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Recent Submissions
Physiochemical and Genomic Characterization of Bacteriophage against Urinary tract Inficting Pseudomonas Aeruginosa to assess its Possibity for Phagr Therapy
(2023) Sapkota, Pragya; Rajani Malla
Introduction: Emerging antibiotic resistance against widespread Uropathogen has grown
to be a significant therapeutic concern for UTI in recent years particularly in low- and
middle-income nations by the reckless uses of antibiotics. In present context, resistance
is seen in almost all antibiotics even in the last line Carbapenems and colistin. Thus, using
phages in therapeutics to fight against antimicrobial resistance can be a solution to the
global threat caused by these resistant bacteria. This study aims to isolate and
characterize the phage both physiochemically and genetically to combat this global issue.
Methodology: Host bacterial species was identified by biochemical test and 16SrRNA
sequencing. Antibiotic susceptibility test was performed by disc diffusion method and
confirmed by Vitek Compact System 2 analyzer. Bacteriophage isolation was done by
Double Layer Agar Assay (DLAA). pH and temperature stability of phage was analyzed.
Latent period, burst size and host range was determined. Whole genomic sequencing of
phage was done. Bioinformatic analysis to assess for any kind of toxin gene or the
virulence gene of the bacterial origin using different tools such as Ugene, RAST, PHASTER
and Proksee.
Results: Lytic bacteriophage against Pseudomonas aeruginosa was isolated. The phage
was maximally stable at temperature 37˚c and pH 7. The latent period was 30 min and
burst size was 96 virions per bacterium. Isolated phage 6661 showed intraspecific host
range with Pseudomonas (P1) whereas no any activities with other Pseudomonas. Protein
profiling of the phage through SDS- PAGE shows the four distinct band of protein in the
gel. The size of DNA was found to be greater than 10kb. Phage length and GC content was
determined by whole genome sequencing which was 43212 kb and 53.79% respectively.
No any toxin or virulence gene was determined on bioinformatic analysis. The isolated
phage significantly reduces the biofilm by 50.73%.
Conclusion: Pseudomonas phage 6661 showed good stability to various physiochemical
factors and on genomic characterization, presence of endolysin absence of toxin or
virulence gene and integrase enzyme favors the therapeutic potential of the virus.
Keywords: Antibiotic resistance, Bacteriophage, Burst size, Colistin, Phage Therapy
Sociology Curriculum (First and Second Semester), 2025
(Faculty of Humanities and Social Sciences, 2025) Sociology Subject Committee, T. U., Kirtipur, Kathmandu; .
Seroprevalence and Serotyping of Dangue in Fection in Nepal
(2019) Dhakal, Inju; Krishna Das Manandhar
Dengue fever represents a serious emerging infectious disease and has become a global
epidemic with almost half of the world’s population at risk of infection. Dengue is
arthropod-borne viral disease caused by RNA virus of the family Flaviviridae and spread by
Aedes mosquitoes mainly, Aedes aegypti and A. albopictus and are prevalent in tropical and
sub-tropical countries. There are 4 distinct, but closely related, serotypes of the virus that
cause dengue (DENV-1, DENV-2, DENV-3 and DENV-4). As It is one of the emerging viral
diseases in Nepal, people living mainly in Terai region are at high risk. Furthermore, cocirculation of multiple
serotypes may worsen any outbreak significantly by
increasing the= risk of patients which might lead to death due to immune pathology
driven complications.
This study aims to determine the antibody titer against dengue serotypes circulating in
Nepalese population by serotype specific In-House ELISA and helps to know whether
Nepalese population have protective antibodies against dengue virus. In this study, In-house
ELISA was performed by using serotype specific antigens for dengue virus. The study
consisted of total sample size of 32 among them 62.5 % were males (n=20) and 37.5% were
females (n=12) with the ratio of male: female as 1.67:1. The age group ranged from 14 years
to 72 years. Antibody production against different serotypes of dengue were found to be
62.5%, 68.75%, 6.25% and 56.25% respectively for DENV-1, DENV-2, DENV-3 and DENV-4
respectively. In case of In-House ELISA, the OD values were found to be higher for DENV-1
antigen and DENV-2 and lower for DENV-3. The statistical analysis was also performed by
unpaired t-test. The p-value was found to be <0.05 for all the controls and samples and there
was significant difference between the mean of the positive and negative samples. Most of
the samples which were positive in In-Bios Kit has shown to have higher antibody titer for all
antigens. The samples which were negative for the kit also showed lower antibody titer. Out
of 32 samples 15 samples were found IgM positive while only two samples were positive for
IgG ELISA, higher IgM titer showed that patients had a recent acute dengue infection and IgG
positive showed that person had an infection sometimes in past. Calculating the ratio
between IgG and IgM, it was found that most of cases were of primary infection. Molecular
serotyping was done by Nested Reverse Transcriptase PCR (RT-PCR), using dengue specific
primers for part of envelope region, which showed the circulation of DENV-1 in the year 2016
in NEPAL. The developed In-House ELISA was able to determine the antibody produced against theserotypes of dengue virus (DENV 1-4) in Nepalese population. Till now ELISA kits for NS1antigen and antibody detection (IgG and IgM) kits are available, however, no any serotypespecific antibody detection ELISA has been developed. Hence this study might be useful inserotype identification which is important in epidemiological and pathological analysis.
Key Words: DENV, RT-PCR, In-House ELISA, Serotyping