Significant variability exists in the cytotoxicity of global methicillin-resistant Staphylococcus aureus lineages

Staphylococcus aureus is a major human pathogen where the emergence of antibiotic resistant lineages, such as methicillin-resistant S. aureus (MRSA), is a major health concern. While some MRSA lineages are restricted to the healthcare setting, the epidemiology of MRSA is changing globally, with the rise of specific lineages causing disease in healthy people in the community. In the past two decades, community-associated MRSA (CA-MRSA) has emerged as a clinically important and virulent pathogen associated with serious skin and soft-tissue infections (SSTI). These infections are primarily cytotoxin driven, leading to the suggestion that hypervirulent lineages/multi-locus sequence types (STs) exist. To examine this, we compared the cytotoxicity of 475 MRSA isolates representing five major MRSA STs (ST22, ST93, ST8, ST239 and ST36) by employing a monocyte-macrophage THP-1 cell line as a surrogate for measuring gross cytotoxicity. We demonstrate that while certain MRSA STs contain highly toxic isolates, there is such variability within lineages to suggest that this aspect of virulence should not be inferred from the genotype of any given isolate. Furthermore, by interrogating the accessory gene regulator (Agr) sequences in this collection we identified several Agr mutations that were associated with reduced cytotoxicity. Interestingly, the majority of isolates that were attenuated in cytotoxin production contained no mutations in the agr locus, indicating a role of other undefined genes in S. aureus toxin regulation.

Bisdemethoxycurcumin Reduces Methicillin-Resistant Staphylococcus aureus Expression of Virulence-Related Exoproteins and Inhibits the Biofilm Formation

Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen of nosocomial infection, which is resistant to most antibiotics. Presently, anti-virulence therapy and anti-biofilm therapy are considered to be promising alternatives. In the current work, we investigated the influence of bisdemethoxycurcumin (BDMC) on the virulence-related exoproteins and the biofilm formation using a reference strain and clinic isolated strains. Western blotting, quantitative RT-PCR, and tumor necrosis factor (TNF) release assay were performed to assess the efficacy of BDMC in reducing the expression of Staphylococcus enterotoxin-related exoproteins (enterotoxin A, enterotoxin B) and α-toxin in MRSA. The anti-biofilm activity of BDMC was evaluated through a biofilm inhibition assay. The study suggests that sub-inhibitory concentrations of BDMC significantly inhibited the expression of sea, seb, and hla at the mRNA level in MRSA. Moreover, the expression of virulence-related exoproteins was significantly decreased by down-regulating accessory gene regulator agr, and the inhibition of biofilms formation was demonstrated by BDMC at sub-inhibitory concentrations. Consequently, the study suggests that BDMC may be a potential natural antibacterial agent to release the pressure brought by antibiotic resistance.

Labdane Diterpenoids from Salvia tingitana Etl. Synergize with Clindamycin against Methicillin-Resistant Staphylococcus aureus

Quorum-sensing (QS) is a regulatory mechanism in bacterial communication, important for pathogenesis control. The search for small molecules active as quorum-sensing inhibitors (QSI) that can synergize with antibiotics is considered a good strategy to counteract the problem of antibiotic resistance. Here the antimicrobial labdane diterpenoids sclareol (1) and manool (2) extracted from Salvia tingitana were considered as potential QSI against methicillin-resistant Staphylococcus aureus. Only sclareol showed synergistic activity with clindamycin. The quantification of these compounds by LC–MS analysis in the organs and in the calli of S. tingitana showed that sclareol is most abundant in the flower spikes and is produced by calli, while manool is the major labdane of the roots, and is abundant also in the leaves. Other metabolites of the roots were abietane diterpenoids, common in Salvia species, and pentacyclic triterpenoids, bearing a γ-lactone moiety, previously undescribed in Salvia. Docking simulations suggested that 1 and 2 bind to key residues, involved in direct interactions with DNA. They may prevent accessory gene regulator A (AgrA) binding to DNA or AgrA activation upon phosphorylation, to suppress virulence factor expression. The antimicrobial activity of these two compounds probably achieves preventing upregulation of the accessory gene regulator (agr)-regulated genes.

Effects of Staphylococcus aureus hemolysin toxins on blood cells and association with skin and soft tissue infections

Staphylococcus aureus (S. aureus) is gram positive, catalase positive cocci which belongs to the family of Staphylococcaceae and is long known as clinical and foodborne pathogen. The emergence of multidrug resistance strain of S. aureus which is methicillin resistant S. aureus (MRSA) challenges the health care system because it can cause wide variety of hospital and community acquired skin and soft tissue infections which are difficult to treat. The virulence of S. aureus is because of different factors which includes toxins, enzymes and superantigens. S. aureus produce variety of exotoxins, enterotoxins and exfoliative toxins which contributes to the virulence of S. aureus. Hemolysin toxins produce by S. aureus strains are associated with different skin and soft tissue infections (SSTIs) and can cause the lysis of RBCs. Hemolysins are regulated by accessory gene regulator (agr) and is required for the enhanced expression of virulence factors secreted by S. aureus. Hemolysins have leucolytic activity and can help in iron scavenging from host. The most important toxin is alpha hemolysin which can induce the apoptosis and cause the lysis of epithelial cells, erythrocytes and keratinocytes. Human immune cells are affected by beta hemolysin and gamma hemolysin is a biocomponent toxin. Delta hemolysin is low molecular weight exotoxin which belongs to the class of phenol soluble modulins. Keywords: MRSA, Exotoxins, Hemolysins, SSTIs

Impact of agr Functionality on the Outcome of Patients with Methicillin-Susceptible Staphylococcus aureus Bacteremia

Few studies have examined the association between methicillin-susceptible Staphylococcus aureus (MSSA) infection and accessory gene regulator ( agr ) functionality. We evaluated the association between agr dysfunction and mortality in patients with MSSA bacteremia.

Gram-positive bacteria secrete RNA aptamers to activate human STING for IL-1β release.

Molecular mechanisms through which Gram-positive bacteria induce the canonical inflammasome are poorly understood. Here, we studied the effects of Group B streptococci (GBS) and Staphylococcus aureus (SA) on inflammasome activation in human macrophages. Dinucleotide binding small RNA aptamers released by SA and GBS were shown to trigger increased IL-1β generation by inflammasomes. The stimulator of interferon genes-STING as a central mediator of innate immune responses has been identified as the key target of pathogenic RNA. Multi-lamellar lipid bodies (MLBs) produced by SA function as vehicles for the RNA aptamers. Notably, expression of RNA aptamers is controlled by an accessory gene regulator quorum sensing system of the bacteria. These findings have been translated to patients with Gram-positive sepsis showing hallmarks of MLB-RNA-mediated inflammasome activation. Together our findings may provide a new perspective for the pathogenicity of Gram-positive bacterial infection in man.

Significant Variability exists in the Toxicity of Global Methicillin-resistant Staphylococcus aureus Lineages.

Staphylococcus aureus is a major human pathogen where the emergence of antibiotic resistant lineages, such as methicillin-resistant S. aureus (MRSA), is a major health concern. While some MRSA lineages are restricted to the healthcare setting, the epidemiology of MRSA is changing globally, with the rise of specific lineages causing disease in healthy people in the community. In the past two decades, community-associated MRSA (CA-MRSA) has emerged as a clinically important and virulent pathogen associated with serious skin and soft-tissue infections (SSTI). These infections are primarily toxin driven, leading to the suggestion that hyper-virulent lineages/multi-locus sequence types (STs) exist. To examine this, we compared the toxic activity of 475 MRSA isolates representing five major MRSA STs (ST22, ST93, ST8, ST239 and ST36) by employing a monocyte-macrophage THP-1 cell line as a surrogate for measuring gross cytotoxicity. We demonstrate that while certain MRSA STs contain highly toxic isolates, there is such variability within lineages to suggest that this aspect of virulence should not be inferred from the genotype of any given isolate. Furthermore, by interrogating the accessory gene regulator (Agr) sequences in this collection we identified several Agr mutations that were associated with reduced toxicity. Interestingly, the majority of isolates that were attenuated in toxin production contained no mutations in the agr locus, indicating a role of other undefined genes in S. aureus toxin regulation.

Bacterial Targets of Antibiotics in Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most prevalent bacterial pathogens and continues to be a leading cause of morbidity and mortality worldwide. MRSA is a commensal bacterium in humans and is transmitted in both community and healthcare settings. Successful treatment remains a challenge, and a search for new targets of antibiotics is required to ensure that MRSA infections can be effectively treated in the future. Most antibiotics in clinical use selectively target one or more biochemical processes essential for S. aureus viability, e.g., cell wall synthesis, protein synthesis (translation), DNA replication, RNA synthesis (transcription), or metabolic processes, such as folic acid synthesis. In this review, we briefly describe the mechanism of action of antibiotics from different classes and discuss insights into the well-established primary targets in S. aureus. Further, several components of bacterial cellular processes, such as teichoic acid, aminoacyl-tRNA synthetases, the lipid II cycle, auxiliary factors of β-lactam resistance, two-component systems, and the accessory gene regulator quorum sensing system, are discussed as promising targets for novel antibiotics. A greater molecular understanding of the bacterial targets of antibiotics has the potential to reveal novel therapeutic strategies or identify agents against antibiotic-resistant pathogens.

Comparative profiling of biofilm-production, quorum sensing system and virulence genes in human and ovine non-aureus staphylococci

Background: This study assessed the genetic characteristics shared by non-aureus staphylococci (NAS) responsible for human infections and those causing mastitis in dairy ewes. In a collaboration between animal and human health care professionals, we collected and identified 125 ovine and 70 human NAS isolates and compared them for biofilm production, presence of autolysins, microbial surface components recognizing adhesive matrix molecules (MSCRAMMS), pyrogenic toxins, and agr alleles regulating quorum-sensing systems. Ovine NAS included: S. epidermidis (57), S. chromogenes (29), S. haemolyticus (17), S. simulans (8), S. caprae (6), S. warneri (5), S. saprophyticus, S. intermedius, and S. muscae (1 each) while human NAS included: S. haemolyticus (28), S. epidermidis (26), S. hominis (4), S. lugdunensis (4), S. capitis (3), S. warneri (2), S. xylosus, S. pasteuri, and S. saprophyticus subsp. bovis (1 each).Results: Based on colony characteristics on Congo Red Agar, 4 (3.2%) ovine, and 49 (70%) human isolates produced biofilm. Few S. epidermidis isolates harbored the icaA/D genes coding for the polysaccharide intercellular adhesin (PIA) and the bhp, aap, and embp genes coding biofilm accumulation proteins. PCR amplification of the genes coding for autolysins (atlE and aae), microbial surface components recognizing adhesive matrix molecules (MSCRAMMs, sdrG and sdrF), enterotoxins (sea, seb, sec, sed, and see), and the toxic shock syndrome toxin (tsst), revealed that 40%, 39.2%, 47.2% and 52.8% of the sheep isolates carried atlE, aae, sdrF and sdrG, respectively, against 37.1%, 42.8%, 32.8%, and 60% of human isolates. Enterotoxins and tsst were not detected. Fifty-nine sheep isolates (all S. epidermidis, 1 S. chromogenes, and 1 S. haemolyticus) and 27 human NAS (all S. epidermidis and 1 S. warneri) were positive for the accessory gene regulator (agr), responsible for the regulation of virulence factors: agr-3se (57.8%) followed by agr-1se (36.8%) predominated in sheep, while agr-1se (65.4%), followed by agr-2se (34.6%) predominated in humans.Conclusions: This comparative study provided a detailed characterization of the putative virulence genes present in human and ovine NAS and indicated that the ability to form biofilms, observed mainly in human S. epidermidis, could be a major virulence factor facilitating colonization, infection, diffusion, and resistance.