Activity of lipid-loaded lectin against co-infection of Candida albicans and Staphylococcus aureus using the zebrafish model.

AIM: Nosocomial infection caused by mixed species of methicillin-resistant Staphylococcus aureus (MRSA) and Candida albicans (CA) is difficult to manage with existing antimicrobials, particularly in the presence of mixed-species biofilm. This study evaluates the activity of cationic lipid, specifically functionalized with lectin, against mixed biofilms of MRSA and CA and their effectiveness in vivo using the zebrafish model. METHODS AND RESULTS: The present study demonstrates for the first time the antimicrobial activity of 2-((N-[2-hydroxyethyl]palmitamido)methyl)-1-methylpyridin-1-ium iodide (cN16E) against MRSA and mixed species of MRSA + CA. The cN16E functionalized with Butea monosperma seed lectin (BMSL) showed a lower minimum inhibitory concentration (MIC) as compared with cN16E. BMSL-cN16E (BcN16E) exhibited strong membrane-damaging activity at a lower concentration than cN16E. Crystal violet assay showed that BcN16E inhibits mixed-species biofilm at the concentration of 15.63 µM, which is four-fold lower than the MIC. Especially, BcN16E was found to be effective in disturbing mature mixed biofilm at 31.25 µM, which is two-fold lower than the MIC, suggesting true antibiofilm activity without pressurizing the microorganisms. The treatment with BcN16E significantly reduced the exopolysaccharide synthesis (> 78%), cell surface hydrophobicity (> 70 %), hyphae formation, staphyloxanthin biosynthesis (> 41 %), and antioxidant enzyme and hemolysin activity (> 70 %). Notably, BcN16E was efficient in reducing the in vivo colonization of bacterial and fungal burden in the blood and muscle tissues of zebrafish. CONCLUSION: Antimicrobial and antibiofilm efficacy of BcN16E against MRSA, and mixed species of MRSA + CA were demonstrated. Importantly, BcN16E treatment rescued Zebrafish coinfected with mixed species of MRSA + CA. Significance and Impact of the study: The results highlight that antimicrobial loaded on lectin provides an additional advantage to recognize microorganism surface glycans and maximize drug delivery to treat polymicrobial infections caused by MRSA and CA.

Hybrid of Metapenaeus dobsoni lectin and platinum nanoparticles exert antimicrobial and immunostimulatory effects to reduce bacterial bioburden in infected Nile tilapia.

A novel antibacterial immunostimulant using Platinum nanoparticles (PtNPs) and lectin from Metapenaeus dobsoni (Md-Lec) was developed. The Md-Lec and PtNPs (Pt-lec) hybrid formed through non-covalent interaction exhibits antimicrobial activity against fish specific pathogens by affecting membrane integrity and producing excess reactive oxygen species. The therapeutic efficacy of Pt-lec was demonstrated through rescuing Aeromonas hydrophila infected Nile Tilapia. Pt-lec prevents the infection spreading and reduces the bacterial bioburden in less than 12 h, and as a result of this the fish were restored to normalcy. To assess immunostimulation, we studied the expression of three different immune related genes, namely LEC, Myd88 and COX-2 in the gills, liver, spleen and kidney of fish under various experimental conditions. Our results showed that Pt-lec treatment appeared to be better when compared to lectin alone in enhancing the expression of Myd88 and COX-2, but LEC was not as expected. These results suggest that Pt-lec has the ability to protect Nile Tilapia against bacterial infection by restricting bacterial bioburden through their direct effects on the bacterial membrane and indirectly through their effects on host immune-related gene expression. This hybrid could have potential "green" application in fish farming in rescuing infected animals when compared to widely and unregulated antibiotics.

Phytolectin conjugated positively charged fatty acid amide impairs virulence factors and inhibits cross-kingdom biofilm formation of Candida albicans and uropathogenic Escherichia coli.

AIM: Polymicrobial biofilm encasing cross-kingdom micro-organisms are apparent in medicine, which imposes serious resistance to conventional antimicrobial treatment. The objective of the study was to explore Butea monosperma seed lectin (BMSL) conjugated antimicrobial lipid, 2-((N-[2-hydroxyethyl]palmitamido)methyl)-1-methylpyridin-1-ium iodide (cN16E) to inhibit mixed-species biofilm of uropathogenic Escherichia coli-Candida albicans. METHODS AND RESULTS: Antimicrobial activity and antibiofilm of cN16E and cN16E-BMSL conjugate (BcN16E) were analysed against single- and mixed microbial cultures. The minimum inhibitory concentration (MIC) indicates that the MIC of cN16E-BMSL conjugate (BcN16E) against cohabiting UPEC-C. albicans was eightfold lower than the cN16E. BcN16E affects membrane integrity to elicit antimicrobial activity. BcN16E inhibits the dual-species biofilm even with 16 times lower MIC of cN16E. BcN16E impairs the biofilm-associated virulence factors which include extracellular polysaccharides, cell surface hydrophobicity, swimming, swarming motilities, hyphal filamentous morphology, curli formation and haemolysin activity. As a proof of concept, we demonstrated BcN16E ability to inhibit dual-species biofilm formation on a urinary catheter. CONCLUSION: The study revealed that the BcN16E is better than cN16E in impairing biofilm-associated virulence factors and exerting antimicrobial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings emphasize that phytolectin has the potential to enhance the anti-virulence strategies of antimicrobials against cross-kingdom biofilm-related infections.

Phytolectin nanoconjugates in combination with standard antifungals curb multi-species biofilms and virulence of vulvovaginal candidiasis (VVC) causing Candida albicans and non-albicans Candida.

Vulvovaginal candidiasis (VVC) is a commonly occurring yeast infection caused by Candida species in women. Among Candida species, C. albicans is the predominant member that causes vaginal candidiasis followed by Candida glabrata. Biofilm formation by Candida albicans on the vaginal mucosal tissue leads to VVC infection and is one of the factors for a commensal organism to get into virulent form leading to disease. In addition to that, morphological switching from yeast to hyphal form increases the risk of pathogenesis as it aids in tissue invasion. In this study, jacalin, a phytolectin complexed copper sulfide nanoparticles (NPs) have been explored to eradicate the mono and mixed species biofilms formed by fluconazole-resistant C. albicans and C. glabrata isolated from VVC patients. NPs along with standard antifungals like micafungin and amphotericin B have been evaluated to explore interaction behavior and we observed synergistic interactions between them. Microscopic techniques like light microscopy, phase contrast microscopy, scanning electron microscopy, confocal laser scanning microscopy were used to visualize the inhibition of biofilm by NPs and in synergistic combinations with standard antifungals. Real-time PCR analysis was carried out to study the expression pattern of the highly virulent genes which are responsible for yeast to hyphal switch, drug resistance and biofilm formation upon treatment with NPs in combination with standard antifungals. The current study shows that lectin-conjugated NPs with standard antifungals might be a different means to disrupt the mixed species population of Candida spp. that causes VVC. LAY SUMMARY: The present study focuses on exploiting the high biding affinity between the cell surface glycans present in Candida cells and the plant lectin, Jacalin. Jacalin serves as a 'Trojan Horse' wherein the lectin-coupled nanoparticles show a high efficacy when compared with the unconjugated nanoparticles. The present approach also improves the anti-biofilm activity of the antifungal drugs against drug-resistant Candida strains.

Shrimp lectin (Md-Lec) conjugated copper sulfide nanoparticles enhance the elimination of aquatic pathogens in infected Nile tilapia (Oreochromis niloticus).

Lectins are known for their ability to bind to cell surface glycans, and are useful to develop a glycan-targeted drug delivery system. This study aimed to evaluate the capacity of pectin capped copper sulfide nanoparticles (pCuS NPs) to modulate the antibacterial activity of a lectin, Md-Lec, purified from the shrimp, Metapenaeus dobsoni. Fluorescence spectroscopy revealed that Md-Lec has the ability to form a complex with pCuS NPs. Haemagglutination assay showed that the carbohydrate binding site of the lectin was preserved even after complexing with pCuS. The minimum inhibitory concentrations (MICs) obtained for Md-Lec and pCuS NPs against the tested aquatic pathogens were 50 µg ml-1 and 12.5 µM, respectively. Interestingly, the MIC of Md-Lec-pCuS NPs complex was four fold lower than that of pCuS, which was attributed to the bacterial cell surface glycan recognization activity of Md-Lec. Zone of inhibition assay showed that the zone size was highest for the lectin conjugated nanoparticles. Mechanistic study revealed that Md-Lec-pCuS NPs affect the bacterial membrane integrity and produce a large volume of reactive oxygen species to kill the bacteria. The practical aspect of using this lectin-pCuS NPs complex was evaluated by treating bacteria infected Nile tilapia (Oreochromis niloticus). The bacterial load was much less in the lectin-pCus NPs complex treated fish; moreover, the fish fully recovered from the infection. It was concluded that the conjugate of antibacterial lectin and NPs is more effective than the individual components.

Remarkable Effect of Jacalin in Diminishing the Protein Corona Interference in the Antibacterial Activity of Pectin-Capped Copper Sulfide Nanoparticles.

Herein, we report a new strategy based on jacalin functionalization to diminish the impact of biological fluids in the antibacterial applications of nanoparticles (NPs). Precoating pectin-capped copper sulfide NPs (pCuS) with bovine serum albumin produced a protein corona, which affects the antibacterial activity of pCuS. It was found that the minimum inhibitory concentration (MIC) increases fourfold because of the formation of the protein corona. Interestingly, the pCuS functionalized with jacalin enhance the targeting capabilities through bacterial cell surface glycan recognition with no interference from the protein corona. The MIC of pCuS decreases 16-fold on functionalization with jacalin. Mechanistic studies indicated that the pCuS functionalized with jacalin impede the protein corona interference and induce bacterial cell death by impairing the GSH/reactive oxygen species balance and disrupting the bacteria cell membrane. As a proof of concept, we used a bacteria-infected zebrafish animal model to demonstrate the interference of biological fluids in the antibacterial activity of NPs. Infected zebrafish treated with 1× MIC of pCuS failed to recover from the infection, but 4× MIC rescues the fish. The requirement of a high dose of NPs to treat the infection confirms the interference of biological fluids in nanotherapeutic applications. At the same time, the jacalin-pCuS complex rescues the infected fish at 16-fold lesser MIC. The results obtained from this study suggest that jacalin-mediated NP targeting may have broad implications in the development of future nanomedicine.

Sub lethal levels of platinum nanoparticle cures plasmid and in combination with carbapenem, curtails carbapenem resistant Escherichia coli.

Drug resistance traits are rapidly disseminated across bacteria by horizontal gene transfer, especially through plasmids. Plasmid curing agents that are active both in vitro and in vivo will resensitize Multi Drug Resistant (MDR) bacteria to antimicrobial agents. Pectin capped platinum nanoparticles (PtNPs) at sub MIC (20 µM) concentration was effective, in causing loss of Extended Spectrum Beta Lactamase (ESBL) harboring plasmid as evidenced by, absence of plasmid in agarose gel and by a concomitant (16-64 fold) drop in MIC for cell wall inhibitors ceftriaxone and meropenem, in carbapenem resistant Escherichia coli (CREC). Interestingly, the plasmid cured strain exhibited small colony morphology and displayed slower growth both in vitro and in vivo. Complementation of cured strain with plasmid from the wild type strain restored resistance towards meropenem and ceftriaxone. Relative to wild type, plasmid cured strain displayed 50% reduction in biofilm formation. Plasmid curing also occurred in vivo in infected zebrafish with curing efficiency of 17% for nanoparticle + meropenem treatment. PtNPs + meropenem reduced bioburden of CREC in infected zebrafish by 2.4 log CFU. Mechanistic studies revealed that nanoparticle interacted with cell surface and perturbed inner membrane integrity. PtNPs did not induce ROS, yet it caused plasmid DNA cleavage, as evidenced by gyrase inhibition assay. Our study for the first time reveals that PtNPs as plasmid curing agent can resensitize MDR bacteria to selective antimicrobial agents in vivo.

Effective elimination of biofilm formed with waterborne pathogens using copper nanoparticles.

In this paper, the self assembling properties of taurolipids were used to prepare stable copper nanoparticles (CuNPs), and demonstrated the ability of CuNPs to eradicate the biofilms formed by waterborne pathogens. The synthesized CuNPs display wine red color and exhibited surface plasmon resonance with a maximum at 590 nm. Transmission electron microscopy showed that the CuNPs are well-dispersed with spherical morphology and the size range between 5 and 12 nm. The powder X-ray diffraction study revealed that the CuNPs was free from copper oxide impurities and crystalline with the face centered cubic structure. The CuNPs exhibited excellent anti-biofilm activity against water borne pathogens such as Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, and Shigella flexneri. Light microscopy and scanning electron microscopy (SEM) study revealed that CuNPs eliminates the mature biofilm at the minimum biofilm eradication concentration of 12.5 µM. The antimicrobial activity of the CuNPs was observed at the minimum inhibitory concentration of 25 µM, indicating the reported CuNPs exhibit true anti-biofilm effect. Fluorescence microscopy and SEM study proved that CuNPs kills the bacteria through membrane damage. The possibility to use CuNPs in cleaning biofilm formed on storage containers was demonstrated through removing the mature biofilm formed on a glass pipe.

Preparation of self-assembled platinum nanoclusters to combat Salmonella typhi infection and inhibit biofilm formation.

In this work, phytoprotein functionalized platinum nanoparticles (PtNCs) were synthesized using the proteins from fresh green spinach leaves. Transmission electron microscopy showed that PtNCs were spherical shape with size ∼5 nm, which self assembled into spherical platinum nanoclustures (PtNCs) with size within the range of 100-250 nm. The presence of elemental platinum was confirmed by EDX analysis. FTIR studies confirm that the PtNCs were stabilized by the protein. As prepared PtNCs inhibits the growth of the food borne pathogen, Salmonella typhi with minimum inhibitory concentration (MIC) of 12.5 µM. Light microscopy evidenced that the PtNCs can damage the established biofilms. Antibacterial mechanistic study revealed that PtNCs damages the S. typhi membranes, which was confirmed by scanning electron microscopy and further by fluorescence microscopy using acridine orange/propidium iodide dual staining assay. Besides membrane damage, PtNCs also triggered the intracellular ROS-mediated oxidative damage over the antioxidant defense and kills S. typhi. The hemolytic test showed low cytotoxicity of PtNCs at 100 µM (four times higher the MIC). Finally, the therapeutic efficacy of PtNCs was validated in S. typhi infected zebrafish animal model and the obtained results are discussed.

Jacalin-copper sulfide nanoparticles complex enhance the antibacterial activity against drug resistant bacteria via cell surface glycan recognition.

In any therapeutic modality the usage of drug in high doses often leads to serious side-effects. Herein, we demonstrated a method to enhance the antibacterial efficacy of CuS NPs at lower concentration through interacting with jackfruit seed lectin, jacalin. Fluorescence quenching studies revealed that jacalin form complex with CuS NPs and the association constant was 1.91 × 104 M-1. Upon complex with jacalin, the bacterial minimum inhibitory concentration (MIC) of CuS NPs drastically decreases from 12.5 µM to 0.78 µM. The addition of jacalin specific sugar, galactose to jacalin-CuS NPs complex (JCuS NPs) reverses the MIC from 0.78 µM to 25 µM. Mechanistic study suggests that JCuS NPs kills bacteria in part by reactive oxygen species and membrane damage, but galactose prevents the action of JCuS NPs at 0.78 µM. JCuS NPs successfully reduce (14 fold) A. hydrophila colonization in an infected zerbra fish and rescue them completely from the infection, but galJCuS NPs and CuS NPs were ineffective at 0.78 µM. Collectively, our studies demonstrates that the enhance antibacterial activity of JCuS NPs is likely due to the interaction between the galactose binding site of jacalin and the bacterial strains, as a result NPs are targeted and delivered sufficiently.

Synthesis, supramolecular organization and thermotropic phase behaviour of N-acyltris(hydroxymethyl)aminomethane.

Herein, we reported the supramolecular organization of N-acyltris(hydroxymethyl)aminomethane (NATM) in the solid state as well as in aqueous solution. Single crystal X-ray diffraction revealed that NATM adopts a fully interdigitized structure. The thermodynamic parameters associated with thermotropic phase behaviour of NATM was determined by differential scanning calorimetry. The molecular packing and phase state of the NATM analyzed by laurdan and prodan fluorescence supports the formation of an interdigitized phase in aqueous solution. The potential application of the self-assembled NATM vesicles was demonstrated through entrapping model drug, Rhodamine B.