Distribution of Esp Virulent Gene Among Staphylococcus aureus Isolates in Clinical Samples from Ekiti State University Teaching Hospital, Ado - Ekiti, Nigeria

Infection with Staphylococcus aureus has been considered a major problem in hospitals. The clinical importance of S. aureus is attributed to notable virulence factors and genetic diversity. The study aimed to investigate the distribution of (Enterococal Surface Protein) esp virulent gene and different genotypes in some clinical isolates obtained from Ekiti State University Teaching Hospital, Ado Ekiti. A total of 100 clinical isolates were collected and screened out of which 75 samples with good S. aureus yield were used for this study. 34 (45.3%) were from males and 41 (54.7%) were from females. The isolates were drawn from across several age groups and sample types. Result of antibiotic susceptibility test against 75 S aureus isolate showed 64.0%, 58.7%, 54.7%, and 52.0% of the isolates were sensitive to ciprofloxacin, streptomycin, rocephin, and perfloxacin respectively. Conversely 88.0% of the isolate shows resistance to ampiclox 78.7% to zinnacef, 70.7% to amoxicillin, 64.0% to erythromycin, 64.0% to gentamycin and 56.0% to septrin. Using Polymerase Chain Reaction (PCR), virulent genes esp formed clusters in S. aureus isolates used in this study. Virulence diversity was observed among isolates. The genetic typing was carried out by Random Amplified Polymorphic DNA (RAPD-PCR) using OPT06 and OPHO6, primers. In conclusion there exist different genotypes among the S. aureus isolates used in this study revealing high level of genetic diversity occurrence among S. aureus isolates. The DNA fingerprint revealed high genetic diversity among isolates irrespective of their sources.


Introduction
The most common cause of hospital acquired infections is Staphylococcus aureus.The variety of illnesses that staphylococci can cause in people includes necrotizing pneumonia, toxic shock syndrome, osteomyelitis, endocarditis, and abscesses in organ tissues [1].Specific strains of the adaptable pathogen S. aureus have been found in a variety of pathological-anatomical locales and disease entities.Numerous virulence factors, including cell wall-anchored proteins, secreted toxins, capsular-and exopolysaccharides, iron-transport systems, and modulators of host immunological activities, in addition to genes for antibiotic resistance, contribute to this versatility [2].
The bacterial chromosome contains the genetic information for the great majority of staphylococcal virulence factors, including surface proteins, sortase, -haemolysin, exotoxins, and agr regulation.Adhesins, surface proteins such protein A, and, in the case of toxins, enterotoxins of S. aureus (SE) and β-Hemolysin (Hlb) are among the virulence factors that play a significant role in pathogenicity [30] Bacterial colonization and adhesion to host cells and extracellular components are aided by surface proteins.Toxic shock syndrome toxin-1 (TSST-1), Panton Valentine leukocidin (PVL), and other cellular proteins, proteases, and toxins that prevent phagocytosis and prevent the host from actively resisting infection through a particular immune response [4], these genes are present in all staphylococcal strains and are not connected to mobile DNA elements [5] Through a global regulation system made up of the accessory regulatory gene (Agr) and the sigma factor (σB), S. aureus has the capacity to regulate the expression of virulence factors in accordance with the environmental conditions in which it is found [6].The ability of S. aureus to adapt to various microenvironments with various environmental, nutritional, and stress conditions may result in the acquisition of genes coding for virulence factors that allow its survival in addition to gene regulation [7].It has been shown that one mobile genetic element can mobilize multiple virulence factor-coding genes.For instance, in the bacteriophage βC-φs, the staphylokinase gene and complement inhibitor protein are mobilized alongside enterotoxin genes and Panton-Valentine leucocidin [8] The human pathogen Staphylococcus aureus is persistent and causes a number of diseases with a wide range of clinical manifestations and severity [1].In response to host signals, including as those found in target tissues and those associated to innate defenses triggered throughout the infectious phase, various networks of virulence genes are probably activated.This expectation shows up in vitro as a pattern of virulence determinant expression that is reliant on growth phase and is set up by global regulatory components [9].The organism produces fibrinogen-binding, fibronectin-binding, and collagen-binding proteins as well as other cell-wall proteins with sticky properties during the exponential growth phase; this expression may facilitate colonization's early stages in the host.Cell-wall protein expression is suppressed as the growth cycle moves from the exponential to the stationary phase, while the production of extracellular toxins and enzymes takes center stage.These exotoxins may promote local invasion and spread of infection by their proteolytic activities and toxic effects on host cells [1].
Genetic study is crucial for understanding the genotype of S. aureus because it has been documented that it affects the complications, severity, and mortality of infection.A Comprehensive gel documentation of DNA fingerprint profile of Staphylococcus aureus isolates which is very important to show comparison of genetic diversity, virulence factors and degree of pathogenicity among isolates has never be carried out in the study area using clinical isolates.This study was therefore carried out to identify and differentiate the genetic diversity, antibiotic resistance and virulence factors of 75 Staphylococcus aureus isolates using their genomic DNA.The current study used a more sensitive genetic PCR and DNA sequencing analyses approaches to estimate virulence mutants in Staphylococcus aureus.

Collections, isolation and purification of Staphylococcus aureus isolates from clinical samples and storage
This study was carried out in Ekiti State University Teaching Hospital Ado-Ekiti, Ekiti State, Nigeria.Ethical approval for collection of sample was obtained from the same institution.One hundred (100) different isolates of S. aureus from clinical samples ranging from urine, urethra, HVS, sputum and wound cultures was obtained from Ekiti State University Teaching Hospital Ado-Ekiti, Ekiti State, Nigeria.These isolates were identified by conventional microbiological methods, including gram staining, catalase and coagulase test.Seventy five (75) of these isolates were collected each into 1.5 ml Eppendorf tubes and store at 4 o C until genomic DNA extraction and purification.

Genomic DNA Isolations and Purification from Staphylococcus aureus isolates
Genomic DNA Isolations and Purification from Staphylococcus aureus isolates was done according to the method described by Onasanya et al. (2003).0.3g of washed bacterial cell was suspended in 200µl of CTAB buffer (50mM Tris, pH 8.0); 0.7 mm NaCl; 10 mm EDTA; 2% hexadecyltrimethyl ammonium bromide and 0.1% 2-mercaptoethanol), followed by the addition of 100 µl of 20% SDS.It was incubated at 65 o C for 30 min.DNA was purified by two extractions with phenol: chloroform: isoamylalcohol (24:25:1) and centrifuged at 14,000 rpm at 4 o C for 10 mins.DNA was precipitated from supernatant with equal volume of absolute ethanol and centrifuged at 10,000 rpm at 4 o C for 10 mins.DNA pellet was washed with 70% ethanol, dried at room Temperature, re-suspended in 200 µl of sterile distilled water.
DNA concentration was measured using a spectrophotometer at 260 nm.DNA degradation was checked by electrophoresis on a 1% agarose gel in TAE buffer (45 mm Tris-acetate, 1 mm EDTA, pH 8.0).
 Amplification products was maintained at 4 0 C until electrophoresis.

Separation of PCR Products
The amplification products were resolved by electrophoresis in a 1.4% agarose gel using TAE buffer (45 mM Trisacetate, 1 mM EDTA, pH 8.0) at 100 V for 2 hours.A 1 kb ladder was included as molecular size marker.Gels were visualized by staining with ethidium bromide solution (0.5μg/ml).Banding patterns were photographed over UV light using UVP-computerized gel photo documentation system.Genotypes and genetic virulence factors relationship determinant analysis (Band Scoring) was done using gel documentation system.

Statistical Analysis
Genomic Data using Genotyping Software was carried out to reveal different Genotypes and genetic virulence factors relationship among 75 Staphylococcus aureus isolates.The results were presented in tables, figures and charts.
Figure 11 shows Cluster of genetic diversity among 75 S. aureus isolates amplified DNA OPT06 PCR-RAPD products analyzed by electrophoresis.DNA fingerprint of 75 S. aureus isolates using OPT06 primer revealed existence of different genotypes among the isolates; some isolates were identical.Figure 12 shows the DNA fingerprint of 75 S. aureus genomic DNA as revealed by OPH06-PCR analysis and Figure 13 shows Cluster of genetic diversity among 75 S. aureus isolates amplified DNA OPH06 PCR-RAPD products analyzed by electrophoresis.DNA fingerprint of 75 S. aureus isolates using OPH06 primer revealed existence of different genotypes among the isolates; some isolates were identical.
Figure 14 shows cluster of genetic diversity among 75 S. aureus isolates as revealed by 64 RAPD markers.The results obtained showed that 75 S. aureus isolates were distinct with high level of genetic diversity as revealed by 64 RAPD PCR makers; only two sets of isolates Sa54, Sa55 and Sa66, Sa67 are identical respectively.Figure 15 shows the PCR profile of virulence target gene detection by 16 specific primers used for preliminary PCR amplification and screening of S. aureus isolates genomic DNA. Figure 16 shows the PCR detection of esp virulence gene at 932 base pairs in 75 S. aureus isolates genomic DNA and Figure 17 shows the esp virulence gene cluster in 75 S. aureus isolates genomes.

Discussion
Staphylococcus aureus is a gram-positive bacterium that can occasionally cause pus and abscesses, sepsis, and even fatal septicemia in humans.It can be found in the axillae, nose, groin, perineal area (in males), mucous membranes, mouth, mammary glands, hair, and intestinal, genitourinary, and upper respiratory tracts.Numerous virulence factors that promote colonization and disease in the host influence S. aureus capacity to cause disease.[11] This study was carried out to determine the distribution of esp virulent gene among Staphylococcus aureus isolates from clinical samples in Ekiti State University Teaching Hospital, Ado-Ekiti, Ekiti State, Nigeria.The highest frequency of isolates of Staphylococcus aureus (56%) in the present study was observed in the 21-40 years age group, followed by 0-20 years age group in which neonates and infants were included.This finding is similar to a previous study by [12] Neonatal immunity is thought to be underdeveloped during this time, making newborns prone to bacterial illnesses and extremely contagious, especially when hospitalized.
From the study, male subjects were more infected with S. aureus (54.7%) compare with female subjects (45.3%).This finding is in agreement with previous report [12]; [13] However, it is not clearly understood why males were more infected than females in the present study.Staphylococcus aureus was found to be more associated with Urine (38.7 %%), Semen (24%) and HVS (20%) respectively.A study by [15] at Ilorin, Nigeria reported wound infections of 38% as the highest frequency of S. aureus isolates.This contradicts the finding of the present study where ear swab had the highest isolate of 30.7%.
Over time, Staphylococcus aureus develops highly quick and effective resistance to several antibiotics.The highest frequency of S. aureus occurred with susceptibility to antimicrobial agent Ciprofloxacin (64%) followed by Streptomycin (58.7%) while the least was Ampliclox (12%).The low percentage sensitivity of S. aureus observed in the present study against the following drugs; Zinnacef, Amoxicillin, Erythromycin and Gentamicin were in agreement with the reports published by previous authors from Nigeria [12]; 14]; [15]; [16]; [17] The greater level of resistance may be related to the medications' earlier exposure to isolates that may have a higher propensity to acquire resistance.Antibiotic abuse is rampant in this setting due to self-medication, which is frequently accompanied by insufficient dosage, noncompliance with therapy, and consumer access to antibiotics over the counter with or without a prescription [18] in the current investigation, the sensitivity of Staphylococcus aureus to Gentamicin was 36%.This contrasts well with studies that some authors have published.In underdeveloped nations like Nigeria, where there are no regulatory laws in this regard, it has been noted that the indiscriminate use of antibiotics without prescription has rendered the routinely used antibiotics utterly ineffective in the treatment of Staphylococcus aureus infections [18] The enterococcal surface protein (esp), a high-molecular-weight surface protein with no known function, is much more common in isolates of Enterococcus faecalis that are the result of infections [19] In this study, the esp virulent gene was detected by PCR at 932 base pairs in S. aureus isolates genomic DNA (figure 10 and 11).According to [12], there exist different genotypes among the S. aureus isolates used in the study revealing high level of genetic diversity occurrence among S. aureus isolates.The DNA fingerprint revealed high genetic diversity among isolates irrespective of their sources 12 A clinical strain of E. faecalis genome underwent random sequencing, and the esp gene was discovered there [20].esp strongly suggests a function in the pathogenesis of E. faecalis because it has been preferentially found in infectionderived E. faecalis strains but not in other less pathogenic enterococcal species [20].Staphylococcus epidermidis, a nonpathogenic bacterium typically found in human nose and oral cavities, secretes esp, a type of serine protease.
Staphylococcus aureus, a bacterium that can be found on the skin and in the nasal passages of up to 25% of healthy individuals and animals, is closely related to S. epidermis.Some S. aureus strains produce a heat-stable toxin that causes disease and is challenging to eradicate because it can build a biofilm.In this study, the esp gene thought only to be present in Staphylococcus epidermidis, E. faecalis and enterococcal species, was found to be present in Staphylococcus aureus isolated from clinical isolates.The esp virulent gene was detected by PCR at 932 base pairs in S. aureus isolates genomic DNA (figure 10 and 11).
The esp was originally isolated and biochemically studied by [21], who found that it can break down a number of proteins with a high predilection for cleavage following glutamic acid residues, including fibrinogen and complement protein C5.Therefore, it was asserted that this process allowed S. epidermidis to sidestep the complement defense system.In actuality, another team of scientists backed up this assertion [21].[22] reported that the gene structure of Esp and the amino acid sequence of its mature form showed close resemblance with one of the S. aureus serine proteases, which have been long regarded as crucial virulence factors that obliterate blood proteins and tissues, thus contributing to increased invasiveness [22].
Esp, as it turned out, prevented S. aureus colonization and infection by preventing the development of biofilms, according to [23].Esp was found to degrade S. aureus biofilm by causing S. aureus to change from the sessile to the planktonic form, even though the exact mechanism by which Esp breaks the S. aureus biofilm is still unknown and does not resemble any bacterial interference mechanisms like growth inhibition and bactericidal activity.Esp-containing supernatant incubation for 16 hours produced a startling four-fold reduction in S. aureus biofilm.Esp differs from other biofilm breakers in that it releases bacteria from the biofilm rather than killing the bacteria inside it.This is one way in which esp differs from other biofilm breakers.As a result, S. aureus won't be able to develop a resistance to esp [23].
Esp demonstrates traits of surface protein receptors identified as microbial surface components, identifying adhesive matrix molecules, and attaching to host components to begin colonization [24].Many of these proteins have a modular structure and contain a number of tandem repeat domains, which are most likely the result of numerous instances of duplication and/or recombination.esp may have a direct ligand-binding activity to the extracellular matrix under in vivo settings or may indirectly play a function in influencing the ligand-binding activity of other molecules, even though we have no evidence that it contains domains for binding to host factors.If this is the case, esp's major function in the host-bacterium interaction would not be to facilitate E. faecalis adhesion to plastic biomaterials.Esp has been suggested to improve the hydrophobicity of cell surfaces and promote hydrophobic interactions [20].Our results demonstrate that the presence of Esp in the cell surface could increase hydrophobicity, adherence to abiotic surfaces, and biofilm formation.
It is recognized that S. aureus surface proteins play a significant role in transmitting infectious illnesses to people, ranging from mild skin infections to life-threatening infections [25].SdrD, Spa, IsdA, and Emp are required for the development of an abscess [26].Because of its significance in several aspects of infection (such as anti-inflammatory and antiangiogenic characteristics and abscess formation), esp has attracted the attention of researchers [27].By forming complexes with pathogen-specific antibodies, complement factor 3d, or VH3-type IgM on the surface of B cells, immunoglobulin-binding proteins Spa and Sbi aid in immune evasion [27].Our results unequivocally show that the newly discovered esp in S. aureus breaks down various surface proteins and toxins, including coagulase and -toxin, indicating that Esp might be useful for the prevention or treatment of infectious illnesses brought on by this pathogen.esp should only be used therapeutically, since its safety has not yet been shown, therefore this should be taken into consideration.An alternate strategy could be the creation of a vaccination that specifically targets proteins that are broken down by esp and also aid in the biofilm formation and colonization of human skin and the human nasal cavity by S. aureus.

Conclusion
In this study, the esp virulent gene was detected by PCR at 932 base pairs in S. aureus isolates genomic DNA (figure 14 and 15).DNA fingerprint revealed high genetic diversity among isolates irrespective of their sources.These isolates had a wide range of virulence genotypes.According to estimates, biofilm-related infections account for about 65% of nosocomial infections in the developed world.Since the usage of medical implants is projected to rise in the near future, this serves as a warning flag.In this setting, bacterial molecules involved in host protein-mediated attachment as well as others, like esp, involved in adhesion to inorganic surfaces and biofilm formation could become promising therapeutic targets in control programs for eliminating persistent enterococcal infections linked to the presence of biofilms.

 1
cycle of 94 o C for 3 min. 45 cycles of 94 o C for 1 min for denaturation, 40 0 C for 1 min for annealing of primer and 72 o C for 2 min for extension.

Figure 7
Figure 7 Cluster showing genetic diversity among 75 S. aureus isolates amplified DNA

Figure 9 Figure 10
Figure 9 Cluster showing genetic diversity among 75 S. aureus isolates amplified DNA

Figure 11 PCRTable 5 Figure 12 Figure 13
Figure 11 PCR Profile virulence target gene detection by 16 specific primers used for preliminary PCR amplification and screening of S. aureus isolates genomic DNA

Table 1
Demographic Characteristics of the Subjects

Table 4
Identity of virulence target gene specific primers used for preliminary PCR amplification and screening of S.