Coagulase Gene Polymorphism of Staphylococcus Aureus Isolates Collected From Human Clinical Samples in Southeastern Iran

The high risk of staphylococcal infections in pediatric, surgical, chemotherapy and ICU wards is indicative of the need for research in regard to bacterial placement in the skin and various organs of healthy individuals and patients according to the method of transmission of a bacterium (1). Given the placement of bacteria in the nose and skin of healthy people, staphylococcal infections can spread due to direct contact between hospital staff and patients in hospitals (2). Prevention and control of Staphylococcus aureus infections are initially dependent on the risk of infection (3). In addition, the analysis of isolates by means of separable DNA typing methods and understanding the transmission of bacterial infections is one of the factors contributing to the prevention and control of S. aureus infections (4). As approaches for detecting the origin of infection, previously conducted research has shown that isolated genotypic characteristics can work properly to find the source of infection as a step forward in controlling the infection (5). The coa gene, which codes for coagulase, is polymorphismic. Coagulase is an important criterion for identifying S. aureus isolates (6). Although pulsed field gel electrophoresis (PFGE) is used as a standard method for genotyping bacteria, the addition of coa gene has more advantages than the mentioned method due to lower cost, not needing specific skill and expertise, high speed and high repeatability; it, additionally, is used more frequently in researches (7,8). In Iran, especially Zabol, southeastern Iran, there is no information on the genetic differences between S. aureus isolates collected from different hospitals and patients with skin lesions and urine samples, especially on the basis of coa gene polymorphism.

collected clinically from 2 hospitals in Zabol (Imam Khomeini and Amiralmomenin) using coa gene and compare them with other regions of Iran. In addition, in this study, the coa gene polymorphism in S. aureus isolates collected from skin lesions and urine samples was investigated using restriction fragment length polymorphism (RFLP) method.

Materials and Methods
Bacterial Isolates In this study, 120 samples were collected from skin lesions and urine samples belonging to patients hospitalized in Zabol from May to November 2014. The samples were then transferred to the Microbiology Department of the Medical School of Zabol University of Medical Sciences and were identified as S. aureus by different biochemical tests such as gram staining, catalase, coagulase, DNase and mannitol fermentation.
All S. aureus isolates were DNase and coagulase positive and fermented mannitol. S. aureus ATCC 25923 and S. epidermidis ATCC 12228 were used as positive and negative controls respectively. Genomic DNA was extracted by boiling method and polymerase chain reaction (PCR) was performed on coa gene.

DNA Extraction
All isolates were swabbed on Trypticase Soy Agar (TSA) (BD, Germany), while the surface of the agar medium was covered with standard vancomycin disks and incubated overnight. The bacterial colonies from the edges of the inhibition zone were then resuspended in sterile distilled water and matched to 0.5 McFarland standards (approximately 10 8 CFU/mL). The bacterial suspension was heated at 95°C for 15 minutes and cooled at room temperature. The cured lysis (2 μL) was used as a DNA template for all isolates when PCR tests were carried out (9).
Polymerase Chain Reaction PCR of the coa gene was carried out using primers forward: ATA GAG ATG CTG GTA CAG G and reverse: GCT TCC GAT TGT TCG ATG C. Reactions were prepared in a final volume of 15 μL, including 2 μL of pattern DNA, 7.5 μL of 2×MasterMix, 1 μL of each primer and 3.5 μL of distilled water. The amplification program included an initial denaturation step of 3 minutes at 94°C followed by 30 cycles of 20 seconds at 94°C, 15 seconds at 55°C and 15 seconds at 72°C and a final extension step of 2 minutes at 72°C.

Restriction Enzyme Digestion
Generally, 10 μL of PCR product was incubated with 1 μL of Alu1 endonuclease (Fermentas, USA), 2 μL of restriction buffer and 17 μL of distilled water for 16 hours at 37°C. Agarose Gel Electrophoresis Digested fragments and PCR products were separated in 2% agarose gels (Gibco, USA). A 100-bp ladder (Fermentas, USA) was used as a standard molecular marker for the calculation of the sizes of the coa and Alu1generated coa fragments. Gels were visualized under UV light after staining with ethidium bromide.
The data were analyzed using chi-square and Fisher exact test in SPSS 16.0, with a significance level of 0.05.

Results
A total of 120 clinical samples from 2 major hospitals were used in this study. Fifty-six specimens (46.6%) from these samples were identified as infected with S. aureus in microbiological studies. From the total of 56 S. aureus isolated in this study, 30 (53.5%) and 26 (46.4%) isolates recovered from skin lesions and urine samples, respectively.
RFLP was used to determine the polymorphism of the coagulase gene from all the isolates of S. aureus collected in this study. Five different sizes, ranging from approximately 600 to 850 bp were observed. Twentytwo isolates contained 700 bp and 14, 8, 6 and 6 isolates contained 650 bp, 600 bp, 800 bp and 850 bp fragment relevant to coa gene in the PCR (Figure 1).
Among the isolates recovered from skin lesions, PCR product of 650 bp was the most common one seen in 10 isolates, followed by 700 bp (nine isolates), 600 bp (five isolates), 800 and 850 bp (each one with 3 isolates). After enzymatic digestion with AluI in the PCR, 4 types of coa patterns ranging from 100 to 400 bp were observed. These types were designated as P1-P4 and the P1 type was the most predominantly noticed product sizes, accounting altogether for 35.71% of the total coa-positive isolates. As shown in Figure 2, the isolates with patterns P1 and P2 had DNA fragments of 250 and 400 bp, 250 and 350 bp, respectively. AluI digestion of the PCR products also yielded 3 PCR fragments in P3 type and 1 PCR fragment in P4 type. As one can clearly observe in Figure 2, 20 isolates with 2 PCR fragments of 250 and 400 bp (P1 type), 14 isolates with 2 fragments of 250 and 350 bp (P2 type), 19 isolates with 3 PCR fragments of 100, 220 and 380 bp (P3 type) and 3 isolates with 1 PCR fragment of 220 bp genotype (P4 type) were noticed product sizes in all isolates ( Figure  2 and Table 1).
Of 56 S. aureus isolates, type P1 was the most common type and was seen in 35.71% of the isolate and P4 type was seen in 5.35% of the isolates.
In isolates recovered from skin lesions, P1 type with 13 isolates (43.33%) was the most frequent and the lowest frequency belonged to P2 type with 8 isolates (26.66%). P4 type was not observed in any isolates of skin lesions. P3 type predominated in 10 isolates of urine sample (38.46%) and P4 type was observed only in 3 isolates (11.53%) of urine samples (Table 1).

Discussion
The coa gene in S. aureus isolates has various numbers of degenerate repeats, which are clearly polymorphic in both number and sequence (10). Therefore, the coa typing method has been reported to be a rapid, inexpensive and appropriate method for genotyping S. aureus strains in epidemiological studies (11).
In our study, PCR amplification of the coa gene resulted in the identification of five different amplicons (600, 650, 700, 800 and 850 bp) and an amplicon with 700 bp length as the most frequent type. The polymorphism of this gene is due to repetitions of 3' elements of the  coa gene in various strains (12). Previously published data from Iran have shown the presence of different coa types (13,14). Talebi-Satlou et al conducted a similar study on S. aureus isolates associated with skin and urinary tract infections in Urmia, Iran, and showed four coa types with 410, 530, 700 and 790 bp length (13). They also reported the coa type with 700 bp as the most common type, which is consistent with our results. However, in contrast to their study that determined 2 RFLP patterns for the dominant coa type, we could classify the predominant coa type in four subtypes (P1-P4), which indicate great heterogeneity among our isolates. It is noteworthy that the coa type with 700 bp length also was reported as a dominant type in another study from Iran (15).  (19). The variation of the PCR products found by the amplification of the 3´end of coa gene reflects variations in sequence of the coa gene of S. aureus isolates. The presence of 5 different sizes in PCR products can indicate the variation in the repeat sequence of 81 bp in the isolates of the present study. This diversity is likely to be related to the presence of immigrants from neighboring countries in Zabol, southeastern Iran. A similar study by other researchers confirms the variation in the base pair size of the coa gene (20,21). Probably, the elimination or replacement of nucleotides within the coa gene will result in such a variety, which may also change the antigenic properties of the enzyme at the 3´end of coa gene (22). This is probably one of the reasons for the antigenic diversity and the persistence of S. aureus infections. The coa gene at 3´end sequence contains a sequence of repetitive element with 81 bp in length, varying among different strains (8). In some studies, the size difference of 10 to 20 bp for the PCR products of the coa gene has been proposed (17). The results of this study showed that the 650 bp and 700 bp were predominant in the isolates of skin lesions and urine samples respectively, indicating that some genotypes are unique for a particular site of infection. Considering this finding, it may be suggested that a specific subset of S. aureus strain is well-adapted in various parts of human body in different region of Iran. However, expanded genetic analyses are necessary to generate more evidence for this finding. Based on the results of a study conducted on coa gene polymorphism of S. aureus isolates collected from buffalo milk, four PCR products of 600, 700, 760 and 850 bp were observed, 3 of which were similar to the results of the present research (22). In a study which was conducted on 21 of S. aureus isolates recovered from cow's breast inflammation by Sanjiv et al, in India, 3 PCR products of 600, 680 and 850 bp (8) were found, 2 of which were similar to those of the current study.
This distribution might be explained by the coevolution of the hosts and the pathogens, and also differences in reservoirs and imply that the successful transfer of bacteria between bovine mastitis milk, raw meat and human is not a frequent occurrence (20,23).
These characteristics show the necessity of comprehensive studies on epidemiological and ecological profiles of a specific origin before applying control programs.
The results of the present study showed that coa typing can be used along with other molecular methods as an appropriate method in epidemiological researches to control and monitor hospitals and community-acquired infections, in distinguishing S. aureus isolates collected from clinical samples.

Ethical Approval
We hereby declare that all ethical standards have been respected in the preparation of the article.