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.

Coxiella burnetii is widespread in ticks (Ixodidae) in the Xinjiang areas of China

“…As described in previous studies [ 58 – 61 ], primers were designed with conserved regions of the C. burnetii IS1111 (This is a multi-copy gene encoding a transposase) and 16S rRNA gene sequences (Table 3 ); the expected product from the C. burnetii primers used for IS1111 amplification was 517 bp, and for the 16S rRNA primers, it was 592 bp. The 25 μL of PCR mixture comprised 2 μL of DNA sample, 12.5 μL of DreamTaq Green PCR Master Mix (2×) (Thermo Fisher Scientific, Lithuania, MA, USA), 8.5 μL of ddH 2 O, and 1 μL of 10 μΜ forward primer and 10 μΜ reverse primer (TSINGKE Biotech, Xian, China).…”

“…As described in previous studies [58][59][60][61], primers were designed with conserved regions of the C. burnetii IS1111 (This is a multi-copy gene encoding a transposase) and 16S rRNA gene sequences (Table 3); the expected product from the C. burnetii primers used for IS1111 amplification was 517 bp, and for the 16S rRNA μΜ forward primer and 10 μΜ reverse primer (TSINGKE Biotech, Xian, China). A negative control was prepared with double-distilled water, positive control for C. burnetii from H. asiaticum preserved by our laboratory.…”

Coxiella burnetii is widespread in ticks (Ixodidae) in the Xinjiang border areas of China

“…As described in previous studies [22][23][24][25], primers were designed with conserved regions of the C. burnetii IS1111 and 16S rRNA gene sequences (Table 1); the expected product from the C. burnetii primers used for IS1111 amplification was 517 bp, and for the 16S rRNA primers, it was 592 bp. The 72°C for 1 min, and the final step at 72°C for 5 min.…”

Correlating Genotyping Data of Coxiella burnetii with Genomic Groups

“…However, differences in the size estimates provided by different capillary electrophoresis equipment can make comparisons between different laboratories difficult [ 32 ]. Other, less commonly used, genotyping methods exist for C. burnetii , such as SNP genotyping [ 36 ], 16S sequencing [ 37 ], or IS 1111 -based genotyping [ 38 ].…”