Mother-to-newborn transmission of mycobacterial L-forms and Vδ2 T-cell response in placentobiome of BCG-vaccinated pregnant women

Abstract

The ability of bacteria to exist as a population of self-replicating forms with defective or entirely missing cell wall (L-forms) is an adaptive mechanism for their survival and reproduction under unfavorable conditions. Bacterial mother-to-fetus transfer is a universal phenomenon in the animal kingdom. However, data about vertical transfer of L bacterial forms are extremely scarce. Bacille Calmette-Guérin is an attenuated strain of M. bovis and the only licensed vaccine used for tuberculosis prevention. We already have shown that filterable L-forms of BCG exist freely in the vaccine and are able to reproduce and to form colonies. The present study was focused on the placental microbiome in the context of mother's BCG vaccination. Here we report an isolation of filterable mycobacterial L-form cultures from gestational tissues and blood of healthy newborns delivered by healthy BCG-vaccinated mothers after normal pregnancy. Of note, vertically transmitted mycobacterial L-forms as a part of placentobiome of the pregnant women didn't influence the number of resident pathogen-reactive Vδ2 cells. Placenta colonization with mycobacterial L-forms occurs by maternal blood-to-decidua transfer very early in gestation. Together, these data showed that BCG L-forms have the capacity to pass trans-placental barrier and that maternal BCG vaccination affects the placentobiome.

Figure 1

Light microscopy of representative L-form cultures from placental samples: (A) Initial phase of L-form development. Arrows point to dark L- form granules associated with placental cells during primary incubation in broth (ZN stained smear, PL-6). (B) Development of pure L-form culture in broth after filtration through bacterial filter with 0.2 µm pore size. Native smear –cluster of spherical L-form cells with different size (PL-6). (C) ZN stained smear from a broth sub-culture. Arrows point to red stained rods occurring in result to reversion of L-forms to acid fast mycobacteria after further sub-cultivation (PL-6). (D) Typical L-form colonies with “fried eggs” shape after sub-cultivation in semisolid medium (PL-6). (E) L-type colonies with granular consistence (PL-15). (F) L-type tiny colonies (PL-16). Magnification: A, B, C - 1000x; D, E, F- 400x.

Figure 2

Light microscopy of L-form cultures from cord blood samples. (A) Development of pure L-form culture in broth after filtration through 0.2 µm bacterial filter. Clusters of spherical L-form cells with different size - native smear (CB6). (B) ZN stained smear from a broth culture. Arrows point to red stained rods, acid fast mycobacteria reverted from L-forms after further sub-cultivation in broth (CB6). (C) L-form colonies with “fried eggs” shape after sub-cultivation in semisolid medium. (D) Tiny L-form “fried eggs” colonies after sub-cultivation in semisolid medium (CB10). Magnification: A, B - 1000x; C, D- 400x.

Figure 3

TEM of representative L-form culture, isolated from а placental sample (PL-12) in broth: (A) Cluster of small spherical L-bodies with low electron density, some of them with extremely small size - under 100 nm; (B) Small electron-dense and shapeless L-bodies associated with multiple vesicular elements; (C) Large L-body with condensed cytoplasmic material at the cell periphery. Spherical body inside the cell, invagination of cytoplasmic membrane and releasing of small elementary body are seen (EB); (D) Large “mother” cell (MC) containing shapeless and round elementary bodies of varying electron density and size. MC is in process of extrusion of elementary bodies by formation of protrusion (PR). Polymorphic L-form elements are seen around the MC; (E,F). Spherical or ellipsoid electron dense CWD cells of different size with well-expressed granular consistence.

Figure 5

Light microscopy of L-form cultures from decidua (D) and maternal blood samples (B). (A) Initial development of L- forms (dark granules) associated with decidua cells in broth (D34). (B) Dark L-form granules along decidua cell debris in broth (D31). (C) Native smear from a pure broth L-form culture, developed after filtration through bacterial filter with 0.2 µm pore size. Cluster of spherical cells with different size (D31). (D–F). Native smears with spherical and granular L-form cells in broth L-form cultures from maternal blood samples B9, B31 and B34 respectively; (G–I) (L)-form growths after sub-cultivation in semisolid medium. Granular L-form growth of B9 (G), typical L-form “fried eggs” colonies of D8 (H) and D9 (I). Magnification: A, B, C, D, F- 1000x; G, H, I - 400x.

Figure 4

TaqMan PCR for IS1610 detection in filterable L-forms isolated from gestational tissues, cord blood and maternal blood samples. Amplifications were performed with chromosomal DNA from single L-forms colonies. DNA from M. tuberculosis H37Rv and M. bovis BCG was used as positive controls. MQ water was used instead of DNA template (NTC). (A) Graph with raw data (Ct values) of the placenta (PL) and cord blood (CB) samples, positive for mycobacterial L-forms; (B) Graph with raw data (Ct values) of the decidua (D) and maternal blood (B) samples, positive for mycobacterial L-forms; (C) Report from representative PCR run of DNA from L-forms colonies derived from term placenta and cord blood samples. Tr-trophoblasts.

Figure 6

The presence of mycobacterial L-forms in the placentobiome of BCG-vaccinated pregnant women has no an impact on the numbers of resident T cells, γδ T cells and phospho-reactive Vδ2 cells. Pl – placenta.