Effects of blocking toll-like receptor-2 (TLR-2) on the survival of Staphylococcus aureus (S. aureus) and cytokine production in peritoneal macrophages of Swiss albino mice were analysed. Macrophages were infected with S. aureus in the presence and absence of anti-TLR-2 antibody. Tumour necrosis factor-a (TNF-a) interleukin-6 (IL-6), interferon-gamma (IFN-c), interleukin-1b (IL-1b), interleukin-12 (IL-12) and interleukin-10 (IL-10) concentrations were measured. Expressions of TLR-2, NF-jB, MyD 88 were analysed by Western Blot. Expression of TLR-2 was increased in S. aureus-infected macrophages with respect to control and was MyD 88 independent. TLR2 blocking significantly reduced TNF-a, IL-6, IL-1b and IL-10 and increased IFN-c and IL-12 production. Decreased catalase activity and increased superoxide dismutase (SOD) by S. aureus with concomitant increase in H 2 O 2 and nitric oxide (NO) were observed in the case of prior TLR-2 blocking. To understand whether catalase contributing in the intracellular survival, was of bacterial origin or not, 3-amino, 1, 2, 4-triazole (ATZ) was used to inhibit specifically macrophage-derived catalase. Catalase enzyme activity from the whole staphylococcal cells in the presence of ATZ suggested that the released catalase were of extracellular origin. From the intracellular survival assay, it was evident that pretreatment of macrophages with ATZ reduces the bacterial burden in macrophages when infected with the recovered bacteria only from the anti-TLR-2 antibody-treated macrophages after phagocytosis. Catalase protein expression from the whole staphylococcal cells recovered after phagocytosis also indicated the catalase release from S. aureus. Capturing of S. aureus via TLR-2 induces inflammatory reactions through activation of NF-jB-signalling pathways which was MyD88-independent.

In this study, our objective was to determine whether a synergistic antimicrobial combination in vitro would be beneficial in the downregulation of pneumococcal virulence genes and whether the associated inflammation of the lung tissue induced by multidrug-resistant Streptococcus pneumoniae infection in vivo needs to be elucidated in order to consider this mode of therapy in case of severe pneumococcal infection. We investigated in vivo changes in the expression of these virulence determinants using an efficacious combination determined in previous studies. BALB/c mice were infected with 10 6 CFU of bacteria. Intravenous levofloxacin at 150 mg/kg and/or ceftriaxone at 50 mg/kg were initiated 18 h postinfection; the animals were sacrificed 0 to 24 h after the initiation of treatment. The levels of cytokines, chemokines, and C-reactive protein (CRP) in the serum and lungs, along with the levels of myeloperoxidase and nitric oxide the inflammatory cell count in bronchoalveolar lavage fluid (BALF), changes in pneumolysin and autolysin gene expression and COX-2 and inducible nitric oxide synthase (iNOS) protein expression in the lungs were estimated. Combination therapy downregulated inflammation and promoted bacterial clearance. Pneumolysin and autolysin expression was downregulated, with a concomitant decrease in the expression of COX-2 and iNOS in lung tissue. Thus, the combination of levofloxacin and ceftriaxone can be considered for therapeutic use even in cases of pneumonia caused by drug-resistant isolates.

ObjectivesEmergence of multidrug resistance among Streptococcus pneumoniae (SP), has limited the available options used to treat infections caused by this organism. The objective of this study was to compare the role of monotherapy and combination therapy with ampicillin (AMP) and azithromycin (AZM) in eradicating bacterial burden and down regulating lung inflammation in a murine experimental pneumococcal infection model.MethodsBalb/C mice were infected with 106 CFU of SP. Treatments with intravenous ampicillin (200 mg/kg) and azithromycin (50 mg/kg) either alone or in combination was initiated 18 h post infection, animals were sacrificed from 0 – 6 h after initiation of treatment. AMP and AZM were quantified in serum by microbiological assay. Levels of TNF-α, IFN-γ IL-6, and IL-10 in serum and in lungs, along with myeloperoxidase, inflammatory cell count in broncho alveolar lavage fluid, COX-2 and histopathological changes in lungs were estimated.ResultsCombination therapy down regulated lung inflammation and accelerated bacterial clearance. This approach also significantly decreased TNF-α, IFN-γ, IL-6 and increased IL-10 level in serum and lungs along with decreased myeloperoxidase, pulmonary vascular permeability, inflammatory cell numbers and COX-2 levels in lungs.ConclusionsCombinatorial therapy resulted in comparable bactericidal activity against the multi-drug resistant isolate and may represent an alternative dosing strategy, which may help to alleviate problems with pneumococcal pneumonia.

We hypothesized that if internalization of Staphylococcus aureus could be blocked by using cytochalasin D (an inhibitor of phagocytosis and phagolysosome fusion), then the intracellular entry and survival of the pathogen in host's phagocytic cells recruited to the inflammatory site can be restricted. At the same time, if we use antimicrobial agents (e.g., ciprofloxacin and azithromycin) having potent intracellular and extracellular microbicidal activity against the bacterium that have not entered into the phagosome and remains adhered to the phagocytic cell membrane, then they can be eradicated from the site of infection without compromising the host cell. To validate this, role of ciprofloxacin (CIP) and azithromycin (AZM) in eliminating S. aureus by suppressing the phagocytic activity of macrophages with cytochalasin D before infection was investigated. CIP and AZM were used either alone or in combination with cytochalasin D. Supernatant and lysate obtained from the culture of macrophages were used for quantification of reactive oxygen species, lysozymes, antioxidant enzymes, and cytokines produced. Azithromycin was better than ciprofloxacin in combination with cytochalasin D for eradicating S. aureus and regulating cytokine release. Further studies are required for ensuring proper delivery of this combination at the site of infection.

Interaction with the live Staphylococcus aureus promotes secretion of interleukin-8 (IL-8), although the expressions of functional CXCR1 (IL-8RA) in murine macrophages have not been identified. Expression of CXCR1 was induced in S. aureus-infected macrophages, whereas, CXCR1 was undetectable in control macrophages. CXCR1 blocking significantly reduced the phagocytosis of S. aureus and TNF-α, IL-6, IL-1β, IFN-γ, IL-12, and IL-8 production and increased release of MIP-2 and soluble TNF-R1. Increased bacterial catalase and decreased superoxide dismutase (SOD) activities by S. aureus with concomitant decrease in hydrogen peroxide (H2O2), superoxide anion, and nitric oxide (NO) release were observed in case of prior CXCR1 blocking. In the presence of cytochalasin D, S. aureus-mediated induction of IL-8 was inhibited concomitant with decreased bacterial count suggesting that internalization of S. aureus was necessary for induction of IL-8. Shedding of TNF-R1 due to CXCR1 blocking after S. aureus inoculation was critical for neutralization of TNF-α signaling and arrests the inflammation.