P. Balamurugan

Melatonin improves bioreductant capacity and silver nanoparticles synthesis using Catharanthus roseus leaves

Melatonin is a natural hormone found in a variety of living organisms, including plants, and possesses the potential to act as a growth promoter and stress alleviator. The present study aims in understanding the effect of melatonin in Catharanthus roseus (C. roseus; medicinal plant used in anticancer therapy) leaf extracts for enhanced silver nanoparticle synthesis. Our results indicated that the supplementation of 300 μM melatonin to C. roseus leaves improves silver nanoparticles (AgNPs) synthesis. The phytosynthesized AgNPs were characterized using UV-Vis spectroscopy, FE-TEM, Zetasizer, XRD, AAS and FTIR spectroscopy. Moreover, the AgNPs were found to be smaller in size (ranging from 10–25 nm) with potent antibacterial activity (MIC and MBC values against the most prevalent urinary tract pathogens, Escherichia coli and Staphylococcus aureus, being 15.6 ± 0.0 μg mL−1 and 31.25 ± 0.0 μg mL−1 respectively). Furthermore, LC-MS/MS analysis of the melatonin treated C. roseus leaf extracts indicated the presence of significant biomolecules; 6-acetyl morphine (used as an analgesic), rauwolscine and fisetin (anticancer compounds). Melatonin treatment of C. roseus leaves also enhanced its antioxidant potential and total chlorophyll content. Thus, melatonin serves as a potential agent to improve C. roseus growth and therapeutic potential.

Development of a biofilm inhibitor molecule against multidrug resistant Staphylococcus aureus associated with gestational urinary tract infections.

Urinary Tract Infection (UTI) is a globally widespread human infection caused by an infestation of uropathogens. Eventhough, Escherichia coli is often quoted as being the chief among them, Staphylococcus aureus involvement in UTI especially in gestational UTI is often understated. Staphylococcal accessory regulator A (SarA) is a quorum regulator of S. aureus that controls the expression of various virulence and biofilm phenotypes. Since SarA had been a focussed target for antibiofilm agent development, the study aims to develop a potential drug molecule targeting the SarA of S. aureus to combat biofilm associated infections in which it is involved. In our previous studies, we have reported the antibiofilm activity of SarA based biofilm inhibitor, (SarABI) with a 50% minimum biofilm inhibitory concentration (MBIC50) value of 200 μg/mL against S. aureus associated with vascular graft infections and also the antibiofilm activity of the root ethanolic extracts of Melia dubia against uropathogenic E. coli. In the present study, in silico design of a hybrid molecule composed of a molecule screened from M. dubia root ethanolic extracts and a modified SarA based inhibitor (SarABIM) was undertaken. SarABIM is a modified form of SarABI where the fluorine groups are absent in SarABIM. Chemical synthesis of the hybrid molecule, 4-(Benzylamino)cyclohexyl 2-hydroxycinnamate (henceforth referred to as UTI Quorum-Quencher, UTIQQ) was then performed, followed by in vitro and in vivo validation. The MBIC50 and MBIC90 of UTIQQ were found to be 15 and 65 μg/mL, respectively. Confocal laser scanning microscopy (CLSM) images witnessed biofilm reduction and bacterial killing in either UTIQQ or in combined use of antibiotic gentamicin and UTIQQ. Similar results were observed with in vivo studies of experimental UTI in rat model. So, we propose that the drug UTIQQ would be a promising candidate when used alone or, in combination with an antibiotic for staphylococcal associated UTI.

Combinatorial Effects of Aromatic 1,3-Disubstituted Ureas and Fluoride on In vitro Inhibition of Streptococcus mutans Biofilm Formation.

Dental caries occur as a result of disequilibrium between acid producing pathogenic bacteria and alkali generating commensal bacteria within a dental biofilm (dental plaque). Streptococcus mutans has been reported as a primary cariogenic pathogen associated with dental caries. Emergence of multidrug resistant as well as fluoride resistant strains of S. mutans due to over use of various antibiotics are a rising problem and prompted the researchers worldwide to search for alternative therapies. In this perspective, the present study was aimed to screen selective inhibitors against ComA, a bacteriocin associated ABC transporter, involved in the quorum sensing of S. mutans. In light of our present in silico findings, 1,3-disubstituted urea derivatives which had better affinity to ComA were chemically synthesized in the present study for in vitro evaluation of S. mutans biofilm inhibition. The results revealed that 1,3-disubstituted urea derivatives showed good biofilm inhibition. In addition, synthesized compounds exhibited potent synergy with a very low concentration of fluoride (31.25–62.5 ppm) in inhibiting the biofilm formation of S. mutans without affecting the bacterial growth. Further, the results were supported by confocal laser scanning microscopy. On the whole, from our experimental results we conclude that the combinatorial application of fluoride and disubstituted ureas has a potential synergistic effect which has a promising approach in combating multidrug resistant and fluoride resistant S. mutans in dental caries management.

Synergistic activity of quorum sensing inhibitor, pyrizine-2-carboxylic acid and antibiotics against multi-drug resistant V. cholerae

Antibiotic resistance is one of the most pivotal health menaces in the human population worldwide. Use of conventional antibiotics at high concentration for prolonged periods facilitates a selective pressure on the bacterial strain to develop resistance to these antibiotics. The ascent of multi-drug resistance in Vibrio cholerae strains is a key issue for alternative drug development against cholera. A drug that could curb the virulence of V. cholerae is an elective choice for treatment. A few research studies have been carried out to discover such alternative medication that has an anti-virulent property rather than antibacterial activity. Combinatorial antibiotic treatments along with anti-biofilm compounds have diverse effects on bacterial growth i.e. it can be additive, synergistic or antagonistic. In the present investigation, combinatorial action of 2,3-pyrazine dicarboxylic acid derivatives (QSIVc targeted against the quorum regulator, LuxO) along with antibiotics was evaluated. The results indicated that QSIVc could inhibit biofilm at a very low concentration (IC50 varying between 1 μM and 70 μM). The synergism of the compounds with the reported antibiotics (doxycycline, tetracycline, chloramphenicol and erythromycin) was evaluated by the checkerboard method. The fractional inhibitory concentration (FIC) values varied from 0.0 to 0.5 for each combination. The ΔE value (750–1900), according to the bliss independence model showed that the QSIVc synergistically enhanced the inhibitory effect of the reported antibiotics against the multidrug resistant clinical isolates of V. cholerae.

Antimicrobial and Antibiofilm Potential of Acyclic Amines and Diamines against Multi-Drug Resistant Staphylococcus aureus

Multi-drug resistant Staphylococcus aureus (MDRSA) remains a great challenge despite a decade of research on antimicrobial compounds against their infections. In the present study, various acyclic amines and diamines were chemically synthesized and tested for their antimicrobial as well as antibiofilm activity against MDRSA. Among all the synthesized compounds, an acyclic diamine, (2,2′-((butane-1,4-diylbis(azanediyl)bis(methylene))diphenol) designated as ADM 3, showed better antimicrobial activity (minimum inhibitory concentration at 50 μg/mL) and antibiofilm activity (MBIC50 at 5 μg/mL). In addition, ADM 3 was capable of reducing the virulence factors expression (anti-virulence). Confocal laser scanning microscope analysis of the in vitro tested urinary catheters showed biofilm reduction as well as bacterial killing by ADM 3. On the whole, our data suggest that acyclic diamines, especially ADM 3 can be a potent lead for the further studies in alternative therapeutic approaches.

Staphylococcus aureus Quorum Regulator SarA Targeted Compound, 2-[(Methylamino)methyl]phenol Inhibits Biofilm and Down-Regulates Virulence Genes

Staphylococcus aureus is a widely acknowledged Gram-positive pathogen for forming biofilm and virulence gene expressions by quorum sensing (QS), a cell to cell communication process. The quorum regulator SarA of S. aureus up-regulates the expression of many virulence factors including biofilm formation to mediate pathogenesis and evasion of the host immune system in the late phases of growth. Thus, inhibiting the production or blocking SarA protein might influence the down-regulation of biofilm and virulence factors. In this context, here we have synthesized 2-[(Methylamino)methyl]phenol, which was specifically targeted toward the quorum regulator SarA through in silico approach in our previous study. The molecule has been evaluated in vitro to validate its antibiofilm activity against clinical S. aureus strains. In addition, antivirulence properties of the inhibitor were confirmed with the observation of a significant reduction in the expression of representative virulence genes like fnbA, hla and hld that are governed under S. aureus QS. Interestingly, the SarA targeted inhibitor showed negligible antimicrobial activity and markedly reduced the minimum inhibitory concentration of conventional antibiotics when used in combination making it a more attractive lead for further clinical tests.

Synergism Between Quorum Sensing Inhibitors and Antibiotics: Combating the Antibiotic Resistance Crisis

With the alarming increase in the antibiotic resistance, there is an immediate need for alternative therapeutic strategies to combat this ever-changing bacterial battle. Combinatorial therapies have gained attention owing to their multiple targeted actions. The use of antibiotic is inevitable and antibiotics in combinations have been in use to treat drug resistant infections. Nevertheless, the multidrug resistant strains have found their own mechanisms to surpass such combinatorial treatments. Quorum sensing (QS) inhibition is considered to be the silver lining but is yet to find its way to commercial use. Hence, to combat the antibiotic resistance crisis, the synergy of QSIs and antibiotics is one of the possible revolutionary approaches. In this chapter, we have highlighted the importance and need for the synergy approach with the successful in vitro and in vivo studies that can possibly be extended to the commercial use.

Modulating the Global Response Regulator, LuxO of V. cholerae Quorum Sensing System Using a Pyrazine Dicarboxylic Acid Derivative (PDCApy): An Antivirulence Approach

Vibrio cholerae is a Gram-negative pathogen which causes acute diarrhoeal disease, cholera by the expression of virulence genes through quorum sensing (QS) mechanism. The QS circuit of V. cholerae is controlled by the global quorum regulator, LuxO, which at low cell density (LCD) state produces major virulence factors such as, toxin co-regulated pilus (TCP) and cholera toxin (CT) to mediate infection. On the contrary, at the high cell density (HCD) state the virulent genes are downregulated and the vibrios are detached from the host intestinal epithelial cells, promoted by HapA protease. Hence, targeting the global regulator LuxO would be a promising approach to modulate the QS to curtail V. cholerae pathogenesis. In our earlier studies, LuxO targeted ligand, 2,3 pyrazine dicarboxylic acid (PDCA) and its derivatives having desired pharmacophore properties were chemically synthesized and were shown to have biofilm inhibition as well as synergistic activity with the conventionally used antibiotics. In the present study, the QS modulatory effect of the PDCA derivative with pyrrolidine moiety designated as PDCApy against the V. cholerae virulence gene expression was analyzed at various growth phases. The data significantly showed a several fold reduction in the expression of the genes, tcp and ct whereas the expression of hapR was upregulated at the LCD state. In addition, PDCApy reduced the adhesion and invasion of the vibrios onto the INT407 intestinal cell lines. Collectively, our data suggest that PDCApy could be a potential QS modulator (QSM) for the antivirulence therapeutic approach.