Previous in vitro long-term cultivation studies of Treponema pallidum subsp. pallidum (T. pallidum) strains have indicated potential differences in the length of generation times among individual strains. In the present study, we have determined in vitro growth rates of seven T. pallidum strains including three from the Nichols-like cluster (DAL-1, Madras, and Haiti B) and four from the SS14-like cluster (Mexico A, SS14, Grady, and Philadelphia 1). Despite the observed considerable variability, the results of standard subcultures identified growth differences between the two clusters during the long-term cultivation. Furthermore, in vitro monocultures with defined inoculum revealed differences among individual strains. During three week-long binary co-cultivations of seven strains (n = 21), different growth rates were confirmed for individual strains (p < 0.001) using PCR amplicon sequencing of genomic regions differentiating treponemal-pairs. The order of strains by decreasing growth rate in vitro was DAL-1, Madras, Mexico A, Haiti B, SS14, Grady, and Philadelphia 1. While the generation time of strain DAL-1 was 32.97 h, the slowest strain, Philadelphia 1, had generation time 43.5 h. These experiments revealed significant physiological differences between the T. pallidum strains, which may also be involved in the variable presentation of syphilis symptoms observed in previous decades.

Department of Biology Faculty of Medicine Masaryk University Kamenice 5 Brno 625 00 Czech Republic

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