Ruth C. Massey

Staphylococcus aureus clumping factor B (ClfB) promotes adherence to human type I cytokeratin 10: implications for nasal colonization.

Staphylococcus aureus is an important cause of sepsis in both community and hospital settings, a major risk factor for which is nasal carriage of the bacterium. Eradication of carriage by topical antibiotics reduces sepsis rates in high‐risk individuals, an important strategy for the reduction of nosocomial infection in targeted patient populations. Understanding the mechanisms by which S. aureus adheres to nasal epithelial cells in vivo may lead to alternative methods of decolonization that do not rely on sustained antimicrobial susceptibility. Here, we demonstrate for the first time that the S. aureus surface‐expressed protein, clumping factor B (ClfB), promotes adherence to immobilized epidermal cytokeratins in vitro. By expressing a range of S. aureus adhesins on the surface of the heterologous host Lactococcus lactis, we demonstrated that adherence to epidermal cytokeratins was conferred by ClfB. Adherence of wild‐type S. aureus was inhibited by recombinant ClfB protein or anti‐ClfB antibodies, and S. aureus mutants defective in ClfB adhered poorly to epidermal cytokeratins. Expression of ClfB promoted adherence of L. lactis to human desquamated nasal epithelial cells, and a mutant of S. aureus defective in ClfB had reduced adherence compared with wild type. ClfB also promoted adherence of L. lactis cells to a human keratinocyte cell line. Cytokeratin 10 molecules were shown by flow cytometry to be exposed on the surface of both desquamated nasal epithelial cells and keratinocytes. Cytokeratin 10 was also detected on the surface of desquamated human nasal cells using immunofluorescence, and recombinant ClfB protein was shown to bind to cytokeratin K10 extracted from these cells. We also showed that ClfB is transcribed by S. aureus in the human nares. We propose that ClfB is a major determinant in S. aureus nasal colonization.

The ica Operon and Biofilm Production in Coagulase-Negative Staphylococci Associated with Carriage and Disease in a Neonatal Intensive Care Unit

ABSTRACT Coagulase-negative staphylococci (CoNS) are a major cause of sepsis in the neonatal intensive care unit (NICU). We evaluated the hypothesis that the ica operon and biofilm production are associated with CoNS disease in this setting. CoNS associated with bacteremia or blood culture contamination and from the skin of infants with CoNS bacteremia or healthy controls were obtained during a prospective case-control study on a busy NICU. A total of 180 strains were identified, of which 122 (68%) were Staphylococcus epidermidis and the remainder were S. capitis (n = 29), S. haemolyticus (n = 11), S. hominis (n = 9), S. warneri (n = 8), and S. auricularis (n = 1). The presence of the genes icaA, icaB, icaC, and icaD was determined by PCR, and biofilm production was examined using qualitative (Congo red agar [CRA]) and quantitative (microtiter plate) techniques. There were no significant differences in the presence of the ica operon or CRA positivity among the four groups of strains. However, quantitative biofilm production was significantly greater in strains isolated from either the blood or the skin of neonates with S. epidermidis bacteremia. We conclude that the quantity of biofilm produced may be associated with the ability to cause CoNS infection. This conclusion suggests that the regulation of biofilm expression may play a central role in the disease process.

Fibronectin-binding protein A of Staphylococcus aureus has multiple, substituting, binding regions that mediate adherence to fibronectin and invasion of endothelial cells

Invasive Staphylococcus aureus infection frequently involves bacterial seeding from the bloodstream to other body tissues, a process necessarily involving interactions between circulating bacteria and vascular endothelial cells. Staphylococcus aureus fibronectin‐binding protein is central to the invasion of endothelium, fibronectin forming a bridge between bacterial fibronectin‐binding proteins and host cell receptors. To dissect further the mechanisms of invasion of endothelial cells by S. aureus, a series of truncated FnBPA proteins that lacked one or more of the A, B, C or D regions were expressed on the surface of S. aureus and tested in fibronectin adhesion, endothelial cell adhesion and invasion assays. We found that this protein has multiple, substituting, fibronectin‐binding regions, each capable of conferring both adherence to fibronectin and endothelial cells, and endothelial cell invasion. By expressing S. aureus FnBPA on the surface of the non‐invasive Gram‐positive organism Lactococcus lactis, we have found that no other bacterial factor is required for invasion. Furthermore, we have demonstrated that, as with other cell types, invasion of endothelial cells is mediated by integrin α5β1. These findings may be of relevance to the development of preventive measures against systemic infection, and bacterial spread in the bacteraemic patient.

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.

Genes encoding a cellulosic polymer contribute toward the ecological success of Pseudomonas fluorescens SBW25 on plant surfaces

Pseudomonas fluorescens SBW25 is a Gram‐negative bacterium that grows in close association with plants. In common with a broad range of functionally similar bacteria it plays an important role in the turnover of organic matter and certain isolates can promote plant growth. Despite its environmental significance, the causes of its ecological success are poorly understood. Here we describe the development and application of a simple promoter trapping strategy (IVET) to identify P. fluorescens SBW25 genes showing elevated levels of expression in the sugar beet rhizosphere. A total of 25 rhizosphere‐induced (rhi) fusions are reported with predicted roles in nutrient acquisition, stress responses, biosynthesis of phytohormones and antibiotics. One rhi fusion is to wss, an operon encoding an acetylated cellulose polymer. A mutant carrying a defective wss locus was competitively compromised (relative to the wild type) in the rhizosphere and in the phyllosphere, but not in bulk soil. The rhizosphere‐induced wss locus therefore contributes to the ecological performance of SBW25 in the plant environment and supports our conjecture that genes inactive in the laboratory environment, but active in the wild, are likely to be determinants of fitness in natural environments.

Phenotypic switching of antibiotic resistance circumvents permanent costs in Staphylococcus aureus

Bacterial antibiotic resistance is often associated with a fitness cost in the absence of the antibiotic [1,2]. We have examined a resistance mechanism in Staphylococcus aureus that negates these costs. Exposure to gentamicin both in vitro and in vivo has been reported to result in the emergence of a gentamicin-resistant small colony variant (SCV)[3-8]. We show that the emergence of SCVs following exposure to gentamicin results from a rapid switch and that bacteria exposed to cycles of gentamicin followed by antibiotic-free medium repeatedly switched between a resistant SCV and a sensitive parental phenotype (revertants). The fitness of revertants relative to S. aureus with stable gentamicin resistance was greater in drug-free media, which suggests that S. aureus has evolved an inducible and reversible resistance mechanism that circumvents a permanent cost to fitness.

Staphylococcus aureus Host Cell Invasion and Virulence in Sepsis Is Facilitated by the Multiple Repeats within FnBPA

Entry of Staphylococcus aureus into the bloodstream can lead to metastatic abscess formation and infective endocarditis. Crucial to the development of both these conditions is the interaction of S. aureus with endothelial cells. In vivo and in vitro studies have shown that the staphylococcal invasin FnBPA triggers bacterial invasion of endothelial cells via a process that involves fibronectin (Fn) bridging to α5β1 integrins. The Fn-binding region of FnBPA usually contains 11 non-identical repeats (FnBRs) with differing affinities for Fn, which facilitate the binding of multiple Fn molecules and may promote integrin clustering. We thus hypothesized that multiple repeats are necessary to trigger the invasion of endothelial cells by S. aureus. To test this we constructed variants of fnbA containing various combinations of FnBRs. In vitro assays revealed that endothelial cell invasion can be facilitated by a single high-affinity, but not low-affinity FnBR. Studies using a nisin-inducible system that controlled surface expression of FnBPA revealed that variants encoding fewer FnBRs required higher levels of surface expression to mediate invasion. High expression levels of FnBPA bearing a single low affinity FnBR bound Fn but did not invade, suggesting that FnBPA affinity for Fn is crucial for triggering internalization. In addition, multiple FnBRs increased the speed of internalization, as did higher expression levels of FnBPA, without altering the uptake mechanism. The relevance of these findings to pathogenesis was demonstrated using a murine sepsis model, which showed that multiple FnBRs were required for virulence. In conclusion, multiple FnBRs within FnBPA facilitate efficient Fn adhesion, trigger rapid bacterial uptake and are required for pathogenesis.

Antagonistic coevolution with parasites increases the cost of host deleterious mutations

The fitness consequences of deleterious mutations are sometimes greater when individuals are parasitized, hence parasites may result in the more rapid purging of deleterious mutations from host populations. The significance of host deleterious mutations when hosts and parasites antagonistically coevolve (reciprocal evolution of host resistance and parasite infectivity) has not previously been experimentally investigated. We addressed this by coevolving the bacterium Pseudomonas fluorescens and a parasitic bacteriophage in laboratory microcosms, using bacteria with high and low mutation loads. Directional coevolution between bacterial resistance and phage infectivity occurred in all populations. Bacterial population fitness, as measured by competition experiments with ancestral genotypes in the absence of phage, declined with time spent coevolving. However, this decline was significantly more rapid in bacteria with high mutation loads, suggesting the cost of bacterial resistance to phage was greater in the presence of deleterious mutations (synergistic epistasis). As such, resistance to phage was more costly to evolve in the presence of a high mutation load. Consistent with these data, bacteria with high mutation loads underwent less rapid directional coevolution with their phage populations, and showed lower levels of resistance to their coevolving phage populations. These data suggest that coevolution with parasites increases the rate at which deleterious mutations are purged from host populations.

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.

Interference competition and parasite virulence.

Within–host competition between parasites, a consequence of infection by multiple strains, is predicted to favour rapid host exploitation and greater damage to hosts (virulence). However, the inclusion of biological variables can drastically change this relationship. For example, if competing parasite strains produce toxins that kill each other (interference competition), their growth rates and virulence may be reduced relative to single–strain infections. Bacteriocins are antimicrobial toxins produced by bacteria that target closely related strains and species, and to which the producing strain is immune. We investigated competition between bacteriocin–producing, insect–killing bacteria (Photorhabdus and Xenorhabdus) and how this competition affected virulence in caterpillars. Where one strain could kill the other, and not vice versa, the non–killing strain was competitively excluded, and insect mortality was the same as that of the killing strain alone. However, when caterpillars were multiply infected by strains that could kill each other, we did not observe competitive exclusion and their virulence was less than single–strain infections. The ubiquity and diversity of bacteriocins among pathogenic bacteria suggest mixed infections will be, on average, less virulent than single infections.