Preclinical characterization and in silico safety assessment of three virulent bacteriophages targeting carbapenem-resistant uropathogenic Escherichia coli.

Phage therapy has recently been revitalized in the West with many successful applications against multi-drug-resistant bacterial infections. However, the lack of geographically diverse bacteriophage (phage) genomes has constrained our understanding of phage diversity and its genetics underpinning host specificity, lytic capability, and phage-bacteria co-evolution. This study aims to locally isolate virulent phages against uropathogenic Escherichia coli (E. coli) and study its phenotypic and genomic features. Three obligately virulent Escherichia phages (øEc_Makalu_001, øEc_Makalu_002, and øEc_Makalu_003) that could infect uropathogenic E. coli were isolated and characterized. All three phages belonged to Krischvirus genus. One-step growth curve showed that the latent period of the phages ranged from 15 to 20 min, the outbreak period ~ 50 min, and the burst size ranged between 74 and 127 PFU/bacterium. Moreover, the phages could tolerate a pH range of 6 to 9 and a temperature range of 25-37 °C for up to 180 min without significant loss of phage viability. All phages showed a broad host spectrum and could lyse up to 30% of the 35 tested E. coli isolates. Genomes of all phages were approximately ~ 163 kb with a gene density of 1.73 gene/kbp and an average gene length of ~ 951 bp. The coding density in all phages was approximately 95%. Putative lysin, holin, endolysin, and spanin genes were found in the genomes of all three phages. All phages were strictly virulent with functional lysis modules and lacked any known virulence or toxin genes and antimicrobial resistance genes. Pre-clinical experimental and genomic analysis suggest these phages may be suitable candidates for therapeutic applications.

Genomic characterization of three bacteriophages targeting multidrug resistant clinical isolates of Escherichia, Klebsiella and Salmonella.

Application of bacteriophages (phages) to treat complex multidrug-resistant bacterial infection is gaining traction because of its efficacy and universal availability. However, as phages are specific to their host, a diverse collection of locally isolated phage from various geographical locations is required to formulate a wide host range phage cocktail. Here, we report morphological and genomic features of three newly isolated phages from river water of the urban region in Kathmandu, Nepal, targeting three different bacteria (Escherichia coli, Klebsiella pneumoniae and Salmonella enterica.) from the Enterobacteriaceae family. Morphological identification and genome analysis indicated that two phages (Escherichia phage vB_EcoM_TU01 and Klebsiella phage vB_KpnP_TU02) were strictly lytic and free from integrases, virulence factors, toxins and known antimicrobial resistance genes, whereas Salmonella phage vB_SalS_TU03 was possibly a temperate phage. The genomic features of these phages indicate that natural phages are capable of lysing pathogenic bacteria and may have potential in bacterial biocontrol.

Isolation and Characterization of Lytic Bacteriophage Against Multi-drug Resistant Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen frequently causing healthcare-associated infections. The apocalyptic rise of antimicrobial resistance has rekindled interest in age-old phage therapy that uses phages (viruses that infect bacteria) to kill the targeted pathogenic bacteria. Because of its specificity, phages are often considered as potential personalized therapeutic candidate for treating bacterial infections. METHODS: In this study, we isolated and purified lytic phages against multi-drug resistant P. aeruginosa using soft agar overlay technique. Phage characteristics like thermal and pH stability, latent period and burst size were determined using one-step growth assay while multiple host range spectrum was determined by spot assay. The phages were further characterized using protein profiling. RESULTS: Three Pseudomonas phages (øCDBT-PA31, øCDBT-PA56 and øCDBT-PA58) were isolated from the holy rivers of Kathmandu valley. Among 3 phages, øCDBT-PA31 demonstrated multiple host range and could lyse multi-drug resistant strain of P. aeruginosa. Further, øCDBT-PA31 showed latent period of 30 minutes with corresponding burst sizes of 423-525 PFU/cell. Interestingly, øCDBT-PA31 also tolerated a wide range of adverse conditions, such as high temperature (50°C) and pH 3-11. Further, protein profiling revealed that øCDBT-PA31 has 4 and øCDBT-PA11 had 3 distinct bands in the gradient gel ranging from approximately 3.5-29 kilodaltons (kDa) suggesting them to be morphologically distinct from each other. CONCLUSIONS: As multi-drug resistant bacteria are emerging as a global problem, lytic phages can be an alternative treatment strategy when all available antibiotics fail.

Pharmacokinetics and Pharmacodynamics of a Novel Virulent Klebsiella Phage Kp_Pokalde_002 in a Mouse Model.

Phage therapy is one of the most promising alternatives to antibiotics as we face global antibiotic resistance crisis. However, the pharmacokinetics (PK) and pharmacodynamics (PD) of phage therapy are largely unknown. In the present study, we aimed to evaluate the PK/PD of a locally isolated virulent novel øKp_Pokalde_002 (Podoviridae, C1 morphotype) that infects carbapenem-resistant Klebsiella pneumoniae (Kp56) using oral and intraperitoneal (IP) route in a mouse model. The result showed that the øKp_Pokalde_002 rapidly distributed into the systemic circulation within an hour via both oral and IP routes. A higher concentration of phage in plasma was found after 4 h (2.3 x 105 PFU/ml) and 8 h (7.3 x 104 PFU/ml) of administration through IP and oral route, respectively. The phage titer significantly decreased in the blood and other tissues, liver, kidneys, and spleen after 24 h and completely cleared after 72 h of administration. In the Kp56 infection model, the bacterial count significantly decreased in the blood and other organs by 4-7 log10 CFU/ml after 24 h of øKp_Pokalde_002 administration. Elimination half-life of øKp_Pokalde_002 was relatively shorter in the presence of host-bacteria Kp56 compared to phage only, suggesting rapid clearance of phage in the presence of susceptible host. Further, administration of the øKp_Pokalde_002 alone in healthy mice (via IP or oral) did not stimulate pro-inflammatory cytokines (TNF-α and IL-6). Also, treatment with øKp_Pokalde_002 resulted in a significant reduction of pro-inflammatory cytokines (TNF-α and IL-6) caused by bacterial infection, thereby reducing the tissue inflammation. In conclusion, the øKp_Pokalde_002 possess good PK/PD properties and can be considered as a potent therapeutic candidate for future phage therapy in carbapenem-resistant K. pneumoniae infections.

Maculosin, a non-toxic antioxidant compound isolated from Streptomyces sp. KTM18.

CONTEXT: Streptomyces species are prolific sources of bioactive secondary metabolites known especially for their antimicrobial and anticancer activities. OBJECTIVE: This study sought to isolate and characterize antioxidant molecules biosynthesized by Streptomyces sp. KTM18. The antioxidant potential of an isolated compound and its toxicity were accessed. MATERIALS AND METHODS: The compound was purified using bioassay-guided chromatography techniques. Nuclear magnetic resonance (NMR) experiments were carried out for structure elucidation. The antioxidant potential of the isolated compound was determined using DPPH free radical scavenging assay. The toxicity of the isolated compound was measured using a brine shrimp lethality (BSL) assay. RESULTS: Ethyl acetate extract of Streptomyces sp. KTM18 showed more than 90% inhibition of DPPH free radical at 50 µg/mL of the test concentration. These data were the strongest among 13 Streptomyces isolates (KTM12-KTM24). The active molecule was isolated and characterized as maculosin (molecular formula, C14H16N2O3 as determined by the [M + H]+ peak at 261.1259). The DPPH free radical scavenging activity of pure maculosin was higher (IC50, 2.16 ± 0.05 µg/mL) than that of commercial butylated hydroxyanisole (BHA) (IC50, 4.8 ± 0.05 µg/mL). No toxicity was observed for maculosin (LD50, <128 µg/mL) in brine shrimp lethality assay (BSLA) up to the compound's antioxidant activity (IC50) concentration range. The commercial standard, berberine chloride, showed toxicity in BSLA with an LD50 value of 8.63 ± 0.15 µg/mL. CONCLUSIONS: Maculosin may be a leading drug candidate in various cosmetic and therapeutic applications owing to its strong antioxidant and non-toxic properties.

Therapeutic Efficacy of Bacteriophage Therapy to Treat Carbapenem Resistant Klebsiella Pneumoniae in Mouse Model.

BACKGROUND: Global emergence of carbapenem-resistant Klebsiella pneumoniae is a major public health concern. Phage therapy - application of lytic phage to kill pathogenic bacteria - is considered as one of the promising alternatives to tackle this antibiotic crisis in recent days. This study aimed to isolate, characterize and evaluate therapeutic efficacy of a novel K. pneumoniae phage in mouse model. METHODS: A novel lytic bacteriophage (phage) Kp_Pokalde_002 was isolated against carbapenem-resistant K. pneumoniae (Kp56) and characterized. Safety parameters of the phage were evaluated by bioinformatic analysis of its genome. A lethal dose (~1×107 CFU/mouse) of Kp56 was determined and administrated in the mice. The infected mice were treated with phage Kp_Pokalde_002 at a multiplicity of infection (MOI) 1.0 (~1×107 PFU/mouse) via both oral and intraperitoneal (IP) routes. RESULTS: Isolated phage comprised an icosahedral capsid with a short tail. Based on genome analysis, the phage was strictly lytic belonging the Podoviridae family (T7-like viruses) and free from any virulent and antibiotic-resistant genes. The phage was stable up to 60 °C for 30 minutes and effective between pH 4 to 11 (optimum pH 9). The phage exhibited a short latent period (20 minutes) with burst size of 121 phage particles per infected cell. The infected mice were rescued with the phage therapy via both oral and IP route. Significant reduction of bacterial load (3-7 log10 CFU/ml) in the blood and lung was observed in the treatment group. CONCLUSIONS: We provide an evidence of successful phage therapy against carbapenem-resistant K. pneumoniae infected mouse model using locally isolated lytic phage.

Prospects of Indole derivatives as methyl transfer inhibitors: antimicrobial resistance managers.

BACKGROUND: It is prudent that novel classes of antibiotics be urgently developed to manage the WHO prioritized multi-drug resistant (MDR) pathogens posing an unprecedented medical crisis. Simultaneously, multiple essential proteins have to be targeted to prevent easy resistance development. METHODS: An integration of structure-based virtual screening and ligand-based virtual screening was employed to explore the antimicrobial properties of indole derivatives from a compound database. RESULTS: Whole-genome sequences of the target pathogens were aligned exploiting DNA alignment potential of MAUVE to identify putative common lead target proteins. S-adenosyl methionine (SAM) biosynthesizing MetK was taken as the lead target and various literature searches revealed that SAM is a critical metabolite. Furthermore, SAM utilizing CobA involved in the B12 biosynthesis pathway, Dam in the regulation of replication and protein expression, and TrmD in methylation of tRNA were also taken as drug targets. The ligand library of 715 indole derivatives chosen based on kinase inhibition potential of indoles was created from which 102 were pursued based on ADME/T scores. Among these, 5 potential inhibitors of MetK in N. gonorrhoeae were further expanded to molecular docking studies in MetK proteins of all nine pathogens among which 3 derivatives exhibited inhibition potential. These 3 upon docking in other SAM utilizing enzymes, CobA, Dam, and TrmD gave 2 potential compounds with multiple targets. Further, docking with human MetK homolog also showed probable inhibitory effects however SAM requirements can be replenished from external sources since SAM transporters are present in humans. CONCLUSIONS: We believe these molecules 3-[(4-hydroxyphenyl)methyl]-6-(1H-indol-3-ylmethyl)piperazine-2,5-dione (ZINC04899565) and 1-[(3S)-3-[5-(1H-indol-3-ylmethyl)-1,3,4-oxadiazol-2-yl]pyrrolidin-1-yl]ethanone (ZINC49171024) could be a starting point to help develop broad-spectrum antibiotics against infections caused by N. gonorrhoeae, A. baumannii, C. coli, K. pneumoniae, E. faecium, H. pylori, P. aeruginosa, S. aureus and S. typhi.

Complete Genome Sequence of Myophage Ec_Makalu_002, Which Infects Uropathogenic Escherichia coli.

We isolated phage Ec_Makalu_002, which infects uropathogenic strains of Escherichia coli Here, we report its complete genome sequence, annotated features, and relatedness to other phages.

A Review on the Effect of Plant Extract on Mesenchymal Stem Cell Proliferation and Differentiation.

Stem cell has immense potential in regenerative cellular therapy. Mesenchymal stem cells (MSCs) can become a potential attractive candidate for therapy due to its remarkable ability of self-renewal and differentiation into three lineages, i.e., ectoderm, mesoderm, and endoderm. Stem cell holds tremendous promises in the field of tissue regeneration and transplantation for disease treatments. Globally, medicinal plants are being used for the treatment and prevention of a variety of diseases. Phytochemicals like naringin, icariin, genistein, and resveratrol obtained from plants have been extensively used in traditional medicine for centuries. Certain bioactive compounds from plants increase the rate of tissue regeneration, differentiation, and immunomodulation. Several studies show that bioactive compounds from plants have a specific role (bioactive mediator) in regulating the rate of cell division and differentiation through complex signal pathways like BMP2, Runx2, and Wnt. The use of plant bioactive phytochemicals may also become promising in treating diseases like osteoporosis, neurodegenerative disorders, and other tissue degenerative disorders. Thus, the present review article is aimed at highlighting the roles and consequences of plant extracts on MSCs proliferation and desired lineage differentiations.

Emergence of dengue in Nepal.

Dengue virus is a major health problem in Nepal. The endogenous dengue appeared in 2006 in the country with reported outbreaks in 2010, 2013 and 2016. Eleven years vertical data show there were sporadic cases in all the years and mostly adults between 25 and 40 years of age were infected with dengue virus. Compared with primary infections, secondary infections were observed in relatively larger numbers during the period of 2008-2016. Most of the cases had symptoms of dengue fever; while 7 and 19 cases demonstrated dengue hemorrhagic fever/dengue shock syndrome in 2010 and 2013 respectively. The proportion of dengue hemorrhagic fever amongst all cases of dengue fever was 2.5:4.7% in 2010 and 2013. We found there is shift of serotype from dengue virus serotype-1 (DENV-1) in 2010, DENV-2 in 2013 and DENV-1 in 2016. We feel there is urgent need for better community, hospital and laboratory based surveillance system capable of monitoring the circulating dengue virus (DENV) serotypes in different districts of Nepal. With improvement in surveillance system and efficient management of cases, the case fatality rate due to severe dengue can be reduced.

Isolation and Physicochemical Characterization of Laccase from Ganoderma lucidum-CDBT1 Isolated from Its Native Habitat in Nepal.

At present, few organisms are known to and capable of naturally producing laccases and white rot fungi are one such group. In the present study, three fungal species, namely, Ganoderma lucidum-CDBT1, Ganoderma japonicum, and Lentinula edodes, isolated from their native habitat in Nepal were screened for laccase production, and G. lucidum-CDBT1 was found to express highest levels of enzyme (day 10 culture media showed 0.92 IU/mg total protein or 92 IU/mL laccase activity with ABTS as substrate). Lignin extracted from rice straw was used in Olga medium for laccase production and isolation from G. lucidum-CDBT1. Presence of lignin (5 g/L) and copper sulfate (30 µM) in the media increased the extracellular laccase content by 111% and 114%, respectively. The laccase enzyme produced by G. lucidum-CDBT1 was fractionated by ammonium sulfate and purified by DEAE Sepharose anion exchange chromatography. The purified enzyme was found to have a molecular mass of 43 kDa and exhibits optimal activity at pH 5.0 and 30°C. The isolated laccase was thermally stable for up to 70°C for 1 h and exhibited broad pH stability. The kinetic constants, Km , Vmax, and Kcat, determined using 2,2'-azinobis-(-3-ethylbenzothiazoline-6-sulfonic acid) as substrate were found to be 110 µM, 36 µmol/min/mg, and 246 min-1, respectively. The isolated thermostable laccase will be used in future experiments for delignification process.

Re-emergence of dengue virus serotype 2 strains in the 2013 outbreak in Nepal.

BACKGROUND & OBJECTIVES: Epidemiological interventions and mosquito control are the available measures for dengue control. The former approach uses serotype and genetic information on the circulating virus strains. Dengue has been frequently reported from Nepal, but this information is mostly lacking. The present study was done to generate a comprehensive clinical and virological picture of a dengue outbreak in Nepal during 2013. METHODS: A hospital-based study involving patients from five districts of Nepal was carried out. Demographic information, clinical details and dengue serological status were obtained. Viral RNA was characterized at the molecular level by reverse-transcription polymerase chain reaction (RT-PCR), nucleotide sequencing and phylogenetic analysis. RESULTS: From among the 2340 laboratory-confirmed dengue cases during the study period, 198 patients consented for the study. Clinically they had fever (100%), headache (59.1%), rashes (18.2%), retro-orbital pain (30.3%), vomiting (15.1%), joint pain (28.8%) and thrombocytopenia (74.3%). Fifteen (7.5%) of them had mucosal bleeding manifestations, and the rest were uncomplicated dengue fever. The patients were mostly adults with a mean age of 45.75 ± 38.61 yr. Of the 52 acute serum samples tested, 15 were positive in RT-PCR. The causative virus was identified as DENV serotype 2 belonging to the Cosmopolitan genotype. INTERPRETATIONS & CONCLUSIONS: We report here the involvement of DENV serotype 2 in an outbreak in Nepal in 2013. Earlier outbreaks in the region in 2010 were attributed to serotype 1 virus. As serotype shifts are frequently associated with secondary infections and severe disease, there is a need for enhancing surveillance especially in the monsoon and post-monsoon periods to prevent large-scale, severe dengue outbreaks in the region.

Molecular studies of E. coli mercuric reductase gene (merA) and its impact on human health.

Bacterial plasmids encode resistance systems for toxic metal ions including Hg2+ functioning by energy-dependent efflux of toxic ions. The inducible mercury resistance (mer) operon encodes both a mercuric ion uptake and a detoxification enzymes. In Gram-negative bacteria especially in E. coli, a periplasmic protein, MerP, an inner- membrane transport protein, MerT, and a cytoplasmic enzyme, mercuric reductase (the MerA protein), are responsible for the transport of mercuric ions into cell and their reduction to elemental mercury, Hg0. Phytoremediation involves the use of plants to extract, detoxify and/or sequester environmental pollutants from soil and water. Transgenic plants cleave mercury ions from methyl-mercury complexes; reduce mercury ions to the metallic form; take up metallic mercury through their roots; and evolve less toxic elemental mercury. PCR were performed to detect 1695 bp of mercuric reductase gene (merA), which is mainly responsible for the conversion of mercuric (Hg+2) and mercurous (Hg+1) ions into non-toxic elemental mercury. PCR products of putative merA genes from environmental E. coli strains were purified and cloned into a plant expression vector pRT100. The construct will be transformed in calli of Nicotiana plants.