Application of Genomic Approaches to Bacterial Pathogenesis and Mechanisms of Antimicrobial Resistance

National Institute Of Allergy And Infectious Diseases

Investigators

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Abstract

MDR gram-negative bacterial pathogens undergo selection and evolution in the natural context of antibiotic treatment in a human host, though important features of host context are often not included in studies of AMR. Additionally, other features underlying bacterial resilience in the context of infection including the ability to evade host defenses often synergize with specific AMR mechanisms and consequently have linked evolutionary relationships. Our work employs a systems biology approach to study the evolutionary mechanisms by which resistance - defined broadly to include resistance to antibiotics and to host defenses - emerges in the natural context of host infection. Current work is organized around three primary projects: Project 1: Mechanisms by which mismatch repair (MMR) deficiencies can facilitate rapid evolution of antimicrobial resistance in P. aeruginosa. P. aeruginosa is an important pathogen responsible for significant nosocomial morbidity and mortality. We previously demonstrated that evolved MMR deficiencies may be dynamically exploited by P. aeruginosa to facilitate rapid acquisition of mutations mediating resistance to two critical broad-spectrum antibiotics, ceftazidime-avibactam (CZA), and ceftolozane-tazobactam (C/T), in the context of acute clinical infection (Khil et al, mBio, 2019). Then, using a combination of directed in vitro microevolution and high throughput genomic and transcriptomic analysis, we characterized the detailed mutational and transcriptional events underlying the development of CZA resistance in wild type (WT) and MMR-deficient P. aeruginosa and identified a number of potential novel resistance mechanisms unique to the MMR deficient isolates. Work performed in 2022 included screening a number of potential novel resistance genes identified in the study above through the introduction of mutations into a common genetic background using a two allele exchange system followed by antibiotic susceptibility testing. This work identified the MexVW efflux pump as a previously unappreciated mediator of resistance to CZA and C/T (Dulanto Chiang et al, submitted 2022). Ongoing work seeks to characterize the structure and function of this pump. RNA-seq experiments were performed to characterize the expression of the mexV and mexW genes, and strategies were devised to express the MexV and MexW proteins in cell lines for purification to facilitate cryo-EM structural studies; purification and reconstitution of the complex in collaboration with Susan Buchanan's lab in NIDDK is ongoing. Other work in 2022 has involved initiating a comprehensive genomic study of antimicrobial resistance in a 20-year collection of P. aeruginosa bacteremia isolates (350) from the NIH Clinical Center. This work is focusing particularly on mechanisms and targets that we have defined in our separate in vitro evolution work above, and for evidence of hypermutation. Potential novel targets will be cloned into the isogenic lab strain system for further study. This collection will also allow us to look historically at changes in the prevalence of different resistance mechanisms, including examining for MexVW associated mutations. All isolates will undergo genomic sequencing and characterization of antimicrobial resistance mechanisms. The genomes of the first 50 of the isolates have been sequenced and assembled and sequencing of the remainder is ongoing. Project 2: In vivo evolution of an emerging zoonotic pathogen Bordetella hinzii in an immunocompromised host. This project applies system biology approaches, including genomics, transcriptomics, and metabolomics to understanding the adaptive evolution in the emerging pathogen Bordetella hinzii following presumptive zoonotic transfer from an animal reservoir to an individual with IL-12RB1 deficiency. Initial work (Launay et al, Nature Communications, 2021) demonstrated that a mutation in the DNA Pol III epsilon proofreading subunit resulted in a replicative DNA proofreading deficiency and drove genetic divergence among the isolates over the course of 45 months of persistent infection. Within these proofreading-deficient lineages, secondary compound hypermutators with complex alterations in mutational spectra emerged and dominated clinical cultures for a period of 12 months, demonstrating their superior in vivo fitness. Evidence of mutational targeting and positive selection was present in multiple sequential enzymes of the tricarboxylic acid cycle and gluconeogenesis pathways, suggesting specialized metabolic adaptation to the host environment. To study the transcriptional landscape of adaptation in these isolates, >100 transcriptomes were sequenced with Illumina short reads and a subset underwent nanopore-based direct long read RNA sequencing. Work completed during FY 2022 focused on detailed metabolic phenotyping of the entire set of isolates and computational integration with large scale transcriptional mapping to characterize how the metabolome was reprogrammed during host adaptation in terms of underlying gene expression and mutations in individual enzymes. The preliminary finding from this work is that the efficiency of amino acid and dipeptide transport/metabolism appears to have been substantially modified during the course of host adaptation. Current ongoing experiments are designed to quantify the fitness consequences of these adaptations in physiologically appropriate media environments using a bioreactor. A second project to study within-host evolution and adaptation of Burkholderia vietnamiensis isolates from another patient with IL-12RB1 deficiency has been initiated and will be pursued in parallel with the B. hinzii analysis. Genomic sequencing of 180 B. vietnamiensis isolates has been completed for this project and genomic analysis of host adaptation is ongoing. Project 3: Comprehensive whole genome sequencing and genomic analysis of a historical collection of clinical Bacteroides fragilis group (BFG) isolates spanning decades. Members of the BFG are important constituents of the human microbiota, but they can also behave as significant pathogens in certain contexts. Historically, antimicrobial susceptibility patterns in BFG isolates were largely predictable, allowing effective use of empiric treatment regimens. Alarming increases in AMR have recently necessitated reconsideration of empiric strategies. To understand the genomic basis of these AMR trends, we have initiated an effort to sequence a large group of clinical BFG isolates spanning a period of five decades. Previous involved long-read nanopore-based sequencing of 386 BFG genomes facilitating end-to-end contiguous assemblies of chromosomes, episomes, and plasmids. Detailed phylogenetic reconstruction and exhaustive annotation of AMR elements in both genome and plasmids has been performed. Work completed in FY 2022 involved using nanopore sequencing in combination with recently developed computational approaches to characterize the 6mA, 5mC, and 4mC methylomes of 260 BFG isolates selected on the basis of AMR phenotype. This work has revealed that single BFG species harbor hundreds of DNA methylation motifs, with most individual motif combinations occurring uniquely in single isolates, implying immense unsampled combinatoric diversity within BFG epigenomes. Additionally, we have refined existing computational approaches to mine methylase genes from the sequenced genomes and identified more than 6000 methyltransferase genes within the genome set, explaining this profound diversity of methylation motifs. Many of the observed methylated motifs are located adjacent to, or within, the gene bodies of AMR genes. Further work will study whether AMR phenotypes are regulated by methylation.