Introduction

The fundamental goal of the skin is to keep microbial populations on its surface under control and prevent diseases from colonizing the underlying tissue [1]. A wound is a disruption in the skin’s protective action [2]. Staphylococcus aureus is the most frequent opportunistic bacteria, causing many superficial and life-threatening infections [3]. It can cause various disorders, including skin and soft tissue infections (SSTIs), invasive infections, and toxin-mediated disorders [4]. Since it produces several virulence factors and acquires multidrug resistance (MDR) to various antibacterial agents, it is a major infectious agent in communities and hospitals [5].

S. aureus has an incredible ability to develop resistance rapidly. Environmental factors and cell membrane disruption or DNA damage can influence the fast development of antibiotic resistance [6]. More than 90% of S. aureus is resistant to penicillin, which remains a global issue [7]. Methicillin-resistant S. aureus (MRSA) is a common inhabitant of a large part of the healthy population and can cause a wide range of illnesses, from minor skin infections to life-threatening diseases [8] The MDR is defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories [9, 10]. The MDR of S. aureus strains has been linked to longer hospital stays, higher mortality rates, and concomitant costs [11].

The presence of many virulence factors, such as surface proteins, biofilms, exoenzymes, exotoxins, and exfoliative toxins, is linked to the ability of S. aureus to cause different infections. All these factors allow bacteria to attach to tissues, causing pathogenesis, and to penetrate the immune system, causing toxicity [12]. One of the virulence factors of S. aureus is a cytolytic, pore-forming toxin, such as α-hemolysin, which is involved in the pathogenesis of S. aureus [13]. Many S. aureus strains, particularly MRSA, release one or more distinct staphylococcal exotoxins, including staphylococcal enterotoxins [14], the most important pathogenic components belonging to the superantigen family [15].

The ability of the microorganism to successfully persist within the hospital and community and several cell wall-associated adhesive molecules, such as fnb (encoding fibronectin-binding protein) is responsible for the possibility of severe animal and human diseases [16, 17]. The ability of S. aureus to build biofilms is linked to the antimicrobial resistance mechanism. Invasion isolates are more likely to form biofilm than healthy individual carriage isolates [18] The polysaccharide intercellular adhesin (PIA) is the most important component of biofilm [19, 20]. The N-acetylglucosamyl transferase enzyme responsible for PIA synthesis is known to be encoded by icaA [21].

S. aureus persists and spreads by acquiring antibiotic resistance genes. Identification of S. aureus virulence genes is important for evaluation of disease development. This study focused on S. aureus virulence genes and to detect their correlation to antimicrobial resistance patterns.

Materials and methods

Study area, design, and population

A cross-sectional study was carried out from November 2019 to September 2021 in Minia University Hospital (Minia, Egypt). Wound samples were collected from the Department of Plastic and Reconstructive Surgery. The samples were properly labeled, indicating the source, sex, and age of the patient. Ethical clearance for the study was granted by Minia University Hospital.

Collection of wound pus samples

Bacterial samples were collected from patients having wound infections present on admission to the outpatient clinic and cultured onto nutrient agar, mannitol salt agar, and DNase agar. All media were produced by Oxoid (England) and prepared according to the manufacturer’s instructions. The cultures were incubated at 37 °C for 24 h to be examined the next day.

Isolation and identification of wound bacterial isolates

The primary identification of bacterial isolates was based on colonial appearance, pigmentation, morphology, Gram staining, and biochemical characteristics. The biochemical tests applied were the standard catalase test, coagulase (tube and slide) test, and DNase test (Fig. 1). For the extended storage of bacterial isolates, preservation in 20% glycerol vials at − 70 °C was carried out.

Fig. 1
figure 1

Flow chart of study procedures

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Antibiotic sensitivity testing

Antimicrobial sensitivity was determined by the Kirby-Bauer agar disc diffusion method according to the Clinical Laboratory Standard Institute (CLSI; 2018). Antibiotic discs were used with the following drug concentrations: linezolid (30 μg), tetracycline (30 μg), chloramphenicol (30 μg), rifampin (5 μg), piperacillin (100 μg), amoxicillin/clavulanic acid (30 μg), ampicillin/sulbactam (20 μg), levofloxacin (5 μg), gentamycin (10 μg), vancomycin (30 μg), oxacillin (1 μg), and ciprofloxacin (5 μg) were applied onto Müller-Hinton agar (Himedia). The plates were aerobically incubated at 37 °C for 24 h, and the diameter of the inhibition zones was measured (in mm). The results were compared to that of the CLSI.

DNA extraction and detection of virulence genes

DNA was extracted using a DNA extraction kit (Qiagen, Germany), and the procedures were carried out according to the manufacturer’s instructions. The oligonucleotide primers used in this study were for the detection of genes encoding α-hemolysin (hla), staphylococcal enterotoxin A (sea), intracellular adhesion A (icaA), and fibronectin-binding protein A (FnbA). Table 1 lists the primer sequences (Metabione, Germany) of this study, and Table 2 presents the conditions of the polymerase chain reaction (PCR) products. The PCR products were resolved by electrophoresis on 1% agarose gel, and electrophoresis was carried out at a constant current of 50 mA for 30 min. DNA bands were visualized by ethidium bromide staining and ultraviolet transillumination light. The size of the fragments was determined by comparing their migration to a 100 bp ladder as a standard.

Table 1 The list of primers sequences
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Table 2 Conditions of PCR products
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Statistical analysis

Statistical analyses were performed using χ2 using SPSS version 16 (SPSS, Inc., Chicago, IL, USA). A χ2 test was used to test the association between S. aureus virulence genes and participant's gender and age as well as with the antibiotic resistance profile. Similarly, the association between the antibiotic resistance profile with participant's gender and age groups was detected. The results were considered statistically significant when P ≤ 0.05.

Results

Prevalence of S. aureus isolates according to gender, age, and sample source

A total of 59 S. aureus isolates were detected from 51 different wound samples. The incidence of S. aureus was much higher in males [n = 36 (70.6%)] than in females [n = 15 (29.4%)]. Patients were classified into different age groups from 1 month to 60 years (mean ± standard deviation, 28.98 ± 16.95). The highest prevalence of S. aureus was observed in the age group between 1 and 20 years (45.1%), followed by patients in the age group between 41 and 60 years (29.4%) and finally patients in the age group from 21 to 40 years (25.5%). Figure 2 shows that the highest number of samples was from accidental wounds, such as animal bites, occupational injuries, a sharp tool, or car accidents (n = 38; 74.5%), followed by seven samples of burn infection (burning agents, such as flame, scald, electrical, boiled water, and chemical reagent; 13.7%). Three samples were from surgical wounds (5.9%) and three samples were from ulcers and abscess discharge (5.9%).

Fig. 2
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Prevalence of S. aureus isolated from patients from different types of wound infections

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Antimicrobial sensitivity testing

Table 3 shows that MRSA isolates accounted for 91.5%, whereas methicillin-susceptible S. aureus (MSSA) isolates accounted for 8.5%. S. aureus had low resistance to chloramphenicol (10.2%), vancomycin (13.5%), and linezolid (16.9%). Moderate (intermediate) resistance was recorded against gentamycin (33.9%), levofloxacin and ciprofloxacin (both 35.6%), rifampin (37.3%), and tetracycline (62.7%). High resistance was observed against oxacillin, amoxicillin/clavulanic acid, ampicillin/sulbactam (all 91.5%), and piperacillin (100%). The MDR in S. aureus isolates was 54.2%. S. aureus had R0 = 0%, R1 = 20.3%, R2 = 23.7%, R3 = 16.9%, R4 = 13.6%, R5 = 16.9%, R6 = 1.7%, R7 = 3.4%, and R8 = 1.7% (R0 represents the number of isolates sensitive to all antimicrobial classes tested, whereas R = 1, 2, 3, 4, 5, 6, 7, and 8 represent isolates resistant to 1, 2, 3, 4, 5, 6, 7, and 8 antibiotic classes, respectively). No statistically significant difference was detected between the resistance profile of tested antibiotics and participant’s gender or age group (p > 0.05) (Tables 4 and 5).

Table 3 Antibiotic sensitivity profile of S. aureus isolates
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Table 4 Correlation between antibiotic resistance profile and patients’ gender
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Table 5 Correlation between antibiotic resistance profile and patients age groups
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Detection of virulence genes

To test the virulence genes of the isolates in this study, hla, sea, icaA, and fnbA were detected by PCR amplification. Table 6 shows that sea was the most predominant in 72.9% of the isolates. icaA was found in 49.2% of the isolates, followed by hla (37.3% of the isolates) and fnbA (13.6% of the isolates). Amplicon sizes of 209, 120, 770, and 1279 bp were considered positive for the presence of hla, sea, icaA, and fnbA, respectively. Figure 3 shows hla, sea, icaA, and fnbA PCR amplification products among S. aureus isolates, respectively. sea was the commonest virulence gene among MRSA and vancomycin-resistant S. aureus (VRSA) isolates (72.2% and 62.5%, respectively). However, sea and icaA were the commonest genes among MSSA isolates (80%; Table 6).

Table 6 Frequencies of virulence genes among MRSA, MSSA and VRSA strains
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Fig. 3
figure 3

Detection of amplification product of different virulence genes: A hla gene by PCR; lane 1: negative control, lane 2: positive control and lanes 3 to 10: positive PCR products (209 bp); B sea gene by PCR; lane 1: positive control, lane 2: negative control and lanes 3 to 11: positive PCR products (120 bp); C icaA gene by PCR; lane 1: positive control, lane 2: negative control and lanes 3 to 10: positive PCR products (770 bp); D fnbA gene by PCR; lanes 1 to 7: positive PCR products (1279 bp), lane 8: positive control and lane 9: negative control

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A significant correlation was observed between virulence genes (hla, sea and icaA) and patients age groups (P < 0.05). While no statistically significant difference was detected between the tested virulence genes and participant’s gender (Table 7).

Table 7 Correlation between virulence genes and patients gender and age groups
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sea was the commonest virulence gene among antibiotic-resistant and antibiotic-sensitive isolates, followed by icaA, hla, and fnbA. The highest distribution of sea was among the ciprofloxacin (95.2%)-, gentamycin (89.9%)-, and tetracycline (75.7%)-resistant isolates. At the same time, the highest distribution of sea was among oxacillin (79.9%)-, linezolid (75.5%)-, and rifampin (73.5%)-sensitive isolates (Table 8).

Table 8 Correlation between S. aureus virulence genes and antibiotic resistance
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A statistically significant correlation (P < 0.05) was detected between the presence and absence of hla and sea and piperacillin, gentamicin, and levofloxacin resistance and sensitivity. However, a significant difference in the distribution of icaA was found among β-lactam-resistant and β-lactam-sensitive isolates. fnbA was significantly associated with piperacillin and ciprofloxacin resistance and sensitivity (Table 8). Table 9 shows that sea and icaA had the highest coexistence (40.7%), followed by sea and hla (21.9%).

Table 9 Coexistence of virulence genes among S. aureus isolates
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Discussion

S. aureus is the commonest pathogenic bacteria found in different wound specimens [22, 23]. Muluye et al. [24] stated that the prevalence of S. aureus in males and females was 38.1% and 28.7%, respectively. The first result was much lower than in this study, whereas the second was similar to this study. Patients were classified into different age groups from 1 month to 60 years. The highest prevalence of S. aureus (45.1%) and examined virulence genes were observed in the age group between 1 and 20 years. In the same time, there is a significant association between tested virulence genes and patients age groups. Torpy et al. [25] stated that the high prevalence of S. aureus in the age group between 1 and 20 years was because most young males (< 20 years) in the country have traditionally worked in occupations such as agriculture, construction, transportation, and industries, all of which are likely to expose them to trauma and different wound infections.

In this study, the highest prevalence of S. aureus was found in trauma and accidental wound infections, similar to other studies [26, 27], suggesting that the rate of S. aureus isolates in open wound infection was 76.9%, similar to these findings.

The predominant isolate S. aureus was sensitive to vancomycin (100%) [28], supporting the findings that considered vancomycin as one of the drugs with high susceptibility pattern against S. aureus. The same study revealed that S. aureus showed a high level of resistance to penicillin and oxacillin (84.6% and 76.9%, respectively). Although these results were lower, they supported this study. They considered piperacillin and oxacillin as drugs with high resistance patterns against S. aureus along with ampicillin/sulbactam and amoxicillin/clavulanic acid.

Linezolid is an efficient antibiotic for treating S. aureus infections among four burn centers [23], in agreement with this study. The sensitivity rate of chloramphenicol against S. aureus was 71.2%, similar to another study [29] that showed a 68.4% sensitivity rate for chloramphenicol. The notable sensitivity of S. aureus to vancomycin, linezolid, and chloramphenicol could be linked to a lower use of these antibiotics due to their shortage availability in the market, high costs, and toxic side effects [30].

Many studies showed a high MRSA prevalence in wound infections [31] and reported a high rate of MRSA and VRSA (44.6% and 61.5%, respectively). The finding for MRSA was lower than in this study, whereas the findings for VRSA were much higher. Moreover, the MRSA results in this study disagreed with Bessa et al. [22], who suggested that 21.8% of S. aureus was resistant to oxacillin. This study also revealed a remarkable increase in MRSA compared to a previous study by Ahmed et al. [32], who reported a 24% MRSA prevalence in the same hospital 10 years ago. This raised the alarm about the escalating and noticeable increase in MRSA prevalence in Egypt. The increase of MRSA in wound infections has contributed to high treatment costs and longer hospital stays, which have major implications for infection management, particularly in developing countries. These findings contribute to a worrying situation in the Minia Government regarding MRSA expansion. The necessity for more detailed molecular epidemiologic surveillance studies on MRSA and VRSA in the next years is critical.

The MDR of S. aureus isolates was 54.2%, similar to other studies [33, 34], which reported 54.9% and 47.9%, respectively. However, another study [28] stated that S. aureus showed 94.8%, higher than this study. Low activity of commonly used antibiotics, such as amoxicillin/clavulanic acid, ampicillin/sulbactam, oxacillin, and piperacillin, may be due to increased consumption of a particular class of antibiotics, resulting in resistance due to mutation(s) at drug target sites or the disruption of drug accumulation in the cytoplasm caused by cell wall rearrangement [31,32,33,34,35,36]. As a result, they are no longer effective in treating wound infections.

The incidence of some major virulence indicators of S. aureus in wound specimens was examined in this study. This study concentrated on a small number of genes linked to S. aureus pathogenicity. These genes (hla, sea, icaA, and fnbA) were chosen because they were the most frequent in aggressive isolates. These targeted genes spread across the isolates after PCR amplification. Furthermore, the bulk of the isolates demonstrated a wide range of gene combinations, indicating that the study sample has a level of genetic diversity.

Antimicrobial resistance and virulence factor genes showed significant relationships in this study. This finding could be explained by the proximity location of the resistance gene to the virulence gene [31, 37].

The predominant virulence and inducible resistance genes in MRSA and MSSA isolates were related to sea [38, 39]. All previous studies supported this study because sea is the commonest among MRSA and MSSA isolates. Cavalcante et al. [40] reported that the prevalence of sea in S. aureus isolates collected from infected skin lesions of atopic dermatitis children was 76.4% in total S. aureus isolates, 73.9% in MRSA isolates, and 78.1% in MSSA isolates, in agreement with this study. Li et al. [41] reported that the frequency of sea in S. aureus isolates from SSTIs in children was 0%, which was totally opposite to this study.

PCR investigation revealed that hla was found in 30.5% of 85 S. aureus isolated from various clinical sources [42], close to the present findings. The prevalence of icaA in MRSA was 60.3% [43], which was slightly higher than the present data. The prevalence of fnbA was 4.9% and 19.9% in MRSA and MSSA strains, respectively [44]. The prevalence of fnbA in MRSA was close to this study, whereas fnbA in MSSA was much lower than in the present data. Another study [45] suggested that the incidence of fnbA in wound swabs was 28.8%, which was slightly higher than the present results. The prevalence of fnbA and icaA in burn units was 2.9% and 44.9%, respectively [46]. fnbA was slightly lower than in this study, whereas the percentage of icaA was similar to the present data. The incidence of fnbA in MRSA and MSSA strains was 15.5% and 36.9%, respectively. However, the incidence of icaA in MRSA and MSSA was 84.5% and 78.3%, respectively [38]. The percentages of fnbA were similar to this study. However, the percentage of icaA was much higher in MRSA but was similar to the present data in MSSA. The prevalence of sea was 11.8% in amoxicillin/clavulanic acid and oxacillin susceptibility samples, 9.2% in rifampin, 0% in penicillin, 88.2% in chloramphenicol, and 100% in vancomycin [47]. The first three percentages were much lower than in this study. However, the percentages of chloramphenicol and vancomycin were slightly higher than in this study. The percentage of the coexistence of sea and hla was 36.9% [17], which was slightly higher than in this study.

The limitation of this study was the inability to detect more virulence genes and express the chosen virulence factors by molecular typing of the isolates (Additional file 1, Additional file 2).

Conclusions

Within the limitations of the current study, it can be concluded that the challenging, increasingly difficult, and widespread bacterial resistance to antibiotics has developed, the incidence of infections caused by MDR S. aureus has increased. The prevalence of CA-MRSA was high among patients with various wound infections. Bacterial resistance profile was the least against vancomycin and linezolid effective antibiotics. The correlation between CA-MRSA strain virulence genes distribution and antibiotic resistance profile showed high incidence of sea and icaA genes. All virulence genes were significantly distributed among piperacillin resistant isolates. β-lactam resistant isolates showed a significant correlation with IcaA virulence gene. After the emergence of high percentage of sea among ciprofloxacin resistant isolates, we expect that more genes will appear in future studies regarding S. aureus virulence genes. Therefore, the spread of bacterial resistance must be monitored in hospitals by using antibacterial agents properly to avoid more complications and to keep the empirical medications as effective as they are.