Antibiotics Resistance and Adhesive Properties of Clinical Staphylococcus aureus Isolated from Wound Infections

Staphylococcus aureus (S. aureus) is a ubiquitous pathogen responsible for several severe infections. This study aimed to investigate the adhesive properties and antibiotic resistance among clinical S. aureus isolated from Hail Hospital Province, Kingdom of Saudi Arabia (KSA), using molecular approaches. This study was conducted according to the ethical committee at Hail’s guidelines on twenty-four S. aureus isolates. A polymerase chain reaction (PCR) was performed to identify genes encoding the β-lactamase resistance (blaZ), methicillin resistance (mecA), fluoroquinolone resistance (norA), nitric oxide reductase (norB), fibronectin (fnbA and fnbB), clumping factor (clfA) and intracellular adhesion factors (icaA and icaD). This qualitative study tested adhesion based on exopolysaccharide production on Congo red agar (CRA) medium and biofilm formation on polystyrene by S. aureus strains. Among 24 isolates, the cna and blaz were the most prevalent (70.8%), followed by norB (54.1%), clfA (50.0%), norA (41.6%), mecA and fnbB (37.5%) and fnbA (33.3%). The presence of icaA/icaD genes was demonstrated in almost all tested strains in comparison to the reference strain, S. aureus ATCC 43300. The phenotypic study of adhesion showed that all tested strains had moderate biofilm-forming capacity on polystyrene and represented different morphotypes on a CRA medium. Five strains among the twenty-four harbored the four genes of resistance to antibiotics (mecA, norA, norB and blaz). Considering the genes of adhesion (cna, clfA, fnbA and fnbB), these genes were present in 25% of the tested isolates. Regarding the adhesive properties, the clinical isolates of S. aureus formed biofilm on polystyrene, and only one strain (S17) produced exopolysaccharides on Congo red agar. All these results contribute to an understanding that the pathogenesis of clinical S. aureus isolates is due to their antibiotic resistance and adhesion to medical material.


Introduction
Epidemiological surveys in clinical settings have revealed a significant increase in hospital infections [1,2]. This rise is linked to the use of immunosuppressive medications in treatment. Staphylococcus is the most common genus among the microorganisms involved [3]. Several Staphylococcus species are now responsible for severe infections with high morbidity and mortality [4]. The Staphylococcus genus contains 47 species and 24 subspecies, 17 of which are found in humans [2]. Its other species can be found in animals or food [5]. Three species are primarily responsible for human pathology among those isolated in humans: Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus [6]. Of these, S. aureus is prevalent. These species are frequently found on human skin and in the nostrils. They are both commensal bacteria and important human pathogens. They are involved in various pathologies, including 1 to 5% of community infections and up to 30% of hospital infections [7].
Staphylococcus aureus is a bacterium that can infect both humans and animals. It can also result in food poisoning. Other sources of contamination include hospital equipment and surfaces and the food industry [4,8]. This germ has recently gained notoriety due to its resistance to antimicrobials, particularly methicillin (β-Lactamines). The presence of the exact clone of S. aureus in the anterior part of the nose and on the skin is frequently detected in colonized people, indicating an endogenous source of bacteria that can cause infections [4] or spread to other patients [9,10]. Hospital staff represent a second reservoir for S. aureus which can be transmitted to patients [11]. S. aureus strains' high pathogenicity is related to their ability to resist multiple antimicrobials and adapt to changing environmental conditions [12]. This species' virulence is associated with numerous virulence factors encoded by genes on the chromosome or plasmid 13, as well as with the combined action of various bacterial surface components [11,12]. This bacterial genus' pathogenicity is due to its ability to adhere to host cells [13].
The formation of a biofilm contributes to the resistance to the immune defenses and antimicrobial agents [14]. Many genes are involved in adhesion and biofilm formation [15]. The ica ADBC gene, which produces biofilm formation by PIA, is found in all strains of S. aureus. Stress conditions such as anaerobic conditions, extreme temperature, ethanol, and antimicrobials regulate the ica gene. The ica ADBC locus is required for Staphylococcus biofilm formation [16]. Other proteins, such as FnbpA and Fnbp B, are required for biofilm formation. Indeed, there are two main FnBPs in S. aureus, FnBPA, and FnBPB. They are encoded by two closely related genes, fnbA, and fnbB, respectively [17].
Due to the increase in the rate of nosocomial infections with S. aureus strains and the prevalence of the multidrug-resistant strains, we aimed in this study to look into the biofilm potency and antibiotic resistance profile, and to explain theses virulence factors by the study of the distribution of the genes responsible for these mechanisms in clinical S. aureus strain genomes.

Tested Strains and Culture Conditions
This study was carried out by the Ethics Committee at Hail Affairs (reference: H-08-L-074). Patient privacy and data confidentiality were protected following the specifications of Helsinki Declaration. Patients developing wound infections were the subjects of this study, which was conducted in March 2021. For this, swab samples were collected from the depths of the wound using a sterile cotton swab under aseptic conditions. The isolates were transferred into a sterile nutrient broth in a test tube and were delivered to the microbiology laboratory at King Khalid Hospital in Hail, Saudi Arabia within 1 h. Twenty-four (24) S. aureus strains were studied in total.
Blood and MacConkey agar plates (Bio-rad, Marnes-la-Coquette, France) were used to plate wound samples, which were then incubated at 37 • C for 24 h. Subculturing on mannitol salt agar was used to confirm the purity of the suspected S. aureus isolates (Bio-rad, Marnes-la-Coquette, France). Catalase, coagulase, and DNase enzymes were identified during the strain identification process. The reference strain of S. aureus ATCC 43300 was used as a quality control.

Study of Exopolysaccharide Production
As previously described, the slime production of the ability pathogenic bacteria was evaluated by culturing each bacterium on Congo red agar (CRA) [18,19]. After aerobic incubation at 37 • C for 24 h, the results were interpreted as follows: very black and black colonies were considered to be regular slime-producing strains. Almost black, very red, red, and Bordeaux-colored colonies were classified as non-slime-producing strains [20,21].

Quantitative Biofilm Production Assay by S. aureus Cells
A semi-quantitative technique performed using 96-well polystyrene plates (Nunc, Roskilde, Denmark) was used to produce biofilm by S. aureus strains in trypticase soya broth (TSB, Bio-rad, Marnes-la-Coquette, France) using the crystal violet 1% (CV) staining assay [22,23]. Bacterial cells were cultured for 24 h at 37 • C in TSB supplemented with 2% glucose (w/v). The optical density after the CV staining was measured at 570 nm (OD 570 ) and the biofilm formation was interpreted as highly positive (OD 570 ≥ 1), having low positivity (0.1 ≤ OD 570 < 1), and negative (OD 570 < 0.1) [24]. The detection of icaA and icaD genes in the tested strains' genomes was also performed using the protocol described previously [25]. All the primers used, the PCR conditions, and the amplicon size are reported in Table 1. The strain of S. aureus ATCC 43300 was used as a positive control. The PCR conditions of cna, fnbA, and fnbB genes were accomplished as described elsewhere [24,26]. The clf A gene (1000 bp) responsible for the binding to fibrinogen was amplified by PCR according to the protocol previously described [27]. All PCR primers sequences are listed in Table 1. Table 1. Primers used for detection of adhesion genes.

PCR Amplification of Efflux Pump Genes
Amplification conditions are summarized in Table 2. The annealing temperature was about 45 • C for nor A and 53 • C for nor B. The strain of S. aureus ATCC 43300 was used as a positive control. Table 2. Primers used for the detection of genes encoding antimicrobials resistance.

Qualitative and Quantitative Study of Biofilm Formation
The capacity of clinical S. aureus strains to produce biofilm was estimated qualitatively by the culture on the CRA medium and quantitatively by the ability to create a biofilm on a polystyrene surface. In addition, three morph types were defined according to their color on CRA were obtained: non-slime-producing S. aureus strains characterized by Bordeaux (70.8%) and almost black colonies (25.0%). In contrast, a black colony characterizing slimepositive bacteria was obtained only for strain S17 of S. aureus (4.1%) ( Figure 1, Table 3).

norB
AGCGCGTTGTCTATCTTTCC GCAGGTGGTCTTGCTGATAA 213 [31] blaZ ACTTCAACACCTGCTGCTTTC TGACCACTTTTATCAGCAACC 172 [32] 2.6. Detection of mec A, blaZ, norA and norB Genes Molecular detection of mec A and bla Z genes was performed using the forward and reverse primers presented in Table 2. PCR assessments were performed according to the specifications of Geha et al. [28] and Martineau et al. [29], respectively. The strain of S. aureus ATCC 43300 was used as a positive control.

Qualitative and Quantitative Study of Biofilm Formation
The capacity of clinical S. aureus strains to produce biofilm was estimated qualitatively by the culture on the CRA medium and quantitatively by the ability to create a biofilm on a polystyrene surface. In addition, three morph types were defined according to their color on CRA were obtained: non-slime-producing S. aureus strains characterized by Bordeaux (70.8%) and almost black colonies (25.0%). In contrast, a black colony characterizing slime-positive bacteria was obtained only for strain S17 of S. aureus (4.1%) ( Figure 1, Table 3).  The main results showed that all S. aureus tested strains (also S17 very black on CRA) exhibited moderate biofilm formation on polystyrene (0.1 < OD 570 < 1) compared to the reference strain S. aureus ATCC 43300 (OD 570 = 1.89 ± 0.13) (Figure 2, Table 3).  The main results showed that all S. aureus tested strains (also S17 very black on CRA) exhibited moderate biofilm formation on polystyrene (0.1 < OD570 < 1) compared to the reference strain S. aureus ATCC 43300 (OD570 = 1.89 ± 0.13) (Figure 2, Table 3).  According to our results, 19 S. aureus strains (79.1%) were positive for both icaA (188 bp) and icaD (198 bp) genes encoding the intracellular adhesins A and D compared to the positive control strain of S. aureus ATCC 43300 for both tested genes (Supplementary Material Figure S1, Table 3). In addition, all the primers used in the experiment exhibited specificity, with a single band. Therefore, only five clinical strains (20.8%) were expected from this correlation (Table 3).

Distribution of Adhesion Genes
This study emphasized four biofilm-related genes encoding fibronectin-binding proteins A and B (fnbA and fnbB), bound coagulase (clf A), and collagen adhesin gene (cna) involved in S. aureus cell attachment and multiplication. All S. aureus strains expressed biofilm genes. As shown in Table 4, the fnbA (259 bp) and fnbB (523 bp) encoding fibronectin were detected in 33.3% and 37.5%, respectively, of the total strains (Supplementary Material Figure S2). The clf A (288 bp) and cna (192 bp) genes encoding collagen were amplified in 50.0% and 70.8% of the total tested strains (Supplementary Material Figure S3).

Distribution of Antibiotic Resistance Genes
The β-lactamases blaZ gene (blaZ), methicillin resistance determinant (mecA), fluoroquinolones resistance gene (norA), and nitric oxide reductase (norB) gene were studied for all S. aureus isolates. All these results are presented in Supplementary Material Figure S4 and Table 5.
Based on the results of the phenotypic profile of antibacterial resistance, the clinical strains of S. aureus S12, S22, and S2 were more resistant to the tested antibiotics, with percentages of resistance of 63.6%, 54.5%, and 45.4%, respectively. Compared to the more sensitive strains, S10 was very susceptible to the action of the antibiotics (0.9% of resistance), followed by isolate S17 (9.1% of resistance) (Supplementary Material Table S1).

Discussion
The skin surface can be affected by several microorganisms that cause wound infection. S. aureus is a pathogenic bacterium that is resistant to penicillin due to the production of β-lactamase and which contributes to the inhibition of its antibacterial activity. S. aureus causes many problems in hospitals and is usually resistant to antimicrobials [3,32].
In the present study, antibiotic susceptibility results were reported to be influenced by various factors, such as the expression of the antimicrobials resistance genes. Our results showed that the blaZ gene was widely spread among S. aureus strains (70.83%), followed by norA, norB, and mecA genes. The norB gene has a single antimicrobial resistance mechanism. At the same time, mecA and blaZ possess several antibiotic resistance mechanisms [3,17,32].
Efflux pump complexes and resistance-conferring antibiotic subunits, as well as protein(s) (norA and norB), are the most prevalent mechanisms of antibiotic resistance. Other instruments include the antibiotic resistance gene or operon (mecA and blaZ) and antibiotic inactivation enzyme (SAT4 and mphC) [31].
S. aureus strains express resistance genes from external sources [33,34]. This can be natural or due to antimicrobial abuse and misuse, leading to chromosomal mutation and antibiotic selection. Antibiotic-resistant strains are receiving significant consideration in the contemporary era [26]. Resistance to many antimicrobial agents causes critical problems in treating S. aureus infection [35,36]. For example, methicillin resistance contributes to the inhibition of the synthesis of the cell wall.
S. aureus strains can live in biofilms in their natural environment, where planktonic cells proliferate and accumulate in multilayer cells. This structure can protect microorganisms from the action of antimicrobials, extracellular enzymes, and stress factors [24].
The results of the biofilm formation using the crystal violet method showed that all tested S. aureus strains formed a biofilm on the polystyrene. Mathur et al. [37] demon-strated that 14.47% of isolates from blood, medical devices, and skin surfaces formed a strong biofilm, 39.4% formed a moderate biofilm, and 46% of the strains were not biofilm producers [37].
The ability of clinical S. aureus isolates to produce exopolysaccharides on CRA plates showed that only one strain was able to produce mucus compared to the results obtained by Arciola et al., in which 57.5% of the isolates had mucus [20].
Bacterial biofilm formation is encoded by adhesions responsible for the pathogenicity of S. aureus strains [20]. Therefore, in this study, we focused on detecting genes coding for them.
The ica A and ica D genes responsible for the synthesis of polysaccharide intercellular adhesin (PIA) were detected in all strains. The presence of icaA/icaD genes was detected in 19/24 S. aureus strains (79.16%). Arciola et al. [26] found that 60.86% of S. aureus strains harbor icaA and icaD genes. In previous research, it has been demonstrated that 98% of clinical isolates of S. aureus have icaA, and 96% have icaD [38].
In the study of Rohde et al. [25], all tested S. aureus strains, including slime-negative isolates, expressed the icaA gene. This was contrary to the research of Arciola et al. [26], who correlate the presence of icaA/icaD genes to the production of exopolysaccharides.
Adhesin genes (cna, fnbA, fnbB and clf A) were expressed in six S. aureus strains (25%). This demonstrated that clf A and clf B proteins were implicated in the pathogenesis of S. aureus, conferring endocarditis, bacteremia, or pyonephrosis [20]. Our results are similar to those reported by Arciola et al. [26], who noted that 84/191 clinical S. aureus strains (44%) expressed two adhesins genes (fnbA and cna). The cna gene was detected in 70.83% of the tested strains in the present work.

Conclusions
This work provides new information on S. aureus isolated from wound surfaces. In fact, S. aureus strains are biofilm producers. They show high percentages of genes responsible for its adhesion and resistance to antimicrobials. These genes can make strains capable of colonizing many human organs. In further work, we envisage studying the effect of natural and synthetic compounds on the biofilm and antimicrobial resistance of S. aureus strains using molecular and in silico approaches.