Absolute dependence on mecA gene as the defining standard in determining the resistance of S. aureus to methicillin became the subject of distrust by many researchers. The present study aimed to determine the frequency of mecA gene in methicillin resistant S. aureus (MRSA) isolates using polymerase chain reaction and to correlate its presence to conventional method. In this regard, two hundred S. aureus isolates were collected from patients with different diseases attending different hospitals in Shandi City, Sudan. Phenotypic Kirby-Bauer method confirmed the existence of methicillin resistant S. aureus in 61.5% of the subjected isolates with MICs ranging from 4 μg/mL to 256 μg/mL when using E-test. However, when amplifying a 310 bp fragment of the mecA gene by PCR, twelve out of the 123 MRSA isolates (9.8%) were mecA negative, whereas all the 77 methicillin sensitive S. aureus (MSSA) were mecA negative. In conclusion, this study drew attention to the credibility of the mecA gene and its usefulness in the detection of all MRSA strains without referring to the traditional methods. Hence, it is highly recommended to consider alternative mechanisms for β-lactam resistance that may compete with mecA gene in the emergence of MRSA phenomenon in the community.
BackgroundThe therapy of visceral leishmaniasis (VL) is limited by resistance, toxicity and decreased bioavailability of the existing drugs coupled with dramatic increase in HIV-co-infection, non-availability of vaccines and down regulation of cell-mediated immunity (CMI). Thus, we envisaged combating the problem with plant-derived antileishmanial drug that could concomitantly mitigate the immune suppression of the infected hosts. Several plant-derived compounds have been found to exert leishmanicidal activity via immunomodulation. In this direction, we investigated the antileishmanial activity of eugenol emulsion (EE), complemented with its immunomodulatory and therapeutic efficacy in murine model of VL.Methodology/Principal FindingsOil-in-water emulsion of eugenol (EE) was prepared and size measured by dynamic light scattering (DLS). EE exhibited significant leishmanicidal activity with 50% inhibitory concentration of 8.43±0.96 μg ml-1 and 5.05±1.72 μg ml─1, respectively against the promastigotes and intracellular amastigotes of Leishmania donovani. For in vivo effectiveness, EE was administered intraperitoneally (25, 50 and 75 mg/kg b.w./day for 10 days) to 8 week-infected BALB/c mice. The cytotoxicity of EE was assessed in RAW 264.7 macrophages as well as in naive mice. EE induced a significant drop in hepatic and splenic parasite burdens as well as diminution in spleen and liver weights 10 days post-treatment, with augmentation of 24h-delayed type hypersensitivity (DTH) response and high IgG2a:IgG1, mirroring induction of CMI. Enhanced IFN-γ and IL-2 levels, with fall in disease-associated Th2 cytokines (IL-4 and IL-10) detected by flow cytometric bead-based array, substantiated the Th1 immune signature. Lymphoproliferation and nitric oxide release were significantly elevated upon antigen revoke in vitro. The immune-stimulatory activity of EE was further corroborated by expansion of IFN-γ producing CD4+ and CD8+ splenic T lymphocytes and up-regulation of CD80 and CD86 on peritoneal macrophages. EE treated groups exhibited induction of CD8+ central memory T cells as evidenced from CD62L and CD44 expression. No biochemical alterations in hepatic and renal enzymes were observed.ConclusionsOur results demonstrate antileishmanial activity of EE, potentiated by Th1 immunostimulation without adverse side effects. The Th1 immune polarizing effect may help to alleviate the depressed CMI and hence complement the leishmanicidal activity.
BackgroundExploration of immunomodulatory antileishmanials of plant origin is now being strongly recommended to overcome the immune suppression evident during visceral leishmaniasis (VL) and high cost and toxicity associated with conventional chemotherapeutics. In accordance, we assessed the in vitro and in vivo antileishmanial and immunomodulatory potential of ethanolic fractions of Azadirachta indica leaves (ALE) and seeds (ASE).MethodsA. indica fractions were prepared by sequential extraction of the powdered plant parts in hexane, ethanol and water. Erythrosin B staining was employed to appraise the anti-promastigote potential of ALE and ASE. Cytostatic or cytocidal mode of action was ascertained and alterations in parasite morphology were depicted under oil immersion light microscopy. Study of apoptotic correlates was performed to deduce the mechanism of induced cell death and anti-amastigote potential was assessed in Leishmania parasitized RAW 264.7 macrophages. In vivo antileishmanial effectiveness was evaluated in L. donovani infected BALB/c mice, accompanied by investigation of immunomodulatory potential of ALE and ASE. Adverse toxicity of the bioactive fractions against RAW macrophages was studied by MTT assay. In vivo side effects on the liver and kidney functions were also determined. Plant secondary metabolites present in ALE and ASE were analysed by Gas chromatography–mass spectrometry (GC-MS).ResultsALE and ASE (500 μg ml−1) exhibited leishmanicidal activity in a time- and dose-dependent manner (IC50 34 and 77.66 μg ml−1, respectively) with alterations in promastigote morphology and induction of apoptosis. ALE and ASE exerted appreciable anti-amastigote potency (IC50 17.66 and 24.66 μg ml−1, respectively) that was coupled with profound in vivo therapeutic efficacy (87.76% and 85.54% protection in liver and 85.55% and 83.62% in spleen, respectively). ALE exhibited minimal toxicity with selectivity index of 26.10 whereas ASE was observed to be non-toxic. The bioactive fractions revealed no hepato- and nephro-toxicity. ALE and ASE potentiated Th1-biased cell-mediated immunity along with upregulation of INF-γ, TNF-α and IL-2 and decline in IL-4 and IL-10 levels. GC-MS analysis revealed several compounds that may have contributed to the observed antileishmanial effect.ConclusionDual antileishmanial and immunostimulatory efficacy exhibited by the bioactive fractions merits their use alone or as adjunct therapy for VL.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0788-3) contains supplementary material, which is available to authorized users.
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