Genetically divergent methicillin-resistant Staphylococcus aureus and sec-dependent mastitis of dairy goats in Taiwan

Important pathogenic species of coagulase-positive and negative Staphylococcus (CPS and CNS, respectively) are S. aureus and S. epidermidis, respectively, which are frequently isolated from the environment and infected animals and humans [1, 2]. S. aureus can cause severe blood infections and necrotizing fasciitis in humans, wound infection and mastitis in cattle, horses, pigs, and goats, exudative epidermitis in pigs, pyoderma in horses, dogs, and cats, pyemic sheep [2, 3]. As an opportunistic pathogen, CNS occasionally cause subclinical or clinical mastitis. For example, in cattle mastitis can be caused by S. capitis, S. chromogenes, S. cohnii, S. epidermidis, S. haemolyticus, S. hominis, S. simulans, S. warneri, S. hyicus, and S. caseolyticus; in goats by S. caprae; and in cattle and sheep by S. xylosus [2]. However, CNS can damage breast tissue to increase the somatic cell counts and decrease milk quality and production [4]. The identification of CNS species is essential to determine their pathogenicity and to develop management practices to prevent mastitis. Due to difficult and expensive procedures for identifying these organisms, many laboratories do not perform these assays.

As opportunistic pathogens, S. aureus and S. epidermidis can also cause bacteremia, subacute endocarditis, and can form biofilms on heart valve prostheses, shunts, total joint arthroplasty or surgical sutures in humans [5]. Some S. aureus isolates possess a staphylococcal enterotoxin (SE), which is a virulence factor for foodborne disease and can form several products from SE genes, designates sea - seu [6, 7]. These SE genes are associated with host-specific infection, such as seb for foodborne infection in humans, sea for bovine infection [8], and sec for mastitis in goats and bovine [9]. However, more than 10 SE genes were identified in isolates obtained from goats [7]. As the most prevalent pathogen in both hospitals and communities, S. aureus can become methicillin (oxacillin)-resistant S. aureus (MRSA/ORSA) by the introduction of an exogenous mobile staphylococcal chromosomal cassette, mec (SCCmec), encoding a low-affinity penicillin-binding protein 2a responsible for methicillin/oxacillin resistance [10–14]. Based on variations in the ccr operon and mec complexes, at least seven SCCmec types have been identified [14–16]. Moreover, methicillin-resistant CNS has been identified in animals [17].

The extensive use of antibiotics in hospitals and animals has increased the emergence of MDR Staphylococcus [1]. Since the emergence of MRSA isolated from humans in the 1960s [18], MRSA has been frequently isolated in hospitals, dogs, and horses as a zoonotic pathogen [17, 19, 20]. Our recent study characterized MDR S. aureus from dairy goats in 2006-2007 and no MRSA was isolated [21]. The current study investigated the prevalent Staphylococcus spp. and characterized the MDR-S. aureus and MRSA isolates collected from goats in 2008 by nucleotide sequencing, pulse-field gel electrophoresis (PFGE) analysis and polymerase chain reaction (PCR) analysis to elucidate the origins of MRSA isolates and the SE types associated with mastitis in goats.

CNS strains S. lentus, S. epidermidis, S. xylosus, and S. caprae, and S. aureus were identified as the predominant species infecting goats in this study. S. aureus and CNS differed in prevalence and bodily location in goats (Table 1). Additionally, CNS and S. aureus accounted for 38.2 and 11% of infections, respectively, in the milk of goats with mastitis [23], and 2.6% (35/1,372) and 17.3% (237/1,372), respectively, in the milk of dairy cows and goats in Taiwan [24]. In animals, the prevalence of S. aureus differed among countries, such as 2.2% in the United Kingdom [25], 6.7% in Canada [26], 9.1% in USA [27], 24.2% in Ethiopia [28], 29.6% in Korea [29], and 32.9% in Uruguay [30]. However, S. aureus accounts for 18.7% (64/342) of staphylococcal infections in goats [7]. In Taiwan, S. aureus infection in goats increased from 1.7% (12/706) [24] up to 2.5% (86/3,427) [31] and 4.9% (27/555) in this study. Sanitation may be another important factor that leads to an increase in S. aureus infection. Indeed, sanitation could reduce the S. aureus infection from 6.6 to 0.6% or from 14.6 to 3.0% [32, 33]. In Taiwan, S. aureus infection in milk ranged from 0-5.2% among farms [21]. In the present study, the prevalence ranged from 3.1 to 6.8% for S. aureus and from 11.2 to 31.8% for CNS among farms, suggesting that CNS infection is more common in goats.

Early reports indicated that staphylococcal infections in goat bodily regions differed and ranged from 6.1% (21/342) in the armpits up to 70.8% (242/342) on the skin of udders and mamilla, and contributed to only 0.7% contamination of the apparatus [7]. Although lacking statistical analysis in this study, Staphylococcus was identified in 42.3% (58/137) and 5.8% (8/137) of milk and udders, respectively (Table 1), suggesting that Staphylococcus is a major bacterial cause of mastitis in goats that can then infect humans through unsanitary milk. As an important virulence factor causing foodborne disease in humans, predominant SE genes are associated with outbreaks in certain countries, such as sea in France [34], seb in eastern Slovakia, Tehran, and Japan [35–37], sec-2 and sec-3 in Taiwan [38]. Furthermore, particular sec types is also associated with food-related Staphylococcus spp. [39], in S. aureus, and CNS isolated from the milk of sheep [7, 40], and in S. aureus collected from sheep or cows with mastitis [41]. Additionally, sea was determined to be the major type in S. aureus from cows with mastitis [8]. As shown in Table 3, sec and sea were the major SE genotypes found in goats and cows, respectively, and sec was associated with mastitis in goats. These data confirmed the importance of the sec gene involved in goat mastitis pathogenesis [42]. In cattle, the SEC toxin (not toxic shock syndrome toxin type 1 or TSST-1) was previously shown to significantly increase somatic cell counts and enhance the severity of the mastitis in acute mastitis [43]. Although SE genes have been found in CNS species, such as S. xylosus, S. warneri, and S. chromogenes isolated from cows and goats [7, 40, 44, 45], they were only identified in CPS but not CNS isolated previously in Taiwan [46, 47] and in the present study (Table 3). As an opportunistic pathogen that can cause mastitis in cattle and goats [1, 21, 48, 49], S. aureus infection can also cause clinical symptoms in cattle [50] but is typically asymptomatic in goats [21, 48].

Using penicillin, ampicillin, and tetracycline antibiotics to treat bacterial infections in animals often increases resistance to these antibiotics. In cattle, S. aureus or other Staphylococcus spp. causing mastitis were more resistance to penicillin and ampicillin, streptomycin, tetracycline, and oxytetracycline [1, 24, 49, 51]. Our data showed an increased resistance to penicillin, ampicillin, cloxacillin, and cephalothin from 2006 to 2008, in addition to the appearance of MRSA isolates from goats in 2008 (Table 2). Furthermore, the MRSA isolates identified in this study belonged to the major nosocomial SCCmec types: SCCmec type II and III [52, 53]. Zoonotic transfer of MRSA has been reported between horses and humans in the USA [20, 54], between cattle and humans in Korea [55], and between livestock and humans in Taiwan [56, 57]. Our data indicate that MRSA isolates may have been acquired from humans or transmitted from different goat breeding farms.

PFGE analysis is typically performed to trace the pathogens responsible for outbreaks. Containing a thick cell wall, S. aureus must be treated with lysostaphin, not lysozyme, to break the pentaglycine linkage within the peptidoglycan [58, 59]. For genomic analysis of S. aureus, genomic DNA cannot be digested by restriction enzyme SmaI and diverse genomic variations in size [60] limit the utility of PFGE analysis. However, pulsotypes appear to correlate with human disease. Pulsotype D S. aureus is associated with more severe symptoms than pulsotype type C bacteria that only cause mild symptoms [50]. In Taiwan, MRSA accounts for 53-83% of S. aureus isolates from hospitals [61] and the major pulsotypes of human MRSA are pulsotype A, followed by types C and D [22]. In this study, pulsotype A was also the most prevalent type of goat MRSA isolate (Figure 1, Table 4). Additionally, PFGE analysis also revealed diverse sources of MRSA in Farm A and a single origin in Farms B and D (Table 4).

The current study was the first report of the appearance of MRSA strains and sec-associated mastitis in goats from Taiwan. Analysis of SCCmec types and pulsotypes revealed that the genetically diverse MRSA strains might have been acquired from humans or transferred from different goat breeding farms.

Chishih Chu is a Professor in the Department of Microbiology, Immunology, and Biopharmaceuticals. Changyou Yu is a Professor in the Department of Veterinary Medicine. Yanhaui Lee was a Master student in the Department of Veterinary Medicine. Yaochi Su is an Associate Professor in the Department of Veterinary Medicine, National Chiayi University, Taiwan, Republic of China.