Introduction

Staphylococcus aureus infection is a critical threat that can induce a wide variety of diseases for humans; it could acquire and produce a wide variety of virulence elements and able to gain anti-microbial resistance factors. The mobile genetic components of S. aureus are implicated in the virulence dissemination, resistance to anti-microbial genes that include pathogenicity islands, plasmids, transposons, resistance to bacteriophages, and chromosome cassettes were reported (Hanssen & Sollid, 2006; Holtfreter et al., 2007). MRSA is considered as a significant nosocomial infection all over the world. MRSA can be considered the most significant problem for both hospitals and public peoples.

The mecA gene of S. aureus contained inside the mobile SCC element, named SCCmec (Hiramatsu, Cui, Kuroda, & Ito, 2001). The ultimate basis of β-lactam resistance S. aureus is PBP2A protein. PBP has low affinity to β-lactams, and sustain transpeptidation activity due to the presence of these antibiotics in lethal dose (Hartman & Tomasz, 1984). mecA encodes PBP2A protein, a gene that placed exclusively in the mobile genetic element for SCCmec (Beck, Berger-Bächi, & Kayser, 1986). SCCmec types are variable in size range between 21 and 67 kb. Hospital acquired-MRSA strains (HA-MRSA) carry varies types of SCCmec genes (I, II and III), while community acquired-MRSA (CA-MRSA) strains had SCCmec genes of types IV and V (Batabyal, Kundu, & Biswas, 2012; Wu, Li, Liu, Xue, & Zhao, 2015). mec gene complex is possessed in all strains of MRSA (Shore & Coleman, 2013). mecA gene and its regulatory genes mecI and mecR1 are contained in the mec gene complex, depending on types of SCCmec (Deurenberg et al., 2007). Type I, II, and III of SCCmec genes were recognized and detected in HA-MRSA isolates, while IV type was detected in CA-MRSA strains (Sousa & Lencastre, 2003) and hospital-(some) associated MRSA (Ito et al., 2004).

The chromosome cascade of CA-MRSA contained SCCmec type V and was recognized in Australia (Ma et al., 2002). Using PCR and molecular studies, HA-MRSA infections all over the world is due to the epidemiology of MRSA, which can be detected by Pulsed-field gel electrophoresis (PFGE) (Murchan et al., 2003).

Coagulase factor is considered one of the main phenotypic is determining factors of S. aureus, which occurs in numerous allelic forms due to the genetic variation in its 3’-end coding region (Goh, Byrne, Zhang, & Chow, 1992). These varia­tions resulted in polymorphic properties among strains (Sanjiv, Kataria, Sharma, & Singh, 2008). The characteristic factor of S. aureus coagulase gene lies in the region that containing multiple repeated strands with 81bp length in the 3' region of the coagulase gene. These repeats number and gene restriction location differ between S. aureus strains (Omar, Ali, Harfoush, & Khayat, 2014). The epidemiological studies focused on the analysis of the Coa gene have revealed that S. aureus isolates could be divided into several subtypes (Javid et al., 2018). The current study aimed to recognize and confirm the distribution of SCCmec genes in clinical MRSA isolates collected from Taif hospitals (Saudi Arabia) using molecular PCR technology and study the correlation between SCCmec and Coa gene repeats.

Materials and Methods

Sampling

Phenotypic methods used for isolation and confirmation of MRSA. Thirty-three MRSA strains were kindly obtained from King Faisal hospital, KSA, from different clinical sources. The isolates were obtained from blood, wound swap, catheter, and swap from September 2019 till January 2020. Identification of the clinical isolates as MRSA was determined by oxacillin and cefoxitin (5 μg and 30μg) discs using BD Phoenix™Automated Microbiology System, regarding CLSI (Clinical and Laboratory Standards Institute).

Anti-microbial susceptibility and MIC tests

Established Phoenix™Automated Microbiology System determined the antibiotic profile of MRSA clinical isolates. Mueller-Hinton agar (Oxoid Limited, Basingstoke, Hampshire, UK) was used for disk diffusion test with the following antibiotic discs: Amoxicillin-clavulanate, Ampicillin, Cefotaxime, Cefoxitin, Ciprofloxacin, Imipenem, Erythromycin, Gentamycin Oxacillin, Penicillin G, Trimethoprim-Sulphamethoxazole and Vancomycin, in a concentration of 25, 10, 30, 30, 5, 10, 15, 10, 5, 6 μg, (1.25 + 23.75 mg) and 30 μg respectively for each antibiotic.

DNA extraction of MRSA strains

Colonies of S. aureus MRSA were cultivated on trypticase soy broth and incubate at 37°C for 24 hrs. For prompt DNA extraction, 50-100 colonies were dissolved in 500 µL of DEPC water and boiled for 8 minutes. The mixture was centrifuged at 14000 rpm for 15 minutes. The supernatant was taken for a polymerase chain reaction.

Molecular Characterization of SCCmec genes and coagulase specific repeats using PCR technology

Extracted DNA was measured and used for PCR amplification on a Bio-Rad light cycler PCR machine. Specific primers for mec genes (mec specific genes type I, II, III, Iva, IVb, IVc, IVd, V and mecA 147) and coagulase repeats were designed as shown in Table 1, using specific Taqman primer designer program. Primers were manufactured by Macrogen (Macrogen Company, GAsa-dong, Geumcheon-gu. Korea). PCR was prepared in a volume of 25 µl, 5 µl DNA template, 10 pM of forward and reverse primer (1 µl), and 12.5 µl master mix. Using sterilized deionized water, the volume was adjusted. The polymerase chain reaction was done in the Thermal Cycle machine (Bio-Rad T100TM). DNA was rapidly extracted from bacterial suspension according to the extraction method. The PCR cycle sequence was 95°C for 5 minutes one cycle. Followed by 40 cycles, each starts with denaturation (94°C for 50 seconds), annealing (55°C for 1 min) and extension for 90 seconds at 72°C. A final extension terminates PCR for 10 minutes at 72°C. As a reference, expression of mecA 147 was run on the same samples used. PCR contents were run and electrophoresed on 2% agarose TBE (Tris-Borate-EDTA) containing gel stained with ethidium bromide. Under UV light, PCR products were visualized and copied in gel documentation system machine.

Statistical analysis

Presented data are means ± standard error for 33 MRSA isolates. Data analyses were done using (ANOVA) by Bonferroni test for SPSS software (SPSS, IBM, and Chicago, IL, USA). Values with P < 0.05 were considered statistically significant.

Results and Discussion

Distribution of S. aureus MRSA among isolates

Isolates from patients in hospitals showed the following prevalence and distributions: 9 isolates were from the swap, 18 isolates were from wound swap, three samples were from blood, and last three isolates were from the catheter (Table 2).

MRSA is a significant problem that affects healthcare facilities (Hal, Stark, Lockwood, Marriott, & Harkness, 2007). It caused an increase in the infection rates in hospitals and the community. This leads to a significant and dramatical increase in morbidity and mortality rates due to invasive MRSA infection (Lodise & McKinnon, 2005; Nimmo et al., 2006).

In the current study, MRSA isolates were more abundant in wound swap (54.54%) followed by swap (27.3%) then blood and catheter (9% each). These findings partially agree with others (Pandey, Raza, & Bhatta, 2013) as they reported the changes in MRSA to be swap 28.7%, wound 25%, blood 3% and 1% from urine.

Anti-microbial susceptibility test and MIC

The results of the antibiogram test of MRSA isolates are presented in Table 3. Almost all the isolates of S. aureus (100%) were identified as MRSA based on their anti-microbial resistance to oxacillin and Cefotexim. Moreover, based on resistance against different antibiotic Amoxycillin –clavulanate, Ampicillin, and Cefotaxime were (100%). Clinical isolates showed 45.5 % resistant to ciprofloxacin and erythromycin. Meanwhile, the resistant against Imipenem and Penicillin G was (100%). Finally, the resistant against Trimethoprim-sulphamethoxazole were (18.18%) for examined isolates. Minimal inhibitory concentration Ampicillin–clavulanate (˃4/2) 63.3%, 2/1(27.27%) and ≤ 1/0.5(9%). Ampicillin, ˃1 (100%). Cefotaxime ˃ 32 (45.45%), ≤ 8 (45.45%), 16 (9%). Ciprofloxacin ˃2 (54.54%), ≤ 0.5 (45.45%). Erythromycin, ˃4 (54.54%), ≤ 0.25 (45.45%) and 0.5 (9%). Gentamycin, ≤ 2 (72.72%), ˃8 (27.27%). Imipenem, ≤ 2 (100%).Oxacillin, ˃ 2 (100%). Penicillin G, ˃ 0.25 (100%). Finally, Trimethoprim-sulfamethoxazole, ≤ 1/19 (81.81%) and ˃4/76 (18.18%).

The anti-microbial susceptibility patterns revealed that all isolates of MRSA are resistant to both Cefotaxime and Oxacillin. The resistant rate to Amoxycillin –clavulanate, Ampicillin and Cefotaxime were 100%, ciprofloxacin and erythromycin were 45.5%, Impanel and Penicillin G were 100%, and Trimethoprim-sulphamethoxazole was 18.18%. These results are parallel in some parts with others as the resistant were 43.47% for erythromycin, 53% for gentamicin and 49% for ciprofloxacin. The activities of erythromycin, gentamicin, ciprofloxacin, clindamycin, and trimethoprim-sulfamethoxazole were low against examined MRSA isolates, suggesting that these antibacterials are not good and suitable to be used as therapies in this area (Miller et al., 2005; Sadeghi & Mansouri, 2014).

Identification of SCCmec types in S. aureus isolates and coagulase amplified fragments (81pb)

A total of 33 S.aureus communities MRSA were used for detection of SCCmec types; all 33 strains showed positive expression for the mecA gene (100%) (Table 4). Of the 33 clinical isolates, the results identified six clinical cases (11.11%) of type I SCCmec, from the swap and wound swap. Fifteen clinical isolates of type II SCCmec (45.45%), from wound and blood. Fifteen clinical isolates of type III SCCmec (45.45%), from wound swap and blood. For SCCmec type IV genes, all isolates were negative to type IV (a), while 21 isolates (63.63%) were type IVb and six isolates were type IVd from the swap and wound swap (11.11%), and finally, three isolates (5.55%) were type V from blood and catheter. Of note, nine isolates (27.27%) contained three different SCCmec genes (type II, III and IVb). In parallel, three isolates (9%) contained two different SCCmec genes (type II and IV b&d).

PCR products and results of coagulase gene amplification of 81 pb repeats are shown in Table 4. Wound samples 15 (83.3%) showed four repeats of 324pb amplified fragments. At the same time, 3 out of 18 wound samples (16.6%) showed five repeats 405bp amplified fragments. For swap isolates, six samples (50%) showed four repeats, and the remaining six samples showed five repeats (50%).

Isolates obtained from catheter and blood showed four repeats for coagulase repeats (81bp).

SCCmec expression constitutes five types, that distributed in are MRSA isolates. SCCmec type IV was first known in 2002 (Ma et al., 2002; Okuma et al., 2002). After that, it is believable that SCCmec type IV has acquired additional genes due to the massive use of antibiotics in hospitals (Okuma et al., 2002). In the current study, types of SCCmec that were recognized are I, II and III. Moreover, subtypes for IVa and IVb in SCCmec do not contain genes for antibiotic resistance (Wisplinghoff et al., 2003), as SCCmec type IVc was the most prominent (74%), as SCCmec subtype IVc was fusidic acid-resistant. All data was correlated to be resistant to antibiotics type that differed according to regional hospital and acquired resistant to antibiotics protocol used in these countries. In our results, the incidence of SCCmec types (I, II, III and V) were 54.54%, 72.72%, 54.54% and 18.18% respectively. These results approved that SCCmec type II showed a high level of resistance to many anti-microbial agents (Hesje, Sanfilippo, Haas, & Morris, 2011). In another study, SCC type V isolates were more susceptible to multiple antibiotics compared to I, II and III (Maree, Daum, Boyle-Vavra, Matayoshi, & Miller, 2007) that agree with current reported findings.

Coagulase producing S. aureus is the main criterion used to discriminate pathogenic S. aureus from other staphylococci. Coagulases consist of three distinct regions: (i) the N terminus region containing the prothrombin-binding site, (ii) a highly conserved central part, and (iii) 27-amino-acid-residue sequences repeated that encoded by 81-bp tandem units located in C-terminal region.

The C-terminal region comprises 5, 6, or 8 repeats of 27 amino acids (Palma, Shannon, Quezada, Berg, & Flock, 2001). In the current study, the repeats found were 4 and 5 repeats in all samples, with a percentage of 4.84% and 18.18% respectively. We found it has no discriminatory power in typing of MRSA strains not enough methods need to further methods for typing as sequencing analysis of the repeats that may be useful in molecular typing.

Conclusion

The current study confirmed that the virulence and distribution of MRSA were increasing and gave different antibiogram activities from different global region. Coa gene gave no detectable differences among MRSA types.