Study on the prevalence of methicillin-resistant Staphylococcus aureus infection, antibiotic resistance pattern, biofilms genes, and antibiotic resistance genes from clinical samples

1. Introduction

It is estimated that between 20% and 25% of the healthy adult population are colonized by S. aureus, a Gram-positive human commensal on a regular basis. Additionally, up to 60% of individuals may experience sporadic colonization ( 1 ). It causes a wide range of infections, including bacteremia, septicemia, and infections of the skin, soft tissue, bone, and pneumonia. In rehabilitated cases, HIV cases, and people with underlying conditions like diabetes, these infections can lead to further complications and mortality. The disease is transmitted through direct contact and/ or objects. ( 2 ). The emergence of Methicillin-Resistant Staphylococcus aureus (MRSA) occurred shortly after the introduction of methicillin, a new antibiotic, in 1960. Methicillin was employed as a therapeutic agent for S. aureus strains exhibiting resistance to penicillin. This resistance was attributed to the penicillinase product called beta-lactamases, which inactivate penicillin. The global observation of S. aureus methicillin resistance indicates that it is resistant to all penicillinase-resistant penicillins and cephalosporins. ( 3 , 4 ). MRSA strains have the capacity to produce a specific protein, designated as penicillin-bindingprotein (PBP), which exhibits reduced affinity for certain classes of penicillin. The PBP gene designated as mecA, can be isolated and utilized for the storage of the information. The Methicillin resistance gene (mecA) is transferred by a small genetic part called the staphylococcal cassette chromosome mec (SCCmec) ( 5 ). Penicillin resistance occurs in certain strains of S. aureus primarily due to the production of a protein called beta-lactamase, which is encoded by a specific gene called blaZ. The genes blaZ, blaI, and blaR1 are located in a single locus ( 6 ). Four distinct variants of the blaZ product have been identified through serotyping and the observation of variations in hydrolysis of selected-lactam substrates (A, B, C, D). The A, C, and D types are often observed on plasmids. In contrast, the B variant is located on the chromosome. However, in contrast to the chromosome-based mecA gene, the blaZ gene is carried by a plasmid ( 7 ). The capacity of bacteria to be identified and eliminated by the innate immune system is impeded by the formation of biofilms ( 8 ). The initial stage in the formation of a biofilm by S. aureus is the adhesion of the bacteria to different surfaces and subsequent growth on host tissues. S. aureus possesses a multitude of surface-bound molecules that facilitate its adhesion to other molecules. These substances, also designated microbial surface components, are capable of recognizing and attaching to different types of protein substrate. Notable examples of these proteins include fibronectin-binding proteins A and B, clumping factors A and B, collagen-binding protein, bone sialoprotein binding protein, and fibrinogen binding protein. ( 9 , 10 ). FnBPA adhesions stick to receptors on cells in the blood vessels, which can precipitate cardiac complications heart problems and infections in cardiac devices by activating platelets. This represents a crucial phase in the development of blood clots and their attachment to artificial parts inside the body ( 11 ). The collagen adhesin (cna) gene is responsible for the production of the cna protein, which facilitates the attachment of S. aureus to collagen tissues and cartilage. Studies have demonstrated that antibiotic resistance does not impede the capacity of MRSA strains to form biofilms ( 12 ). The objective of the present study is to examine the prevalence of biofilm genes (cna and fnbA) and antimicrobial resistance genes (mecA and blaZ) in addition to theantimicrobial resistance patterns of MRSA in Karaj, Iran.

2. Materials and Methods

In this study, 78 strains of S. aureus were collected over a six-month period from clinical samples including urine and wounds from two laboratories in Karaj. The samples were transported using the Tryptic Soy Broth (TSB) transport medium was used to transfer these samples and delivered to the laboratory within a maximum of two hours. The samples were cultured on blood agar and mannitol salt agar from (Ibresco, Iran) were used to culture these samples in plates at 37°C for 24-48 hours. The S. aureus strain was then isolated by standard microbiological methods,including: catalase, coagulase, mannitol fermentation and DNase tests.

2.1. Detection of S. Aureus Specific Nuc Gene

The DNA was extracted from the samples using the boiling method. All isolated strains were subjected to analysis via the polymerase chain reaction(PCR) method. Specifically, nuc primers (F: 5 AGCGATTGATGGTGATACGG-3 and R: 5-ATACGCTAAGCCACGTCCAT-3) were employed for the identification of S. aureus strains ( 13 ). The PCR was conducted in a total volume of 25 μl ,comprising 14 μl master amplicon (YTA, Tehran, Iran), 1 pmol of each forward and reverse primer,and 8 μl of distilled water containing 2 μl of template DNA. The following PCR parameters were employed for the 35 -cycle PCR: an initial denaturation at 94°C for 2 minutes, denaturation at 94°C for 1 minute, annealing at 55°C for 0.5 minutes, extension at 72°C for 2 minutes and a final extension at 72°C for 5 minutes ( 13 ). The PCR results (226 bp) were visualized using 1.5% agarose gel electrophoresis (YTA, Tehran, Iran).

2.2. Antimicrobial Susceptibility of S. Aureus Isolates

To confirm the precise identity of the MRSA isolates, the presence of the mecA gene was verified through the use of by amplification PCR. Subsequently, the investigation was conducted to investigate the susceptibility patterns of the following 10 antimicrobial agents, representing a diverse range of antimicrobial classes. The susceptibility of the samples to the discs of Tetracycline (30 µg), Oxacillin (1 µg), Doxycycline (30 µg), Erythromycin (15 µg), Trimethoprim/sulfamethoxazole (25 µg), Amikacin (15 µg), Penicillin (10 µg), Vancomycin (30 µg), Ciprofloxacin (5 µg) and Gentamicin (10 µg) (Padtan Teb, Iran). The antibiotic susceptibility patterns of S. aureus isolates were determined by the Kirby-Bauer method, and the results were interpreted in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines ( 14 ).

2.3. DNA Extraction

The DNA was extracted from the samples using the boiling method. A loopful of bacterial colonies was suspended in 300 µl of sterile distilled water and then heated for 20 minutes. The resulting liquid part was then used as a DNA sample in the PCR mixture after being spun it in a centrifuge for 15 minutes at a fast speed of 13,000 rpm ( 16 ).

2.4. Detection of Genes

PCR and electrophoresis techniques were employed to determine the presence of mecA, blaZ, cna, and fnbA genes. A multiplex PCR was conducted on the in two cna and fnbA genes: The resulting PCR products were visualized with 3% agarose gel (YTA, Tehran, Iran) for the amplified genes,which contained Safe Stain (YTA, Tehran, Iran), The molecular approach was optimized using S.aureus ATCC 25923 as the control strain. The primer sequences and PCR conditions utilized for the detection of genes are presented in Table 1. The amplification conditions for the mecA gene were as follows: denaturation for two minutes at 94°C, followed by 40 cycles of 94°C for two minutes, 57°C for 60 seconds, 72°C for 2 minutes, and finally extension at 72°C ( 10 ). The amplification conditions for the blaZ gene were as follows: the process commenced with heating at 94°C for 5 minutes, followed by 35 cycles of heating at 94°C for 30 seconds, cooling at 55°C for 30 seconds, and heating again at 72°C for 30 seconds.

Subsequently, a final heating step at 72°C for 10 minutes ( 11 ) is performed. The amplification conditions for the fnbA and cna genes were as follows:an initial denaturation was performed, wherein the temperature was first set at 95°C for two minutes. Subsequently 50 cycles were conducted,wherein the temperature was initially set at 95°C for 30 seconds, then at 55°C for 1 minute, and finally at 72°C for 1 minute with 1 minute for the final step ( 12 , 14 ).

2.5. Statistical Analysis

The results were analyzed using the Pearson's chi-square test or Fisher's exact test was used to analyze the results and a value of P<0.05 was considered statistically significant (SPSS 25.0, SPSS Inc. Chicago, IL, USA).

3. Results

3.1. Antibiotic Susceptibility

Of the 63 clinical samples in which the presence of the mecA gene was confirmed, 40 (63.5%) samples were urine samples and 23 (36.5%) were wound samples. The overall resistance of MRSA isolates to antimicrobial agents was 100% for oxacillin; 95.2% for penicillin; 31.7% for ciprofloxacin, tetracycline and doxycycline. The resistance rates for erythromycin; amikacin; gentamicin; trimethoprim-sulfamethoxazole; and vancomycin were 30.2%, 27%, 23.8%, 17.5%, and 3.2%, respectively. The overall antibiotic susceptibility pattern of strains to antimicrobial agents is illustrated in Table 2. Figure 1 illustrates the prevalence of antibiotic resistance in clinical samples.

Figure 1. Percentage of antibiotic resistance in clinical samples

3.2. Gene pattern characterization

In this study, the presence of antibiotic resistance and biofilm -related genes was evaluated in S. aureus isolates by PCR method. All isolates were subjected to investigation with regard to the presence of the cna, fnbA, mecA and blaZ genes. The prevalence of the cna, fnbA, mecA and blaZ genes was determined to be as 66.7%, 81%, 46% and 95.2% respectively. The result of PCR for the cna, fnbA, mecA and blaZ genes from S. aureus isolates are presented in Figure 2.

Figure 2. Amplification of mecA, fnbA, can and blaZ genes from S. aureus isolates. Lane M, DNA marker (100 bp); Lane1 and 2, blaZ (518 bp); Lane 3 and 4, fnbA (268 bp); Lane 5 and 6, fnbA and cna (128 and 192 bp).

The frequency of the blaZ gene was observed in urine and wound samples at 97.5% and 91.3%, respectively. No significant relationship was observed between the frequency of this gene and the clinical samples (P>0.05). The frequency of the mecA gene was observed in urine and wound samples at 50% and 39.1%, respectively. No significant relationship was found between the frequency of this gene and clinical samples (P>0.05). Additionally, the frequency of the fnbA gene was observed in urine and wounds at 80% and 82.6%, respectively. The frequency of the cna gene was observed to be present in urine and wounds at the rate of 67% and 69%, respectively. A significant relationship was observed between the frequency of this gene and wound samples (P>0.05). In isolates with the fnbA, 81% also exhibited the cna gene, demonstrating a significant relationship between these frequencies. The blaZ gene was present in 95.2% of the samples, indicating a significant relationship between the presence of this gene and the production of beta-lactamase enzyme in S. aureus isolates (P<0.05). A significant correlation was observed between gentamicin and tetracycline antibiotic resistance with cna and the fnbA genes (P<0.05).

4. Discussion

S. aureus is a common bacterium that colonizes the human skin and mucous membranes. However, S. aureus is also a major causative agent of hospitaland community-associated infection that can result in life- threatening disease ( 17 ). The control of antibiotic-resistant S. aureus strains have been dependent on three factors: ensuring proper hand hygiene among healthcare workers, restriction of antibiotic use, and prompt identification and isolation of infected patients ( 18 , 19 ). In this study, 63 isolates of MRSA were examined. Of these, 60 (95.2%) exhibited the blaZ genes, while 51 (81%) demonstrated the fnbA gene. Additionally, the frequency of the cna gene was observed in 42 isolates (66.7%). The results of the antibiotic resistance pattern showed that all isolates were resistant to penicillin, while the lowest resistance was observed with vancomycin. In the study by Gomes et al. ( 20 ) in Brazil, 56 strains of S. aureus resistant to methicillin were isolated from blood cultures. Of these, 86% of the samples were positive for the presence of the blaZ gene, and 84% of the isolates had the mecA gene. In Egypt, Amr and Al Gammal ( 21 ) reported that of 114 S. aureus strains, 90 (78.9%) were MRSA, and 10 strains (8.8%) were resistant to vancomycin. Of the MRSA strains, 88 out of 90 carried the mecD gene, and all 10 vancomycin-resistant isolates were positive for both the mecA and vanA genes. Kim and Lee ( 22 ) conducted a study in Korea in which oral saliva samples were collected from a total of 112 patients with dental diseases, including 80 outpatients in dental hospitals and 32 patients in dental clinics. Among these, 37 S. aureus strains were positive for the blaZ gene: 27 strains from hospital patients and 10 from clinic patients. In India, Naseer and Jayaraj ( 23 ) reported that out of 360 S. aureus strains, only 7 (1.9%) were vancomycin-resistant and positive for the presence of the mecA gene. In a study conducted by Mohammadi et al. ( 24 ) at a burn center in Tehran, of 83 S. aureus isolates, 74.7% were positive for the cna gene, and 42.1% had the fnbA gene. Khasawneh et al. ( 25 ) reported that out of 57 S. aureus isolates, 22 were MRSA. The prevalence of the cna and fnbA genes in MRSA isolates was 40.9% and 81.8%, respectively. Similar studies indicate that the prevalence of resistant S. aureus is highly dependent on geographical region, biological patterns, and regional antibiotic usage, contributing to variations in study outcomes. Therefore, monitoring shifts in antibiotic resistance patterns over time may greatly aid in treating S. aureus infections. To prevent drug resistance, performing an antibiogram test prior to antibiotic use is recommended PCR testing on penicillin-resistant strains may be necessary to detect beta-lactamase-producing organisms, allowing for more accurate reporting. It is also recommended to limit non-prescription antibiotic use in unnecessary cases to reduce antibiotic resistance levels.

Acknowledgment

We would like to thank members of SabaBiomedicals Science-Based Company for their kind support.

Authors' Contribution

A comprehensive literature review and research, conceptualization, methodology, supervision, project administration, writing-reviewing and editing, methodology, investigation, studies analysis: Z.H., A.S.B., F.B., Writing original draft preparation, writing-reviewing and editing, and methodology: B.J.L., P.M.G investigation. Validation and Reviewing: M.H.K., A.S.B.

Ethics

All experimental procedures were carried out with the utmost respect for the principles of ethical research, ensuring the welfare and safety of the participants.

Conflict of Interest

The authors declare that they have no conflicts of interest to disclose.

Data Availability

The data that support the findings of this study are available on request from the corresponding author.

References

1. Gaddafi MS, Yakubu Y, Junaidu AU, Bello MB, Bitrus AA, Musawa AI, Garba B, Lawal H. Occurrence of Methicillin-resistant Staphylococcus aureus (MRSA) From Dairy Cows in Kebbi, Nigeria. Iranian Journal of Veterinary Medicine. 2023; 17(1): 19-26.
2. Foroutan S, Eslampour MA, Emaneini M, Jabalameli F, Akbari G. Characterization of Biofilm Formation Ability, Virulence Factors and Antibiotic Resistance Pattern of Staphylococcus aureus Isolates from Subclinical Bovine Mastitis. Iranian Journal of Veterinary Medicine. 2022; 16(2):144-154.
3. Azizkhani M, Tooryan F. Methicillin resistant Staphylococcus aureus (MRSA) in pastry cream products sold in Amol (Iran). Iranian Journal of Veterinary Medicine. 2018; 12(2): 167-174.
4. Azizkhani M, Akhondzadeh Basti A, Tooryan F. Comparing inhibitory potential of Eugenia caryophyllus and Origanum compactum against the growth and gene expression of enterotoxins in Staphylococcus aureus ATCC 29213. Iranian Journal of Veterinary Medicine. 2017; 11(4): 299-311.
5. Azizkhani M, Jafari F, Haghighi P, Dehghan M. Evaluating Contamination Level of Raw and Roasted Nuts Distributed in Commercial Markets in Mazandaran Province, Iran. Iranian Journal of Veterinary Medicine. 2020; 14(2):167-176.
6. Takayama Y, Tanaka T, Oikawa K, Fukano N, Goto M, Takahashi T. Prevalence of blaZ gene and performance of phenotypic tests to detect penicillinase in Staphylococcus aureus isolates from Japan. Annals of laboratory medicine. 2018; 28;38(2):155-59.
7. Olsen JE, Christensen H, Aarestrup FM. Diversity and evolution of blaZ from Staphylococcus aureus and coagulase-negative staphylococci. Journal of Antimicrobial Chemotherapy. 2006; 1;57(3):450-60.
8. Foster TJ. Immune evasion by staphylococci. Nature reviews microbiology. 2005; 3(12):948-58.
9. Kot B, Sytykiewicz H, Sprawka I. Expression of the biofilm-associated genes in methicillin-resistant Staphylococcus aureus in biofilm and planktonic conditions. International journal of molecular sciences. 2018; 19(11):3487.
10. Kadkhoda H, Ghalavand Z, Nikmanesh B, Kodori M, Houri H, Maleki DT, et al. Characterization of biofilm formation and virulence factors of Staphylococcus aureus isolates from paediatric patients in Tehran, Iran. Iranian Journal of Basic Medical Sciences. 2020; 23(5):691.
11. Foster TJ, Höök M. Surface protein adhesins of Staphylococcus aureus. Trends in microbiology. 1998; 6(12):484-488.
12. Shahmoradi M, Faridifar P, Shapouri R, Mousavi SF, Ezzedin M, Mirzaei B. Determining the biofilm forming gene profile of Staphylococcus aureus clinical isolates via multiplex colony PCR method. Reports of biochemistry & molecular biology. 2019; 7(2):181.
13. Abdulrahman RF. DETECTION OF Staphylococcus aureus FROM LOCAL AND IMPORTED chicken in duhok province/kurdistan region of iraq using conventional and molecular methods. Basrah Journal of Veterinary Research. 2020; 19(1)
14. CLSI. Performance Standards for Antimicrobial Susceptibility Testing CLSI Supplement M100. Wayne, PA: Clinical and Laboratory Standards Institute; 2022.
15. Rahimi F, Khashei S, Katouli M. Prevalence, diversity, and antimicrobial susceptibility proles of methicillin-resistant Staphylococcus aureus among patients with diabetic foot infections in a referral hospital in Tehran, Iran. Research Square. 2023; 19(3):e142081.
16. Fatholahzadeh B, Emaneini M, Gilbert G, Udo E, Aligholi M, Modarressi MH, Nouri K, Sedaghat H, Feizabadi MM. Staphylococcal cassette chromosome mec (SCC mec) analysis and antimicrobial susceptibility patterns of methicillin-resistant Staphylococcus aureus (MRSA) isolates in Tehran, Iran. Microbial drug resistance. 2008; 14(3):217-20.
17. Rahimi H, Saei HD, Ahmadi M. Nasal carriage of Staphylococcus aureus: Frequency and antibiotic resistance in healthy ruminants. Jundishapur journal of microbiology. 2015; 8(10):e22413.
18. Campoccia D, Speziale P, Ravaioli S, Cangini I, Rindi S, Pirini V, Montanaro L, Arciola CR. The presence of both bone sialoprotein-binding protein gene and collagen adhesin gene as a typical virulence trait of the major epidemic cluster in isolates from orthopedic implant infections. Biomaterials. 2009; 30(34): 6621-6628.
19. Atchade E, De Tymowski C, Grall N, Tanaka S, Montravers P. Toxic Shock Syndrome: A Literature Review. Antibiotics. 2024; 13(1):96.
20. Gomes RMF, Bomfim MRQ, Trindade MJV, Farias LM, Santos SG. Potential Spread of Methicillin-Resistant Staphylococcus aureus Recovered from Patients with Bloodstream Infection. Chemo Open Access. 2015; 4(2):149.
21. Amr GE, Al Gammal S. Emergence of vancomycin resistant Staphylococcus aureus isolated from patients in ICUs of Zagazig University Hospitals. The Egyptian Journal of Medical Microbiology (EJMM). 2017; 26(2):1-7.
22. Kim GY, Lee CH. Antimicrobial susceptibility and pathogenic genes of Staphylococcus aureus isolated from the oral cavity of patients with periodontitis. J Periodontal Implant Sci. 2015; 45(6): 223-28.
23. Naseer BS, Jayaraj YM. Identification of Multi-Resistant Staphylococcus aureus in Clinical Specimens. Res J Med Sci. 2010; 4(3): 204-7.
24. Mohammadi A, Goudarzi M, Dadashi M, Soltani M, Goudarzi H, Hajikhani B. Molecular detection of genes involved in biofilm formation in Staphylococcus aureus strains isolates: evidence from shahid motahari hospital in Tehran. Jundishapur Journal of Microbiology. 2020; 13(7):e102058.
25. Khasawneh AI, Himsawi N, Abu-Raideh J, Salameh MA, Al-Tamimi M, Mahmoud SAH, Saleh T. Status of biofilm-forming genes among Jordanian nasal carriers of methicillin-sensitive and methicillin-resistant Staphylococcus aureus. Iranian Biomedical Journal. 2020; 24(6):386.