Bordetella pertussis exhibits genomic diversity within patients and laboratory culture

Abstract

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.

Keywords: Bordetella pertussis; genetic polymorphisms; genome assembly; genome duplication; genome typing; genomic rearrangements; genomic variations; pulsed-field gel electrophoresis; serial passage; whole genome sequencing.

Fig 1

Genome sequence variation among isolates from the same patient. (A) Specimen collection and processing (image courtesy of CDC Public Health Image Library). (B) Histogram depicting the number of isolates per set. Eighty-nine percent of sets included at least four isolates. (C) Rare SNPs within isolate sets from 149 patients analyzed. Seventy-seven percent of sets had zero SNP differences within sets; 11% of sets had one to three SNP differences; and only 12% had more than three SNPs within sets. Reported values represent maximum pairwise SNP distances.

Fig 2

Genome variations among isolates from the same patient. (A) The PFGE profile of J767 contained an additional band (bottom and red arrow) that differentiated it from the profile of CDC237 and the rest of that patient’s set (J763, J764, J765, and J766). (B) WGS identified the variation in J767 as a regional duplication (blue arrow). An increase in coverage is also observed with J766 (yellow arrow) but did not reach 2× coverage. (C) The banding pattern of the PFGE profiles of a patient set from CO differed by two bands (bottom and red arrows), consistent with CDC structures 237 and 300. (D) WGS identified the variation detected in panel C as a regional genomic inversion. In panel B, genome coordinates are indicated in the x-axis; the y-axis indicates the number of times each position on the genome coordinate is observed.

Fig 3

Dynamics of genome rearrangement during laboratory culture. The plots compare coverages for different passages (P01, P02, P03…) of isolates J767 (A), J766 (B), and J818 (C) using the same lineage per isolate. The plots show increases in depth in different regions in all three isolates. Zooming in at the increased coverage (inset, top right of each panel) confirmed the presence of a duplication stably maintained from passage 1 of J767 (A) and the progressive accumulation of a subpopulation of isolate J766 containing a duplication (B) distinct from that of J767. (C) WGS analysis of the serial passage of the pair of isolates J818/J820 initially investigated for a regional inversion revealed a regional duplication not previously observed at passage 8. Genome coordinates are in kilobases (kb) as indicated in the x-axis. The y-axis indicates the number of times each position on the genome coordinate is observed.