Background Antibiotic resistances of pathogens and breast cancer warrant the search for new alternative strategies. Phytoextracts can eradicate microbe-borne diseases as well as cancer with lower side effects compared to conventional antibiotics. Aim Unripe and ripe Azadirachta indica (neem) seed extracts were explored as potential antibiofilm and anticancer agents in combating multidrug-resistant infectious bacteria as well as anticancer agents against the MDR breast cancer cell lines. Methods Shed-dried neem seeds (both unripe and ripe) were pulverized and extracted using methanol. The chemical components were identified with FTIR and gas chromatography - mass spectrometry. Antibiofilm activity of neem seed extracts were assessed in terms of minimum biofilm inhibitory concentration (MBIC), minimum biofilm eradication concentration (MBEC), and fluorescence microscopic studies on Staphylococcus aureus and Vibrio cholerae. Bacterial cells were studied by fluorescence microscopy using acridine orange/ethidium bromide as the staining agents. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were evaluated to observe the antibacterial activities. Cytotoxicity of the extracts against human blood lymphocytes and the anticancer activity against drug-resistant breast cancer cell lines were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and fluorescence-activated cell sorting (FACS) studies. Results 4-Ethyl-2-hydroxy-2-cyclopentene-1-one, phthalic acid, and 2-hexyl-tetrahydro thiophane were the major compounds in unripe neem seed, whereas 3,5-dihydroxy-6-methyl-2,3-dihydro-4-H-pyran-4-one and 4-ethylbenzamide were predominant in ripe neem seed. Triazine derivatives were also common for both the extracts. MBIC values of unripe and ripe neem seed extracts for S. aureus are 75 and 100 µg/mL, respectively, and for V. cholerae, they are 100 and 300 µg/mL, respectively. MBEC values of unripe and ripe seed extracts are 500 and 300 µg/mL, respectively for S. aureus and for V. cholerae the values are 700 and 500 µg/mL, respectively. Fluorescence microscopic studies at 16 and 24 h, after bacterial culture, demonstrate enhanced antibiofilm activity for the ripe seed extract than that of the unripe seeds for both the bacteria. MTT assay reveals lower cytotoxicity of both the extracts towards normal blood lymphocytes, and anticancer activity against breast cancer cell line (MDA-MB-231) with superior activity of ripe seed extract. FACS studies further supported higher anticancer activity for ripe seed extract. Conclusions Methanolic extract of neem seeds could substantially inhibit and eradicate biofilm along with their potent antibacterial and anticancer activities. Both the extracts showed higher antibiofilm and antibacterial activity against S. aureus (gram-positive) than V. cholerae (gram-negative). Moreover, ripe seed extract showed higher antibiofilm and anticancer activity than unripe extracts. Graphical Abstract

Vibrio cholerae, the Gram-negative bacterium causing lethal diarrheal disease cholera, forms biofilm on solid surfaces to gain adaptive advantage for successful survival in aquatic reservoirs. Expression of exopolysaccharide (EPS), an extracellular matrix material, has been found critical for biofilm-based environmental persistence. In a subset of epidemic-causing V. cholerae, absence of flagellum but not motility was identified to induce elevated exopolysaccharide expression. Identification of the role played by quorum sensing autoinducer molecules, i.e., cholera autoinducer 1 (CAI-1) and autoinducer 2 (AI-2) as well as central regulator LuxO on EPS expression in the subset was explored. Deletion mutations were introduced in vital genes responsible for synthesizing CAI-1 (cqsA), AI-2 (luxS), flagellum (flaA), LuxO (luxO), flagellar motor (motX), and VpsR (vpsR) in the model strain MO10. Subsequent phenotypic alterations in terms of colony morphology, EPS expression, biofilm formation, and transcription level of relevant genes were analyzed. Autoinducer cross-feeding experiment confirmed the role of autoinducers in EPS signaling. Results reveal that autoinducers and flagellum are the two major EPS signaling units in this subset where one unit becomes predominant for EPS production in absence of the other. Moreover, either unit exerts negative influence on EPS induction by the other. Both the EPS signaling cascades are independent of LuxO contribution and essentially involve sodium-driven flagellar motor and VpsR. A cell density and flagellum-mediated, but LuxO-independent, EPS signaling mechanism is considered to be functional in these organisms that confers their survival fitness.

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Polycyclic aromatic hydrocarbons (PAHs) belong to a large group of organic pollutant which considers as a potential health hazard to living beings. Herein, naphthalene biodegradation potential by free and immobilized Pseudomonas putida strain KD10 and Pseudomonas sp. consortium were studied. Additionally, naphthalene 1, 2-dioxygenase (nahAc) was sequenced and analyzed, which reveals two altered amino acid residues. However, the altered amino acid residues are not present in the vicinity of the active site. The gas-phase binding free energy (ΔGLondon) of the mutant variant of naphthalene 1, 2-dioxygenase was -7.10 kcal mol-1 which closely resembles the wild type variant. Naphthalene biodegradation rate by Pseudomonas putida strain KD10 was 79.12 mg L-1 day-1 and it was significantly elevated up to 123 mg L-1 day-1 by the immobilized Pseudomonas sp. consortium. The half-life (t1/2) for naphthalene biodegradation was 3.1 days with the inhibition constant (ki), substrate saturation constant (ks) and maximum specific degradation rate constant (qmax) of 1268 mg L-1, 395.5 mg L-1 and 0.65 h-1, respectively, for the Pseudomonas putida strain KD10. However, the t1/2 value was significantly reduced to 2 days along with ki, ks and qmaxvalues of 1475 mg L-1, 298.8 mg L-1 and 0.71 h-1, respectively, by the immobilized Pseudomonas sp. consortium. The GC-MS data suggest that KD10 might follow D-gluconic acid mediated meta-cleavage pathway of catechol biodegradation. It is concluded that naphthalene biodegradation performance by immobilized Pseudomonas sp. consortium was superior to free or immobilized Pseudomonas putida KD10. Microbial consortium immobilization could be a useful tool for water quality management and environmental remediation.HighlightsSuperior naphthalene biodegradation by Pseudomonas sp. consortium immobilized in calcium alginate beads.A common mutation prone amino acid stretch inside chain A of naphthalene 1, 2-dioxygenase has been identified.A new naphthalene biodegradation pathway by Pseudomonas putida strain KD10 has been proposed.

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