111 This work is licensed under a Creative Commons Attribution 4.0 International License IHJPAS. 37 (2) 2024 Ibn Al-Haitham Journal for Pure and Applied Sciences Journal homepage: jih.uobaghdad.edu.iq PISSN: 1609-4042, EISSN: 2521-3407 Detection of Integron Classes and Agr Group in Staphylococcus aureus Isolated from Different Clinical Samples Rasha Zaid Tariq Ahmed 1 and Rana Mujahid Abdullah 2* 1,2 Department of Biology College of Education Pure Science (Ibn Al-Haitham), University of Baghdad, Baghdad, Iraq. *Corresponding Author. Received: 16 April 2023 Accepted: 22 May 2023 Published: 20 April 2024 doi.org/10.30526/37.2.3419 Abstract Fifty isolates of Staphylococcus aureus were obtained out of 220 samples collected from clinical sources after a diagnosis of the bacteria, and then a sensitivity test was performed for the isolates, and the minimum inhibitor concentration of 15 antibiotics in the bacterial isolates under study was determined using the Vitek -2 Compact System device. All isolates showed 100% resistance to Benzylpenicillin at (>=0.5-0.12) and the resistance to Oxacillin was 76% at (>= 1- 4), Ciprofloxacin and Moxifloxacin was 42, 40% at (>=2-8), Gentamycin (20%) at >=16, Erythromycin by (78%) at >=8, Clindamycin (by 74%) at (>=8-16), Tetracycline by 40% at (>=2-16), Fusidic Acid by (14%) at (>=8-32), and Rifampicin by (6% ) at of (>=32 and Trimethoprim/ Sulfamethoxal by 14% at a concentration of (>=80-320). The isolates also showed less resistance to Vancomycin, Linezolid, and Teicoplanin (5, 2, and 2%), respectively, at a concentration of >=32 and all isolates were sensitive to Tigecycline (100%). Genomic DNA was extracted for the bacterial isolates under study and revealed that the bacteria possessed the integron. The results showed that S. aureus bacteria had the genes of the integron (intI1, intI2, intI3) using a specific promoter. The results showed that the most commonly found genes in S. aureus bacteria were the intI1 gene by 41 (82%), followed by the intI2 gene (5%). All isolates gave a negative result for the intI3 gene. The result of molecular typing using the agr method showed that the percentage of the presence of the genes agr1, agr2, and agr3 for S. aureus bacteria was 88, 6, and 35%, respectively, and all the isolates under study showed that they did not contain the agr4 gene. Keywords: Staphylococcus aureus, integron class І. П. Ш, agr group. 1.Introduction The epidemiology of Staphylococcus aureus bacteria is one of the most important aspects that must be taken care of and taken care of because of its importance to the individual and population in order to control its distribution, confront the disease and prevent it, these bacteria work to settle different areas of the body such as the nose, pharynx, airways, armpits and skin and found about 25-30% endemic healthy people and this percentage increases in people working in hospitals about 80% [1]. Integron is defined as elements of animated DNA that have https://jih.uobaghdad.edu.iq/index.php/j/index#1609-4042 https://jih.uobaghdad.edu.iq/index.php/j/index#2521-3407 https://orcid.org/0009-0006-2231-6235 mailto:rasha_zaid@ihcoedu.uobaghdad.edu.iq https://orcid.org/0000-0001-6017-2005 mailto:dr.ranamujahid@gmail.com https://orcid.org/0009-0006-2231-6235 mailto:rasha_zaid@ihcoedu.uobaghdad.edu.iq https://orcid.org/0000-0001-6017-2005 mailto:dr.ranamujahid@gmail.com https://orcid.org/0009-0006-2231-6235 mailto:rasha_zaid@ihcoedu.uobaghdad.edu.iq https://orcid.org/0000-0001-6017-2005 mailto:dr.ranamujahid@gmail.com https://orcid.org/0009-0006-2231-6235 mailto:rasha_zaid@ihcoedu.uobaghdad.edu.iq https://orcid.org/0000-0001-6017-2005 mailto:dr.ranamujahid@gmail.com https://orcid.org/0009-0006-2231-6235 mailto:rasha_zaid@ihcoedu.uobaghdad.edu.iq https://orcid.org/0000-0001-6017-2005 mailto:dr.ranamujahid@gmail.com https://orcid.org/0009-0006-2231-6235 mailto:rasha_zaid@ihcoedu.uobaghdad.edu.iq https://orcid.org/0000-0001-6017-2005 mailto:dr.ranamujahid@gmail.com https://orcid.org/0009-0006-2231-6235 mailto:rasha_zaid@ihcoedu.uobaghdad.edu.iq https://orcid.org/0000-0001-6017-2005 mailto:dr.ranamujahid@gmail.com https://orcid.org/0009-0006-2231-6235 mailto:rasha_zaid@ihcoedu.uobaghdad.edu.iq https://orcid.org/0000-0001-6017-2005 mailto:dr.ranamujahid@gmail.com IHJPAS. 37 (2) 2024 112 the ability to implant and move in large pieces such as a plasmid and chromosome and acquire the exchange of gene bands that are mobile genetic elements that are characterized by having a recombination site and a single gene its size ranges between 500-1000 base pairs and usually gene strands merge within the integron between two genetic union sites (attI, attC) or exist in the form of a circular strand of DNA independently and the gene strands have the ability to move within the same genome of the organism or the genome of another organism. The gene strands have antibiotic resistance genes found within the recombination site (attC) that confer resistance to many antibiotics, including β-lactam antibiotics, aminoglycosides, rifampin, erythromycin, lincomycin, quinolones, chloramphenicol, trimethoprim [2]. One of the most common species found in gram-negative and gram-positive bacteria, including Staphylococcus aureus, which was considered important in its resistance to antibiotics through vertical gene transfer [3] is the Class 1 integron. This class is found at 9% in the genome of bacteria and is more prevalent and common among bacteria with clinical sources [2]. This class is directly related to plasmids and transposons, including (Tn3 and Tn402) which play an important role in transferring class I integron within the genome of one organism or transferring it to the genome of another organism [4]. Many studies and research indicate depending on the integrase gene (intI) the presence of the first class integrons within three genetic link sites (attI 1, attC, Secondary site) which is associated with different genomes and this class is characterized by the ability to acquire gene bands responsible for resistance to many antibiotics and that the gene bands of the first class contain aadA resistance genes that code resistance to many antibiotics, including Streptomycin and Trimethoprim and have been observed in Staphylococcus aureus bacteria [2; 4; 5]. Studies indicated that Gram-positive bacteria, including S. aureus integron class 1, contain genes resistant to heavy metals and antibiotics and have the ability to move from chromosome to plasmid and vice versa, causing a genetic mutation after implantation at the target sequence site, and encoding the integron primarily in antibiotic-resistant Staphylococcus aureus [6]. Class 2 integron is structurally and organizationally similar to the first class and it is directly associated with the Transposon family, which includes (Tn7, Tn1825, Tn4132) that carry the region of attI2 genetic reconnection and the promoter pc, which is found within the Transposon family and there are within the Transposon genes (tnsD, tnsC, tnsB, tnsA, tnsE) that mediate the transfer of the second class integron to the bacteria chromosome. Studies indicated that the percentage of amino acid homogenization within the genes of the second class IintI2 is less than 50% compared to intI1 genes that cannot complete the recombination process due to its loss of function and its production of an ineffective and short polypeptide [2]. Studies indicated that the gene bands of the second class intI2 carry the genes dihydrofolate reductase (dfrA1), streptothricin acetyltransferase (sat1), and aminoglycoside adenyltransferase (aadA1), which mediate resistance to trimethoprim, streptothrivin, and streptomycin, and the gene bands of the second category integron also contain a gene blaoxa2 resistant to β -lactam such as the oxacillin and ampicillin found in S. aureus bacteria [6]. It was found in gene bands intI2 carrying the gene ereA mediates resistance to erythromycin and that this is a common type in gram-positive and gram-negative bacteria and is characterized by its ability to resist many antibiotics [7]. [3] explained that the genes of the integron first class are carriers of the aadA gene responsible for resistance to aminoglycosides, while the class 3 integron is organizationally and structurally similar to the first class and the second class, and its presence is associated with microorganisms isolated from soil and fresh water and is similar in function to the genes of the IHJPAS. 37 (2) 2024 113 first class intI1. It has the ability to acquire gene bands at the site of genetic recombination; intI3 is less compared to the first and second-class integrons. The third class was first identified in Japan in 1993, where it was found in Klebsiella pneumoniae bacteria, after which it was identified in a few microorganisms such as Acinetobacter spp., Escherichia coli, Seratia marcescens, and Salmonella spp. isolated from the environment, and it is reported that the percentage of the presence of class III in gram-negative bacteria by 0–10% was resistant to Ceftazidime and Sulbactam-Cefoperazone. Studies have also indicated that its presence in bacteria isolated from animals is 7% [2]. The regulator of virulence factors Agr was first described in 1988 and codes for many virulence factors produced by S. aureus bacteria, such as hemolysin, toxic shock syndrome, staphylokinase enzyme, protein regulation, and biofilm [8; 9]. The helper genome, which differs from the basic genome, carries the basic genes that encode many activities such as growth and nutrition. The accessory genome includes mobile genetic elements (such as transposons, plasmids, phages, insertion sequences, pathogenicity islands, and genomic islands). Moving elements play an important role in regulating many virulence factors, such as transposons and plasmids. They carry genes that encode antibiotic resistance, and pathogenicity islands include Vsa1 and Vsa2. About 50% of the virulence factors responsible for encoding many intestinal toxin genes (Seb, toxic shock, and genomic syndrome) are encoded by genomic islands, which encode many virulence and antibiotic resistance factors. The bacterial bacteriophage encodes many of the virulence factors of these bacteria, such as exfoliative toxin, Panton-Valentine leucocidin (PVL), and some intestinal toxins, and the regulator of virulence factors Agr is the most studied virulence regulator and depends on the mechanism of quorum sensing, which works to regulate many virulence factors and toxins. The agr regulator consists of two units encoded by operon p1 and operon p2, where p2 encodes four types of proteins: AgrA, AgrB, AgrC, and AgrD, and the AgrD protein encodes the quorum sensing in S. aureus bacteria, which is a pentagonal structure AIP (autoinducing peptide) secretes to the outside through the phosphorylation of the AgrC protein and then activates AgrA, which works in a cooperative manner with the regulator SarA and encodes to produce operon p2 and p3, thus producing high levels of RNAIII catalyzed by the production of many virulent agents, including a-hemolycin, leukocyte toxin, lipase, and fibronectin-binding protein. At the end of the stationary phase and the logarithmic phase, as well as the production of RNAII, amplifying the quorum sensing signal [10, 11; 12; 13], The current study used the agr method to detect integron I, II, and III as well as genotype S. aureus bacteria with multiple antibiotic resistance. 2. Materials and Methods 2.1. Isolation of bacteria 220 samples were collected from different clinical sources: 30 samples of urinary tract infection, 50 samples of wounds, 40 samples of burns, 30 samples of sputum, and 70 samples of the cervix from several hospitals in the city of Baghdad: educational laboratories, Baghdad Teaching Hospital, Al-Saheed Ghazi Al-Hariri Hospital for Specialized Surgery, Burns and Wounds Hospital, and Medical City, during the period from 1/10/2022 to 30/1/2023. 2.2. Diagnosis of isolates 2.2.1. Culture diagnosis Bacterial isolates were cultured on the Mannitol salt agar and Blood agar [14]. IHJPAS. 37 (2) 2024 114 2.2.2. Gram Stain Smears from bacterial isolates growing on mannitol salt agar were prepared at 24 hours of age, stained with gram stain, to determine whether the bacteria were Gram-positive or Gram- negative, and examined with light microscopy to observe the shape of the cells, their interaction, and their arrangement with the gram stain [15]. 2.2.3. Biochemical test Including: the Oxidase test [16], Catalase test [15], Coagulase test [17] and the final diagnosis of the isolates was done using the Vitek-2 device and according to the manufacturer's instructions. 2.3. Bacterial sensitivity testing was carried out using the Vitek-2 system This is to determine the resistance of Staphylococcus aureus to 15 antibiotics, which included Linezolid, Ciprofloxacin, Gentamicin (Benzylpenicillin, Oxacillin, Fusidic Acid, Ciprofloxacin, Moxifloxacin, Erythromycin, Teicoplanin, Rifampicin Clindamycin, Vancomycin, Tetracycline, Tigecycline, Trimethoprim/ Sulfamethoxazole). 2.4. DNA isolation A Genomic DNA Extraction kit prepared by Zymo (USA) was used to extract DNA for the bacterial isolates under study according to the manufacturer's instructions. 2.5. DNA Examination The concentration and purity of DNA extracted from isolated samples using a nanodrop spectrophotometer are detected by determining the concentration of DNA, measuring its purity, and reading its absorption at a wavelength between 260 and 280 nm. 2.6. Molecular diagnosis of the integron genes of S.aureus bacteria This study was conducted using PCR technology to detect the integron genes (intI1, intI2, intI3) and the agr genes (agr1, agr2, agr3, agr4), as shown in Table 1. Table 1. Oligonucleotide primer sequence and Size amplicon. Reference Product Size(bp) Initial Sequence From 5′ to 3′ Target gene Ye et al. [6] 280 F-5′- CCTCCCGCACGATGATC-3 IntiI R-5′- TCCACGCATCGTCAGGC -3′ Ye et al. [6] 233 F-5′- TTATTGCTGGGATTAGGC -3 IntiII F-5′- ACGGCTACCCTCTGTTATC -3′ Ye et al. [6] 600 R-5′-AGTGGGTGGCGAATGAGTG-3 IntiIII F-5′-TGTTCTTGTATCGGCAGGTG-3 Gilot et al. [18] 441 R-5′-ATGCACATGGTGCACATGC -3 Agr1 F-5′- GTCACAAGTACTATAAGCTGCGA-3 Gilot et al. [18] 575 R-5′-ATGCACATGGTGCACATGC -3 Agr2 F-5′-TATTACTAATTGAAAAGTGGCCATAGC -3′ Gilot et al. [18] R-5′-ATGCACATGGTGCACATGC -3 Agr3 323 F-5′-GTAATGTAATAGCTTGTATAATAATACCCAG -3 Gilot et al. [18] 659 R-5′-ATGCACATGGTGCACATGC -3 F-5′-GTAATGTAATAGCTTGTATAATAATACCCAG -3 Agr4 R-5′-CGATAATGCCGTAATACCCG -3 The reaction mixture was prepared to diagnose the genes: 25 μl for each gene (GO Taq Green Master Mix μl, 1.5 μl from DNA template, 1 μl from Primer Forward, and 1 μl from Reverse, 16 μl from ion-free distilled water), according to the manufacturer, Integrated DNA Technologies Company, Canada. The reaction conditions were determined according to the following program with modifications, as shown in Tables 2 and 3. IHJPAS. 37 (2) 2024 115 Table 2. The optimum condition of detection integrons gene No Stage Temperature Time No. cycles 1 Initial Denaturation ᵒC94 5 min 1 2 Denaturation ᵒC 94 Sec45 35 3 Annealing 45sec IntI1 ᵒC58 IntI2 ᵒC60 IntI3 ᵒC58 4 Extension ᵒC72 1min. 5 Extension -2 ᵒC72 7 min. 1 Table 3. The optimum condition of detection agr gene. No. Phase Tm (ᵒC) Time No. of cycle 1 Initial Denaturation 94ᵒC 5 min. 1 cycle 2 Denaturation -2 94ᵒC 30 sec. 35 cycle 3 Annealing 57 ᵒC 90 sec. 4 Extension-1 72ᵒC 60 sec. 5 Extension -2 72ᵒC 7 min. 1 cycle 2.7. The reaction products were separated using Agarose: Bio Basic INC (Canada) with a concentration of 2% containing 5 μl of Red Safe Nucleic Acid Staining Solution and using DNA ladder (100-1500) base pairs with a voltage difference of 5 volts for an hour was imaging using UV light Optima (Japan). 2.8. Phylogenetic tree Construction Photo captions were used to calculate the molecular size to detect bands resulting from PCR reactions and compare them to the size of the DNA Ladder [19]. 3. Results and Discussion Identification was made based on the morphological characteristics of isolates when cultured on blood agar. The bacterial colonies appeared convex in shape, shiny with smooth, rounded edges, and white , tending to lead to a golden yellow and fully β-hemolysis type [20] when grown on mannitol salt agar containing 7.5% NaCl. Bacterial colonies appear with a non-zigzag edge, a rounded shape, and are yellow. They are fermented for mannitol with a diameter ranging from 0.1–0.8 mm, which acts to decrease the pH and thus leads to the appearance of a yellow color. This is considered a differential examination between the species of staphylococci [21]. Microscopic examination of slides prepared colonies stained with gram-stain showed that gram- positive form clusters resembling bunches of grapes with a dark purple color, and non-spores- forming [22], and when performing biochemical tests approved for the diagnosis of S.aureus bacteria, Oxidase test, Catalase test and Coagulase test were used, all isolates gave a negative result for the Oxidase test, all isolates showed a positive result for the Catalase test and all isolates were given initially diagnosed as a positive result of the Coagulase test conducted by the tube method when a layer appears inside the tube indicates the ability of bacteria to produce the plasma coagulant enzyme and works to convert fibrinogen to fibrin [23]. After conducting laboratory tests, culture diagnosis, and using the Vitek-2 Compact System, 50 isolates of S. aureus bacteria were obtained out of a total of 220 isolated samples from different clinical cases, both genders and different ages, and from several hospitals in Baghdad. The results of the study showed that the highest percentage of S. aureus isolates was from burns (15 isolates) at 30%, followed by wounds (12 isolates) at 24%, urine (10 isolates) at 20%, and the lowest percentage IHJPAS. 37 (2) 2024 116 of infection isolated from the cervix (8 isolates) at 16% and sputum (5 isolates) at 10%, as shown in Table 4. Table 4. Source, Numbers and Percentages of S.aureus isolates. Source of isolate Number of isolates Percentage% Urine 10 20 Wounds 12 24 Cervicitis 8 16 Burns 15 30 Sputum 5 10 Total number 50 100 The results were in agreement with the findings of local studies [24] and [25] in their study conducted in China that the highest rate of injury from burns is 60%, and the result is also consistent with the results of [26], who showed that the highest percentage of injury was isolated from burns at 51 (53.13%), and the result was close to the findings of [27] and [28], who showed that the percentage of these bacteria isolated from burns and wounds was 40.36%, respectively. The result differed from the [29] study; the percentage of staphylococcal bacteria isolates from wound samples was 57.1%, the lowest rate of infection, which amounted to 7% of urinary tract infections, and the result agreed with the results [30], who recorded the highest rate of infection in males at 90% compared to females at 60%. Staphylococcus aureus is one of the most pathogenic bacteria that can infect the community and cause infections in hospitals because of its ability to transmit infection and its multiple resistances to antibiotics [31]. The isolates showed varying antibiotic resistance when using 15 antibiotics using the Vitek-2 system. All isolates showed 100% resistance to benzylpenicillin at the minimum inhibitor concentration (>=0.5–0.12). The resistance to Oxacillin was 76% at concentration (>=1-4), and for each of the two antibiotics Ciprofloxacin and Moxifloxacin by 42%, 40% at concentration (>=2-8), Gentamycin by 20% at a concentration of >=16, Erythromycin by 78% at a concentration of >=8, and Clindamycin by 74% at concentration of (>=8-16), Tetracycline by 40% at concentration of (>=2-16), Fusidic acid by 14% at a concentration of (>=8-32), Rifampicin by 6% at a concentration of (>=32), and Trimethoprim/Sulfamethoxal by 14% at a concentration of (>=80-320). The isolates showed the least resistance to Vancomycin, Linezolid, and Teicoplanin (5, 2, and 2%), respectively, at the minimum inhibitor concentration (>=32), as shown in Table 4. The results of the current study agreed with many studies, including [32] study which recorded the percentage of resistance o4. aureus to Benzyl penicillin (100%) and the study differed with the results of [33] who found resistance of S. aureus isolates to benzyl penicillin by 66.8%. The current study showed that the percentage of resistance to oxacillin was 76%, and it was closely compatible with the studies of [34], [35], [36], and [37], who recorded the percentages (90, 81.92, 62.7, 90%), respectively. The result of the current study agreed with the results of [39] Abdlel-Mongy et al. (2018), who found that the resistance rate of erythromycin was 78%, and the result of the current study differed from the results reached by [40] that they conducted on 200 isolates of Staphylococcus aureus. The resistance rate for this antibiotic was 29.5%, and the current study showed that the resistance rate of Clindamycin was 74%. The result agreed closely with the study carried out by [41] and the study carried out by [42], who showed that the percentage of resistance to Clindamycin reached 68.8% and 80%, respectively, and studies indicated that Clindamycin is used to treat skin injuries in people who IHJPAS. 37 (2) 2024 117 are allergic to penicillin [43]. S. aureus isolates showed resistance to Gentamicin by 20%. [40] stated that the percentage of resistance to Gentamicin was 7.5% and the results of the study disagreed to the study carried out by [44], they found a resistance rate to Gentamicin was 48.48%. The study showed that the percentage of resistance to Ciprofloxacin and Moxifloxacin reached 42 and 40%, respectively, The result of the study showed that the resistance rate of S. aureus to tetracycline was 40%. [37] indicated in their study that the percentage of resistance to tetracycline reached 46%. The percentage of resistance to fusidic acid, trimethoprim, and sulfamethoxal in the current study was 14%, and the result was fairly close to the result of the study carried out by [49], who found a resistance rate of 6.70%. The current study showed that the percentage of resistance to Rifampicin was 6%, and [27] and [52] found the percentage of resistance to Rifampicin to be (47, 61.8%), respectively, which is higher than the findings of this study. Rifampicin combined with vancomycin, is used to treat MRSA-resistant Staphylococcus. The reason for the resistance of S. aureus bacteria to rifampicin, which works to inhibit the synthesis of RNA polymerase, is the occurrence of chromosomal mutations encoded by the rpoB gene that prevent the antibiotic from binding to the target site [53]. The current study showed the least resistance to the antibiotics Vancomycin, Linezolid, and Teicoplanin by (5,2, 2%), respectively. [40] found in their study that the sensitivity percentages of S. aureus to antibiotics (Linezolid, Tigecycline, and Teicoplanin) were 100%, which was closely consistent with the current study and differed from the [54] study, which showed the percentage of resistance to Linezolid was 16.9%. Vancomycin was an effective antibiotic for staphylococcus resistant to MRSA. The isolates under study showed that the least resistance to vancomycin was 5%. [51] showed that the sensitivity percentage of S. aureus bacteria to Vancomycin was 100%, and the result of the current study differed from the study carried out by [55] and [54], who found the percentage of resistance to vancomycin to be 10 and 13.5%, respectively. Table 5. Shows the percentage of the minimum inhibition values of S.aureus bacteria. Antibiotic Resistance Resistant Values of MICs No. Isolates Percentage % Benzylpenicillin 50 100 0.5 -0.12 ≤ Oxacillin 38 76 1-4 ≤ Gentamicin 10 20 16 ≤ Ciprofloxacin 21 42 2-8 ≤ Moxifloxacin 20 40 2-8 ≤ Erythromycin 39 78 8≤ Clindamycin 37 74 8-16≤ Linezolid 1 2 ≤32 Teicoplanin 1 2 ≤32 Vancomycin 2 5 32≤ Tetracycline 20 40 2 -16≤ Fusidic Acid 7 14 8- 32≤ Rifampicin 3 6 ≤32 Trimethoprim/Sulfamethoxazole 7 14 80- 320 ≤ The intI1 gene was detected in S.aureus isolates and the percentage was 82% and when comparing the multiplied bands with the volumetric index, it was found that the resulting bands with a molecular weight of 280 base pairs as shown in Figure 2 A and B. The result showed the detection of the intI2 gene that the percentage of S.aureus possession of the intI2 gene was 5% IHJPAS. 37 (2) 2024 118 and when comparing the multiplied bands with the volumetric index, it was found that the resulting beams have a molecular weight of 233 as in Figure 2 and when investigating the intI3 gene, the result of the current study showed a negative result for all isolates as shown in Table 6. Table 6. Shows the distribution of the integrons between S.aureus isolates No Source Number of isolates with integrons gene IntI1 No. (%) IntI2 No. (%) IntI3 No. (%) 1 UTI 10(20) 0 0 2 Wounds 11(22) 0 0 3 Cervix 6(12) 0 0 4 Burns 10(20) 2(10) 0 5 Sputum 4(8) 0 0 Total No. 41(82) 2(10) 0 Figure 2. A- Electrophoresis of PCR product for S.aureus isolates on agarose gel at a concentration of 2% and a potential difference of 5 V for 60 min for the M path represents the volumetric index (1500-100 ) ,280 base pairs) A: Pathways (12, 19, 26, 31, 33, 40, 48) of intI1 for positive isolates Figure 2. B- Electrophoresis of PCR product for S.aureus isolates on agarose gel at a concentration of 2% and a potential difference of 5 V for 60 min for the M pathway represents the volumetric index (1500-100), Pathways (36- 40) for intI2 positive isolates, 233 base pairs. The results were close to the findings of [7] who found the percentage of integron 1 was 101 isolates (72.6%) out of a total of 139 isolates and the percentage of intI2 in S.aureus isolates was IHJPAS. 37 (2) 2024 119 49 isolates (35.2%). The study carried out by [56] also indicated that the first class integron in S. aureus bacteria isolated from different clinical cases in Iran by (56.3%) and the second class integron by (18.7%) and the result of the third class integron was negative for all isolates. [3] who found that the percentage of S.aureus had of the intI1 gene was (46.6%) and the intI2 gene was (3.4%). [57] were showed in their study at China that the percentage of S. aureus bacteria had the intI1 gene was (85.1%) and the percentage of intI2 and intI3 were negative for all isolates. [45] showed that the possession percentage of S.aureus class integrin 1 was (24.8%) and all isolates showed negative result of class IntI 3 and studies carried out by [7] indicated that the genes of the first class integron carry genes (aadA, aadB), which are responsible for resistance to aminoglycosides antibiotics such as (Gentamicin, tobramycin, Kanamycin, Streptomycin) and also carry the genes dfrA1 and dfrA11 mediate resistance to trimethoprim and also carry blaoxa2 genes that code resistance to β-lactam and carry genes (catB3, catB8, cmlA6) responsible for resistance to chloramphenicol antibiotic. Studies indicated that the genes of the second class of integron that carry the genes of dihydrofolate reductase (dfrA1) acetyltransferase (sat1) and aminoglycoside adenyltransferase (aadA1) responsible for resistance to trimethoprim, streptothrivin, streptomycin, and the genes of the second class integron also contain the gene blaoxa2 responsible for resistance to β-lactam antibiotics such as oxacillin and ampicillin, which was found in S. aureus bacteria [6]. [58] explained that the genes of the class 1 integron that carry the sul1/qacEΔ1 gene responsible for resistance to antibiotics, erythromycin, ciprofloxacin trimethoprim/ sulfamethoxazole. The result of electrophoresis when detecting AGR A genes showed that the percentage of agr1, agr2, and agr3 presence for S. aureus bacteria was 88, 6, and 35%, respectively, and by comparing the multiplied bands with the volumetric index, it was found that the resulting bands had a molecular weight of 441, 575, and 323, base pairs, as shown in Table 7. and Figure 3. Table 7. Shows the distribution of agr in relation to the source of isolates No Source Agr group Agr1 No. (%) Agr2 No. (%) Agr3 No. (%) Agr4 1 UTI 10 1 8 - 2 Wounds 11 1 10 - 3 Cervix 5 - 7 - 4 Burns 13 1 5 - 5 Sputum 5 - 5 - Total No. 44(88) 3(6) 35(70) - Figure 3. Electrophoresis of PCR product of genes (agr1 441, agr2 575, agr3 323, agr4 659) base pair for S.aureus isolates on agarose gel at a concentration of 2% and a potential difference of 5 V for 60 minutes. Pathway M represents volumetric index 1500-100 base pairs, pathways (1-30) for agr gene of positive isolates. IHJPAS. 37 (2) 2024 120 The current study agreed with the results of [59] Rasheed and Hussein (2020), who showed that the percentage of the agr presence in staphylococcal bacteria MRSA was 51 (76.1%), 4 (5.62%), and 8 (10.52%), respectively, out of a total of 67 isolates, and all isolates showed a negative result for the agr4 gene. [13] showed that S. aureus had agr1 of 16 (29.09%), agr2 of 30 isolates (54.54%), agr3 of 6 isolates (10.9%), and agr4 of 3 isolates (5.45%) out of 55 isolates. [60] found that the rate of regulation of the virulence genes agr1 in the resistance S. aureus was 14 (50%), agr2 was 3 (10.7%), agr3 was 10 (35.8%), and agr4 was 1 (3.5%) of a total of 28 isolates. The current result differed from the study carried out by [61], who showed that the percentage of Agr1 presence in bacteria was 5% and the percentage of Agr3 presence was 55%. [62] showed that the presence rate of Agr1 was 131 isolates (78.4%), Agr2 was 17 isolates (10.2%), and Agr3 reached 8 isolates (4.8%). [28] showed that the presence rate of Agr1 reached 82 isolates (55%), Agr2 was 37 isolates (25%), Agr3 reached 10 isolates (7%) and Agr4 was 21 isolates (14%). Studies have indicated that the AGR regulator codes for several genes responsible for virulence in S. aureus bacteria (such as a and β-hemolysin, leucocidin, lipase, toxic shock syndrome tsst, coagulase, and fibronectin-binding protein [63, 64]. Studies have indicated that each group of agr regulators mediates several diseases, such as agr4 encoding exfoliative toxin (eta etb) and impetigo, as well as agr1 and agr2 encoding many enterotoxin genes (Seg, Sei, Sem, Seo), leukytox-lethal genes LukF-PV and LukS-PV, and toxic shock syndrome genes (tst) regulated by agr1, agr2, and agr3 [65, 66]. [67] shows that bacterial resistance to MRSA is regulated by Agr2. Many researchers have confirmed the use of the agr method as a method of genotyping [60]. Studies have indicated that the typing method has become important in working to find genetic relationships between bacterial strains, classify bacteria on the basis of their epidemiology, identify methods and sources of infection, distinguish bacterial strains with high virulence and treat them to prevent their spread [68]. The genetic relationship was found and identified by typing S. aureus under study by using the agr method, where the sequences of this method were found spread in multiple regions of the bacterial genome agr1, agr2, agr3, agr4. The results of the current study showed the existence of genetic relationship between bacterial isolates isolated from different clinical sources. The results of the current study showed the presence of four clones, which is clonal A includes 5 (Figure 4) bacterial isolates found between them with genetic convergence and in one clonal and isolated these isolates from patients in Baghdad Teaching Hospital and the source from which these isolates were isolated was from cases of wounds and urine and these isolates had the genes intI1, agr3 and resistance to antibiotics and clonal B includes 7 bacterial isolates found between them genetic convergence and isolated from patients in the hospital burns and wounds /Medical City and the source isolates burns and wounds and had the genes (intI1, agr1) and integron intI1 and clonal C includes 28 bacterial isolates found between them genetic convergence and isolated from Baghdad Teaching Hospital, Burns and Wounds Hospital and Al-Shaheed Ghazi Al-Hariri Hospital for Specialized Surgery and the source of isolates burns, wounds and sputum characterized by having agr1. agr2. agr3. intI1., int2. The clonal D includes 7 bacterial isolates found between them genetic convergence and in one clonal isolated from several hospitals in the Medical city and the source of isolates were cervical, sputum and burns and it is characterized by having genes (agr1, agr3, int1). and found three isolates of bacteria that were not related to each other and were isolated from private clinics. it is characterized by having genes (agr1, agr2, agr3, int1). The result of the current study as shown in Table 8. appeared that isolates have agr1, agr3 genes and they are IHJPAS. 37 (2) 2024 121 characterized by their resistance to many antibiotics The results showed that Integron 1 is characterized by its resistance to antibiotic Tetracycline, Vancomycin, Ciprofloxacin, Moxifloxan). Figure 4. Dendogram of S.aureus isolates using Past Jaccard/up GMA. Table 8. Distribution of integron and agr among S.aureus isolates. NO. Intl1 Intl2 agrtype Resistance profile 1 + - 1 Cip.Oxa.Ben 2 + - 1.3 Tet.Clind.Eryth.oxa.Ben 3 + - 1.3 Tet.Clind.Eryth.oxa.Ben 4 + - 1.3 Tet.Clind.Eryth.oxa.Ben 5 + - 1 Tet.Clind.Eryth.oxa.Ben.Genta.Vanco.Teico.Linez.Cip.Moxi 6 + - 1.2.3 Tet.Clind.Eryth.oxa.Ben.Genta.Cip.Moxi 7 + - 1.3 Tet.Clind.Eryth.oxa.Ben.Genta.Cip.Moxi 8 + - 1.3 Tet..Eryth.oxa.Ben.Genta.Cip.Moxi 9 + - 1.3 Tet..Eryth.oxa.Ben.Genta.Cip.Moxi 10 + - 1.3 Tet..Eryth.oxa.Ben.Genta.Cip.Moxi.Trime 11 + - 1.3 Clind.Eryth. Ben. 12 - - 1.3 .Eryth.oxa.Ben.Genta.Cip.Moxi 13 + - 1.3 Tet..Eryth.oxa.Ben.Genta.Cip.Moxi.Trime.clind 14 + - 1.3 Tet..Eryth.oxa.Ben.Cip.Clind.Moxi 15 + - 3 Tet..Eryth.oxa.Ben. Cip.Moxi. 16 + - 1.3 Clind.Tet.Eryth.oxa.Ben 17 + - 1.3 Tet..Eryth.oxa.Ben.Genta.Cip.Moxi. 18 + - 1.3 Tet..Eryth.oxa.Ben..Cip.Moxi.Trime.clind 19 - - 1.3 Clind,Eryth.Ben 20 + - 1 Tet..Eryth.oxa.Ben. Cip.Moxi.Trime.clind 21 + - 1.2 Fusidic.Ben.oxa 22 + - 1 Clind,Eryth.Ben 23 + - 3 Oxa.Ben 24 + - 3 Clind.Eryth.Ben 25 + - 3 Oxa.Ben 26 - - 3 Clind.Eryth.Ben 27 + - 1.3 Clind,Eryth.Ben 28 + - 1.3 Oxa.Ben 29 + - 1.3 Clind,Eryth.Ben 30 + - 1 Oxa.Ben.Clind 31 - - 1.3 Clind,Eryth.Ben 32 + - 1 Clind,Eryth.Ben 33 - - 1.3 Oxa.Ben 34 + - 1.3 Clind,Eryth.Ben 35 + - 2 Oxa.Ben IHJPAS. 37 (2) 2024 122 NO. Intl1 Intl2 agrtype Resistance profile 36 + + 1 Clind,Eryth.Ben 37 - - 1 Oxa.Ben 38 - - 1 Clind,Eryth.Ben.oxa 39 + - 1.3 Tet..Eryth.oxa.Ben. Cip.Moxi..clind 40 + - 1.3 Tet..Eryth.oxa.Ben. Cip.Moxi..clind.vanco 41 - + 1.3 Tet..Eryth.oxa.Ben. Cip.Moxi..clind 42 + - 1 Clind,Eryth.Ben.oxa 43 + - 1.3 Tet..Eryth.oxa.Ben. Cip.Moxi.Trime.clind.Genta.Fusidic.Rifam 44 + - 1.3 .Tet..Eryth.oxa.Ben. Cip.Moxi..clind.Genta.Fusidic. 45 + - 1.3 .Tet..Eryth.oxa.Ben...clind.Genta.Fusidic. 46 + - 1 Clind,Eryth.Ben 47 + - 1.3 .Eryth.oxa.Ben..Cip.Moxi.clind 48 - - 1 Ben.Oxa 49 + - 1.3 clind..Eryth.oxa.Ben.Cip.Moxi 50 + - 1 clind..Eryth.oxa.Ben.Cip.Moxi ● Oxa:Oxacillin, Genta: Gentamicin, Cip:Ciprofloxacin, Moxi: Moxifloxacin, Eryth: Erythromycin, Ben: Benzylpenicillin, Clind: Clindmycin, Linez: Linezolid, Teico Teicoplanin, Vanco: Vancomycin, Tet: Tetracyclin, Tige: Tigecycline, Fusid: Fusidicacid,Rifam: Rifampicin. Trime: Trimethoprim/ Sulfamethoxazle 4. Conclusions Staphylococcus aureus isolates were more prevalent in burn samples, according to the research findings. The results of the detection of the genes of IntI1 were major among these isolates and were characterized by their resistance to antibiotics such as Tetracycline, Vancomycin, Ciprofloxacin, and Moxifloxacin. 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