Suppressive effect of therapeutic antibiotic regimen on antipneumococcal Th1/Th17 responses in neonatal mice.

BACKGROUND: Antibiotics are commonly used in human neonates, but their impact on neonatal T cell immunity remains poorly understood. The aim of this study was to investigate the impact of the antibiotic piperacillin with the beta-lactamase inhibitor tazobactam on neonatal CD4+ and CD8+ T cell responses to Streptococcus pneumoniae. METHODS: Splenic and lung cells were isolated from the neonatal mice receiving piperacillin and tazobactam or saline (sham) and cultured with S. pneumoniae to analyze T cell cytokine production by ELISA and flow cytometry. RESULTS: Antibiotic exposure to neonatal mice resulted in reduced numbers of CD4+/CD8+ T cells in the spleen and lungs compared to control mice. Upon in vitro stimulation with S. pneumoniae, splenocytes and lung cells from antibiotic-exposed mice produced lower levels of IFN-γ (Th1)/IL-17A (Th17) and IL-17A cytokines, respectively. Flow cytometric analysis revealed that S. pneumoniae-stimulated splenic CD4+ T cells from antibiotic-exposed mice expressed decreased levels of IFN-γ and IL-17A compared to control mice, whereas lung CD4+ T cells produced lower levels of IL-17A. However, no significant difference was observed for IL-4 (Th2) production. CONCLUSIONS: Neonatal mice exposure to piperacillin and tazobactam reduces the number of CD4+ and CD8+ T cells, and suppresses Th1 and Th17, but not Th2, responses to S. pneumoniae. IMPACT: Exposure of neonatal mice with a combination of piperacillin and tazobactam reduces CD4+/CD8+ T cells in the spleen and lungs. Antibiotic exposure suppresses neonatal Th1 and Th17, but not Th2, responses to Streptococcus pneumoniae. Our findings may have important implications for developing better therapeutic strategies in the neonatal intensive care unit.

Evolution via recombination: Cell-to-cell contact facilitates larger recombination events in Streptococcus pneumoniae.

Homologous recombination in the genetic transformation model organism Streptococcus pneumoniae is thought to be important in the adaptation and evolution of this pathogen. While competent pneumococci are able to scavenge DNA added to laboratory cultures, large-scale transfers of multiple kb are rare under these conditions. We used whole genome sequencing (WGS) to map transfers in recombinants arising from contact of competent cells with non-competent 'target' cells, using strains with known genomes, distinguished by a total of ~16,000 SNPs. Experiments designed to explore the effect of environment on large scale recombination events used saturating purified donor DNA, short-term cell assemblages on Millipore filters, and mature biofilm mixed cultures. WGS of 22 recombinants for each environment mapped all SNPs that were identical between the recombinant and the donor but not the recipient. The mean recombination event size was found to be significantly larger in cell-to-cell contact cultures (4051 bp in filter assemblage and 3938 bp in biofilm co-culture versus 1815 bp with saturating DNA). Up to 5.8% of the genome was transferred, through 20 recombination events, to a single recipient, with the largest single event incorporating 29,971 bp. We also found that some recombination events are clustered, that these clusters are more likely to occur in cell-to-cell contact environments, and that they cause significantly increased linkage of genes as far apart as 60,000 bp. We conclude that pneumococcal evolution through homologous recombination is more likely to occur on a larger scale in environments that permit cell-to-cell contact.

Correction: Naturally Acquired Human Immunity to Pneumococcus Is Dependent on Antibody to Protein Antigens.

[This corrects the article DOI: 10.1371/journal.ppat.1006137.].

Naturally Acquired Human Immunity to Pneumococcus Is Dependent on Antibody to Protein Antigens.

Naturally acquired immunity against invasive pneumococcal disease (IPD) is thought to be dependent on anti-capsular antibody. However nasopharyngeal colonisation by Streptococcus pneumoniae also induces antibody to protein antigens that could be protective. We have used human intravenous immunoglobulin preparation (IVIG), representing natural IgG responses to S. pneumoniae, to identify the classes of antigens that are functionally relevant for immunity to IPD. IgG in IVIG recognised capsular antigen and multiple S. pneumoniae protein antigens, with highly conserved patterns between different geographical sources of pooled human IgG. Incubation of S. pneumoniae in IVIG resulted in IgG binding to the bacteria, formation of bacterial aggregates, and enhanced phagocytosis even for unencapsulated S. pneumoniae strains, demonstrating the capsule was unlikely to be the dominant protective antigen. IgG binding to S. pneumoniae incubated in IVIG was reduced after partial chemical or genetic removal of bacterial surface proteins, and increased against a Streptococcus mitis strain expressing the S. pneumoniae protein PspC. In contrast, depletion of type-specific capsular antibody from IVIG did not affect IgG binding, opsonophagocytosis, or protection by passive vaccination against IPD in murine models. These results demonstrate that naturally acquired protection against IPD largely depends on antibody to protein antigens rather than the capsule.

Protective role of the capsule and impact of serotype 4 switching on Streptococcus mitis.

The polysaccharide capsule surrounding Streptococcus pneumoniae is essential for virulence. Recently, Streptococcus mitis, a human commensal and a close relative of S. pneumoniae, was also shown to have a capsule. In this study, the S. mitis type strain switched capsule by acquisition of the serotype 4 capsule locus of S. pneumoniae TIGR4, following induction of competence for natural transformation. Comparison of the wild type with the capsule-switching mutant and with a capsule deletion mutant showed that the capsule protected S. mitis against phagocytosis by RAW 264.7 macrophages. This effect was enhanced in the S. mitis strain expressing the S. pneumoniae capsule, which showed, in addition, increased resistance against early clearance in a mouse model of lung infection. Expression of both capsules also favored survival in human blood, and the effect was again more pronounced for the capsule-switching mutant. S. mitis survival in horse blood or in a mouse model of bacteremia was not significantly different between the wild type and the mutant strains. In all models, S. pneumoniae TIGR4 showed higher rates of survival than the S. mitis type strain or the capsule-switching mutant, except in the lung model, in which significant differences between S. pneumoniae TIGR4 and the capsule-switching mutant were not observed. Thus, we identified conditions that showed a protective function for the capsule in S. mitis. Under such conditions, S. mitis resistance to clearance could be enhanced by capsule switching to serotype 4, but it was enhanced to levels lower than those for the virulent strain S. pneumoniae TIGR4.

Chemical debridement of contaminated titanium surfaces: an in vitro study.

OBJECTIVE: To compare the efficacy of different chemical solutions when used for chemical debridement of biofilm contaminated titanium surfaces in an in-vitro experimental study. MATERIALS AND METHODS: Commercially pure titanium discs with a diameter of 6.2 mm and height of 2 mm, mirror-polished with a measured surface amplitude value SA = 0.037 µm ± 0.009 were used as test-surfaces. A biofilm was simulated with multi-layers of Staphylococcus epidermidis ATCC359844 covering the entire titanium surface. The chemical agents tested were: 3% H2O2, 0.2% Chlorhexidine, 24% EDTA-gel, 3% H2O2 mixed with 1.6 g/L TiO2 and sterile saline solution. The decontamination effect was evaluated by optical density analysis using spectrophotometry and with scanning electron microscopy (SEM) images of the remaining biofilm. RESULTS: The suspensions of 3% H2O2 and 1.6 g/L TiO2 or 3% H2O2 alone were the most effective in removing S. epidermidis biofilms (p < 0.05), whereas 0.2% chlorhexidine or 24% EDTA gel had no significant effects. SEM images of the remaining biofilms supported the quantitative results indicating the higher efficacy of 3% H2O2 and 1.6 g/L TiO2 or 3% H2O2 alone. It also revealed that EDTA, despite a non-significant effect on reducing the amount of established biofilms, was able to alter the biofilm architecture, as demonstrated by increased interspaced regions. CONCLUSIONS: In this in vitro study the decontamination potential of a suspension of 3% H2O2 and 1.6 g/L TiO2 or 3% H2O2 alone were encouraging. Whether such procedures would have a similar effect in vivo remains to be determined.

Markerless Genome Editing in Competent Streptococci.

Selective markers employed in classical mutagenesis methods using natural genetic transformation can affect gene expression, risk phenotypic effects, and accumulate as unwanted genes during successive mutagenesis cycles. In this chapter, we present a protocol for markerless genome editing in Streptococcus mutans and Streptococcus pneumoniae achieved with an efficient method for natural transformation. High yields of transformants are obtained by combining the unimodal state of competence developed after treatment of S. mutans with sigX-inducing peptide pheromone (XIP) in a chemically defined medium (CDM) or of S. pneumoniae with the competence-stimulating peptide (CSP) together with use of a donor amplicon carrying extensive flanking homology. This combination ensures efficient and precise integration of a new allele by the recombination machinery present in competent cells.

Extracellular identification of a processed type II ComR/ComS pheromone of Streptococcus mutans.

The competence-stimulating peptide (CSP) and the sigX-inducing peptide (XIP) are known to induce Streptococcus mutans competence for genetic transformation. For both pheromones, direct identification of the native peptides has not been accomplished. The fact that extracellular XIP activity was recently observed in a chemically defined medium devoid of peptides, as mentioned in an accompanying paper (K. Desai, L. Mashburn-Warren, M. J. Federle, and D. A. Morrison, J. Bacteriol. 194:3774-3780, 2012), provided ideal conditions for native XIP identification. To search for the XIP identity, culture supernatants were filtered to select for peptides of less than 3 kDa, followed by C(18) extraction. One peptide, not detected in the supernatant of a comS deletion mutant, was identified by tandem mass spectrometry (MS/MS) fragmentation as identical to the ComS C-terminal sequence GLDWWSL. ComS processing did not require Eep, a peptidase involved in processing or import of bacterial small hydrophobic peptides, since eep deletion had no inhibitory effect on XIP production or on synthetic XIP response. We investigated whether extracellular CSP was also produced. A reporter assay for CSP activity detection, as well as MS analysis of supernatants, revealed that CSP was not present at detectable levels. In addition, a mutant with deletion of the CSP-encoding gene comC produced endogenous XIP levels similar to those of a nondeletion mutant. The results indicate that XIP pheromone production is a natural phenomenon that may occur in the absence of natural CSP pheromone activity and that the heptapeptide GLDWWSL is an extracellular processed form of ComS, possibly the active XIP pheromone. This is the first report of direct identification of a ComR/ComS pheromone.

ResistoXplorer: a web-based tool for visual, statistical and exploratory data analysis of resistome data.

The study of resistomes using whole metagenomic sequencing enables high-throughput identification of resistance genes in complex microbial communities, such as the human microbiome. Over recent years, sophisticated and diverse pipelines have been established to facilitate raw data processing and annotation. Despite the progress, there are no easy-to-use tools for comprehensive visual, statistical and functional analysis of resistome data. Thus, exploration of the resulting large complex datasets remains a key bottleneck requiring robust computational resources and technical expertise, which creates a significant hurdle for advancements in the field. Here, we introduce ResistoXplorer, a user-friendly tool that integrates recent advancements in statistics and visualization, coupled with extensive functional annotations and phenotype collection, to enable high-throughput analysis of common outputs generated from metagenomic resistome studies. ResistoXplorer contains three modules-the 'Antimicrobial Resistance Gene Table' module offers various options for composition profiling, functional profiling and comparative analysis of resistome data; the 'Integration' module supports integrative exploratory analysis of resistome and microbiome abundance profiles derived from metagenomic samples; finally, the 'Antimicrobial Resistance Gene List' module enables users to intuitively explore the associations between antimicrobial resistance genes and the microbial hosts using network visual analytics to gain biological insights. ResistoXplorer is publicly available at http://www.resistoxplorer.no.

Effects of Expression of Streptococcus pneumoniae PspC on the Ability of Streptococcus mitis to Evade Complement-Mediated Immunity.

Streptococcus pneumoniae and Streptococcus mitis are genetically closely related and both frequently colonise the naso-oropharynx, yet S. pneumoniae is a common cause of invasive infections whereas S. mitis is only weakly pathogenic. We hypothesise that sensitivity to innate immunity may underlie these differences in virulence phenotype. We compared the sensitivity of S. pneumoniae and S. mitis strains to complement-mediated immunity, demonstrating S. mitis strains were susceptible to complement-mediated opsonophagocytosis. S. pneumoniae resistance to complement is partially dependent on binding of the complement regulator Factor H by the surface protein PspC. However, S. mitis was unable to bind factor H. The S. pneumoniae TIGR4 strain pspC was expressed in the S. mitis SK142 strain to create a S. mitis pspC+ strain. Immunoblots demonstrated the S. mitis pspC+ strain expressed PspC, and flow cytometry confirmed this resulted in Factor H binding to S. mitis, reduced susceptibility to complement and improved survival in whole human blood compared to the wild-type S. mitis strain. However, in mouse models the S. mitis pspC+ strain remained unable to establish persistent infection. Unlike S. pneumoniae strains, culture in serum or blood did not support increased CFU of the S. mitis strains. These results suggest S. mitis is highly sensitive to opsonisation with complement partially due to an inability to bind Factor H, but even when complement sensitivity was reduced by expression of pspC, poor growth in physiological fluid limited the virulence of S. mitis in mice.

Impact of narrow-spectrum penicillin V on the oral and faecal resistome in a young child treated for otitis media.

OBJECTIVES: Antibiotic overuse has led to the global emergence of antimicrobial-resistant bacteria, and children are among the most frequent users of antibiotics. Most studies with broad-spectrum antibiotics show a severe impact on resistome development in patients. Although narrow-spectrum antibiotics are believed to have fewer side effects, their impact on the microbiome and resistome is mostly unknown. The aim of this study was to investigate the impact of the narrow-spectrum antibiotic phenoxymethylpenicillin (penicillin V) on the microbiome and resistome of a child treated for acute otitis media. METHODS: Oral and faecal samples were collected from a 1-year-old child before (Day 0) and after (Days 5 and 30) receiving penicillin V for otitis media. Metagenomic sequencing data were analysed to determine taxonomic profiling using Kraken and Bracken software, and resistance profiling using KMA in combination with the ResFinder database. RESULTS: In the oral samples, antimicrobial resistance genes (ARGs) belonging to four classes were identified at baseline. At Day 5, the abundance of some ARGs was increased, whereas some remained unchanged and others could no longer be detected. At Day 30, most ARGs had returned to baseline levels or lower. In the faecal samples, seven ARGs were observed at baseline and five at Day 5. At Day 30, the number of ARGs had increased to 21. CONCLUSIONS: Following penicillin V, we observed a remarkable enrichment of the aecal resistome, indicating that even narrow-spectrum antibiotics may have important consequences in selecting for a more resistant microbiome.

Antibiotic Stewardship in Premature Infants: A Systematic Review.

INTRODUCTION: Antibiotic treatment in premature infants is often empirically prescribed, and practice varies widely among otherwise comparable neonatal intensive care units. Unnecessary and prolonged antibiotic treatment is documented in numerous studies. Recent research shows serious side effects and suggests long-term adverse health effects in prematurely born infants exposed to antibiotics in early life. One preventive measure to reduce unnecessary antibiotic exposure is implementation of antibiotic stewardship programs. Our objective was to review the literature on implemented antibiotic stewardship programs including premature infants with gestational age ≤34 weeks. METHODS: Six academic databases (PubMed [Medline], McMaster PLUS, Cochrane Database of Systematic Reviews, UpToDate, Cochrane Central Register of Controlled Trials, and National Institute for Health and Care Excellence) were systematically searched. PRISMA guidelines were applied. RESULTS: The search retrieved 1,212 titles of which 12 fitted inclusion criteria (11 observational studies and 1 randomized clinical trial). Included articles were critically appraised. We grouped the articles according to common area of implemented stewardship actions: (1) focus on reducing initiation of antibiotic therapy, (2) focus on shortening duration of antibiotic therapy, (3) various organizational stewardship implementations. The heterogeneity of cohort composition, of implemented actions and of outcome measures made meta-analysis inappropriate. We provide an overview of the reduction in antibiotic use achieved. CONCLUSION: Antibiotic stewardship programs can be effective for premature newborns especially when multifactorial and tailored to this population, focusing on reducing initiation or on shortening the duration of antibiotic therapy. Programs without specific measures were less effective.

Cyclic Di-adenosine Monophosphate Regulates Metabolism and Growth in the Oral Commensal Streptococcus mitis.

Cyclic di-adenosine monophosphate (c-di-AMP) has emerged as an important bacterial signaling molecule that functions both as an intracellular second messenger in bacterial cells and an extracellular ligand involved in bacteria-host cross-talk. In this study, we identify and characterize proteins involved in controlling the c-di-AMP concentration in the oral commensal and opportunistic pathogen Streptococcusmitis (S. mitis). We identified three known types of c-di-AMP turnover proteins in the genome of S. mitis CCUG31611: a CdaA-type diadenylate cyclase as well as GdpP-, and DhhP-type phosphodiesterases. Biochemical analyses of purified proteins demonstrated that CdaA synthesizes c-di-AMP from ATP whereas both phosphodiesterases can utilize c-di-AMP as well as the intermediary metabolite of c-di-AMP hydrolysis 5'-phosphadenylyl-adenosine (pApA) as substrate to generate AMP, albeit at different catalytic efficiency. Using deletion mutants of each of the genes encoding c-di-AMP turnover proteins, we show by high resolution MS/MS that the intracellular concentration of c-di-AMP is increased in deletion mutants of the phosphodiesterases and non-detectable in the cdaA-mutant. We also detected pApA in mutants of the DhhP-type phosphodiesterase. Low and high levels of c-di-AMP were associated with longer and shorter chains of S. mitis, respectively indicating a role in regulation of cell division. The deletion mutant of the DhhP-type phosphodiesterase displayed slow growth and reduced rate of glucose metabolism.

AI-2/LuxS is involved in increased biofilm formation by Streptococcus intermedius in the presence of antibiotics.

Bacteria utilize quorum-sensing communication to organize their behavior by monitoring the concentration of bacterial signals, referred to as autoinducers (AIs). The widespread detection of AI-2 signals and its enzymatic synthase (LuxS) in bacteria suggests that AI-2 is an inter- and intraspecies communication signal. We have previously shown that antibiotic susceptibility is affected by AI-2 signaling in Streptococcus anginosus. Since chronic infections involve persistent biofilms resilient to antibiotic treatment, we explored the role of AI-2/LuxS in Streptococcus intermedius biofilm formation and cell viability when the organism was exposed to sub-MICs of ampicillin, ciprofloxacin, or tetracycline. The S. intermedius wild type (WT) and its isogenic luxS mutant, strain SI006, were exposed to sub-MICs of ampicillin, ciprofloxacin, or tetracycline. Biofilms were formed on polystyrene discs in microtiter plates. To assess planktonic cell viability, the ATP microbial viability assay was performed and the numbers of CFU were determined. For complementation assays, the AI-2 precursor dihydroxy pentanedione (DPD) was used as a supplement for SI006. Relative luxS expression was quantified by real-time PCR. The sub-MICs of all three antibiotics increased biofilm formation in S. intermedius WT. However, biofilm formation by SI006 was either unaffected or reduced (P < or = 0.05). Bacterial viability tests of biofilm and planktonic cell cultures indicated that SI006 was more susceptible to antibiotics than the WT. DPD complemented the luxS mutant phenotype. Real-time PCR revealed modest yet significant changes in luxS expression in the presence of antibiotic concentrations that increased biofilm formation. In conclusion, in S. intermedius, AI-2/LuxS was involved in antibiotic susceptibility and increased biofilm formation at sub-MICs of antibiotic.

Furanones, potential agents for preventing Staphylococcus epidermidis biofilm infections?

OBJECTIVES: Staphylococcus epidermidis is often associated with biofilm infections related to medical implants. The aim of the present study was to find furanones that decrease biofilm formation without irritative or genotoxic effects, or effects on S. epidermidis growth. METHODS: After screening including bioluminescence and biofilm assays, 2 furanones out of 11 were chosen for further studies. MIC values of the two furanones were established to determine whether biofilm inhibition effects were ascribed to inhibition of bacterial growth. To further investigate interference with communication, the effect of the furanones was tested in the presence of the autoinducer-2 precursor (S)-4,5-dihydroxy-2,3-pentanedione. The furanones were tested for possible irritative effects by the Hen's egg test chorioallantoic membrane procedure. Finally, potential genotoxic effects in mice were assessed by a membrane array, and effects on global gene expression were investigated by using a microarray representing 30,000 genes of the mouse genome. RESULTS: From the bioluminescence assay, 4 furanones out of 11 were chosen for further biofilm analyses. Biofilm formation by S. epidermidis was significantly decreased by the four furanones tested at concentrations not affecting microbial growth. Two furanones were chosen for further studies: one that decreased biofilm statistically more than the others and one containing two bromo substituents. The two furanones were found to be non-irritative and non-genotoxic at the concentrations used. CONCLUSIONS: Furanones may inhibit biofilm formation through interference with quorum sensing and thus represent promising agents for protecting surfaces from being colonized by S. epidermidis.

Competence in Streptococcus pneumoniae and Close Commensal Relatives: Mechanisms and Implications.

The mitis group of streptococci comprises species that are common colonizers of the naso-oral-pharyngeal tract of humans. Streptococcus pneumoniae and Streptococcus mitis are close relatives and share ~60-80% of orthologous genes, but still present striking differences in pathogenic potential toward the human host. S. mitis has long been recognized as a reservoir of antibiotic resistance genes for S. pneumoniae, as well as a source for capsule polysaccharide variation, leading to resistance and vaccine escape. Both species share the ability to become naturally competent, and in this context, competence-associated killing mechanisms such as fratricide are thought to play an important role in interspecies gene exchange. Here, we explore the general mechanism of natural genetic transformation in the two species and touch upon the fundamental clinical and evolutionary implications of sharing similar competence, fratricide mechanisms, and a large fraction of their genomic DNA.

Conserved Pheromone Production, Response and Degradation by Streptococcus mutans.

Streptococcus mutans, a bacterium with high cariogenic potential, coordinates competence for natural transformation and bacteriocin production via the XIP and CSP pheromones. CSP is effective in inducing bacteriocin responses but not competence in chemically defined media (CDM). This is in contrast to XIP, which is a strong inducer of competence in CDM but can also stimulate bacteriocin genes as a late response. Interconnections between the pathways activated by the two pheromones have been characterized in certain detail in S. mutans UA159, but it is mostly unknown whether such findings are representative for the species. In this study, we used bioassays based on luciferase reporters for the bacteriocin gene cipB and the alternative sigma factor sigX to investigate various S. mutans isolates for production and response to CSP and XIP pheromones in CDM. Similar to S. mutans UA159, endogenous CSP was undetectable in the culture supernatants of all tested strains. During optimization of the bioassay using the cipB reporter, we discovered that the activity of exogenous CSP used as a standard was reduced over time during S. mutans growth. Using a FRET-CSP reporter peptide, we found that S. mutans UA159 was able to degrade CSP, and that such activity was not significantly different in isogenic mutants with deletion of the protease gene htrA or the competence genes sigX, oppD, and comR. CSP cleavage was also detected in all the wild type strains, indicating that this is a conserved feature in S. mutans. For the XIP pheromone, endogenous production was observed in the supernatants of all 34 tested strains at peak concentrations in culture supernatants that varied between 200 and 26000 nM. Transformation in the presence of exogenous XIP was detected in all but one of the isolates. The efficiency of transformation varied, however, among the different strains, and for those with the highest transformation rates, endogenous XIP peak concentrations in the supernatants were above 2000 nM XIP. We conclude that XIP production and inducing effect on transformation, as well as the ability to degrade CSP, are conserved functions among different S. mutans isolates. Understanding the functionality and conservation of pheromone systems in S. mutans may lead to novel strategies to prevent or treat unbalances in oral microbiomes that may favor diseases.

In Vitro Performance of Bioinspired Phenolic Nanocoatings for Endosseous Implant Applications.

In the quest for finding new strategies to enhance tissue integration and to reduce the risk of bacterial colonization around endosseous implants, we report the application of auto-oxidative phenolic coatings made of tannic acid and pyrogallol to titanium surfaces. The functionalized surfaces were screened for their biological performance using cultures of primary human osteoblasts and biofilm-forming bioluminescent staphylococci S. epidermidis Xen43 and S. aureus Xen29. No toxic effect of the coatings on osteoblasts was detected. While tannic acid coatings seemed to induce a delay in osteoblast maturation, they revealed anti-inflammatory potential. Similar effects were observed for pyrogallol coatings deposited for 24 h. Thin pyrogallol coatings deposited for 2 h seemed to promote osteoblast maturation and revealed increased calcium deposition. The effects on osteoblast were found to be related to the release of phenolic compounds from the surfaces. While the phenolic coatings could not inhibit staphylococcal biofilm formation on the titanium surfaces, released phenolic compounds had an inhibitory effect the growth of planktonic bacteria. In conclusion, the assessed coating systems represent a versatile functionalization method which exhibit promising effects for endosseous implant applications.

Relative Contributions of Extracellular and Internalized Bacteria to Early Macrophage Proinflammatory Responses to Streptococcus pneumoniae.

Both intracellular immune sensing and extracellular innate immune sensing have been implicated in initiating macrophage proinflammatory cytokine responses to Streptococcus pneumoniae The S. pneumoniae capsule, a major virulence determinant, prevents phagocytosis, and we hypothesized that this would reduce activation of host innate inflammatory responses by preventing activation of intracellular proinflammatory signaling pathways. We investigated this hypothesis in human monocyte-derived macrophages stimulated with encapsulated or isogenic unencapsulated mutant S. pneumoniae Unexpectedly, despite strongly inhibiting bacterial internalization, the capsule resulted in enhanced inflammatory cytokine production by macrophages infected with S. pneumoniae Experiments using purified capsule material and a Streptococcus mitis mutant expressing an S. pneumoniae serotype 4 capsule indicated these differences required whole bacteria and were not due to proinflammatory effects of the capsule itself. Transcriptional profiling demonstrated relatively few differences in macrophage gene expression profiles between infections with encapsulated S. pneumoniae and those with unencapsulated S. pneumoniae, largely limited to reduced expression of proinflammatory genes in response to unencapsulated bacteria, predicted to be due to reduced activation of the NF-κB family of transcription factors. Blocking S. pneumoniae internalization using cytochalasin D had minimal effects on the inflammatory response to S. pneumoniae Experiments using murine macrophages indicated that the affected genes were dependent on Toll-like receptor 2 (TLR2) activation, although not through direct stimulation of TLR2 by capsule polysaccharide. Our data demonstrate that the early macrophage proinflammatory response to S. pneumoniae is mainly dependent on extracellular bacteria and reveal an unexpected proinflammatory effect of encapsulated S. pneumoniae that could contribute to disease pathogenesis.IMPORTANCE Multiple extra- and intracellular innate immune receptors have been identified that recognize Streptococcus pneumoniae, but the relative contributions of intra- versus extracellular bacteria to the inflammatory response were unknown. We have shown that intracellular S. pneumoniae contributes surprisingly little to the inflammatory responses, with production of important proinflammatory cytokines largely dependent on extracellular bacteria. Furthermore, although we expected the S. pneumoniae polysaccharide capsule to block activation of the host immune system by reducing bacterial internalization and therefore activation of intracellular innate immune receptors, there was an increased inflammatory response to encapsulated compared to unencapsulated bacteria, which is likely to contribute to disease pathogenesis.

Antibacterial Surface Coating for Bone Scaffolds Based on the Dark Catalytic Effect of Titanium Dioxide.

Biomaterials which promote tissue integration and resist microbial colonisation are required in bone tissue engineering to prevent biomaterial-associated infections. Surface modification of established materials for bone tissue engineering, such as TiO2, have emerged as promising anti-infective strategies. Interestingly, the antibacterial activity of TiO2 in the form of particles can be enhanced by combining it with H2O2, even in the absence of irradiation. However, it remains unknown whether TiO2 surfaces elicit a similar effect. In this study, the antibacterial effect of porous TiO2 scaffolds generated by the catalytic decomposition of H2O2 in the absence of light (dark catalysis) was investigated. Porous ceramic foams were fabricated and sol-gel coated for high catalytic activity. Degradation of methylene blue in the presence of 3% H2O2 increased by 80% for the sol-gel-coated surfaces. The degradation kinetics indicate that intermediate free radicals that form at the liquid-TiO2 interface are responsible for the oxidative behavior of the surface. TiO2 surfaces were further pretreated with 30% H2O2 for prolonged oxidative behavior. The biological response toward such surfaces was assessed in vitro. S. epidermidis biofilms formed on modified surfaces showed reduced viability compared to nonmodified surfaces. Further, the same surface modification showed no cytotoxic effects on MC3T3 preosteoblasts. However, the results from the conducted genotoxicity assay were inconclusive, and further studies are needed to exclude ROS-mediated DNA damage. To conclude, this study provides evidence that a simple surface modification based on the dark catalytic effect of TiO2 can be used to create antibacterial surface properties for ceramic bone scaffolds.