Molecular Detection of Virulence Genes, Biofilm Formation, and Antibiotic Resistance in Pathogenic Staphylococcus aureus from Taif Hospitals

The bacterium Staphylococcus can cause various health problems, particularly in hospitalized patients. Therefore, the current study aimed to isolate methicillin-resistant Staphylococcus aureus (MRSA) strains, test their capability to form a biofilm, and detect genes related to virulence and biofilm formation. Bacterial isolates were collected from the King Faisal Specialist Hospital and Children’s hospital in Taif Governorate, Saudi Arabia, and identified using primers for mecA and nuc1. They were tested for resistance against twelve widely distributed antibiotics and biofilm formation capability. The MRSA isolates were tested for fnbA, fnbB


INTRODUCTION
Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most pathogenic microbes that cause diseases in humans and animals.2][3] Indiscriminate use of antibiotics has resulted in an increase in antibiotic-resistant bacteria, owing to which patients fail to respond to antibiotic treatment.][9] S. aureus is cluster-shaped and has the ability to form a three-dimensional biofilm that surrounds the cluster of cells, allowing them to resist unfavorable conditions.MRSA can develop antibiotic resistance owing to its biofilm formation capability and hence poses a great threat to hospitalized patients. 2,9The biofilm is an organized structure and a major virulence factor that is important in protecting Staphylococcus cells from exposure to antibiotics. 10Biofilmforming bacteria infect biofilms in most human infections, with Staphylococcus aureus being the most harmful biofilm-producing species. 7,8MRSA biofilms can spread to the hippocampus, and are not affected by antibiotics as the biofilm acts as a protective shield that increases resistance to antibiotics and other immune factors. 9,11RSA causes chronic infections owing to its ability to resist various antibiotics by forming a biofilm on artificial heart valves, catheters, and medically implanted prostheses. 12,13The spread of MRSA along with other staphylococcal diseases has led to a significant increase in the use of antibiotics at an estimated annual cost of $450 million, with increased disease rates associated with biofilm-mediated infection. 14Therefore, an understanding of the evolution ofstaphylococcal biofilms at the molecular level is necessary to generate new treatment strategies for biofilmassociated infections and reduce the burden caused by these pathogens.Therefore, this study aimed to isolate antibiotic-resistant staphylococci and study their genetic details in 50 relation to the capability of biofilm formation.

Collection of Staphylococcus isolates
The study protocol was approved by Taif University Medical Ethics Review Board (Project No. 1-437-5371) in accordance with the guidelines for human protection.Samples were collected from patients at King Faisal Hospital and Children's hospital in Taif City, Saudi Arabia, from October 2020 to November 2021, with documented patient consent.Approximately 100 bacterial isolates were collected and identified using a fully automated VITEK-2 COMPACT microbiology system (Bio Mtrieux, Inc., Durham, NC, USA).

DNA isolation
DNA was extracted from the isolates Detection of the mecA, SCCmec, and fibronectinbinding protein genes PCR was performed using Go Taq ® Green Master Mix (Promega, USA), according to the manufacturer's instructions.The primers used and conditions for each gene are listed in Table 1. 2,17mplicons were observed after electrophoresis on 1.5% agarose gel using a 100 bp DNA ladder (Fermentas, Lithuania, USA).

Data analyses
Pearson's simple linear correlation coefficient (r) and their significance (P) were assessed using SPSS 20.

Isolation of antibiotic-resistant bacteria
Approximately 100 clinical samples including urine and stool swabs were collected and analyzed for antibiotic-resistant bacteria.Of the 100 cultures tested, 24 bacterial isolates resistant to multiple antibiotics were identified as Staphylococcus and tested for biofilm formation, antibiotic resistance, and detection of virulence and biofilm genes.These isolates were assigned codes S1-S24.

Antibiotic susceptibility testing
In total, 12 antibiotics were tested, and the isolates showed high variability of resistance.Most isolates showed highest resistance to cefrizine and cefepime (96%).The isolates also showed high resistance to amoxicillin and ampicillin (92%), followed by aztreonam (83%).
All the isolates were sensitive to chloramphenicol.The isolates showed low resistance to lincomycin and gentamicin (8%) (Figure 1 and Table 2).The most sensitive isolates were S11, S16, and S18, which were resistant to amoxicillin, cefepime, cefrizine, and aztreonam.Isolates S15 and S17 showed the highest resistance to most of the antibiotics tested.The other isolates showed moderate resistance.

Determination of slime production
Phenotypic slime production was assessed by culturing isolates on CRA plates.Among the isolates, S1, S2, S13, S14, S15, and S17 were slime-producers, developing almost black colonies.The remaining isolates were considered as non-producers because they produced white colonies on CRA plates (Figure 2 and Table 3).

PCR analysis for mecAI, ncu1, and SCCmec genes
The PCR amplification products of mecA and SCCmec in S. aureus isolates are shown in Figure 3 and Table 4.All isolates carried mecA I with a size of approximately 162 bp.The ncu1 amplicon, approximately 301 bp in size, was found   in most isolates except S11.This suggested that all isolates were MRSA, except S11, which is a weak isolate incapable of biofilm formation.Moreover, PCR amplicons of SCCmec were produced in all isolates, as shown in Figure 3.Most isolates had SCCmec III with a size of approximately 243 bp; however, isolates S16, S18, S19, S20, S21, S22, S23, and S24 did not contain SCCmec III.The SCCmec V amplicon, with a size of approximately 325 bp, was found in most tested isolates except in S11, S16, and from S18 to S24.

Detection of fnbA and fnbB
The presence of adhesive genes was confirmed by 127 bp and 524 bp bands of fnbA and fnbB, respectively.Almost all isolates were found to possess genes for both the homologous fibronectin-binding proteins fnbA and fnbB, except S3 and S11, which contained only the fnbA (Table 4 and Figure 2).

DISCUSSION
Biofilm formation is characteristic of several bacterial species and is related to their virulence.Chronic bacterial infections are closely related to biofilm formation. 18,19The current study showed that two isolates, S15 and S17, had high capability to form biofilms.Moreover, 55% of the isolates were medium-grade positive for biofilm formation.1][22] MRSA is characterized by its adhesiveness, an important trait for infecting humans.8][9] Several genes responsible for biofilm formation have been characterized, example, genes encoding adhesion molecules in S. aureus. 23,24In this study, a polystyrene microtiter plate was used to detect biofilm formation.Molecular methods to detect the presence of biofilm-forming genes require the development of biofilm and polysaccharide adhesion between bacterial cells, which is brought about by genes encoding intracellular adhesion enzymes. 8,9The expression of genes related to biofilm formation is regulated by multiple genes, such as fnbA and fnbB, which may interact with each other and regulate biofilm formation.fnbA and fnbB contribute to the invasion and adhesion of this bacterial species, and therefore, may be related to its ability to form biofilms. 25 The prevalence of Staphylococcus carrying these genes have been previously observed, 26,27 and the differences in availability of the genes might be due to varied primer sequences or the location of these genes in the bacterial chromosome.In the current study, 96% of the Staphylococcus isolates carried fnbB and 92% had fnbA, which is in accordance with previous studies. 25The presence of fnbB may be related to the ability to form biofilms.The antibiotic resistance gene, mecA, has recently become prevalent; however, it is not indigenous to S. aureus and has been acquired recently from unknown sources. 17The mecA gene product is a penicillin-binding protein (PBP), specifically BP2a.S. aureus produces four PBPs, 10 which are cytoplasmic membrane-fixing enzymes involved in cell wall formation. 4,28,29Eleven species containing SCCmec have been assigned to the Staphylococcus species. 4,30][32] All the isolates carried mecA and 96% of them had ncu1.Moreover, 79% of the isolates carried SCCmecIII, and 71% had SCCmecV.Several subtypes of SCCmec, including IIA to E, IVa to IVg, and VT have been reported. 2Two possible explanations for the ability of Staphylococcus species to colonize synthetic materials, such as catheters or hospital plastics, are the production of polysaccharide slime by Staphylococcus isolates and the presence of adhesives on biomaterial surfaces to host matrix proteins that are absorbed in vivo. 6,9

CONCLUSION
PCR is an easy, fast, and cheap way to characterize pathogenic S. aureus isolates capable of forming biofilms and carrying related genes such as fnbA and fnbB.In addition, MRSA isolates are resistant to many antibiotics and can cause health issues.The genes mecA and SCCmec are virulence markers in Staphylococcus isolates.

ACKNOWLEDGMENTS
The author extends his appreciation to King Faisal Hospital, Health Affairs and Children's Hospital at Taif, Saudi Arabia, for facilitating the collection of bacterial samples and antibiotic resistance assessment.

FUNDING
None.

DATA AVAILABILITY
All datasets generated or analyzed during this study are included in the manuscript.

ETHICS STATEMENT
This study was approved by Taif University Medical Ethics Review Board (Project No. 1-437-5371).

Figure 2 .
Figure 2. Colorimetric scale for colony analysis of slime production by Staphylococcus aureus 288 S15 and S11 using Congo Red agar assay.A: slime-producing strain (almost black); B: non-289 producing strain (white)

Table 1 .
Primer sequences and amplicon sizes of tested genes fibronectin-binding protein mecA and SCCmec

Table 2 .
The number of isolates and antibiotic resistance profile of Staphylococcus aureus isolates

Table 3 .
Biofilm formation, grade of biofilm and production of the slime of Staphylococcus aureus isolates

Table 4 .
The number of Staphylococcus aureus isolates that habitat the Biofilm and SCCmec genes