Biology subjects

Newbrough, A.

Publications and source records attributed to Newbrough, A..

3 recordsLinked to original sources

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↗

bioRxiv2024

Blood cultures at baseline and during persistent candidemia contain populations of genetically diverse Candida albicans strains that may differ in echinocandin tolerance and virulence

It is unknown whether within-patient Candida albicans diversity is common during bloodstream infections (BSIs). We determined whole genome sequences of 10 C. albicans strains from blood cultures (BCs) in each of 4 patients. BCs in 3 patients contained mixed populations of strains that differed by large-scale genetic variants, including chromosome (Chr) 5 or 7 aneuploidy (n=2) and Chr1 loss of heterozygosity (n=1). Chr7 trisomy (Tri7) strains from patient MN were attenuated for hyphal and biofilm formation in vitro compared to euploid strains, due at least in part to NRG1 over-expression. Nevertheless, representative Tri7 strain M1 underwent filamentation during disseminated candidiasis (DC) in mice. M1 was more fit than euploid strain M2 during DC and mouse gastrointestinal colonization, and in blood ex vivo. M1 and M2 exhibited identical echinocandin minimum inhibitory concentrations, but M2 was more tolerant to micafungin in vitro. Furthermore, M2 was more competitive with M1 in mouse kidneys following micafungin treatment than it was in absence of micafungin. Tri7 strains represented 74% of patient MNs baseline BC population, but after 1d and 3d of echinocandin treatment, euploid strains were 93% and 98% of the BC population, respectively. Findings suggest that echinocandin tolerant, euploid strains were a subpopulation to more virulent Tri7 strains at baseline and then were selected upon echinocandin exposure. In conclusion, BCs in at least some patients are comprised of diverse C. albicans populations not recognized by the clinical lab, rather than single strains. Clinical relevance of C. albicans diversity and antifungal tolerance merits further investigation.

microbiology↗

bioRxiv2022

Within-host genotypic and phenotypic diversity of contemporaneous carbapenem-resistant Klebsiella pneumoniae from blood cultures of patients with bacteremia

Carbapenem-resistant Klebsiella pneumoniae (CRKP) are major pathogens globally. It is unknown whether bloodstream infections (BSIs) by CRKP and other bacteria are commonly caused by single organisms or mixed microbial populations. We hypothesized that contemporaneous CRKP from blood cultures of individual patients are genetically and phenotypically distinct. We determined short-read whole genome sequences of 10 strains from single colonies from CRKP-positive blood cultures in each of 6 patients (Illumina HiSeq). All strains were sequence type (ST)-258 K. pneumoniae that were unique by core genome single nucleotide polymorphism phylogeny, antibiotic resistance and virulence genes, capsular polysaccharide (CPS) gene mutations, and/or plasmid loss. Strains from each of 3 patients that differed in antibiotic resistance, virulence and/or CPS gene content underwent long-read sequencing for genome completion (Oxford Nanopore), and were tested for phenotypes in vitro and pathogenicity during mouse BSIs. Genetically distinct strains within individual patients exhibited significant differences in carbapenem, beta-lactam/beta-lactamase inhibitor and other antibiotic responses, CPS production, mucoviscosity, and susceptibility to serum killing. In 2 patients, strains differed significantly in their ability to infect organs and cause mortality in mice. In conclusion, we identified genotypic and phenotypic variant ST258 K. pneumoniae strains from blood cultures of individual patients, which were not detected by the clinical laboratory at time of BSI diagnosis. The data support a new paradigm of CRKP population diversity during BSIs. If validated for other BSIs, within-host bacterial diversity may have profound implications for medical, microbiology laboratory and infection prevention practices, and for understanding emergence of antibiotic resistance and pathogenesis. IMPORTANCEIn processing positive microbiologic cultures, standard clinical laboratory practice is to test a single bacterial strain from each morphologically distinct colony. We performed comprehensive whole genome sequence analyses on 10 carbapenem-resistant Klebsiella pneumoniae (CRKP) strains from positive blood cultures from each of 6 patients. Our findings that all strains were genetically unique and that genetic variants manifested differences in phenotypes like antibiotic responsiveness and virulence suggest that CRKP bloodstream infections may be commonly caused by mixed bacterial populations. Results raise questions about laboratory protocols and treatment decisions that are directed against a single strain. The observation that pan-genome analyses revealed inter-strain differences that were not evident by studying core genomes has important implications for investigating nosocomial outbreaks and transmission. Data also suggest a model of pathogenesis of CRKP infections, in which environmental pressures in vivo may select for outgrowth of variants that manifest antibiotic resistance, tolerance or specific virulence attributes.

microbiology↗