ORCID Profile
0000-0002-5469-3397
Current Organisations
Tribhuvan University
,
CSIRO
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Publisher: Unpublished
Date: 2014
Publisher: Elsevier BV
Date: 03-2022
Publisher: Cold Spring Harbor Laboratory
Date: 28-04-2023
DOI: 10.1101/2023.04.27.538627
Abstract: Staphylococcus aureus colonizes 30% of the human population, but only a few clones cause severe infections. S. aureus’ virulence varies and partly depends on the presence of prophages, viral DNA embedded in the S. aureus core genome, such as hlb-converting prophage (ϕSa3int). Human-adapted S. aureus often harbours a ϕSa3int group of prophages preferentially integrated into their β-hemolysin ( hlb ) gene that encodes human immune evasion cluster (IEC) genes. Exotoxins and immune modulatory molecules encoded by this prophage can inhibit human innate immunity increasing S. aureus pathogenicity. This study aims to investigate the genomic and phenotypic plasticity of S. aureus and changes in its extracellular proteome after the acquisition of ϕSa3int prophage. To achieve this, we used S. aureus strains isolated from the sinus cavities of a patient with severe chronic rhinosinusitis (CRS) at two different time points ( S. aureus SA222 and S. aureus SA333) and hybrid sequenced the strains using short-read Illumina and long-read Oxford nanopore technology. In silico analysis showed the presence of a ϕSa3int prophage in the later isolate but not in the earlier isolate while most of the core genes remained identical. Using mitomycin C, we induced the ϕSa3int prophage, and transduced it into the Sa3int-prophage-free SA222 isolate to obtain a laboratory generated ‘double lysogen’. We confirmed the successful lysogenisation with culture methods (spot assay, blood agar) and also by sequencing. We compared growth kinetics, biofilm biomass and metabolic activity between parent and the lysogen by establishing growth curves, crystal violet and resazurin assays. Exoproteins were identified and quantified using mass spectrophotometry. Integration of ϕSa3int prophage in SA222 down-regulated the beta-hemolysin expression of the lysogen . In silico analysis of the S. aureus genome confirmed the insertion of a ∼43.8 kb ϕSa3int prophage into hlb gene. Insertion of prophage DNA did not alter the growth kinetics, biofilm formation, adhesion to primary human nasal epithelial cells and the metabolic activity in a biofilm. However, the acquisition of ϕSa3int prophage significantly changed the expression of various secreted proteins, both bacterial and prophage-encoded. Altogether, thirty-eight exoproteins were significantly differentially regulated in the laboratory created lysogen, compared to its recipient strain SA222. Among these proteins, there was significant upregulation of 21 exoproteins (55.3 %) including staphylokinase (sak), SCIN (scn), and intercellular adhesion protein B (icaB) and downregulation of 17 exoproteins (44.7 %), including β-hemolysin (hlb/sph) and outer membrane porin (phoE). Most of the upregulated proteins were involved in immunomodulation that help S. aureus escape human innate immunity and help cause chronic infection. These findings may contribute to the development of novel approaches to render S. aureus susceptible to the immune response by blocking prophage-associated defence mechanisms. A ϕSa3int prophage preferentially integrates into the β-haemolysin gene ( hlb ) gene thereby disrupting the beta-hemolysin function. A ∼43.8 kb ϕSa3int prophage acquisition by S. aureus has no impact on its growth kinetics, biofilm formation and adhesion to primary human nasal epithelial cells (HNECs). The presence of a ϕSa3int group prophage likely enhances Staphylococcus aureus’ human immune evasion capability as the prophage encodes a complete set of immune evasion cluster (IEC) genes. Targeted identification of virulence factors in addition to species and strain identification may lead to better-personalized therapy as not all S. aureus carry the same virulence genes.
Publisher: Frontiers Media SA
Date: 16-08-2021
DOI: 10.3389/FCIMB.2021.684704
Abstract: 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 10 5 PFU/ml) and 8 h (7.3 x 10 4 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 log 10 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.
Publisher: Research Square Platform LLC
Date: 21-03-2022
DOI: 10.21203/RS.3.RS-1363627/V2
Abstract: 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 erse 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 spp. ) 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 have potential to be used therapeutically.
Publisher: Research Square Platform LLC
Date: 15-09-2022
DOI: 10.21203/RS.3.RS-2058457/V1
Abstract: The presence of pathogenic and spoilage microorganisms in meat and its product remains a significant concern for suppliers, consumers and public health officials worldwide. Frequent contamination of antibiotic resistant microorganism in meat and meat products poses an important public health issue. In view of this situation, the present study was conducted from August 31 st to September 20 th , 2010 to study the microbial quality of slaughterhouses and to determine the prevalence of antibiotic resistant member of Enterobacteriaceae. For this water and swab s les were taken from 30 slaughterhouses of Khusibun, Kathmandu. Water s les were processed to determine MPN of coliforms and swab s les were analyzed for the presence of members Enterobacteriaceae and Salmonella species . The research showed that the water used in cleaning of slaughter houses was heavily contaminated with coliforms among which two were Enterobacter spp., eleven were Citrobacter spp. and five were E. coli . From the swab taken from the different areas of the slaughterhouses different members of Enterobacteriaceae were isolated among which two were Salmonella typhi and three were E. coli . From the study it was found the all Salmonella typhi were sensitive to Chor henicol, Ciprofloxacin, Ofloxacin and Nalidixic acid and resistant towards Amoxycillin. In case of E. coli isolates, 87.5% was sensitive towards Chlor henicol and 12.5% was resistant towards it. In the case of Ciprofloxacin 62.5% was sensitive and 37.5% was resistant. In case of Ofloxacin 87.5% was sensitive and 12.5% was resistant. In case of Nalidixic acid 75% was sensitive and 25% was resistant and for Amoxycillin 62.5% was sensitive and 37.5% was resistant.
Publisher: Elsevier BV
Date: 06-2022
DOI: 10.1016/J.PATHOL.2021.08.014
Abstract: Infectious diseases caused by antibiotic-resistant bacteria in planktonic and biofilm form are difficult to treat with conventional antibiotics. Silver nanoparticles (Ag NPs) can be used as alternatives to antibiotics and can alter the susceptibility of bacteria to antibiotics. Here, the antibacterial properties of 16 different antibiotics and Ag NPs, alone and in combination, were tested against clinical isolates of Pseudomonas aeruginosa (n=3), Staphylococcus aureus (n=3) and methicillin-resistant Staphylococcus aureus (MRSA) (n=2) isolated from chronic rhinosinusitis (CRS) patients. The microdilution method and resazurin assay were used to determine the minimum inhibitory concentration and minimum biofilm eradication concentration for planktonic and biofilm forms, respectively. Results showed that Ag NPs and gentamicin combinations had synergistic antibacterial activity against P. aeruginosa planktonic and biofilm forms and MRSA biofilms. Furthermore, additive effects against biofilms were seen for combinations of Ag NPs with tobramycin or ciprofloxacin against P. aeruginosa with mupirocin against MRSA and with augmentin, doxycycline, azithromycin and clindamycin against S. aureus. Moreover, additive effects against planktonic forms were observed for combinations of Ag NPs with tobramycin, ciprofloxacin, imipenem, ceftazidime and aztreonam against P. aeruginosa with gentamicin or linezolid against MRSA and with doxycycline or clindamycin against S. aureus. In conclusion, Ag NP-antibiotic combinations can result in enhanced antimicrobial action against P. aeruginosa, MRSA and S. aureus clinical isolates in planktonic and biofilm forms and can be used in the context of CRS with confirmed infection.
Publisher: Wiley
Date: 16-08-2023
DOI: 10.1111/IMM.13655
Abstract: Chronic rhinosinusitis (CRS) represents chronic inflammation of the sinus mucosa characterised by dysfunction of the sinuses' natural defence mechanisms and induction of different inflammatory pathways ranging from a Th1 to a Th2 predominant polarisation. Recalcitrant CRS is associated with Staphylococcus aureus dominant mucosal biofilms however, S. aureus colonisation of the sinonasal mucosa has also been observed in healthy in iduals challenging the significance of S. aureus in CRS pathogenesis. We aimed to investigate the relationship between CRS key inflammatory markers, S. aureus biofilm properties/virulence genes and the severity of the disease. Tissue s les were collected during endoscopic sinus surgery from the ethmoid sinuses of CRS patients with (CRSwNP) and without (CRSsNP) nasal polyps and controls ( n = 59). CD3+ T‐cell subset frequencies and key inflammatory markers of CD4+ helper T cells were determined using FACS analysis. Sinonasal S. aureus clinical isolates were isolated ( n = 26), sequenced and grown into biofilm in vitro, followed by determining their properties, including metabolic activity, biomass, colony‐forming units and exoprotein production. Disease severity was assessed using Lund–Mackay radiologic scores, Lund–Kennedy endoscopic scores and SNOT22 quality of life scores. Our results showed that S. aureus biofilm properties and CRS severity scores correlated positively with total CD4+ T‐cell frequencies but looking into CD4+ T‐cell subsets showed an inverse correlation with Th1 and Th17 cell frequencies. CD4+ T‐cell frequencies were higher in patients harbouring lukF.PV‐ positive S. aureus while its regulatory and Th17 cell subset frequencies were lower in patients carrying sea− and sarT/U‐ positive S. aureus. Recalcitrant CRS is characterised by increased S. aureus biofilm properties in relation to increased total CD4+ helper T‐cell frequencies and reduced frequencies of its Th1, Th17 and regulatory T‐cell subsets. These findings offer insights into the pathophysiology of CRS and could lead to the development of more targeted therapies.
Publisher: Springer Science and Business Media LLC
Date: 19-05-2022
DOI: 10.1007/S00203-022-02948-0
Abstract: 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 erse 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.
Publisher: Oxford University Press (OUP)
Date: 12-2023
DOI: 10.1093/BIOINFORMATICS/BTAC776
Abstract: In recent years, there has been an increasing interest in bacteriophages, which has led to growing numbers of bacteriophage genomic sequences becoming available. Consequently, there is a need for a rapid and consistent genomic annotation tool dedicated for bacteriophages. Existing tools either are not designed specifically for bacteriophages or are web- and email-based and require significant manual curation, which makes their integration into bioinformatic pipelines challenging. Pharokka was created to provide a tool that annotates bacteriophage genomes easily, rapidly and consistently with standards compliant outputs. Moreover, Pharokka requires only two lines of code to install and use and takes under 5 min to run for an average 50-kb bacteriophage genome. Pharokka is implemented in Python and is available as a bioconda package using ‘conda install -c bioconda pharokka’. The source code is available on GitHub (bouras13 harokka). Pharokka has been tested on Linux-64 and MacOSX machines and on Windows using a Linux Virtual Machine.
Publisher: Cold Spring Harbor Laboratory
Date: 04-2023
DOI: 10.1101/2023.03.29.534842
Abstract: Chronic Rhinosinusitis (CRS) is an inflammatory condition of the paranasal sinus mucosa. Despite being a common health issue, the exact cause of CRS is yet to be understood. However, research suggests that Staphylococcus aureus , particularly in the biofilm form, drives the disease. This study aimed to investigate the impact of long-term exposure to secreted factors of Staphylococcus aureus biofilm (SABSF), harvested from clinical isolates of non-CRS carriers and CRS patients, on the nasal mucosa in a rat model. Wistar rats were randomised (n=5/group) to receive daily intranasal instillations of 40 μL (200 μg/μL) SABSF for 28 days or vehicle control with S. aureus isolated from the sinuses of a non-CRS carrier, a type 2 endotype CRS with nasal polyps (CRSwNP) patient, and a non-type 2 endotype CRS without nasal polyps (CRSsNP) patient. The sinonasal s les of the rats were then analysed through histopathology and transcriptome profiling. The results showed that all three intervention groups displayed significant lymphocytic infiltration (p≤0.05). However, only the SABSF collected from the CRSwNP patient caused significant mucosal damage, mast cell infiltration, and goblet cell hyperplasia compared to the control. The transcriptomics results indicated that SABSF significantly enriched multiple inflammatory pathways and showed distinct transcriptional expression differences between the control group and the SABSF collected from CRS patients (p≤0.05). Additionally, the SABSF challenges induced the expression of IgA and IgG but not IgE. In conclusion, this in vivo study indicates that long-term exposure to SABSF leads to an inflammatory response in the nasal mucosa with increased severity for S. aureus isolated from a CRSwNP patient. The findings of this study shed light on the role of S. aureus in the development of CRS and could inform future research and treatment efforts.
Publisher: Microbiology Society
Date: 15-12-2021
Abstract: Prophages affect bacterial fitness on multiple levels. These include bacterial infectivity, toxin secretion, virulence regulation, surface modification, immune stimulation and evasion and microbiome competition. Lysogenic conversion arms bacteria with novel accessory functions thereby increasing bacterial fitness, host adaptation and persistence, and antibiotic resistance. These properties allow the bacteria to occupy a niche long term and can contribute to chronic infections and inflammation such as chronic rhinosinusitis (CRS). In this study, we aimed to identify and characterize prophages present in Staphylococcus aureus from patients suffering from CRS in relation to CRS disease phenotype and severity. Prophage regions were identified using PHASTER. Various in silico tools like ResFinder and VF Analyzer were used to detect virulence genes and antibiotic resistance genes respectively. Progressive MAUVE and maximum likelihood were used for multiple sequence alignment and phylogenetics of prophages respectively. Disease severity of CRS patients was measured using computed tomography Lund–Mackay scores. Fifty-eight S. aureus clinical isolates (CIs) were obtained from 28 CRS patients without nasal polyp (CRSsNP) and 30 CRS patients with nasal polyp (CRSwNP). All CIs carried at least one prophage (average=3.6) and prophages contributed up to 7.7 % of the bacterial genome. Phage integrase genes were found in 55/58 (~95 %) S. aureus strains and 97/211 (~46 %) prophages. Prophages belonging to Sa3int integrase group (phiNM3, JS01, phiN315) (39/97, 40%) and Sa2int (phi2958PVL) (14/97, 14%) were the most prevalent prophages and harboured multiple virulence genes such as sak, scn, chp, luk E/D, sea . Intact prophages were more frequently identified in CRSwNP than in CRSsNP ( P =0.0021). Intact prophages belonging to the Sa3int group were more frequent in CRSwNP than in CRSsNP ( P =0.0008) and intact phiNM3 were exclusively found in CRSwNP patients ( P =0.007). Our results expand the knowledge of prophages in S. aureus isolated from CRS patients and their possible role in disease development. These findings provide a platform for future investigations into potential tripartite associations between bacteria-prophage-human immune system, S. aureus evolution and CRS disease pathophysiology.
Publisher: Cold Spring Harbor Laboratory
Date: 28-03-2023
DOI: 10.1101/2023.03.28.534496
Abstract: Chronic rhinosinusitis (CRS) is a common chronic sinonasal mucosal inflammation associated with Staphylococcus aureus biofilm and relapsing infections. This study aimed to determine rates of S. aureus persistence and pathoadaptation in CRS patients by investigating the genomic relatedness and antibiotic resistance/tolerance in longitudinally collected S. aureus clinical isolates. A total of 68 S. aureus isolates were sourced from 34 CRS patients at least six months apart. Isolates were grown into 48-hour biofilms and tested for tolerance to antibiotics. A hybrid sequencing strategy was used to obtain high-quality reference-grade assemblies of all isolates. Single nucleotide variants (SNV) ergence in the core genome and sequence type clustering were used to analyse the relatedness of the isolate pairs. Single nucleotide and structural genome variations, plasmid similarity, and plasmid copy numbers between pairs were examined. Our analysis revealed that 41% (14/34 pairs) of S. aureus isolates were persisters, while 59% (20/34 pairs) were non-persisters. Persister isolates showed episode-specific mutational changes over time with a bias towards events in genes involved in adhesion to the host and mobile genetic elements such as plasmids, prophages, and insertion sequences. A significant increase in the copy number of conserved plasmids of persister strains (p .05) was seen, indicating a role of the “mobilome” in promoting persistence. This was accompanied by a significant increase in biofilm tolerance against all tested antibiotics (p .001), which was linked to a significant increase in biofilm biomass (p .05) over time, indicating a biofilm central pathoadaptive process in persisters. In conclusion, our study provides important insights into the mutational changes underlying S. aureus persistence in CRS patients highlighting pathoadaptive mechanisms in S. aureus persisters culminating in increased biofilm biomass linked to an increase in plasmid copy number over time.
Publisher: Australasian College of Health Service Management
Date: 29-11-2020
Abstract: Social media is a common platform that enables its users to share opinions, personal experiences, perspectives with one another instantaneously, globally. It has played a paramount role during pandemics such as COVID-19 and unveiled itself as a crucial means to communicate between the sources and the in iduals. However, it also has become a place to disseminate misinformation and fake news rapidly. Infodemic, a plethora of information, some authentic some not makes it even harder to general people to receive factual and trustworthy information when required, has grown to be a major risk to public health and social media is developing as a trendy platform for this infodemic. This commentary aims to explore how social media has affected the current situation. We also aim to share our insight to control this misinformation. This commentary contributes to evolving knowledge to counter fake news or health-related information shared over various social media platforms.
Publisher: Nepal Health Research Council
Date: 23-04-2021
DOI: 10.33314/JNHRC.V19I1.3282
Abstract: 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.Keywords: Bacteriophage klebsiella pneumonia phage therapy
Location: Australia
No related grants have been discovered for Roshan Nepal.