Comprehensive genomics reveals novel sequence types of multidrug resistant Klebsiella oxytoca with uncharacterized capsular polysaccharide K- and lipopolysaccharide O-antigen loci from the National Hospital of Uganda.

The Klebsiella oxytoca complex comprises diverse opportunistic bacterial pathogens associated with hospital and community-acquired infections with growing alarming antimicrobial resistance. We aimed to uncover the genomic features underlying the virulence and antimicrobial resistance of isolates from Mulago National Hospital in Uganda. We coupled whole genome sequencing with Pathogenwatch multilocus sequence typing (MLST) and downstream bioinformatic analysis to delineate sequence types (STs) capsular polysaccharide K- and O-antigen loci, along with antimicrobial resistance (AMR) profiles of eight clinical isolates from the National Referral Hospital of Uganda. Our findings revealed that only two isolates (RSM6774 and RSM7756) possess a known capsular polysaccharide K-locus (KL74). The rest carry various unknown K-loci (KL115, KL128, KLI52, KL161 and KLI63). We also found that two isolates possess unknown loci for the lipopolysaccharide O-antigen (O1/O2v1 type OL104 and unknown O1). The rest possess known O1 and O3 serotypes. From MLST, we found four novel sequence types (STs), carrying novel alleles for the housekeeping genes glyceraldehyde-6-phosphate dehydrogenase A (gapA), glucose-6-phosphate isomerase (pgi), and RNA polymerase subunit beta (rpoB). Our AMR analysis revealed that all the isolates are resistant to ampicillin and ceftriaxone, with varied resistance to other antibiotics, but all carry genes for extended-spectrum beta-lactamases (ESBLs). Notably, one strain (RSM7756) possesses outstanding chromosomal and plasmid-encoded AMR to beta-lactams, cephalosporins, fluoroquinolones and methoprims. Conclusively, clinical samples from Mulago National Referral Hospital harbor novel STs and multidrug resistant K. oxytoca strains, with significant public health importance, which could have been underrated.

Microbial chassis as the platform for production of dihydroxy xanthophyll-based carotenoids: an overview of recent advances in biomanufacturing.

Physiological and environmental cues prompt microbes to synthesize diverse carotenoids, including dihydroxy xanthophylls, facilitating their adaptation and survival. Lutein and its isomeric counterpart, zeaxanthin, are notable dihydroxy xanthophylls with bioactive properties such as antioxidative, anti-inflammatory, anticancer, and neuroprotective effects, particularly beneficial for human ocular health. However, global natural resources for co-producing lutein and zeaxanthin are scarce, with zeaxanthin lacking commercial sources, unlike lutein sourced from marigold plants and microalgae. Traditionally, dihydroxy xanthophyll production primarily relies on petrochemical synthetic routes, with limited biological sourcing reported. Nonetheless, microbiological synthesis presents promising avenues as a commercial source, albeit challenged by low dihydroxy xanthophyll yield at high cell density. Strategies involving optimization of physical and chemical parameters are essential to achieve high-quality dihydroxy xanthophyll products. This overview briefly discusses dihydroxy xanthophyll biosynthesis and highlights recent advancements, discoveries, and industrial benefits of lutein and zeaxanthin production from microorganisms as alternative biofactories.

A sophisticated virulence repertoire and colistin resistance of Citrobacter freundii ST150 from a patient with sepsis admitted to ICU in a tertiary care hospital in Uganda, East Africa: Insight from genomic and molecular docking analyses.

Sepsis and multidrug resistance comprise a complex of factors attributable to mortality among intensive care unit (ICU) patients globally. Pathogens implicated in sepsis are diverse, and their virulence and drug resistance remain elusive. From a tertiary care hospital ICU in Uganda, we isolated a Citrobacter freundii strain RSM030 from a patient with sepsis and phenotypically tested it against a panel of 16 antibiotics including imipenem levofloxacin, cotrimoxazole and colistin, among others. We sequenced the organism's genome and integrated multilocus sequencing (MLST), PathogenFinder with Virulence Factor analyzer (VFanalyzer) to establish its pathogenic relevance. Thereafter, we combined antiSMASH and PRISM genome mining with molecular docking to predict biosynthetic gene clusters (BGCs), pathways, toxin structures and their potential targets in-silico. Finally, we coupled ResFinder with comprehensive antibiotic resistance database (CARD) to scrutinize the genomic antimicrobial resistance profile of the isolate. From PathogenFinder and MLST, this organism was confirmed to be a human pathogen (p = 0.843), sequence type (ST)150, whose virulence is determined by chromosomal type III secretion system (T3SS) (the injectosome) and plasmid-encoded type IV secretion system (T4SS), the enterobactin biosynthetic gene cluster and biofilm formation through the pgaABCD operon. Pathway and molecular docking analyses revealed that the shikimate pathway can generate a toxin targeting multiple host proteins including spectrin, detector of cytokinesis protein 2 (Dock2) and plasmalemma vesicle-associated protein (PLVAP), potentially distorting the host cell integrity. From phenotypic antibiotic testing, we found indeterminate results for amoxicillin/clavulanate and levofloxacin, with resistance to cotrimoxazole and colistin. Detailed genome analysis revealed chromosomal beta lactam resistance genes, i.e. blaCMY-79, blaCMY-116 and blaTEM-1B, along with multiple mutations of the lipopolysaccharide modifying operon genes PmrA/PmrB, pmrD, mgrA/mgrB and PhoP/PhoQ, conferring colistin resistance. From these findings, we infer that Citrobacter freundii strain RSM030 is implicated in sepsis and resistance to standard antibiotics, including colistin, the last resort.

Complete genome sequence of Bacillus subtilis strain MARUCo01 isolated from marine sediments of the Indian Ocean in Bagamoyo, Tanzania.

Bacillus subtilis has emerged as a species with potential for versatile nonribosomal peptides and polyketides of therapeutic importance, including antibiotics. From our molecular bioprospecting project, we report a full genome of Bacillus subtilis strain MARUCo01 locally isolated from sediments of the Indian Ocean along the coast of Bagamoyo in Tanzania.

Dynamic metabolomic crosstalk between Chlorella saccharophila and its new symbiotic bacteria enhances lutein production in microalga without compromising its biomass.

The microalgae Chlorella saccharophila UTEX247 was co-cultured with its symbiotic indigenous isolated bacterial strain, Exiguobacterium sp., to determine the possible effects of bacteria on microalgae growth and lutein productivity. Under optimal conditions, the lutein productivity of co-culture was 298.97 µg L-1 d-1, which was nearly 1.45-fold higher compared to monocultures i.e., 103.3 µg L-1 d-1. The highest lutein productivities were obtained in co-cultures, accompanied by a significant increase in cell biomass up to 0.84-fold. These conditions were analyzed using an untargeted metabolomics approach to identify metabolites enhancing valuable renewables, i.e., lutein, without compromising growth. Our qualitative metabolomic analysis identified nearly 30 (microalgae alone), 41 (bacteria alone), and 75 (co-cultures) metabolites, respectively. Among these, 46 metabolites were unique in the co-culture alone. The co-culture interactions significantly altered the role of metabolites such as thiamine precursors, reactive sugar anomers like furanose and branched-chain amino acids (BCAA). Nevertheless, the central metabolism cycle upregulation depicted increased availability of carbon skeletons, leading to increased cell biomass and pigments. In conclusion, the co-cultures induce the production of relevant metabolites which regulate growth and lutein simultaneously in C. saccharophila UTEX247, which paves the way for a new perspective in microalgal biorefineries.

Complete genome sequence data of Priestia megaterium strain MARUCO02 isolated from marine mangrove-inhabited sediments of the Indian Ocean in the Bagamoyo Coast.

Priestia is a genus of biotechnologically important bacteria adapted to thrive in a wide range of environmental conditions including the marine sediments. Here, we screened and isolated a strain from the Bagamoyo marine mangrove-inhabited sediments and then employed whole genome sequencing to recover and define its full genome. De novo-assembly with Unicycler (v. 0.4.8) and annotation with Prokaryotic Genome Annotation Pipeline (PGAP) revealed that that its genome contains one chromosome (5,549,131 bp), with a GC content of 37.62%. Further analysis showed that the genome contains 5,687 coding sequences (CDS), 4 rRNAs, 84 tRNAs, 12 ncRNAs, and at least 2 plasmids (1,142 bp and 6,490 bp). On the other hand, antiSMASH-based secondary metabolite analysis revealed that the novel strain (MARUCO02) contains gene clusters for biosynthesis of MEP-DOXP-dependent versatile isoprenoids (eg. carotenoids), siderophores (synechobactin and schizokinen) and polyhydroxyalkanoates (PHA). The genome dataset also informs about the presence genes encoding enzymes required for generation of hopanoids, compounds that confer adaption to harsh environmental conditions including industrial cultivation recipes. Our data from this novel Priestia megaterium strain MARUCO02 can be used for reference and in genome-guided selection of strains for production of isoprenoids as well as industrially useful siderophores and polymers, amenable for biosynthetic manipulations in a biotechnological process.

Omics for Bioprospecting and Drug Discovery from Bacteria and Microalgae.

"Omics" represent a combinatorial approach to high-throughput analysis of biological entities for various purposes. It broadly encompasses genomics, transcriptomics, proteomics, lipidomics, and metabolomics. Bacteria and microalgae exhibit a wide range of genetic, biochemical and concomitantly, physiological variations owing to their exposure to biotic and abiotic dynamics in their ecosystem conditions. Consequently, optimal conditions for adequate growth and production of useful bacterial or microalgal metabolites are critically unpredictable. Traditional methods employ microbe isolation and 'blind'-culture optimization with numerous chemical analyses making the bioprospecting process laborious, strenuous, and costly. Advances in the next generation sequencing (NGS) technologies have offered a platform for the pan-genomic analysis of microbes from community and strain downstream to the gene level. Changing conditions in nature or laboratory accompany epigenetic modulation, variation in gene expression, and subsequent biochemical profiles defining an organism's inherent metabolic repertoire. Proteome and metabolome analysis could further our understanding of the molecular and biochemical attributes of the microbes under research. This review provides an overview of recent studies that have employed omics as a robust, broad-spectrum approach for screening bacteria and microalgae to exploit their potential as sources of drug leads by focusing on their genomes, secondary metabolite biosynthetic pathway genes, transcriptomes, and metabolomes. We also highlight how recent studies have combined molecular biology with analytical chemistry methods, which further underscore the need for advances in bioinformatics and chemoinformatics as vital instruments in the discovery of novel bacterial and microalgal strains as well as new drug leads.