Antimicrobial Susceptibility of Staphylococcus aureus and Characterization of Methicillin-Resistant Staphylococcus aureus Isolated from Bovine Mastitis in Korea

Abstract

A total of 402 Staphylococcus aureus isolates from bovine mastitis milk collected during 2003–2009 in Korea were tested for susceptibility to 20 antimicrobial agents. All S. aureus isolates were susceptible to 11 of 20 antimicrobials tested; no resistance was observed against pirlimycin, telithromycin, novobiocin, penicillin/novobiocin, quinupristin/dalfopristin, clindamycin, rifampin, ciprofloxacin, trimethprim/sulfamethoxazol, vancomycin, and linezolid. Over 66% of the S. aureus isolates were resistant to penicillin. Resistance was also seen for gentamicin (11.9%), erythromycin (7.7%), methicillin (oxacillin and cefoxitin, 6.2%), and tetracycline (4.2%). No noticeable change was observed in penicillin, gentamicin, and erythromycin resistance over the 7-year period. Tetracycline resistance appeared to decrease consistently, whereas methicillin resistance was observed from 2005. About 2.7% (11/402) were resistant to three or more antimicrobials. Genotyping of the 17 methicillin-resistant S. aureus (MRSA) isolated from each cow revealed two staphylococcal cassette chromosome mec (SCCmec) types (IV and IVa), three spa types (t286, t324, and untypable), and two sequence types (ST1 and ST72). Eleven of 17 (64.7%) MRSA strains belonged to SCCmec IVa-t324-ST72. The rest of strains belonged to SCCmec IVa-t286-ST1 (n = 3) and SCCmec IV-untypable-ST72 (n = 3). None of the MRSA carried the Panton-Valentine leukocidin gene. These characteristics are the same as those found in community-acquired (CA) MRSA strains prevalent in humans in Korea. Three pulsed-field gel electrophoresis types (A–C) were observed among the 17 MRSA strains examined, and 14 strains belonged to the same pulsed-field gel electrophoresis pattern regardless of their geographical origin and year of isolation. The results of this study provide evidence of CA-MRSA infection in dairy cattle for the first time in Korea.

Introduction

S taphylococcus aureus is one of the most contagious pathogens causing bovine mastitis (Kerro-Dego et al., 2002). Antimicrobial resistance in S. aureus is of grave concern for public health. Especially, methicillin-resistant S. aureus (MRSA) strains have long been a serious problem in hospitals worldwide since the 1980s. There has been another increasing concern that MRSA has emerged in community setting and animals (Van Loo et al., 2007). Recently, community-acquired (CA)-MRSA strains have emerged as a major concern with respect to MRSA infections (Tristan et al., 2007). Also, there is increasing number of reports on MRSA infections in animals (Juhász-Kaszanyitzky et al., 2007; Van Loo et al., 2007).

In Korea, over 60% and 5.9% of S. aureus isolated from hospitals and community, respectively, were reported to be MRSA (Kim et al., 2007). MRSA has been also isolated from various nonhuman sources including hospitalized dogs, bovine milk, and meats in Korea (Lee, 2003; Kwon et al., 2006; Lim et al., 2010). In addition, potential transmission of MRSA between humans and animals was suggested by recent studies in Korea: the predominant human types of hospital-associated (HA)-MRSA and CA-MRSA in Korea were, respectively, isolated from hospitalized dogs and chickens (Kwon et al., 2006) and beef and pork (Lim et al., 2010).

Several studies have reported antimicrobial resistance in S. aureus isolated from bovine mastitis in Korea (Kang et al., 1995, 2001; Moon et al., 2005), and MRSA isolates from mastitic milk of dairy cattle were first reported in 2003 (Lee, 2003). Since then only a few studies have reported MRSA isolates from bovine in Korea (Kwon et al., 2005; Moon et al., 2007). In addition, those studies were all conducted with milk samples collected before 2004, and updated data are needed. Therefore, the aims of this study were (1) to determine antimicrobial susceptibility of S. aureus isolated from mastitic bovine milk collected during 2003–2009 throughout Korea and (2) to further study on the molecular characteristics of the MRSA isolates and compare the epidemiological relatedness among them.

Materials and Methods

Bacterial isolates

A total of 402 isolates originated from 128 dairy cattle farms were selected from 909 S. aureus isolated from bovine mastitis in Korea during 2003–2009. Among S. aureus isolated in 2003 and 2004, two to four isolates per farm that had no genetic relationship were selected based on the previous data of restriction fragment length polymorphism analysis for the strains in our laboratory (Moon et al., 2007). Among S. aureus isolated in 2005–2009, two to four isolates (2005–2007) and six to eight isolates (2007 and 2008) per farm were selected based on their differences in antimicrobial resistance patterns, which were obtained from the previous data of antimicrobial susceptibility test during routine procedures for mastitis diagnosis in our laboratory. The numbers of S. aureus isolates/originated farms selected by year were 63/25 in 2003, 66/19 in 2004, 62/27 in 2005, 52/16 in 2006, 57/26 in 2007, 50/7 in 2008, and 52/8 in 2009. The mean number of isolates per year was 58 (ranging from 50 to 63).

The milk samples used in this study were collected from individual quarters of lactating cows as part of routine diagnostic samples submitted for determination of etiology of mastitis, and only samples with somatic cell counts >200,000 cells/mL were subjected to bacteriological examination. Each S. aureus isolate represented a single strain from a single mammary quarter of a cow. Primary culture of milk samples was performed as described by the National Mastitis Council (NMC, 1999), and isolates presumed as S. aureus using standard microbiological procedures were identified using the VITEK system (BioMérieux). Finally, they were confirmed to be S. aureus by polymerase chain reaction (PCR) (Pérez-Roth et al., 2001).

Susceptibility testing

Using the standard Kirby-Bauer disk diffusion method (Bauer et al., 1966), susceptibility of the isolates were determined against 20 antimicrobial agents commonly used for treatment of bovine mastitis in Korea or considered as important agents for humans as follows: antimicrobials used for treatment of bovine mastitis included in this study were ceftiofur, cephalothin, ciprofloxacin, erythromycin, gentamicin, novobiocin, penicillin, penicillin/novobiocin, pirlimycin, tetracycline, and trimethoprim/sulfamethxazol. Antimicrobials not used for treatment of bovine mastitis but important for humans were cefoxitin, chloramphenicol, clindamycin, linezolid. oxacillin, quinupristin/dalfopristin, rifampin, telithromycin, and vancomycin. One antimicrobial from each subclass of antimicrobials was selected based on Clinical and Laboratory Standards Institute (CLSI, 2008b) standard classification, and isolates showing resistance to three or more antimicrobial subclasses were considered multidrug resistant. All the antimicrobials were purchased from BBL (Becton Dickinson), except ceftiofur, penicillin/novobiocin, and pirlimycin (Oxoid).

Confirmed S. aureus isolates were inoculated into Mueller-Hinton broth (Becton Dickinson) and incubated overnight at 37°C. The turbidity of the suspensions were adjusted to a 0.5 McFarland standard and streaked onto Mueller-Hinton agar (Becton Dickinson) plates. Antimicrobial disks were added on the plates and they were incubated aerobically at 35°C ± 2°C for 16–18 h. The results were recorded as susceptible, intermediate, or resistant by measurement of the inhibition zone diameter according to the interpretive standards of Animal CLSI M31-A3 (ceftiofur, penicillin/novobiocin, pirlimycin) (CLSI, 2008a) and M100-S19 (all the antimicrobials except ceftiofur, penicillin/novobiocin, and pirlimycin) (CLSI, 2008b). Results for novobiocin were interpreted according to the criteria provided by the manufacturer (BBL; Becton Dickinson). Isolates with intermediate zones of resistance were not considered to be resistant in this study. The reference strain S. aureus ATCC 25923 was used as the quality control organism and included with each batch of isolates tested. Data from all quality control organisms were within appropriate CLSI quality control ranges.

Minimum inhibitory concentration

Of the 25 MRSA isolates phenotypically resistant to oxacillin and/or cefoxitin by disk diffusion methods, 17 strains originating from different cows were selected for further characterization. The minimum inhibitory concentration (MIC) values of oxacillin and cefoxitin for the 17 strains selected were determined using the E-test strips (AB Biodisk).

Staphylococcal cassette chromosome mec typing

The types of staphylococcal cassette chromosome mec (SCCmec) gene complex of the strains were determined by a multiplex PCR described previously (Okuma et al., 2002; Oliveira and de Lencastre, 2002). Unique SCCmec type patterns not consistent with those described by Oliveira and de Lancastre (2002) were further characterized by use of the method of Okuma et al. (2002) to determine the ccr gene and mec gene complex.

Multilocus sequence typing

Multilocus sequence typing (MLST) was carried out according to the method described by Enright et al. (2000). Primers were used for the amplification and sequence analyses of the seven housekeeping genes, arcC, aroE, glpF, gmk, pta, tpi, and yqiL. PCR products were purified and the products were then directly sequenced at Macrogen Seoul, Korea. Allele numbers were assigned using the MLST website (www.mlst.net).

Pulsed-field gel electrophoresis

Preparation of bacterial DNA and digestion with SmaI have been described previously (McDougal et al., 2003). Pulsed-field gel electrophoresis (PFGE) was performed with CHEP-Mapper apparatus (Bio-Rad) and a voltage of 6 V/cm ramped with an initial switching time of 5 sec and a final switching time of 40 sec. SeaKem Gold agarose 1% gel was run for 21 h at 14°C in a 0.5 × TBE buffer (50 mM Tris, 50 mM boric acid, and 0.2 mM ethylenediaminetetraacetic acid [pH 8.0]). PFGE patterns were assigned with a capital letter. Strains different in one band were assigned different capital letters.

Staphylococcal protein gene typing

spa typing was performed as described by Shopsin et al. (1999). Briefly, spa gene was amplified using primers spa 1095u (5′-AGACGATCCTTCGGTGAGC-3′) and spa 1517d (5′-CAGCAGTAGTGCCGTTTG-3′). Sequence analyses of extracted PCR products were performed at Macrogen. Sequence data were analyzed using Ridom StaphType software (www.spaserver.ridom.de). The spa repeats and spa types were determined and assigned a numeric code using a previously described method (Harmsen et al., 2003).

Detection of staphylococcal toxin genes

Presence of Panton-Valentine leukocidin (PVL) toxin genes were determined by PCR as described by Lina et al. (1999). The primer sequences for the PVL genes were as follows: for luk-PV-1, 5′-ATCATTAGGTAAAATGTCTGGACATGATCCA-3′; for luk-PV-2, 5′-GCATCAASTGTATTGGATAGCAAAAGC-3′. PCR products were resolved by electrophoresis through 1.5% agarose gels (Invitrogen).

Results

All S. aureus isolates were susceptible to 11 of 20 antimicrobials tested; no resistance was observed against pirlimycin, telithromycin, novobiocin, penicillin/novobiocin, quinupristin/dalfopristin, clindamycin, rifampin, ciprofloxacin, trimethprim/sulfamethoxazol, vancomycin, and linezolid. Nine antimicrobials to which S. aureus isolates showed resistance were penicillin, gentamicin, erythromycin, tetracycline, cefoxitin, oxacillin, ceftiofur, cephalothin, and chloramphenicol. Around 10% of the isolates showed resistance to these antimicrobials except penicillin, to which about 66% of S. aureus showed resistance. Excluding penicillin, the highest percentage of resistance was seen for gentamicin (11.9%), followed by erythromycin (7.7%), cefoxitin (6.2%), and tetracycline (4.2%). Mostly similar percentages of resistance to penicillin, gentamicin, and erythromycin were observed throughout the 7-year period of the study. Tetracycline resistance has tended to decrease gradually until 2007 and no resistance was observed afterward. Resistance to methicillin and ceftiofur emerged in 2005, whereafter their resistances remained with fluctuations (Table 1).

Table 1.

Antimicrobial Resistance of Staphylococcus aureus Isolated from Mastitic Bovine Milk Samples (Staphylococcal Cassette Chromosome ≥200,000/mL) Collected During 2003–2009 in Korea

				Percentage (no.) of resistant isolates
Antimicrobial subclass	Antimicrobials	Disk content	Zone diameter of resistance	2003 (n = 63)	2004 (n = 66)	2005 (n = 62)	2006 (n = 52)	2007 (n = 57)	2008 (n = 50)	2009 (n = 52)	Total (n = 402)
Pencillins	Penicillin	10 U	≤28	66.7 (42)	51.5 (34)	70.9 (44)	73.1 (38)	38.6 (22)	92.0 (46)	78.8 (41)	66.4 (267)
Aminoglycosides	Gentamicin	10 μg	≤12	9.5 (6)	15.2 (10)	11.3 (7)	34.6 (18)	0.0 (0)	12.0 (6)	1.9 (1)	11.9 (48)
Macrolides	Erythromycin	15 μg	≤13	4.8 (3)	3.0 (2)	12.9 (8)	9.6 (5)	15.8 (9)	0.0 (0)	7.7 (4)	7.7 (31)
Tetracyclines	Tetracycline	30 μg	≤14	7.9 (5)	9.1 (6)	8.1 (5)	1.9 (1)	0.0 (0)	0.0 (0)	0.0 (0)	4.2 (17)
Cephamycin	Cefoxitin	30 μg	≤21	0.0 (0)	0.0 (0)	6.5 (4)	9.6 (5)	7.0 (4)	24.0 (12)	0.0 (0)	6.2 (25)
Pencillinnase-stable penicillins	Oxacillin	1 μg	≤10	0.0 (0)	0.0 (0)	6.5 (4)	9.6 (5)	0.0 (0)	10.0 (5)	0.0 (0)	3.4 (14)
Cephalosporins III	Ceftiofur	30 μg	≤17	0.0 (0)	0.0 (0)	6.5 (4)	7.7 (4)	0.0 (0)	2.0 (1)	0.0 (0)	2.2 (9)
Ketolides	Telithromycin	15 μg	≤18	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)
Cephalosporins I	Cephalothin	30 μg	≤14	0.0 (0)	0.0 (0)	6.5 (4)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.9 (4)
Phenicols	Chloramphenicol	30 μg	≤12	3.2 (2)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.5 (2)
Penicillins/other class combinations	Penicillin/novobiocin	10 U/30 μg	≤14	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)
Streptogramins	Quinupristin/dalfopristin	15 μg	≤15	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)
Lincosamides	Clindamycin	2 μg	≤14	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)
	Pirlimycin	2 μg	≤12	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)
Ansamycins	Rifampin	5 μg	≤16	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)
Fluoroquinolones	Ciprofloxacin	5 μg	≤15	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)
Folate pathway inhibitors	Trimethprim/sulfamethoxazol	1.25 μg/23.75 μg	≤10	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)
Glycopeptides	Vancomycin	30 μg	≤14^a	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)
Oxazolidinones	Linezolid	30 μg	≤20^a	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)
Others	Novobiocin	30 μg	≤14	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)

Considered as intermediate or resistant because only the range of susceptibility against vancomycin (≥15) and linezolid (≥21) was designated by the CLSI (2008b).

Antimicrobial resistance pattern of 402 S. aureus isolates are presented in Table 2. About 26% (103/402) of the isolates were susceptible to all antimicrobials tested in this study. The majority of the isolates (49.2%) showed resistance only to penicillin. About 2.7% (11/402) of S. aureus displayed resistance to three or more antimicrobials, and they were all MRSA except only one strain.

Table 2.

Number of Isolates Resistant to the Given Number of Antimicrobials and the Resistance Patterns Observed Among Staphylococcus aureus Isolates from Mastitic Bovine Milk Samples Collected During 2003–2009 in Korea

No. of antimicrobials	Resistance pattern	No. of isolates (%)
0	Pansusceptible	103 (25.6)	103 (25.6)
1	E	14 (3.5)	230 (57.2)
	GM	14 (3.5)
	TE	4 (1.0)
	P	198 (49.2
2	P, CM	2 (0.5)	58 (14.4)
	P, E	8 (2.0)
	P, FOX	10 (2.5)
	P, GM	24 (6.0)
	P, TE	9 (2.2)
	P, FOX	5 (1.2)
3	P, FOX, XNL	1 (0.2)	2 (0.5)
	P, GM, E	1 (0.2)
4	P, GM, E, FOX	1 (0.2)	2 (0.5)
	P, GM, FOX, XNL	1 (0.2)
5	P, GM, E, FOX, XNL	3 (0.7)	3 (0.7)
7	P, GM, E, TE, FOX, CF, XNL	4 (1.0)	4 (1.0)
Total		402 (100.0)

CF, cephalothin; CM, chloramphenicol; E, erythromycin; FOX, cefoxitin; GM, gentamicin; P, penicillin; TE, tetracycline; XNL, ceftiofur.

Seventeen MRSA strains from different cows originating from eight farms were further characterized and the results are summarized in Table 3. All the strains showed cefoxitin MIC of ≥8 μg/mL, whereas their MIC for oxacillin ranged from 4 to >16 μg/mL. Three MRSA isolated in 2005 all showed resistance to gentamicin, erythromycin, and tetracycline besides β-lactam antimicrobials. Genotyping of the 17 MRSA strains by MLST and spa and SCCmec typing revealed 2 sequence types (ST1, ST72), 3 spa types (t286, t324, and untypable), and 2 SCCmec types. The majority (11/17, 64.7%) of MRSA strains belonged to SCCmec type IVa-t324-ST72. The rest of strains belonged to SCCmec IVa-t286-ST1 (n = 3) and SCCmec IV-untypable-ST72 (n = 3). None of the isolates carried the PVL gene. Three PFGE types (A–C) were observed among the 17 MRSA strains examined, and 14 strains belonged to the same PFGE pattern regardless of their geographical origin and year of isolation.

Table 3.

Characterization of Methicillin-Resistant Staphylococcus aureus Strains Isolated from Bovine Mastitis During 2003–2009 in Korea (n = 17)

			MIC (μg mL)
Strain ID	Farms	Years	OX	FOX	PVL	PFGE types	SCCmec	Antimicrobial resistance pattern	spa type	MLST
05-GW-544	GW-1	2005	16	16	—	A	IVa	GM, E, TE, XNL	T286	ST1
05-GW-546	GW-1	2005	16	24	—	B	IVa	GM, E, TE, XNL	T286	ST1
05-KG-623	KG-1	2005	32	32	—	B	IVa	GM, E, TE, XNL	T286	ST1
06-KG-2-1578	KG-2	2006	4	16	—	C	IVa	GM, E, XNL	T324	ST72
06-KG-3-1593	KG-3	2006	6	12	—	C	IVa	GM, XNL	T324	ST72
06-KG-3-1586	KG-3	2006	4	12	—	C	IVa	GM, E	T324	ST72
07-CC-1-20	CC-1	2007	4	8	—	C	IVa	—	T324	ST72
07-KB-1-48	KB-1	2007	4	8	—	C	IV	—	UT	ST72
07-KB-1-49	KB-1	2007	4	8	—	C	IV	—	UT	ST72
07-KB-1-50	KB-1	2007	4	8	—	C	IV	—	UT	ST72
08-CC-2-108	CC-2	2008	6	16	—	C	IVa	—	T324	ST72
08-CC-2-109	CC-2	2008	12	16	—	C	IVa	—	T324	ST72
08-CC-2-110	CC-2	2008	16	16	—	C	IVa	—	T324	ST72
08-CC-2-112	CC-2	2008	4	16	—	C	IVa	—	T324	ST72
08-CC-2-113	CC-2	2008	4	8	—	C	IVa	—	T324	ST72
08-CC-2-114	CC-2	2008	4	12	—	C	IVa	—	T324	ST72
08-KG-4-166	KG-4	2008	4	16	—	C	IVa	—	T324	ST72

MIC, minimum inhibitory concentration; MLST, multilocus sequence typing; OX, oxacillin; PFGE, pulsed-field gel electrophoresis; PVL, Panton-Valentine leukocidin; SCCmec, staphylococcal cassette chromosome mec; spa, staphylococcal protein A; UT, untypable.

Discussion

In this study, considerable rates (>66%) of resistance were observed only to penicillin and around 10% to other antimicrobials such as gentamicin, erythromycin, cefoxitin, oxacillin, and tetracycline. Similar to our result, particularly high resistance (>50%) to penicillin but <10% to other antimicrobials was also reported from Korea (Moon et al., 2005, 2007) and other countries such as the United States (Erskine et al., 2002), Finland (Pitkälä et al., 2004), Turkey (Güler et al., 2005), and Iran (Gooraninejad et al., 2007). Much lower proportion of penicillin resistance, which was still the most prevalent in S. aureus isolates from bovine mastitis, was reported from Sweden (7.1%) (Bengtsson et al., 2009), United States (12.8%) (Anderson et al., 2006), Kenya (23.6%) (Shitandi and Sternesjö, 2004), and Japan (27.4%) (Yoshimura et al., 2002). In contrast, there are reports that other antimicrobials, such as erythromycin and tetracycline (China) (Li et al., 2009), streptomycin and polymixin (Ethiopia) (Getahun et al., 2008), amoxicillin (Italy) (Moroni et al., 2006), and dicloxacillin and cefozidim (Mexico) (Ochoa-Zarzosa et al., 2008), showed much lower activity than or equally ineffective to penicillin and in S. aureus isolated from bovine mastitis. Although we found no noticeable increase in antimicrobial resistance of S. aureus isolates from mastitis over the past 7 years, penicillin resistance rates observed in this study are fairly higher than those reported earlier in Korea: <40% (Kang et al., 1995) and 48% (Kang et al., 2001) of S. aureus isolates from bovine mastitis during 1992–1993 and 1999–2000, respectively.

Aside from penicillin, gentamicin resistance was found to be the most prevalent in S. aureus isolates among 19 antimicrobials tested. Gentamicin resistance (11.9%) observed in this study is fairy higher than those from other countries such as the United States (1.1%) (Erskine et al., 2002), Japan (3.9%) (Yoshimura et al., 2002), Finland (0%) (Pitkälä et al., 2004), and Iran (0%) (Gooraninejad et al., 2007). A recent report from China exceptionally showed higher gentamicin resistance than ours (28%) (Li et al., 2009). Our result is also higher than those reported by previous studies from Korea: 3.8% (Kang et al., 1995), 5.3% (Kang et al., 2001), and 9.2% (Moon et al., 2005). Meanwhile, Heo et al. (2008) reported that two of four S. aureus isolated from beef were resistant to gentamicin, suggesting high gentamicin resistance in animals in Korea. This is also a concern to be heeded considering the importance of gentamicin in human medicine.

Methicillin resistance (6.2%) observed in this study was higher than those reported by previous studies on S. aureus isolates from bovine mastitis in Korea: 0.18% (Kwon et al., 2005), 3.1% (Moon et al., 2005), and 2.7% (Moon et al., 2007). Although it is difficult to make comparisons because of differences in methodologies for sampling or detection, our result is also higher than those reported from other countries including Urguay (2.0%) (Gianneechini et al., 2002), United States (0.6%) (Erskine et al., 2002), Switzerland (1.4%) (Huber et al., 2010), and many other European countries (Pitkälä et al., 2004; Güler et al., 2005; Bengtsson et al., 2009), where none of S. aureus showed resistance to oxacillin. Recently, however, higher prevalence of MRSA in bovine mastitis milk was reported from European countries such as Hungary (7.2%) (Juhász-Kaszanyitzky et al., 2007), Belgium (9.3%) (Vanderhaeghen et al., 2010), and Turkey (17.2%) (Türkyilmaz et al., 2010).

SCCmec elements are highly diverse in their structural organization and genetic content and it is common practice to define MRSA clones by the combination of SCCmec type and the chromosomal background such as ST in which SCCmec resides (Enright et al., 2002). In this study, 2 SCCmec types (IV and IVa), 2 sequence types (ST1, ST72), and 3 spa types (t286, t324, and untypable) were revealed by genotyping of the 17 MRSA strains. Eleven of the 17 MRSA originating from five farms belonged to SCCmec type IVa-t324-ST72 with PVL negative, which has been reported to be the most predominant clonal type circulating in the community in South Korea (Kim et al., 2007; Park et al., 2007; Bae et al., 2010; Lee et al., 2010). We also found three MRSA originating from two farms that belonged to SCCmec IVa-t286-ST1, which has been isolated from neonates in hospital in Korea (Ko et al., 2006; Bae et al., 2010). This MRSA clone might also be distributed in the community in Korea and primary obstetrics clinics may be a reservoir for the clone (Bae et al., 2010). SCCmec IV type was observed only in three MRSA strains, which were all isolated in a farm in 2007. This result seems to reflect the fact that type IVa SCCmec is common in CA-MRSA strains in Korea but type IV is not (Park et al., 2007).

Presence of livestock-associated MRSA strains in bovine mastitis have been reported in other countries such as Germany, Belgium, and Switzerland (Monecke et al., 2007; Huber et al., 2010; Vanderhaeghen et al., 2010). HA-MRSA strains in mastitis of dairy cows have been also reported from Japan (Hata et al., 2010) and Turkey (Türkyilmaz et al., 2010). There are a limited number of publications on MRSA infections in dairy cattle with mastitis in Korea, and so far no study reported CA-MRSA clone prevalent in the community in Korea. One study reported a new SCCmec complex subtype IVg MRSA from bovine mastitis milk, but it had no genetic relatedness with CA-MRSA that had caused human infection in Korea (Kwon et al., 2005). Thus, this is the first report of CA-MRSA in dairy cattle with mastitis in Korea. Previous studies have suggested that cattle, horse, and swine farms were significant sources for CA-MRSA (Van Loo et al., 2007; van Rijen et al., 2008). In Korea, HA-MRSA clones of ST5-SCCmec type II and ST5-SCCmec type III were detected from hospitalized dogs and chickens, respectively (Kwon et al., 2006). However, CA-MRSA from animal sources has not been reported until recently when ST72-SCCmec type IVa-PVL–negative strains were first reported from beef and pork (Lim et al., 2010). One reason for this may be due to the fact that few have looked for CA-MRSA in animal isolates.

Our finding suggests that human MRSA strains have entered into dairy herds in Korea. Epidemiological studies on S. aureus and antimicrobial use in Korea have reported that ST72-SCCmec IVa MRSA strains were not frequently isolated until 2005 (Kim et al., 2008), suggesting that IVa strains have emerged recently in Korea. This is supported by our finding that ST72-SCCmec IVa strains were detected only from samples collected since 2005. These strains have been recently shown to be spreading in the community as well as in animals and animal products in Korea (Kim et al., 2007; Park et al., 2007; Lim et al., 2010), posing a major concern in the public health in Korea.

In this study, 3 of 17 (17.6%) MRSA showed multiple resistances to gentamicin, erythromycin, and tetracycline besides resistance to β-lactams. Similarly, Moon et al. (2007) reported that 2 of 21 MRSA were resistant to more than three of the non–β-lactam antimicrobials including gentamicin and erythromycin. Multidrug resistance in MRSA was also reported from Turkey (Türkyilmaz et al., 2010), where all or most of the MRSA isolated from bovine mastitis showed resistance to erythromycin, clindamycin, gentamicin, and tetracycline (Türkyilmaz et al., 2010). However, these findings differ from the result of a previous report from Korea (Kwon et al., 2005), in which none of the MRSA isolated from bovine mastitis milk showed multidrug resistance. These disparities might be partly due to the differences in SCCmec types among the MRSA strains. It is known that CA-MRSA strains characterized by the presence of type IV or V SCCmec elements are susceptible to multiple antibiotics other than β-lactams because they have smaller number of resistance genes compared with HA-MRSA (Francis et al., 2005).

In this study, an identical PFGE type was observed among 14 of 17 MRSA strains originated from six different farms during 2006–2008, and also an identical type of PFGE was found in different individual within a herd. Our data suggest the potential transmission of MRSA strains within a herd and between herds. Especially, all MRSA strains originating from the same farm showed the same spa type, ST, and PFGE except for MRSA strains originating from Farm GW-1. This indicates a clonal spread of MRSA among cows within farms, suggesting that the development and spread of MRSA within farms may have been strongly associated with risk factors at the farms, such as poor management and existence of carriers of the same MRSA strain. Further investigation is needed to elucidate the farm factors and MRSA transmission route, and integrated management of MRSA-positive farms is necessary for prevention of further spread of MRSA.

In conclusion, no noticeable increase in antimicrobial resistance of S. aureus isolates from mastitis was found over the past 7 years. However, the proportion of penicillin resistance and MRSA observed in this study seemed to be higher than those reported by previous studies in Korea. CA-MRSA clone, the most prevalent in the community in Korea, was detected from mastitis milk of dairy cattle, suggesting the transmission of MRSA between dairy cattle and humans.

Footnotes

Acknowledgment

This work was supported by the National Veterinary Research and Quarantine Service, Ministry for Food, Agriculture, Forestry, and Fisheries, Republic of Korea.

Disclosure Statement

No competing financial interests exist.

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