Human eye, which is constantly exposed to the external environment, is a unique organ serving as the window of our body. Ocular disease with its complications, due to microorganisms, is a significant health problem worldwide particularly in the least income countries [1]. Conjunctivitis, blepharitis and dacryocystitis are considered the most common manifestations of external eye infections [2,3]. These pathogenic microorganisms include bacteria, fungi, viruses and parasites [2]. Drug resistant S. aureus strains are seen in a few S. aureus strains [4] developed. The strains of Staph. aureus that was resistant to antibiotics containing beta-lactams, such as Penicillins, Amoxicillin, Methicillin, Ampicillin, Cephalosporins, Oxacillin and others [5,6]. The inclination is to the right. Antibiotic resistance was acquired by aureus, resulting in a worldwide clonedistribution of antimicrobial resistance expressions. Instead of MRSA strains, there are numerous bacterial diseases that cause mortality in the public and clinics [7]. S. aureus infection, like MRSA strains, has been around for a long time [7]. Since the indiscriminate use of antibiotics is not a standard process, hospital facilities are not sanitary and patients and health personnel are overburdened Infectious bacteria, such as S.auroreus, are spread more easily [8]. As a result, it makes sense to assess the state of microbial resistance to the most widely used antibiotics for treating S. aureus-caused eye infections [9]. In recent decades, encapsulation of antimicrobial medicines innanoparticle systems have emerged as a promising carrier approach for increasing therapeutic efficacy while decreasing unwanted side effects. (Troncar) Antibiotic treatment through NPS offers numerous benefits,  including  controlled  and uniform dispersion in the target area, higher solubility, longer release, improved patient compliance, fewer side effects and improved cellular internalization [10].

The cross-sectional study was conducted at Al-Zharr Hospital and private clinics in Kut city of Wasit province in Iraq during the period from December 2022 to May 2023. approval has been obtained from Wasit health Director to conduct the study 

Using simple random sampling technique, patients of either gender were enrolled for obtaining specimens from eye infections. The sample size was calculated using the formula [11]:

    Z₁₋ₐ/₂² * p(1 - p)
n = ──────────────────
           d²

Standard normal variate Z1-α/2 was 1.96 at 5% type 1 error (p = 0.05) and 2.58 at 1% type 1 error (p = 0.01). As p<0.05 are typically regarded as significant in research, 1.96 was utilised in the formula in which p was the population expected proportion based on previous studies [12] and d was the absolute inaccuracy or precision determined by the researchers. 

Briefly, the patient was requested to look up while lowering the eyelid down and the sample was collected from one or both eyes based on the nature of the infection. Sterile cotton swab that had been premoistened with sterile physiological saline was used gently to collect eye discharge. Swab was rubbed softly over the lower conjunctival sac from medial to lateral side and back again [13]. Then simple were cultured on several different media, such as blood agar, Mac Conkey agar and mannitol salt agar the latter being a selective medium for S. aureus. The samples were incubated at 37°C for 24 hours after which cultured bacteria were isolated and identified according to colony morphology, shape, size, colour and pigment production.

Deoxyribonucleic acid (DNA) extractions were carried out using a commercial kit (Presto™ Mini gDNA Bacteria Kit, Geneaid, Thailand) to obtain DNA templates for use in PCR assays. The DNA of P. aeruginosa isolates was extracted as per the manufacturer’s instructions. 

For cell harvesting pre-lyses, the bacterial strains were cultured on mannitol salt agar for 18 hours at 37°C. Then, they were harvested by centrifugation for 1 minute at a speed of 14,000rpm, with the supernatant being discarded.

Further, 20µL of proteinase potassium (K) (solution and 180µL of buffer Guanidinium thiocyanate (GT) were added to the pellet and mixed, with the sample tubes inverted every 3 minutes for the duration of the incubation period.

After mixing for 10 seconds with 200µL of buffer Guanidine Brochloride (GB), the cell lysate was incubated for 10 minutes at 70°C, with sample tubes mixed by inversion every 3 minutes to induce lysis. The elution buffer was pre-heated 200 l/sample at 70°C for DNA elution.

For DNA binding, the lysate samples were treated with 200µL of 100% ethanol and thoroughly mixed by shaking. The mixture was transferred to a spin column in a 2ml collection tube and placed in a new 2ml collection tube for the genome DNA (GD) column.

For DNA elution, the spin column was placed in a 1.5 microcentrifuge tube and 100µL of pre-heated elution buffer was added to the middle of the column matrix. After letting the mixture stand for 3 minutes to ensure that all of the elution buffer had been absorbed, the spin column was centrifuged for 30 seconds at 14,000rpm to elute the purified DNA. The extracted DNA was stored in the freezer at -20°C until use.

The concentration and purity of the DNA was measured by using an instrument (Nano Drop) and agarose gel electrophoresis.

During the process, 1μl of the extracted DNA was added to the instrument in order to detect DNA concentration and purity by analysing the optimal degree )OD( (260/280 ratio to verify the protein and DNA concentration. 

For agarose gel electrophoresis, 1x Tris-borate-EDT (TBE) buffer was placed in the electrophoresis tank, after which the agarose tray was immersed in the electrophoresis tank. It was ensured that the buffer was roughly several millilitres above the agarose surface. Each well was filled with 5μl of the sample and 2μl of dye and the tank was then filled and closed. Electrophoresis was performed using 70 volt/cm of gel run swat electrophoresis. With the use of gel paper, the agarose was extracted from the tank and visualised. 

For the optimization of the primers used, 2.5µl of the master mix was mixed with 5-6µl of DNA, along with 1µl of the forward and revers primers. Optimisation was programmed for SeA, Seb, Sec, Sed, Tst, eta, mecA and Etb gens and primer of gene grades were chosen and the annealing temperature of PCR were set at 55°C, 58°C and 52°C. 

Detection of SeA, Seb, Sec, Sed, Tst, eta, mec A and Etb genes was carried out by mixing 12.5ml master mix, 5-6ml DNA, 1mL each of forward and reverse primers and nuclease-free deionised water to a final volume of 20mL, as per the manufacturers’ instructions. PCR cycling programme parameters used in the reaction for the detection of the genes of interest were noted (Table 1).

Table 1: The Sequence and Source of the Gene Primers Used in the Study

1 = Forward, 2 = Reverse

Data was analysed using SPSS 20. Chi-square test was used to analyse the data. p<0.001 was considered statistically significant [13].

Antimicrobial Susceptibility Test

Antibiotic resistance phenotypes (Methicillin/Oxacillin sensitivity test): All isolates of S. aureus were checked for the sensitivity to 1 μg Oxacillin disc and 5 μg Methicillin disc (Difco) by the disk diffusion method that instructed by NCCLS. The resistance breakpoints were ≥12 mm to ≤10 mm for 1 μg Oxacillin and ≥14 mm to ≤10 mm for 5 μg Methicillin. The capacity of extra antibiotic discs to inhibit MRSA or MSSA was estimated according to the instructions provided by NCCLS using commercially available discs that include: Augmenitin (AC 30 µg), tetracycline (T,30 µg), erythromycin (E,15 µg), ceftizoxime (CEF 20 µg), ciprofloxacin (Ci 5 µg), clindamycin (CC, 2 µg), clarithromycin (Cl 15 µg) and vancomycin (V, 30 µg). The zone of inhibition produced by S. aureus against each antibiotic was measured and interpreted as resistant and susceptible according to standards of Clinical Laboratory and Standards Institute [14].

Prevalence of S. Aureus Among Various Oral Infections

Out of (154) different eye infection samples, we were able to extract 63 (40.9%) pure S. aureus. Culture, microscopic inspection, biochemical testing and API staph system identification kits were used to identify these isolates. Table 1 shows the prevalence of S. aureus among. various eye infections. The current study show the highest percentage of S. aureus infections was observed in conjunctivitis 23 (36%), followed by blepharoconjunctivitis and the frequency of S. aureus was 15 (24%), then blepharitis 10 (16%), dacryocystitis 8 (13%) While other external eye infections was 7 (8%) in external eye infections (Table 2).

Table 2: Prevalence of S. Aureus Among Various Eye Infections

* Highly Significant Difference (p<0.01)

The present study's findings revealed that S. aureus isolated from eye infections, which may be caused by conjunctivitis, can operate as a reservoir for opportunistic microorganisms. If antibiotics are used to treat periodontal disease or other infections, they can lead to an increase in Staphylococcus spp. in the eye. S. aureus strains can cause antibiotic resistance is widespread and can Periodontitis develops as a result of antibiotic therapy. The fact that S. aureus is more prevalent in the oral cavity might result in a more severe illness. The current percentages of isolated S. aureus are consistent with those reported by (15), who found that conjunctivitis was 36 (33.8%), followed by blepharoconjunctivitis and blepharitis at 19(26.8%) and 12(16.9%), respectively. Also, accord with the findings of (16) who found a prevalence of S. aureus in the eye drops of 21% and eye swabs of 11% in 110 patients attending a eye hospital with a variety of oral illnesses. 13 Salivary carriages of S. aureus was detected in 41% of patients with decreased salivary flow rates attending an eye medicine clinic, with concentrations ranging from 3.7x101 to 5.2x103 cfu ml. Because of the variety of the normal eye flora and the healthy carriage of S. aureus in specific patient groups, the case for S. aureus in the etiology of eye dysaesthesia and mucositis is difficult. However, given the high rates of S. aureus recovery in patients with oral mucosal symptoms such as pain, burning, erythema and swelling, physicians should consider the potential of this pathogen playing a role in eye mucosal illness.

Prevalence of Methicillin Resistant S. Aureus (MRSA)

In the current study all 63 coagulase positive isolates of S. aureus were subjected to disc diffusion method to 5 μg Methicillin disc and 1 μg Oxacillin disc to determine MRSA; the test results discovered that 37(58.73%) of isolated S. aureus were MRSA strain Figure 1.

Figure 1: Detection of (MRSA)

The Susceptibility of MRSA and MSSA Isolates to Antimicrobial Agents

The Susceptibility of MRSA and MSSA isolates to antimicrobial agents where shown in the Figure 2,3. The results of current study showed the rate of MRSA was 37/63 (58.73%) from various eye infection is lower than the rate reported from Iraq in previous reports in which MRSA was isolated from 85% of health workers in Basrah city [17], also it is very lower than that reported by Hussein [18], among health care workers in Kurdistan region of Iraq, in 2015 where the MRSA prevalence was 53% On the other hand, study in Iran was 69% [19], while in a study conducted in India, the percentage was much lower 16.6% [20], MRSA prevalence 51.4% at the Korean hospital from the Staph aureus collected from blood and nasal colonizers [21]. In general, MRSA was highly prevalent in Asian countries. In the German study there was a decrease in MRSA rate [22]. In Turkey 2017, high rates of Staph aureus highly resiste to penicillin and ampicillin [23]. A study in Isfahan Iran, in 2018 showed that MRSA was 51.9% among oral infection patients and 16% among health workers [24]. HA-MRSA occurred at a higher rate than CA-MRSA in the world, but in Iraq the rates were similar for the HA-MRSA and CA-MRSA (19.4% and 17%, respectively), as mentioned by [16]. This result can be explained by long hospitalization, random use of antibiotics, lack of awareness and receiving antibiotics before coming to hospital, which are some of the potential predisposing factors for the appearance of MRSA in the hospital and community. Results of current study differs from that reported in the United States of America where a high incidence of MRSA occurred in a hospital-acquired S. aureus infection (HA-MRSA) (59%), compared to a community-acquired infection of S. aureus (17%) [19]. This difference can be explained by the CA-MRSA biology appearing to be different from the HA-MRSA and the MSSA, which may allow CA-MRSA to cause diseases other than those expected from MSSA ]24] regarding Susceptibility of MRSA and MSSA isolates to antimicrobial agents the results showed that MRSA appeared more resistant to antibiotic than MSSA, Wang also found higher antibiotic resistance rat in MRSA compared to MSSA except with Trimethoprim/Sulfamethoxazole [25]. Multi-Drug Resistance (MDR) was more evident among the MRSA than MSSA (26), MRSA in this study were Multi Drug Resistant (MDR), this result was similar to previous research [26]. As this study they reported high resistance to Cifoxitin (100%) amoung MRSA isolates. highest susceptibility for Vancomycin & Genatmycin [27].

Figure 2: The Susceptibility of MRSA Isolates to Antimicrobial Agents

Figure 3: Virulence Genes Distribution of MRSA Isolates

MRSA is resistant to all types of antibiotics containing β -lactam [28], the resistance is conducted with low affinity for β-lactam antibiotics resulting in resistance to all β-lactams antibiotics or due enzymes that hydrolytically destroy β-lactams, MRSA may contain one or both of these mechanisms [29].

Virulence Gens of MSSA

Detection of the MRSA virulence genes by PCR technique All MRSA isolates were tested for seven genes (TssT, Sea, Seb, Sec, Sed, Eta and Etb gene) as well as mecA gen. (Figure 3,4). All isolates and 100% contained Eta and mecA genes, followed by SeA (94.5%), Seb (39.68%), Sec (34.92%), Sed (28.57%), Tsst (34.92%) and Etb (17.46%) that most of MRSA samples harbored at least two virulence gene, multiple toxin gene combinations were also observed. MRSA isolates recovered from eye infections samples contained all genes, MRSA isolate from conjunctivitis samples harbored seven gene, while there blepharoconjunctivitis samples contained mecA, Sea, Seb, Sec, TSST and Eta gene. blepharitis continued mecA, Sea, Seb, TSST and Eta gene; while their other external eye infections contained MecA, Sea and Tst, hospital wards MRSA contained mecA, Sea, Seb and Eta gene, MRSA isolated from Tooth impaction harboring MecA, Sea, Eta gene genes. 

Figure 4: Agarose Gel Electrophoresis Image Shows PCR Product of (Eta & Sea) Genes Analysis of S. Aureus. Lane M Marker ladder (100-500bp), Lanes (1-10): gene of S. Aureus Isolate with 163bp

This result agreed with many studies that showed all MRSA isolate harboring the MecA gene [30], other studies considered that Methicillin resistance can happen in MecA absence, MRSA could have another mechanism (s) for resistance; e.g., altered target site or maybe reduced drug accumulation. MecA gene absence may also be due to a technical error upon detection [31]. Staph aureus could be containing Several Enterotoxins (SEs) that could cause poisoning symptoms when taken. [32] Staph aureus enterotoxin also may be implicated as virulence factors in some cases of toxic shock- like syndromes. In a local study, Sea, Seb rate was (86.78%, 52.2% respectively) [33]. In general, the Sea gene was the most common compared to the Seb, Sec and Sed genes and this corresponds to what was stated in this study results, The TSST coded by Tst gene [34]. The Tsst gene was detected in (35.13%) MRSA isolates in this study, The percentage was close to these studies by [35] (33%,32.6%) respectively, Concerning the Sec gene, Delta hemolysins is 26 peptide amino acids encoded by the gene, the mechanism of secreting delta toxin was not yet been understood [36], high prevalent of Eta gene reported in this study (100%), The result was similar to and (100%), the current study showed the percentage Etb were (18.91%) the result agree with Ezeamagu [37] who reported the Eta was the lower percentage 10 % among the virulence gens in MRSA. MRSA genes variation could be observed in different countries, in the same country, in different cities, or within the same city, in different hospitals, even within a single hospital in different parts. papers discussed above, that possible carriage of MRSA and contaminated hospital environment led to the development of infection.

The prevalence rate of Methicillin –Resistance Staphlococcus aureus isolates among external eye infections was high..Increased resistance rate to ampicillin, penicillin, erythromycin, trimethoprim-sulphamethoxazole, tobramycin and tetracycline was observed. Ciprofloxacin and gentamicin were found to have better activity against Methicillin –Resistance Staphlococcus aureus isolated from external ocular infections.

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