Colony formation in solid medium by the relapsing fever spirochetes Borrelia hermsii and Borrelia turicatae

Abstract

Relapsing fever (RF) in North America is caused primarily by the spirochete Borrelia hermsii and is associated with the bite of its tick vector Ornithodoros hermsi. Although this spirochete was known long before the discovery of the Lyme disease (LD) spirochete, Borrelia burgdorferi, basic methods to facilitate the study of B. hermsii have lagged behind. One important technique to expedite the study of the molecular biology and pathogenesis of B. hermsii would be a reliable method to grow and clone these bacteria in solid medium, which we now describe. We have defined the solidifying agent, plating temperature, oxygen concentration, and pH for the efficient plating of two species of RF spirochetes, B. hermsii and Borrelia turicatae. Importantly, this technique allowed us to successfully isolate virulent, clonal cell lines of spirochetes, and to enumerate and isolate viable B. hermsii from infected mouse blood and tick tissues. Our results also demonstrate the value of testing a range of several environmental variables to increase the efficiency of bacterial isolation, which may be helpful for researchers working on other prokaryotes that are intractable for in vitro growth.

Assessing the Contribution of an HtrA Family Serine Protease During Borrelia turicatae Mammalian Infection

“…Further studies are required, however, to assess if in vitro sensitivity to elevated culture temperature is unique to B. turicatae or if this is common among TBRF spirochetes. Next, to evaluate susceptibility of btpA mutants to oxidizing agents, a semi-solid media plating-based approach was utilized (Raffel et al, 2018; Bourret et al, 2019). Bourret et al recently showed that TBRF spirochetes are significantly more resistant to H 2 O 2 relative to B. burgdorferi , hypothesizing that BtpA could be involved in this differential susceptibility (Bourret et al, 2019).…”

“…One mL aliquots were transferred to 5-mL polypropylene culture tubes (Midwest Scientific, Valley Park, MO) and cultured in the presence or absence of H 2 O 2 , t -butyl peroxide, or diethylamine NONOate (Cayman Chemical, Ann Arbor, MI) for 2 h under aerobic conditions at 34°C. Following incubation, serial dilutions of the cultures were prepared in mBSK and were plated on semi-solid mBSK media with 12% rabbit serum in 6-well culture plates as described previously (Raffel et al, 2018). Colony forming units (CFUs) were enumerated after 8–10 days of incubation.…”

The relapsing fever spirochete Borrelia turicatae persists in the highly oxidative environment of its soft‐bodied tick vector

“…One milliliter aliquots of cells were transferred to 5 ml polypropylene culture tubes (MIDSCI) and incubated at 34°C under microaerobic conditions for 2 h in the presence or absence of either H 2 O 2 (Sigma, St. Louis, MO) or diethylamine NONOate (Cayman Chemical). Following treatment, serial dilutions of cells were prepared in mBSK and were plated on semi‐solid mBSK media as previously described (Raffel, Williamson, Schwan, & Gherardini, ). Plates were incubated at 34°C under microaerobic conditions for 7–14 days, and colony forming units (CFUs) were enumerated.…”

Genetic Manipulation of Borrelia

“…The availability of genetic tools and complete genomic sequences for multiple RF species (Lescot et al, 2008;Elbir et al, 2012;Miller et al, 2013;Barbour and Campeau Miller, 2014;Barbour, 2016;Wilder et al, 2016;Elbir et al, 2017;Kuleshov et al, 2020) has permitted investigation of genetically modified RF spirochetes in their respective tick vectors or rodent hosts, and informative comparisons with other Borrelia, as detailed in Radolf and Samuels (2021). As with LD spirochetes, there is considerable variation among RF strains in the efficiency with which they can be grown, transformed or recovered as colonies in solid medium (Raffel et al, 2018). A noteworthy feature of genetic studies with RF spirochetes is retention of infectivity following prolonged cultivation (Lopez et al, 2008), suggesting a more stable segmented genome or a stronger requirement for plasmidencoded functions during in vitro growth, relative to the LD Borrelia.…”

“…Spirochetes in the genus Borrelia have been known to cause human disease for well over a century, but in-depth investigations of these pathogenic bacteria awaited many developments, including the advent of molecular genetics. Since transformation of Borrelia burgdorferi was first described in 1994 (Samuels et al, 1994b;Samuels, 1995), a molecular genetic approach has been applied with growing success to study the biology and pathogenic potential of these tick-borne spirochetes (Tilly et al, 2000;Rosa et al, 2005;Battisti et al, 2008;Fine et al, 2011;Brisson et al, 2012;Raffel et al, 2014;Krishnavajhala et al, 2017;Raffel et al, 2018;Samuels et al, 2018). Both in vitro and in vivo analyses have thus been conducted to identify and describe the basic functions of many Borrelia genes and gene products and their contributions to the relationship of these important pathogens with their tick vectors and mammalian hosts.…”