Justine K. Rudkin

Methicillin Resistance Alters the Biofilm Phenotype and Attenuates Virulence in Staphylococcus aureus Device-Associated Infections

Clinical isolates of Staphylococcus aureus can express biofilm phenotypes promoted by the major cell wall autolysin and the fibronectin-binding proteins or the icaADBC-encoded polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG). Biofilm production in methicillin-susceptible S. aureus (MSSA) strains is typically dependent on PIA/PNAG whereas methicillin-resistant isolates express an Atl/FnBP-mediated biofilm phenotype suggesting a relationship between susceptibility to β-lactam antibiotics and biofilm. By introducing the methicillin resistance gene mecA into the PNAG-producing laboratory strain 8325-4 we generated a heterogeneously resistant (HeR) strain, from which a homogeneous, high-level resistant (HoR) derivative was isolated following exposure to oxacillin. The HoR phenotype was associated with a R602H substitution in the DHHA1 domain of GdpP, a recently identified c-di-AMP phosphodiesterase with roles in resistance/tolerance to β-lactam antibiotics and cell envelope stress. Transcription of icaADBC and PNAG production were impaired in the 8325-4 HoR derivative, which instead produced a proteinaceous biofilm that was significantly inhibited by antibodies against the mecA-encoded penicillin binding protein 2a (PBP2a). Conversely excision of the SCCmec element in the MRSA strain BH1CC resulted in oxacillin susceptibility and reduced biofilm production, both of which were complemented by mecA alone. Transcriptional activity of the accessory gene regulator locus was also repressed in the 8325-4 HoR strain, which in turn was accompanied by reduced protease production and significantly reduced virulence in a mouse model of device infection. Thus, homogeneous methicillin resistance has the potential to affect agr- and icaADBC-mediated phenotypes, including altered biofilm expression and virulence, which together are consistent with the adaptation of healthcare-associated MRSA strains to the antibiotic-rich hospital environment in which they are frequently responsible for device-related infections in immuno-compromised patients.

Methicillin resistance and the biofilm phenotype in Staphylococcus aureus.

Antibiotic resistance and biofilm-forming capacity contribute to the success of Staphylococcus aureus as a human pathogen in both healthcare and community settings. These virulence factors do not function independently of each other and the biofilm phenotype expressed by clinical isolates of S. aureus is influenced by acquisition of the methicillin resistance gene mecA. Methicillin-sensitive S. aureus (MSSA) strains commonly produce an icaADBC operon-encoded polysaccharide intercellular adhesin (PIA)-dependent biofilm. In contrast, the release of extracellular DNA (eDNA) and cell surface expression of a number of sortase-anchored proteins, and the major autolysin have been implicated in the biofilm phenotype of methicillin-resistant S. aureus (MRSA) isolates. Expression of high level methicillin resistance in a laboratory MSSA strain resulted in (i) repression of PIA-mediated biofilm production, (ii) down-regulation of the accessory gene regulator (Agr) system, and (iii) attenuation of virulence in murine sepsis and device infection models. Here we review the mechanisms of MSSA and MRSA biofilm production and the relationships between antibiotic resistance, biofilm and virulence gene regulation in S. aureus.

Predicting the virulence of MRSA from its genome sequence

Microbial virulence is a complex and often multifactorial phenotype, intricately linked to a pathogens evolutionary trajectory. Toxicity, the ability to destroy host cell membranes, and adhesion, the ability to adhere to human tissues, are the major virulence factors of many bacterial pathogens, including Staphylococcus aureus. Here, we assayed the toxicity and adhesiveness of 90 MRSA (methicillin resistant S. aureus) isolates and found that while there was remarkably little variation in adhesion, toxicity varied by over an order of magnitude between isolates, suggesting different evolutionary selection pressures acting on these two traits. We performed a genome-wide association study (GWAS) and identified a large number of loci, as well as a putative network of epistatically interacting loci, that significantly associated with toxicity. Despite this apparent complexity in toxicity regulation, a predictive model based on a set of significant single nucleotide polymorphisms (SNPs) and insertion and deletions events (indels) showed a high degree of accuracy in predicting an isolates toxicity solely from the genetic signature at these sites. Our results thus highlight the potential of using sequence data to determine clinically relevant parameters and have further implications for understanding the microbial virulence of this opportunistic pathogen.

Methicillin Resistance Reduces the Virulence of Healthcare-Associated Methicillin-Resistant Staphylococcus aureus by Interfering With the agr Quorum Sensing System

The difficulty in successfully treating infections caused by methicillin-resistant Staphylococcus aureus (MRSA) has led to them being referred to as highly virulent or pathogenic. In our study of one of the major healthcare-associated MRSA (HA-MRSA) clones, we show that expression of the gene responsible for conferring methicillin resistance (mecA) is also directly responsible for reducing the ability of HA-MRSA to secrete cytolytic toxins. We show that resistance to methicillin induces changes in the cell wall, which affects the bacterias agr quorum sensing system. This leads to reduced toxin expression and, as a consequence, reduced virulence in a murine model of sepsis. This diminished capacity to cause infection may explain the inability of HA-MRSA to move into the community and help us understand the recent emergence of community-associated MRSA (CA-MRSA). CA-MRSA typically express less penicillin-binding protein 2a (encoded by mecA), allowing them to maintain full virulence and succeed in the community environment.

Offsetting virulence and antibiotic resistance costs by MRSA.

The prevalence of diverse MRSA (methicillin-resistant Staphylococcus aureus) types in both hospital and community settings is a major health problem worldwide. Here we compare hospital-acquired MRSAs with large type II SCCmec elements with those prevalent in both hospital and community settings with smaller type IV SCCmec elements. We find that the type II but not the type IV SCCmec element causes the bacteria to reduce their levels of costly toxin expression. We compare the relative growth rates of these MRSA types and show that the type II SCCmec carrying MRSAs are more affected than those carrying type IV elements and from this we hypothesize that offsetting the costs associated with antibiotic resistance and toxin expression is why the type II are confined to hospital environments where antibiotic use, the prevalence of immunocompromised individuals and vector-mediated transmission is high. In contrast, those MRSAs that are also successful in the community can maintain their high levels of toxin expression due to a lower fitness burden associated with the smaller SCCmec element.

From genotype to phenotype: can systems biology be used to predict Staphylococcus aureus virulence?

With the advent of high-throughput whole-genome sequencing, it is now possible to sequence a bacterial genome in a matter of hours. However, although the presence or absence of a particular gene can be determined, we do not yet have the tools to extract information about the true virulence potential of an organism from sequence data alone. Here, we focus on the important human pathogen Staphylococcus aureus and present a framework for the construction of a broad systems biology-based tool that could be used to predict virulence phenotypes from S. aureus genomic sequences using existing technology.

An Essential Role for Coagulase in Staphylococcus aureus Biofilm Development Reveals New Therapeutic Possibilities for Device-Related Infections

High-level resistance to antimicrobial drugs is a major factor in the pathogenesis of chronic Staphylococcus aureus biofilm-associated, medical device-related infections. Antimicrobial susceptibility analysis revealed that biofilms grown for ≤ 24 hours on biomaterials conditioned with human plasma under venous shear in iron-free cell culture medium were significantly more susceptible to antistaphylococcal antibiotics. Biofilms formed under these physiologically relevant conditions were regulated by SaeRS and dependent on coagulase-catalyzed conversion of fibrinogen into fibrin. In contrast, SarA-regulated biofilms formed on uncoated polystyrene in nutrient-rich bacteriological medium were mediated by the previously characterized biofilm factors poly-N-acetyl glucosamine, fibronectin-binding proteins, or autolytic activity and were antibiotic resistant. Coagulase-mediated biofilms exhibited increased antimicrobial resistance over time (>48 hours) but were always susceptible to dispersal by the fibrinolytic enzymes plasmin or nattokinase. Biofilms recovered from infected central venous catheters in a rat model of device-related infection were dispersed by nattokinase, supporting the important role of the biofilm phenotype and identifying a potentially new therapeutic approach with antimicrobials and fibrinolytic drugs, particularly during the early stages of device-related infection.

Oxacillin Alters the Toxin Expression Profile of Community-Associated Methicillin-Resistant Staphylococcus aureus

ABSTRACT The emergence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is a growing cause for concern. These strains are more virulent than health care-associated MRSA (HA-MRSA) due to higher levels of toxin expression. In a previous study, we showed that the high-level expression of PBP2a, the alternative penicillin binding protein encoded by the mecA gene on type II staphylococcal cassette chromosome mec (SCCmec) elements, reduced toxicity by interfering with the Agr quorum sensing system. This was not seen in strains carrying the CA-MRSA-associated type IV SCCmec element. These strains express significantly lower levels of PBP2a than the other MRSA type, which may explain their relatively high toxicity. We hypothesized that as oxacillin is known to increase mecA expression levels, it may be possible to attenuate the toxicity of CA-MRSA by using this antibiotic. Subinhibitory oxacillin concentrations induced PBP2a expression, repressed Agr activity, and, as a consequence, decreased phenol-soluble modulin (PSM) secretion by CA-MRSA strains. However, consistent with other studies, oxacillin also increased the expression levels of alpha-toxin and Panton-Valentine leucocidin (PVL). The net effect of these changes on the ability to lyse diverse cell types was tested, and we found that where the PSMs and alpha-toxin are important, oxacillin reduced overall lytic activity, but where PVL is important, it increased lytic activity, demonstrating the pleiotropic effect of oxacillin on toxin expression by CA-MRSA.

Redeploying β-Lactam Antibiotics as a Novel Antivirulence Strategy for the Treatment of Methicillin-Resistant Staphylococcus aureus Infections.

Innovative approaches to the use of existing antibiotics is an important strategy in efforts to address the escalating antimicrobial resistance crisis. We report a new approach to the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections by demonstrating that oxacillin can be used to significantly attenuate the virulence of MRSA despite the pathogen being resistant to this drug. Using mechanistic in vitro assays and in vivo models of invasive pneumonia and sepsis, we show that oxacillin-treated MRSA strains are significantly attenuated in virulence. This effect is based primarily on the oxacillin-dependent repression of the accessory gene regulator quorum-sensing system and altered cell wall architecture, which in turn lead to increased susceptibility to host killing of MRSA. Our data indicate that &bgr;-lactam antibiotics should be included in the treatment regimen as an adjunct antivirulence therapy for patients with MRSA infections. This would represent an important change to current clinical practice for treatment of MRSA infection, with the potential to significantly improve patient outcomes in a safe, cost-effective manner.

Bacterial toxins: Offensive, defensive, or something else altogether?

The secretion of proteins that damage host tissue is well established as integral to the infectious processes of many bacterial pathogens. However, recent advances in our understanding of the activity of toxins suggest that the attributes we have assigned to them from early in vitro experimentation have misled us into thinking of them as merely destructive tools. Here, we will discuss the multifarious ways in which toxins contribute to the lifestyle of bacteria and, by considering their activity from an evolutionary perspective, demonstrate how this extends far beyond their ability to destroy host tissue.