Staphylococcus aureus is a common Gram-positive bacterium known for its dual nature as a commensal organism and an opportunistic pathogen capable of causing a wide range of infections. While skin and soft tissue infections are the most prevalent manifestations, recent studies show a growing trend of respiratory tract infections caused by S. aureus, especially among pediatric populations and hospitalized patients [1,2]. Upper respiratory tract colonization by S. aureus occurs in approximately 30% of individuals, particularly in the anterior nares and nasopharynx. Although this colonization is often asymptomatic, it is widely recognized as a precursor to invasive infection [3,4]. Studies conducted by Okesola and Ige [5] and Özyılmaz et al. [6] have also confirmed the significance of S. aureus among respiratory pathogens in both community and hospital settings. The respiratory tract’s microbial ecosystem is complex and dynamic. Chonmaitree et al. [7] and Kramer et al. [8] described the presence of pathogens such as S. pneumoniae, H. influenzae and S. aureus as part of the nasopharyngeal flora in children, which can lead to infection under certain predisposing conditions like viral co-infection or immune suppression [9,10]. Factors such as age, gender, smoking exposure and underlying diseases like diabetes and asthma influence the colonization and infection rates of S. aureus [11-13]. Furthermore, S. aureus is equipped with an array of virulence factors including coagulase, β-hemolysin, DNase, gelatinase and toxins, which facilitate tissue invasion and immune evasion [14-16]. Its capacity to form biofilms on epithelial surfaces and medical devices significantly contributes to persistent infections [17-19]. These biofilms also enhance resistance to antimicrobial therapy, complicating treatment and increasing morbidity. Antibiotic resistance among S. aureus strains, particularly methicillin-resistant S. aureus (MRSA) and vancomycin-resistant strains (VRSA), has been extensively documented [20-22]. The rise in multidrug-resistant strains has prompted the need for advanced diagnostic techniques. Molecular tools like polymerase chain reaction (PCR) targeting 16S rRNA and femA genes have been adopted for accurate and rapid identification of S. aureus and its resistance markers [23-25]. While the 16S rRNA gene is widely used as a universal bacterial marker, the femA gene is particularly associated with methicillin resistance and is a reliable molecular indicator for MRSA detection. Despite these advances, data on the molecular characteristics and resistance profiles of S. aureus from pediatric respiratory infections remain limited in Iraq. In particular, there is a need to understand the virulence factors and biofilm formation capabilities of local isolates to guide effective infection control strategies and treatment protocols. This study aims to isolate and identify Staphylococcus aureus strains from respiratory samples of children under 12 years old in Kirkuk city and to assess their antibiotic susceptibility patterns, virulence characteristics and biofilm production.

This cross-sectional study was conducted in Kirkuk city, Iraq, from October 1, 2023, to May 1, 2024. A total of 90 respiratory samples were collected from children under 12 years of age attending private consultation clinics with signs of respiratory tract infections. Informed consent was obtained from the parents or guardians of each child and the study protocol was approved by the local ethics committee. Nasopharyngeal swabs were collected using sterile cotton swabs under aseptic conditions. The specimens were immediately transferred to the microbiology laboratory in sterile transport media and processed within one hour of collection to ensure sample integrity. All glassware and media were sterilized by autoclaving at 121°C for 15 minutes at 15 psi pressure. Heat-sensitive solutions were sterilized through 0.22 μM membrane filtration. Chemical reagents including catalase and oxidase solutions, methyl red, VP reagents and MacFarland standards were prepared fresh according to Brown and Smith. Samples were inoculated on blood agar, MacConkey agar and mannitol salt agar and incubated aerobically at 37°C for 24–48 hours. Primary identification was based on colony morphology, pigmentation and hemolysis. Bacterial isolates were subjected to Gram staining and further identified using standard biochemical tests including:

• Catalase, oxidase, coagulase, indole, urease and citrate tests

• Triple Sugar Iron Agar (TSIA) slant, Voges-Proskauer (VP) and Methyl Red (MR) tests

• Additional tests like gelatin liquefaction, mannitol fermentation and Kligler iron agar were also employed

Phenotypic identification of S. aureus virulence factors was performed using:

• Hemolysis on blood agar to determine β-hemolysis 

• DNase production using DNase agar

• Gelatinase and protease detection on gelatin and skim milk agar 

• Lecithinase and lipase production on egg yolk and Tween 20 medium 

• Urease and β-lactamase testing using Christensen’s agar and iodometric methods respectively

Biofilm production of S. aureus isolates was assessed by:

• Tissue Culture Plate (TCP) Method – quantitative and most reliable [17]

• Tube Method – qualitative observation of film lining the tube

• Congo Red Agar (CRA) Method – based on colony morphology [26]

The antibiotic sensitivity pattern of S. aureus was evaluated using the Kirby-Bauer disk diffusion method on Mueller-Hinton agar following Clinical and Laboratory Standards Institute (CLSI) guidelines. Inocula were standardized to 0.5 McFarland and results were interpreted using CLSI breakpoints for commonly used antibiotics.

Statistical Analysis

Data were entered into Microsoft Excel 2019 and analyzed using SPSS version 26.0. Descriptive statistics were used to summarize the data as frequencies and percentages. Chi-square (χ²) tests were used to assess associations between categorical variables such as age, sex, risk factors and bacterial isolation. A p-value less than 0.05 was considered statistically significant. Antibiotic resistance profiles were compared using percentage distributions and biofilm formation was statistically analyzed across detection methods using chi-square tests.

Out of the 90 respiratory samples collected from children under 12 years as shown in table 1, 85 (94.44%) yielded positive bacterial growth, while 5 (5.56%) were culture-negative. This high positivity rate reflects the frequent colonization or infection of the upper respiratory tract among pediatric patients, possibly due to immature immunity or exposure to crowded environments.

Table 2 shows a total of 128 bacterial isolates were recovered from the 85 positive cultures. The most frequently isolated pathogen was Staphylococcus aureus (46.88%), followed by Staphylococcus epidermidis (14.06%), Escherichia coli (10.94%) and Streptococcus pneumoniae (7.81%). These findings align with studies showing that S. aureus remains a dominant pathogen in respiratory infections among children.

Among the 85 positive cultures (table 3), 41 (48.24%) were pure S. aureus, 19 (22.35%) were mixed cultures involving S. aureus and 25 (29.41%) were free of S. aureus. This supports the predominance of S. aureus as either a primary or co-pathogen.

A higher frequency of S. aureus infections was observed among older children (ages 7–12, table 4), likely due to increased social exposure and environmental contacts. The highest prevalence was seen in the 10–12 age group.

Among the 60 patients with S. aureus, 36 (60%) were males and 24 (40%) were females, indicating a slightly higher susceptibility or exposure in boys (table 5).

Analysis of risk factors revealed that 36.67% of the S. aureus-infected patients shown in table 6 were smokers (passive/household exposure), 38.33% had type 2 diabetes, 66.67% had respiratory tract infections, 16.67% had asthma and 5% had a history of malignancy.

All S. aureus isolates were coagulase, DNase, gelatinase and urease positive. Beta-hemolysis was detected in 95%, capsule production in 83.33%, lecithinase in 86.67%, lipase in 73.33% and protease in 35% of isolates (table 7).

A high rate of resistance was observed (table 8): 85% of isolates were methicillin-resistant, 75% were vancomycin-resistant and 70% produced beta-lactamase.

Biofilm assessment revealed that 45% of isolates shown in table 9 were strong producers by the Tissue Culture Plate (TCP) method, 41.67% by tube method and 28.33% by CRA method. CRA also showed the highest intermediate activity (45%).

Table 1: Nasopharyngeal swab culture results

Table 2: Distribution of bacterial isolates

Table 3: Culture types in the study

Table 10 presents the antibiotic sensitivity pattern of Staphylococcus aureus isolates, showing varying levels of susceptibility to the tested agents. The highest sensitivity was observed with levofloxacin (85%), followed by azithromycin, ceftazidime and imipenem (each 75%), indicating that fluoroquinolones and third-generation cephalosporins remain relatively effective options for empirical treatment. Moderate sensitivity was noted for rifampin and erythromycin (66.67%), while cefixime and cefotaxime demonstrated limited effectiveness with just over half of the isolates being sensitive (53.33%). Alarmingly, cefepime and tetracycline showed only 50% effectiveness, suggesting rising resistance levels. Most concerning, all isolates (100%) were resistant to amoxicillin, confirming widespread β-lactamase activity and the ineffectiveness of this commonly used antibiotic. These findings underscore the need for routine antibiotic susceptibility testing and prudent antibiotic use to guide effective treatment and curb resistance development.

Table 4: Age-wise distribution of S. aureus infection

Table 5: Gender-wise distribution of S. aureus infection

Table 6: Risk factors among patients with S. aureus infection

Table 7: Distribution of S. aureus virulence factors

Table 8: Antibiotic resistance as virulence factors

Table 9: Biofilm formation by different methods

Chi-square = 7.71, p-value = 0.034 → Significant correlation between method and biofilm strength.

Table 10. Antibiotic Sensitivity Pattern of S. aureus Isolates 

The current study analyzed nasopharyngeal swabs from 90 children under 12 years old for bacterial pathogens, focusing on Staphylococcus aureus (S. aureus) isolation, virulence factors, antibiotic resistance and biofilm production. A high positivity rate (94.44%) for bacterial cultures was recorded, indicating a significant burden of bacterial colonization among symptomatic pediatric patients. Among the isolates, S. aureus was predominant (46.88%), followed by S. epidermidis (14.06%) and E. coli (10.94%). This aligns with studies by Ahmed and Salim [1] and Zelelie and Mekonnen [2], who reported S. aureus as the most common upper respiratory pathogen in children. Similar findings were reported by Kousalya et al. [3] and Mohammed [4], further confirming the clinical relevance of S. aureus in pediatric nasopharyngeal infections. The infection rate of S. aureus increased with age within the under-12 group and 60% of infections occurred in males. This male predominance is consistent with Batton et al. [11] and Mohammed [4], who attributed the disparity to behavioral and physiological differences, including higher exposure to environmental risk factors and smoking prevalence in male guardians, which may influence household exposure. The presence of risk factors was notable: 66.67% had concurrent respiratory infections, 38.33% had type 2 diabetes and 36.67% were exposed to smoking. These findings are consistent with Choi et al. [12] and Olsen et al. [13], who demonstrated that smoking enhances mucosal susceptibility to bacterial adhesion. Furthermore, Smit et al. [27] and Kanafani et al. [28] highlighted the role of chronic conditions like diabetes and asthma in increasing S. aureus colonization, supporting our results. Staphylococcus aureus demonstrated high sensitivity to levofloxacin (85%), azithromycin, ceftazidime and imipenem (each 75%), whereas it showed marked resistance to amoxicillin, cefepime and tetracycline (≥50%). These findings reflect those of Al-Zoubi et al. [20] and Moghnieh et al. [21], who reported significant resistance to penicillin-class antibiotics. Mama et al. [22] also reported high resistance to β-lactams and erythromycin, reinforcing the importance of empirical treatment revisions. All S. aureus isolates expressed coagulase, DNase, gelatinase and urease. Notably, 95% demonstrated β-hemolysis, 86.67% lecithinase and 35% protease activity. These findings are consistent with the studies by Ibrahim et al. [14] and Al-Jundiy [15], who found high expression rates of virulence enzymes linked to tissue invasion and immune evasion. Among the S. aureus isolates, 85% exhibited methicillin resistance, 75% vancomycin resistance and 70% β-lactam resistance. These findings align with Lin et al. [29] and Al-Zoubi et al. [20], who described the rise of multidrug-resistant MRSA strains and the critical need for surveillance and antimicrobial stewardship. The increasing emergence of vancomycin-resistant S. aureus (VRSA) is particularly alarming due to treatment failures and limited alternatives. The highest rate of strong biofilm formation was observed using the tissue culture plate (TCP) method (45%), followed by tube method (41.67%) and Congo red agar (CRA) (28.33%). These results are comparable with Knobloch et al. [17] and Oliveira et al. [18], supporting TCP as the gold standard due to its sensitivity and reproducibility. Our findings underscore that biofilm-producing S. aureus strains pose therapeutic challenges due to their enhanced resistance and persistence., stated Neamah et al. [24] and Kaur et al. [25]. This study highlights the high prevalence of S. aureus among children with respiratory symptoms in Kirkuk, with significant virulence potential, antibiotic resistance and biofilm-forming ability.

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