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Article

Study of Class 1, 2, and 3 Integrons, Antibiotic Resistance Patterns, and Biofilm Formation in Clinical Staphylococcus aureus Isolates from Hospital-Acquired Infections

by
Eman E. Hegazy
1,*,
Wageih Salem ElNaghy
1,*,
Marwa M. Shalaby
1,
Sarah M. Shoeib
2,
Nashwa S. M. Abdeen
2,
Mohamed H. Fouda
2,
Ola A. Elshora
2,
Mohammed H. Elnaggar
3,
Waleed Elrefaey
3,
Rasha Youssef Hagag
3,
Ahmed A. Elhadidy
3,
Mohamed A. Elsebaey
3,
Mohamed A. Eltomey
4,
Ahmed Mohamed El Nakib
5,
Mai Nabil Ageez
6 and
Maha S. Elnady
1
1
Department of Medical Microbiology and Immunology, Faculty of Medicine, Tanta University, Tanta 31527, Egypt
2
Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta 31527, Egypt
3
Department of Internal Medicine, Faculty of Medicine, Tanta University, Tanta 31527, Egypt
4
Department of Radiology and Imaging, Faculty of Medicine, Tanta University, Tanta 31527, Egypt
5
Department of Tropical Medicine, Faculty of Medicine, Mansoura University, Mansoura35511, Egypt
6
Department of Obstetrics and Gynecology, Faculty of Medicine, Tanta University, Tanta 31527, Egypt
*
Authors to whom correspondence should be addressed.
Pathogens 2025, 14(7), 705; https://doi.org/10.3390/pathogens14070705
Submission received: 26 May 2025 / Revised: 29 June 2025 / Accepted: 15 July 2025 / Published: 17 July 2025
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Versions Notes

Abstract

Antibiotic resistance and biofilm formation complicate Staphylococcus aureus infections, raising concerns for global health. Understanding antimicrobial resistance and biofilm formation in these pathogens is essential for effective infection management. The current research aimed to assess antibiotic resistance patterns, biofilm formation, and the occurrence of integron classes 1, 2, and 3 in clinical S. aureus isolates. The disc diffusion method tested antibiotic susceptibility. MRSA strains were identified by cefoxitin disc diffusion, and the mecA gene by PCR. The D-test also assessed macrolide–lincosamide–streptogramin B. A microtiter plate assay assessed biofilm formation. By PCR, integron classes were examined. Of the 63 S. aureus isolates, 25 were MSSA and 38 were MRSA. Pus (39.5%) was the most prevalent clinical source of MRSA isolates, while blood (24%) was the predominant source of MSSA isolates. MRSA isolates were more resistant to clindamycin, ciprofloxacin, ofloxacin, levofloxacin, tetracycline, and doxycycline than MSSA isolates. In total, 76.2% of the isolates produced biofilm. Biofilm-producing isolates were more resistant to cefoxitin and clindamycin. The isolates had 33.3% cMLSB resistance. The intI1 gene was found in 21 S. aureus isolates (33.3%), whereas the intI2 or intI3 genes were not detected. Our findings demonstrate the need for strict infection control to prevent the spread of resistant bacteria.

1. Introduction

Staphylococcus aureus (S. aureus) is a common human colonizer of skin and mucosal membranes. It is an opportunistic pathogen and a significant human pathogen capable of causing various infections, including superficial and systemic diseases, which lead to exceptionally high morbidity and mortality rates [1,2].
As one of the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species), these organisms are known for their multidrug resistance and association with nosocomial infections worldwide [3]. S. aureus has developed resistance to many antibiotics, like aminoglycosides, β-lactams, and macrolide–lincosamide–streptogramin B, either through chromosomal mutations or by acquisition of resistance determinant from other bacteria. Consequently, the World Health Organization (WHO) has listed S. aureus as a high-priority antimicrobial-resistant pathogen [4,5].
Methicillin-resistant S. aureus (MRSA) is a strain of S. aureus that exhibits resistance to beta-lactam antibiotics. Its multidrug resistant phenotype reduces treatment alternatives and increases human susceptibility to severe infections [6]. This resistance is attributed to target modification mediated by the mecA or mecC genes found within a mobile genetic element called staphylococcal cassette chromosome mec (SCCmec). These genes encode penicillin-binding protein 2a (PBP2a), which has low affinity for β-lactam antibiotics [7].
The mecA gene can be acquired by mobile genetic elements (MGEs) that can transfer around inside a genome or move from one species to another [8]. MGEs, such as plasmids, transposons, and integrons, are responsible for the acquisition and dissemination of antimicrobial resistance genes through horizontal gene transfer [9]. Four classes categorize integrons, and antimicrobial resistance has been associated with integron classes 1–3 [10]. Among these, class 1 and class 2 integrons are the most predominant and have been identified in both Gram-negative and Gram-positive bacteria, including S. aureus [11].
Multidrug-resistant (MDR) S. aureus strains that can form biofilms contribute substantially to treatment failure and the persistence of infections. Biofilms provide a protective shield for the bacteria against antibiotics and the immune responses of the host [12].
Biofilms are complex microbial communities that adhere to surfaces and are surrounded in an extracellular matrix that facilitates horizontal gene transfer of resistance determinants [13]. MDR S. aureus strains with biofilm-forming capacity are particularly associated with severe clinical infections [14].
Integrons have been thoroughly studied in Gram-negative bacteria, but recent studies have begun to emphasize their significance in Gram-positive species, especially S. aureus [15]. The presence of integrons may be associated with increased antibiotic resistance and an enhanced capacity for biofilm formation in S. aureus. Our objective was to evaluate antibiotic resistance patterns, biofilm formation, and the presence of integron classes 1, 2, and 3 in clinical isolates of S. aureus.

2. Materials and Methods

2.1. Study Design and Sample Collection

This hospital-based cross-sectional study investigated 63 non-repetitive Staphylococcus aureus isolates obtained from patients admitted to different intensive care units and clinical departments at Tanta University Hospitals. Various consecutive clinical samples were aseptically collected from hospitalized patients showing signs and symptoms of hospital-acquired infections, according to the infection site, over the course of eight months, from October 2024 to May 2025, including respiratory samples (sputum, endotracheal tube [ETT] aspirates), blood, urine, and pus. The pus was from surgical wound infections (post-caesarean sections, myomectomy, hysterectomy) and burn infections. All samples were transferred immediately to the Medical Microbiology Department laboratory for processing.

2.2. Isolation and Identification of S. aureus

All clinical samples were cultivated on blood agar, nutrient agar, and mannitol salt agar (Oxoid, Hampshire, UK), then incubated aerobically at 37 °C for 24–48 h. Blood samples were processed using the BacT/ALERT 3D 60 automated culture system (bioMérieux, Marcy-l’Étoile, France). Standard biochemical tests were employed for identifying all S. aureus isolates [16]. The identification of species was verified using an automatic VITEK2 system for Gram-positive identification (bioMérieux, Marcy-l’Étoile, France) in line with the manufacturer’s guidelines. All confirmed S. aureus isolates were preserved at −80 °C in brain heart infusion broth supplemented with 20% glycerol (Merck, Darmstadt, Germany) for evaluation of antibiotic susceptibility testing, biofilm formation, and molecular study. For bacterial revival, one loopful was streaked over blood agar and incubated aerobically for 18–24 h at 37 °C.

2.3. Antimicrobial Susceptibility Testing

The Kirby–Bauer disc diffusion method, recommended by CLSI 2025 [17], was used to assess the susceptibility of S. aureus isolates on Mueller–Hinton agar plates (HiMedia, Mumbai, India) to the following antibiotics: erythromycin (15 µg), cefoxitin (30 µg), clindamycin (2 µg), ofloxacin, ciprofloxacin. (5 µg), doxycycline, tetracycline (30 µg), linezolid (30 µg), gentamicin (10 µg), and trimethoprim/sulfamethoxazole (1.25/23.75 µg) (HiMedia, Mumbai, India). Vancomycin susceptibility was determined by minimum inhibitory concentration (MIC) testing using E test strips (bioMérieux, Marcy-l’Étoile, France) according to the manufacturer’s instructions, with CLSI-defined breakpoints: sensitive (≤2 µg/mL) as shown in (Figure 1), intermediate (4–8 µg/mL), and resistant (≥16 µg/mL). MRSA isolates were identified by evaluating their resistance to cefoxitin (FOX, 30 µg) disc. Isolates exhibiting an inhibition zone of less than 21 were classified as MRSA [17]. The S. aureus ATCC 25923 strain was used as quality control.

2.4. Phenotypic Detection of Inducible Clindamycin Resistance

To evaluate if the erythromycin resistant isolates exhibited inducible clindamycin resistance, the D-zone test described in CLSI guidelines [17] was applied. On Mueller–Hinton agar plates inoculated with 0.5 McFarland fresh bacterial suspensions, an erythromycin (15 µg) disc was positioned 15 mm (edge to edge) from the clindamycin (2 µg). After 18 h of incubation at 37 °C, the plates were inspected. The inhibitory zone sizes were interpreted according to the following: flattening the sensitive zone of inhibition to clindamycin (known as the D-zone) near the erythromycin disc, indicative of a positive result for inducible clindamycin resistance (iMLSB phenotype) as shown in (Figure 1). The isolate exhibited erythromycin resistance and clindamycin sensitivity, characterized by circular zones of inhibition, indicative of the MS phenotype. Strains exhibiting resistance to both antibiotics were considered to possess constitutive macrolide–lincosamide–Streptogramin B resistance (cMLSB phenotype).

2.5. Biofilm Formation by Tissue Culture Plate Technique

Staphylococcus aureus isolates were evaluated for biofilm formation as previously explained [18]. Each isolate was first cultured overnight in Trypticase soy broth (HiMedia, Mumbai, India) tryptic soy broth. We adjusted the bacterial suspensions to match a standard turbidity of 0.5 McFarland, then a volume of 100 μL from each suspension was transferred into the wells of a sterile 96-well microtiter plate except for the last column, which is used as a negative control. Then we incubated the plate for 24 h. Following incubation, the wells were carefully evacuated and rinsed with saline to eliminate any non-adherent cells. Biofilm visualization was achieved by staining the wells with 150 μL of 0.2% crystal violet for 15 min at room temperature. We discarded the excess stain and then the wells were cleaned with water. After air drying at room temperature, the retained crystal violet was solubilized with 95% ethanol. The assay was performed in triplicate. An ELISA reader recorded the optical density (OD) of every well at 620 nm. The mean OD values were determined for all examined isolates and negative controls. Based on previously defined standards, the isolates were classified into four groups: non biofilm producers (ODs ≤ ODc) and weak (ODc ≤ ODs ≤ 2× ODc), moderate (2× ODc ≤ ODs ≤ 4× ODc), and strong biofilm producers (ODs > 4× ODc) [19]. ODc describes the OD of the negative control, and ODs describe the OD of the experimental samples.

2.6. Molecular Detection of MRSA and Three Integron Classes by PCR

MRSA isolates based on phenotypic testing (resistant to cefoxitin) have been verified by amplifying the mecA gene. Additionally, all S. aureus isolates underwent amplification of intI1, intI2, and intI3. DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Amplification was performed in accordance with the procedure described by Zhang et al. and Zomorodi et al. [20,21]. The S. aureus ATCC 25923 strain served as a positive control, while distilled water was utilized as a negative control in substitute of the DNA template. The PCR products were visualized and photographed under UV light following electrophoresis for 45 min at 100 V through a 1% agarose that contained ethidium bromide (1 μg/mL) (Figure 2). The utilized primers, the amplicon sizes, and the thermal cycling program are included in Table S1 in the Supplementary Materials.

2.7. Statistical Analysis of the Data

The IBM SPSS 20.0 software (IBM Corp., Armonk, NY, USA, 2011) processed the data entered into the computer. 2011 (IBM Corp, Armonk, NY, USA). Category data were expressed as numbers and percentages. The two groups were compared using a chi-squared test. The Fisher exact test or Monte Carlo adjustment was employed when more than 20% of expected cells counted less than 5. The Kolmogorov–Smirnov test evaluated continuous data normality. Range, mean, standard deviation, median, and interquartile range were used for quantitative data. To compare two normally distributed quantitative data sets, Student’s t-test was used. Result significance was determined at the 5% level. The crude odds ratio (COR) and 95% confidence interval (CI) were computed. Significant variables associated with biofilm production, including type of specimen, mec A, and MLSB phenotypes, were entered into backward Wald binary logistic regression models, with the adjusted odds ratio (AOR) and 95% CI used to investigate the independent determinants of biofilm production. No multicollinearity was detected.

3. Results

3.1. Demographic Data and Clinical Source of MRSA and MSSA Isolates

The basic patient characteristics for isolated MRSA and MSSA are presented in Table 1. We evaluated 63 non-repetitive clinical S. aureus isolates, including 38 (60.3%) MRSA and 25 (39.7%) MSSA isolates from various hospital-acquired infections. The mean age of patients with MRSA was 41.47 ± 14.62 years, whereas those with MSSA were significantly older (49.24 ± 11.68 years, p = 0.030). No statistically significant difference in gender distribution was detected between MRSA and MSSA cases. The most frequent clinical source of MRSA isolates was pus (39.5%), whereas blood (24%) was the predominant source for MSSA isolates.

3.2. Antimicrobial Resistance Patterns Among MRSA and MSSA

The resistance patterns of 63 S. aureus isolates to various antimicrobials and the differences in resistance between MRSA and MSSA are shown in Table 2. All isolates exhibited susceptibility to linezolid and vancomycin. Among the 63 S. aureus isolates, resistance rates were observed as follows: 79.4% to erythromycin, 66.7% to ciprofloxacin, 60.3% to cefoxitin, and 58.7% to levofloxacin. Clindamycin showed the least resistance. MRSA isolates exhibited markedly greater resistance than MSSA isolates for the following antibiotics: clindamycin, ciprofloxacin, ofloxacin, levofloxacin, tetracycline, and doxycycline.

3.3. MLSB Phenotypes and Biofilm Formation

Biofilm formation and MLSB phenotypes among S. aureus isolates are shown in Table 3. The D-test results indicated that constitutive macrolide–lincosamide–streptogramin B (cMLSB) resistance was demonstrated in 21 isolates (33.3%, 21/63), while inducible MLSB (iMLSB) resistance was observed in 17 cases (27%, 17/63). Macrolide–streptogramin B (MS) resistance accounted for 20.6% of isolates (13/63), while 19% (12/63) were sensitive. Biofilm production was detected in 76.2% of the isolates, with 30.2% classified as strong biofilm formers.

3.4. Correlation Between Biofilm Formation and Antibiotic Resistance

Isolates that produce biofilms exhibited significantly greater resistance to cefoxitin (70.8% compared to 26.7%, p = 0.002) and clindamycin (39.6% compared to 6.7%, p = 0.024). No significant differences in resistance to levofloxacin, ciprofloxacin, and gentamycin were observed between biofilm-producing and non-biofilm-producing isolates. (Table 4).

3.5. Characterization of intI1 Gene-Positive S. aureus Isolates

Class 1 integrons were the most prevalent and accounted for 33.3% (21/63), whereas the intI2 or intI3 were not detected. The intI1 gene was detected in 55.3% (21/38) of MRSA isolates, while 44.7% (17/38) were negative for this gene. Additionally, the gene was not detected in any MSSA isolates. All intI1 gene-positive isolates were MRSA and biofilm producers, with most categorized as strong biofilm formers. These isolates exhibited a multidrug-resistant pattern, with frequent resistance to cefoxitin, erythromycin, clindamycin, ciprofloxacin, ofloxacin, levofloxacin, tetracycline, doxycycline, and trimethoprim/sulfamethoxazole (Table 5).

3.6. Logistic Regression Analysis for Predictors of Biofilm Production

The multivariate logistic regression analysis showed significant correlation between biofilm formation and certain microbiological and genotypic factors, particularly the type of specimen: isolates from pus displayed significantly higher biofilm formation p = 0.02), while other specimen types (ETT, blood, sputum) did not reach statistical significance. Regarding the presence of mec A and intI1 genes, the results show a significant correlation between biofilm formation and the presence of mec A gene, with biofilm producers having a much higher rate of this gene compared to non-producers (p = 0.049). intI1-positive isolates all produced biofilm (100%), preventing the calculation of an odds ratio (zero value for non-biofilm producers). In terms of MLSB phenotypes, cMLSB phenotypes were significantly associated with biofilm production p = 0.04) (Table 6).

4. Discussion

Over the past decade, MRSA infections have escalated to epidemic levels worldwide, creating considerable therapeutic difficulties due to the growing complexity of treatment. S. aureus is classified as a high priority for the discovery and production of novel antibiotics by the World Health Organization [22,23]. The current research investigated the prevalence of Class 1, 2, and 3 integrons, biofilm formation, and antimicrobial resistance patterns in clinical Staphylococcus aureus isolated from hospital-acquired infections in a tertiary care hospital in Egypt.
In this study, a total of 63 S. aureus strains were examined. Most strains were isolated from pus samples, followed by blood, with endotracheal aspirates being least frequent (14.3%). The prevalence rate of MRSA in the current study was 60.3%. Our findings agree with those of Zomorodi et al. and Mohammadi et al., who reported that the prevalence of MRSA was 59.6% and 59.1%, respectively [21,24]. Naimi and colleagues in Afghanistan, along with Dendi et al. in Algeria, reported that 56% and 50% of isolated S. aureus strains were MRSA, respectively [25,26].
Significant regional variations exist; for instance, Mahfouz et al. reported a substantially higher MRSA prevalence of 94.5% [27]. Additionally, El Maghraby et al. [28], Amr and Gammal [29], and Saeed et al. [30] found that MRSA accounted for 85%, 78%, and 76% of their S. aureus isolates, respectively. Moreover, studies from Nepal and Eritrea documented rates of 75% and 72%, respectively [31,32]. Variations in antibiotic prescribing practices, infection control measures, and healthcare infrastructure across regions may account for these discrepancies. Conversely, lower MRSA prevalence was reported in other Egyptian studies [33,34,35]. This indicates that there is variety even within a single nation, potentially due to variations in study populations or methodologies. Additionally, Jomehzadeh N. Emrani reported a lower prevalence, finding that 48.3% of the isolated S. aureus were MRSA [19], while Moghaddam et al. and Qodrati et al. observed that 39.4% and 37.5% of examined S. aureus isolates were MRSA, respectively [36,37]. These discrepancies may result from local factors, such as climate or microbe prevalence, while others are likely attributable to differing preventative techniques, topical and systemic treatments, sample regimens, and study durations [38].
Regarding the susceptibility pattern of S. aureus, the current investigation revealed that S. aureus isolates exhibited the highest resistance rates to erythromycin (79.4%), ciprofloxacin (66.7%), cefoxitin (60.3%), levofloxacin (58.7%), and tetracycline (55.6%). S. aureus isolates were resistant to doxycycline and ofloxacin (50.8% each), trimethoprim-sulfamethoxazole (44.4%), gentamicin (38.1%), and clindamycin (31.7%). However, these isolates were all sensitive to linezolid and vancomycin (100%).
In agreement with the findings of the present study, Zomorodi et al. reported that their isolates exhibited the highest resistance to erythromycin and ciprofloxacin, with frequencies of 86.3% and 66.1%, respectively. Furthermore, 59.6% were classified as MRSA. All isolates demonstrated susceptibility to linezolid [21].
In contrast, Adesoji et al. found that clinical S. aureus isolates were completely resistant (100%) to erythromycin but showed the least resistance to ofloxacin (27.5%) and gentamicin (20%) [39]. In addition, Kashef et al. [40] found that tested S. aureus isolates had a greater resistance rate to the fluoroquinolone class (76.3%). Staphylococci features and antimicrobial resistance profiles vary depending on geographical area and antibiotic use [41].
The current investigation revealed significant variations in susceptibility levels to commonly used antibiotics (clindamycin, ciprofloxacin, ofloxacin, levofloxacin, tetracycline, and doxycycline) between MRSA and MSSA isolates. The findings of the present investigation were analogous to those of La Vecchia et al. [42]. MRSA strains were more resistant to clindamycin, macrolides, quinolones, and tetracycline than were MSSA isolates.
Clindamycin is considered a viable option for the management of infections caused by S. aureus due to its oral and parenteral availability, 90% oral bioavailability, low cost, and excellent tissue penetration. It also can suppress the production of S. aureus toxins [43].
The D test results indicate that the cMLSB phenotype was the most common macrolide resistance mechanism, found in 33.3% of cases; the iMLSB phenotype followed at 27.0% and the MS phenotype at 20.6%, while 19.0% of the isolates were completely susceptible to MLSB antibiotics. These phenotypic patterns draw attention to the frequency of MLSB-mediated resistance mechanisms in the examined S. aureus isolates. Our results correspond with various reports indicating that cMLSB is the predominant macrolide resistance mechanism among macrolide-resistant S. aureus isolates [28,44,45,46,47].
A primary cause of antibiotic resistance is the defiant features of the biofilms formed by these bacteria [14]. The capacity of S. aureus to produce biofilms markedly enhances antibiotic resistance and persistent infections [48].
Biofilm formation was evaluated phenotypically using tissue culture plate assays. Among the analyzed isolates, 48 (76.2%) exhibited biofilm production. The level of biofilm formation was categorized as follows: 19 (30.2%) were classified as strong producers, 17 (27.0%) as moderate producers, and 12 (19.0%) as weak producers. These results are consistent with other investigations evaluating biofilm formation by S. aureus isolates [14,49], while other studies show a higher number of biofilm producers. For instance, Abdelraheem et al. [50], Karki et al. [51], El Maghraby et al. [28], and Bimanand et al. [52] determined biofilm in 81.6%, 86.3%, 94.5%, and 96%, respectively, of their isolates. Conversely, Nasr et al. found that 46% of S. aureus isolates produced biofilm [53].
The current study showed that S. aureus strains that produce biofilms were much more resistant to cefoxitin and clindamycin than those that do not produce biofilms. This correlation highlights the function of biofilms in augmenting antibiotic tolerance through reduced drug penetration and metabolic dormancy. This finding agrees with Jomehzadeh & Emrani, and Banerjee et al., who identified significant disparities in susceptibility rates to routinely utilized antibiotics between biofilm-forming and non-biofilm-forming bacteria [19,54].
Integrons are essential genetic elements that retain cassettes with antibiotic resistance genes, contributing significantly to multidrug resistance (MDR) and complicating treatment options, leading to deterioration of patient outcomes [11]. In the present investigation, 21/63 (33.3%) of the S. aureus isolates contained the intI1 gene. This finding was lower than other findings in Iran, China, and Egypt, which found that the incidence of intI1 was 39.6%, 42.5%, and 46.6% of clinical S. aureus isolates, respectively [55,56,57]. This geographical variation may result from differences in antibiotic prescription practices, infection control measures, or genetic characteristics of prevalent S. aureus strains. While the incidence of intI1 was low in the current study, nearly all S. aureus isolates possessed the intI1 gene and were identified as MRSA and MDR, which underscores the significance of this relationship when constraining treatment options.
However, this frequency exceeded that documented by Mohammadi et al. and Deng et al., who found that 24.8% and 31.6% of tested S. aureus isolates carried class 1 integrons, respectively [24,58]. Conversely, Maratha et al. and Mostafa et al. reported that 71% and 72.6% of MRSA isolates harbored the intI1 gene, respectively [11,59]. In contrast, a recent study in Iran found that only 7.6% of S. aureus isolates had the intI1 gene. All tested S. aureus isolates in our investigation were negative for the intI2 and intI3 genes. This finding is consistent with previous reports [21,59]. However, in earlier studies, class 2 integrons were detected in 3.4% and 35.2% of isolates, respectively [11,57]. The variation in integron prevalence underscores the importance of integrating molecular surveillance of integrons into antibiotic resistance monitoring programs to enhance treatment regimens.
The results show a significant correlation between biofilm formation and the presence of intI1 and mecA genes in S. aureus isolates, with biofilm producers having a much higher rate of these genes compared to non-producers (p = 0.002). This finding is well matched with those reported by Pozzi et al. and Aghmiyuni et al., who emphasized that the existence of mecA was linked with enhanced biofilm formation [60,61]. This agrees with research revealing a correlation between biofilm development and antibiotic resistance in S. aureus, frequently promoted by the mecA gene [62]. In line with our results, there was a significant correlation between class 1 integrons and biofilm formation [13]. This observation is consistent with existing literature indicating that biofilm communities can enhance horizontal gene transfer among bacteria because of their closely packed structure [63]. However, we emphasize that this association does not essentially indicate a causal link between intI1 and biofilm development, as our study did not analytically evaluate any mechanistic connections. To justify these preliminary results and clarify the fundamental mechanisms, additional studies with more detailed molecular analysis are required.
However, isolates from pus displayed significantly higher biofilm formation p = 0.02), while other specimen types (ETT, blood, sputum) did not reach statistical significance. This finding suggests that biofilm formation is more likely caused by genetic variables than by the place of infection. These results underscore the need to focus on genetic variables in the management of biofilm-associated infections.
Our study has a few limitations. It is a single-center study with a small sample size, which limits the generalizability of our findings. More extensive genotyping would add significant epidemiological value. Future research should include gene cassette sequencing, SCCmec typing, and multilocus sequence analysis with multicenter studies to provide deeper epidemiological insights.

5. Conclusions

The prevalence of MRSA isolates is alarming. Furthermore, there is a rising prevalence of cMLSB and iMLSB resistance phenotypes. Most S. aureus strains with the intI1 gene were MRSA and MDR. Ultimately, these results highlight the persistent issue of antimicrobial resistance and require a reassessment of approaches for treatment of S. aureus infections. Our findings further emphasize the need for strict infection control and prompt antimicrobial stewardship activities that can prevent the dissemination of resistant bacteria.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pathogens14070705/s1, Table S1: Primers Sequences, Amplicon Sizes, and Thermal Cycling Programs for mecA, intI1, intI2, and intI3 Genes.

Author Contributions

Conceptualization, E.E.H., M.S.E., M.M.S. and N.S.M.A.; Data curation, E.E.H., O.A.E., S.M.S. and N.S.M.A.; Formal analysis, M.M.S., M.H.E. and M.N.A.; Investigation, W.S.E. and M.S.E.; Methodology, E.E.H., M.S.E. and S.M.S.; Resources, M.H.F., R.Y.H., A.A.E., N.S.M.A., M.S.E. and W.E.; Software, M.H.F., M.A.E. (Mohamed A. Eltomey), A.A.E., M.A.E. (Mohamed A. Elsebaey), W.E., A.M.E.N. and M.S.E.; Supervision, E.E.H., W.S.E. and S.M.S.; Validation, M.N.A., M.H.E. and W.S.E.; Visualization, S.M.S. and R.Y.H.; Writing—original draft, E.E.H. and M.S.E.; Writing—review & editing, E.E.H. and M.S.E. All authors have read and agreed to the published version of the manuscript.

Funding

This study was conducted without external or internal institutional funding (complete self-funding).

Institutional Review Board Statement

This research adhered to the Declaration of Helsinki guidelines and received approval from the Medical Ethics Committee at the Faculty of Medicine, Tanta University, Egypt (approval code 36264PR1037/1/25).

Informed Consent Statement

Written informed consent was obtained from all subjects.

Data Availability Statement

Data can be obtained from the corresponding author upon request.

Conflicts of Interest

The authors disclose no conflicts of interest.

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Pathogens 14 00705 g001
Figure 1. (a) D zone test for inducible clindamycin resistance; (b) Vancomycin E-test showing a sensitive isolate of S. aureus (MIC = 2 µg/mL).
Figure 1. (a) D zone test for inducible clindamycin resistance; (b) Vancomycin E-test showing a sensitive isolate of S. aureus (MIC = 2 µg/mL).
Pathogens 14 00705 g001
Pathogens 14 00705 g002
Figure 2. (a) Gel electrophoresis of PCR amplification of the mecA gene. Lane 1: 100 pb DNA ladder marker. Lane 2: mecA-positive control (585 bp). Lane 3, 4, 5, 6, 7, 8, 9, 11 = mecA-positive isolates Lane 10 = mecA-negative isolate. (b). Agarose gel electrophoresis of PCR products of the intI1 gene amplicon among S. aureus, positive isolates gave a single band of 280 bp. Lane 1: 100 pb DNA ladder marker Lane 2: intI1 -positive control (280 bp). Lane 3, 4, 7, 8, 9, 10, 13, 14, 15, 16 = intI1-positive isolates Lane 5, 6, 11, 12, 17, 18 = intI1-negative isolate.
Figure 2. (a) Gel electrophoresis of PCR amplification of the mecA gene. Lane 1: 100 pb DNA ladder marker. Lane 2: mecA-positive control (585 bp). Lane 3, 4, 5, 6, 7, 8, 9, 11 = mecA-positive isolates Lane 10 = mecA-negative isolate. (b). Agarose gel electrophoresis of PCR products of the intI1 gene amplicon among S. aureus, positive isolates gave a single band of 280 bp. Lane 1: 100 pb DNA ladder marker Lane 2: intI1 -positive control (280 bp). Lane 3, 4, 7, 8, 9, 10, 13, 14, 15, 16 = intI1-positive isolates Lane 5, 6, 11, 12, 17, 18 = intI1-negative isolate.
Pathogens 14 00705 g002
Table 1. Comparison between MRSA and MSSA isolates according to demographic and clinical characteristics.
Table 1. Comparison between MRSA and MSSA isolates according to demographic and clinical characteristics.
MRSA
(n = 38)		MSSA
(n = 25)		Test of Sig.p
Age (years)
Min.–Max18–7027–68t =
2.227 *		0.030 *
Mean ± SD41.47 ± 14.6249.24 ± 11.68
Median (IQR)41 (28–53)51 (40–60)
Sex
Male23 (60.5%)13 (52.0%)χ2= 0.4480.503
Female15 (39.5%)12 (48.0%)
Type of specimen
Blood7 (18.4%)6 (24.0%)χ2= 4.591MC p = 0.340
ETT4 (10.5%)5 (20.0%)
Urine7 (18.4%)5 (20.0%)
Pus15 (39.5%)4 (16.0%)
Sputum5 (13.2%)5 (20.0%)
ETT: Endotracheal tube. IQR: Interquartile range. SD: Standard deviation. t: Student t-test. χ2: Chi-squared test. MC: Monte Carlo p: p value for comparing between MRSA and MSSA *: Statistically significant at p ≤ 0.05
Table 2. Antimicrobial resistance patterns among MRSA and MSSA clinical isolates.
Table 2. Antimicrobial resistance patterns among MRSA and MSSA clinical isolates.
MRSA
(n = 38)		MSSA
(n = 25)		χ2p
Antibiotics
Cefoxitin (FOX)38 (100.0%)0 (0.0%)63.000 *<0.001 *
Linezolid (LZD)0 (0.0%)0 (0.0%)––
Vancomycin 0 (0.0%)0 (0.0%)––
Erythromycin (ERY)30 (78.9%)20 (80.0%)0.0100.920
Clindamycin (CLD)18 (47.4%)2 (8.0%)10.786 *0.001 *
Ciprofloxacin (CIP)29 (76.3%)13 (52.0%)4.012 *0.045 *
Gentamycin (GEN)17 (44.7%)7 (28.0%)1.7910.181
Ofloxacin (OFX)24 (63.2%)8 (32.0%)5.857 *0.016 *
Levofloxacin (LEV)27 (71.1%)10 (40.0%)5.999 *0.014 *
Tetracycline (TET)30 (78.9%)5 (20.0%)21.221 *<0.001 *
Doxycycline (DO)28 (73.7%)4 (16.0%)20.076 *<0.001 *
Trimethoprim/sulfamethoxazole (SXT)20 (52.6%)8 (32.0%)2.6000.107
χ2: Chi-squared test p: p value for comparing between MRSA and MSSA *: Statistically significant at p ≤ 0.05.
Table 3. Distribution of biofilm production and MLSB phenotypes among S. aureus isolates.
Table 3. Distribution of biofilm production and MLSB phenotypes among S. aureus isolates.
No. (%)
Biofilm
   Non-biofilm producer15 (23.8%)
   Biofilm producer48 (76.2%)
    Weak12 (19.0%)
    Moderate17 (27.0%)
    Strong19 (30.2%)
 MLSB phenotypes
   MS13 (20.6%)
   cMLSB21 (33.3%)
   iMLSB17 (27.0%)
   Sensitive12 (19.0%)
Table 4. Correlation of antibiotic resistance with the biofilm production.
Table 4. Correlation of antibiotic resistance with the biofilm production.
Biofilm Productionχ2p
Non-Biofilm Producer
(n = 15)		Biofilm Producer
(n = 48)
ResistanceSensitivityResistanceSensitivity
Antibiotic resistance
Cefoxitin (FOX)4 (26.7%)11 (73.3%)34 (70.8%)14 (29.2%)9.314 *0.002 *
Erythromycin (ERY)11 (73.3%)4 (26.7%)39 (81.3%)9 (18.8%)0.437FE p = 0.489
Clindamycin (CLD)1 (6.7%)14 (93.3%)19 (39.6%)29 (60.4%)5.715 *FE p = 0.024 *
Ciprofloxacin (CIP)10 (66.7%)5 (33.3%)32 (66.7%)16 (33.3%)0.001.000
Gentamycin (GEN)3 (20.0%)12 (80.0%)21 (43.8%)27 (56.3%)2.7340.098
Ofloxacin (OFX)7 (46.7%)8 (53.3%)25 (52.1%)23 (47.9%)0.1340.714
Levofloxacin (LEV)9 (60.0%)6 (40.0%)28 (58.3%)20 (41.7%)0.0130.909
Tetracycline (TET)6 (40.0%)9 (60.0%)29 (60.4%)19 (39.6%)1.9290.165
Doxycycline (DO)6 (40.0%)9 (60.0%)26 (54.2%)22 (45.8%)0.9180.338
Trimethoprim/sulfamethoxazole (SXT)4 (26.7%)11 (73.3%)24 (50.0%)24 (50.0%)2.5200.112
χ2: Chi-squared test FE p: Fisher’s exact test p-value * Statistically significant at p ≤ 0.05.
Table 5. Resistance profile, clinical source, and biofilm production among intI1 gene-positive S. aureus isolates.
Table 5. Resistance profile, clinical source, and biofilm production among intI1 gene-positive S. aureus isolates.
Isolate No.Clinical SourceMLSB PhenotypeBiofilm
Production		Antibiotic Resistance Profile
1PuscMLSBStrongFOX, ERY, CLD, CIP, OFX, TET, DO, SXT
3BloodcMLSBStrongFOX, ERY, CLD, CIP, OFX, GEN, DO, SXT
5PuscMLSBStrongFOX, ERY, CLD, CIP, GEN, DO, SXT
6ETTcMLSBStrongFOX, ERY, CLD, CIP, GEN, OFX, LEV, DO, SXT
8PuscMLSBWeakFOX, ERY, CLD, CIP, GEN, OFX, LEV, DO, SXT
10UrinecMLSBModerateFOX, ERY, CLD, LEV, DO, SXT
12PuscMLSBStrongFOX, ERY, CLD, OFX, DO, SXT
13BloodcMLSBModerateFOX, ERY, CLD, OFX, TET
15PuscMLSBStrongFOX, ERY, CLD, CIP, GEN, LEV, OFX, TET, DO, SXT
20PuscMLSBStrongFOX, ERY, CLD, CIP, GEN, OFX, TET, SXT
23UrinecMLSBModerateFOX, ERY, CLD, TET, DO, SXT
27PuscMLSBStrongFOX, ERY, CLD, CIP, OFX, LEV, TET, SXT
32PuscMLSBWeakFOX, ERY, CLD, CIP, GEN, LEV, TET, SXT
39UrineMSStrongFOX, ERY, CIP, GEN, OFX, LEV, TET, DO, SXT
42UrineiMLSBModerateFOX, ERY CIP, GEN, OFX, LEV, TET, DO, SXT
44SputumiMLSBStrongFOX, ERY, CIP, GEN, OFX, LEV, TET, DO
47ETTMSStrongFOX, ERY, CIP, OFX, LEV, TET
50BloodiMLSBWeakFOX, CIP, LEV, TET, DO
54SputumSensitiveWeakFOX, CIP, OFX, LEV, TET, DO
60PusSensitiveStrongCIP
63BloodSensitiveStrongFOX, CIP, OFX, LEV, TET, DO
Table 6. Logistic regression analysis for predictors of biofilm production.
Table 6. Logistic regression analysis for predictors of biofilm production.
VariablesBiofilm ProductionBivariate AnalysisLogistic Regression Analysis *
Non-Biofilm Producer (n = 15)Biofilm Producer (n = 48)p ValueCOR (95% CI)p ValueAOR (95% CI)
Type of specimen
Urine6 (50%)6 (50%) Ref.
ETT3 (33.3%)6 (66.7%)0.42 (0.3–11.9)
Blood3 (23.1%)10 (76.9%)0.23.3 (0.6–18.5)
Pus2 (10.5%)17 (89.5%)0.028.5 (1.3–54.1)
Sputum1 (10%)9 (90%)0.079 (0.9–94.9)
intI1 gene
Negative15 (35.7%)27 (64.3%)0.002*Ref.
Positive021 (100%) Undefined
mec A gene
Negative11 (44%)14 (56%)0.002 *Ref.0.049Ref.
Positive4 (10.5%)34 (89.5%) 6.7 (1.8–24.6) 4.2 (1.009–17.4)
MLSB phenotypes
MS7 (53.8%)6 (46.2%) Ref. Ref.
cMLSB1 (4.8%)20 (95.2%)0.00723.3 (2.4–229.3)0.0412.2 (1.1–133)
iMLSB3 (17.6%)14 (82.4%)0.055.4 (1.04–28.5)0.084.7 (0.8–26.2)
Sensitive4 (33.3%)8 (66.7%)0.32.3 (0.5–11.8)0.51.7 (0.3–9.6)
Constant
% correctly predicted
Model ꭓ2, p-value		−0.6
76.2
16.2, 0.003
*: Factors included in regression analysis included type of specimen, mecA, and MLSB phenotypes. COR: Crude odds ratio, AOR: Adjusted odds ratio, CI: Confidence interval.
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Hegazy, E.E.; ElNaghy, W.S.; Shalaby, M.M.; Shoeib, S.M.; Abdeen, N.S.M.; Fouda, M.H.; Elshora, O.A.; Elnaggar, M.H.; Elrefaey, W.; Hagag, R.Y.; et al. Study of Class 1, 2, and 3 Integrons, Antibiotic Resistance Patterns, and Biofilm Formation in Clinical Staphylococcus aureus Isolates from Hospital-Acquired Infections. Pathogens 2025, 14, 705. https://doi.org/10.3390/pathogens14070705

AMA Style

Hegazy EE, ElNaghy WS, Shalaby MM, Shoeib SM, Abdeen NSM, Fouda MH, Elshora OA, Elnaggar MH, Elrefaey W, Hagag RY, et al. Study of Class 1, 2, and 3 Integrons, Antibiotic Resistance Patterns, and Biofilm Formation in Clinical Staphylococcus aureus Isolates from Hospital-Acquired Infections. Pathogens. 2025; 14(7):705. https://doi.org/10.3390/pathogens14070705

Chicago/Turabian Style

Hegazy, Eman E., Wageih Salem ElNaghy, Marwa M. Shalaby, Sarah M. Shoeib, Nashwa S. M. Abdeen, Mohamed H. Fouda, Ola A. Elshora, Mohammed H. Elnaggar, Waleed Elrefaey, Rasha Youssef Hagag, and et al. 2025. "Study of Class 1, 2, and 3 Integrons, Antibiotic Resistance Patterns, and Biofilm Formation in Clinical Staphylococcus aureus Isolates from Hospital-Acquired Infections" Pathogens 14, no. 7: 705. https://doi.org/10.3390/pathogens14070705

APA Style

Hegazy, E. E., ElNaghy, W. S., Shalaby, M. M., Shoeib, S. M., Abdeen, N. S. M., Fouda, M. H., Elshora, O. A., Elnaggar, M. H., Elrefaey, W., Hagag, R. Y., Elhadidy, A. A., Elsebaey, M. A., Eltomey, M. A., El Nakib, A. M., Ageez, M. N., & Elnady, M. S. (2025). Study of Class 1, 2, and 3 Integrons, Antibiotic Resistance Patterns, and Biofilm Formation in Clinical Staphylococcus aureus Isolates from Hospital-Acquired Infections. Pathogens, 14(7), 705. https://doi.org/10.3390/pathogens14070705

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