Function analysis of choline binding domains (CBDs) of LytA, LytC and CbpD in biofilm formation of Streptococcus pneumoniae.

Biofilm formation is an important strategy for the colonization of Streptococcus pneumoniae, which can increase the capacity to evade antibiotic and host immune stress. Extracellular choline-binding proteins (CBPs) are required for successful biofilm formation, but the function of extracellular CBPs in the process of biofilm formation is not fully understood. In this study, we tend to analyze the functions of LytA, LytC and CbpD in biofilm formation by in vitro studies with their choline-binding domains (CBDs). Biofilm formation of S. pneumoniae was enhanced when cultured in medium supplemented with CBD-C and CBD-D. Parallel assays with ChBp-Is (choline binding repeats with different C-terminal tails) and character analysis of CBDs reveal a higher isoelectric point (pI) is related to promotion of biofilm formation. Phenotype characterization of biofilms revel CBD-C and CBD-D function differently, CBD-C promoting the formation of membrane-like structures and CBD-D promoting the formation of regular reticular structures. Gene expression analysis reveals membrane transport pathways are influenced with the binding of CBDs, among which the phosphate uptake and PTS of galactose pathways are both up-regulated under conditions with CBDs. Further, extracellular substances detection revealed that extracellular proteins increased with CBD-A and CBD-D, exhibiting as increase in extracellular high molecular weight proteins. Extracellular DNA increased under CBD-A but decreased under CBD-C and CBD-D; Extracellular phosphate increased under CBD-C. These support the alterations in membrane transport pathways, and reveal diverse reactions to extracellular protein, DNA and phosphate of these three CBDs. Overall, our results indicated extracellular CBP participate in biofilm formation by affecting surface charge and membrane transport pathways of pneumococcal cells, as well as promoting reactions to extracellular substances.

Vitamin K3 inhibits FtsZ assembly, disrupts the Z-ring in Streptococcus pneumoniae and displays anti-pneumococcal activity.

The respiratory pathogen, Streptococcus pneumoniae has acquired multiple-drug resistance over the years. An attractive strategy to combat pneumococcal infection is to target cell division to inhibit the proliferation of S. pneumoniae. This work presents Vitamin K3 as a potential anti-pneumococcal drug that targets FtsZ, the master coordinator of bacterial cell division. Vitamin K3 strongly inhibited S. pneumoniae proliferation with a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) of 6 µg/ml. Vitamin K3 disrupted the Z-ring localization in both S. pneumoniae and Bacillus subtilis within 30 min of treatment, while the membrane integrity and nucleoid segregation remain unchanged. Several complementary experiments showed that Vitamin K3 inhibits the assembly of purified S. pneumoniae FtsZ (SpnFtsZ) and induces conformational changes in the protein. Interestingly, Vitamin K3 interfered with GTP binding onto FtsZ and increased the GTPase activity of FtsZ polymers. The intrinsic tryptophan fluorescence of SpnFtsZ revealed that Vitamin K3 delays the nucleation of FtsZ polymers and reduces the rate of polymerization. In the presence of a non-hydrolyzable analog of GTP, Vitamin K3 did not show inhibition of FtsZ polymerization. These results indicated that Vitamin K3 induces conformational changes in FtsZ that increase GTP hydrolysis and thereby, destabilize the FtsZ polymers. Together, our data provide evidence that Vitamin K3 derives its potent anti-pneumococcal activity by inhibiting FtsZ assembly.

Streptococcus pneumoniae metal homeostasis alters cellular metabolism.

Streptococcus pneumoniae colonizes the human nasopharyngeal mucosa and is a leading cause of community-acquired pneumonia, acute otitis media, and bacterial meningitis. Metal ion homeostasis is vital to the survival of this pathogen across diverse biological sites and contributes significantly to colonization and invasive disease. Microarray and qRT-PCR analysis revealed an upregulation of an uncharacterized operon (SP1433-1438) in pneumococci subjected to metal-chelation by N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN). Supplementation of zinc, cobalt, and nickel following TPEN treatment significantly abrogated induction. BLASTP comparisons and protein topology analysis predicted this locus to encode components of ATP binding cassette (ABC) transporters involved in multidrug resistance (SP1434-1435) and energy-coupling factor (ECF) transporters (SP1436-1438). Inductively coupled plasma mass spectrometry (ICP-MS) analysis identified differences in intracellular metal content in a Δ1434-8 mutant strain compared to parental T4R. Further, analysis of the secreted metabolome of WT and Δ1434-8 strains identified significant changes in pneumococcal glycolytic and amino acid metabolic pathways, indicating a shift towards mixed acid fermentation. Additionally, proteomic analysis revealed differentially expressed proteins in the Δ1434-8 mutant strain, with nearly 20% regulated by the global catabolite repressor, CcpA. Based on these findings, we propose that the transporters encoded by SP1433-1438 are involved in regulating the central metabolism of S. pneumoniae and contributing to bacterial survival during metal stress.

Truncated Pneumolysin from Streptococcus pneumoniae as a TLR4-Antagonizing New Drug for Chronic Inflammatory Conditions.

Microbial proteins have recently been found to have more benefits in clinical disease treatment because of their better-developed strategy and properties than traditional medicine. In this study, we investigated the effectiveness of a truncated peptide synthesized from the C-terminal sequence of pneumolysin, i.e., C70PLY4, in Streptococcus pneumoniae, in treating chronic inflammatory conditions. It has been shown that C70PLY4 significantly blocks the transendothelial migration of neutrophils and attenuates the formation of atherosclerotic plaque and the secretion of soluble forms of the intercellular adhesion molecule-1 (ICAM-1), the vascular cell adhesion molecule 1 (VCAM-1), and E-selectin in high-fat-diet/streptozotocin-induced inflammatory rats. The mechanism and the docking simulation analysis further indicated that C70PLY4 might serve as a Toll-like receptor 4 (TLR4) antagonist by competing for the binding site of MD2, an indispensable protein for lipopolysaccharide (LPS)-TLR4 interaction signaling, on the TLR4 structure. Moreover, compared to the full-length PLY, C70PLY4 seems to have no cytotoxicity in human vascular endothelial cells. Our study elucidated a possible therapeutic efficacy of C70PLY4 in reducing chronic inflammatory conditions and clarified the underlying mechanism. Thus, our findings identify a new drug candidate that, by blocking TLR4 activity, could be an effective treatment for patients with chronic inflammatory diseases.

Polyurethane-biomacromolecule combined foam dressing containing asiaticoside: fabrication, characterization and clinical efficacy for traumatic dermal wound treatment.

Polyurethane combined (PUC) foam dressings with various biomacromolecules were fabricated with the adsorption of asiaticoside and silver nanoparticles for traumatic wound treatment. Biomacromolecules had varying effects on physicochemical and mechanical properties of PU foam. With 2% incorporation, starches, high molecular weight chitosan and gelatin provided stiffer and more porous foams while carboxymethylcellulose had the highest compression strength but the lowest water vapor transmission. High water absorption was from foams with carboxymethylcellulose, alginate, hydroxypropyl methylcellulose and low molecular weight chitosan. Increasing the concentrations up to 12% had more prominent effect. However, powdery surface was noticed with poorer tensile properties that 6% incorporation was selected. FTIR spectra and DSC thermograms suggested interaction of PU formulation with biomacromolecules. EDS analysis confirmed existence of active compounds while acceptable stability was from sterilized PUC foam with alginate. On healthy volunteers, this selected foam dressing caused no skin irritation and retained moisture comparable to commercial product. In patients with traumatic dermal wounds, healing improvement with shorter wound closure time, higher reepithelialization and less pain score were from the selected foam dressing compared to standard gauze soaked with chlorhexidine. This PU-alginate combined foam dressing adsorbed with asiaticoside and silver nanoparticles proved advantages for traumatic dermal wound management.

The mechanism of iron-compensation for manganese deficiency of Streptococcus pneumoniae.

Given their involvement in catalysis, infection, and biofilm formation, Fe and Mn are essential for bacterial survival and virulence. In this study, we found that Streptococcus pneumoniae (S. pneumoniae) could grow in the Mn-deficient medium (MDCM). Furthermore, findings showed that the Fe concentration in the bacterium increased when the Mn concentration decreased. In addition, it was noted that supplementing MDCM with Fe resulted in the recovery of bacterial growth. Quantitative proteomics using stable-isotope dimethyl labeling was performed to investigate the adaptive growth mechanism of S. pneumoniae under Mn-deficient conditions. It was found that the expression levels of 25 proteins were downregulated, whereas those of 54 proteins were upregulated in S. pneumoniae grown in MDCM. It was also noted that several of the downregulated proteins were involved in cell energy metabolism, amino acid synthesis, and reduction of oxidation products. More importantly, several ATP-binding cassette transporters related to Fe uptake, such as PiuA, PiaA, PitA, and SPD_1609, were overexpressed for increased Fe uptake from the MDCM. The results suggest that Mn deficiency disturbs multiple metabolic processes in S. pneumoniae. Furthermore, it causes a compensatory effect of Fe for Mn, which is beneficial for the survival of the bacterium in extreme environments. SIGNIFICANCE: The relationship between manganese and iron metabolism in S. pneumoniae has not been clearly revealed. In this paper, we suggest that Mn limitation disturbs multiple metabolic processes and evidently decreases the ATP level in the bacterium. In order to survive in this extreme environment, bacteria upregulated three type of Fe ion transporters PiuABC (heme), PiaABC (ferrichrome) and PitABC (Fe3+) to uptake enough Fe ions to response to Mn deficiency. Therefore, this study reveals a bacterial mechanism of Fe compensation for Mn, and provides new insight for investigating the relativeness of Fe and Mn metabolism of bacteria.

Engineering detoxified pneumococcal pneumolysin derivative ΔA146PLY for self-biomineralization of calcium phosphate: Assessment of their protective efficacy in murine infection models.

Vaccine design ushered in the era of nanotechnology, as the vaccine is being developed toward particulate formulation. We have previously shown that the attenuated pneumolysin mutant (ΔA146PLY) was a safe and effective pneumococcal vaccine candidate. Here, to further optimize the formulation, we fused calcium phosphate (CaP) binding domains with ΔA146PLY so that the biocompatible CaP can mineralize with the protein automatically, allowing simple production of nanoparticle antigen during preparation. We fabricated four different nanoparticles, and then we compared the characteristics of different CaP-ΔA146PLY nanoparticles and demonstrated the influence of CaP binding domains on the size, shape and surface calcium content of the nanoparticles. It was found that these self-biomineralized CaP-ΔA146PLY nanoparticles varied in their capacity to induce BMDCs and splenocytes production of cytokines. We further demonstrated that, compared to free proteins, nanoparticle antigens induced more efficient humoral and cellular immune responses which was strong enough to protect mice from both pneumonia and sepsis infection. Also, the integration of CaP to protein has no significant impairment on body weight of animals, and subcutaneous injection of ΔA146PLY-peptides@CaP nanoparticles did not lead to permanent formation of nodules in the skin relative to Alum adjuvant formulated antigens. Together, our data sufficiently suggest that soluble ΔA146PLY vaccine candidate could be processed into nanoparticles by self-biomineralization of CaP, the immunogenicity of which could be efficiently improved by the CaP binding domains and biomineralization.

Biophysical Characterization and Thermal Stability of Pneumococcal Histidine Triad Protein D in the Presence of Zinc and Manganese.

The pneumococcal histidine triad protein D (PhtD) is believed to play a central role in pneumococcal metal ion homeostasis and has been proposed as a promising vaccine candidate against pneumococcal disease. To investigate for potential stabilizers, a panel of physiologically relevant metals was screened using the thermal shift assay and it was found that only Zn2+ and Mn2+ were able to increase PhtD melting temperature. Differential scanning calorimetry analysis revealed a sequential unfolding of PhtD and the presence of at least 3 independent folding domains that can be stabilized by Zn2+ and Mn2+. UV spectroscopy and fluorescence quenching studies showed significant Zn2+-induced tertiary structure changes in PhtD characterized by decreased accessibility of inner tryptophan residues to the aqueous solvent. Isothermal titration calorimetry data show no apparent binding to Mn2+ but revealed a Zn2+:PhtD exothermic interaction stoichiometry of 3:1 with strong enthalpic contribution, suggesting that 3 of the 5 histidine triads are accessible binding sites for Zn2+. Only Zn+2, but not Mn+2, was able to increase the thermal stability of PhtD in the presence of aluminum hydroxide adjuvant, making it a promising stabilizer excipient candidate in vaccine products containing PhtD.

Pyruvate oxidase of Streptococcus pneumoniae contributes to pneumolysin release.

BACKGROUND: Streptococcus pneumoniae is one of the leading causes of community acquired pneumonia and acute otitis media. Certain aspects of S. pneumoniae's virulence are dependent upon expression and release of the protein toxin pneumolysin (PLY) and upon the activity of the peroxide-producing enzyme, pyruvate oxidase (SpxB). We investigated the possible synergy of these two proteins and identified that release of PLY is enhanced by expression of SpxB prior to stationary phase growth. RESULTS: Mutants lacking the spxB gene were defective in PLY release and complementation of spxB restored PLY release. This was demonstrated by cytotoxic effects of sterile filtered supernatants upon epithelial cells and red blood cells. Additionally, peroxide production appeared to contribute to the mechanism of PLY release since a significant correlation was found between peroxide production and PLY release among a panel of clinical isolates. Exogenous addition of H2O2 failed to induce PLY release and catalase supplementation prevented PLY release in some strains, indicating peroxide may exert its effect intracellularly or in a strain-dependent manner. SpxB expression did not trigger bacterial cell death or LytA-dependent autolysis, but did predispose cells to deoxycholate lysis. CONCLUSIONS: Here we demonstrate a novel link between spxB expression and PLY release. These findings link liberation of PLY toxin to oxygen availability and pneumococcal metabolism.

iTRAQ-Based Proteomics Revealed the Bactericidal Mechanism of Sodium New Houttuyfonate against Streptococcus pneumoniae.

Sodium new houttuyfonate (SNH), an addition product of active ingredient houttuynin from the plant Houttuynia cordata Thunb., inhibits a variety of bacteria, yet the mechanism by which it induces cell death has not been fully understood. In the present study, we utilized iTRAQ-based quantitative proteomics to analyze the protein alterations in Streptococcus pneumoniae in response to SNH treatment. Numerous proteins related to the production of reactive oxygen species (ROS) were found to be up-regulated by SNH, suggesting that ROS pathways may be involved as analyzed via bioinformatics. As reported recently, cellular reactions stimulated by ROS including superoxide anion (O2(•-)), hydrogen peroxide (H2O2), and hydroxyl radicals (OH(•)) have been implicated as mechanisms whereby bactericidal antibiotics kill bacteria. We then validated that SNH killed S. pneumoniae in a dose-dependent manner accompanied by the increasing level of H2O2. On the other hand, the addition of catalase, which can neutralize H2O2 in cells, showed a significant recovery in bacterial survival. These results indicate that SNH indeed induced H2O2 formation to contribute to the cell lethality, providing new insights into the bactericidal mechanism of SNH and expanding our understanding of the common mechanism of killing induced by bactericidal agents.

Transcriptome analysis of Streptococcus pneumoniae treated with the designed antimicrobial peptides, DM3.

In our previous studies, we generated a short 13 amino acid antimicrobial peptide (AMP), DM3, showing potent antipneumococcal activity in vitro and in vivo. Here we analyse the underlying mechanisms of action using Next-Generation transcriptome sequencing of penicillin (PEN)-resistant and PEN-susceptible pneumococci treated with DM3, PEN, and combination of DM3 and PEN (DM3PEN). DM3 induced differential expression in cell wall and cell membrane structural and transmembrane processes. Notably, DM3 altered the expression of competence-induction pathways by upregulating CelA, CelB, and CglA while downregulating Ccs16, ComF, and Ccs4 proteins. Capsular polysaccharide subunits were downregulated in DM3-treated cells, however, it was upregulated in PEN- and DM3PEN-treated groups. Additionally, DM3 altered the amino acids biosynthesis pathways, particularly targeting ribosomal rRNA subunits. Downregulation of cationic AMPs resistance pathway suggests that DM3 treatment could autoenhance pneumococci susceptibility to DM3. Gene enrichment analysis showed that unlike PEN and DM3PEN, DM3 treatment exerted no effect on DNA-binding RNA polymerase activity but observed downregulation of RpoD and RNA polymerase sigma factor. In contrast to DM3, DM3PEN altered the regulation of multiple purine/pyrimidine biosynthesis and metabolic pathways. Future studies based on in vitro experiments are proposed to investigate the key pathways leading to pneumococcal cell death caused by DM3.

Linezolid Surveillance Results for the United States (LEADER Surveillance Program 2014).

Thelinezolidexperience andaccuratedetermination ofresistance (LEADER) surveillance program has monitored linezolid activity, spectrum, and resistance since 2004. In 2014, a total of 6,865 Gram-positive pathogens from 60 medical centers from 36 states were submitted. The organism groups evaluated wereStaphylococcus aureus(3,106), coagulase-negative staphylococci (CoNS; 797), enterococci (855),Streptococcus pneumoniae(874), viridans group streptococci (359), and beta-hemolytic streptococci (874). Susceptibility testing was performed by reference broth microdilution at the monitoring laboratory. Linezolid-resistant isolates were confirmed by repeat testing. PCR and sequencing were performed to detect mutations in 23S rRNA, L3, L4, and L22 proteins and acquired genes (cfrandoptrA). The MIC50/90forStaphylococcus aureuswas 1/1 µg/ml, with 47.2% of isolates being methicillin-resistantStaphylococcus aureus Linezolid was active against allStreptococcus pneumoniaestrains and beta-hemolytic streptococci with a MIC50/90of 1/1 µg/ml and against viridans group streptococci with a MIC50/90of 0.5/1 µg/ml. Among the linezolid-nonsusceptible MRSA strains, one strain harboredcfronly (MIC, 4 µg/ml), one harbored G2576T (MIC, 8 µg/ml), and one containedcfrand G2576T with L3 changes (MIC, ≥8 µg/ml). Among CoNS, 0.75% (six isolates) of all strains demonstrated linezolid MIC results of ≥4 µg/ml. Five of these were identified asStaphylococcus epidermidis, four of which containedcfrin addition to the presence of mutations in the ribosomal proteins L3 and L4, alone or in combination with 23S rRNA (G2576T) mutations. Six enterococci (0.7%) were linezolid nonsusceptible (≥4 µg/ml; five with G2576T mutations, including one with an additionalcfrgene, and one strain withoptrAonly). Linezolid demonstrated excellent activity and a sustained susceptibility rate of 99.78% overall.

Copper(II)-Bis(Thiosemicarbazonato) Complexes as Antibacterial Agents: Insights into Their Mode of Action and Potential as Therapeutics.

There is increasing interest in the use of lipophilic copper (Cu)-containing complexes to combat bacterial infections. In this work, we showed that Cu complexes with bis(thiosemicarbazone) ligands [Cu(btsc)] exert antibacterial activity against a range of medically significant pathogens. Previous work using Neisseria gonorrhoeae showed that Cu(btsc) complexes may act as inhibitors of respiratory dehydrogenases in the electron transport chain. We now show that these complexes are also toxic against pathogens that lack a respiratory chain. Respiration in Escherichia coli was slightly affected by Cu(btsc) complexes, but our results indicate that, in this model bacterium, the complexes act primarily as agents that deliver toxic Cu ions efficiently into the cytoplasm. Although the chemistry of Cu(btsc) complexes may dictate their mechanism of action, their efficacy depends heavily on bacterial physiology. This is linked to the ability of the target bacterium to tolerate Cu and, additionally, the susceptibility of the respiratory chain to direct inhibition by Cu(btsc) complexes. The physiology of N. gonorrhoeae, including multidrug-resistant strains, makes it highly susceptible to damage by Cu ions and Cu(btsc) complexes, highlighting the potential of Cu(btsc) complexes (and Cu-based therapeutics) as a promising treatment against this important bacterial pathogen.

Varied metal-binding properties of lipoprotein PsaA in Streptococcus pneumoniae.

Streptococcus pneumoniae is a Gram-positive pathogen responsible for pneumonia, otitis media, and meningitis. Manganese and zinc ions are essential for this bacterium, playing regulatory, structural, or catalytic roles as the critical cofactors in the bacterial proteins and metabolic enzymes. Lipoprotein PsaA has been found to mediate Mn(2+) and Zn(2+) transportation in Streptococcus pneumoniae. In the present work, we conducted a systemic study on the contributions from key amino acids in the metal-binding site of PsaA using various spectroscopic and biochemical methods. Our experimental data indicate that four metal-binding residues contribute unequally to the Mn(2+) and Zn(2+) binding, and His139 is most important for both the structural stability and metal binding of the protein. PsaA-Mn(2+) has a lower thermal stability than PsaA-Zn(2+), possibly due to the different coordination preferences of the metals. Kinetics analysis revealed that PsaA-Mn(2+) binding is a fast first-order reaction, whereas PsaA-Zn(2+) binding is a slow second-order reaction, implying that PsaA kinetically prefers binding Mn(2+) to Zn(2+). The present results provide complementary information for understanding the mechanisms of metal transport and bacterial virulence via lipoproteins in Streptococcus pneumoniae.

AdcA and AdcAII employ distinct zinc acquisition mechanisms and contribute additively to zinc homeostasis in Streptococcus pneumoniae.

Streptococcus pneumoniae is a globally significant human pathogen responsible for nearly 1 million deaths annually. Central to the ability of S. pneumoniae to colonize and mediate disease in humans is the acquisition of zinc from the host environment. Zinc uptake in S. pneumoniae occurs via the ATP-binding cassette transporter AdcCB, and, unusually, two zinc-binding proteins, AdcA and AdcAII. Studies have suggested that these two proteins are functionally redundant, although AdcA has remained uncharacterized by biochemical methods. Here we show that AdcA is a zinc-specific substrate-binding protein (SBP). By contrast with other zinc-binding SBPs, AdcA has two zinc-binding domains: a canonical amino-terminal cluster A-I zinc-binding domain and a carboxy-terminal zinc-binding domain, which has homology to the zinc-chaperone ZinT from Gram-negative organisms. Intriguingly, this latter feature is absent from AdcAII and suggests that the two zinc-binding SBPs of S. pneumoniae employ different modalities in zinc recruitment. We further show that AdcAII is reliant upon the polyhistidine triad proteins for zinc in vitro and in vivo. Collectively, our studies suggest that, despite the overlapping roles of the two SBPs in zinc acquisition, they may have unique mechanisms in zinc homeostasis and act in a complementary manner during host colonization.

Inhibitory effect of polyunsaturated fatty acids on apoptosis induced by Streptococcus pneumoniae in alveolar macrophages.

BACKGROUND & OBJECTIVES: Apoptosis is considered as a major defense mechanism of the body. Multiple pathogens induce macrophage apoptosis as a mode of immune evasion. In earlier studies, n-3 polyunsaturated fatty acids (PUFA) have been reported to be protective against neuronal apoptosis and neuronal degeneration, seen after spinal cord injury. In this study, we tried to evaluate the role of n-3 polyunsaturated fatty acids on the process of macrophage phagocytic activity and apoptosis in mice. METHODS: Mice were divided into three groups (n=60); Group I was fed on sea cod oil; Group II on flaxseed oil supplementation for 9 wk along with standard laboratory chow diet. Group III was fed on standard diet and served as control. After supplementation, phagocytic and apoptotic (morphological staining: acridine orange plus ethidium bromide; H-33342 plus propidium iodide staining and DNA ladder formation) activities of mouse alveolar macrophages were assessed. RESULTS: Alveolar macrophages (obtained from sea cod oil and flaxseed oil fed group mice) showed significant increase in bacterial uptake as well as intracellular killing (P 0.05) of Streptococcus pneumoniae. Significant decrease (P<0.05) in apoptotic cells was observed among alveolar macrophages from sea cod and flaxseed oil fed mice whereas maximum apoptosis was observed in control alveolar macrophages on interaction with bacteria in vitro which was confirmed by DNA laddering. INTERPRETATION & CONCLUSIONS: These findings suggest that dietary supplementation with n-3 polyunsaturated fatty acids to mice led to enhanced phagocytic capability of their alveolar macrophages as well as provided protection against apoptosis upon challenge with S. pneumoniae.

Bacterial cytolysin during meningitis disrupts the regulation of glutamate in the brain, leading to synaptic damage.

Streptococcus pneumoniae (pneumococcal) meningitis is a common bacterial infection of the brain. The cholesterol-dependent cytolysin pneumolysin represents a key factor, determining the neuropathogenic potential of the pneumococci. Here, we demonstrate selective synaptic loss within the superficial layers of the frontal neocortex of post-mortem brain samples from individuals with pneumococcal meningitis. A similar effect was observed in mice with pneumococcal meningitis only when the bacteria expressed the pore-forming cholesterol-dependent cytolysin pneumolysin. Exposure of acute mouse brain slices to only pore-competent pneumolysin at disease-relevant, non-lytic concentrations caused permanent dendritic swelling, dendritic spine elimination and synaptic loss. The NMDA glutamate receptor antagonists MK801 and D-AP5 reduced this pathology. Pneumolysin increased glutamate levels within the mouse brain slices. In mouse astrocytes, pneumolysin initiated the release of glutamate in a calcium-dependent manner. We propose that pneumolysin plays a significant synapto- and dendritotoxic role in pneumococcal meningitis by initiating glutamate release from astrocytes, leading to subsequent glutamate-dependent synaptic damage. We outline for the first time the occurrence of synaptic pathology in pneumococcal meningitis and demonstrate that a bacterial cytolysin can dysregulate the control of glutamate in the brain, inducing excitotoxic damage.

A single-blinded randomized clinical trial comparing polymyxin B-trimethoprim and moxifloxacin for treatment of acute conjunctivitis in children.

OBJECTIVE: To perform a randomized controlled trial comparing moxifloxacin hydrochloride with polymyxin B-trimethoprim for the treatment of acute conjunctivitis. STUDY DESIGN: Patients ages 1-18 years old with acute conjunctivitis had cultures performed and were randomized to receive either moxifloxacin hydrochloride or polymyxin B-trimethoprim ophthalmic solution for 7 days. Response to treatment was determined by phone query on day 4-6 and by examination with post-treatment conjunctival culture on day 7-10. RESULTS: One hundred and twenty-four patients were enrolled. Eighty patients (65%) had recognized pathogens (55 Haemophilus influenzae, 22 Streptococcus pneumoniae, 4 Moraxella catarrhalis) isolated from their conjunctiva. One hundred fourteen (56/62 moxifloxacin and 58/62 polymyxin B-trimethoprim) completed the 4-6 day evaluation, with 43/56 (77%) of the moxifloxacin group and 42/58 (72%) of the polymyxin B-trimethoprim group clinically cured according to parents (noninferiority test P = .04). Eighty-nine (39/56 moxifloxacin and 50/58 polymyxin B-trimethoprim) patients completed the 7-10 day evaluation. Clinical cure was observed in 37/39 (95%) of the moxifloxacin and 49/51 (96%) of the polymyxin B-trimethoprim treated groups (noninferiority test P ≤ .01). Clinical cure rates for culture positive and negative conjunctivitis were not different. There was no statistically significant difference in bacteriologic cure rates between the 2 groups. CONCLUSIONS: Polymyxin B-trimethoprim continues to be an effective treatment for acute conjunctivitis with a clinical response rate that does not differ from moxifloxacin. Use of polymyxin B-trimethoprim for the treatment of conjunctivitis would result in significant cost savings compared with fluoroquinolones.

In vitro and in vivo pharmacokinetic/pharmacodynamic activity of clofoctol.

The aim of the study was to examine the In vitro susceptibility of clinical isolates of respiratory pathogens to clofoctol compared with amoxicillin and erythromycin, and to characterize the pharmacokinetic/pharmacodynamic (PK/PD) relationships of clofoctol using a murine pneumonia infection model. Strains clinically isolated from patients between 2005 and 2009 were used to examine susceptibility: penicillin-susceptible Streptococcus pneumoniae, penicillin-resistant S. pneumoniae, Streptococcus pyogenes, methicillin-susceptible Staphylococcus aureus, methicillin-resistant S. aureus, and Haemophilus influenzae. The In vitro activity of clofoctol against clinical isolates has essentially remained unchanged over recent years. The MIC50 and MIC90 of clofoctol against penicillin-resistant S. pneumoniae are lower than that of amoxicillin and erythromycin. The area under curve/minimum inhibitory concentration (AUC/MIC) ratio is the PK/PD parameter that best correlates with in vivo clofoctol efficacy; the value of AUC/MIC required to achieve the maximum effect in this study was 75.5.

Characterization of central carbon metabolism of Streptococcus pneumoniae by isotopologue profiling.

The metabolism of Streptococcus pneumoniae was studied by isotopologue profiling after bacterial cultivation in chemically defined medium supplemented with [U-(13)C(6)]- or [1,2-(13)C(2)]glucose. GC/MS analysis of protein-derived amino acids showed lack of (13)C label in amino acids that were also essential for pneumococcal growth. Ala, Ser, Asp, and Thr displayed high (13)C enrichments, whereas Phe, Tyr, and Gly were only slightly labeled. The analysis of the labeling patterns showed formation of triose phosphate and pyruvate via the Embden-Meyerhof-Parnas pathway. The labeling patterns of Asp and Thr suggested formation of oxaloacetate exclusively via the phosphoenolpyruvate carboxylase reaction. Apparently, α-ketoglutarate was generated from unlabeled glutamate via the aspartate transaminase reaction. A fraction of Phe and Tyr obtained label via the chorismate route from erythrose 4-phosphate, generated via the pentose phosphate pathway, and phosphoenolpyruvate. Strikingly, the data revealed no significant flux from phosphoglycerate to Ser and Gly but showed formation of Ser via the reverse reaction, namely by hydroxymethylation of Gly. The essential Gly was acquired from the medium, and the biosynthesis pathway was confirmed in experiments using [U-(13)C(2)]glycine as a tracer. The hydroxymethyl group in Ser originated from formate, which was generated by the pyruvate formate-lyase. Highly similar isotopologue profiles were observed in corresponding experiments with pneumococcal mutants deficient in PavA, CodY, and glucose-6-phosphate dehydrogenase pointing to the robustness of the core metabolic network used by these facultative pathogenic bacteria. In conclusion, this study demonstrates the dual utilization of carbohydrates and amino acids under in vitro conditions and identifies the unconventional de novo biosynthesis of serine by pneumococci.