Browsing by Author "Musundi, Sebastian"

Now showing 1 - 4 of 4
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    PublicationOpen Access
    Application of nanopore sequencing to identify antimicrobial resistance 4genes, mobile genetic elements and virulence factors in clinical isolates.
    (BioRxiV, 2023-10-26) Kimani, Racheal; Gitaka, Jesse; Kanoi, Bernard N; Essuman, Suliman; Mbogo, David; Wakaba, Patrick; Musundi, Sebastian
    The global health challenge posed by the emergence of antibiotic resistance pathogen is further exacerbated in African countries by the indiscriminate use of antibiotics, poor surveillance and lack of stewardship programs. To address this issue, we employed the Oxford Nanopore Technologies (ONT) to sequence 17 clinical isolates from a referral hospital in Kenya. Our comprehensive bioinformatics approach facilitated the assembly, identification of sequence types and prediction of antimicrobial resistance genes, mobile genetic elements (plasmids and integrons) and virulence genes. Of the 17 isolates, five were A. baumannii, four E. coli, three S. haemolyticus, three were E. cloacae, while S. aureus and E. faecalis were single isolates. For the detection of AMR genes, A. baumannii isolates harbored genes such as blaOXA-23 which mediates resistance to carbapenems, E. coli and E. cloacae carried blaCTX-M-15 which confers resistance to cephalosporins and S. haemolyticus harbored blaZ, responsible for resistance against penicillins. S. aureus co-haboured mecA and blaZ genes. In addition,, various other different AMR genes to chloramphenicol, macrolides, aminoglycosides, tetracycline were also observed. For plasmid replicons, E. coli carried the most number of plasmids and shared ColRNAI_1 and IncFIB(pB171)_1_pB171 with A. baumannii and IncR_1 with E. cloacae. Many genes encoding various virulence factors including fimA-I and ompA, senB were identified in E. coli, hlgA-C and hla/hly, hlb, hld in S. aureus and efaA, ebpA-C in E. faecalis. In conclusion, most isolates contained a combination of different AMR genes harbored in plasmids and integrons and virulence genes. This study provides significant information on genetic determinants of antibiotic resistant pathogens in clinical isolates and could assist in developing strategies that improve patient treatment.
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    PublicationOpen Access
    Genetic variation of the Plasmodium falciparum circumsporozoite protein in parasite isolates from Homabay County in Kenya
    (Front. Parasitol, 2024-04-08) Maina, Michael; Waweru, Harrison; Musundi, Sebastian; Kuja, Josiah; Kiboi, Daniel; Kanoi, Bernard N.; Gitaka, Jesse
    The Plasmodium falciparum Circumsporozoite Protein (PfCSP) has been used in developing the RTS,S, and R21 malaria vaccines. However, genetic polymorphisms within Pfcsp compromise the effectiveness of the vaccine. Thus, it is essential to continuously assess the genetic diversity of Pfcsp, especially when deploying it across different geographical regions. In this study, we assessed the genetic diversity of the Pfcsp on isolates from Homabay County, a malaria-endemic region in western Kenya, and compared it against other isolates from Kenya. We extracted DNA from 27 microscopically confirmed P. falciparum positive samples and conducted Illumina sequencing to generate paired-end short reads. The sequences were then mapped to the Pf3D7 reference genome, and genetic variation was analyzed using bcftools. Additionally, we retrieved isolates from two other malaria-endemic regions in Kenya, Kisumu (n=58) and Kilifi (n=596), from MalariaGEN version 7 and compared their genetic diversity and natural selection. We also evaluated the predicted binding affinities for HLA class I and II supertype alleles for the identified haplotypes using NetMHCpan and NetMHCIIpan. Our results show that the N-terminal of PfCSP was relatively conserved with a notable mutation at A98G across all isolates. The number of NANP repeats varied across the three Kenyan sites within the central repeat region. Furthermore, the C-terminal region showed polymorphism within the Th2R and Th3R regions. Haplotype network analysis of the Kenyan isolates revealed 69 haplotypes, with the 3D7 reference being found in the most prevalent haplotype. When assessing the predicted binding affinities between supertypes in HLA class I and II with the identified haplotypes, we observed stronger predicted binding affinities to multiple haplotypes except for those containing the 3D7 reference. The results suggest the need to take into account the existing changes occurring in Pfcsp while developing malaria vaccines.
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    PublicationOpen Access
    Identification of conserved cross-species B-cell linear epitopes in human malaria: a subtractive proteomics and immuno-informatics approach targeting merozoite stage proteins
    (Frontiers in Immunology, 2024-02) Musundi, Sebastian; Gitaka, Jesse; Kanoi, Bernard N
    Human malaria, caused byfive Plasmodium species (P. falciparum, P. vivax, P.malariae, P. ovale,andP. knowlesi), remains a significant global health burden.While most interventions targetP. falciparum, the species associated with highmortality rates and severe clinical symptoms, non-falciparum species exhibitdifferent transmission dynamics, remain hugely neglected, and pose a significantchallenge to malaria elimination efforts. Recent studies have reported the presenceof antigens associated with cross-protective immunity, which can potentiallydisrupt the transmission of various Plasmodium species. With the sequencing ofthe Plasmodium genome and the development of immunoinformatic tools, in thisstudy, we sought to exploit the evolutionary history of Plasmodium species toidentify conserved cross-species B-cell linear epitopes in merozoite proteins. Weretrieved Plasmodium proteomes associated with human malaria and applied asubtractive proteomics approach focusing on merozoite stage proteins. Bepipred2.0 and Epidope were used to predict B-cell linear epitopes usingP. falciparumasthe reference species. The predictions were further compared against human andnon-falciparum databases and their antigenicity, toxicity, and allergenicityassessed. Subsequently, epitope conservation was carried out using locallysequencedP. falciparumisolates from a malaria-endemic region in westernKenya (n=27) and Kenyan isolates from MalariaGEN version 6 (n=131). Finally,physiochemical characteristics and tertiary structure of the B-cell linear epitopeswere determined. The analysis revealed eight epitopes that showed high similarity(70-100%) between falciparum and non-falciparum species. These epitopes werehighly conserved when assessed across local isolates and those from theMalariaGEN database and showed desirable physiochemical properties. Ourresults show the presence of conserved cross-species B-cell linear epitopes thatcould aid in targeting multiple Plasmodium species. Nevertheless, validating theirefficacyin-vitroandin-vivoexperimentally is essential
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    PublicationOpen Access
    Metatranscriptomic analysis of wastewater sites reveals a high abundance of antimicrobial resistance genes from hospital wastewater
    (MedRxiV, 2024-02-05) Musundi, Sebastian; Gitaka, Jesse; Kanoi, Bernard N; Apondi, Ernest Wandera; Sagoe, Wanjiru Kate; Wangui, Esther; Ng'ang'a, Samuel
    Objective This study analyzed the metatranscriptome of wastewater samples from different sites with a focus of identifying antibiotic resistance genes and the bacterial community. Methods Twenty-four wastewater samples were collected from a hospital, university sewer, and the influent and effluent of a wastewater treatment plant (WWTP). The metatranscriptome was sequenced to identify antimicrobial resistance genes (ARGs), mobile genetic elements, and bacterial community composition. Results Metatranscriptome analysis revealed varying abundances of ARG transcripts across different sites, with 84, 27, 14, and 9 ARG transcripts identified in wastewater collected from the hospital, university, influent, and effluent of a WWTP, respectively. Notably, hospital wastewater contained clinically relevant beta-lactam ARGs, including bla-NDM-1 and several blaOXA transcripts. Four core ARGs, against sulfonamides sul1 and sul2 and aminoglycosides aph(6)-ld and aph(’3’)-lb were also identified. The predominant bacterial community comprised of Gammaproteobacteria, with priority pathogens like Neisseria gonorrhea and Helicobacter pylori present in hospital wastewater and WWTP influent. Conclusion These findings provide insights into the wastewater resistome and how meta-transcriptomic data can be utilized for the surveillance of antibiotic resistance. Overall, our study highlights the utility of wastewater surveillance in understanding and addressing the dissemination of antibiotic resistance and emphasizes the crucial role of proper wastewater management in protecting public and environmental health.