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. 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. 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 we characterized the detailed mutational and transcriptional events underlying the development of CZA and C/T resistance in wild type (WT) and MMR-deficient P. aeruginosa and identified a number of potential novel resistance mutations unique to the MMR deficient isolates. Work performed in FY23 included ongoing screening of a number of potential novel resistance genes identified in the study above. Specific work focused on completing experiments establishing the MexVW efflux pump as a novel mediator of resistance to CZA and C/T (published in Dulanto et al, PLoS Biology, 2022). Ongoing work seeks to characterize the structure and function of this pump. We have engineered a number of cell lines expressing MexV, MexW, and OprM proteins for purification to facilitate cryo-EM structural studies, and cryo-EM studies of these purified components is currently ongoing in collaboration with Susan Buchanan's lab in NIDDK. Other work in FY23 has involved completion of the initial phases of a large scale comprehensive genomic study of antimicrobial resistance in a 20-year collection of P. aeruginosa bacteremia isolates (320 isolates from 300 unique patients) 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. We will also introduce MexVW deletions into clinical isolates that are have evolved resistance to CZA and C/T naturally in patients to assess the contribution of MexVW to this resistance. All isolates will undergo genomic sequencing and characterization of antimicrobial resistance mechanisms. The genomes of the first 220 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. 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 FY23 focused on continued metabolic phenotyping of the entire set of isolates. The preliminary findings from this work are that (1) the efficiency of metabolism of a variety of nitrogen containing compounds, including amino acids and dipeptides appears to have been substantially modified during the course of host adaptation; (2) subsets of isolates evolved specific amino acid auxotrophies involving methionine and phenylalanine. Current ongoing experiments are designed to quantify the fitness consequences of these adaptations in physiologically appropriate media environments using a bioreactor and test the specific hypotheses that these evolved auxotrophies confer tolerance or persistence to antibiotics and/or improved survivability to host attack within neutrophils. Presentation of this work was made at ASM Microbe in Houston, Texas, June 2023 (Ellis et al, 2023). Work completed in FY23 on a second project to study within-host evolution and adaptation of Burkholderia vietnamiensis isolates from another patient with IL-12RB1 deficiency has involved genomic analysis of 183 B. vietnamiensis isolates including both short-read and long-read sequencing. The main findings include (1) a remarkably complex and dynamic population structure that was present in the intravascular space; (2) superimposed mobile element insertional mutagenesis mediating secondary genomic plasticity, including interruption of genes and positioning of cis-acting IS promoters; and (3) homologous recombination-based events resulting in chromosomal fusions and deletions. Ongoing work aims to understand how these changes may have facilitated adaptive evolution. Presentation of this work was made at ASM Microbe in Houston, Texas, June 2023 (Moller et al, 2023) 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 2023 involved using nanopore sequencing in combination with recently developed computational approaches to complete characterization the 6mA, 5mC, and 4mC methylomes of 268 BFG isolates selected on the basis of AMR phenotype, revealing 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. This work was published as Tisza et al, Nature Communications, 2023. Ongoing work aims to study how AMR gene expression and phenotypes are regulated by methylation.