Genetic Determination and Clonal Relationships of Staphylococcus aureus Isolated from Dairy Cows in Baturraden, Central Java, Indonesia

Staphylococcus aureus is a relatively clonal microorganism that causes subclinical and clinical mastitis. Cases of mastitis in cows at Baturraden are leading to signifi cant and ongoing problems due to reduced production and lower milk quality. This study was designed to identify which of selected virulence determinant genes of S. aureus are involved in the Baturraden infection, and to determine the clonal relationship among these isolates. Seventeen isolates were identifi ed as S. aureus based on their biochemical properties and species specifi city for 23S rRNA and nuc genes. S. aureus isolates were genotypically characterized for the selected virulence determinants: coa, clfA, fnbA, fnbB, cap5, spa IgG and spa Xregion genes. Clonal relationship analysis among isolates was carried out using AFLP and results compared with previously confi rmed relationships between selected S. aureus isolated from other regions. The results show that eight isolates contain all the genes, but six isolates lack fnbB and two isolates lack cap5 genes. AFLP analysis showed that all isolates of S. aureus originating from cows in Baturraden belong to one cluster. This study provides additional knowledge about S. aureus infection in Baturraden cows, including the number of virulence determinant genes that may play a role in pathogenicity.


Introduction
Staphylococcus aureus (S. aureus) is a relatively clonal microorganism that causes subclinical and clinical mastitis (Melles et al. 2009;Salasia et al., 2011). This disease causes signifi cant economic losses in dairy farms due to decrease in milk production, shorter lactation times and increased costs for treatment and stock replacement (Halasa et al., 2007;Nielsen, 2009). S. aureus is also known as an important food-borne pathogen which has the ability to produce various virulence factors and a wide range of extracellular proteins that contribute to the pathogenicity of the organism (Nashev et al., 2004).
Cases of subclinical and clinical mastitis of cows are occuring at Baturraden, one of the national dairy cow centres in Indonesia, causing signifi cant and ongoing problems due to decreased production and lower quality of milk (Agus, 1991). S. aureus is recognized as a major pathogen causing subclinical and clinical intramammary infections in dairy cows in Indonesia (Abrar et al., 2012). Therefore, this site provides an excellent opportunity to investigate the characteristics of Indonesian strains of S. aureus. Tato (2012) has proposed that, in order to develop strategies to control S. aureus infection from various countries or areas, we need the ability to map the virulence brought to you by CORE View metadata, citation and similar papers at core.ac.uk provided by Indonesian Journal of Biotechnology determinants of bacteria, as well as the capability of the bacteria to survive the heavy pressure of various antibiotics, geographical conditions, and adaptation to environments. Salasia et al. (2011) reported that molecular typing of S. aureus and determining the clonal relationship between isolates has proved useful for epidemiological studies. Therefore, mapping the virulence determinant genes and clonal relationship patterns of S. aureus isolates from Baturraden could be important in trying to address the problems there.
The present study was designed to identify which of selected virulence determinant genes of S. aureus act in the pathogenicity of the S. aureus infection in Baturraden, and to determine the clonal relationship among these isolates. The results may contribute not only to knowledge of S. aureus in specific locations, but also to potential control strategies for staphylococcal mastitis in the future.

Milk samples and bacterial isolation
Milk samples were collected aseptically by hand milking into sterile 15 ml tubes from seventeen dairy cows from Balai Besar Pembibitan Ternak Unggul (BBPTU) Baturraden, Central Java, Indonesia. Milk samples were placed in an ice box for transport to the Faculty of Veterinary Medicine, Gadjah Mada University, for isolation of strains. The strains were identified as S. aureus by their properties in mannitol salt agar, Gram staining, and biochemical assays, as described by Ariyanti et al. (2011) and Salasia et al. (2011). The strains were then identifi ed genotypically by PCR amplifi cation of the 23S rRNA and thermonuclease nuc genes . The reaction mixture (30 μL) for PCR contained 1 μL of 10 pmol of the forward and reverse primers (Table  1; Invitrogen, USA), 14 μL PCR mix (Roche, Germany) containing Taq DNA polymerase, MgCl 2 , and dNTPs, 5 μL of DNA template, and 9 μL molecular grade water. The DNA of the isolates was prepared with the DNeasy blood and tissue kit (Qiagen, Germany) following the manufacturer's directions. The amplifi cation of the genes was carried out with a thermal cycler (Mastercycler; Eppendorf, Germany); the thermal cycler programs are shown in Table 1.

Isolation of DNA and purifi cation
Chrosomal DNA from S. aureus was isolated by a DNeasy blood and tissue kit (Qiagen, Germany) according to the manufacturer's protocol. The bacterial strains were cultivated on blood agar (Oxoid, Germany) containing 5% defi brinated sheep blood for 24 h at 37 o C . S. aureus colonies (5-10) were suspended with 180 μL TE buffer (10 mM Tris-HCl and 1 mM EDTA [pH 8]) containing 5 μL lysostaphin (1.8 U/μL; Sigma, USA) in 2-mL microfuge tubes. The suspension was incubated for 1 h at 37 o C, and 25 μL of proteinase K (14.8 mg/ mL; Sigma, USA) and 200 μL of AL buffer (containing reagents AL1 and AL2 from the kit; Qiagen, Germany) was then added. The suspensions were incubated for 30 min at 56 o C, and then for 10 min at 95 o C before being spun at 6,000 × g for a few seconds. Ethanol (420 μL) was added to each sample and the mixture placed in a spin DNeasy column. After centrifugation at 6,000 × g for 1 min, the spin columns were placed in a clean collection tube and the sample was washed twice with 500 μL of AW buffer (Qiagen, Germany). After the second wash and centrifugation at 6,000 × g for 3 min, the DNeasy spin columns were placed in a clean 2-mL microfuge tube, and the DNA was eluted twice with 200 μL and 100 μL of AE buffer (Qiagen, Germany). DNA was stored at -20 o C .

Genotype characterization
A PCR method was used to identify the genetic determinants of selected virulence factors. The oligonucleotide primers used amplifi ed the virulence genes encoded by coa, clfA, fnbA, fnbB, cap5, spa IgG and spa X-region genes. The sequences of the primers and PCR conditions are shown in Table 1.

Amplifi ed Fragment Length Polymorphism (AFLP) analysis
The DNA of the isolates was prepared with the DNeasy blood and tissue kit (Qiagen, Germany) as per by the manufacturer's protocol. An AFLP analysis was performed according to Boerema et al. (2006) and Salasia et al. (2011). Genomic DNA (5 μL) was digested overnight (16 h) at 37 o C with 10 U of HindIII (Invitrogen, USA) and 5 mM spermidine trihydrochloride (Sigma, USA) added to a fi nal volume of 20 μL. The 5 μL of digested DNA was added to a ligation reaction containing 0.2 μg of each adapter oligonucleotide (ADH-1 ACG GTA TGC GAC AG and ADH-2 AGC TCT GTC GCA TAC CGT GAG) (Invitrogen, USA) and 1 U of T4 DNA ligase (Invitrogen, USA) in a fi nal volume of 20 μL, and incubated for 4 h at room temperature (approximately 20 o C). The ligated DNA samples were heated for 10 min at 80 o C to inactivate the T4 ligase and then diluted 1 : 5 in sterile water. PCR reactions were performed in a total volume of 50 μL containing 2.5 μL of template DNA, 200 μM of dNTPs (Roche, Germany), 1 μL of HI-X primer (GGT ATG CGA CAG AGC TTX, where X = A, T, G or C; 100 pmol/μL; Invitrogen, USA), and 1 μL (5 U) of Taq DNA polymerase (Roche, Germany) in 1 × PCR buffer provided by the manufacturer (Roche, Germany). Each HI-X primer was used in four separate PCR reactions. Amplifi cation was performed in a thermal cycler (Eppendorf, Germany). After initial denaturation for 4 min at 94 o C, target gene fragments were amplifi ed for 33 cycles. Each cycle consisted of a denaturation step for 1 min at 94 o C, an annealing step for 1 min at 60 o C, and an extension for 2.5 min at 72 o C. An additional base pair was incorporated into each adapter oligonucleotide in the restriction site in order to eliminate it after  the ligation reaction. The PCR products were separated by gel electrophoresis in a 1.5% (w/v) agarose gel (Roth, Germany) in 0.5 × TBE buffer (containing a mixture of Tris base, boric acid and EDTA). A 1-kb Plus DNA ladder (Invitrogen, USA) was used as a size marker. The resulting bands were visualized using ethidium bromide staining under UV transillumination and the gel images were evaluated visually. AFLP patterns were analyzed using NTSYSpc 2.1 software (Applied Biostatistic, New York). Dendrograms were obtained with the average linkage method (unweighted pair group average method, UGPMA) following Boerema et al. (2006) and Salasia et al. (2011).

Results and Discussion
Seventeen isolates were identifi ed as S. aureus (Table 2). All 17 cultures were Gram positive and positive for catalase, coagulase, VP test, and clumping factor reaction on microscope slides (data not shown). Eight isolates contained all the coa, nuc, clfA, fnbA, fnbB, cap5, spa IgG, and spa X-region genes of various sizes, but six isolates lacked fnbB and two isolates lacked cap5 genes ( Table 2).
The proteins encoded by coa, fnbA, clfA and fnbB, spa IgG and the spa X-region contribute to the avoidance mechanism of S. aureus against the host immune system. These genes, except the coa gene, encode adhesin proteins that can bind to fi brinogen, elastin and fibronectin, mainly on the surface of epithelial cells. This binding is an important stage in the process of colonization and infection through the attachment of bacteria to host cells and extracellular matrix. The ability of S. aureus to bind fi brinogen could inhibit the process of opsonisation of S. aureus (Pratomo et al., 2012).
S. aureus produces an enzyme called coagulase encoded by the coa gene. This is a polypeptide that binds to and activates prothrombin, thereby converting fi brinogen to fi brin and promoting clotting of plasma or blood and leading to the formation of abscesses (Cheng et al., 2010). Seventeen isolates in this study contain this gene, with polymorphism in size; two isolates have sizes of 600 bp and the others are 900 bp (Table 2). da Silva and da Silva (2005) and Saei et al. (2009) reported that the coa gene can be used as an epidemiological marker for typing S. aureus isolates.
Amplification of spa IgG and spa X-region genes in the present study showed the same amplicon sizes as the previous study by . However, the current study showed the polymorphism bands of the spa X-region gene for two isolates with sizes of 100 bp and for the other 15 isolates 200 bp ( Table 2). The spa-X region exists in all strains of S. aureus, where X-region indicates tandem repeats with sizes ranging from 24-26 bp in conserved areas in the genome of S. aureus. Protein A, encoded by spa IgG and spa X-region, acts to bind the Fc region of IgG, which has an important role in the phagocytosis mechanism through the classical pathway of complement activation (Atkins et al., 2008).
Capsular polysaccharides of S. aureus also play an important role in the pathogenesis , causing invasive disease (O'Riordan and Lee, 2004) by protecting the bacteria from phagocytosis of leukocytes and facilitating attachment to host epitel and endotel cells. The study shows that 15 S. aureus isolates (88%) have a cap5 gene. This result agrees with the previous fi nding of Salasia et al. (2011), who reported that most S. aureus isolated from cows have cap5 genes.
Amplification of the 23S rRNA, nuc, clfA, fnbA, fnbB, cap5 and spa IgG genes produced amplicons with approximate sizes of 1250, 1050, 300, 1300, 900, 880 and 900 bp, respectively. The coa gene and the spa X-region showed polymorphism bands. The size of the coa gene in two isolates was 600 bp and in the other 15 isolates was 900 bp; similarly the spa X-region gene showed two sizes: 100 bp in the same isolates as they had smaller size for the coa gene and 200 bp in the other isolates ( Figure 1 and Table 2). AFLP was performed using basic PCR combined with a restriction enzyme and a DNA ligase to cut the genome at specifi c sites and ligate it with the appropriate adapter using a restriction enzyme (Janssen et al., 1996). This study used the primer HI-G (ATG GGT AGC TTG CGA CAG) as used by previous investigations (Boerema et al., 2006;Salasia et al., 2011). To compare the results of the AFLP analysis, we used 10 S. aureus isolates from dairy cows in Yogyakarta (SU2, SU10, SU24, SU25 SU16, and SU28), Solo (SU34 and SU39) and Boyolali (BY5 and BY7) that had been previously generated by   Salasia et al. (2011). These data were used to unravel the relationship between isolates from Baturraden and those from the other regions ( Figure 2). The dendrogram in Figure 2 shows that S. aureus isolated from Baturraden group in one cluster, which has a similarity coefficient of 80%. These isolates had a distant relationship with the ones collected from Yogyakarta and Solo, with a similarity coeffi cient of 62%, but the Baturraden isolates had a closer relationship with those from Boyolali, with a similarity coefficient of 78%.
Clonal analysis of AFLP-based patterns of S. aureus originating from cows in Baturraden showed that there were similarities in some isolates, with values of the similarity coefficient of 100% (BR39a, BR40c, BR00b, BR02a, BR67c, BR82a, BR75a and BR38d). The results showed a close relationship between the S. aureus isolates originating from one location (cluster). S. aureus originating from Boyolali (BY5 and BY7), Solo and Yogyakarta showed different coeffi cients similarity, indicating the different origins of the isolates. Salasia et al. (2011) indicated that the clonal relationships between isolates of S. aureus are typically based on the origin of the isolates. Based on genotypic patterns of S. aureus strains, BR49b and BR49c are genotypically different from the other isolates. It is interesting that these S. aureus strains had 100% coeffi cient similarity, the same coa gene size (600bp), and the same spa-X region gene size (100bp). Boerema et al. (2006) and Salasia et al. (2011) suggested that the variations in the characteristics of genotypic patterns of S. aureus are infl uenced by the relationship between the isolates, and that clustering isolates may be partly derived from a single progenitor (ancestor).
Results obtained in this study provide information and additional knowledge about S. aureus infection in Baturraden cows, including the number of virulence determinant genes that may play a role in pathogenicity. We also present clonal relationship patterns among Baturraden isolates that could be important in trying to address the problems there.