In vivo evolution of an emerging zoonotic bacterial pathogen in an immunocompromised human host

Zoonotic pathogen transfer to humans can create novel agents, but the subsequent genetic events are poorly understood. Prior research on chronic infections in immunocompromised patients has illuminated bacterial pathogenicity, showing within-host evolution influenced by antibiotic stress and immune response. These studies revealed processes like genetic diversification, clonal selection, and trade-offs, with deleterious mutation rates contributing to rapid evolution in some cases. *Bordetella hinzii*, a relative of *Bordetella pertussis*, is an emerging human pathogen causing various infections. This study focuses on *B. hinzii* adaptation in a patient with interleukin-12 receptor beta 1 (IL-12Rβ1) deficiency, impairing the immune response to intracellular pathogens. A previous study on *Salmonella* in an IL-12Rβ1 deficient individual showed elevated spontaneous mutation rates. This work examines a rare case of *B. hinzii* adaptation to a human host, providing insights into host-pathogen interactions in compromised immunity.

The introduction extensively reviews the existing literature on within-host bacterial evolution, focusing on studies of established pathogens in immunocompromised individuals. It highlights the role of selective pressures such as antibiotic use and immune response in driving evolutionary changes. The review also emphasizes the role of mismatch repair (MMR) deficiencies and mobile genetic elements in accelerating evolution. Furthermore, it introduces *Bordetella hinzii* as an emerging human pathogen, discussing its zoonotic origin and the increasing evidence of its ability to cause disease in humans. The review sets the stage for this study by emphasizing the gap in knowledge regarding the evolution of emerging zoonotic pathogens in immunocompromised hosts, specifically focusing on the interplay between host immunodeficiency and pathogen adaptation.

The study involved the analysis of 24 *B. hinzii* isolates cultured from blood and stool samples of an IL-12Rβ1 deficient patient over 45 months. Phenotypic diversity was observed among the isolates, ranging from tiny to large, mucoid colonies. Illumina Whole Genome Sequencing (WGS) and PacBio sequencing were used for genomic analysis. Antimicrobial susceptibility testing was performed using automated broth microdilution methods. Phylogenetic analysis was conducted using maximum likelihood methods. In vitro mutational accumulation (MA) experiments were performed to assess spontaneous mutation rates. dN/dS analysis was used to evaluate selective pressures on coding sequences. The mutational spectra of the isolates were analyzed to identify patterns and potential underlying mechanisms. Comparative genomics was also performed using available *B. hinzii* genomes from NCBI and PATRIC. The reconstruction of the last common ancestor (LCA) was done using two approaches; one using a reference genome and variant calling with Snippy, and another using concatenated core genes and RAxML. Variant calling relative to the LCA and pseudogene identification utilized Snippy, VCFtools, and custom scripts. Homoplasy testing was conducted using the Pa80 homoplasyfinder. Simulations were performed to identify potential targets of selection by randomly redistributing unique mutations across the genome. Longitudinal ATCC accumulation experiments and parallel mutation accumulation experiments with the DNAQ Rugged mutation approach were conducted to measure mutation rates. Variant calling for these experiments employed BWA, samtools, FreeBayes, BCFtools, and VarNeuma. The study followed ethical guidelines, with data deposited in NCBI Sequence Read Archive and Zenodo.

The study found remarkable phenotypic and genetic diversity among *B. hinzii* isolates from the patient. A significant finding was the prevalence of an E96 substitution in the DNA polymerase III ε-subunit (DnaQ E9G) in 20 out of 24 isolates, leading to a proofreading deficiency and significantly higher mutation rates. This hypermutator phenotype facilitated the emergence of multiple lineages with mutations in DNA repair genes and altered mutational spectra. The analysis revealed an enrichment of mutations in genes involved in the tricarboxylic acid (TCA) cycle and gluconeogenesis pathways, suggesting metabolic adaptation to the human host environment. A significant excess of G:C → T:A transversions indicated oxidative stress played a major role in shaping the genetic diversification. The study found that many DnaQ E9G isolates had accumulated non-synonymous mutations in DNA replication and repair genes. The mutational spectra could be clustered into three groups: DnaQ WT, DnaQ E9G, and DnaQ E9G with additional mutations in base excision repair genes (MutM/MutY). The analysis revealed an excess of G:C → T:A transversions, suggesting oxidative stress contributed to the evolution. Many genes were repeatedly mutated in the DnaQ E9G isolates, with significantly elevated dN/dS ratios, suggesting positive selection. Targeted genes included those involved in carbohydrate metabolism, gluconeogenesis, and long-chain fatty acid metabolism. The presence of multiple biotypes within single colonies suggests aggregation of genetically diverse lineages, possibly contributing to the observed diversity. The study also identified pseudogenes, indicating gene inactivation during adaptation. The study concludes that early inactivation of DNA proofreading combined with prolonged oxidative stress facilitated rapid genomic adaptation.

This study's findings address the research question by demonstrating the significant role of a proofreading-deficient DNA polymerase in driving rapid adaptation of *B. hinzii* within a human host. The high mutation rate resulting from the DnaQ E9G mutation provided the raw material for natural selection to act upon, leading to the observed metabolic adaptations and diversification. The significant excess of G:C→T:A transversions suggests a role for oxidative stress in shaping the evolutionary trajectory. The findings emphasize the importance of considering host immune status when studying pathogen evolution, especially after zoonotic transfer. The results contribute to the field by providing a detailed genomic analysis of adaptation in an emerging pathogen, highlighting the complex interplay between pathogen genetics, host immunity, and environmental factors.

This study provides compelling evidence for the crucial role of hypermutation in the adaptation of *B. hinzii* following zoonotic transfer to a human host. The E96 substitution in the DNA polymerase III ε-subunit initiated a cascade of events, including mutations in DNA repair genes, metabolic pathway alterations, and an apparent response to oxidative stress. These findings underscore the influence of host immune deficiency on shaping pathogen evolution. Further research should investigate the specific functional consequences of the identified mutations and explore the broader implications of oxidative stress in driving pathogen adaptation.

While the study provides a comprehensive genomic analysis, functional validation of the identified mutations remains limited. The study is based on a single patient, limiting the generalizability of the findings. The potential for in vitro artifacts in the mutational accumulation experiments should be considered, although the researchers implemented methods to minimize this. The study's focus on a single case limits the extent to which these findings can be broadly generalized. Further investigation across multiple hosts and with larger sample sizes is warranted to confirm the observed patterns and mechanisms.