Bioactive Compound-Loaded Nanocarriers for Hair Growth Promotion: Current Status and Future Perspectives.

Hair loss (alopecia) has a multitude of causes, and the problem is still poorly defined. For curing alopecia, therapies are available in both natural and synthetic forms; however, natural remedies are gaining popularity due to the multiple effects of complex phytoconstituents on the scalp with fewer side effects. Evidence-based hair growth promotion by some plants has been reported for both traditional and advanced treatment approaches. Nanoarchitectonics may have the ability to evolve in the field of hair- and scalp-altering products and treatments, giving new qualities to hair that can be an effective protective layer or a technique to recover lost hair. This review will provide insights into several plant and herbal formulations that have been reported for the prevention of hair loss and stimulation of new hair growth. This review also focuses on the molecular mechanisms of hair growth/loss, several isolated phytoconstituents with hair growth-promoting properties, patents, in vivo evaluation of hair growth-promoting activity, and recent nanoarchitectonic technologies that have been explored for hair growth.

Supersaturation-Based Drug Delivery Systems: Strategy for Bioavailability Enhancement of Poorly Water-Soluble Drugs.

At present, the majority of APIs synthesized today remain challenging tasks for formulation development. Many technologies are being utilized or explored for enhancing solubility, such as chemical modification, novel drug delivery systems (microemulsions, nanoparticles, liposomes, etc.), salt formation, and many more. One promising avenue attaining attention presently is supersaturated drug delivery systems. When exposed to gastrointestinal fluids, drug concentration exceeds equilibrium solubility and a supersaturation state is maintained long enough to be absorbed, enhancing bioavailability. In this review, the latest developments in supersaturated drug delivery systems are addressed in depth.

Identification and validation of reference genes for reliable analysis of differential gene expression during antibiotic induced persister formation in Klebsiella pneumoniae using qPCR.

The study of differential gene expression in persister cells is compounded by ceasure of conventional cellular metabolic pathways during persistence. There is, hence, a requirement to identify and validate suitable reference genes whose expression remains stable during persistence. We evaluated the suitability of five genes viz. dnaJ, groEL, rpoB, kp751, kp4432 as references to study gene expression using real-time polymerase chain reaction (qPCR) during persister cell formation in Klebsiella pneumoniae. Results obtained showed that while dnaJ and groEL suffered from unstable expression; rpoB, kp751 and kp4432 showed stable expression. Further, it was observed that data normalization using either of the stable genes viz. rpoB, kp751, kp4432 alone, resulted in either too low expression levels or too high variation among replicates. Our study indicates the concurrent use of kp4432 and rpoB as reference genes to be the most suitable for reliable analysis of differential gene expression during antibiotic induced persister formation in K. pneumoniae. kp4432 and rpoB encode NAD-dependant phenylacetaldehyde dehydrogenase and DNA-directed RNA polymerase beta subunit respectively. The outcome of this study will increase the utility of qPCR in studying the temporal changes in gene expression during persistence. The study will also aid in understanding mechanisms underlying persister cell formation particularly in K. pneumoniae.

Antibiotic Resistance Profile, Outer Membrane Proteins, Virulence Factors and Genome Sequence Analysis Reveal Clinical Isolates of Enterobacter Are Potential Pathogens Compared to Environmental Isolates.

Outer membrane proteins (OMPs) of gram-negative bacteria play an important role in mediating antibacterial resistance, bacterial virulence and thus affect pathogenic ability of the bacteria. Over the years, prevalence of environmental antibiotic resistant organisms, their transmission to clinics and ability to transfer resistance genes, have been studied extensively. Nevertheless, how successful environmental bacteria can be in establishing as pathogenic bacteria under clinical setting, is less addressed. In the present study, we utilized an integrated approach of investigating the antibiotic resistance profile, presence of outer membrane proteins and virulence factors to understand extent of threat posed due to multidrug resistant environmental Enterobacter isolates. Also, we investigated clinical Enterobacter isolates and compared the results thereof. Results of the study showed that multidrug resistant environmental Enterobacter isolates lacked OmpC, lacked cell invasion abilities and exhibited low reactive oxygen species (ROS) production in neutrophils. In contrast, clinical isolates possessed OmpF, exhibited high invasive and adhesive property and produced higher amounts of ROS in neutrophils. These attributes indicated limited pathogenic potential of environmental Enterobacter isolates. Informations obtained from whole genome sequence of two representative bacterial isolates from environment (DL4.3) and clinical sources (EspIMS6) corroborated well with the observed results. Findings of the present study are significant as it highlights limited fitness of multidrug resistant environmental Enterobacter isolates.

Carbohydrate-Coated Gold-Silver Nanoparticles for Efficient Elimination of Multidrug Resistant Bacteria and in Vivo Wound Healing.

Multidrug resistant (MDR) bacteria have emerged as a major clinical challenge. The unavailability of effective antibiotics has necessitated the use of emerging nanoparticles as alternatives. In this work, we have developed carbohydrate-coated bimetallic nanoparticles (Au-AgNP, 30-40 nm diameter) that are nontoxic toward mammalian cells yet highly effective against MDR strains as compared to their monometallic counterparts (Ag-NP, Au-NP). The Au-AgNP is much more effective against Gram-negative MDR Escherichia coli and Enterobacter cloacae when compared to most of the potent antibiotics. We demonstrate that in vivo, Au-AgNP is at least 11000 times more effective than Gentamicin in eliminating MDR Methicillin Resistant Staphylococcus aureus (MRSA) infecting mice skin wounds. Au-AgNP is able to heal and regenerate infected wounds faster and in scar-free manner. In vivo results show that this Au-AgNP is very effective antibacterial agent against MDR strains and does not produce adverse toxicity. We conclude that this bimetallic nanoparticle can be safe in complete skin regeneration in bacteria infected wounds.

Antibacterial Efficacy of Polysaccharide Capped Silver Nanoparticles Is Not Compromised by AcrAB-TolC Efflux Pump.

Antibacterial therapy is of paramount importance in treatment of several acute and chronic infectious diseases caused by pathogens. Over the years extensive use and misuse of antimicrobial agents has led to emergence of multidrug resistant (MDR) and extensive drug resistant (XDR) pathogens. This drastic escalation in resistant phenotype has limited the efficacy of available therapeutic options. Thus, the need of the hour is to look for alternative therapeutic approaches to mitigate healthcare concerns caused due to MDR bacterial infections. Nanoparticles have gathered much attention as potential candidates for antibacterial therapy. Equipped with advantages of, wide spectrum bactericidal activity at very low dosage, inhibitor of biofilm formation and ease of permeability, nanoparticles have been considered as leading therapeutic candidates to curtail infections resulting from MDR bacteria. However, substrate non-specificity of efflux pumps, particularly those belonging to resistance nodulation division super family, have been reported to reduce efficacy of many potent antibacterial therapeutic drugs. Previously, we had reported antibacterial activity of polysaccharide-capped silver nanoparticles (AgNPs) toward MDR bacteria. We showed that AgNPs inhibits biofilm formation and alters expression of cytoskeletal proteins FtsZ and FtsA, with minimal cytotoxicity toward mammalian cells. In the present study, we report no reduction in antibacterial efficacy of silver nanoparticles in presence of AcrAB-TolC efflux pump proteins. Antibacterial tests were performed according to CLSI macrobroth dilution method, which revealed that both silver nanoparticles exhibited bactericidal activity at very low concentrations. Further, immunoblotting results indicated that both the nanoparticles modulate the transporter AcrB protein expression. However, expression of the membrane fusion protein AcrA did show a significant increase after exposure to AgNPs. Our results indicate that both silver nanoparticles are effective in eliminating MDR Enterobacter cloacae isolates and their action was not inhibited by AcrAB-TolC efflux protein expression. As such, the above nanoparticles have strong potential to be used as effective and alternate therapeutic candidates to combat MDR gram-negative Enterobacterial pathogens.

Retrospective study on clonal relationship of multidrug-resistant Klebsiella spp. indicates closed circulation and initiation of clonal divergence.

PURPOSE: Antibiotic resistance patterns often exhibit geographical variations. Periodic analyses of resistance spectra and phylogenetic trends are important guides for facilitating judicious use of therapeutic interventions. The present study retrospectively analysed the infection trends, resistance patterns, and clonal relationships between isolates of Klebsiella spp. from a tertiary care hospital. METHODOLOGY: Bacterial isolates were collected from January 2013 to June 2014 and their resistance profiles were identified using an automated bacterial identification system. A phylogenetic tree was constructed using housekeeping genes with Molecular Evolutionary Genetic Analysis software. The dN/dS ratio was determined by the Synonymous Non-synonymous Analysis Program while polymorphic sites, and the difference per site was calculated using DNA Sequence Polymorphism software. Statistical Package for Social Science software was used to perform all statistical analyses. KEY FINDINGS: The results of this study indicated the prevalence of community-acquired urinary tract and lower respiratory tract infections caused by Klebsiella spp. among geriatric patients. The occurrence of new allelic profiles, a low dN/dS ratio and the lack of strong evolutionary descent between isolates indicated that mutations play a major role in the evolution of the organism. CONCLUSION: The findings of this study highlight the consequences of antimicrobial agents exerting a silent and strong selective force on the evolution of Klebsiella spp. The expansion of such analyses is of great importance for addressing rapidly emerging antibiotic-resistant opportunistic pathogens.

Draft Genome Sequences of Clinical and Nonclinical Isolates of Klebsiella spp. Exhibiting Nonheritable Tolerance toward Antimicrobial Compounds.

A clinical isolate and a nonclinical isolate of Klebsiella pneumoniae were found to exhibit nonheritable tolerance in response to antimicrobial compounds. The draft genome sequences of both isolates are presented here.

Draft Genome Sequences of Nonclinical and Clinical Enterobacter cloacae Isolates Exhibiting Multiple Antibiotic Resistance and Virulence Factors.

Enterobacter spp. have been implicated as opportunistic pathogens which over the years have gained resistance toward most of the available therapeutic drugs. We sequenced two multidrug-resistant Enterobacter cloacae isolates harboring multiple efflux pump genes. These isolates exhibited strain-specific modulation of efflux pump protein expression.

Antibiotic resistance determinants and clonal relationships among multidrug-resistant isolates of Klebsiella pneumoniae.

In the present study, we performed PCR based screening to determine the presence of antibiotic resistance genes and sequencing to find mutation in QRDR region among fourteen isolates of K. pneumoniae. Association analysis was conducted to detect the co-resistance among the isolates. Multi-locus sequence analysis was carried out to determine the clonal relationship among them. All the K. pneumoniae isolates showed resistance to multiple antibiotics and exhibited cross-resistance to antibiotics. Although few isolates co-harbored variants of ß-lactamase genes, others carried qnrB on plasmid and mutations in Quinolone-Resistant Determining Region (QRDR). This study thus indicates that clonally unrelated K. pneumoniae isolates exhibited co-resistance, harboured multiple antibiotic resistance genes present on the chromosome, plasmids and/or integron Therefore, the data from this study can provide guidelines for the prudent use of antibiotics to avert the impending danger of losing out on the available antibiotics for therapeutic use.

Association study of multiple antibiotic resistance and virulence: a strategy to assess the extent of risk posed by bacterial population in aquatic environment.

The present study explored the association between multiple antibiotic resistance (MAR) index and virulence index to determine what percent of environmental antibiotic-resistant (eARB) bacteria could pose threat as potential pathogen. 16srRNA-based sequencing of 113 non-duplicate isolates identified majority of them to be gram negative belonging to Enterobacter, Pseudomonas, Aeromonas, Proteus, Acinetobacter, and Klebsiella. Statistical comparison of MAR indices of the abovementioned genera indicated differences in the median values among the groups (p < 0.001). Pair-wise multiple comparison by Dunn's method indicated significant difference in MAR indices (p < 0.05), based on which multiple antibiotic resistance phenotype could be ranked in the order Pseudomonas > Klebsiella = Acinetobacter > Proteus > Aeromonas > Enterobacter. Association between MAR index and virulence index revealed that 25% of isolates in the population under study posed high threat to human/animal or both; out of which 75% isolates belonged to genus Pseudomonas. Based on observations of comparative analysis of the six gram-negative genera, it could be concluded that Pseudomonas isolates from environment pose significantly high threat as potential pathogens while Enterobacter isolates posed no threat.

Efflux mediated colistin resistance in diverse clones of Klebsiella pneumoniae from aquatic environment.

The peptide drug colistin is commonly used to treat carbapenem resistant gram negative bacterial infections. In the present study, we report efflux mediated colistin resistance in multidrug resistant Klebsiella pneumoniae isolates belonging to ST200 and ST1296, isolated from a fresh water environment. The isolates exhibited intermediate resistance to human serum, possessed Type 1 fimbriae and harbored blaSHV-34 and blaTEM-1 genes. Our results highlight the evolving nature of these clones in the country. These observations emphasize the need for judicious usage of antibiotics to prevent the imminent danger of losing out on currently available therapeutic options.

Polysaccharide-capped silver Nanoparticles inhibit biofilm formation and eliminate multi-drug-resistant bacteria by disrupting bacterial cytoskeleton with reduced cytotoxicity towards mammalian cells.

Development of effective anti-microbial therapeutics has been hindered by the emergence of bacterial strains with multi-drug resistance and biofilm formation capabilities. In this article, we report an efficient green synthesis of silver nanoparticle (AgNP) by in situ reduction and capping with a semi-synthetic polysaccharide-based biopolymer (carboxymethyl tamarind polysaccharide). The CMT-capped AgNPs were characterized by UV, DLS, FE-SEM, EDX and HR-TEM. These AgNPs have average particle size of ~20-40 nm, and show long time stability, indicated by their unchanged SPR and Zeta-potential values. These AgNPs inhibit growth and biofilm formation of both Gram positive (B. subtilis) and Gram negative (E. coli and Salmonella typhimurium) bacterial strains even at concentrations much lower than the minimum inhibitory concentration (MIC) breakpoints of antibiotics, but show reduced or no cytotoxicity against mammalian cells. These AgNPs alter expression and positioning of bacterial cytoskeletal proteins FtsZ and FtsA. CMT-capped AgNPs can effectively block growth of several clinical isolates and MDR strains representing different genera and resistant towards multiple antibiotics belonging to different classes. We propose that the CMT-capped AgNPs can have potential bio-medical application against multi-drug-resistant microbes with minimal cytotoxicity towards mammalian cells.

Analysis of clonally related environmental Vibrio cholerae O1 El Tor isolated before 1992 from Varanasi, India reveals origin of SXT-ICEs belonging to O139 and O1 serogroups.

In this study, we report the presence of SXT in environmental Vibrio cholerae O1 El Tor strains isolated before 1992 from Varanasi, India. All isolates, except one, were resistant to Tm, and/or Sul, Sm, Fr, Na and Am. None contained plasmids. PCR and DNA sequencing revealed the presence of SXT containing dfrA1 and/or sulII, strAB in six isolates and dfr18, sulII and strAB in five isolates. Three clinical V. cholerae O1 isolated during 1992 contained the antibiotic resistance gene cassette aadA1 in the class 1 integron. Conjugation experiments, followed by PCR analysis of transconjugants, provided evidence of the transferable nature of SXT and associated antibiotic resistance genes, and its integration into the prfC site. Results of phylogenetic analysis of the intSXT gene of clonally similar V. cholerae showed a clear difference between dfr18(+) and dfrA1(+) V. cholerae O1 isolates. This is the first report of the occurrence of SXT harbouring sulII, strAB, dfr18 and/or dfrA1 genes in environmental V. cholerae O1 isolated prior to 1992 from Varanasi, India, and suggests emergence of SXT(+) antibiotic-resistant V. cholerae O139 and O1 from an environmental V. cholerae progenitor by acquisition of SXT and antibiotic-resistant gene clusters.

Vibrio cholerae non-O1, non-O139 strains isolated before 1992 from Varanasi, India are multiple drug resistant, contain intSXT, dfr18 and aadA5 genes.

In this study, we report the presence of the SXT element and Class I integron in Vibrio cholerae non-O1, non-O139 strains isolated from Varanasi, India. Isolates were resistant to cotrimoxazole, trimethoprim and/or streptomycin, furazolidone and ampicillin. None contained plasmids. Polymerase chain reaction (PCR) and DNA sequencing revealed the presence of antibiotic resistance gene cassettes, aadA1, aadA2, aadA5 and dfrA15, in the Class I integron and SXT, an integrative conjugative element containing dfr18, sulII and strAB, in three and six of the isolates respectively. Conjugation experiments, followed by PCR analysis of transconjugants, provided evidence for the transferable nature of intSXT and associated antibiotic resistance gene cassettes. This is the first report of the occurrence of SXT ICE, dfr18, sulII, strAB and aadA5 genes in environmental V. cholerae non-O1, non-O139 strains from Varanasi, India, that had been isolated before 1992.

Application of DNA-based methods in typing Vibrio cholerae strains.

Molecular biology-based techniques based on microbial genotype or DNA sequence have emerged as a basic tool in biological research and in the establishment of large databases of characterized organisms. Genotyping methods have the potential to provide information on subtypes of the organism and their source and/or origin of infection, and to recognize particularly virulent strains of the organism and monitor vaccination programs. Pulsed-field gel electrophoresis, ribotyping, CTX typing, amplified fragment length polymorphism, enterobacterial intergenic consensus sequence-PCR, multilocus sequence typing and microarray methods are more often used for the determination of genetic changes of toxigenic and nontoxigenic Vibrio cholerae strains, origin of infection and relationship between clinical and environmental strains, with the simultaneous detection of the number of copies and types of CTX prophages and genes required for persistence in diverse aquatic environments. This review will discuss DNA-based techniques for the molecular analysis of V. cholerae, its application and future directions.

Concurrent sorption of Zn(II), Cu(II) and Co(II) by Oscillatoria angustissima as a function of pH in binary and ternary metal solutions.

This paper reports biosorption of Zn(II), Cu(II) and Co(II) onto O. angustissima biomass from single, binary and ternary metal solutions, as a function of pH and metal concentrations via Central Composite Design generated by statistical software package Design Expert 6.0. The experimental design revealed that metal interactions could be best studied at lower pH range i.e. 4.0-5.0, which facilitates adequate availability of all the metal ions. The sorption capacities for single metal decreased in the order Zn(II)>Co(II)>Cu(II). In absence of any interfering metals, at pH 4.0 and an initial metal concentration of 0.5 mM in the solution, the adsorption capacities were 0.33 mmol/g Zn(II), 0.26 mmol/g Co(II) and 0.12 mmol/g Cu(II). In a binary system, copper inhibited both Zn(II) and Co(II) sorption but the extent of inhibition of former was greater than the latter; sorption values being 0.14 mmol/g Zn(II) and 0.27 mmol/g Co(II) at initial Zn(II) and Co(II) concentration of 1.5 mM each, pH 4.0 and 1mM Cu(II) as the interfering metal. Zn(II) and Co(II) were equally antagonistic to each others sorption; Zn(II) and Co(II) sorption being 0.23 and 0.24 mmol/g, respectively, at initial metal concentration of 1.5 mM each, pH 4.0 and 1mM interfering metal concentration. In contrast, Cu(II) sorption remained almost unaffected at lower concentrations of the competing metals. Thus, in binary system inhibition dominance observed was Cu(II)>Zn(II), Cu(II)>Co(II) and Zn(II) approximately Co(II), due to this the biosorbent exhibited net preference/affinity for Cu(II) sorption over Zn(II) or Co(II). Hence, the affinity series showed a trend of Cu(II)>Co(II)>Zn(II). In a ternary system, increasing Co(II) concentration exhibited protection against the inhibitory effect of Cu(II) on Zn(II) sorption. On the other hand, the inhibitory effect of Zn(II) and Cu(II) on Co(II) sorption was additive. The model equation for metal interactions was found to be valid within the design space.

Microbial biomass: an economical alternative for removal of heavy metals from waste water.

Today indiscriminate and uncontrolled discharge of metal contaminated industrial effluents into the environment has become an issue of major concern. Heavy metals, being non-biodegradable and persistent, beyond a permissible concentration form unspecific compounds inside the cells thereby causing cellular toxicity. The only alternative to remove them from the wastewater is by immobilizing them. The conventional methods adopted earlier for this purpose included chemical precipitation, oxidation, reduction, filtration, electrochemical treatment, evaporation, adsorption and ion-exchange resins. These methods require high energy inputs especially when it refers to dilute solutions. Here microbial biomass offers an economical option for removing heavy metals by the phenomenon of biosorption. Non-living or dead biomass sequester metal(s) on their cell surface due to certain reactive groups available like carboxyl, amine, imidazole, phosphate, sulphydryl, sulfate and hydroxyl. The process can be made economical by procuring spent biomass from industry or naturally available bulk biomass. A batch or a continuous process of removal of heavy metals directly from effluents can be developed in a fixed bed reactor using the immobilized biomass. Further biosorption potential of the biomass can be improved by various physical and chemical treatments. The availability of variety of microbial biomass and their metal binding potential makes it a economical and sustainable option for developing effluent treatment process for removal and recovery of heavy metals.