Investigation of Methicillin Resistance and Panton-Valentine Leukocidin in Staphylococci Isolated from Bovine Mastitis *

Background: Mastitis, which is inflammation of the mammary gland, is among the most important diseases in dairy herds resulting in reductions of milk yield and milk quality. Although several groups of microorganisms have been reported as etiological agents of mastitis, Staphylococci are the most frequently isolated bacteria from bovine mastitic milk samples. The aim of this study was to isolate the Staphylococcus species from bovine mastitis, investigate the mecA ve pvl genes in isolated species by PCR and determine the antibiotic resistance of methicillin resistant strains to some antibiotics commonly used in veterinary field. Materials, Methods & Results: In the present study, 972 half-udder milk samples (n = 757 CMT positive, n = 215 CMT negative) were used from 251 lactating cows from 34 different enterprises located center town and villages of Ödemiş, İzmir. Ten microliters of each milk sample was inoculated onto Columbia blood agar, containing 7% of sheep blood and incubated under aerobic conditions for 24-48 h at 37°C. The certain identification of Staphylococcus isolates was achieved using CrystalTM Identification Systems Gram-Positive ID kit. Bacterial DNAs were extracted from all strains using boiling method and strains were screened for the presence of 16S rDNA, mecA and pvl genes by PCR. The antimicrobial resistance of MRS species was determined by using disc diffusion method. A total of 182 (18.72%) Staphylococcus strains were isolated from 972 half-udder milk samples. Of 182 Staphylococcus strains, 137 (75.27%) and 45 (24.73%) were detected as CPS and CNS, respectively. Among the 11 different Staphylococcus species, S. intermedius (42.30%) was the most common species isolated, followed by S. aureus (32.97%) and S. saprophyticus (10.99%). The mecA positivity was found in only 4 (2.2%) S. intermedius strains, while pvl toxin gene was determined in none of the strains. Four MR S. intermedius strains were resistant to oxacillin and cefoxitin. The resistance was also found to erythromycin (50%), rifampicin (25%), gentamicin (25%), tetracycline (25%) and trimethoprim/sulfamethoxazole (25%) in the isolates. Discussion: In this study, S. intermedius had the highest isolation rate and this finding was considered remarkable. Generally, in mastitis diagnostics all CPS isolates are classified as S. aureus. In our study, the certain identification of all CPS may explain the high isolation rate of S. intermedius. The sampling method may also be the reason of higher isolation rate for S. intermedius in accordance with the most common ones causing mastitis. All of mecA positive strains were S. intermedius and this was the another remarkable finding of our study. Because similar result was seen in only one study from Korea, while the investigation finding related to MR S. intermedius was not determined in Turkey. However, the mecA positivity found in our study was lower than the other author’s isolation rate. The difference between the sample size, geographical variations and diversity in strains may be the causes of this discrepancy. It was investigated pvl toxin gene in 182 Staphylococcus strains by PCR and found this gene in none of strains. According to this finding, it was considered that pvl gene may have not an efficient role in the pathogenesis of mastitis in terms of sampled animals and sampling area. Antibiotic resistance of 4 MR S. intermedius strains against various antibiotics commonly used in Turkey was investigated. Of methicillin resistant strains, 2, 1 and 1 were resistant to 3, 6 and 5 antibiotics, respectively. It was considered that geographical differences, number of tested isolates and diversity in MR strains may be effective on the antibiotic resistance levels. This is the first study showing the presence of mecA gene in S. intermedius strains isolated from bovine mastitic milk samples in Turkey.


INTRODUCTION
Mastitis, which is defined as an inflammatory reaction of mammary gland, continues to be prominent problem affecting the animal and public health in dairy industry [2,10,12].Among several groups of microorganisms playing role in the etiology of mastitis, Staphylococci are the most frequently isolated bacteria from bovine mastitic milk samples [9,21,24,32].Methicillin resistant Staphylococci (MRS) have been considered as an emerging problem in veterinary medicine in recent years [22,29,33].Methicillin resistance is mediated by the mecA gene located on the chromosome of MRS.MRS are often resistant to antimicrobials both all β-lactams and other than β-lactams are widely used in both human and veterinary medicine [11,25].Panton-Valentine leukocidin (PVL) is a cytotoxin believed by many researchers to be responsible for the severe symptoms of Staphylococcus aureus infections [17,18].However, the pathogenic role of PVL in the pathogenesis of mastitis is still controversial [15,34,38].
In Turkey, the studies investigating the presence of mecA and pvl toxin genes in Staphylococci isolated from mastitic milk samples are limited [7,34].Therefore, this study was aimed to isolate the Staphylococcus species from bovine mastitis, investigate the mecA ve pvl genes in isolated species by Polymerase Chain Reaction (PCR) and determine the antibiotic resistance of methicillin resistant strains to some antibiotics commonly used in veterinary field.To our knowledge, this is the first study investigating the presence of mecA and pvl genes in the Staphylococci isolated from quarter milk samples in İzmir province of Turkey.

Milk samples and bacteriological identification
A total of 972 half-udder milk samples were collected from 251 lactating cows on 34 different private enterprises in the center town and villages of Ödemiş, located in the İzmir province of Turkey.No antibiotics had been applied to the animals in the previous three months.Firstly, California mastitis test (CMT) was applied for each mammary quarter according to the method described by Schalm et al. [28] and scores were evaluated as +1, +2, +3 and negative.Before sampling for bacteriologic examination, the teat ends were cleaned by 70% alcohol and dried.The first streams of foremilk were discharged, and then 10 mL of milk was collected aseptically from each udder half into sterile vials.Samples were immediately transported to the laboratory in a cool box on ice.Ten µL of each milk sample was inoculated onto Columbia blood agar 1 , containing 7% of sheep blood and incubated under aerobic conditions for 24-48 h at 37°C.The infection status of milk samples were determined according to the procedures recommended by the National Mastitis Council [23].Intramammary infection was defined when ≥ 500 cfu/mL colonies were found.Each different colony was examined macroscopically (colony morphology, haemolysis, pigment production) and microscopically (Gram staining).Then oxidase, catalase, slide and tube coagulase and anaerobic fermentations of glucose and mannitol tests were performed to suspected colonies [13].The certain identification of Staphylococcus isolates was achieved using Crystal™ Identification Systems Gram-Positive ID kit 2 .In all tests, methicillin resistant S. aureus (MRSA) ATCC ® 33591 and methicillin sensitive S. aureus (MSSA) ATCC ® 25923 were used as control strains 1 .All isolates identified were stored at -20°C in brain heart infusion broth containing 15% glycerol until further analysis.
Detection of 16S rDNA, mecA and pvl genes in Staphylococcus spp.by PCR DNA was extracted from the positive controls 1 (MRSA ATCC ® 33591 and PVL S. aureus ATCC ® 49775), negative control 1 (MSSA ATCC ® 25923) and all test strains using boiling method.For this purpose, all strains were inoculated onto Trypticase Soy Agar 1 .After the incubation, fresh colonies were suspended in 500 µL of DEPC-treated water (DNase-RNase free).The suspension was held in a 100ºC of water bath for 10 min.After centrifugation at 9,167 g for 5 min, the supernatant containing bacterial DNA was used as a template for subsequent PCR mixture [39].

Isolation and identification findings
A total of 182 (18.72%)Staphylococcus spp.were identified from 972 half-udder milk samples (757 CMT positive, 215 CMT negative) belong to 251 lactating cows by using Crystal™ Identification Systems Gram-Positive ID kit.It was determined that 120 (47.8%) of 251 cows were infected with the least one Staphyloccus species.Of 182 Staphylococcus strains, 137 (75.27%) were found as coagulase-positive Staphylococci (CPS) and 45 (24.73%) as coagulasenegative Staphylococci (CNS).A total of 11 different species were identified in this study.The most frequently isolated species were Staphylococcus intermedius (42.30%), followed by S. aureus (32.97%) and Staphylococcus saprophyticus (10.99%).The identified species and their isolation rates were shown in Table 1.Also, 122 (16.11%) and 60 (27.90%)Staphylococcus species were isolated from 757 CMT positive and 215 CMT negative milk samples, respectively.The distribution of species according to CMT scores was shown in Table 2.  16S rDNA specific bands were seen in all of 182 Staphylococcus isolates obtained from half-udder milk samples, 4 (2.2%) of these harboured the mecA gene.It was also detected all of mecA positive strains were S. intermedius.Thus, the mecA positivity was found to be 5.19% in 77 S. intermedius isolates.Two of methicillin resistant S. intermedius strains were obtained from the same enterprises.In our study, none of the isolates had pvl toxin gene.Amplification of 16S rDNA (420 bp) and mecA (314 bp) genes in S. intermedius strains was shown in Figure 1.

DISCUSSION
The present study investigated the presence of mecA and pvl genes in Staphylococcus spp.isolated from bovine mastitic milk samples by PCR and the antibiotic resistance of methicillin resistant strains to antibiotics commonly used in veterinary field.
Staphylococci are the most common bacterial agents isolated from bovine with mastitis.However, the distribution of species obtained from the studies related to the etiology of Staphylococcal mastitis have shown the variation [14,24,36].Vishnupriya et al. [36] reported that 22 and 74 of 96 Staphylococcus spp.isolated from 158 CMT positive milk samples were CPS and CNS, respectively.In the same study, the most frequently isolated species were found to be S. aureus (n = 20) and Staphylococcus epidermidis (n = 17).In a study, it was reported that 113 (71.5%)Staphylococcus spp.were isolated from 158 CMT positive milk samples, of these 5 and 108 were found to be S. aureus and CNS, respectively.It was also emphasized Staphylococcus haemolyticus and Staphylococcus chromogenes were the most frequently isolated species among the CNS [14].Pehlivanoğlu & Yardımcı [24] from Turkey reported the highest isolation rate belonged to S. aureus (n = 65) and S. intermedius (n = 17) among the 100 Staphylococcus strains isolated from CMT positive half-udder milk samples.In our study, of 182 Staphylococcus isolates obtained from 972 mammary quarter milk samples, 137 (75.27%) were determined to be CPS and 45 (24.73%) to be CNS.Among the 11 different Staphylococcus species, S. intermedius (n = 77; 42.30%) was the most common species isolated, followed by S. aureus (n = 60; 32.97%) and S. saprophyticus (n = 20; 10.99%) (Table 1).Compared with the other investigation results [14,24,36], while the similarity in terms of isolated species was found, the differences were detected in the isolation rates of species.In the present study, the high isolation rate of S. intermedius was remarkable for us.Taponen [31] has emphasized that in mastitis diagnostics all CPS isolates are usually classified as S. aureus and all other isolates as CNS.In our study, the identification of all CPS was achieved by using identification kit.This certain identification may explain the high isolation rate of S. intermedius.Unlike other studies, we also made the bacterial isolation from both CMT positive and CMT negative milk samples.It was considered this sampling method may be the reason of higher isolation rate for S. intermedius in accordance with the most common ones causing mastitis.In the present study, S. saprophyticus was the predominant CNS species and this was another remarkable finding of our study.Taponen [31] has reported that S. saprophyticus may be found in the cows' environment.However, some researchers emphasized that the increase was determined in the isolation rate of this agent from animals with mastitis [3,37].
The California mastitis test has been accepted as a quick and simple screening test to predict somatic cell count from individual quarters or composite milk.However, microbiological growth has been shown in CMT negative mammary quarters as well as CMT positive ones [2].Bhutto et al. [2] detected the bacteriological growth in the 57.6% of CMT negative half-udder milk samples, while bacteriological isolation from CMT negative milk samples was found to be 30.1% in another study [1].We collected 972 half-udder milk samples, of which was 757 CMT positive and 215 CMT negative.The isolation rate of Staphylococcal agents from CMT negative half-udder milk samples was found to be 32.97%, while this rate was 16.11% in CMT positive ones (Table 2).According to some authors, the sensitivity and specificity of CMT are unsteady and also sensitivity of CMT may be low for different pathogens [26,27].
Methicillin resistance is mediated by the mecA gene located on the chromosome of MRS [25].Various researchers have reported the different mecA prevalence in Staphylococci, especially S. aureus, isolated from bovine mastitic milk samples [5,7,8,19,22,24].In a study from Germany, 15 (12.39%) of 121 CNS isolates were determined to be mecA positive [8].Moon et al. [22] found the mecA positivity in 13 and 12 of 835 S. aureus and 763 CNS strains, respectively, while the mecA positivity was found to be 9.2% (n = 15) in 163 S. aureus strains in another study [19].Similarly, the investigation results related to the presence of mecA in Staphylococci are variable in Turkey.In a study, methicillin resistance was found in 20 of 100 Staphylococcus strains [24], while Erdem & Türkyılmaz [7] determined mecA positivity in only two of S. aureus strains isolated from 145 mastitic milk samples.In another investigation, only 4 (6.7%) of 59 S. aureus isolates were found to be MRSA [5].In our study, 16S rDNA specific bands were detected in all of 182 Staphylococcus isolates obtained from half-udder milk samples, 4 (2.2%) of these harboured the mecA gene.All of mecA positive strains were S. intermedius and this result was remarkable for us.When the comprehensive literature investigation was made, the report on MR S. intermedius was seen in only one research from Korea.In this study, Moon et al. [22] reported that only one of 20 MRS strains was S. intermedius.In Turkey, the investigation finding related to MR S. intermedius was not determined.It was considered that our different result may be associated with certain identification of S. intermedius, while CPS isolates are usually classified as S. aureus in several reports [31].The mecA positivity found in our study was lower than the prevalence of mecA gene reported by other authors.The difference between the sample size, geographical variations and diversity in strains may be the causes of this discrepancy.It was also considered the specific and controlled antibiotic usage for the treatment of mastitis in sampling area may be effective on this result.When we conferred with veterinarians working in sampling area, they emphasized that the suitable, controlled and synergical effective antibiotics are usually used according to antibiotic susceptibility test results in Ödemiş.
One of the several virulence factors that are produced by S. aureus is PVL and may contribute to its pathogenicity.Generally, PVL toxin causing pneumonia and necrotizing dermatitis are more widely in human population [17,18].The pvl gene encoding this toxin has been reported in 0-56% of S. aureus strains isolated from animals with mastitis [15,33,34,38].Zecconi et al. [38] determined the pvl gene positivity as 56% in S. aureus strains.In another study, the pvl gene was found in none of 76 S. aureus strains isolated bovine mastitic milk samples [15].In Turkey, Ünal [34] reported the prevalence of pvl toxin gene to be 6.6% in S. aureus isolates, while this gene was determined in none of 16 MRSA strains by Türkyılmaz et al. [33].We investigated pvl toxin gene in 182 Staphylococcus strains by PCR and found this gene in none of strains.According to this finding, it was considered that pvl gene may have not an efficient role in the pathogenesis of mastitis in terms of sampled animals and sampling area.
Methicillin resistant Staphylococci species have been recognized to be resistant to antimicrobials both all β-lactams and other than β-lactams are widely used in both human and veterinary medicine.Various researchers have emphasized that this problem causes an increase in the risk of treatment failure and cost for antimicrobial therapy and hospitalization, while the range of therapeutic options decrease [20,25].In several studies related to the antibiotic resistance in MRSA strains isolated from animals with mastitis, the multiple antibiotic resistance have usually emphasized in strains [16,33,35].We investigated the antibiotic resistance of 4 MR S. intermedius strains against various antibiotics commonly used in Turkey.All of MR S. intermedius strains were resistant to oxacillin and cefoxitin, while

Table 1 .
Identified Staphylococcus species from dairy cow quarter milk samples and their isolation rates.

Table 2 .
Distribution of Staphylococcus species isolated from dairy cow quarter milk samples according to CMT scores.