Mitigating the toxicity of palmitoylated analogue of α-melanocyte stimulating hormone(11-13) by conjugation with gold nanoparticle: characterisation and antibacterial efficacy against methicillin sensitive and resistant Staphylococccus aureus.

In an attempt to develop potent and non-toxic antimicrobial agent, the palmitoylated analogue of α-melanocyte stimulating hormone(11-13), Pal-α-MSH(11-13) was conjugated with gold nanoparticles (GNPs) for the first time and the efficacy of derived complex was investigated against two strains of Staphylococccus aureus. The GNPs were synthesized using tri-sodium citrate as reductant and Pal-α-MSH(11-13) was conjugated thereafter. The particles were characterised by UV-vis spectroscopy, transmission electron microscopy, dynamic light scattering, fourier transform infrared spectroscopy etc. Conjugation occurred via electrostatic interaction between anionic GNPs and cationic Pal-α-MSH(11-13). The zeta potential of GNP-Pal-α-MSH(11-13) was - 26.91, indicating its stability. The antibacterial activity was determined by minimal inhibitory concentration (MIC) and killing kinetics assay, whereas, inhibition of biofilm formation was studied by determining the biofilm biomass by crystal violet dye binding method, viability of biofilm-embedded cells by counting CFUs and metabolic activity by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The toxicity was analysed by hemolysis assay against murine RBCs and cytotoxicity against 3T3 fibroblasts. The MIC was 18 µM for GNP-Pal-α-MSH(11-13) and 12 µM for Pal-α-MSH(11-13). The killing kinetics and biofilm inhibition studies indicated the comparable efficacy of peptide before and after nano-conjugation. Importantly, the conjugation resulted in diminished toxicity, as evidenced by 0.29 ± 0.03% hemolysis and 100% viable fibroblasts at 72 µM compared to the Pal-α-MSH(11-13), showing 74.99 ± 1.59% hemolysis and 59.39 ± 1.06% viable fibroblasts. The nano-fabrication drastically reduced the peptide toxicity without compromising its antibacterial efficacy. The anionicity of the conjugate may be responsible for non-toxicity that makes them suitable for pharmaceutical applications.

Lentic and effluent water of Delhi-NCR: a reservoir of multidrug-resistant bacteria harbouring blaCTX-M, blaTEM and blaSHV type ESBL genes.

Antimicrobial resistance is not restricted to clinics but also spreading fast in the aquatic environment. This study focused on the prevalence and diversity of extended-spectrum ß-lactamase (ESBL) genes among bacteria from lentic and effluent water in Delhi-NCR, India. Phenotypic screening of 436 morphologically distinct bacterial isolates collected from diverse sites revealed that 106 (∼24%) isolates were ESBL positive. Antibiotic profiling showed that 42, 60, 78 and 59% ESBL producing isolates collected from Ghazipur slaughterhouse, Lodhi garden pond, Hauz Khas lake and Jasola wastewater treatment plant, respectively, were multidrug-resistant (MDR). The multiple antibiotic resistance (MAR) index varied from 0.20 to 0.32 among selected locations. The prevalence of ESBL gene variants blaSHV, blaTEM and blaCTX-M were found to be 17.64, 35.29 and 64%, respectively. Furthermore, the analysis of obtained gene sequences showed three variants of blaCTX-M (15, 152 and 205) and two variants of blaTEM (TEM-1 and TEM-116) among ESBL producers. The co-existence of 2-3 gene variants was recorded among 48% ESBL positive isolates. New reports from this study include the blaCTX-M gene in Acinetobacter lwoffii, Enterobacter ludwigii, Exiguobacterium mexicanum and Aeromonas caviae. Furthermore, the identification of blaTEM and blaSHV in an environmental isolate of A. caviae is a new report from India.

Biosynthesis of Silver Nanoparticles Using Culture Supernatant of Shewanella sp. ARY1 and Their Antibacterial Activity.

PURPOSE: In this study, silver nanoparticles (AgNPs) were biosynthesized using culture supernatant of strain Shewanella sp. ARY1, characterized and their antibacterial activity was investigated against Gram-negative bacteria Escherichia coli and Klebsiella pneumoniae. METHODS: The strain Shewanella sp. ARY1 was isolated from river Yamuna, Delhi and used for biosynthesis of AgNPs via extracellular approach. Biosynthesized AgNPs were characterized by UV-Visible (UV-Vis) spectrophotometer, fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), energy dispersive X-ray (EDX), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Antibacterial activity of AgNPs was determined by well diffusion, broth microdilution and streaking plate assay to determine the zone of inhibition (ZOI), minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), respectively. The effect of AgNPs on treated bacteria was investigated by electron microscopy analysis. Further, the biocompatibility of AgNPs was tested against mice erythrocytes (RBC) by hemolytic assay. RESULTS: The UV-Vis spectral analysis revealed absorption maxima at 450 nm which confirmed the formation of AgNPs. The FTIR analysis suggested the involvement of various supernatant biomolecules, as reducing and capping agents in the synthesis of AgNPs. The XRD and EDX analysis confirmed the crystalline and metallic nature of AgNPs, respectively. The TEM and SEM analysis showed nanoparticles were spherical with an average size of 38 nm. The biosynthesized AgNPs inhibited the growth and formed a clear zone of inhibition (ZOI) against tested Gram-negative strains. The MIC and MBC were determined as 8-16 µg/mL and 32 µg/mL, respectively. Further, electron microscopy analysis of treated cells showed that AgNPs can damage the outer membrane, release of cytoplasmic contents, and alter the normal morphology of Gram-negative bacteria, leading to cell death. The hemolytic assay indicated that the biosynthesized AgNPs were biocompatible at low dose concentrations. CONCLUSION: This study demonstrates an eco-friendly process for extracellular synthesis of AgNPs using Shewanella sp. ARY1 and these AgNPs exhibited excellent antibacterial activity, which may be used to combat Gram-negative pathogens.

Plasmid-Mediated Ampicillin, Quinolone, and Heavy Metal Co-Resistance among ESBL-Producing Isolates from the Yamuna River, New Delhi, India.

Antibiotic resistance is one of the major current global health crises. Because of increasing contamination with antimicrobials, pesticides, and heavy metals, the aquatic environment has become a hotspot for emergence, maintenance, and dissemination of antibiotic and heavy metal resistance genes among bacteria. The aim of the present study was to determine the co-resistance to quinolones, ampicillin, and heavy metals among the bacterial isolates harboring extended-spectrum ß-lactamases (ESBLs) genes. Among 73 bacterial strains isolated from a highly polluted stretch of the Yamuna River in Delhi, those carrying blaCTX-M, blaTEM, or blaSHV genes were analyzed to detect the genetic determinants of resistance to quinolones, ampicillin, mercury, and arsenic. The plasmid-mediated quinolone resistance (PMQR) gene qnrS was found in 22 isolates; however, the qnrA, B, C, and qnrD genes could not be detected in any of the bacteria. Two variants of CMY, blaCMY-2 and blaCMY-42, were identified among eight and seven strains, respectively. Furthermore, merB, merP, merT, and arsC genes were detected in 40, 40, 44, and 24 bacterial strains, respectively. Co-transfer of different resistance genes was also investigated in a transconjugation experiment. Successful transconjugants had antibiotic and heavy metal resistance genes with similar tolerance toward antibiotics and heavy metals as did their donors. This study indicates that the aquatic environment is a major reservoir of bacteria harboring resistance genes to antibiotics and heavy metals and emphasizes the need to study the genetic basis of resistant microorganisms and their public health implications.

Anti-Bacterial and Anti-Candidal Activity of Silver Nanoparticles Biosynthesized Using Citrobacter spp. MS5 Culture Supernatant.

The present study described the extracellular synthesis of silver nanoparticles (AgNPs) using environmental bacterial isolate Citrobacter spp. MS5 culture supernatant. To our best knowledge, no previous study reported the biosynthesis of AgNPs using this bacterial isolate. The biosynthesized AgNPs were characterized using different techniques like UV-Vis spectroscopy, fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM) equipped with energy dispersive X-ray (EDX). The analysis of UV-Vis spectra revealed absorption maxima at 415 nm due to surface plasmon resonance (SPR) indicated the formation of AgNPs and FTIR spectrum confirmed the participation of proteins molecule in AgNPs synthesis. XRD and EDX spectrum confirmed the metallic and crystalline nature of AgNPs. TEM and SEM showed spherical nanoparticles with a size range of 5-15 nm. The biosynthesized AgNPs showed effective independent as well as enhanced combined antibacterial activity against extended spectrum ß-lactamase (ESBL) producing multidrug resistant Gram-negative bacteria. Further, effective antifungal activity of AgNPs was observed towards pathogenic Candida spp. The present study provides evidence for eco-friendly biosynthesis of well-characterized AgNPs and their potential antibacterial as well as antifungal activity.

Application of FTIR-ATR spectroscopy to confirm the microwave assisted synthesis and curing of Cashew nut shell liquid derived nanostructured materials.

The present work reports the development of nanostructured material from Cashew nut shell liquid (CNSL, an agro byproduct of cashew industry, 87% cardanol) to evaluate their potential in antibacterial applications as a substitute of petroleum feedstock via an energy-efficient method. The nanostructured material was synthesized by coordination polymerization reaction of cardanol and divalent Mn(II) salt with the aid of microwave irradiations. FTIR spectroscopy was used to confirm the proposed structure of the synthesized materials. FTIR-ATR spectroscopy was employed to verify the curing of material by comparing the spectra of the cured samples with the frequencies of uncured samples. Magnetic moment and UV-visible spectroscopy were used to confirm the proposed structure of the material further. Morphology of the synthesized material was investigated by XRD, optical microscopy, SEM and TEM and thermal behaviour by TGA/DTG/DSC technique. Agar diffusion method was utilized to investigate the antibacterial activity of the synthesized material against bacterial strains E. coli, K. pneumoniae, B. subtilis and S. aureus. N2 adsorption-desorption was investigated to check BET specific surface area and BJH pore size distribution of the same. The results revealed that the synthesized materials were obtained as semicrystalline, porous, thermally stable and nanostructured film forming materials with moderate to good antibacterial activity against different nosocomial bacteria. They can be used as thermally stable antibacterial agents in the field of films/coatings for health care applications.

Prevalence and diversity of blaTEM, blaSHV and blaCTX-M variants among multidrug resistant Klebsiella spp. from an urban riverine environment in India.

In the present study, we have investigated prevalence and diversity of ESBL genes among Klebsiella isolates obtained from highly polluted stretch of river Yamuna, India. Phenotypic screenings of 116 Klebsiella isolates revealed ~30% were positive for ESBL production. Antibiotic profiling showed multidrug resistance phenotype among 90% isolates. Prevalence of blaTEM, blaSHV and blaCTX-M genes were found to be 57, 54 and 48% respectively. Furthermore, we identified eight variants of blaSHV (SHV-1, SHV-11, SHV-27, SHV-28, SHV-38, SHV-61, SHV-144, SHV-148), three each of blaTEM (TEM-1, TEM-116, TEM-206) and blaCTX-M (CTX-M-15, CTX-M-55, CTX-M-188) among Klebsiella spp. Co-occurrence of blaTEM, blaSHV and blaCTX-M (any two or all three) was observed among 45% Klebsiella isolates. Occurrence of blaCTX-M-188 and blaTEM-206 in environmental isolates of K. pneumoniae has not been reported earlier. Identification of blaTEM-206, blaSHV-27 and blaSHV-144 from Klebsiella spp. and blaTEM-116 from K. quasipneumoniae and K. variicola is the first report from India.

Antibiotics, Resistome and Resistance Mechanisms: A Bacterial Perspective.

History of mankind is regarded as struggle against infectious diseases. Rather than observing the withering away of bacterial diseases, antibiotic resistance has emerged as a serious global health concern. Medium of antibiotic resistance in bacteria varies greatly and comprises of target protection, target substitution, antibiotic detoxification and block of intracellular antibiotic accumulation. Further aggravation to prevailing situation arose on observing bacteria gradually becoming resistant to different classes of antibiotics through acquisition of resistance genes from same and different genera of bacteria. Attributing bacteria with feature of better adaptability, dispersal of antibiotic resistance genes to minimize effects of antibiotics by various means including horizontal gene transfer (conjugation, transformation, and transduction), Mobile genetic elements (plasmids, transposons, insertion sequences, integrons, and integrative-conjugative elements) and bacterial toxin-antitoxin system led to speedy bloom of antibiotic resistance amongst bacteria. Proficiency of bacteria to obtain resistance genes generated an unpleasant situation; a grave, but a lot unacknowledged, feature of resistance gene transfer.

Study of pandrug and heavy metal resistance among E. coli from anthropogenically influenced Delhi stretch of river Yamuna

Abstract Escalating burden of antibiotic resistance that has reached new heights present a grave concern to mankind. As the problem is no longer confined to clinics, we hereby report identification of a pandrug resistant Escherichia coli isolate from heavily polluted Delhi stretch of river Yamuna, India. E. coli MRC11 was found sensitive only to tobramycin against 21 antibiotics tested, with minimum inhibitory concentration values >256 µg/mL for amoxicillin, carbenicillin, aztreonam, ceftazidime and cefotaxime. Addition of certain heavy metals at higher concentrations were ineffective in increasing susceptibility of E. coli MRC11 to antibiotics. Withstanding sub-optimal concentration of cefotaxime (10 µg/mL) and mercuric chloride (2 µg/mL), and also resistance to their combinatorial use, indicates better adaptability in heavily polluted environment through clustering and expression of resistance genes. Interestingly, E. coli MRC11 harbours two different variants of blaTEM (blaTEM-116 and blaTEM-1 with and without extended-spectrum activity, respectively), in addition to mer operon (merB, merP and merT) genes. Studies employing conjugation, confirmed localization of blaTEM-116, merP and merT genes on the conjugative plasmid. Understanding potentialities of such isolates will help in determining risk factors attributing pandrug resistance and strengthening strategic development of new and effective antimicrobial agents.

Study of pandrug and heavy metal resistance among E. coli from anthropogenically influenced Delhi stretch of river Yamuna.

Escalating burden of antibiotic resistance that has reached new heights present a grave concern to mankind. As the problem is no longer confined to clinics, we hereby report identification of a pandrug resistant Escherichia coli isolate from heavily polluted Delhi stretch of river Yamuna, India. E. coli MRC11 was found sensitive only to tobramycin against 21 antibiotics tested, with minimum inhibitory concentration values >256µg/mL for amoxicillin, carbenicillin, aztreonam, ceftazidime and cefotaxime. Addition of certain heavy metals at higher concentrations were ineffective in increasing susceptibility of E. coli MRC11 to antibiotics. Withstanding sub-optimal concentration of cefotaxime (10µg/mL) and mercuric chloride (2µg/mL), and also resistance to their combinatorial use, indicates better adaptability in heavily polluted environment through clustering and expression of resistance genes. Interestingly, E. coli MRC11 harbours two different variants of blaTEM (blaTEM-116 and blaTEM-1 with and without extended-spectrum activity, respectively), in addition to mer operon (merB, merP and merT) genes. Studies employing conjugation, confirmed localization of blaTEM-116, merP and merT genes on the conjugative plasmid. Understanding potentialities of such isolates will help in determining risk factors attributing pandrug resistance and strengthening strategic development of new and effective antimicrobial agents.