Detection of coagulase gene in Staphylococcus aureus from several dairy farms in East Java, Indonesia, by polymerase chain reaction

Aim: This study was conducted to study the coagulase (coa) gene-based genetic diversity of Staphylococcus aureus, isolated from different samples of cattle from three different regions in East Java Province, Indonesia. Materials and Methods: A total of 160 raw milk samples collected in East Java Province, Indonesia, were screened for the presence of S. aureus. The presumptive isolates were confirmed by coa test. The confirmed S. aureus isolates were subjected to coa gene polymerase chain reaction. Results: Of 160 different samples, 20 (12.5%) isolates of S. aureus were confirmed by positive coa test. Of 20 S. aureus isolates, 19 (95%) isolates carried coa gene. Six different genotypes of coa gene, i.e., 440 bp, 510 bp, 547 bp, 680 bp, 740 bp, and 820 bp were obtained. One coa genotypes, 510 bp (10 isolates) were observed in polymorphism to be more prevalent than the others, and the genotype was present in at least one isolates from every region. Conclusion: It can be concluded that coa gene is easily epidemiological tool for detection of variation strain from S. aureus.


Introduction
Staphylococcus aureus is the most pathogenic bacteria species of the genus Staphylococcus [1]. S. aureus can be isolated from domestic and food animals and associated with disease such as mastitis [2]. S. aureus secretes two clotting factors, coagulase (coa) protein and von Willebrand factor binding protein [3]. coa protein is an important phenotypic determinant and virulence factor of S. aureus [4]. The ability of its coa to clot plasma is a defining property of S. aureus and distinguished the species from other coa-negative staphylococci [5].
Variable genome structure that is associated with strains variant in the certain area shown by S. aureus was known to be responsible for the emergence of different epidemiological profiles [6]. Staphylocoagulase, as the major phenotypic determinant of S. aureus, exists in various allelic forms caused by the genetic variance in its 3'-end coding region [7]. The variations in its 3' region have resulted in the gene to have polymorphic properties which, therefore, the same analysis result in all strains would not be possible [8].
The distinguishing factor of S. aureus coa gene lies in the heterogeneity of the region containing multiple repeated strands with 81 bp length in the 3' region of the gene. Each S. aureus strains have differences in replication number and gene restriction location [9]. Polymerase chain reaction (PCR) amplification results of this region showed different size and number of DNA bands which can be differentiated further using enzyme restriction [7]. The unique property of staphylocoagulase which can be easily analyzed using a simple technique such as PCR amplification and the availability of this enzyme in all strains of S. aureus made coa gene amplification to be the simplest molecular typing method in S. aureus epidemiological study. Using this epidemiological method, coa gene typing is considered a simple and effective method for typing S. aureus isolates from bovine mastitic milk [10]. Epidemiological studies based on analysis of the coa gene have shown that S. aureus isolates could be divided into a number of subtypes, but only a few are responsible for most cases of bovine mastitis in different geographical areas [11].
In Indonesia, especially in East Java, little is known about the genotypic variance and the distribution of S. aureus isolated from raw cow's milk. Therefore, the aim of this study was to detect the genotype variance of S. aureus isolated from raw cow's milk sample in three regions in East Java based on its coa gene by PCR amplification and to understand the strains distribution. Available at www.veterinaryworld.org/Vol.12/January-2019/9.pdf

Ethical approval
Raw milk were used in this study, hence ethical approval was not necessary. Raw milk samples were collected from three regions in East Java province, Indonesia.

Bacterial isolates
A total of 20 S. aureus isolates from raw milk obtained from several farms in three regions such as Pasuruan region for Nongkojajar and Grati farm, Malang region for Batu farm, and Lumajang region for Senduro farm in East Java, Indonesia, were used in this study that shown in Table-1. The isolation and identification were performed for counting bacteria using conventional phenotyping method involved mannitol salt phenol red agar growth (E. Merck, Darmstadt, Germany), Gram staining, microscopic observation, catalase test, and tube coa test [12].

DNA preparation
All S. aureus isolates were subcultured on MSA and incubated at 37°C for 24 h before DNA extraction. The DNA of all S. aureus isolates in this study was extracted using QIAamp ® DNA Mini Kit (QIAGEN, Singapore) and done using the manufacturer method.

PCR amplification of the coa gene
For PCR amplification, a total of 50 µl reaction mixture contained 28 µl Go taq green master mix (Promega, Germany), 20 µl RNase free water, and 1 µl of each forward and reverse primer was prepared. The primer used for coa gene amplification as described by Hookey et al. [13] was 5'ATA GAG ATG CTG GTA CAG G3'and 5'GCT TCC GAT TGT TCG ATG C3'. A total of 2.5µl of DNA template were added to the mixture. The mixture then amplified using PCR cycler according to the protocol of Akineden et al. [14] with modification as following: Predenaturation at 94°C for 45 s, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 58°C for 1 min, and extension at 72°C for 1 min. The amplification ended by a final extension at 72°C for 2 min. The presence of PCR products was determined by electrophoresis of 10 µl of products in 2% agarose gel with TBE buffer and 100 bp DNA ladder as a marker (Promega, Germany).

Results
The 19 S. aureus isolates produced a single band with variance molecular size ranging from 440 bp to 820 bp (Figure-1). One isolate in this study did not produce the band. The 20% of isolates accounted for 4 of 20 produced a single band of 440 bp length. A single band with a molecular size of 510 bp was produced by most (50%) of the isolates. A single band with 547 bp, 680 bp, and 820 bp length was produced by one (5%) isolate, respectively. The 10% (2/20) isolates produced single band with molecular size 740 bp. According to the size of the product, the S. aureus isolates in this study can be grouped into 7 groups (Table-2).

Discussion
In this study, 20 S. aureus strains were subtyped by coa gene PCR and resulted in single amplicon which indicates no size polymorphisms [15]. Amplification of coa gene showed six different PCR products of 440, 510, 547, 680, 740, and 820 bp. The difference of amplification products reflects the variation in coa gene length among S. aureus strains. Former studies were done by other researchers [13][14][15][16][17][18][19][20] using the same primer pairs and also showed that different coa gene types exist. The reason behind this polymorphism is unclear, but it is likely caused by the insertion, or deletion mutations of some portions in 3' end region of coa gene resulted in a change of the gene size and probably also the antigenic properties of the enzyme. This region of the gene may have an important role in   Available at www.veterinaryworld.org/Vol.12/January-2019/9.pdf antigenic variation and its defense some inhibitory mechanism of anti-coa agents [18]. The coa gene PCR amplification of 20 S. aureus isolates revealed 6 coa PCR types. The most prevalence (50%) coa gene type was 510 bp length which found in at least one isolate of S. aureus from every location. Studies [16,18,21,22] from different countries showed that various coa types can be found in S. aureus isolates from milk samples and some of the genotypes were more prevalent. In a previous study [8], it was reported that predominant types of S. aureus could be varied in different areas and they may be more resistant to neutrophil bactericidal activities than that of the rare types, which indicates that they may have different features that help them to survive host immunity mechanism.
Several coa gene types (547 bp, 680 bp, and 820 bp) were infrequent and only found in a particular location and not present in another location. The infrequent genotypes might be less adapted to the mammary gland therefore easily eliminated from the herd and less easily spread [18]. These different and exclusiveness found among the location may be caused by the pathogen coevolution against its host. The pressure of environments, management, animal trading specific to a certain geographic area would lead to the selection of distinct and genetically adaptable strains [8].
The presence of coa genotypes that differ by geographic location and the genotypes that prevailed in each location could be explained by the pathogens-hosts coevolution and the differences in management, nutrition, locations, reservoir bacteria, and environment. The phenomenon of strain homogeneity among some heads in different region also might be explained by the animal trading among regions, the transmission of the strains among herds with close geographic location, the range of discriminatory power of the typing method used, and hosts adaptation to S. aureus strains that is present in the environment [18]. The finding of the same genotype among distant location farm could be explained by the interregional herd movement; pathogen spread with human as a carrier, or the homoplasy phenomenon that is the independent acquisition of similar structures without commons ancestors [23].
One isolates analyzed in this study found to produce no amplification products. The isolate showed positive results in coa test tube and based on other identification test results phenotypically identified as S. aureus. This contradiction results between traditional and molecular method also reported in a former study [24] where 10 strains that classified as coa negative by coa test tube were found to be positive by a molecular method using PCR. The findings emphasize the use of molecular methods in the identification and detection of S. aureus [24,25].
The PCR amplification results of S. aureus isolated from cow milk from several herds in East Java, Indonesia, showed the genotype variance of S. aureus based on its coa gene polymorphism properties. The results also revealed that the coa gene types of S. aureus strains circulated among herds in the area were varied by 7 types. Among those types, one genotype (510 bp) was predominant, and three genotypes (547 bp, 680 bp, and 820 bp) were unique to particular herds and infrequent. These research results of coa gene amplification by PCR are very useful and relatively simple method for S. aureus genotyping, and further studies using RFLP technique and sequencing methods on large strains collection from various sources could provide a complete picture of a characteristic of S. aureus circulated in the area as well as the epidemiology pattern.

Conclusion
This study has shown that based on its coa gene polymorphism the strains of S. aureus that contaminated milk from several dairy farms in three locations in East Java consists of at least 6 types with one genotype predominant strain. It can be concluded that coa gene is easily epidemiological tool for detection of variation strain from S. aureus. Further researches using RFLP technique and sequencing method on various origin strains might be necessary to understand the epidemiology profile of contamination and infection.