Demonstration of the Phenomena of Microbial Persistence and Reversion with Bacterial L-Forms in Human Embryonic Kidney Cells

Abstract

A series of experiments was undertaken in which human embryonic kidney (HEK) fibroblasts were infected with either relatively stable or stable L-forms of Streptococcus faecalis. The infected cells were maintained by splitting over long periods (1 to 2 months) while samples were withdrawn for culture and electron microscopic studies. Relatively stable S. faecalis L-forms could be cultured from infected cells only during the first week after infection, although L-form-like material was frequently detected at later periods by electron microscopy. HEK cells continued to proliferate and showed no gross histopathology until reversion occurred. At reversion, electron microscopy revealed the presence of intracellular L-forms, transitional variants, and intra-and extracellular bacterial forms. Unlike relatively stable L-forms, stable L-forms were culturable throughout the experimental period. These experiments demonstrate the phenomena of persistence and reversion in vitro. Electron microscopy indicated that the L-forms entered the HEK cells, where they underwent morphological changes. Observations suggested that these altered L-forms (elementary bodies) persisted in the cell until reversion to the parent bacterial form was triggered. Infected cells, when treated with L-form antiserum and stained with fluorescein-conjugated goat anti-rabbit serum, were consistently positive regardless of cultural findings. bacterium (20).

No effects without causes: the Iron Dysregulation and Dormant Microbes hypothesis for chronic, inflammatory diseases

“…To investigate whether dormant bacteria are present, we thus need culture‐independent methods, of which ultramicroscopic (e.g. Domingue et al ., ; Domingue, , ; Domingue & Woody, ; Ewald, ; Green, Heidger Jr & Domingue, , b ; Mattman, ; Potgieter et al ., ) and molecular sequence‐based methods (Amar et al ., ; Cherkaoui et al ., ; Fernández‐Cruz et al ., ; Gaibani et al ., ; Grif et al ., , b ; C.L. Liu et al ., ; Moriyama et al ., ; NIH HMP Working Group et al ., ; Nikkari et al ., ; Sakka et al ., ; Sato et al ., ; Valencia‐Shelton & Loeffelholz, ; Woyke, Doud & Schulz, ) are by far the most common.…”

Bacterial persistence and expression of disease

“…However, the persistence of antigens derived from the L-form were shown in the tissues at 12 weeks postinjection, and the continual rise in antibody titer suggested that the organisms remained in the tissues but could not be cultured in vitro. This same strain was used in a human embryonic cell culture system and could be cultured as an L-form for up to 73 days postinfection (79). As with the stable L-form in cell culture, there was minimal evidence of histopathologic damage caused by this organism.…”

Adaptation of an osmotically fragile L-form of Streptococcus pyogenes to physiological osmotic conditions and its ability to destroy human heart cells in tissue culture

“…Splitting of infected heart cells by scraping, in lieu of trypsin treatment, and the use of multiple refeedings did not result in their recovery. However, human embryonic kidney cells infected with a stable L-form of S. faecalis have been maintained for up to 60 days by splitting cells by scraping with a rubber policeman (8). Also, use of heat-killed b-forms caused some tissue destruction that was reversible only after trypsin treatment.…”