Over the past decade, genomic characterization of Bordetella pertussis isolates recovered from US pertussis patients has unveiled noteworthy structural gene order variations. Whole-genome sequencing (WGS) shows that although B. pertussis exhibits little gene sequence variation, genomes from clinical isolates frequently differ in gene order through rearrangement between insertion sequence elements. To better understand rates of genome rearrangement and single nucleotide polymorphism (SNP) in B. pertussis, intra-patient genomic diversity was examined. Five states submitted, on average, five isolates per patient specimen following culture confirmation to the US Centers for Disease Control and Prevention for molecular characterization. Analysis of 149 patient specimen sets revealed only rare SNP variation, while isolate sets from 12 patients included genomic rearrangements that did not impact vaccine antigen production. To investigate the frequency and stability of such rearrangements during laboratory culture, replicate cultures of two pairs of isolates differing by duplication and inversion were subcultured for eight serial passages. WGS confirmed the initial presence of rare duplication mutations that became dominant in later passages, as well as the stable maintenance of a large, inverted genomic region during passage. These findings suggest that B. pertussis exhibits genomic diversity within a single clinical diagnostic specimen and acquires genomic variations during serial laboratory passages, indicative of bacterial genomic plasticity. Importantly, these rearrangements did not impact the frequency or distribution of SNPs. These results underscore the importance of minimizing the laboratory passaging of clinical isolates used for infectious disease surveillance. IMPORTANCE: The whooping cough-causing bacterium Bordetella pertussis can alter its genetic structure while conserving areas essential for vaccine efficacy. By examining B. pertussis from infected patients, we demonstrate the significance of reducing laboratory manipulation of bacteria to maintain reliable monitoring data from laboratory cultures. Public health officials can use these data to develop efficient disease control tactics and better understand B. pertussis's adaptability. This study highlights the importance of immunizations and the necessity of thorough genetic surveillance in the fight against this chronic and avoidable respiratory illness.

This report describes the complete genome sequence assemblies from four representative isolates of the human pathogen Corynebacterium belfantii. These data provide necessary references to aid accurate sequence-based species discrimination among closely related Corynebacterium spp. pathogens.

Whole-genome sequencing (WGS) of microbial pathogens recovered from patients with infectious disease facilitates high-resolution strain characterization and molecular epidemiology. However, increasing reliance on culture-independent methods to diagnose infectious diseases has resulted in few isolates available for WGS. Here, we report a novel culture-independent approach to genome characterization of Bordetella pertussis, the causative agent of pertussis and a paradigm for insufficient genomic surveillance due to limited culture of clinical isolates. Sequencing libraries constructed directly from residual pertussis-positive diagnostic nasopharyngeal specimens were hybridized with biotinylated RNA "baits" targeting B. pertussis fragments within complex mixtures that contained high concentrations of host and microbial background DNA. Recovery of B. pertussis genome sequence data was evaluated with mock and pooled negative clinical specimens spiked with reducing concentrations of either purified DNA or inactivated cells. Targeted enrichment increased the yield of B. pertussis sequencing reads up to 90% while simultaneously decreasing host reads to less than 10%. Filtered sequencing reads provided sufficient genome coverage to perform characterization via whole-genome single nucleotide polymorphisms and whole-genome multilocus sequencing typing. Moreover, these data were concordant with sequenced isolates recovered from the same specimens such that phylogenetic reconstructions from either consistently clustered the same putatively linked cases. The optimized protocol is suitable for nasopharyngeal specimens with diagnostic IS481 Ct < 35 and >10 ng DNA. Routine implementation of these methods could strengthen surveillance and study of pertussis resurgence by capturing additional cases with genomic characterization.