115 © Published by College of Education for Pure Science (Ibn Al-Haitham), University of Baghdad. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License Investigating the Presence of eap and spa Genes of Staphylococcus aureus and their Relation to Antibiotic Resistance Jilan Ahmed Shakur 1* and Rasmiya Abd Aburesha 2 1,2 Department of Biology, College of Science, University of Baghdad, Baghdad, Iraq.. *Corresponding Author. Received:23 April 2023 Accepted:19 June 2023 Published: 20 July 2024 doi.org/10.30526/37.3.3436 Abstract Staphylococcus aureus is an extracellular and intracellular microbe that can infect different mammalian cells like epithelial, endothelial, and osteoblasts. It is a source of recurrent infection and has persistently caused a variety of human diseases, like skin infections and systemic diseases such as osteomyelitis, endocarditis, pneumonia, bacteremia, sepsis, and toxic shock syndrome. Diagnosed (30) isolates of S. aureus have been taken from urine. A clinical sample has been done according to the culture on mannitol salt agar and biochemical tests, then identification through amplification of the 16srRNA gene by conventional PCR, detection of spa and eap genes by conventional PCR found all isolated to have a spa gene with a percentage of 100% but an eap gene found with a percentage of 53.3%, and detection of their relation with antibiotic resistance found 76.70% of this isolates resistance to different types of antibiotic. Many factors contributed to the rapid development of antimicrobial resistance, including cell membrane disruption, environmental factors, and DNA damage. All of these factors contributed to the rapid development of antibiotic resistance. Staphylococcus aureus is resistant to a lot of commonly used antibiotics, such as Beta- lactams, Tetracycline, Aminoglycosides, Fluoroquinolones, Macrolides, and Chloramphenicol. Keywords :S. aureus, 16srRNAgene, eap gene, spa genes, antibiotic resistance. 1. Introduction Staphylococcus aureus is an extracellular and intracellular microbe that can infect different mammalian cells like epithelial, endothelial, and osteoblasts. It is a source of recurrent infection and a persistent cause of a variety of diseases in humans, like skin infections and systemic diseases such as osteomyelitis, endocarditis, pneumonia, bacteremia, sepsis, and toxic shock syndrome [1, 2]. Many factors helped to develop antimicrobial resistance rapidly, including cell membrane disruption, environmental factors and DNA damage, all of which fastened the development of antibiotic resistance. S. aureus is resistant to a lot of commonly used antibiotics, such as beta- lactamases, tetracycline, aminoglycosides, fluoroquinolones, macrolides, and chloramphenicol [3]. S. aureus has different virulence factors that enable the microorganism to be affected. This causes a wide range of infections because this factor helps the pathogen in attachment to host cells, tissue invasion, and weakness of the host immune system. S. aureus https://creativecommons.org/licenses/by/4.0/ https://orcid.org/ mailto:jelianahmed5@gmail.com https://orcid.org/ mailto:Rasmiaabd8@gmail.com IHJPAS. 2024, 37( 3 ) 116 occurs in 20–50% of humans.The pathogenic capacity is combined with the effects of extracellular factors, toxins, and the invasive properties of the strain [4-6]. 2. Materials and Methods 2.1. Sample collection: 150 clinical samples were collected during the period (February to May 2022) using sterile swaps taken from tonsils and sterile cups for the collected urine samples. All the samples were taken from people returning to Tuz General Hospital in the Salah al-Din Governorate and from patients broadcasting in hospitals. 2.2. Bacterial identification: All clinical samples cultured on the selective media (Mannitol salt agar) that differentiate S. aureus isolates through mannitol fermentation [7, 8], then taking smears of isolates and staining them with Gram's stain investigated through an oil immersion microscope lens, the cells appear under light microscopes. Gram-positive cocci clustered mostly in grape-like irregular clusters, circular clusters, single, paired, or short chains because S. aureus bacteria divided into three planes during binary fission [9,10]. Biochemical tests include The catalase test was distinguished by transporting some pure colonies of bacterial isolates taken from mannitol salt agar media to a clean glass slide by a sterilized loop, then adding a few drops of 3% H2O2 to it. The production of air bubbles is referred to as a positive result that evidences enzyme production [11, 12]. The coagulase test was evaluated to distinguish free plasma coagulase enzymes that lead to coagulation products from interactions between blood and S. aureus bacteria, which indicates a positive result. While the clotting did not emerge at room temperature, that refers to a negative result [13]. 2.2.1. Confirmed this method of identification through detection 16srRNA by PCR (Polymerase chain reaction) includes two steps. 2.2.1.1. Extraction and determine S. aureus DNA: Genomic DNA extraction according to Onasanya et al. 2003 and Geneaid Biotech kit instructions, with some modifications, agarose gel electrophoresis that was stained by adding ethidium bromide was used for determining DNA extraction presence and integrity. The optimum DNA concentration was detected for the PCR, and all the samples were conserved at -20 °C until use. The genomic DNA samples were mixed with the loading buffer (DNA: loading 7/2 v/v) and then loaded inside the wells of the gel [15]. 2.2.1.2. 16srRNA gene amplification: The 16srRNA gene commonly used for identification, classification, and quantitation of microbes that codes for the small subunit of the ribosome found in prokaryotes such as bacteria and archaea has several factors that make it the perfect target to complete phylogeny studies. [16] used a specific primer [17] in the PCR program according to the TRANS protocol detected 16srRNA. Table 1. PCR reaction components for detection 16srRNA according to Trans protocol Primer Type of reaction Master mix volume Volume of F-primer (10pmol/ml) Volume of R-primer (10pmol/ml Nuclease free water DNA Final volume 16s rRNA uniPCR 10 1 1 6 ml 2 ml 20 ml IHJPAS. 2024, 37( 3 ) 117 Table 2. PCR program to amplify 16srRNA Steps oC Min-Sec Cycle Product band Initial denaturation 95 oC 4min 1 257 Denaturation 95 oC 4 min 30 Annealing 53 oC 45 sec Extension 72 oC 45 sec Final extension 72 oC 7 min 1 2.3. Antimicrobial resistance test Detection of S. aureus and determination of the antimicrobial resistance profile of each isolate Antimicrobial resistance testing was carried out by the Kirby Bauer disc diffusion method according to the Clinical Laboratory Standards Institute on Muller Hinton agar [18, 19]. The antibiotics used to determine antibiotic resistance include amoxicillin (AMC 30), chloramphenicol (C10), erythromycin (E10), tetracycline (TE10), penicillin (P10), and vancomycin (VA30), all obtained from the Bioanalyse company. The ranges of antibiotic zone diameter (mm) that are suggested for S. aureus according to research in reference [20] 2.4. Detection of S. aureus virulence factors spa and eap genes Detection of S. aureus spa and eap genes that were encoding for regulating virulence factor was carried out by the amplification of the extraction DNA of S. aureus with the target gene by using PCR, which included using various specific sets of primers encoding for each target gene mixed with the template (DNA sample) and master mix reagents (PCR buffer, MgCl2, Taq polymerase, and dNTPs). The end constituent was deionized water. The mixture was then mixed and centrifuged for 3 seconds to collect the drops from the walls to ensure a final volume of 25 ml. The mixture was transferred to a thermal cycler to start the reaction according to the steps of the specific program [21]. Table 3. PCR reaction components according to (Trans protocol) Primer Type of reaction Master mix volume Volume of F-primer (10pmol/ml) Volume of R- primer (10pmol/ml Nucleas e free water DNA Final volume Spa gene uniPCR 10 1 1 6 ml 2 ml 20 ml Eap gene uniPCR 10 1 1 6 ml 2 ml 20 ml Table 4 explains the PCR program to amplify the spa gene using a specific primer [22]. Table 4. PCR program to amplify spa gene Steps oC Min-Sec Cycle Product band Initial denaturation 95 oC 4min 1 variable Denaturation 95 oC 30 sec 30 Annealing 50 oC 45sec Extension 72 oC 45 sec Final extension 72 oC 7min 1 Table 5 explains the PCR program to amplify the eap gene using a specific primer [23]. IHJPAS. 2024, 37( 3 ) 118 Table 5. PCR program to amplify eap gene Steps oC Min-Sec Cycle Product band Initial denaturation 95 oC 4min 1 230 Denaturation 95 oC 30 sec 30 Annealing 50 oC 45sec Extension 72 oC 45 sec Final extension 72 oC 7min 1 3. Results The number of samples taken from people returning to Tuz General Hospital were125, collected from urinary tract (100) samples with a percentage of 66.6 percent, and from tonsils (25) samples with a percentage of 16.6%. Also collected from patient broadcasts in hospitals were 24 samples with a percentage (16%) from tonsils and one sample with a percentage (0.6%) from the urinary tract, as shown in Table 6. Table 6. the sample source and patient type Patient type Sample source Urinary tract Percentage tonsil percentage Patient return to hospital 100 66.6% 25 16.6% Patient broadcasts in hospitals 1 0.6% 24 16% 3.1. Bacteria identification Staphylococcus aureus isolates identification include Mannitol salt agar culture, biochemical tests, and confirmed primary identification by detection of 16srRNA by PCR (polymerase chain reaction). Only from all samples identified 30 isolates with a percentage of 29.7% of S. aureus bacteria this percentage is shown in Table 7. This result found S. aureus bacteria infect the urinary tract more than the upper respiratory tract. Table 7. positive culture result and negative result Bacterial source Positive result percentage Negative result percentage Urinary tract 30 29.7% 71 70.2% tonsil 0 0 49 100% 3.1.1. Biochemical test All S. aureus bacteria mannitol fermenter appear with golden yellow on mannitol salt agar plates, Gram-positive cocci, catalase positive, coagulase positive, and oxidase, motility negative and more strain hemolysis it is lysis the red blood cell. 3.1.2 molecular detection of Staphylococcus aureus includes two steps, the first one is extraction S. aureus DNA Extracted DNA and purified from all S. aureus using the manufacturer's DNA purification kit (DNA extraction kit) used agarose gel electrophoresis and stained by adding ethidium bromide for determined DNA extraction presence and integrity. The optimum DNA concentration was detected for the PCR. Figure 1 shows DNA extraction. IHJPAS. 2024, 37( 3 ) 119 Figure 1. showed S. aureus DNA extraction visualized by 1 % agarose gel electrophoresis that stained with ethidium bromide by 1xTBE and used UV transilumiator at 350nm. The second step used the PCR technique to amplify the 16srRNA gene was applied on the 30 isolates taken from the urinary tract after primary identification, and all isolates were S. aureus bacteria. Figure 2 clarify 16SrRNA amplification. Figure 2. PCR amplification of 16srRNAgene (257bp) of S. aureus, visualized by 1 % agarose electrophoresis that using TBE to stained by ethidium bromide, for 30 min at 90 Volt. , Lane M: Marker Lane (1-30): Urinary tract sample from people return to Hospital. 3.2. Antimicrobial resistance test by disk diffusion method Antimicrobial resistance testing was performed using the disk diffusion method on 30 S. aureus isolates against six commonly used antibiotics (P, TE, AMC, C, and VA). All S. aureus isolates showed varying resistance percentages to the different antibiotics used in the study, as shown in Figure 3. IHJPAS. 2024, 37( 3 ) 120 Figure3. result of antimicrobial resistance test by disk diffusion method 3.3. PCR reaction result for detected some S. aureus genes Used conventional PCR to investigate about 2 genes (the eap gene and the spa gene). This gene is responsible for the virulence factor in 30 isolates of S. aureus bacteria. All isolates showed a spa gene with a 100% percentage. Figure 4 shows the spa gene in all isolates. Figure 4. PCR amplification of the spa gene of S. aureus, visualized by 1% agarose electrophoresis, using TBE to stain with ethidium bromide for 30 min at 90 volts Lane M: 1500 Marker. The eap gene detected in this study with percentage of 53.3% from all stains, that show in Figure 5. Figure 5. PCR amplification of eap gene (230bp) of S. aureus, visualized by 1 % agarose electrophoresis that stained with ethidium bromide by using TBE, for 30 min at 90 Volt. Lane M: 1500 Marker 0.00% 10.00% 20.00% 30.00% 40.00% 50.00% 60.00% 70.00% 80.00% 90.00% 100.00% E 10 C10 VA 30 AMC30 TE 10 P 10 26.70% 30% 46.70% 23.30% 6.70% 6.70% 73.30% 70% 53.30% 76.70% 93.30% 93.30% % Antibiotics Sensitive Resistance IHJPAS. 2024, 37( 3 ) 121 3.4. Antibiotic resistance relation with spa and eap virulence genes Antimicrobial therapy plays an important role in controlling S. aureus toxicity and infectivity that cause S. aureus harm is stronger this is related to the virulence genes but has become less effective due to widespread drug resistance, the resistance returns to bacterial capsular polysaccharide and some virulence gene like spa gene and eap gene [32, 33]. In this study, it was shown that S. aureus has a high ability to resist some types of antibiotics, such as erythromycin, chloramphenicol, vancomycin, amoxicillin, and penicillin, with percentages of 73.3%, 70.0%, 53.3%, 76.7%, 93.3%, and 93.3, respectively. Because of the S. aureus virulence gene spa, it was found with a 100% considered molecule that binds to Fcγ of human and animal immunoglobulin (Ig), a defense mechanism that hinders the capacity of antibodies with specific binding activities for the S. aureus surface to enable Fc receptor-mediated opsonophagocytosis and bacterial killing that allows S. aureus to avoid innate and adaptive immune responses [34]. While the eap gene found with a percentage of 53.3% is considered a protein, it has two structural homologues, EapH1 and EapH2, which impair the adherence of neutrophils to non-stimulated and TNF-alpha-stimulated endothelial cells, may prevent the release of neutrophil serine proteases, inhibit extracellular killing of bacteria, cause trans-endothelial migration of neutrophils, and reduce inflammation responses because it directly interferes with neutrophil serine protease activity. All these genes of S. aureus bacteria contribute to bacterial escape through immunity defenses and antibiotic resistance [35, 36]. Table 8 shows all the results. Table 8. S. aureus spa and eap virulence genes relation with antibiotic resistance Resistance Antibiotic R % Spa gene % eap gene % erythromycin 10 22 73.3 30 100% 16 53.3% Chloramphenicol 10 21 70.0 30 100% 16 53.3% vancomycin 30 16 53.3 30 100% 16 53.3% Amoxicillin30 23 76.7 30 100% 16 53.3% Tetracycline 10 28 93.3 30 100% 16 53.3% Penicillin 10 28 93.3 30 100% 16 53.3% 4. Discussion The first step in diagnosis, all samples cultured on Mannitol salt agar medium for 24 hours at 37oC are selective and differential growth mediums that contain a high concentration (~7.5%–10%) of NaCl, Mannitol sugar, and phenol red as an indicator for differentiation between two groups of staphylococci [24, 25]. S. aureus growth on the mannitol salt agar and changed the culture color from pink to yellow, this bacteria was isolated from the urinary tract of people returning to Tuz General Hospital and found S. aureus bacteria infect the urinary tract more than the upper respiratory tract. The second step of diagnosis used the PCR technique to amplify the 16srRNA gene, which helped to identify the bacterial genome, it was a highly developed and effective tool that helped to recognize specific bacterial strains, the increasing number of multidrug-resistant (MDR) staphylococcal infections leads to investigating basic questions about the genetic variations because antibiotic resistance evolved within the bacterial population. Therefore, in recent years, 16srRNA gene amplification has become more common as a genetic marker to confirm S. aureus phylogeny and taxonomy identification, which is increasingly prevalent in the clinical environment [17, 26].The most common resistance mechanisms include active efflux of the antibiotic, enzymatic inactivation of the drug, and mutations in the rRNA. For example, IHJPAS. 2024, 37( 3 ) 122 penicillin resistance returns to antibiotic action, plasma membrane, penicillin-less bacterial permeable to the antibiotic, and modification of the receptor site of the bacterial protein [27, 28]. Through genetic study found spa acts as a genetic marker to distinguish S. aureus strains and has an important role in S. aureus resistance to different types of antibiotics, especially erythromycin [29]. The eap gene which is involved in invasion of host tissues, [30] showed in their study eap gene was with a percentage 100%, but the [31] study showed the S. aureus adhesion gene (eap) responsible for antibiotic resistance that led to evolution and development has become hyper virulent and spread in hospitals and communities. Through the relation between antibiotic resistance with spa , eap virulence genes ,found all these genes of S. aureus contribute about bacterial escape through immunity defenses and antibiotic resistance [35, 36] 5. Conclusion The urinary tract system was more infected with S. aureus bacteria compared with the pharynx. The return patient to the hospital was more infected with S. aureus bacteria than patients in the hospital. Through antibiotic resistance tests, S. aureus bacteria found more resistance to three antibiotics TE, P, and AMC. The spa gene is found in all strains of S. aureus bacteria, but the eap gene is not found in all strains, and these genes have an important role in antibiotic resistance. Acknowledgment My sincere thanks to the members and staff of Tuz hospital Laboratories especially (bacteriological staff) for their help in the collection and identification of isolates. Conflict of Interest No, conflict of interest Funding No, Financial support in preparation for the publication is included in the funding section. 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