Virulence Determination in Invasive and Commensal Staphylococcus Epidermidis

The skin is a crucial barrier protecting the body from the outside world. When this barrier is compromised due to injury, it weakens the body’s natural immune system, making it more vulnerable to bacterial infections [1,2]. Staphylococcus epidermidis (S. epidermidis) is a significant component of the human skin microbiota and plays a crucial role in infections related to medical implants. Non-spore-forming staphylococci are commonly found in nature. Humans and animals naturally harbor several species of Staphylococcus on the skin, mucous membranes, digestive tract, and respiratory system. Staphylococci colonize the skin, particularly in moist areas such as the scalp, face, hands, navel, armpits, and perineum. Both intact skin (via sweat ducts or hair follicles) and damaged skin (such as through lesions) provide entry points for staphylococci. Coagulase-Negative Staphylococci (CoNS) have traditionally been considered less harmful than coagulase-positive ones [3,4].

Staphylococcus epidermidis is generally considered a beneficial bacterium with properties such as regulating the immune system [5] and protecting against pathogen colonization [6]. However, it also can cause hospital-acquired infections associated with implanted medical devices [7,8].

Antibiotic resistance poses a serious threat to public health [9]. Methicillin-resistant staphylococci-caused urinary tract infections (UTIs) are a rising issue for many healthcare facilities, mainly because they are linked to the development of biofilms by these isolates on both living and nonliving surfaces [10].

On the other hand, nothing is known about the processes the bacterium uses to adapt to its environment during carriage, which is a need for pathogenicity [11]. The term "virulence factors" is defined broadly to include both genes and proteins that make it easier for an organism to infect and stay alive in a person’s body.

It is evident that most of these elements also play significant roles in S. epidermidis commensal life as a benign resident of human skin. Therefore, they may not be classified as "virulence factors" in the strictest sense. The two most significant virulence factors of S. epidermidis were biofilm formation and multidrug resistance [12]. The molecular pathways underlying virulence, S. epidermidis, is by far the most thoroughly researched member of the CNS.

virulence of Staphylococcus strains attributed to various factors like the clumping factor clfA, IgG-binding region genes, host specificity, and diverse immunological reactions. which found to possess IgG-binding regions, these are crucial in understanding the pathogenicity of Staphylococcus strains [13].

Genetic markers (fdh) that correctly differentiate between infection and contaminant or commensal sources may assist in the diagnosis of S. epidermidis infections [16,17].

The struggle of S. epidermidis isolates methicillin is due to the mecA gene, which is carried by a mobile genetic element called the cluster chromosomal cassette (SCCmec). The mecA gene encodes a modified penicillin-bound protein (PBP2a) with low affinity for beta-lactam antibiotics [18]. Based on the categories of the MEC gene pool and the types of the CCR gene pool, eleven types (from the first to the eleventh) of SCCmec were assigned to Staphylococcus aureus [19]. The mecA gene is responsible for both virulence and methicillin resistance traits observed in S. epidermidis strains isolated from human clinical infections [20,21].

S. epidermidis ability to adhere to various surfaces is due to its extensive repertoire of surface proteins, each of which has unique adhesive properties that contribute to enhancing its cling ability [14] The surface proteins of Staphylococcus epidermis (S. epidermidis), especially SesI, have attracted great attention due to their immunomodulatory properties [13] and their association with invasive isolates [15].

Microorganism antibiotic resistance is a severe public health issue. Numerous antimicrobial resistance strategies have been developed due to the rising infection rates of bacteria resistant to almost all antibiotics [22]. Antimicrobial resistance has also brought attention to how linked people, animals, and the environment are and how important these factors are in the spread of resistance genes [20,23]. The rise of methicillin-resistant S. epidermidis strains and the clinical significance of such strains have presented several therapeutic hurdles in recent decades [24]. Hospitals now have a severe challenge with resistant S. epidermidis [25,26]. In the United States, annually, approximately 100,000 cases of infections are caused by staphylococcal strains that are resistant to treatment. These infections have a mortality rate of around 10% [26]. The main focus of the study has been on virulence factors in invasive and commensal S.epidermidis isolates and opportunistic human disease.

A. Ethical Statement

The College of Science Research Ethics Committee has accepted this work (ref. CSEC/1220/0081). Each participant consented to the researcher obtaining the specimens. According to the Declaration of Helsinki, each participant obtained informed permission.

B. Samples collection

The commensal isolates of S. epidermidis were collected from the skin and urine of healthy individuals. In contrast, invasive isolates were obtained from wounds, urine, and blood of hospital patients in different hospitals in Baghdad. All isolates were identified using biochemical tests and the Vitek 2 system.

C. Assessment of Antibiotic Susceptibility

The antibiotic’s susceptibility was evaluated by employing a disc diffusion technique that followed the established protocols provided by the Clinical and Laboratory Standards Institute (CLSI) [27] (Table 1).

D. Detection of some virulence genes by conventional polymerase chain reaction (PCR)

All S. epidermidis isolates were inspected for the presence of the commensal marker fdh gene, methicillin resistance mecA gene, clumping factor clf gene, IgG binding protein gene, and invasive marker S. epidermidis surface protein sesI gene. Total DNA extraction was obtained by culturing each isolate overnight at 35°C with shaking in BHI broth (BD et al., USA). The PrestoTM Mini gDNA Bacteria Kit (Geneaid, Taiwan) was used to extract the genomic DNA from the S. epidermidis isolates, and the AccuPower® PCR PreMix and Gradient master cycler (Eppendorf, Germany) was used for all amplifications. For PCR, a 50 µl final volume containing master mix 25µl (Roche et al.), upstream primer 10µm 0.5-5.0µl, downstream primer 0.5-5.0µl, DNA template 1-5µl, and nuclease-free water to 50µl was used (Promega kit). The conditions were as follows: one cycle of initial denaturation at 95℃ for 2 minutes, then 30 cycles of denaturation at 95 ℃ for one minute, annealing at a temperature dependent on the gene annealing temperature for 20 sec, elongation at 72 ℃ for 1 minute, and a final extension cycle at 72 ℃ for 5 minutes. Electrophoresis in 2% agarose gel was used to analyze amplicons. The positive and negative controls were S. epidermidis ATCC 35984 and S. aureus ATCC 29213, respectively. The National Center for Biotechnology Information’s GenBank sequence database obtained the fdh, mecA, clf, IgG, and sesI gene sequences. The primers were created by the Microgene company [28] using specific primers, respectively (Table 2).

E. Statistical Analysis

The System—SAS (2018) [29] program was used to detect the effect of different factors on study parameters. This study used the chi-square test to compare percentages (0.05 and 0.01 probability) significantly.

A. Staphylococcus epidermidis isolates diagnostic

One hundred and fifty samples were collected in our study to characterize the human skin microbiome in healthy individuals (HIs) [30], and 150 samples were collected from different body parts of clinical patients. The results showed 30(10%) detection as S. epidermidis was detected by traditional biochemical tests and Vitek 2.

B. Assessment of Antibiotic Susceptibility

In clinical samples, S. epidermidis was the most frequently isolated CONS. Additionally, among the discovered CONS, we noted variations in antibiotic reluctance. S. epidermidis had higher rates of resistance to cefoxitin 13/15 (86.6%), 11/15 (73.3%) in, Oxacillin 15/15(100), 12/15(80%) penicillin15/15 (100%), 11/15(73.3%) in clinical and healthy isolates respectively, while the most effective antibiotic the vancomycin 12/15(80%) 15/15(100%) in clinical and healthy isolates respectively. Figure 1, 2 and Table 3.

C. Detection of the Staphylococcus epidermidis virulence gene by Polymerase chain reactions

S. epidermidis isolates were obtained from clinical and healthy individuals subject to investigation about the virulence genes by using the PCR technique. The results show commensal marker gene (fdh) found in 3/15(20%), 6/15(40%), and the invasive marker gene (sesI) present in 3/15(20%), 1/15(6.6%) from clinical and healthy isolates respectively Figure 3 and Figure 4 Figure 5.

While the virulence factors genes as mecA present in 12/15(80%), 7/15 (46.6%) clf gene were found in 11/15(73.3%), 9/15(60%) in clinical and healthy isolates, respectively Figure 6 and Figure 7, the IgG gene was detected in 14/15(93.3%) Figure 8 in both clinical and healthy individual isolates. Figure 9 Table 4.