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Escherichia coli encounter variety of environmental and processing stresses during their growth, survival, and infection. Herein, the thermotolerance behavior and transcription of virulent genes responsible for the pathogenicity in isolated strains of pathogenic E. coli were evaluated. Among 176 E. coli isolates, 4 isolates (2.27%) were confirmed to be pathogenic E. coli, out of which 2 isolates were positive for EHEC and 2 were positive for EPEC based on their virulence factors. Thermotolerance was induced under thermal adaptation at higher temperature, regardless of the pathotypes. Cells grown and adapted at 42 °C, exhibited highest transcription of genes associated with adhesion (eae), hemolysis (hlyA), and shiga toxin production (stx1). However, expression of these genes was downregulated in cells adapted at lower temperature of 4 °C and 25 °C compared to control. Further, transcription of stx2 was upregulated by 70% and 17% at 4 °C and 25 °C, respectively, while the transcription level was reduced by 44% relative to control at 42 °C. The findings indicate that expression of virulent genes in pathogenic E. coli at elevated temperature do not be depend on thermotolerance of the strain harboring these genes.

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In an era of antibiotic resistance where natural antibiotic substitutes are considered essential, the antimicrobial and antibiofilm activities of Citrus limon extract on strains of pathogenic Escherichia coli isolated from pork were evaluated. The strains which form biofilms were more resistant (MIC50 = 2.5 mgml-1) compared to non-biofilm forming strains (MIC50 = 1.25 mgml-1). Use of C. limon extract at 20 mgml-1 concentration has resulted in inhibition of biofilm formation by 53.96%. Cyclobarbital, 5, 8-dimethoxycumarin, orotic acid and 3-methylsalicylhydrazide were the major phytochemicals in C. limon extract with highest docking affinities against the biofilm associated proteins in E. coli. The results of simulation studies have clearly illustrated the energy stability of the protein-ligand complexes. Absorption, distribution, metabolism, excretion and toxicity (ADMET) profiles revealed that the phytochemicals in C. limon could be used in the drug design studies to preferentially target the specific receptors to combat biofilms associated with E. coli.

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OBJECTIVES: Previously, a series of side chain-modified quinolinyl ß-enaminones was identified to possess significant activity against chloroquine-sensitive or -resistant Plasmodium falciparum and Brugia malayi microfilariae. The present study evaluates in vitro and in vivo activity of the series against Leishmania donovani and reports their mode of action. METHODS: The in vitro activity of 15 quinolinyl ß-enaminone derivatives against Leishmania promastigotes and amastigotes was assessed by luciferase assay. The reduction of organ parasite burden was assessed by Giemsa staining in L. donovani-infected BALB/c mice and hamsters. Intracellular Ca2+ and ATP level in active derivative (3D)-treated promastigotes were determined by fluorescence and luminescence assays. Flow cytometry was performed to determine loss of mitochondrial membrane potential (MMP) using JC-1 dye, reactive oxygen species (ROS) generation using 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) dye, phosphatidylserine externalization by Annexin V-FITC staining and cell-cycle arrest by propidium iodide (PI) staining. RESULTS: Compounds 3A, 3B and 3D showed significant in vitro efficacy against L. donovani with IC50 < 6 µM and mild cytotoxicity (∼75% viability) at 25 µM on J774 macrophages. 3A and 3D at 50 mg/kg and 100 mg/kg reduced parasite burden (>84%) in infected mice and hamsters, respectively, whereas 3D-treated animals demonstrated maximum parasite burden reduction without organ toxicity. Mode-of-action analysis revealed that 3D induced apoptosis by inhibiting mitochondrial complex II, reducing MMP and ATP levels, increasing ROS and Ca2+ levels, ultimately triggering phosphatidylserine externalization and sub-G0/G1 cell-cycle arrest in promastigotes. CONCLUSIONS: Compound 3D-mediated inhibition of L. donovani mitochondrial complex induces apoptosis, making it a promising therapeutic candidate for visceral leishmaniasis.

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An environmentally benign, simple, efficient, and convenient route is described for the synthesis of novel pyrazolo[1,5-a]pyrimidine derivatives under ultrasound irradiation. Condensation of aminopyrazole 5 with formylated active proton compounds (6, 8, E-G, 12, and 15) furnished pyrazolopyrimidine (7, 9, 10, 13, and 16) in high-to-excellent yields. In comparison with conventional methods, ultrasound irradiation offers several advantages, such as shorter reaction time, higher yields, milder conditions, and environmental friendliness. The reaction is clean with excellent yields and reduces the use of solvents. X-ray crystallographic study of compound 7c confirmed the regioselectivity of the reaction. The antibacterial profile of the newly synthesized compounds was evaluated by cup and saucer method.

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