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Our previous studies showed that mycobacterial L-forms persist in the blood of BCG vaccinated people and that BCG vaccine is able to produce, under appropriate conditions, filterable, self-replicating L-bodies with virus-like size. Because filterability is one of the characteristics of L-forms, considerable interest has been shown in their capacity to cross the maternal-fetal barrier. The current study demonstrated isolation of mycobacterial L-form cultures from umbilical cord blood of 5 healthy newborns of healthy mothers vaccinated previously with BCG. The isolated cultures showed distinctive growth characteristics of cell wall deficient L-form bacteria. Transmission electron microscopy demonstrated presence of L-bodies with extremely small size of 100 nm and revealed morphological transformations, typical for L-forms. IS6110 Real Time PCR assay confirmed that all L-form isolates were of mycobacterial origin and belonged to Mycobacterium tuberculosis complex which includes vaccinal BCG substrains. In conclusion, we could suggest that reproductive filterable L-bodies of BCG origin are able to fall in blood circulation of the fetus by vertical transmitted pathway and colonize newborns.

Possible persistence of bacteria in human blood as cell wall deficient forms (L-forms) represents a top research priority for microbiologists. Application of live BCG vaccine and L-form transformation of vaccine strain may display a new intriguing aspect concerning the opportunity for occurrence of unpredictable colonization inside the human body by unusual microbial life forms. L-form cultures were isolated from 141 blood samples of people previously vaccinated with BCG, none with a history of exposure to tuberculosis. Innovative methodology to access the unusual L-form elements derived from human blood was developed. The methodology outlines the path of transformation of non- cultivable L-form element to cultivable bacteria and their adaptation for growth in vitro. All isolates showed typical L-forms growth features ("fried eggs" colonies and biofilm). Electron microscopy revealed morphology evidencing peculiar characteristics of bacterial L-form population (cell wall deficient polymorphic elements of variable shape and size). Regular detection of acid fast bacteria in smears of isolated blood L-form cultures, led us to start their identification by using specific Mycobactrium spp. genetic tests. Forty five of 97 genetically tested blood cultures provided specific positive signals for mycobacteria, confirmed by at least one of the 3 specific assays (16S rRNA PCR; IS6110 Real Time PCR and spoligotyping). In conclusion, the obtained genetic evidence suggests that these L-forms are of mycobacterial origin. As the investigated people had been vaccinated with BCG, we can assume that the identified mycobacterial L-forms may be produced by persisting live BCG vaccine.

Bacterial L-form conversion, or existence without cell walls, is assumed a universal phenomenon in nature. An interesting aspect of this phenomenon is occurrence of L-forms in vaccine strains. Since BCG is currently a widely used and extensively studied live vaccine for tuberculosis, understanding L-form conversion of M. bovis BCG bacilli can provide new insight into behavior of BCG vaccine. In this respect, specific features, concerning the ability of BCG vaccine to produce viable filterable forms and L-forms, were studied by filtration and starvation stress experiments in vitro. The filterable forms obtained after filtration of BCG suspension, grew on Middlebrook 7H9 semisolid agar and formed typical "fried eggs" L-form colonies. Electron microscopy clearly demonstrated presence of L-form elements with size smaller than the size of bacterial filter pores of 0.2 µm in M. bovis BCG strains. Development of L-form subpopulation with typical morphological appearance of self-replicating cell wall-defective forms was observed after filtration, as well as after starvation stress. Specific DNA detection of pncA gene in derived L-form cultures from filterable and stressed BCG strains verified their identity as M. bovis BCG. In conclusion, the results confirm existence of filterable forms in commercial BCG vaccine, which are able to develop L-form population under appropriate conditions. L-form transformation of BCG bacilli displays a new intriguing aspect concerning exhibition of unusual features and atypical behavior of live BCG vaccine. Further research is requested to explore the influence of L-form phenomenon on BCG vaccine effects in vivo.