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| Effects of agr alleles on the hemolytic activities of S. aureus. The overnight cultures of S. aureus strains with the same CFUs were plated on Columbia blood agar plates and grown for 24 h at 37 • C. (A) Hemolytic activities of the four wild agr allelic strains: agrI (Newman), agrII (N315), agrIII (MW2), and agrIV (XQ). (B) Hemolytic activities of the wild Newman strain, Newman agrBDC, and agr genomic in situ replacement mutants of Newman (agrI to agr IV). Hemolytic activities of the wild type, agrBDC mutants and pLI50-agr plasmid complement mutants of Newman (C) and N315 (D). The hemolytic activities of the relative strains were determined by measuring the optical density (OD543).
Source publication
The a ccessory g ene r egulator ( agr ) quorum-sensing system is an important global regulatory system of Staphylococcus aureus and contributes to its pathogenicity. The S. aureus agr system is divided into four agr groups based on the amino acid polymorphisms of AgrB, AgrD, and AgrC. The agr activation is group-specific, resulting in variations in...
Contexts in source publication
Context 1
... amino acid sequences of AgrB, AgrD, and AgrC are variable (Supplementary Figure 1), whereas AgrA, RNAIII, and their promoter regions are highly conserved (Supplementary Figure 2). According to the polymorphisms of AgrB, AgrD, and AgrC, the agr system in S. aureus is divided into four types named agrI, agrII, agrIII, and agrIV. ...
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... hemolytic capabilities of S. aureus vary with their agr types. As shown in Figure 2A, the Newman (agrI) and XQ (agrIV) strains have strong hemolytic FIGURE 1 | The identification of agr allelic mutant in S. aureus Newman. (A) The confirmation of agr allele replacement plasmid by restriction enzyme digestion (take type II agrBDC replacement as an example). ...
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... 5, 6, and 7 are patterns of the PCR amplification products of agrBDC-up (Band5, primers UL + UR), agrBDC-down (Band6, primers DL + DR), and agrBDC-II from N315 (Band7, primers AL + AR) fragments, respectively. (B) The confirmation of agr allelic replacement strains in Newman background by PCR amplification with primers AL + AR (Bands 1, 3, 5, 7) and primers OL + OR (Bands 2, 4, 6, 8). Bands 1-2 refer to the PCR products of NewmanagrBDC mutants, Bands 3-4 are the PCR products of the congenic strain harbor agrBDC-II sequence, bands 5-6 refer to PCR products of the congenic strain with agrBDC-III replacement, and bands 7-8 for agrBDC-IV replacement. ...
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... 1-2 refer to the PCR products of NewmanagrBDC mutants, Bands 3-4 are the PCR products of the congenic strain harbor agrBDC-II sequence, bands 5-6 refer to PCR products of the congenic strain with agrBDC-III replacement, and bands 7-8 for agrBDC-IV replacement. All primers are shown in Supplementary Table 2. activities, the MW2 (agrIII) strain showed weak hemolysis, and the N315 (agrII) strain presented the lowest hemolytic toxicity (Figure 2A). ...
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... assess the effects of different agr alleles on S. aureus hemolytic activity, the agrBDC genes in Newman strain (agrBDC-I) were deleted and in situ substituted with other three agrBDC alleles (agrBDC-II, agrBDC-III, and agrBDC-IV). As expected, the hemolytic activity of the NewmanagrBDC strain was significantly lower than that of the wild strain ( Figure 2B). The knock-in of type I agrBDC genes back to the genome of NewmanagrBDC mutant recovered the hemolytic activity of revertant strain (Figure 2B). ...
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... expected, the hemolytic activity of the NewmanagrBDC strain was significantly lower than that of the wild strain ( Figure 2B). The knock-in of type I agrBDC genes back to the genome of NewmanagrBDC mutant recovered the hemolytic activity of revertant strain (Figure 2B). Contrary to our expectations, no visible hemolysis and very low hemolytic activity were discovered on three congenic replacement strains harboring heterologous agr systems (Figure 2B). ...
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... knock-in of type I agrBDC genes back to the genome of NewmanagrBDC mutant recovered the hemolytic activity of revertant strain (Figure 2B). Contrary to our expectations, no visible hemolysis and very low hemolytic activity were discovered on three congenic replacement strains harboring heterologous agr systems (Figure 2B). The hemolytic activity controlled by agr system seemed to be severely suppressed when the three heterologous agr alleles were in situ recombined into the genome of NewmanagrBDC mutant. ...
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... investigate this further, four recombinant pLI50 plasmids containing types I to IV agrBDCA genes and their promoter sequences were, respectively, transformed into the agrBDC mutant strains of Newman or N315. As shown in Figures 2C,D, the four agr complemented strains showed similar hemolytic activities to each other. The differences in hemolysis across agr groups were significantly weakened in the same Newman or N315 background when compared with the standard agr allelic strains. ...
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... expected, the knockout of agrBDC genes led to significant variations in hemolysis activity, pigment formation, exoprotein expression, and virulence factor expressions of NewmanagrBDC mutant. No discernible difference was observed between wild N315 and N315agrBDCA mutant because of the low activity of the N315 agr system under normal condition (Figures 2-6). However, when the three heterologous agr alleles (agrII, agrIII, and agrIV) were individually introduced into the native agr site of NewmanagrBDC genome, the hemolytic activity, pigment formation, and exoprotein expression of three congenic strains were almost identical to those of NewmanagrBDC mutant. ...
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... Figure 2 | The comparisons of AgrA (A), RNAIII (B), and promoter sequences (C) among the four S. aureus agr allelic strains: agrI (Newman), agrII (N315), agrIII (MW2), and agrIV (XQ). The sequences were obtained from NCBI (National Center for Biotechnology Information) database (http://www.ncbi.nlm.nih.gov) and aligned with the Clustal X and DNAMAN software. ...
Similar publications
Bacteria use quorum sensing (QS) as a communication mechanism that releases small signaling molecules which allow synchronizing a series of activities involved in the pathogenesis, such as the biosynthesis of virulence factors or the regulation of growth of other bacterial species. HQNO is a metabolite of the Pseudomonas aeruginosa -specific QS sig...
Citations
... AgrA expression can influence several virulence factors including pigment formation in S. aureus . Both S. aureus and Staphylococcus lugdunensis with mutant agrA operon were found to have reduced pigmentation as compared to wildtype strains (Aubourg et al., 2022;Tan et al., 2022;Cella et al., 2023). On this basis, we validated the docking analysis of AgrA using staphyloxanthin quantification. ...
... RNA III-independent agr pathway is involved in carotenoid production in S. aureus (Queck et al., 2008). Mutations on agr can lead to variations in virulence factors including loss of pigment production, hemolysin activity, and oxidative stress regulation (Aubourg et al., 2022;Tan et al., 2022). The impact of agr gene expression levels on biofilm formation is, however, less definitive compared to other genes such as icaA and icaR (Arciola et al., 2015). ...
New approaches to combating microbial drug resistance are being sought, with the discovery of biofilm inhibitors considered as alternative arsenal for treating infections. Natural products have been at the forefront of antimicrobial discovery and serve as inspiration for the design of new antibiotics. We probed the potency, selectivity, and mechanism of anti-biofilm activity of modified oxylipins inspired by the marine natural product turneroic acid. Structure-activity relationship (SAR) evaluation revealed the importance of the trans-epoxide moiety, regardless of the position, for inhibiting biofilm formation. trans-12,13-epoxyoctadecanoic acid (1) and trans-9,10 epoxyoctadecanoic acid (4) selectively target the early stage of biofilm formation, with no effect on planktonic cells. These compounds interrupt the formation of a protective polysaccharide barrier by significantly upregulating the ica operon’s transcriptional repressor. This was corroborated by docking experiment with SarA and scanning electron micrographs showing reduced biofilm aggregates and the absence of thread-like structures of extrapolymeric substances. In silico evaluation revealed that 1 and 4 can interfere with the AgrA-mediated communication language in Staphylococci, typical to the diffusible signal factor (DSF) capacity of lipophilic chains.
... Staphylococcus aureus strains can be grouped into four categories based on their agr system (agrI-IV). These groups often exhibit different phenotypes, such as varying biofilm formation capacities [37,38]. Certain chemotypes of L. origanoides with thymol (32.7%) and carvacrol (18.8%) have been found to reduce the expression of agr in S. aureus [35], suggesting that differences in the type of agr among the strains could affect the biofilm biomass production and IC 50 antibiofilm activity of EO. ...
Staphylococcus aureus infections are prevalent in healthcare and community environments. Methicillin-resistant S. aureus is catalogued as a superbug of high priority among the pathogens. This Gram-positive coccus can form biofilms and produce toxins, leading to persistent infection and antibiotic resistance. Limited effective antibiotics have encouraged the development of innovative strategies, with a particular emphasis on resistance mechanisms and/or virulence factors. Medicinal aromatic plants have emerged as promising alternative sources. This study investigated the antimicrobial, antibiofilm, and antihemolysis properties of three different chemotypes of Lippia origanoides essential oil (EO) against susceptible and drug-resistant S. aureus strains. The chemical composition of the EO was analyzed using GC-MS, revealing high monoterpene concentrations, with carvacrol and thymol as the major components in two of the chemotypes. The third chemotype consisted mainly of the sesquiterpene β-caryophyllene. The MIC values for the two monoterpene chemotypes ranged from 62.5 to 500 µg/mL for all strains, whereas the sesquiterpene chemotype showed activity against seven strains at concentrations of 125–500 µg/mL, which is the first report of its anti-S. aureus activity. The phenolic chemotypes inhibited biofilm formation in seven S. aureus strains, whereas the sesquiterpene chemotype only inhibited biofilm formation in four strains. In addition, phenolic chemotypes displayed antihemolysis activity, with IC50 values ranging from 58.9 ± 3.8 to 128.3 ± 9.2 µg/mL. Our study highlights the importance of L. origanoides EO from the Yucatan Peninsula, which has the potential for the development of anti-S. aureus agents.
... Several studies have associated specific diseases with different agr groups, such as invasive infections linked to agr group I strains and non−invasive infections to agr group III strains. Notably, agr group I predominates among S. aureus isolates (Bibalan et al., 2014;Javdan et al., 2019;Tan et al., 2022). Our study observed a prevalence of agr type I (67%, 69/103) in S. aureus isolates from vascular access infections, particularly in MRSA isolates. ...
Introduction
Staphylococcus aureus, is a pathogen commonly encountered in both community and hospital settings. Patients receiving hemodialysis treatment face an elevated risk of vascular access infections (VAIs) particularly Staphylococcus aureus, infection. This heightened risk is attributed to the characteristics of Staphylococcus aureus, , enabling it to adhere to suitable surfaces and form biofilms, thereby rendering it resistant to external interventions and complicating treatment efforts.
Methods
Therefore this study utilized PCR and microtiter dish biofilm formation assay to determine the difference in the virulence genes and biofilm formation among in our study collected of 103 Staphylococcus aureus, isolates from hemodialysis patients utilizing arteriovenous grafts (AVGs), tunneled cuffed catheters (TCCs), and arteriovenous fistulas (AVFs) during November 2013 to December 2021.
Results
Our findings revealed that both MRSA and MSSA isolates exhibited strong biofilm production capabilities. Additionally, we confirmed the presence of agr types and virulence genes through PCR analysis. The majority of the collected isolates were identified as agr type I. However, agr type II isolates displayed a higher average number of virulence genes, with MRSA isolates exhibiting a variety of virulence genes. Notably, combinations of biofilm-associated genes, such as eno−clfA−clfB−fib−icaA−icaD and eno−clfA−clfB−fib−fnbB−icaA−icaD, were prevalent among Staphylococcus aureus, isolates obtained from vascular access infections.
Discussion
These insights contribute to a better understanding of the molecular characteristics associated with Staphylococcus aureus, infections in hemodialysis patients and provided more targeted and effective treatment approaches.
... RNAII could translate into four essential proteins (agrBDCA), while the RNAIII is the small yet effective component regulating the expression of many important virulence factors. The agr locus could be divided into four groups according to sequence diversity of agrB, agrD, and agrC [10][11][12]. It is reported that strains of different agr types often exhibit different phenotypes [11]. ...
... The agr locus could be divided into four groups according to sequence diversity of agrB, agrD, and agrC [10][11][12]. It is reported that strains of different agr types often exhibit different phenotypes [11]. ...
Background
Staphylococcus aureus, one of the most prevalent opportunistic pathogens, mainly colonizes the nasal cavity and is a risk factor for severe infections. Virulence factors and accessory gene regulator (agr) are key to the severity and diversity of staphylococcal infection. In this study, we aimed to characterise S. aureus agr-types and virulence genes and correlated them with genetic background and antibiotic-resistant phenotypes.
Results
Agr types were identified in 704 isolates (98.5%), with only 11 isolates were negative for agr type. Most of our isolates were classified as agr type I, followed by types III, II and IV. The enterotoxin c gene (sec) was detected in 48.6% of isolates, showing the highest prevalence among the five enterotoxin genes detected. The positivity rates for the lukS/F-PV and tsst genes were 4% and 2.2%, respectively, while neither sed nor SasX were detected. ST45, ST59, ST338, ST188, ST6, ST7, ST22, ST25, ST398, and ST944 belonged to agr I group, while ST5 and ST15 belonged to agr II group. ST30 and ST1 were classified into agr III group, and ST121 was assigned into agr IV group. The tsst gene was found exclusively within agr I and III types belonging to ST7 and ST30 isolates, while the lukS/F-PV was predominantly carried by agr I type isolates primarily within CC59 and CC22 clones. Among the methicillin-resistant S. aureus (MRSA) isolates, 89.7% belonged to agr I group, and 97.8% of rifampicin-resistant or intermediate isolates were assigned to agr I group. MRSA isolates harboured more tested virulence genes compared to methicillin-susceptible S. aureus isolates.
Conclusions
We characterized the distributions of agr types and eight major virulence genes of 715 S. aureus isolates, and our findings revealed clear associations between agr types and STs, as well as virulence genes, and drug resistant phenotypes.
... Our data showing the downregulation of exotoxins and increased production of fibronectin binding after growth under LSMMG conditions is therefore consistent with the findings of Rosado et al. [21] at least for the CA-MRSA and MSSA strains evaluated. The S. aureus strain N315 used by Castro et al. [20] is an HA-MRSA strain which in common with many HA-MRSA strains is only weakly cytotoxic [44,45]. For certain HA-MRSA strains, reduced agr activation has been linked to changes in the cell wall architecture associated with acquisition of the mobile genetic element (SCCmec) that confers methicillin resistance [45]. ...
Bacterial contamination during space missions is problematic for human health and damages filters and other vital support systems. Staphylococcus aureus is both a human commensal and an opportunistic pathogen that colonizes human tissues and causes acute and chronic infections. Virulence and colonization factors are positively and negatively regulated, respectively, by bacterial cell-to-cell communication (quorum sensing) via the agr (accessory gene regulator) system. When cultured under low-shear modelled microgravity conditions (LSMMG), S. aureus has been reported to maintain a colonization rather than a pathogenic phenotype. Here, we show that the modulation of agr expression via reduced production of autoinducing peptide (AIP) signal molecules was responsible for this behavior. In an LSMMG environment, the S. aureus strains JE2 (methicillin-resistant) and SH1000 (methicillin-sensitive) both exhibited reduced cytotoxicity towards the human leukemia monocytic cell line (THP-1) and increased fibronectin binding. Using S. aureus agrP3::lux reporter gene fusions and mass spectrometry to quantify the AIP concentrations, the activation of agr, which depends on the binding of AIP to the transcriptional regulator AgrC, was delayed in the strains with an intact autoinducible agr system. This was because AIP production was reduced under these growth conditions compared with the ground controls. Under LSMMG, S. aureus agrP3::lux reporter strains that cannot produce endogenous AIPs still responded to exogenous AIPs. Provision of exogenous AIPs to S. aureus USA300 during microgravity culture restored the cytotoxicity of culture supernatants for the THP-1 cells. These data suggest that microgravity does not affect AgrC-AIP interactions but more likely the generation of AIPs.
... The majority of these virulence genes are regulated by the accessory gene regulator (agr) system which is divided into four (1-4) agr groups. 7,8 The agr region is crucial in pathogenesis and actively controls the expression of virulence factors, heterogeneous resistance in methicillin-resistant S. aureus (MRSA), and biofilm development. [9][10][11] It co-regulates the expression of several exoproteins including α-, β-, γ-haemolysin as well as lipases, phenol-soluble modulins and TSST-1 while down regulating the synthesis of cell-wall-associated proteins such as protein A, coagulase, and fibronectin binding protein. ...
Staphylococcus aureus is a prominent pathogen that causes serious community and hospital-acquired infections globally. Its pathogenicity is attributed to a variety of secreted and cell surface associated proteins that are modulated by the quorum-sensing accessory gene regulator (agr) system. In this study, we investigated the presence of toxin genes and agr involved with S. aureus from clinical samples and apparently healthy individuals. Unequivocal identification of the isolates was obtained with the Vitek 2 system. We screened 70 clinical (CL) and 22 community (C) S. aureus strains for the methicillin resistance (mecA) gene, agr and superantigens (SAg) (enterotoxins and toxic shock syndrome toxin-1) using PCR techniques. A total of 12 clinical isolates were classified as methicillin-resistant S. aureus (MRSA); 89 isolates belonged to one of the four agr groups (agr1-4), and 3 isolates were non-typeable. Of the agr groups, agr1 was the most prominent and mostly consisted of isolates from pus/wounds. The methicillin-susceptible S. aureus (MSSA) isolates were distributed within the four agr groups while MRSA strains were restricted to agr1 and agr3. The most common enterotoxin gene, sei, was likewise more prevalent in MSSA strains than in MRSA strains, where sea predominated. The co-existence of two or more enterotoxins was confirmed in 40% of the isolates. sea occurred through all the agr groups except agr3 and sei was not found in agr1 and agr4. The toxic shock toxin (tst) gene was detected in six MSSA. These findings suggest that MSSA may cause more lethal infections than MRSA because of the increased frequency of toxic genotypes seen in MSSA strains.
... Agr activity in strain UAMS-1 (Agr class III) is relatively low compared to many other clinically relevant S. aureus strains (67,68). To determine whether basal Agr activity impacts nos regulation, the pJBnos1 construct was moved into the USA300 LAC strain AH1263, which has much stronger Agr activity (Agr class I) (69)(70)(71), and JLB316 (isogenic AH1263 agr mutant). Surprisingly, nos promoter activity measured from pJBnos1 (containing the UAMS-1 nos promoter sequence) was significantly more active (~3.5-fold ...
Staphylococcus aureus nitric oxide synthase (saNOS) contributes to oxidative stress resistance, antibiotic tolerance, virulence, and modulation of aerobic and nitrate-based cellular respiration. Despite its involvement in these essential processes, the genetic regulation of nos expression has not been well characterized. 5′ rapid amplification of cDNA ends on nos RNA isolated from S. aureus UAMS-1 (USA200 strain) and AH1263 (USA300 strain) revealed that the nos transcriptional start site mapped to an adenine nucleotide in the predicted Shine-Dalgarno site located 11 bp upstream of the nos ATG start codon, suggesting that the nos transcript may have a leaderless organization or may be subject to processing. The SrrAB two-component system (TCS) was previously identified as a positive regulator of nos RNA levels, and experiments using a β-galactosidase reporter plasmid confirmed that SrrAB is a positive regulator of nos promoter activity. In addition, the quorum-sensing system Agr was identified as a negative regulator of low-oxygen nos expression in UAMS-1, with activity epistatic to SrrAB. Involvement of Agr was strain dependent, as nos expression remained unchanged in an AH1263 agr mutant, which has higher Agr activity compared to UAMS-1. Furthermore, nos promoter activity and RNA levels were significantly stronger in AH1263 relative to UAMS-1 during late-exponential low-oxygen growth, when nos expression is maximal. Global regulators Rex and MgrA were also implicated as negative regulators of low-oxygen nos promoter activity in UAMS-1. Collectively, these results provide new insight into factors that control nos expression.
IMPORTANCE
Bacterial nitric oxide synthase (bNOS) has recently emerged in several species as a key player in resistance to stresses commonly encountered during infection. Although Staphylococcus aureus (sa)NOS has been suggested to be a promising drug target in S. aureus , an obstacle to this in practice is the existence of mammalian NOS, whose oxygenase domain is like bacterial NOS. Increased understanding of the nos regulatory network in S. aureus could allow targeting of saNOS through its regulators, bypassing the issue of also inhibiting mammalian NOS. Furthermore, the observed strain-dependent differences in S. aureus nos regulation presented in this study reinforce the importance of studying bacterial NOS regulation and function at both the strain and species levels.
... For instance, the DEGs (agrB, KQ76_10520; and graS, KQ76_03245) encoding histidine kinases were significantly up-regulated by 2.565-fold and 2.989-fold, respectively (p < 0.05). The accessory gene regulator (agr) quorum-sensing system contributes to its pathogenicity of S. aureus [56]. GraS, the sensor histidine kinase of the GraXRS system, has been suggested to directly activate the response regulator ArlR [53]. ...
The increase in bacterial resistance and the decline in the effectiveness of antimicrobial agents are challenging issues for the control of infectious diseases. Traditional Chinese herbal plants are potential sources of new or alternative medicine. Here, we identified antimicrobial components and action modes of the methanol-phase extract from an edible herb Potentilla kleiniana Wight et Arn, which had a 68.18% inhibition rate against 22 species of common pathogenic bacteria. The extract was purified using preparative high-performance liquid chromatography (Prep-HPLC), and three separated fragments (Fragments 1–3) were obtained. Fragment 1 significantly elevated cell surface hydrophobicity and membrane permeability but reduced membrane fluidity, disrupting the cell integrity of the Gram-negative and Gram-positive pathogens tested (p < 0.05). Sixty-six compounds in Fragment 1 were identified using Ultra-HPLC and mass spectrometry (UHPLC-MS). The identified oxymorphone (6.29%) and rutin (6.29%) were predominant in Fragment 1. Multiple cellular metabolic pathways were altered by Fragment 1, such as the repressed ABC transporters, protein translation, and energy supply in two representative Gram-negative and Gram-positive strains (p < 0.05). Overall, this study demonstrates that Fragment 1 from P. kleiniana Wight et Arn is a promising candidate for antibacterial medicine and food preservatives.
... The results revealed the presence of agrA, agrC, and agrD genes in all isolate that showed a positive ability to form biofilms in the Congo red media and the microtiter plate, (Tan et al., 2022;Dufour et al., 2002). ...
Chalazion is a common infection of the meibomian gland in all ages’ eyelids. The current study sought to isolate and identify bacteria linked with chalazion in patients hospitalized to Mosul hospitals between 10/1/2021 and 1/3/2022. Samples were obtained using swabs soaked in the transport media (amies), and bacteria were identified using traditional methods with API20E. Vitek-2 and molecular approaches based on the 16 S rRNA gene verified the species-level diagnosis. Using BLAST software, the nitrogen base sequences were determined and compared to those of the NCBI. The results showed that Staphylococcus aureus was isolated by 52.2% and 62.5% among women, notably in the age (21-40) years, and by 27.8% for females and 19.2% for the same age group in males, and upper eyelid infection predominated. The infection rate in people with oily skin was 50.9% in females and 34.6% in men, with the presence of the agr A, agrC, and agrD genes in isolates that exhibited a positive result in the Congo red medium and microtiter plate.
The pervasive presence of Staphylococcus epidermidis and other coagulase-negative staphylococci on the skin and mucous membranes has long underpinned a casual disregard for the infection risk that these organisms pose to vulnerable patients in healthcare settings. Prior to the recognition of biofilm as an important virulence determinant in S. epidermidis, isolation of this microorganism in diagnostic specimens was often overlooked as clinically insignificant with potential delays in diagnosis and onset of appropriate treatment, contributing to the establishment of chronic infection and increased morbidity or mortality. While impressive progress has been made in our understanding of biofilm mechanisms in this important opportunistic pathogen, research into other virulence determinants has lagged S. aureus. In this review, the broader virulence potential of S. epidermidis including biofilm, toxins, proteases, immune evasion strategies and antibiotic resistance mechanisms is surveyed, together with current and future approaches for improved therapeutic interventions.
