1 Methicillin-resistant 2 ACCEPTEDjcm.asm.org/content/early/2007/01/31/JCM.02193-06.full.pdf · 2...

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1 Methicillin-resistant Staphylococcus pseudintermedius 1 in a veterinary teaching hospital 2 3 Takashi Sasaki 1 , Ken Kikuchi 1* , Yoshikazu Tanaka 2 , Namiko Takahashi 1 , 4 Shinichi Kamata 2 , and Keiichi Hiramatsu 1 5 6 Department of Infection Control Science, Faculty of Medicine, Juntendo University, 7 Tokyo 113-8421, Japan 1 and Department of Veterinary Hygiene, Veterinary School, 8 Nippon Veterinary & Life Science University, 1-7-1 Kyounan, Musashino, 9 Tokyo 180-8602, Japan 2 . 10 11 Running title: Methicillin-resistant S. pseudintermedius 12 13 14 *Corresponding author. Mailing address: Ken Kikuchi, M.D.,Ph.D. 15 Department of Infection Control Science, Faculty of Medicine, Juntendo University, 16 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan 17 18 Phone: 81-3-3813-3111 ext. 3822 19 Fax: 81-3-3816-2782 20 E-mail: [email protected] 21 22 23 ACCEPTED Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. J. Clin. Microbiol. doi:10.1128/JCM.02193-06 JCM Accepts, published online ahead of print on 31 January 2007 on October 11, 2018 by guest http://jcm.asm.org/ Downloaded from

Transcript of 1 Methicillin-resistant 2 ACCEPTEDjcm.asm.org/content/early/2007/01/31/JCM.02193-06.full.pdf · 2...

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Methicillin-resistant Staphylococcus pseudintermedius 1

in a veterinary teaching hospital 2

3

Takashi Sasaki1, Ken Kikuchi

1*, Yoshikazu Tanaka

2, Namiko Takahashi

1, 4

Shinichi Kamata2, and Keiichi Hiramatsu

1 5

6

Department of Infection Control Science, Faculty of Medicine, Juntendo University, 7

Tokyo 113-8421, Japan1 and Department of Veterinary Hygiene, Veterinary School, 8

Nippon Veterinary & Life Science University, 1-7-1 Kyounan, Musashino, 9

Tokyo 180-8602, Japan2. 10

11

Running title: Methicillin-resistant S. pseudintermedius 12

13

14

*Corresponding author. Mailing address: Ken Kikuchi, M.D.,Ph.D. 15

Department of Infection Control Science, Faculty of Medicine, Juntendo University, 16

2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan 17

18

Phone: 81-3-3813-3111 ext. 3822 19

Fax: 81-3-3816-2782 20

E-mail: [email protected] 21

22

23

ACCEPTED

Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.02193-06 JCM Accepts, published online ahead of print on 31 January 2007

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ABSTRACT 1

We surveyed methicillin-resistant coagulase-positive staphylococci (MRCPS) from 57 2

(26 inpatient and 31 outpatient) dogs and 20 veterinary staff in a veterinary teaching 3

hospital. From the staff, 3 MRCPS were isolated and 2 were methicillin-resistant 4

Staphylococcus aureus (MRSA). In contrast, 18 MRCPS strains were detected in both 5

inpatient (12/26, 46.2%) and outpatient (6/31, 19.4%) dogs. Among them, only one 6

strain was MRSA. Using direct sequencing of sodA and hsp60 genes, the 18 MRCPS 7

strains other than MRSA from a staff and 17 dogs were finally identified as 8

Staphylococcus pseudintermedius, a novel species of Staphylococcus from a cat. All the 9

methicillin-resistant S. pseudintermedius (MRSP) strains were multi-drug resistant to 10

erythromycin, clindamycin, trimethoprim/sulfamethoxazole and levofloxacin. Most of 11

MRSP strains showed high-level resistance to oxacillin (!128 µg/ml, 15/18, 83.3%), 12

and 10 of 15 (66.7%) high-level oxacillin-resistant MRSP strains carried type-III 13

SCCmec. DNA fingerprinting of MRSP strains by pulsed-field gel electrophoresis 14

yielded 8 clusters: clone A with 4 subtypes, clone B with 4 subtypes, clone C with 3 15

subtypes, and 5 other different single clones. MRSP strains from the staff, some 16

inpatient and outpatient dogs shared 3 major clones (clone A, B, C), but the strains of 17

the other 5 different clusters were distributed independently among inpatient or 18

outpatient dogs. This genetic diversity suggested that the MRSP strains were not only 19

acquired in this veterinary teaching hospital but also acquired in primary veterinary 20

clinics in the community. To our knowledge, this is the first report of MRSP in dogs 21

and humans in a veterinary institution. 22

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INTRODUCTION 1

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most 2

important antimicrobial-resistant nosocomial pathogens of humans worldwide, and has 3

also emerged recently in patients without established risk factors in the community (8). 4

MRSA has also been detected in veterinary medicine, especially in cows, poultries, 5

horses and pet dogs and cats (39). In human hospitals, healthcare-associated outbreaks 6

of MRSA are well documented (8). Recently, suspected cases of human-to-animal or 7

animal-to-human transmission of MRSA have been reported sporadically (29, 36, 41, 8

42, 48, 50). In methicillin-resistance of coagulase-positive staphylococci (CPS) other 9

than S. aureus, which are predominant in animals, there have been also a few reports of 10

methicillin-resistant Staphylococcus intermedius (MRSI) from dogs (15, 20, 23, 49). 11

However, MRSI has been uncommon as a nosocomial pathogen in a veterinary hospital. 12

There have been five species of coagulase-positive staphylococci (CPS) other 13

than S. aureus, Staphylococcus intermedius, Staphylococcus schleiferi, Staphylococcus 14

hyicus, Staphylococcus delphini, and Staphylococcus pseudintermedius that has recently 15

been described in cats (11). Since biochemical characteristics of S. schleiferi and S. 16

hyicus are different from those of S. intermedius, S. delphini, and S. psudintermedius 17

(11), it is easy to identify S. schleiferi and S. hyicus. Commercial kits such as rapid 18

ID32 Staph (bioMérieux), VITEK2 ID-GPC and ID-GP identification card 19

(bioMérieux), or Phoenix ID panel PMIC/ID-13 (BD) are also available for 20

identification of the latter two species (26). However, the phenotypic characteristics of S. 21

intermedius, S. delphini, and S. psudintermedius resemble one another, and commercial 22

kits are not available for differentiation of these species (11). Furthermore, 16S rRNA 23

genes of these species are >99% identical, and could not be used to differentiate the 24

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species (1, 11, 43). The discriminating power of sodA or hsp60 sequencing is superior 1

to phenotypic characterization or 16S rRNA sequencing for species identification of 2

staphylococci (24, 25, 38). Although S. intermedius has been reported to be prevalent 3

among dogs, cats, horses, goats, and pigeons (7, 13), these S. intermedius strains with 4

different biotypes might have included S. delphini and S. pseudintermedius. 5

In the present study, we screened CPS from dogs and staffs in a veterinary 6

teaching hospital to determine the prevalence of MRCPS and study their mode of 7

nosocomial spread among dogs and humans. Furthermore, for definite species 8

identification of CPS strains, we performed sodA and hsp60 sequencing. 9

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MATERIALS AND METHODS 1

Sampling from dogs and veterinary staffs, isolation and phenotypic 2

characterization of methicillin-resistant coagulase-positive staphylococci 3

Samples from both nostrils of 57 dogs (26 inpatients and 31 outpatients) and 4

median septum of both nostrils of 20 veterinary staffs were collected by using Seed 5

Swab (Eiken Chemical Co., Ltd., Tokyo, Japan) from January to March 2006 at Nippon 6

Veterinary and Life Science University Hospital. Informed consent of the owners and 7

veterinary staffs were obtained prior sampling. Ethical association in Nippon Veterinary 8

and Life Science University had approved this study. All patient-dogs in the Nippon 9

Veterinary and Life Science University Hospital were transferred from primary care 10

animal hospitals in the community. Swabs were immediately inoculated on 11

mannitol-salt agar (Nissui Co., Ltd., Tokyo, Japan) directly and incubated at 37 ℃ for 12

48 h for selective isolation of staphylococci. Catalase-positive, Gram-positive cocci that 13

were presumptively identified as staphylococci by colonial morphology, were 14

subcultured on trypticase-soy agar II with 5% sheep blood (TSAB; BD Japan, Co., Ltd. 15

Tokyo, Japan). Tube coagulase test using rabbit plasma (Denka Seiken Co., Ltd., Tokyo, 16

Japan) were performed and only coagulase-positive staphylococci (CPS) were selected 17

for further investigations (12). Species identification of CPS was carried out by latex 18

agglutination test for clumping factor and protein A (PS test, Eiken Chemistry Co., Ltd, 19

Tokyo, Japan), DNase test, and rapid ID32 Staph (bioMérieux, Marcy-l`Etoile, France) 20

(12, 26, 37). A strain of S. intermedius identified phenotypically was designated as S. 21

intermedius group (SIG) composed of S. intermedius, S. delphini, and S. 22

psudintermedius presumptively, because it was very difficult to differentiate these three 23

species by biochemical reactions (11). These strains were stored in 10% skim milk at 24

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-85oC until use, and maintained on TSAB. 1

2

Antimicrobial susceptibility testing 3

Methicillin-resistance was determined by measurement of MIC of oxacillin using 4

the agar dilution method for all CPS strains (9). MIC tests for other antimicrobial agents 5

were performed by broth microdilution method using MIC-2000 system (Dry plate 6

Eiken DP22, Eiken). Antibiotics tested were penicillin (PEN), ampicillin (AMC), 7

oxacillin (OXA), cefazolin (CFZ), cefotiam (CTM), cefaclor (CEC), flomoxef (FMX), 8

imipenem (IPM), gentamicin (GEN), arbekacin (ABK), erythromycin (ERY), 9

clindamycin (CLI), minocycline (MIN), levofloxacin (LVX), fosfomycin (FOF), 10

vancomycin (VAN), teicoplanin (TEC) and trimethoprim/sulfamethoxazole (SXT). The 11

MICs were interpreted as susceptible or resistant according to CLSI (formerly National 12

Committee for Clinical Laboratory Standards) recommendations (9). Breakpoints for 13

human coagulase-negative staphylococci were applied to SIG strains by 14

recommendation of Bemis (4). Oxacillin-resistance was classified as low-level (MIC; 15

0.5-64 µg/ml) or high-level (MIC; !128 µg/ml). 16

17

DNA extraction for amplification 18

A single colony was suspended to a McFarland 1.0 standard in 100 µl of TE 19

buffer (20 mM Tris, 2 mM EDTA, pH 7.5) with 10 U of achromopeptidase (Wako 20

Chemical, Co. Ltd., Osaka, Japan), and the suspension was incubated at 55 ℃ for 10 21

min. After centrifugation at 18,500 X g for 5 min, the supernatants were used as crude 22

DNA extracts for PCR (22). 23

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Genetic identification of coagulase-positive staphylococci 1

To confirm species identification by phenotypic characterization, we conducted 2

molecular identification of staphylococci by detection of femB gene encoding for an 3

enzyme important in cross-linking peptidoglycan in Staphylococcus aureus (19). Direct 4

sequencing of sodA and hsp60 genes was performed for differentiation of SIG strains 5

(24, 25, 38). Sequencing reactions were performed by using a Big Dye Terminator 6

(version 1.1) cycle sequencing kit (Applied Biosystems) with an ABI Prism 3100 7

genetic analyzer (Applied Biosystems, Foster City, CA). Multiple alignment was 8

carried out by the CLUSTAL X program (46). The construction of the unrooted 9

phylogenetic tree was performed by the neighbor-joining method (40). 10

11

Detection of mecA and SCCmec typing 12

mecA was determined for all the CPS to confirm methicillin-resistance, and 13

SCCmec typing was performed for all mecA-positive CPS strains by amplification of 14

the regions within SCCmec, ccr region (three classes of ccr), and mec region (IS1272, 15

mecI-R1, mecA), by PCR as described previously (17, 18, 30). 16

17

Detection of enterotoxins, exfoliative toxins, TSST-1 and the Panton-Valentine 18

leukocidin (PVL) genes 19

Localization of 11 exotoxin genes, encoding for staphylococcal enterotoxins 20

SEA (sea), SEB (seb), SEC (sec), SED (sed), SEE (see), SEG (seg), SEH (seh), SEI 21

(sei), SEJ (sei); exfoliative toxin A, B (ETA; eta, ETB; etb); TSST-1 (tst); 22

Panton-Valentine leukocidin (lukS and lukF) were detected by PCR as reported 23

previously (3, 28, 33). 24

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Pulsed field gel electrophoresis (PFGE) analysis 1

Chromosomal DNAs of the CPS strains digested with Sma I, Asc I and Xma I

2

(New England Biolabs, Beverly, MA) were analyzed by pulsed-field gel electrophoresis 3

(PFGE) as described previously (37) with minor modification. The PFGE conditions for 4

Sma I- and Xma I or Asc I-digested DNAs were as follows: switch time, 3.0 s - 9.0 s; 5

and run time, 9 h and switch time, 8.0 s - 45.0 s; and run time, 12 h (for Sma I and Xma 6

I), switch time, 5.0 s - 40.0 s; run time, 22 h (for Asc I), included angle, 120°; voltage 7

was 6 V/cm. The buffer temperature was maintained at 11.3 ℃. The PFGE patterns 8

were interpreted according to the criteria of Tenover (45). Isolates showing six or fewer 9

fragment differences were considered to be subtypes of a pulse type (45). 10

11

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RESULTS 1

Surveillance of coagulase-positive staphylococci from dogs and veterinary staffs 2

In 20 veterinary staffs, CPS strains were isolated from 6 staffs (30.0%) and 3 of 6 3

were methicillin-resistant CPS (MRCPS) (Table 1). Among 3 MRCPS strains, 2 were 4

MRSA. Another MRCPS strain was identified as S. intermedius phenotypically and 5

categorized as SIG. This strain was methicillin-resistant SIG (MRSIG). 6

In 57 dogs, CPS strains were isolated from 30 dogs (52.6%) were isolated and 18 7

of 30 were MRCPS. S. aureus strains were isolated 5 dogs (8.8%), and SIG strains were 8

isolated from 25 dogs (43.9%). 20% (1 of 5) of S. aureus strains and 68% (17 of 25) of 9

SIG strains showed methicillin-resistance. 10

11

Species identification of methicillin-resistant S. intermedius group (MRSIG) strains 12

based on molecular phylogenetic analysis. 13

All of the 18 MRCPS strains other than MRSA in the present study were 14

phenotypically identified as MRSIG. The sodA sequence-based analysis revealed that 15

all the 18 strains had only 96 to 97 % sequence similarities with Staphylococcus 16

delphini CIP 103732T, and were quite different from Staphylococcus intermedius CIP 17

8160T with 91 - 92 % similarities. Furthermore, there were only 93 to 94 % similarities 18

of hsp60 gene between these 18 strains with both S. delphini CIP 103732T and S. 19

intermedius ATCC 29663T, respectively. These 18 strains belonged to the same cluster 20

of sodA and hsp60 gene sequence and the sequence similarities were above 99 % with 21

Staphylococcus pseudintermedius LMG 22219 T

for both sodA gene (Fig. 1) and hsp60 22

gene (Fig. 2). Thus, all of the 18 MRSIG strains were identified as S. pseudintermedius 23

by phylogenetic analysis of sodA and hsp60 genes. 24

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Antimicrobial susceptibility profiles 1

Among 3 MRSA strains isolated in the present study, 2 strains from a veterinary 2

staff and a dog were resistant to all !-lactams, minocycline, erythromycin, clindamycin, 3

and levofloxacin, but susceptible to vancomycin, trimethoprim/sulfamethoxazole, 4

teicoplanin, and gentamicin. In contrast, one strain from a staff showed susceptibility to 5

all antimicrobial agents other than !-lactams. 6

On the other hand, all of methicillin-resistant Staphylococcus pseudintermedius 7

(MRSP) strains were divided into 2 antibiogram-types by oxacillin-susceptibility (Table 8

2.). The majority of MRSP strains (15/18, 83.3%) showed high-level resistance to 9

oxacillin, and the rests of them (3/18, 16.7%; 1 from inpatient dog, 2 from outpatient 10

dogs) were low-level resistant (2-4 µg/ml) despite of mecA-positive. These 3 strains 11

were more susceptible to cefazolin, cefotiam, cefaclor, and fosfomycin than high-level 12

oxacillin-resistant strains were, nevertheless they were resistant to minocycline. All of 13

the MRSP strains were resistant to erythromycin, clindamycin, 14

trimethoprim/sulfamethoxazole, gentamicin (except for one strain which was 15

intermediate) and levofloxacin. Most of dogs with MRSP nasal carriage (14/17; 82.4%) 16

had received antimicrobial agents as cefmetazole, cefaclor, enrofloxacin, or fosfomycin, 17

within the previous six months. 18

19

Molecular characterization of MRCPS strains 20

Table 3 shows genotypic characteristics of 21 MRCPS strains analyzed in this 21

study. Two MRSA strains with high-level resistance to oxacillin isolated from a staff 22

and a dog belonged to type-II SCCmec, and had tst, sec, seg, sei genes. The PFGE 23

patterns of these two strains (X1 and X2) were closely related to each other (Fig. 3). 24

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The remaining multi-drug susceptible MRSA strain belonged to type-IV SCCmec, and 1

had no toxin genes. The PFGE pattern of this strain was different from those of the 2

other two strains. 3

For MRSP strains, 10 of 18 strains (55.6%) carried type-III SCCmec element, one 4

strain (5.6 %) had type-V SCCmec element, and 7 strains (38.9 %) were non-typeable. 5

PFGE analysis of Asc I-digested DNA was less discriminative than that of Sma 6

I-digested DNA because all the MRSP strains were considered to be subtypes within the 7

same cluster (Fig.4). The PFGE patterns of Sma I-digested DNA from the MRSP strains 8

were differentiated into 8 clones (A to H). Clones A to C were divided into several 9

subtypes as A1 to A4, B1 to B4, and C1 to C3, respectively (Fig. 4). Only two pairs of 10

strains shared identical patterns (A1 and D) each other. The genomic DNAs from strain 11

NVAU06046 and NVAU06038 could not be digested by Sma I. The neoschizomer Xma 12

I, which cuts the same recognition sequence of Sma I but at a different position, also 13

could not digest the two DNAs (data not shown). Clones with A to E PFGE types 14

showed high-level resistance to oxacillin. PFGE types A and B were predominant in 15

inpatient dogs (7 of 11; 63.6%). One MRSP strain NVAU06013 isolated from a 16

veterinary staff, also belonged to clone A with type-III SCCmec. Both PFGE types B 17

and C were detected in inpatient and outpatient dogs. In contrast, PFGE types D to H 18

were only isolated from outpatient dogs, and PFGE types F and G were only found in 19

the inpatient dogs. 20

PFGE type H strain with non-typeable (NT) SCCmec, PFGE type F strain with 21

type-V SCCmec, and PFGE type G strain with non-typeable SCCmec showed low-level 22

resistance to oxacillin. No toxin genes were detected in the MRSP strains. 23

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DISCUSSION 1

To our knowledge, this is the first report on the prevalence of methicillin-resistant 2

Staphylococcus pseudintermedius (MRSP) in dogs and humans in a veterinary 3

institution. In dogs, the majority of CPS from nasal, oral, ear commensal flora or 4

pyoderma specimens have been reported to be S. intermedius (7, 39). In this study, there 5

was a small rate of S. aureus carriage in dogs (only 5/57, 8.8%) as compared to that in 6

humans; only one MRSA strain was detected from a dog with MRSA infection. In dogs, 7

S. intermedius is considered to be more predominant than S. aureus (7, 39), but we 8

could not find any methicillin-resistant S. intermedius in this study. Surprisingly, all the 9

MRSIG strains were identified as S. pseudintermedius using sodA or hsp60 sequencing. 10

Meyer described that different biotypes of S. intermedius strains showed only 50-65% 11

DNA homology using DNA-DNA reassociation tests, indicating that they belonged to 12

different species (31). Other authors also reported that S. intermedius isolates from 13

different sources could be genotypically or phenotypically differentiated (6, 13, 14). In 14

the phylogenetic point of view, these results suggest that S. pseudintermedius is not a 15

novel emerging species among dogs, but might have been recognized as one of the 16

different biotypes of S. intermedius. 17

SIG strains did not seem to be found frequently in humans (only one MRSP was 18

isolated in this study). This human MRSP strain showed similar susceptibility patterns 19

and similar genotype (PFGE pattern and type-III SCCmec) to those of dog-derived 20

MRSP strains. This suggests dog-to-human transmission. Similar reports have been 21

published previously (16, 21, 44). 22

The prevalence of methicillin-resistant SIG isolates from healthy or sick dogs has 23

still been uncommon in previous reports (15, 23, 47, 49), however, the rate of 24

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methicillin-resistance among SIG isolates in the present study was higher (17/25, 68%) 1

than those of previous reports. The use of cephalosporins, carbapenems, and 2

fluoroquinolones is a well-known risk factor for acquisition of MRSA in humans (51). 3

Since broad-spectrum antimicrobials were used for 14 of 17 dogs with MRSP in this 4

study, antimicrobial selective pressure might have contributed to this prevalence of 5

methicillin-resistance. 6

There were no types-I, II or IV SCCmec in MRSP strains. In Japan, the majority 7

of human healthcare-associated MRSA strains carry type-II SCCmec, and type-III 8

SCCmec is detected in major HA-MRSA strains from South America, East Europe, and 9

Asian countries other than Japan and South Korea (10, 35). No type-III SCCmec has 10

been reported in Japanese MRSA strains (10). In the evolutionary point of view, the 11

difference of SCCmec element between MRSA and MRSP suggests that acquisition and 12

spread of mecA gene in Staphylococcus aureus and Staphylococcus pseudintermedius 13

had occurred via independent transmission route. 14

A minority of MRSP strains (3 of 18) with low-level oxacillin-resistance showed 15

different PFGE patterns from those of any high-level oxacillin-resistant strains. Kania 16

reported that 22 (40%) of 55 methicillin-susceptible S. intermedius determined by disk 17

diffusion method had mecA gene (20). Furthermore, Bemis described that the 18

breakpoint of oxacillin for S. aureus was too low for the detection of oxacillin 19

resistance in S. intermedius and S. schleiferi strains (4). These results suggest that 20

low-level oxacillin-resistance in S. intermedius and S. schleiferi strains might be 21

underestimated or unrecognized in the veterinary laboratories. Heterogeneous 22

resistance is well known in many species of mecA-positive staphylococci, but the 23

mechanism of the different resistance level has not been elucidated yet. Future studies 24

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are needed to clarify this mechanism. 1

In our study, all staphylococcal enterotoxin genes, toxic shock syndrome toxin-1, 2

Pantone-Valentine leukocidin, and exfoliative toxin genes were not detected in the 3

MRSP strains. Becker described that 33 of 281 (11.3%) S. intermedius strains have SEC 4

genes by the same PCR system used in this study (2). The reason for this discrepancy is 5

uncertain, but it may be due to the difference in species; ours were identified as S. 6

pseudintermedius, a distinct species from S. intermedius. Further testing of the 7

enterotoxigenic potential among S. intermedius, S. delphini, and S. psudintermedius 8

should be carried out. 9

The genomic DNAs from strains NVAU06046 and NVAU06038 could not be 10

digested by Sma I and Xma I, however Asc I-digested DNAs of both strains yield 11

banding patterns denoted as A6. This result might be due to unknown 12

restriction/methylation system in the recognition sequence of Sma I and Xma I site. 13

Although Bens reported similar findings (5), the DNA modification enzyme in our 14

strains was different from the ones sited in his report due to protection of both Sma I 15

and Xma I digestion. 16

The PFGE diversities of MRSP strains suggest that they were not only acquired 17

in this veterinary teaching hospital but also acquired in primary veterinary clinics in the 18

community. Although other comparable data of MRSP strains are not available, 19

Middleton reported that MRSA isolates in 7 veterinary teaching hospitals showed large 20

genetic diversities (32). 21

MRSA in the veterinary regions have been described in several reports (27, 29, 22

36, 41, 42, 48, 49). In this study, 3 MRSA strains isolated from two staffs and a dog 23

were also isolated. They had profiles similar to the MRSA strains which have been 24

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recently predominant in healthcare-associated or community-acquired MRSA in Japan 1

(22, 34, 37). Loeffler also reported that MRSA in veterinary hospital showed the 2

common population in the United Kingdom (29). Molecular epidemiology suggests that 3

sources of MRSA isolates from dogs are derived from human. 4

In conclusion, the prevalence of not MRSI but MRSP carriage in dogs in this 5

veterinary teaching hospital was unexpected. Because S. pseudintermedius is a new 6

species established in 2005 (11). Further investigations to reclassify canine 7

coagulase-positive staphylococci based on genetic identification such as sodA or hsp60 8

sequencing should be carried out. 9

10

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ACKNOWLEDGMENTS 1

This work was partially supported by a Grant-in-Aid for 21st Century COE Research 2

from The Ministry of Education, Science, Sports, Culture and Technology of Japan. 3

We thank M. Bonkobara, T. Yougo and T. Washizu for their kind help in collecting 4

specimens. 5

6

7

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FIGURE LEGENDS 1

2

Figure 1. Phylogenetic tree (unrooted) based on partial sodA gene sequences of 3

Staphylococcus intermedius group isolated in the present study. The tree was 4

constructed by the neighbor-joining method, using CLUSTAL X. 5

6

Figure 2 Phylogenetic tree (unrooted) based on partial hsp60 gene sequences of 7

Staphylococcus intermedius group isolated in the present study. The tree was 8

constructed by the neighbor-joining method, using CLUSTAL X. 9

10

Figure 3. PFGE patterns of Sma I digested MRSA strains in the veterinary hospital. 11

Low molecular weight marker DNA λ is shown on the left side. 3 lanes on the right 12

display results obtained with strains. Each lane shows a different PFGE type. 13

14

Figure 4. PFGE patterns of MRSP strains digested with Sma I and Asc I in the 15

veterinary hospital. Low molecular weight marker DNA λ are shown on the left. 16

Lanes on the right side display results obtained with strains. Each lane shows a different 17

PFGE type. 18

19

20

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TABLE 1. Carriage of methicillin-resistant staphylococci in veterinary staffs and dogs

Group No. tested No. (%) of humans or dogs with

S. aureus MRSA SIG* MRSIG** CPS*** MRCPS****

Veterinary staffs 20 5 (25.0) 2 (10.0) 1 (5.0) 1 (5.0) 6 (30.0) 3 (15.0)

Dog

Inpatient 26 2 (7.7) 1 (3.8) 13 (50.0) 11 (42.3) 15 (57.7) 12 (46.2)

Outpatient 31 3 (9.7) 0 (0) 12 (38.7) 6 (19.4) 15 (48.4) 6 (19.4)

Total 57 5 (8.8) 1 (1.75) 25 (43.9) 17 (29.8) 30 (52.6) 18 (31.6)

*SIG, Staphylococcus intermedius group

**MRSIG, methicillin-resistant Staphylococcus intermedius group

***CPS, coagulase-positive staphylococci

****MRCPS, methicillin-resistant coagulase-positive staphylococci (MRSA+MRSIG)

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TABLE 2. MICs for antimicrobial agents against methicillin-resistant Staphylococcus pseudintermedius in the veterinary teaching hospital

range MIC50 MIC90 range MIC50 MIC90

OXA >128 >128 >128 2-4 2 4

AMC 8->16 >16 >16 8->16 8 >16

CFZ 4->32 4 8 !0.5-2 !0.5 2

CTM !0.5->32 4 8 !0.5-2 !0.5 2

CEC 32->32 >32 >32 2-32 2 32

FMX !0.5 !0.5 !0.5 !0.5 !0.5 !0.5

IPM !0.12 !0.12 !0.12 !0.12 !0.12 !0.12

GEN 16->16 16 >16 8-16 8 16

ABK !0.5 !0.5 !0.5 !0.5 !0.5 !0.5

MIN !0.5 !0.5 !0.5 8-16 8 16

ERY >8 >8 >8 >8 >8 >8

CLI >4 >4 >4 >4 >4 >4

VAN !0.5-1 1 1 !0.5-1 !0.5 1

TEC !0.5 !0.5 !0.5 !0.5 !0.5 !0.5

FOF 32->32 32 >32 !8 !8 !8

SXT >76/4 >76/4 >76/4 >76/4 >76/4 >76/4

LVX 8->8 8 >8 8->8 8 >8a MRSP, Methicillin-resistant Staphylococcus pseudintermedius

H, high-level resistance to OXA (MIC; "128 µg/ml) ; L, low-level resistance to OXA (MIC; !64 µg/ml)cOXA, oxacillin; AMC, ampicillin; CFZ, cefazolin; CTM, cefotiame; CEC, cefaclor

FMX, flomoxef; IPM, imipenem; GEN, gentamicin; ABK, arbekacin; MIN, minocycline

ERY, erythromycin; CLI, clindamycin; VAN, vancomycin; TEC, teicopranin

FOF, fosfomycin; SXT, sulfamethoxazole/trimethoprim; LVX, levofloxacin

H-MRSPa (n=15) L-MRSP

a (n=3)Antimicrobial agent

b

MIC (µg/ml) of antimicrobial agent against

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TABLE 3. Molecular characterization of methicillin-resistant coagulase-positive staphylococci

Resistance to SCCmec Toxin type PFGE patterne

OXAc typed Sma I Asc I

NVAU06003 MRSA Human H II tst, sec, seg, sei X1 ND

NVAU06030 MRSA Dog-i H II tst, sec, seg, sei X2 ND

NVAU06032 MRSA Human H IV none Y ND

NVAU06013 MRSP Human H III none A4 a1

NVAU06021 MRSP Dog-o H III none B2 a4

NVAU06029 MRSP Dog-o H III none C2 a1

NVAU06002 MRSP Dog-o H III none D a1

NVAU06018 MRSP Dog-o H III none D a1

NVAU06037 MRSP Dog-o H NT none E a3

NVAU06046 MRSP Dog-o L NT none H a6

NVAU06016 MRSP Dog-i H III none A2 a1

NVAU06017 MRSP Dog-i H III none A3 a1

NVAU06022 MRSP Dog-i H III none B3 a3

NVAU06031 MRSP Dog-i H III none C1 a1

NVAU06039 MRSP Dog-i H III none C3 a1

NVAU06045 MRSP Dog-i L V none F a5

NVAU06043 MRSP Dog-i H NT none A1 a1

NVAU06044 MRSP Dog-i H NT none A1 a1

NVAU06034 MRSP Dog-i H NT none B1 a1

NVAU06033 MRSP Dog-i H NT none B4 a2

NVAU06038 MRSP Dog-i L NT none G a6

a MRSP, Methicillin-resistant Staphylococcus pseudintermedius

b "Dog-o" and "Dog-i" indicates outpatient and inpatient dogs, respectively.

cH, high-level resistance to OXA (MIC; !128 µg/ml) L, low-level resistance to OXA (MIC; 0.5 - 64 µg/ml)

dNT, Non-typeable

e ND, Not done

Strain no. Speciesa Groupb

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