Biology subjects

Olaitan, A. O.

Publications and source records attributed to Olaitan, A. O..

4 recordsLinked to original sources

bioRxiv2026

Development of a CRISPR-Cas12a Assay for Detection of the PnimBG Mutation Associated with Heme-Dependent Metronidazole Resistance in Clostridioides difficile

Objective Heme-dependent metronidazole (MTZ) resistance has emerged in Clostridioides difficile and is primarily mediated by a T-to-G single-nucleotide polymorphism (SNP) in the promoter of nimB (PnimBG). We aimed to develop a CRISPR-Cas12a-based assay for rapid detection of the PnimBG mutation associated with MTZ resistance. Methods A series of primers and CRISPR RNAs (crRNAs) were designed and optimized to identify the most effective combination for discriminating between PnimBG (MTZ-resistant) and PnimBT (MTZ-susceptible) alleles. Detection was evaluated by visual fluorescence using blue-light and UV imaging systems, as well as by quantitative fluorescence measurements. Results The CRISPR-Cas12a assay accurately differentiated PnimBG and PnimBT alleles in C. difficile. In a collection of clinical isolates, the assay correctly identified all 11 MTZ-susceptible isolates carrying the PnimBT allele and all 10 MTZ-resistant isolates carrying the PnimBG allele. Fluorescence signals were readily detected both visually and quantitatively. Furthermore, colony PCR using crude colony lysates yielded results comparable to those obtained with purified genomic DNA, enabling rapid colony-based detection without conventional DNA extraction. Conclusions The CRISPR-Cas12a assay enables rapid molecular detection of the PnimBG mutation associated with heme-dependent MTZ resistance in C. difficile. This approach could complement conventional susceptibility testing for MTZ resistance detection.

microbiology↗

bioRxiv2022

Decoding a cryptic mechanism of metronidazole resistance among globally disseminated fluoroquinolone-resistant Clostridioides difficile

Severe outbreaks and deaths have been linked to the emergence and global spread of fluoroquinolone-resistant Clostridioides difficile over the past two decades. At the same time, metronidazole, a nitro-containing antibiotic, has shown decreasing clinical efficacy in treating C. difficile infection (CDI). Most metronidazole-resistant C. difficile exhibit an unusual resistance phenotype that can only be detected in susceptibility tests utilizing molecularly intact heme. Here we describe the mechanism underlying this trait, which we discovered using molecular genetics, phylogenetics, and population analyses. Most metronidazole-resistant strains evolved a T to G mutation, we term PnimBG, in the -10 regulatory promoter of the 5-nitroimidazole reductase nimB, resulting in the gene being constitutively transcribed. Silencing or deleting nimB eliminated metronidazole resistance. We identified the protein as a heme-dependent nitroreductase that degraded nitro-drugs to an amine lacking antimicrobial activity. We further discovered that the metronidazole-resistant PnimBGmutation was strongly associated with the Thr82Ile substitution conferring fluoroquinolone resistance in epidemic strains. Re-analysis of published genomes from global isolates confirmed that all but one encoding PnimBG also carried the Thr82Ile mutation. Our findings suggest that fluoroquinolone and metronidazole resistance co-mediated the pandemic of healthcare-associated C. difficile that are associated with poorer treatment outcomes in CDI patients receiving metronidazole.

microbiology↗

bioRxiv2022

The Ferrous Iron Transporter FeoB1 is Essential for Clostridioides difficile Toxin Production and Pathogenesis in Mice

Ferrous iron is the dominant form of iron in the oxygen-limited large intestine, the site for Clostridioides difficile infection (CDI). We investigated the extent to which C. difficile requires the ferrous iron transporter 1 (FeoB1), a main permease for iron acquisition, using in vitro and in vivo approaches. Construction of feoB1 deletion mutant in C. difficile R20291 (i.e., R20291{Delta}feoB1) decreased intracellular iron content by [~]25%. This was accompanied by downregulation of tcdA and tcdB genes and reduced synthesis of TcdA and TcdB toxins, which was reflected by a [~]1000-fold reduction in cytopathy against Vero cells. Complementation with WT feoB1 restored toxin production. Transcriptional and biochemical analyses revealed {Delta}feoB1 altered metabolic pathways that negatively impact toxin production, including downregulation of the oxidative branch of the Kreb cycle and an associated cellular accumulation of pyruvate. Hence FeoB1 influences multiple bioenergetic and redox pathways, which in turn inhibits toxin biosynthesis. R20291{Delta}feoB1 was avirulent in mice with underlying colitis. In this model, mice are prone to develop severe CDI, but mice infected with the mutant lacked diarrheal symptoms, had lesser inflammatory responses, bacterial bioburdens and cecal toxin titers. These results strongly imply that ferrous iron acquisition and homeostasis is central to toxin metabolism and virulence. Hence targeting FeoB1 could be a potential therapeutic strategy to impede the colonization and pathogenesis of C. difficile, even in the setting of colitis where intestinal bleeding may occur and CDI is more severe.

microbiology↗

bioRxiv2020

Ebselen Exhibits Antimicrobial Activity Against Clostridioides difficile By Disrupting Redox Associated Metabolism

High recurrence rates and spread of antibiotic-resistant strains necessitate the need for alternative therapeutics for Clostridioides difficile infections (CDIs). Ebselen, a reactive organoselenium compound inhibits C. difficile virulence toxins TcdA and TcdB, by covalently binding to their cysteine protease domains. Ebselen is thought to lack antibacterial activity against C. difficile cells and its anti-toxin action is suggested to be solely responsible for its efficacy. However, C. difficile has several essential cysteine-containing enzymes that could be potential sites for covalent modification by ebselen; hence, we re-evaluated its anti-C. difficile properties. In BHI agar, ebselen inhibited almost all C. difficile strains (MICs of 2-8 {micro}g/ml), with ribotype 078 being intrinsically resistant (MIC>64 {micro}g/ml). Wilkins-Chalgren and Brucella agars are recommended media for anaerobic susceptibility testing. Ebselen was either slightly attenuated by pyruvate found in Wilkins-Chalgren agar or obliterated by blood in Brucella agars. Transcriptome analysis showed ebselen altered redox-associated processes, cysteine metabolism and significantly enhanced the expression of Stickland proline metabolism to likely regenerate NAD+ from NADH. Intracellularly cells increased the uptake of cysteines, depleted non-protein thiols and disrupted NAD+/NADH redox ratio. Growth inhibitory concentrations of ebselen also reduced toxin and spore production. Taken together, ebselen has bactericidal activity against C. difficile, with multiple mechanisms of action that negatively impacts toxin production and sporulation. To harness the polypharmacological properties of ebselen, chemical optimization is warranted, especially to obtain derivatives that could be effective in severe CDI, where intestinal bleeding could occur.

microbiology↗