Repurposing of FDA approved drugs against uropathogenic Escherichia coli: In silico, in vitro, and in vivo analysis.

Urinary tract infections (UTIs) are a serious health concern worldwide. Treatment of UTIs is becoming a challenge as uropathogenic Escherichia coli (UPEC), which is the most common etiological agent, has developed resistance to the main classes of antibiotics. Small molecules that interfere with metabolic processes rather than growth are attractive alternatives to conventional antibiotics. Repurposing of already known drugs for treating infectious diseases could be an attractive avenue for finding novel therapeutics against infections caused by UPEC. Virtual screenings enable the rapid and economical identification of target ligands from large libraries of compounds, reducing the cost and time of traditional drug discovery. Moreover, the drugs that have been approved by the FDA have low cytotoxicity and good pharmacological characteristics. In this work, we targeted the HisC enzyme of the histidine biosynthetic pathway as enzymes of this pathway are absent in humans. We screened the library of FDA-approved drugs against HisC via molecular docking, and four hits (Docetaxel, Suramin, Digitoxin, and Nystatin) showing the highest binding energy were selected. These were further tested for antibacterial activity, which was observed only for Docetaxel (MIC value of 640 µg/ml); therefore, Docetaxel was further tested for its efficacy in vivo in murine catheter UTI model and antibiofilm activity using crystal violet staining and scanning electron microscopy. Docetaxel inhibited biofilm formation and reduced the bacterial load in urine, kidney, and bladder. Docking studies revealed that Docetaxel acts by blocking the binding site of HisC to the native substrate by competitive inhibition. Docetaxel may be a potential new inhibitor for UPEC with antibacterial and antibiofilm capability.

Characterization, genome analysis and in vitro activity of a novel phage vB_EcoA_RDN8.1 active against multi-drug resistant and extensively drug-resistant biofilm-forming uropathogenic Escherichia coli isolates, India.

AIM: We aimed to study host range, stability, genome and antibiofilm activity of a novel phage vB_EcoA_RDN8.1 active against multi-drug resistant (MDR) and extensively drug-resistant (XDR) biofilm-forming uropathogenic Escherichia coli isolates. METHODS AND RESULTS: A novel lytic phage vB_EcoA_RDN8.1 active against UPEC strains resistant to third-generation cephalosporins, fluoroquinolones, aminoglycosides, imipenem, beta-lactamase inhibitor combination and polymyxins was isolated from community raw sewage water of Chandigarh. It exhibited a clear plaque morphology and a burst size of 250. In the time-kill assay, the maximum amount of killing was achieved at MOI 1.0. vB_EcoA_RDN8.1 belongs to the family Autographiviridae, has a genome size of 39.5 kb with a GC content of 51.6%. It was stable over a wide range of temperatures and pH. It was able to inhibit biofilm formation which may be related to an endolysin encoded by ORF 19. CONCLUSIONS: The vB_EcoA_RDN8.1 is a novel lytic phage that has the potential for inclusion into phage cocktails being developed for the treatment of urinary tract infections (UTIs) caused by highly drug-resistant UPEC. SIGNIFICANCE AND IMPACT OF THE STUDY: We provide a detailed characterization of a novel lytic Escherichia phage with antibiofilm activity having a potential application against MDR and XDR UPEC causing UTIs.

Human Papilloma Virus-Associated Oral Pharyngeal Squamous Cell Carcinoma: Prevalence, Prevention, and Awareness of Vaccination in the Indian Population.

Human papilloma virus (HPV), one of the most common sexually transmitted infections, plays a pivotal role in head and neck cancer, primarily oral and oropharyngeal squamous cell carcinomas. HPV is a vaccine-preventable disease that also contributes to cervical cancer. Although HPV vaccination effectively protects the individual against all HPV-associated human carcinomas, the awareness of HPV vaccination and its acceptance is poor in developing nations like India. India has a very high burden of oral cancer, and, unfortunately, the morbidity and mortality rates are also high as the cancer is often detected at an advanced stage. In this review, we explore the prevalence of HPV-associated head and neck squamous cell carcinoma among the Indian population and the awareness of HPV vaccination among Indian youth. Since the prognosis for HPV-associated head and neck squamous cell carcinoma is good, early diagnosis of the cancer is crucial in improving the outcome of the treatment modalities. Efforts are needed to create and increase awareness of HPV vaccination. Routine screening for HPV infection in oral mucosa can prevent the silent epidemic from taking the lives of many young people.

Computational Guided Drug Targets Identification against Extended-Spectrum Beta-Lactamase-Producing Multi-Drug Resistant Uropathogenic Escherichia coli.

Urinary tract infections (UTIs) are one of the most frequent bacterial infections in the world, both in the hospital and community settings. Uropathogenic Escherichia coli (UPEC) are the predominant etiological agents causing UTIs. Extended-spectrum beta-lactamase (ESBL) production is a prominent mechanism of resistance that hinders the antimicrobial treatment of UTIs caused by UPEC and poses a substantial danger to the arsenal of antibiotics now in use. As bacteria have several methods to counteract the effects of antibiotics, identifying new potential drug targets may help in the design of new antimicrobial agents, and in the control of the rising trend of antimicrobial resistance (AMR). The public availability of the entire genome sequences of humans and many disease-causing organisms has accelerated the hunt for viable therapeutic targets. Using a unique, hierarchical, in silico technique using computational tools, we discovered and described potential therapeutic drug targets against the ESBL-producing UPEC strain NA114. Three different sets of proteins (chokepoint, virulence, and resistance genes) were explored in phase 1. In phase 2, proteins shortlisted from phase 1 were analyzed for their essentiality, non-homology to the human genome, and gut flora. In phase 3, the further shortlisted putative drug targets were qualitatively characterized, including their subcellular location, broad-spectrum potential, and druggability evaluations. We found seven distinct targets for the pathogen that showed no similarity to the human proteome. Thus, possibilities for cross-reactivity between a target-specific antibacterial and human proteins were minimized. The subcellular locations of two targets, ECNA114_0085 and ECNA114_1060, were predicted as cytoplasmic and periplasmic, respectively. These proteins play an important role in bacterial peptidoglycan biosynthesis and inositol phosphate metabolism, and can be used in the design of drugs against these bacteria. Inhibition of these proteins will be helpful to combat infections caused by MDR UPEC.

Vibrio Phage VMJ710 Can Prevent and Treat Disease Caused by Pathogenic MDR V. cholerae O1 in an Infant Mouse Model.

Cholera, a disease of antiquity, is still festering in developing countries that lack safe drinking water and sewage disposal. Vibrio cholerae, the causative agent of cholera, has developed multi-drug resistance to many antimicrobial agents. In aquatic habitats, phages are known to influence the occurrence and dispersion of pathogenic V. cholerae. We isolated Vibrio phage VMJ710 from a community sewage water sample of Manimajra, Chandigarh, in 2015 during an outbreak of cholera. It lysed 46% of multidrug-resistant V. cholerae O1 strains. It had significantly reduced the bacterial density within the first 4-6 h of treatment at the three multiplicity of infection (MOI 0.01, 0.1, and 1.0) values used. No bacterial resistance was observed against phage VMJ710 for 20 h in the time-kill assay. It is nearest to an ICP1 phage, i.e., Vibrio phage ICP1_2012 (MH310936.1), belonging to the class Caudoviricetes. ICP1 phages have been the dominant bacteriophages found in cholera patients' stools since 2001. Comparative genome analysis of phage VMJ710 and related phages indicated a high level of genetic conservation. The phage was stable over a wide range of temperatures and pH, which will be an advantage for applications in different environmental settings. The phage VMJ710 showed a reduction in biofilm mass growth, bacterial dispersal, and a clear disruption of bacterial biofilm structure. We further tested the phage VMJ710 for its potential therapeutic and prophylactic properties using infant BALB/c mice. Bacterial counts were reduced significantly when phages were administered before and after the challenge of orogastric inoculation with V. cholerae serotype O1. A comprehensive whole genome study revealed no indication of lysogenic genes, genes associated with possible virulence factors, or antibiotic resistance. Based on all these properties, phage VMJ710 can be a suitable candidate for oral phage administration and could be a viable method of combatting cholera infection caused by MDR V. cholerae pathogenic strains.

In Vitro and In Vivo Studies of Heraclenol as a Novel Bacterial Histidine Biosynthesis Inhibitor against Invasive and Biofilm-Forming Uropathogenic Escherichia coli.

Globally, urinary tract infections (UTIs) are one of the most frequent bacterial infections. Uropathogenic Escherichia coli (UPEC) are the predominant etiological agents causing community and healthcare-associated UTIs. Biofilm formation is an important pathogenetic mechanism of UPEC responsible for chronic and recurrent infections. The development of high levels of antimicrobial resistance (AMR) among UPEC has complicated therapeutic management. Newer antimicrobial agents are needed to tackle the increasing trend of AMR and inhibit biofilms. Heraclenol is a natural furocoumarin compound that inhibits histidine biosynthesis selectively. In this study, for the first time, we have demonstrated the antimicrobial and antibiofilm activity of heraclenol against UPEC. The drug reduced the bacterial load in the murine catheter UTI model by ≥4 logs. The drug effectively reduced bacterial loads in kidney, bladder, and urine samples. On histopathological examination, heraclenol treatment showed a reversal of inflammatory changes in the bladder and kidney tissues. It reduced the biofilm formation by 70%. The MIC value of heraclenol was observed to be high (1024 µg/mL), though the drug at MIC concentration did not have significant cytotoxicity on the Vero cell line. Further molecular docking revealed that heraclenol binds to the active site of the HisC, thereby preventing its activation by native substrate, which might be responsible for its antibacterial and antibiofilm activity. Since the high MIC of heraclenol is not achievable clinically in human tissues, further chemical modifications will be required to lower the drug's MIC value and increase its potency. Alternatively, its synergistic action with other antimicrobials may also be studied.

Complete Genome Sequence of Salmonella Phage vB_SenA_SM5, Active against Multidrug-Resistant Salmonella enterica Serovar Typhi Isolates.

Phage vB_SenA_SM5, active against multiple isolates of multidrug-resistant Salmonella enterica serovar Typhi, was isolated from the sewage water of a tertiary-care referral hospital in Chandigarh, India. It has a 154.4-kb-long double-stranded DNA genome, belongs to the family Ackermannviridae, and is closest to Salmonella phage Chennai, which was isolated in southern India.

Genome Analysis and Antibiofilm Activity of Phage 590B against Multidrug-Resistant and Extensively Drug-Resistant Uropathogenic Escherichia coli Isolates, India.

Urinary tract infections (UTIs) are among the most common bacterial infections in humans. Uropathogenic Escherichia coli (UPEC), which are the most frequent agents causing community as well as hospital-acquired UTIs, have become highly drug-resistant, thus making the treatment of these infections challenging. Recently, the use of bacteriophages (or 'phages') against multidrug-resistant (MDR) and extensively drug-resistant (XDR) microorganisms has garnered significant global attention. Bacterial biofilms play a vital role in the pathogenesis of UTIs caused by UPEC. Phages have the potential to disrupt bacterial biofilms using lytic enzymes such as EPS depolymerases and endolysins. We isolated a lytic phage (590B) from community sewage in Chandigarh, which was active against multiple MDR and XDR biofilm-forming UPEC strains. During whole-genome sequencing, the 44.3 kb long genome of phage 590B encoded 75 ORFs, of which 40 were functionally annotated based on homology with similar phage proteins in the database. Comparative analysis of associated phage genomes indicated that phage 590B evolved independently and had a distinct taxonomic position within the genus Kagunavirus in the subfamily Guernseyvirinae of Siphoviridae. The phage disrupted biofilm mass effectively when applied to 24 h old biofilms formed on the Foley silicon catheter and coverslip biofilm models. To study the effect of intact biofilm architecture on phage predation, the biofilms were disrupted. The phage reduced the viable cells by 0.6-1.0 order of magnitude after 24 h of incubation. Regrowth and intact bacterial cells were observed in the phage-treated planktonic culture and biofilms, respectively, which indicated the emergence of phage-resistant bacterial variants. The phage genome encoded an endolysin which might have a role in the disruption and inhibition of bacterial biofilms. Moreover, the genome lacked genes encoding toxins, virulence factors, antibiotic resistance, or lysogeny. Therefore, lytic phage 590B may be a good alternative to antibiotics and can be included in phage cocktails for the treatment of UTIs caused by biofilm-forming MDR and XDR UPEC strains.

Whole genome sequencing and in vitro activity data of Escherichia phage NTEC3 against multidrug-resistant Uropathogenic and extensively drug-resistant Uropathogenic E. coli isolates.

This data article describes the whole-genome sequencing and in vitro activity data of Escherichia phage NTEC3 isolated from a community sewage sample in Chandigarh, India. The phage NTEC3 was active against multi-drug-resistant (MDR) and extensively drug-resistant (XDR) biofilm-forming Uropathogenic Escherichia coli (UPEC) strains. The genome of this phage was linear, double-stranded, and 44.2 kb long in size. A total of 72 ORFs (open reading frames) were predicted and 30 ORFs were encoded for functional proteins. The phage belonged to the Kagunavirus genus of the Siphoviridae family. Phylogenetic analysis using DNA polymerase was performed to understand the phage evolutionary relationships. Genes encoding for lysogeny, virulence, toxins, antibiotic resistance, and the CRISPR/CRISPR-like system were not found during screening. The annotated genome was deposited in Genbank under the accession number OK539620.

Study on Blood Serum Levels of Heavy and Trace Metals in Chronic Non-Healing Wounds.

Wound healing is a complex, highly regulated process that is important in sustaining the skin barrier function. The etiologic relation of specific metals is not adequately described for chronic non-healing wounds. The aim of this study was to estimate heavy and trace metals in chronic non-healing wound and their association with wound healing. The levels of zinc, selenium, copper, magnesium, chromium, cadmium, iron, and lead were estimated in serum of chronic non-healing wound patients using atomic absorption spectrophotometry. The tests were carried out in 50 patients with chronic non-healing wound and thirty healthy volunteers as control. The serum levels of elements namely zinc, selenium, copper, magnesium, and chromium were significantly reduced in chronic non-healing wounds (P < .001) as compared to control. Lead and cadmium levels had shown the significantly increasing trend in chronic non-healing wound cases (P < .001). The present study demonstrated a significant decrease in serum, levels of selenium, zinc, copper, magnesium, iron, and chromium levels in patients with chronic non-healing wound indicating an association between these elements and wound healing. To summarize the findings of our research, hence trace elements were decreasing in chronic non-healing wound patients suggesting their role in wound healing.

Characterization and in vitro activity of a lytic phage RDN37 isolated from community sewage water active against MDR Uropathogenic E. coli.

PURPOSE: Due to the emergence of multidrug-resistant Uropathogenic E. coli (MDR-UPEC) strains, alternatives to antibiotics like phage therapy have been sought. The present study was planned to characterize and test the activity of a phage (RDN37) which was isolated from community sewage water of Chandigarh and was found to be active against MDR-UPEC. MATERIALS AND METHODS: We studied the morphology of the phage by transmission electron microscopy and determined one-step growth curve analysis and stability of the phage at various temperature and pH ranges. PCR amplification and Sanger sequencing were performed to confirm the phage family. Genome sequences from 12 related phages (BLASTn identity >95%) were obtained from the NCBI database in GenBank format. A phylogenetic analysis was conducted using the neighbour-joining method in ClustalX2 and MEGAX. Host range and lytic activity were tested by spot assay and time-kill experiment, respectively. RESULTS: Phage RDN37 had a large burst size and belonged to the Myoviridae family as per transmission electron microscopy and Sanger sequencing results. It was stable over wide range of temperature (-20°,4°, 25°, 37 °C) and pH (6,7,8). The phylogenetic analysis of amplified PCR product (major coat protein gp23) grouped the phage RDN37 with Escherichia phage vB_EcoM_IME537 (MT179807) isolated from community sewage water in China. RDN37 phage was active against MDR-UPEC strains resistant to third generation cephalosporins, aminoglycosides, carbapenems, fluoroquinolones and cotrimoxazole. The multiplicity of infection (MOI) of 0.01 was found to be optimum to reduce the bacterial cell density in the time-kill assay. CONCLUSIONS: RDN37 is a stable lytic phage with large burst size, specific to E. coli, has a therapeutic potential to treat UTI caused by highly drug resistant UPEC. A cocktail of multiple phages will be required to overcome its narrow host range.

Complete Genome Sequence of Escherichia Phage 590B, Active against an Extensively Drug-Resistant Uropathogenic Escherichia coli Isolate.

Escherichia phage 590B, which was isolated from community sewage water in Chandigarh, India, exhibited lytic activity against an extensively drug-resistant uropathogenic Escherichia coli isolate. The genome of the phage is linear, double stranded, and 44.39 kb long. Phage 590B is a member of the Siphoviridae family and is closest to phage vB_EcoS_XY2, which was isolated in China.

Draft Genome Sequence of Escherichia Phage PGN829.1, Active against Highly Drug-Resistant Uropathogenic Escherichia coli.

The Escherichia phage PGN829.1 was isolated from sewage of a tertiary care referral hospital in North India. It lyses multiple strains of highly drug-resistant uropathogenic E. coli. It belongs to the family Podoviridae. Its genome is closest to that of Escherichia phage Vb_EcoP_PhAPEC7.

A non-surgical method for induction of lung cancer in Wistar rats using a combination of NNK and high dietary fats.

Lung cancer is one of the most common malignant neoplasms all over the world. Smoking and a number of constituents of tobacco are responsible for development of lung tumours; however, the deleterious effects of tobacco-derived carcinogen, nitrosamine 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (nicotine-derived nitrosamine ketone (NNK)) remain unmatched. We report the development of a novel rodent model by administering multiple doses of NNK to male Wistar rats and feeding them with high-fat and low-protein diet. Tumour cells in lungs were observed in approximately 98 % rats after 8 months of NNK treatment, as evident by histopathological analysis. This rodent model showed slow progression of lung tumours which has helped us to assess early indicators of oxidative damage in lungs by studying the levels of lipid peroxidation and antioxidant parameters. LPO was elevated by 46.94 %, SOD, CAT, GSH and GR activity was decreased by 48.67 %, 22.04 %, 21.46 % and 20.85 %, respectively in serum of NNK treated rats when compared with control. These findings suggest that increased oxidative stress can represent a risk factor for the development of chronic disease in early future. This new animal model is an attempt to greatly facilitate studies of the pathophysiology, biochemistry and therapy of lung cancer.

Elevated expression of complement C3 protein in chemically induced hepatotumorogenesis in Wistar rats: a correlative proteomics and histopathological study.

Liver cancer remains the leading cause of cancer-related mortality worldwide. Early detection of liver cancer is problematic due to the lack of a marker with high diagnosis sensitivity and specificity. The present study was designed to determine the differently expressed proteins at early stage in the serum of animals with liver cancer vis-à-vis controls and figure out the function of the proteins. One-dimensional electrophoresis (1D), two-dimensional electrophoresis (2DE) and liquid chromatography mass spectrometry (LC-MS/MS) were used to screen the serum proteins of liver cancer induced in animals by diethyl nitrosamine (DEN)+2-acetyl amino fluorine (2-AAF). From optimized 2DE image and computer assisted PD Quest analysis were found to be differentially expressed spots when the serum from normal and treated animals were compared. Among these, one spot was selected whose expression level was higher in DEN+2-AAF treated animal sera than in adjacent normal animal sera. The target spot was excised from the 2D gel of liver cancer sera and the peptide mass fingerprinting as obtained LC-MS/MS analysis after digesting the chosen protein spot. This was identified to be complement C3 protein. The changes in complement C3 expression level were validated by Western blot analysis. We reported that the changes in complement C3 concentration start at very early stage of tumorogenesis. The fully grown tumors were developed at 120 days and hepatotumorogenesis was confirmed by histopathological examination. This protein may therefore represent a powerful tool in search for candidate biomarkers for HCC.

DEN+2-AAF-induced multistep hepatotumorigenesis in Wistar rats: supportive evidence and insights.

Diethylnitrosamine (DEN), found in many commonly consumed foods, has been reported to induce cancers in animals and humans. Several models have been developed to study multistage carcinogenesis in rat liver; these include the Solt-Farber-resistant hepatocyte model. In the Solt-Farber model, the initiation consists of either a necrogenic dose of a hepatocarcinogen or a non-necrogenic dose in conjunction with partial hepatectomy (PH). We report a novel protocol for tumor induction in liver which eliminates the need for PH. Male Wistar rats were injected with single i.p. dose of DEN (200 mg/kg body weight), controls received saline only. After 1 week of recovery, the DEN-treated animals were administered with the repeated doses of 2-acetyamino fluorine (150 mg/kg body weight) orally in 1 % carboxymethyl cellulose that served as promoting agent. Thirty days after the DEN administration, hepatocellular damage was observed as evident by histopathological analysis. The marker enzyme analysis showed elevated levels of serum AST, ALT, and alkaline phosphatase and a decrease in the levels of liver superoxide dismutase and catalase. The oxidative stress in liver was confirmed by elevated levels of lipid peroxidation and a decrease in antioxidant parameters.

Proteomic analysis of differentially expressed proteins in lung cancer in Wistar rats using NNK as an inducer.

Lung cancer is one of the commonest cancers detected worldwide with a high mortality rate. The responsible factors affecting survival include delayed prognosis, and lack of effective treatments. To help improve the disease management, there is a need for better screening and development of specific markers that help in the early diagnosis. Analysis of differentially expressed proteins in cancer cells in comparison to their normal counterparts using proteome profiling revealed identification of new biomarkers as therapeutic targets. Therefore, an animal model for lung cancer was developed and monitored by histopathological evaluation. Lung tissue proteins were isolated, solubilized and resolved on 2D gel electrophoresis using broad pH range IPG strips (pH 3-10). Liquid chromatography and mass spectrometry (LC-MS/MS) revealed 66 proteins to be differentially expressed in cancer tissue as compared to normal. The study identified and characterized three of these proteins, namely peroxiredoxin-6, ß-actin and collagen α-1 (VI) as potentially prospective biomarkers for early detection of lung cancer.