Culyba, M. J. | Search bioRxiv

bioRxiv2026

High-depth whole genome sequencing of blood culture plates reveals evolutionary dynamics in cases of persistent bacteremia due to methicillin-resistant Staphylococcus aureus

Within a single bacterial strain, DNA sequence variation is expected between individual clones. Whole genome sequencing (WGS) can be applied to clinical cultures to detect this polyclonal variation, enabling tracking of within-host evolution and transmission. Culture isolates from infected patients are often sequenced as individual colonies (c-seq). To increase the sensitivity of variant detection, cultures can also be sequenced to a high depth of coverage as a pool (p-seq), but the utility of this approach is not clear for most clinical specimens. To understand the performance of high-depth WGS in bacteremia, we applied p-seq to blood culture plates for 10 patients with persistent bacteremia due to methicillin-resistant Staphylococcus aureus. As a comparison, for six patients, we also applied c-seq to five colonies (c5-seq) from the same plates. p-seq was more sensitive than c5-seq for detecting low frequency variant alleles; however, the most important factor for new variant detection was the number of culture plates analyzed rather than the sequencing method used. We also used these data to construct Muller plots for three patients with especially diverse infecting populations, which enabled visualization of rapid evolutionary dynamics in response to antibiotic exposures. We identified 204 unique variant alleles, and our analysis provides additional evidence for parallel evolution of several different genes during S. aureus bacteremia. Overall, these data provide a detailed view of evolutionary dynamics during clinical cases of MRSA bacteremia and describe the merits and limitations of a c-seq versus p-seq strategy for analyzing blood culture plates using WGS. ImportanceAs bacterial whole genome sequencing (WGS) is increasingly used as a research tool for clinical samples, it is important to understand the pros and cons of different culture sampling methodologies. Here, we analyzed cases of persistent bacteremia due to methicillin-resistant Staphylococcus aureus by applying WGS to either each of five individual colonies isolated on blood culture plates (c5-seq) or the pooled bacterial population on each plate (p-seq). We found that c5-seq was a more practical and informative method to understand evolutionary dynamics.

microbiology↗

bioRxiv2025

Emergence of mutHV76G among longitudinal carbapenem resistant Klebsiella pneumoniae causing long-term colonization and recurrent infection disrupts DNA mismatch repair and results in a hypermutator phenotype

Although hypermutation due to Mut protein mutations that disrupt DNA mismatch repair has been characterized in some bacteria, its mechanisms and consequences in Klebsiella pneumoniae remain poorly defined. We analyzed 11 longitudinal KPC-3 carbapenemase-producing, ST258 K. pneumoniae isolates collected over [~]4 years from an immunocompromised patient with chronic colonization and recurrent infections. After [~]3.3 years, isolates developed ceftazidime-avibactam (CZA)-resistance with restored carbapenem susceptibility, coinciding with emergence of a V76G substitution in a highly-conserved motif in the core of MutH endonuclease. Compared with earlier isolates, mutHV76G-carrying isolates showed greater within-host genomic diversification (69-179 vs. 2-12 SNP differences) and acquired blaKPC-3L169P, encoding an KPC {Omega}-loop substitution that mediates CZA resistance and re-establishes carbapenem susceptibility. mutHV76G isolates exhibited stepwise increases in meropenem-vaborbactam (MVB) and cefiderocol minimum inhibitory concentrations, plausibly linked to substitutions in KPC, OmpK36 porin, CirA iron transporter and/or EnvZ kinase. Clinical mutHV76G isolates and CRISPR-engineered mutHV76G mutants were hypermutators based on rifampin mutational frequencies. Using isogenic mutant and parent strains, we confirmed that mutHV76G accelerated evolution of CZA and MVB resistance in vitro and in vivo, promoted transfer and uptake of resistance plasmids, and improved bacterial fitness during mouse infections. Resistance evolution in mice recapitulated clinical trajectories, with blaKPC-3 and ompK36 mutations emerging under CZA and MVB exposure, respectively. Phenotypes of mutHV76G and mutH-null strains were comparable, indicating that the V76G substitution largely abrogates MutH function. Our findings reveal MutH-mediated hypermutation as an adaptive mechanism in K. pneumoniae, enabling rapid antibiotic resistance development and plasmid acquisition without fitness cost. ImportanceHypermutator bacteria pose a formidable clinical threat by rapidly evolving antibiotic resistance and adapting within the human host. Klebsiella pneumoniae is a major cause of multidrug-resistant infections, yet the contribution of hypermutation to its evolution remains poorly characterized. Analyzing K. pneumoniae isolates collected over [~]4 years from a chronically infected/colonized patient, we demonstrate that emergence of a mutation in mutH (mutHV76G), a DNA mismatch repair gene, results in hypermutation phenotypes and rapid accumulation of gene mutations. Both clinical and lab-engineered mutHV76G mutant strains rapidly acquire resistance or reduced susceptibility to new antibiotics like ceftazidime-avibactam, meropenem-vaborbactam and cefiderocol, due to mutations in carbapenemase (blaKPC-3), porin (ompK36) and other genes. mutHV76G-driven hypermutation also enhances horizontal transfer of resistance plasmids and improves K. pneumoniae fitness during mouse infections. This study is important for understanding K. pneumoniae hypermutation as a potent mediator of antibiotic resistance and other phenotypes relevant to human infections.

microbiology↗