Genotyping and Quantifying Lyme Pathogen Strains by Deep Sequencing of the Outer Surface Protein C (ospC) Locus.

A mixed infection of a single tick or host by Lyme disease spirochetes is common and a unique challenge for the diagnosis, treatment, and surveillance of Lyme disease. Here, we describe a novel protocol for differentiating Lyme strains on the basis of deep sequencing of the hypervariable outer surface protein C locus (ospC). Improving upon the traditional DNA-DNA hybridization method, the next-generation sequencing-based protocol is high throughput, quantitative, and able to detect new pathogen strains. We applied the method to more than one hundred infected Ixodes scapularis ticks collected from New York State, USA, in 2015 and 2016. An analysis of strain distributions within individual ticks suggests an overabundance of multiple infections by five or more strains, inhibitory interactions among coinfecting strains, and the presence of a new strain closely related to Borreliella bissettiae A supporting bioinformatics pipeline has been developed. The newly designed pair of universal ospC primers target intergenic sequences conserved among all known Lyme pathogens. The protocol could be used for culture-free identification and quantification of Lyme pathogens in wildlife and potentially in clinical specimens.

Lyme disease spirochaete Borrelia burgdorferi does not require thiamin.

Thiamin pyrophosphate (ThDP), the active form of thiamin (vitamin B1), is believed to be an essential cofactor for all living organisms1,2. Here, we report the unprecedented result that thiamin is dispensable for the growth of the Lyme disease pathogen Borrelia burgdorferi (Bb)3. Bb lacks genes for thiamin biosynthesis and transport as well as known ThDP-dependent enzymes4, and we were unable to detect thiamin or its derivatives in Bb cells. We showed that eliminating thiamin in vitro and in vivo using BcmE, an enzyme that degrades thiamin, has no impact on Bb growth and survival during its enzootic infectious cycle. Finally, high-performance liquid chromatography analysis reveals that the level of thiamin and its derivatives in Ixodes scapularis ticks, the enzootic vector of Bb, is extremely low. These results suggest that by dispensing with use of thiamin, Borrelia, and perhaps other tick-transmitted bacterial pathogens, are uniquely adapted to survive in tick vectors before transmitting to mammalian hosts. To our knowledge, such a mechanism has not been reported previously in any living organisms.