 by means of the MLST database does not indicate
any relationship to profiles of prevalent clonal complexes
of methicillin-susceptible S. aureus (13), of epidemic
healthcare-associated MRSA, or of lukS-lukF–containing
community-associated MRSA from Europe.
MRSA exhibiting ST398 may colonize and cause
infections in humans and in certain animal species such as
dogs, horses, and pigs. The isolation of MRSA ST398
showing the same characteristics from a wound infection
in a dog and from nasal colonization of the staff of a vet-
erinary practice where this dog had been treated suggests
that interspecies transmission may occur. The differences
in spa types between the isolates containing the same PCR
results for SCCmec can be explained by a single genetic
event. Because isolates taken at the same time from nasal
colonization in veterinary staff of the same practice exhib-
it either spa-type t011 or t034, this difference does not jus-
tify discrimination between the two types. Of particular
concern was the subsequent detection of MRSAST398 not
only in outpatients but also in inpatients with ventilator-
associated pneumonia in the same hospital unit at about the
same time (Table).
Future recording of MRSA ST398 from infected and
colonized humans (especially when detected by screening
at admission to hospitals) will require a thorough analysis
with respect to association with animals and routes of
transmission. Tracing MRSA carriers among contacts
should also include pet animals, horses, and other live-
stock. Because of the time and labor needed to complete
MLST, spa-typing combined with BURP analysis of types
is an efficient tool for recognizing this clonal lineage.
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 2, February 2007257
Figure. Repeats of the X-region in methicillin-resistant
Staphylococcus aureus of clonal lineage ST398.
MRSA ST398, Central Europe
258 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 2, February 2007
Furthermore, detection of MRSA by appropriate methods
should be implemented into antimicrobial resistance sur-
veillance programs in veterinary medicine.
Prof Witte is head of the laboratory for nosocomial infec-
tions at the Robert Koch Institute, Wernigerode Branch. He also
is a professor on the medical faculty of Magdeburg University.
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Address for correspondence: Wolfgang Witte, Robert Koch Institute,
Wernigerode Br, Burgstraße 37, 38855 Wernigerode, Germany; email: