The present study is focused on the placental microbiome - new and hot topic of research - in the context of mother’s BCG vaccination. Here we report for the first time to our knowledge an isolation of mycobacterial L-form cultures from gestational tissues of healthy newborns delivered by healthy BCG-vaccinated mothers after normal pregnancy. Since BCG vaccination is currently recommended in 156 countries37 and mandatory in 64 of them38, this study opens the question whether the vaccination in the childhood of the woman has an impact on her placental microbiome during pregnancy. Although the effectiveness of the BCG vaccine has been debated for decades, the possible trans-placental transfer of BCG L-forms as a phenomenon affecting fetal and/or neonatal immune system is not described so far.

Our study clearly demonstrates four major findings: 1) presence of mycobacterial L-forms in the placenta and cord blood of healthy newborn babies delivered from healthy BCG-vaccinated mothers; 2) vertically transmitted mycobacterial L-forms originated from the maternal BCG vaccine have a capacity to revert back to walled mycobacteria; 3) placenta colonization with mycobacterial L-forms occurs by maternal blood-to-decidua transfer very early in gestation (1st trimester) and 4) mycobacterial L-forms as a part of placentobiome of BCG-vaccinated pregnant women did not influence the number of placental pathogen-reactive Vδ2 cells.

L-forms differ fundamentally from normal state of bacteria. They exist without cell walls, assume mostly spherical shapes and are capable of multiplying through unusual modes, such as irregular binary fission, budding, protrusion-extrusion of elementary bodies and granules from large bodies, multiple divisions with intracellular fragmentation of cytoplasm, or combination of all types39. Recent experiments by Loessner’s group have shown that L-forms are an independent form of life that can multiply indefinitely as the authors said “an alternative form of bacterial life”40. Beyond doubt, our morphological examination of isolated L-form cultures and especially TEM observation on placental L-forms, point to an amazing cell division mechanism in which new L-form vesicles are formed (by membrane constriction i.e. invagination) and develop within large ‘mother’ cell. When the mother cells enlarge enough a release of daughter cells (new L-forms) occurs (see Fig. 3). Our data fits well with recently published time-lapse confocal laser scanning microscopy data of the growth and multiplication of GFP-labelled L-forms showing different steps of L-form propagation as a novel model for the growth and division of L-forms of Listeria, B. subtilis, E. coli, S. aureus, and C. glutamicum 41,42. In line with other researchers and our previous studies, we found here that L-forms in gestational tissues and blood exist as unusual morphological units, such as large and elementary bodies, vesicles, granules, filterable forms, and display typical L-form growth. Although polymorphic, L-forms usually form colonies with unique shape of “fried eggs”, which are accepted by many authors as a convincing criterion for L-form growth7,33,39,43,44. Crucially, IS6110 Real Time PCR assay confirmed that L-form isolates from gestational tissues and maternal/cord blood are mostly of mycobacterial origin. Our recent publications showed that mycobacterial L-forms persist in blood of BCG-vaccinated people and that filterable, self-replicating bodies with virus-like size of 100 nm are able to cross the materno-fetal barrier, and by falling in fetus blood circulation to colonize the newborn10,11. Keeping in mind that L-form cultures were isolated after filtration of broths, inoculated with gestational tissue, maternal blood or cord blood samples, we suggest that very small L-bodies (filterable forms) are able to pass through pores with 0.2 µm size, germinate again and to launch a new life L-cycle. The filterable forms have been considered as minimal reproductive cells, which can be formed from large L-bodies in all possible ways. It is believed that such filterable bodies contain a bacterial genome and minimal metabolic capability sufficient to initiate reproduction7,39,45. One of characteristics that distinguish L-forms from walled bacteria is loss of peptidoglycans (PG), respectively PG -associated molecules and their functions46,47. Of interest, some authors accept that electron-dense spots found in L-form membrane regions, observed by TEM also in our study, are accumulated PG precursor molecules made in the cell cytoplasm which normally would be inserted into the existing PG meshwork outside of the membrane48. In general, L-forms are thought to be unstable and genetically identical to their parent strain. They retain the ability to revert back to the classical walled form, suggesting that the ability to rebuild a cell wall de novo is also a common property of bacteria49,50. Indeed, isolated filterable L-forms in our study were unstable and reversible and we successfully visualized CDW bacteria together with their walled counterparts - ZN-positive typical rod-shaped mycobacterial bacilli in the cultures of placental and cord blood isolates.

Demonstration that BCG bacilli can convert to CWD forms inside resting or pre- activated macrophages in vivo suggests that this phenomenon could significantly enhance BCG survival and persistence ability4,33. Moreover, the well-packed BCG L-forms could be released to the extracellular space probably exploiting apoptotic-like pathway for subsequent rounds of new entry and uptake by macrophages4. Thus, the L-form state of M. bovis could be regarded as an important factor for the long-term survival strategy of this pathogen. Actually, little is known how long M. bovis BCG as a live strain can survive in the vaccinated persons. There are reports about detection and isolation of BCG bacilli from patients with AIDS many years after their vaccination51,52,53. Recently published data showed that conversion of bacteria to L-forms, may often result in chronic infections, since L-forms remain slumbered for long periods in the tissues becoming sequestered in protective regions of the body7. Placenta is considered as such a protective and immune privileged organ, where an immune tolerance must be established in the course of normal pregnancy. The placenta forms a barrier between mother and fetus and in this respect it provides immunologic control on the transfer of gas, nutrients, pathogens and antigens from mother to fetus. In human invasive implantation and hemochorial type of placenta, placental trophoblast cells come in close contact with the maternal tissues forming two materno-fetal interfaces. One materno-fetal interface is that wherein the maternal decidua contacts with the interstitial placental cytotrophoblasts. The other one is formed between the placental chorionic villi (covered by syncytiotrophoblasts) and the maternal blood and increases in volume as pregnancy progresses. We still know very little about the identity and number of microbes that traverse the placenta, whether they persist in the infant or whether their presence has short- or long term health consequences. Much of the current knowledge about placental barrier comes from the understanding of its impermeability to many large molecules and circulating pathogens54. Different pathogens can infect and cross the placenta include viruses (e.g. Cytomegalovirus, HIV), protozoa (e.g. Toxoplasma, Trypanosoma, Leishmania, Plasmodium), and bacteria (e.g. Treponema, Brucella, Listeria)55,56. Among macroparasites, some helminths have also been observed to cross the placental barrier (e.g. Toxocora57,58 and Trichinella59). Silica nanoparticles smaller than 300 nm have been shown to penetrate the placental barrier in mice60. Nano-sized vesicles of 30–100 nm in diameter (exosomes) and their trafficking within the placental micro-environment have been recognized to play a role in mediating embryo-maternal interactions61,62. It has been shown that exosomes, released by BCG-infected macrophages contain mycobacterial components62. Summing up the accounted data above and dual materno-fetal contact we could suggest couple of routes for vertical transmission of mycobacterial L-forms such as hematogenous transfer across the placenta by direct infection of trophoblasts or through intercellular communication, placental transfer of the infected immune cells from the maternal blood stream and/or transfer of mycobacterial L-forms from infected maternal decidua. The later pathway is supported by our finding that in half of the paired maternal blood-decidua samples, the decidua was already colonized with mycobacterial L-variants. Moreover, two of the positive paired samples were completed with positive trophoblasts as well showing that L-forms were transmitted to early placenta (trophoblasts) through maternal decidua. Although we succeeded to isolate mycobacterial L-forms from gestational tissues as early as 6 weeks of pregnancy would be interesting to screen more triple samples maternal blood-decidua-trophoblasts in order to find out convincing data about direct and/or mediated by the maternal decidua L-forms infection of trophoblasts.

In agreement with previous studies we found that in human term placenta γδ T cells account for 5–10% of CD3+ lymphocytes63. About 10% of the resident γδ T cells are phosphoreactive Vδ2 cells, which is in line with data showing that the majority of the placental γδ T cells are Vd132. Our results demonstrate that the presence of mycobacterial L-forms in the placentobiome of BCG-vaccinated pregnant women had no impact on the number of placental Vδ2 cells. We could not exclude, however, a possible reflection on their phenotype. In normal state mycobacteria four different phosphoantigens termed TUBag1 to TUBag4 have been isolated and identified from the mycobacterial wall64 suggesting that the maintenance of specific Vδ2 immunity necessarily needs BCG bacilli to produce these immunogenic cell wall-associated compounds65. When bacteria shed their cell wall, they might also lose factors contributing to their specific ‘pathogen-associated molecular pattern’ important for recognition of the invader by the (innate) immune system of the hosts66,67,68. Data from experimental infections with L-forms demonstrated their atypical interactions with phagocyte cells and incomplete and ineffective phagocytosis69,70,71,72. As the bacterial cell wall is highly immunogenic, several groups have tried to elucidate whether or not CWD bacteria evoke an immune response and, if so, how this response differs from that triggered by bacteria harboring a cell wall (reviewed in)73.

Some authors have found that the L-forms were pathogenic only when reverted in the host to bacterial forms74. As discussed by Errington et al. these reports show controversial results and most of the work is difficult to interpret in the light of our modern understanding of immune mechanisms, particularly innate immune responses8. Thus, although not proven yet it is reasonable to assume that when BCG bacilli transform in CWD forms, probably they cease and/or decrease their immunogenicity but still could provide a reservoir hidden from the immune system and resistant to treatment with cell-wall-specific antibiotics.

Despite large amounts of literature published on L-forms, atypical bacterial forms have been neglected by clinicians for very long time because of difficulty to identify and prove them. However, a lot of papers and reviews44 , 6 , 7 support the concept that L-forms can be induced in vivo, can persist there for a significant span of time and can be the cause for latent, chronic and relapsing/recurrent infections, as well as for diseases of unknown infectious-allergic or autoimmune origin. Combination of modern imaging techniques and molecular approaches, such as generation of stable L-form lines and fluorescently stained L-form cells, an array-based transcriptomics of parent and L-form cells have been applied in order to better understand L-form conversion and the molecular and genetic changes accompanying this unusual transition40 , 8,42. The change from the normal form to the L-form is accompanied by drastic changes in cell metabolism and gene activity, suggesting that L-forms can adapt to their cell wall deficiency by adjusting expression levels of genes important for survival and adaptation to this unusual life style41 , 75.

In summary, this study identifies novel data about mycobacterial L-forms colonization of gestational tissues (placenta, decudua) and cord blood of healthy newborns delivered by healthy BCG-vaccinated pregnant women and provides the first formal demonstration that maternal BCG vaccination affects the placentobiome. Work on L-forms in cord blood provides food for thought in terms of the safety of the use of cord blood stem cells for bone marrow transplantation. Our research is ongoing to define how maternal decidua mediates the process of mycobacterial L-forms colonization of placenta. With the results here we hope to stimulate a research on L-forms in the placentobiome and their short- and long-term effects on the immunity of the fetus/newborn as well as the relationship of these cryptic organisms with autoimmune diseases in adulthood.