Antibacterial synergy between rutin and florfenicol enhances therapeutic spectrum against drug resistant Aeromonas hydrophila.

Emergence of antibiotic resistant bacteria has necessitated the drive to explore competent antimicrobial agents or to develop novel formulations to treat infections including Aeromonas hydrophila. The present study investigates the synergistic antibacterial effects of citrus flavonoid rutin and florfenicol (FF) against A. hydrophila in vitro and in vivo. Rutin is extracted and purified from Citrus sinensis peel through preparative HPLC and characterized through TLC, GC-MS and 1H and 13C NMR analyses. Though rutin did not display significant antibacterial activity, it modulated FF activity resulting in four-fold reduction in the MIC value for FF. The anti-biofilm potential of synergistic association of rutin and FF was validated by protein analysis, quantification of exopolysaccharide (EPS) and microscopy studies using sub-MIC doses. Besides antibacterial action, in vivo studies showed that Rutin/FF combination enhanced host immunity by improving blood cell count, anti-protease, and lysozyme activities as well as decreased the oxidative stress and the pathological changes of tilapia Oreochromis niloticus against A. hydrophila infection. No significant DNA damages or clastogenic effects were detected in tilapia challenged with A. hydrophila under Rutin/FF treatment. It is shown that an acute-phase Lipopolysaccharide binding protein (LBP) enhances the innate host defence against bacterial challenge. Semi quantitative RT-PCR and western blot results revealed the significant increase of LBP in the supernatant of tilapia monocytes/macrophages challenged with A. hydrophila upon treatment. The study findings substantiate that the combination of natural molecules with antibiotics may open up possibilities to treat MDR strains.

Approaches for the simultaneous detection of thiamphenicol, florfenicol and florfenicol amine using immunochemical techniques.

Thiamphenicol and florfenicol are antibacterial agents permitted for use as veterinary drugs in animals used for food production. However, as the EU has established maximum residue limits for both and the metabolite florfenicol amine, there is a requirement to monitor animal food products for their residues. In this study antisera were generated which can simultaneously detect thiamphenicol, florfenicol and florfenicol amine in an immunoassay. Details of the various coupling techniques employed to prepare immunogens and enzyme labels are provided and the antibodies produced have been assessed, in homologous and heterologous ELISA formats, with respect to sensitivity and specificity. It was found that while the antisera raised to thiamphenicol and florfenicol generally performed better in a heterologous set up, those raised to florfenicol amine were not only less affected by the assay format but also produced the most sensitive antibodies to all three target analytes. Antisera matched previous sensitivity (IC50 < 1 ng mL⁻¹) but had improved cross-reactivity (>100%) to thiamphenicol and florfenicol.

Simultaneous determination of thiamphenicol, florfenicol and florfenicol amine in swine muscle by liquid chromatography-tandem mass spectrometry with immunoaffinity chromatography clean-up.

A rapid and sensitive liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method to quantify thiamphenicol (TAP), florfenicol (FF), and florfenicol amine (FFA) in swine muscle is described. An immunoaffinity chromatography (IAC) column based on polyclonal antibodies and protein A-sepharose CL 4B was used to clean-up extracted samples. IAC optimized conditions were found that allowed the IAC to be reused for selective binding of TAP, FF, and FFA. The dynamic column capacity was more than 512ng/mL of gel after being used for 15 cycles. From fortified swine muscle samples at levels of 0.4-50ng/g, the average recoveries were 85.2-98.9% with intra- and inter-day variations less than 9.8% and 12.4%, respectively. The limit of quantitation ranged from 0.4 to 4.0microg/kg.

Molecular features determining lymphocyte reactivity in allergic contact dermatitis to chloramphenicol and azidamphenicol.

BACKGROUND: We report on two cases of allergic contact dermatitis to chloramphenicol and azidamphenicol respectively, with in vivo and in vitro lymphocyte reactivity to both compounds. The molecular features determining lymphocyte reactivity were explored because chloramphenicol, azidamphenicol, and thiamphenicol exhibit almost identical chemical structures. METHODS: With chloramphenicol, azidamphenicol, and the chemically related thiamphenicol, we performed patch tests and lymphocyte transformation tests with both patients. Furthermore, the interleukin-5 and interferon-gamma concentrations in the cultures of peripheral blood mononuclear cells of one patient were determined. RESULTS: Patch tests showed delayed hypersensitivity reactions to chloramphenicol and azidamphenicol, but not to thiamphenicol. These results were confirmed by lymphocyte transformation tests with peripheral blood mononuclear cells of the patients, showing a proliferative T-cell response to azidamphenicol and chloramphenicol. Moreover, lymphocytes from one patient secreted large amounts of interleukin-5, but not of interferon-gamma upon coculture with azidamphenicol. CONCLUSIONS: Since lymphocyte reactivity was observed to chloramphenicol and azidamphenicol, but not to thiamphenicol, the epitope(s) recognized by the allergen-reactive T cells may be formed by the nitro-group of the benzene ring shared by chloramphenicol and azidamphenicol.