Hypoxia-Mediated Impairment of the Mitochondrial Respiratory Chain Inhibits the Bactericidal Activity of Macrophages

In infected tissues oxygen tensions are low. As innate immune cells have to operate under these conditions, we analyzed the ability of macrophages (M phi) to kill Escherichia coli or Staphylococcus aureus in a hypoxic microenvironment. Oxygen restriction did not promote intracellular bacterial growth but did impair the bactericidal activity of the host cells against both pathogens. This correlated with a decreased production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates. Experiments with phagocyte NADPH oxidase (PHOX) and inducible NO synthase (NOS2) double-deficient M phi revealed that in E. coli- or S. aureus-infected cells the reduced antibacterial activity during hypoxia was either entirely or partially independent of the diminished PHOX and NOS2 activity. Hypoxia impaired the mitochondrial activity of infected M phi. Inhibition of the mitochondrial respiratory chain activity during normoxia (using rotenone or antimycin A) completely or partially mimicked the defective antibacterial activity observed in hypoxic E. coli-or S. aureus-infected wild-type M phi, respectively. Accordingly, inhibition of the respiratory chain of S. aureus-infected, normoxic PHOX-/- NOS2(-/-) M phi further raised the bacterial burden of the cells, which reached the level measured in hypoxic PHOX-/- NOS2(-/-) M phi cultures. Our data demonstrate that the reduced killing of S. aureus or E. coli during hypoxia is not simply due to a lack of PHOX and NOS2 activity but partially or completely results from an impaired mitochondrial antibacterial effector function. Since pharmacological inhibition of the respiratory chain raised the generation of ROI but nevertheless phenocopied the effect of hypoxia, ROI can be excluded as the mechanism underlying the antimicrobial activity of mitochondria.

Moderate intensity static magnetic field has bactericidal effect on E. coli and S. epidermidis on sintered hydroxyapatite

The application of electromagnetic field in the context of bacteria associated infections on biomaterial surfaces has not been extensively explored. In this work, we applied a moderate intensity static magnetic field (100 mT) to understand the adhesion and growth behavior of both gram positive (S. epidermidis) and gram negative bacteria (E. coli) and also to investigate bactericidal/bacteriostatic property of the applied electromagnetic field. An in-house built magnetometer was used to apply static homogeneous magnetic field during a planned set of in vitro experiments. Both the sintered hydroxyapatite (HA) and the control samples seeded with bacteria were exposed to the magnetic field (100 mT) for different timescale during their log phase growth. Quantitative analysis of the SEM images confirms the effect of electromagnetic field on suppressing bacterial growth. Furthermore, cell integrity and inner membrane permeabilization assays were performed to understand the origin of such effect. The results of these assays were statistically analyzed to reveal the bactericidal effect of magnetic field, indicating cell membrane damage. Under the investigated culture conditions, the bactericidal effect was found to be less effective for S. Epidermidis than E. coli. (c) 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 2012:100B:12061217, 2012.

Bactericidal/permeability increasing protein: A multifaceted protein with functions beyond LPS neutralization

Bactericidal permeability increasing protein (BPI), a 55-60kDa protein, first reported in 1975, has gone a long way as a protein with multifunctional roles. Its classical role in neutralizing endotoxin (LPS) raised high hopes among septic shock patients. Today, BPI is not just a LPS-neutralizing protein, but a protein with diverse functions. These functions can be as varied as inhibition of endothelial cell growth and inhibition of dendritic cell maturation, or as an anti-angiogenic, chemoattractant or opsonization agent. Though the literature available is extremely limited, it is fascinating to look into how BPI is gaining major importance as a signalling molecule. In this review, we briefly summarize the recent research focused on the multiple roles of BPI and its use as a therapeutic.

Bactericidal effect of silver-reinforced carbon nanotube and hydroxyapatite composites

Bacterial infection remains an important risk factor after orthopedic surgery. The present paper reports the synthesis of hydroxyapatite-silver (HA-Ag) and carbon nanotube-silver (CNT-Ag) composites via spark plasma sintering (SPS) route. The retention of the initial phases after SPS was confirmed by phase analysis using X-ray diffraction and Raman spectroscopy. Energy dispersive spectrum analysis showed that Ag was distributed uniformly in the CNT/HA matrix. The breakage of CNTs into spheroid particles at higher temperatures (1700 degrees C) is attributed to the Rayleigh instability criterion. Mechanical properties (hardness and elastic modulus) of the samples were evaluated using nanoindentation testing. Ag reinforcement resulted in the enhancement of hardness (by similar to 15%) and elastic modulus (similar to 5%) of HA samples, whereas Ag reinforcement in CNT, Ag addition does not have much effect on hardness (0.3 GPa) and elastic modulus (5 GPa). The antibacterial tests performed using Escherichia coli and Staphylococcus epidermidis showed significant decrease (by similar to 65-86%) in the number of adhered bacteria in HA/CNT composites reinforced with 5% Ag nanoparticles. Thus, Ag-reinforced HA/CNT can serve as potential antibacterial biocomposites.

Amine-functionalized multiwall carbon nanotubes impart osteoinductive and bactericidal properties in poly(epsilon-caprolactone) composites

Poly(epsilon-caprolactone) (PCL) is an aliphatic polyester widely used for biomedical applications but lacks the mechanical properties desired for many load-bearing orthopedic applications. The objective of this study was to prepare and characterize PCL composites incorporating multiwall carbon nanotubes (MWNTs) with different surface functional groups. PCL composites were prepared by melt-mixing with three different types of MWNTs: pristine (pMWNT), amine functionalized (aMWNT), and carboxyl functionalized (cMWNT). Melt rheology and scanning electron microscopy indicated good dispersion of the nanotubes in the matrix. Tensile strength and elastic modulus of the polymer was significantly increased by the incorporation of MWNTs and further enhanced by favorable interactions between PCL and aMWNTs. Thermal analysis revealed that MWNTs act as heterogeneous nucleation sites for crystallization of PCL and increase polymer crystallinity. Incorporation of functionalized MWNTs increased the surface water wettability of PCL. Osteoblast proliferation and differentiation was significantly enhanced on functionalized composites. aMWNT composites also exhibited the best bactericidal response. This study demonstrates that surface functionalization of MWNTs profoundly influences the properties of PCL and amine-functionalization offers the optimal combination of mechanical properties, osteogenesis and antimicrobial response. These results have important implications for designing nanocomposites for use in orthopedics.

Hot pressed silver doped hydroxyapatite biomaterials with bactericidal properties against magnetotactic bacteria

Hydroxyapatite (HA) is widely being researched for hard tissue replacement for its good osseointegration and biocompatibility property. However, the inferior antibacterial property of HA often results in infection at host site, and this leads to rejection of the implant. The antibacterial property of silver (Ag) is well known and in the past decade or so, the application of Ag is reinvented in medicinal applications like catheters, vascular grafts and orthopaedic implants. In this respect, the present work reports the synthesis of Ag doped HA using hot pressing in argon atmosphere. This work also reports the effect of HA-Ag composition on bacterial colonisation during in vitro study. The bactericidal property of Ag doped HA has been investigated against magnetotactic bacteria, a `magnetite' containing bacteria. Magnetotactic bacteria were seeded onto pellets, and the adhesion of bacteria was evaluated using scanning electron microscopy. It was confirmed that incorporation of Ag in HA leads to improved bactericidal property.

First-in-Class Inhibitors of Sulfur Metabolism with Bactericidal Activity against Non-Replicating M-tuberculosis

Development of effective therapies to eradicate persistent, slowly replicating M. tuberculosis (Mtb) represents a significant challenge to controlling the global TB epidemic. To develop such therapies, it is imperative to translate information from metabolome and proteome adaptations of persistent Mtb into the drug discovery screening platforms. To this end, reductive sulfur metabolism is genetically and pharmacologically implicated in survival, pathogenesis, and redox homeostasis of persistent Mtb. Therefore, inhibitors of this pathway are expected to serve as powerful tools in its preclinical and clinical validation as a therapeutic target for eradicating persisters. Here, we establish a first functional HTS platform for identification of APS reductase (APSR) inhibitors, a critical enzyme in the assimilation of sulfate for the biosynthesis of cysteine and other essential sulfur-containing molecules. Our HTS campaign involving 38?350 compounds led to the discovery of three distinct structural classes of APSR inhibitors. A class of bioactive compounds with known pharmacology displayed potent bactericidal activity in wild-type Mtb as well as MDR and XDR clinical isolates. Top compounds showed markedly diminished potency in a conditional Delta APSR mutant, which could be restored by complementation with Mtb APSR. Furthermore, ITC studies on representative compounds provided evidence for direct engagement of the APSR target. Finally, potent APSR inhibitors significantly decreased the cellular levels of key reduced sulfur-containing metabolites and also induced an oxidative shift in mycothiol redox potential of live Mtb, thus providing functional validation of our screening data. In summary, we have identified first-in-class inhibitors of APSR that can serve as molecular probes in unraveling the links between Mtb persistence, antibiotic tolerance, and sulfate assimilation, in addition to their potential therapeutic value.

A bactericidal homodimeric phospholipases A(2) from Bungarus fasciatus venom

Group IIA secretory phospholipases A(2) (sPLA(2)-II) is generally known to display potent grampositive bactericidal activity, while group IA sPLA(2) (sPLA(2)-I) reportedly is not. In this work, a novel sPLA(2)-I named BFPA was identified from Bungarus fas

The bactericidal effect of dendritic copper microparticles, contained in an alginate matrix, on Escherichia coli

Although the bactericidal effect of copper has been known for centuries, there is a current resurgence of interest in the use of this element as an antimicrobial agent. During this study the use of dendritic copper microparticles embedded in an alginate matrix as a rapid method for the deactivation of Escherichia coli ATCC 11775 was investigated. The copper/alginate produced a decrease in the minimum inhibitory concentration from free copper powder dispersed in the media from 0.25 to 0.065 mg/ml. Beads loaded with 4% Cu deactivated 99.97% of bacteria after 90 minutes, compared to a 44.2% reduction in viability in the equivalent free copper powder treatment. There was no observed loss in the efficacy of this method with increasing bacterial loading up to 10(6) cells/ml, however only 88.2% of E. coli were deactivated after 90 minutes at a loading of 10(8) cells/ml. The efficacy of this method was highly dependent on the oxygen content of the media, with a 4.01% increase in viable bacteria observed under anoxic conditions compared to a >99% reduction in bacterial viability in oxygen tensions above 50% of saturation. Scanning electron micrographs (SEM) of the beads indicated that the dendritic copper particles sit as discrete clusters within a layered alginate matrix, and that the external surface of the beads has a scale-like appearance with dendritic copper particles extruding. E. coli cells visualised using SEM indicated a loss of cellular integrity upon Cu bead treatment with obvious visible blebbing. This study indicates the use of microscale dendritic particles of Cu embedded in an alginate matrix to effectively deactivate E. coli cells and opens the possibility of their application within effective water treatment processes, especially in high particulate waste streams where conventional methods, such as UV treatment or chlorination, are ineffective or inappropriate.

Bactericidal/permeability-increasing protein attenuates systemic inflammation and acute lung injury in porcine lower limb ischemia-reperfusion injury.

OBJECTIVE: To investigate the role of recombinant bactericidal/permeability-increasing protein (rBPI21) in the attenuation of the sepsis syndrome and acute lung injury associated with lower limb ischemia-reperfusion (I/R) injury. SUMMARY BACKGROUND DATA: Gut-derived endotoxin has been implicated in the conversion of the sterile inflammatory response to a lethal sepsis syndrome after lower torso I/R injury. rBPI21 is a novel antiendotoxin therapy with proven benefit in sepsis. METHODS: Anesthetized ventilated swine underwent midline laparotomy and bilateral external iliac artery occlusion for 2 hours followed by 2.5 hours of reperfusion. Two groups (n = 6 per group) were randomized to receive, by intravenous infusion over 30 minutes, at the start of reperfusion, either thaumatin, a control-protein preparation, at 2 mg/kg body weight, or rBPI21 at 2 mg/kg body weight. A control group (n = 6) underwent laparotomy without further treatment and was administered thaumatin at 2 mg/kg body weight after 2 hours of anesthesia. Blood from a carotid artery cannula was taken every half-hour for arterial blood gas analysis. Plasma was separated and stored at -70 degrees C for later determination of plasma tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 by bioassay, and IL-8 by enzyme-linked immunosorbent assay (ELISA), as a markers of systemic inflammation. Plasma endotoxin concentration was measured using ELISA. Lung tissue wet-to-dry weight ratio and myeloperoxidase concentration were used as markers of edema and neutrophil sequestration, respectively. Bronchoalveolar lavage protein concentration was measured by the bicinclinoic acid method as a measure of capillary-alveolar protein leak. The alveolar-arterial gradient was measured; a large gradient indicated impaired oxygen transport and hence lung injury. RESULTS: Bilateral hind limb I/R injury increased significantly intestinal mucosal acidosis, intestinal permeability, portal endotoxemia, plasma IL-6 concentrations, circulating phagocytic cell priming and pulmonary leukosequestration, edema, capillary-alveolar protein leak, and impaired gas exchange. Conversely, pigs treated with rBPI21 2 mg/kg at the onset of reperfusion had significantly reduced intestinal mucosal acidosis, portal endotoxin concentrations, and circulating phagocytic cell priming and had significantly less pulmonary edema, leukosequestration, and respiratory failure. CONCLUSIONS: Endotoxin transmigration across a hyperpermeable gut barrier, phagocytic cell priming, and cytokinemia are key events of I/R injury, sepsis, and pulmonary dysfunction. This study shows that rBPI21 ameliorates these adverse effects and may provide a novel therapeutic approach for prevention of I/R-associated sepsis syndrome.