Methicillin Resistance Elements in the Canine Pathogen Staphylococcus pseudintermedius and Their Association with the Peptide Toxin PSM-mec.

Staphylococcus pseudintermedius is a frequent cause of infections in dogs. Infectious isolates of this coagulase-positive staphylococcal species are often methicillin- and multidrug-resistant, which complicates therapy. In staphylococci, methicillin resistance is encoded by determinants found on mobile genetic elements called Staphylococcal Chromosome Cassette mec (SCCmec), which, in addition to methicillin resistance factors, sometimes encode additional genes, such as further resistance factors and, rarely, virulence determinants. In this study, we analyzed SCCmec in a collection of infectious methicillin-resistant S. pseudintermedius (MRSP) isolates from predominant lineages in the United States. We found that several lineages characteristically have specific types of SCCmec elements and Agr types and harbor additional factors in their SCCmec elements that may promote virulence or affect DNA uptake. All isolates had SCCmec-encoded restriction-modification (R-M) systems of types I or II, and sequence types (STs) ST84 and ST64 had one type II and one type I R-M system, although the latter lacked a complete methylation enzyme gene. ST68 isolates also had an SCCmec-encoded CRISPR system. ST71 isolates had a psm-mec gene, which, in all but apparently Agr-dysfunctional isolates, produced a PSM-mec peptide toxin, albeit at relatively small amounts. This study gives detailed insight into the composition of SCCmec elements in infectious isolates of S. pseudintermedius and lays the genetic foundation for further efforts directed at elucidating the contribution of identified accessory SCCmec factors in impacting SCCmec-encoded and thus methicillin resistance-associated virulence and resistance to DNA uptake in this leading canine pathogen.

Virulence Mechanisms of Staphylococcal Animal Pathogens.

Staphylococci are major causes of infections in mammals. Mammals are colonized by diverse staphylococcal species, often with moderate to strong host specificity, and colonization is a common source of infection. Staphylococcal infections of animals not only are of major importance for animal well-being but have considerable economic consequences, such as in the case of staphylococcal mastitis, which costs billions of dollars annually. Furthermore, pet animals can be temporary carriers of strains infectious to humans. Moreover, antimicrobial resistance is a great concern in livestock infections, as there is considerable antibiotic overuse, and resistant strains can be transferred to humans. With the number of working antibiotics continuously becoming smaller due to the concomitant spread of resistant strains, alternative approaches, such as anti-virulence, are increasingly being investigated to treat staphylococcal infections. For this, understanding the virulence mechanisms of animal staphylococcal pathogens is crucial. While many virulence factors have similar functions in humans as animals, there are increasingly frequent reports of host-specific virulence factors and mechanisms. Furthermore, we are only beginning to understand virulence mechanisms in animal-specific staphylococcal pathogens. This review gives an overview of animal infections caused by staphylococci and our knowledge about the virulence mechanisms involved.

Extensive remodelling of the cell wall during the development of Staphylococcus aureus bacteraemia.

The bloodstream represents a hostile environment that bacteria must overcome to cause bacteraemia. To understand how the major human pathogen Staphylococcus aureus manages this we have utilised a functional genomics approach to identify a number of new loci that affect the ability of the bacteria to survive exposure to serum, the critical first step in the development of bacteraemia. The expression of one of these genes, tcaA, was found to be induced upon exposure to serum, and we show that it is involved in the elaboration of a critical virulence factor, the wall teichoic acids (WTA), within the cell envelope. The activity of the TcaA protein alters the sensitivity of the bacteria to cell wall attacking agents, including antimicrobial peptides, human defence fatty acids, and several antibiotics. This protein also affects the autolytic activity and lysostaphin sensitivity of the bacteria, suggesting that in addition to changing WTA abundance in the cell envelope, it also plays a role in peptidoglycan crosslinking. With TcaA rendering the bacteria more susceptible to serum killing, while simultaneously increasing the abundance of WTA in the cell envelope, it was unclear what effect this protein may have during infection. To explore this, we examined human data and performed murine experimental infections. Collectively, our data suggests that whilst mutations in tcaA are selected for during bacteraemia, this protein positively contributes to the virulence of S. aureus through its involvement in altering the cell wall architecture of the bacteria, a process that appears to play a key role in the development of bacteraemia.

Extensive re-modelling of the cell wall during the development of Staphylococcus aureus bacteraemia.

The bloodstream represents a hostile environment that bacteria must overcome to cause bacteraemia. To understand how the major human pathogen Staphylococcus aureus manages this we have utilised a functional genomics approach to identify a number of new loci that affect the ability of the bacteria to survive exposure to serum, the critical first step in the development of bacteraemia. The expression of one of these genes, tcaA, was found to be induced upon exposure to serum, and we show that it is involved in the elaboration of a critical virulence factor, the wall teichoic acids (WTA), within the cell envelope. The activity of the TcaA protein alters the sensitivity of the bacteria to cell wall attacking agents, including antimicrobial peptides, human defence fatty acids, and several antibiotics. This protein also affects the autolytic activity and lysostaphin sensitivity of the bacteria, suggesting that in addition to changing WTA abundance in the cell envelope, it also plays a role in peptidoglycan crosslinking. With TcaA rendering the bacteria more susceptible to serum killing, while simultaneously increasing the abundance of WTA in the cell envelope, it was unclear what effect this protein may have during infection. To explore this, we examined human data and performed murine experimental infections. Collectively, our data suggests that whilst mutations in tcaA are selected for during bacteraemia, this protein positively contributes to the virulence of S. aureus through its involvement in altering the cell wall architecture of the bacteria, a process that appears to play a key role in the development of bacteraemia.

Exacerbation of allergic rhinitis by the commensal bacterium Streptococcus salivarius.

Allergic rhinitis (AR)-commonly called hay fever-is a widespread condition that affects the quality of life of millions of people. The pathophysiology of AR remains incompletely understood. In particular, it is unclear whether members of the colonizing nasal microbiota contribute to AR. Here, using 16S ribosomal RNA sequencing, we show that the nasal microbiome of patients with AR (n = 55) shows distinct differences compared with that from healthy individuals (n = 105), including decreased heterogeneity and the increased abundance of one species, Streptococcus salivarius. Using ex vivo and in vivo models of AR, we demonstrate that this commensal bacterium contributes to AR development, promoting inflammatory cytokine release and morphological changes in the nasal epithelium that are characteristic of AR. Our data indicate that this is due to the ability of S. salivarius to adhere to the nasal epithelium under AR conditions. Our study indicates the potential of targeted antibacterial approaches for AR therapy.

Identification and characterization of the pathogenic potential of phenol-soluble modulin toxins in the mouse commensal Staphylococcus xylosus.

In contrast to the virulent human skin commensal Staphylococcus aureus, which secretes a plethora of toxins, other staphylococci have much reduced virulence. In these species, commonly the only toxins are those of the phenol-soluble modulin (PSM) family. PSMs are species-specific and have only been characterized in a limited number of species. S. xylosus is a usually innocuous commensal on the skin of mice and other mammals. Prompted by reports on the involvement of PSMs in atopic dermatitis (AD) and the isolation of S. xylosus from mice with AD-like symptoms, we here identified and characterized PSMs of S. xylosus with a focus on a potential involvement in AD phenotypes. We found that most clinical S. xylosus strains produce two PSMs, one of the shorter α- and one of the longer ß-type, which were responsible for almost the entire lytic and pro-inflammatory capacities of S. xylosus. Importantly, PSMα of S. xylosus caused lysis and degranulation of mast cells at degrees higher than that of S. aureus δ-toxin, the main PSM previously associated with AD. However, S. xylosus did not produce significant AD symptoms in wild-type mice as opposed to S. aureus, indicating that promotion of AD by S. xylosus likely requires a predisposed host. Our study indicates that non-specific cytolytic potency rather than specific interaction underlies PSM-mediated mast cell degranulation and suggest that the previously reported exceptional potency of δ-toxin of S. aureus is due to its high-level production. Furthermore, they suggest that species that produce cytolytic PSMs, such as S. xylosus, all have the capacity to promote AD, but a high combined level of PSM cytolytic potency is required to cause AD in a non-predisposed host.

Commensal Staphylococcus epidermidis contributes to skin barrier homeostasis by generating protective ceramides.

Previously either regarded as insignificant or feared as potential sources of infection, the bacteria living on our skin are increasingly recognized for their role in benefitting human health. Skin commensals modulate mucosal immune defenses and directly interfere with pathogens; however, their contribution to the skin's physical integrity is less understood. Here, we show that the abundant skin commensal Staphylococcus epidermidis contributes to skin barrier integrity. S. epidermidis secretes a sphingomyelinase that acquires essential nutrients for the bacteria and assists the host in producing ceramides, the main constituent of the epithelial barrier that averts skin dehydration and aging. In mouse models, S. epidermidis significantly increases skin ceramide levels and prevents water loss of damaged skin in a fashion entirely dependent on its sphingomyelinase. Our findings reveal a symbiotic mechanism that demonstrates an important role of the skin microbiota in the maintenance of the skin's protective barrier.

Rapid pathogen-specific recruitment of immune effector cells in the skin by secreted toxins.

Swift recruitment of phagocytic leucocytes is critical in preventing infection when bacteria breach through the protective layers of the skin. According to canonical models, this occurs via an indirect process that is initiated by contact of bacteria with resident skin cells and which is independent of the pathogenic potential of the invader. Here we describe a more rapid mechanism of leucocyte recruitment to the site of intrusion of the important skin pathogen Staphylococcus aureus that is based on direct recognition of specific bacterial toxins, the phenol-soluble modulins (PSMs), by circulating leucocytes. We used a combination of intravital imaging, ear infection and skin abscess models, and in vitro gene expression studies to demonstrate that this early recruitment was dependent on the transcription factor EGR1 and contributed to the prevention of infection. Our findings refine the classical notion of the non-specific and resident cell-dependent character of the innate immune response to bacterial infection by demonstrating a pathogen-specific high-alert mechanism involving direct recruitment of immune effector cells by secreted bacterial products.

Bacterial virulence plays a crucial role in MRSA sepsis.

Bacterial sepsis is a major global cause of death. However, the pathophysiology of sepsis has remained poorly understood. In industrialized nations, Staphylococcus aureus represents the pathogen most commonly associated with mortality due to sepsis. Because of the alarming spread of antibiotic resistance, anti-virulence strategies are often proposed to treat staphylococcal sepsis. However, we do not yet completely understand if and how bacterial virulence contributes to sepsis, which is vital for a thorough assessment of such strategies. We here examined the role of virulence and quorum-sensing regulation in mouse and rabbit models of sepsis caused by methicillin-resistant S. aureus (MRSA). We determined that leukopenia was a predictor of disease outcome during an early critical stage of sepsis. Furthermore, in device-associated infection as the most frequent type of staphylococcal blood infection, quorum-sensing deficiency resulted in significantly higher mortality. Our findings give important guidance regarding anti-virulence drug development strategies for the treatment of staphylococcal sepsis. Moreover, they considerably add to our understanding of how bacterial sepsis develops by revealing a critical early stage of infection during which the battle between bacteria and leukocytes determines sepsis outcome. While sepsis has traditionally been attributed mainly to host factors, our study highlights a key role of the invading pathogen and its virulence mechanisms.

Pathogenicity and virulence of Staphylococcus aureus.

Staphylococcus aureus is one of the most frequent worldwide causes of morbidity and mortality due to an infectious agent. This pathogen can cause a wide variety of diseases, ranging from moderately severe skin infections to fatal pneumonia and sepsis. Treatment of S. aureus infections is complicated by antibiotic resistance and a working vaccine is not available. There has been ongoing and increasing interest in the extraordinarily high number of toxins and other virulence determinants that S. aureus produces and how they impact disease. In this review, we will give an overview of how S. aureus initiates and maintains infection and discuss the main determinants involved. A more in-depth understanding of the function and contribution of S. aureus virulence determinants to S. aureus infection will enable us to develop anti-virulence strategies to counteract the lack of an anti-S. aureus vaccine and the ever-increasing shortage of working antibiotics against this important pathogen.

Contribution of Staphylococcal Enterotoxin B to Staphylococcus aureus Systemic Infection.

Staphylococcal enterotoxin B (SEB), which is produced by the major human pathogen, Staphylococcus aureus, represents a powerful superantigenic toxin and is considered a bioweapon. However, the contribution of SEB to S. aureus pathogenesis has never been directly demonstrated with genetically defined mutants in clinically relevant strains. Many isolates of the predominant Asian community-associated methicillin-resistant S. aureus lineage sequence type (ST) 59 harbor seb, implying a significant role of SEB in the observed hypervirulence of this lineage. We created an isogenic seb mutant in a representative ST59 isolate and assessed its virulence potential in mouse infection models. We detected a significant contribution of seb to systemic ST59 infection that was associated with a cytokine storm. Our results directly demonstrate that seb contributes to S. aureus pathogenesis, suggesting the value of including SEB as a target in multipronged antistaphylococcal drug development strategies. Furthermore, they indicate that seb contributes to fatal exacerbation of community-associated methicillin-resistant S. aureus infection.

Role of Phenol-Soluble Modulins in Staphylococcus epidermidis Biofilm Formation and Infection of Indwelling Medical Devices.

Phenol-soluble modulins (PSMs) are amphipathic, alpha-helical peptides that are secreted by staphylococci in high amounts in a quorum-sensing-controlled fashion. Studies performed predominantly in Staphylococcus aureus showed that PSMs structure biofilms, which results in reduced biofilm mass, while it has also been reported that S. aureus PSMs stabilize biofilms due to amyloid formation. We here analyzed the roles of PSMs in in vitro and in vivo biofilms of Staphylococcus epidermidis, the leading cause of indwelling device-associated biofilm infection. We produced isogenic deletion mutants for every S. epidermidis psm locus and a sequential deletion mutant in which production of all PSMs was abolished. In vitro analysis substantiated the role of all PSMs in biofilm structuring. PSM-dependent biofilm expansion was not observed, in accordance with our finding that no S. epidermidis PSM produced amyloids. In a mouse model of indwelling device-associated infection, the total psm deletion mutant had a significant defect in dissemination. Notably, the total psm mutant produced a significantly more substantial biofilm on the implanted catheter than the wild-type strain. Our study, which for the first time directly quantified the impact of PSMs on biofilm expansion on an implanted device, shows that the in vivo biofilm infection phenotype in S. epidermidis is in accordance with the PSM biofilm structuring and detachment model, which has important implications for the potential therapeutic application of quorum-sensing blockers.

Resistance to leukocytes ties benefits of quorum sensing dysfunctionality to biofilm infection.

Social interactions play an increasingly recognized key role in bacterial physiology1. One of the best studied is quorum sensing (QS), a mechanism by which bacteria sense and respond to the status of cell density2. While QS is generally deemed crucial for bacterial survival, QS-dysfunctional mutants frequently arise in in vitro culture. This has been explained by the fitness cost an individual mutant, a 'quorum cheater', saves at the expense of the community3. QS mutants are also often isolated from biofilm-associated infections, including cystic fibrosis lung infection4, as well as medical device infection and associated bacteraemia5-7. However, despite the frequently proposed use of QS blockers to control virulence8, the mechanisms underlying QS dysfunctionality during infection have remained poorly understood. Here, we show that in the major human pathogen Staphylococcus aureus, quorum cheaters arise exclusively in biofilm infection, while in non-biofilm-associated infection there is a high selective pressure to maintain QS control. We demonstrate that this infection-type dependence is due to QS-dysfunctional bacteria having a significant survival advantage in biofilm infection because they form dense and enlarged biofilms that provide resistance to phagocyte attacks. Our results link the benefit of QS-dysfunctional mutants in vivo to biofilm-mediated immune evasion, thus to mechanisms that are specific to the in vivo setting. Our findings explain why QS mutants are frequently isolated from biofilm-associated infections and provide guidance for the therapeutic application of QS blockers.

Pathogen elimination by probiotic Bacillus via signalling interference.

Probiotic nutrition is frequently claimed to improve human health. In particular, live probiotic bacteria obtained with food are thought to reduce intestinal colonization by pathogens, and thus to reduce susceptibility to infection. However, the mechanisms that underlie these effects remain poorly understood. Here we report that the consumption of probiotic Bacillus bacteria comprehensively abolished colonization by the dangerous pathogen Staphylococcus aureus in a rural Thai population. We show that a widespread class of Bacillus lipopeptides, the fengycins, eliminates S. aureus by inhibiting S. aureus quorum sensing-a process through which bacteria respond to their population density by altering gene regulation. Our study presents a detailed molecular mechanism that underlines the importance of probiotic nutrition in reducing infectious disease. We also provide evidence that supports the biological significance of probiotic bacterial interference in humans, and show that such interference can be achieved by blocking a pathogen's signalling system. Furthermore, our findings suggest a probiotic-based method for S. aureus decolonization and new ways to fight S. aureus infections.

Basis of Virulence in Enterotoxin-Mediated Staphylococcal Food Poisoning.

The Staphylococcus aureus enterotoxins are a superfamily of secreted virulence factors that share structural and functional similarities and possess potent superantigenic activity causing disruptions in adaptive immunity. The enterotoxins can be separated into two groups; the classical (SEA-SEE) and the newer (SEG-SElY and counting) enterotoxin groups. Many members from both these groups contribute to the pathogenesis of several serious human diseases, including toxic shock syndrome, pneumonia, and sepsis-related infections. Additionally, many members demonstrate emetic activity and are frequently responsible for food poisoning outbreaks. Due to their robust tolerance to denaturing, the enterotoxins retain activity in food contaminated previously with S. aureus. The genes encoding the enterotoxins are found mostly on a variety of different mobile genetic elements. Therefore, the presence of enterotoxins can vary widely among different S. aureus isolates. Additionally, the enterotoxins are regulated by multiple, and often overlapping, regulatory pathways, which are influenced by environmental factors. In this review, we also will focus on the newer enterotoxins (SEG-SElY), which matter for the role of S. aureus as an enteropathogen, and summarize our current knowledge on their prevalence in recent food poisoning outbreaks. Finally, we will review the current literature regarding the key elements that govern the complex regulation of enterotoxins, the molecular mechanisms underlying their enterotoxigenic, superantigenic, and immunomodulatory functions, and discuss how these activities may collectively contribute to the overall manifestation of staphylococcal food poisoning.

Antimicrobial Peptide Resistance Mechanism Contributes to Staphylococcus aureus Infection.

Antimicrobial peptides (AMPs) constitute an important part of innate host defense. Possibly limiting the therapeutic potential of AMPs is the fact that bacteria have developed AMP resistance mechanisms during their co-evolution with humans. However, there is no direct evidence that AMP resistance per se is important during an infection. Here we show that the Staphylococcus aureus Pmt ABC transporter defends the bacteria from killing by important human AMPs and elimination by human neutrophils. By showing that Pmt contributes to virulence during skin infection in an AMP-dependent manner, we provide evidence that AMP resistance plays a key role in bacterial infection.

Phenol-Soluble Modulin Toxins of Staphylococcus haemolyticus.

Coagulase-negative staphylococci (CoNS) are important nosocomial pathogens and the leading cause of sepsis. The second most frequently implicated species, after Staphylococcus epidermidis, is Staphylococcus haemolyticus. However, we have a significant lack of knowledge about what causes virulence of S. haemolyticus, as virulence factors of this pathogen have remained virtually unexplored. In contrast to the aggressive pathogen Staphylococcus aureus, toxin production has traditionally not been associated with CoNS. Recent findings have suggested that phenol-soluble modulins (PSMs), amphipathic peptide toxins with broad cytolytic activity, are widespread in staphylococci, but there has been no systematic assessment of PSM production in CoNS other than S. epidermidis. Here, we identified, purified, and characterized PSMs of S. haemolyticus. We found three PSMs of the ß-type, which correspond to peptides that before were described to have anti-gonococcal activity. We also detected an α-type PSM that has not previously been described. Furthermore, we confirmed that S. haemolyticus does not produce a δ-toxin, as results from genome sequencing had indicated. All four S. haemolyticus PSMs had strong pro-inflammatory activity, promoting neutrophil chemotaxis. Notably, we identified in particular the novel α-type PSM, S. haemolyticus PSMα, as a potent hemolysin and leukocidin. For the first time, our study describes toxins of this important staphylococcal pathogen with the potential to have a significant impact on virulence during blood infection and sepsis.

Different drugs for bad bugs: antivirulence strategies in the age of antibiotic resistance.

The rapid evolution and dissemination of antibiotic resistance among bacterial pathogens are outpacing the development of new antibiotics, but antivirulence agents provide an alternative. These agents can circumvent antibiotic resistance by disarming pathogens of virulence factors that facilitate human disease while leaving bacterial growth pathways - the target of traditional antibiotics - intact. Either as stand-alone medications or together with antibiotics, these drugs are intended to treat bacterial infections in a largely pathogen-specific manner. Notably, development of antivirulence drugs requires an in-depth understanding of the roles that diverse virulence factors have in disease processes. In this Review, we outline the theory behind antivirulence strategies and provide examples of bacterial features that can be targeted by antivirulence approaches. Furthermore, we discuss the recent successes and failures of this paradigm, and new developments that are in the pipeline.

Toxin Mediates Sepsis Caused by Methicillin-Resistant Staphylococcus epidermidis.

Bacterial sepsis is a major killer in hospitalized patients. Coagulase-negative staphylococci (CNS) with the leading species Staphylococcus epidermidis are the most frequent causes of nosocomial sepsis, with most infectious isolates being methicillin-resistant. However, which bacterial factors underlie the pathogenesis of CNS sepsis is unknown. While it has been commonly believed that invariant structures on the surface of CNS trigger sepsis by causing an over-reaction of the immune system, we show here that sepsis caused by methicillin-resistant S. epidermidis is to a large extent mediated by the methicillin resistance island-encoded peptide toxin, PSM-mec. PSM-mec contributed to bacterial survival in whole human blood and resistance to neutrophil-mediated killing, and caused significantly increased mortality and cytokine expression in a mouse sepsis model. Furthermore, we show that the PSM-mec peptide itself, rather than the regulatory RNA in which its gene is embedded, is responsible for the observed virulence phenotype. This finding is of particular importance given the contrasting roles of the psm-mec locus that have been reported in S. aureus strains, inasmuch as our findings suggest that the psm-mec locus may exert effects in the background of S. aureus strains that differ from its original role in the CNS environment due to originally "unintended" interferences. Notably, while toxins have never been clearly implied in CNS infections, our tissue culture and mouse infection model data indicate that an important type of infection caused by the predominant CNS species is mediated to a large extent by a toxin. These findings suggest that CNS infections may be amenable to virulence-targeted drug development approaches.

Bacterial Abscess Formation Is Controlled by the Stringent Stress Response and Can Be Targeted Therapeutically.

Cutaneous abscess infections are difficult to treat with current therapies and alternatives to conventional antibiotics are needed. Understanding the regulatory mechanisms that govern abscess pathology should reveal therapeutic interventions for these recalcitrant infections. Here we demonstrated that the stringent stress response employed by bacteria to cope and adapt to environmental stressors was essential for the formation of lesions, but not bacterial growth, in a methicillin resistant Staphylococcus aureus (MRSA) cutaneous abscess mouse model. To pharmacologically confirm the role of the stringent response in abscess formation, a cationic peptide that causes rapid degradation of the stringent response mediator, guanosine tetraphosphate (ppGpp), was employed. The therapeutic application of this peptide strongly inhibited lesion formation in mice infected with Gram-positive MRSA and Gram-negative Pseudomonas aeruginosa. Overall, we provide insights into the mechanisms governing abscess formation and a paradigm for treating multidrug resistant cutaneous abscesses.