Staphylococcus aureus sarA Regulates Inflammation and Colonization during Central Nervous System Biofilm Formation

Infection is a frequent and serious complication following the treatment of hydrocephalus with CSF shunts, with limited therapeutic options because of biofilm formation along the catheter surface. Here we evaluated the possibility that the sarA regulatory locus engenders S. aureus more resistant to immune recognition in the central nervous system (CNS) based on its reported ability to regulate biofilm formation. We utilized our established model of CNS catheter-associated infection, similar to CSF shunt infections seen in humans, to compare the kinetics of bacterial titers, cytokine production and inflammatory cell influx elicited by wild type S. aureus versus an isogenic sarA mutant. The sarA mutant was more rapidly cleared from infected catheters compared to its isogenic wild type strain. Consistent with this finding, several pro-inflammatory cytokines and chemokines, including IL-17, CXCL1, and IL-1β were significantly increased in the brain following infection with the sarA mutant versus wild type S. aureus, in agreement with the fact that the sarA mutant displayed impaired biofilm growth and favored a planktonic state. Neutrophil influx into the infected hemisphere was also increased in the animals infected with the sarA mutant compared to wild type bacteria. These changes were not attributable to extracellular protease activity, which is increased in the context of SarA mutation, since similar responses were observed between sarA and a sarA/protease mutant. Overall, these results demonstrate that sarA plays an important role in attenuating the inflammatory response during staphylococcal biofilm infection in the CNS via a mechanism that remains to be determined.     

Identification of Intracellular Bacteria in Adenoid and Tonsil Tissue Specimens: The Efficiency of Culture Versus Fluorescent In Situ Hybridization (FISH)

Monocyte/macrophage cells from human nasopharyngeal lymphoid tissue can be a source of bacteria responsible for human chronic and recurrent upper respiratory tract infection. Detection and characterization of pathogens surviving intracellularly could be a key element in bacteriological diagnosis of the infections as well as in the study on interactions between bacteria and their host. The present study was undertaken to assess the possibility of isolation of viable bacteria from the cells expressing monocyte/macrophage marker CD14 in nasopharyngeal lymphoid tissue. Overall, 74 adenotonsillectomy specimens (adenoids and tonsils) from 37 children with adenoid hypertrophy and recurrent infections as well as 15 specimens from nine children with adenoid hypertrophy, which do not suffer from upper respiratory tract infections (the control group), were studied. The suitability of immunomagnetic separation for extraction of CD14⁺ cells from lymphoid tissue and for further isolation of the intracellular pathogens has been shown. The coexistence of living pathogens including Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pyogenes with the bacteria representing normal nasopharyngeal microbiota inside CD14⁺ cells was demonstrated. Twenty-four strains of these pathogens from 32.4 % of the lysates of CD14⁺ cells were isolated. Concurrently, the fluorescent in situ hybridization (FISH) with a universal EUB388, and the species-specific probes demonstrated twice more often the persistence of these bacterial species in the lysates of CD14⁺ cells than conventional culture. Although the FISH technique appears to be more sensitive than traditional culture in the intracellular bacteria identification, the doubts on whether the bacteria are alive, and therefore, pathogenic would still exist without the strain cultivation.     

Small colony variants of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> — <Emphasis Type="Italic">review</Emphasis>

Bacterial variants of Staphylococcus aureus called small colony variants (SCVs) originate by mutations in metabolic genes, resulting in emergence of auxotrophic bacterial subpopulations. These variants are not particularly virulent but are able to persist viable inside host cells. SCVs show their characteristic auxotrophic growth deficiency and depressed α-cytotoxin activity. Environmental pressure such as antibiotics, select for isogenic SCV cells that are frequently found coexisting with their parent wild-type strains in a mixed bacterial culture. SCV strains often grow on blood agar as non-pigmented or pinpoint pigmented colonies and their key biochemical tests are often non-reactive. Their altered metabolism or auxotrophism can result in long generation time and thus SCV phenotype, more often than not SCV can be overgrown by their wild-type counterparts and other competitive respiratory flora. This could affect laboratory detection. Thus, molecular methods, such as 16S rRNA partial sequencing or amplification of species-specific DNA targets (e.g. coagulase, nuclease) directly from clinical material or isolated bacterial colonies, become the method of choice. Patients at risk of infection by S. aureus SCVs include cystic fibrosis patients (CF), patients with skin and foreign-body related infections and osteomyelitis, as they suffer from chronic staphylococcal infections and are subject to long-term antibiotic therapy. Molecular evidence of SCV development has not been found except for some random mutations of the thymidylate synthase gene (thyA) described in SCV S. aureus strains of CF patients. These variants are able to bypass the antibiotic effect of folic acid antagonists such as sulfonamides and trimethoprim. Resistance to gentamicin and aminoglycosides in the hemin or menadione auxotrophic SCVs was hypothesized as being due to decreased influx of the drugs into cells as a result of decreased ATP production and decreased electrochemical gradient on cell membranes.     

In or out: Phagosomal escape of Staphylococcus aureus

Staphylococcus aureus is internalised by host cells in vivo, and recent research results suggest that the bacteria use this intracellularity to persist in the host and form a reservoir for recurrent infections. However, in different cells types, the pathogen resorts to alternative strategies to survive phagocytosis and the antimicrobial mechanisms of host cells. In non‐professional phagocytes, S. aureus either escapes the endosome followed by cytoplasmic replication or replicates within autophagosomes. Professional phagocytes possess a limited capacity to kill S. aureus and hence the bacteria, well equipped with immune evasive mechanisms, replicate within the cells, eventually lyse out of the cells and thus persist in a continuous cycle of phagocytosis, host cell death, and bacterial release.     

The YfiBNR Signal Transduction Mechanism Reveals Novel Targets for the Evolution of Persistent Pseudomonas aeruginosa in Cystic Fibrosis Airways

The genetic adaptation of pathogens in host tissue plays a key role in the establishment of chronic infections. While whole genome sequencing has opened up the analysis of genetic changes occurring during long-term infections, the identification and characterization of adaptive traits is often obscured by a lack of knowledge of the underlying molecular processes. Our research addresses the role of Pseudomonas aeruginosa small colony variant (SCV) morphotypes in long-term infections. In the lungs of cystic fibrosis patients, the appearance of SCVs correlates with a prolonged persistence of infection and poor lung function. Formation of P. aeruginosa SCVs is linked to increased levels of the second messenger c-di-GMP. Our previous work identified the YfiBNR system as a key regulator of the SCV phenotype. The effector of this tripartite signaling module is the membrane bound diguanylate cyclase YfiN. Through a combination of genetic and biochemical analyses we first outline the mechanistic principles of YfiN regulation in detail. In particular, we identify a number of activating mutations in all three components of the Yfi regulatory system. YfiBNR is shown to function via tightly controlled competition between allosteric binding sites on the three Yfi proteins; a novel regulatory mechanism that is apparently widespread among periplasmic signaling systems in bacteria. We then show that during long-term lung infections of CF patients, activating mutations invade the population, driving SCV formation in vivo. The identification of mutational “scars” in the yfi genes of clinical isolates suggests that Yfi activity is both under positive and negative selection in vivo and that continuous adaptation of the c-di-GMP network contributes to the in vivo fitness of P. aeruginosa during chronic lung infections. These experiments uncover an important new principle of in vivo persistence, and identify the c-di-GMP network as a valid target for novel anti-infectives directed against chronic infections.     

Intracellular Staphylococcus aureus employs the cysteine protease staphopain A to induce host cell death in epithelial cells

Staphylococcus aureus is a major human pathogen, which can invade and survive in non-professional and professional phagocytes. Uptake by host cells is thought to contribute to pathogenicity and persistence of the bacterium. Upon internalization by epithelial cells, cytotoxic S. aureus strains can escape from the phagosome, replicate in the cytosol and induce host cell death. Here, we identified a staphylococcal cysteine protease to induce cell death after translocation of intracellular S. aureus into the host cell cytoplasm. We demonstrated that loss of staphopain A function leads to delayed onset of host cell death and prolonged intracellular replication of S. aureus in epithelial cells. Overexpression of staphopain A in a non-cytotoxic strain facilitated intracellular killing of the host cell even in the absence of detectable intracellular replication. Moreover, staphopain A contributed to efficient colonization of the lung in a mouse pneumonia model. In phagocytic cells, where intracellular S. aureus is exclusively localized in the phagosome, staphopain A did not contribute to cytotoxicity. Our study suggests that staphopain A is utilized by S. aureus to exit the epithelial host cell and thus contributes to tissue destruction and dissemination of infection.     

Combined Use of Bacteriophage K and a Novel Bacteriophage To Reduce Staphylococcus aureus Biofilm Formation

Biofilms are major causes of impairment of wound healing and patient morbidity. One of the most common and aggressive wound pathogens is Staphylococcus aureus, displaying a large repertoire of virulence factors and commonly reduced susceptibility to antibiotics, such as the spread of methicillin-resistant S. aureus (MRSA). Bacteriophages are obligate parasites of bacteria. They multiply intracellularly and lyse their bacterial host, releasing their progeny. We isolated a novel phage, DRA88, which has a broad host range among S. aureus bacteria. Morphologically, the phage belongs to the Myoviridae family and comprises a large double-stranded DNA (dsDNA) genome of 141,907 bp. DRA88 was mixed with phage K to produce a high-titer mixture that showed strong lytic activity against a wide range of S. aureus isolates, including representatives of the major international MRSA clones and coagulase-negative Staphylococcus. Its efficacy was assessed both in planktonic cultures and when treating established biofilms produced by three different biofilm-producing S. aureus isolates. A significant reduction of biofilm biomass over 48 h of treatment was recorded in all cases. The phage mixture may form the basis of an effective treatment for infections caused by S. aureus biofilms.     

Staphylococcus aureus SigB activity promotes a strong fibronectin–bacterium interaction which may sustain host tissue colonization by small‐colony variants isolated from cystic fibrosis patients

Genes encoding cell‐surface proteins regulated by SigB are stably expressed in Staphylococcus aureus small‐colony variants (SCVs) isolated from cystic fibrosis (CF) patients. Our hypothesis is that CF‐isolated SCVs are locked into a colonization state by sustaining the expression of adhesins such as fibronectin‐binding proteins (FnBPs) throughout growth. Force spectroscopy was used to study the fibronectin–FnBPs interaction among strains varying for their SigB activity. The fibronectin–FnBPs interaction was described by a strength of 1000 ± 400 pN (pulling rate of 2 μm s^?1), an energetic barrier width of 0.6 ± 0.1 Å and an off‐rate below 2 × 10^?4 s^?1. A CF‐isolated SCV highly expressed fnbA throughout growth and showed a sustained capacity to bind fibronectin, whereas a prototypic strain showed a reduced frequency of fibronectin‐binding during the stationary growth phase when its fnbA gene was down‐regulated. Reduced expression of fnbA was observed in sigB mutants, which was associated with an overall decrease adhesion to fibronectin. These results suggest that the fibronectin–FnBPs interaction plays a role in the formation of a mechanically resistant adhesion of S. aureus to host tissues and supports the hypothesis that CF‐isolated SCVs are locked into a colonization state as a result of a sustained SigB activity.     

Location of Pathogenic Bacteria during Persistent Infections: Insights from an Analysis Using Game Theory

Bacterial persistent infections are responsible for a significant amount of the human morbidity and mortality. Unlike acute bacterial infections, it is very difficult to treat persistent bacterial infections (e.g. tuberculosis). Knowledge about the location of pathogenic bacteria during persistent infection will help to treat such conditions by designing novel drugs which can reach such locations. In this study, events of bacterial persistent infections were analyzed using game theory. A game was defined where the pathogen and the host are the two players with a conflict of interest. Criteria for the establishment of Nash equilibrium were calculated for this game. This theoretical model, which is very simple and heuristic, predicts that during persistent infections pathogenic bacteria stay in both intracellular and extracellular compartments of the host. The result of this study implies that a bacterium should be able to survive in both intracellular and extracellular compartments of the host in order to cause persistent infections. This explains why persistent infections are more often caused by intracellular pathogens like Mycobacterium and Salmonella. Moreover, this prediction is in consistence with the results of previous experimental studies.     

A privileged intraphagocyte niche is responsible for disseminated infection of Staphylococcus aureus in a zebrafish model

The innate immune system is the primary defence against the versatile pathogen, Staphylococcus aureus. How this organism is able to avoid immune killing and cause infections is poorly understood. Using an established larval zebrafish infection model, we have shown that overwhelming infection is due to subversion of phagocytes by staphylococci, allowing bacteria to evade killing and found foci of disease. Larval zebrafish coinfected with two S. aureus strains carrying different fluorescent reporter gene fusions (but otherwise isogenic) had bacterial lesions, at the time of host death, containing predominantly one strain. Quantitative data using two marked strains revealed that the strain ratios, during overwhelming infection, were often skewed towards the extremes, with one strain predominating. Infection with passaged bacterial clones revealed the phenomenon not to bedue to adventitious mutations acquired by the pathogen. After infection of the host, all bacteria are internalized by phagocytes and the skewing of population ratios is absolutely dependent on the presence of phagocytes. Mathematical modelling of pathogen population dynamics revealed the data patterns are consistent with the hypothesis that a small number of infected phagocytes serve as an intracellular reservoir for S. aureus, which upon release leads to disseminated infection. Strategies to specifically alter neutrophil/macrophage numbers were used to map the potential subpopulation of phagocytes acting as a pathogen reservoir, revealing neutrophils as the likely ‘niche’. Subsequently in a murine sepsis model, S. aureus abscesses in kidneys were also found to be predominantly clonal, therefore likely founded by an individual cell, suggesting a potential mechanism analogous to the zebrafish model with few protected niches. These findings add credence to the argument that S. aureus control regimes should recognize both the intracellular as well as extracellular facets of the S. aureus life cycle.