Factors affecting the multiplication and subculture of Treponema pallidum subsp. pallidum in a tissue culture system

Abstract

Limited multiplication of Treponema pallidum subsp. pallidum (Nichols strain) can be obtained in the presence of SflEp rabbit epithelial cell cultures, but continuous culture has not yet been achieved. In the system currently employed, growth is exponential for the first 10 to 15 days of culturing, after which multiplication and the percentage of motile organisms decrease. In an effort to identify culture conditions which may adversely affect treponemal viability and growth, eight culture parameters were monitored over a 12-day period of incubation. Several of these parameters, including pH, redox potential, dissolved oxygen concentration, and glucose levels were found to change dramatically during the course of incubation, indicating that they may be responsible for the cessation of treponemal multiplication. The feasibility of extending the period of growth by subculturing was also investigated. In preparation for planned serial subcultivation experiments, several subculture procedures were tested and found to be effective in allowing the transfer of T. pallidum from 3-day-old primary cultures to secondary cultures without loss of motility or growth potential. Increases of up to 55-fold were observed in secondary cultures, but increased growth due to subculturing was not a consistent finding. Use of subculture intervals of .6 days resulted in a progressive decrease in treponemal multiplication in secondary cultures, although retention of motility was extended in the subcultures compared with motility in the primary cultures. These results indicate that the lack of continued multiplication of T. pallidum in subcultures is not due to damage to the treponemes during subculture. Prolonged multiplication of T. pallidum may be obtained through the stabilization of culture conditions by either performing subcultures at regular intervals or by medium replacement techniques. It was also found that primary T. pallidum cultures could be established by using as the inoculum treponemes that had been stored at -70°C in a medium containing 15% glycerol.

Successful isolation of Treponema pallidum strains from patients’ cryopreserved ulcer exudate using the rabbit model

“…4 Strain typing was performed on testes samples that yielded PCR+ results. 5 Weakly positive TP-PA titer +/-1:80 6 Isolate could not be typed due to low number of spirochetes after rabbit propagation 7 Strain type observed with residual lesion swab specimen in NRS https://doi.org/10.1371/journal.pone.0227769.t002…”

“…Research on the utility of various culture media and methods for in vitro propagation have yielded inconsistent results over the years. Sustained passage has been limited or unattainable, with low yields, contamination, and/or loss of viability, virulence and pathogenicity being reported [1][2][3][4][5][6][7]. However, a recent study suggests that in vitro propagation of T. pallidum is possible using a microaerobic, nutrient-defined rabbit cell culture system, with sustained propagation of viable treponemes for >6 months [8].…”

The genome of Treponema pallidum: new light on the agent of syphilis

“…T. pallidum is also unusual in terms of its degree of dependence on the host. It is an obligate parasite of humans and is one of the few medically important bacteria that has not been cultured continuously in vitro [10,11]. Limited multiplication can be obtained in a tissue culture system [12], but the standard means of propagating T. pallidum is through the intratesticular infection of rabbits.…”

Similarity between the 38-kilodalton lipoprotein of Treponema pallidum and the glucose/galactose-binding (MglB) protein of Escherichia coli

“…pallidum (T pallidum) (45). The inability to cultivate T. pallidum continuously in vitro (61,87) and the lack of a genetic exchange system for analysis of the organism continue to hamper progress in elucidating the physiology, ultrastructure, and molecular biology of this important human pathogen (59,87).…”