Phylogenetic inference of Coxiella burnetii by 16S rRNA gene sequencing

Abstract

Coxiella burnetii is a human pathogen that causes the serious zoonotic disease Q fever. It is ubiquitous in the environment and due to its wide host range, long-range dispersal potential and classification as a bioterrorism agent, this microorganism is considered an HHS Select Agent. In the event of an outbreak or intentional release, laboratory strain typing methods can contribute to epidemiological investigations, law enforcement investigation and the public health response by providing critical information about the relatedness between C. burnetii isolates collected from different sources. Laboratory cultivation of C. burnetii is both time-consuming and challenging. Availability of strain collections is often limited and while several strain typing methods have been described over the years, a true gold-standard method is still elusive. Building upon epidemiological knowledge from limited, historical strain collections and typing data is essential to more accurately infer C. burnetii phylogeny. Harmonization of auspicious high-resolution laboratory typing techniques is critical to support epidemiological and law enforcement investigation. The single nucleotide polymorphism (SNP) -based genotyping approach offers simplicity, rapidity and robustness. Herein, we demonstrate SNPs identified within 16S rRNA gene sequences can differentiate C. burnetii strains. Using this method, 55 isolates were assigned to six groups based on six polymorphisms. These 16S rRNA SNP-based genotyping results were largely congruent with those obtained by analyzing restriction-endonuclease (RE)-digested DNA separated by SDS-PAGE and by the high-resolution approach based on SNPs within multispacer sequence typing (MST) loci. The SNPs identified within the 16S rRNA gene can be used as targets for the development of additional SNP-based genotyping assays for C. burnetii.

Fig 1. 16S rRNA gene sequence of the C. burnetii Nine Mile reference strain.

Five SNPs and one insertion polymorphism identified among the 55 C. burnetii 16S rRNA gene sequences are highlighted in green. The nine variable regions corresponding to those reported in the variability map of the E.coli 16S rRNA gene [34, 40] are highlighted in yellow. Bi-directional primers used to generate 16S rRNA sequences are boxed and priming sites are underlined.

Fig 2. Insertion polymorphism identified in the 16S rRNA gene of C. burnetii Mauriet.

(A) DNA sequences obtained using primers E8F and E341R designate an adenine insertion at nucleotide position 213 which is confirmed in the electropherograms generated by Sequencher™ analysis software. (B) Sequence alignment shows 16S rRNA genes of C. burnetii Z3055 (GenBank accession number NZ_LK937696) and 3262 (GenBank accession number CP013667) also possess this insertion which is absent in the Nine Mile reference (Ref) strain sequence.

Fig 3. 16S rRNA gene SNP-based phylogeny of C. burnetii.

This rooted phylogenetic tree was created from pairwise distances observed from 16S rRNA gene SNP sites. The five SNPs and one insertion identified among the 55 16S rRNA gene sequences gave rise to six groups (color coded). Based on sequence alignment to the 16S rRNA gene of C. burnetii Nine Mile (bold), SNPs at positions 64, 164, 622, 830, and 1022 and an insertion at positon 213 (in parentheses) gave rise to the six distinct SNP signatures, which are displayed on their respective branches.

Fig 4. MST loci SNP-based phylogeny of C. burnetii.

This rooted phylogenetic tree was created from pairwise distances observed from 14 SNP sites within MST loci. SNP positions in C. burnetii Nine Mile (bold) are outlined in Hornstra et al. [19]. The predicted genomic groups from this phylogenetic analysis are circled in blue and the 16S rRNA gene-based groups are color coded for comparison.