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.
Transition of bacteria to cell wall deficient L-forms in response to stress factors has been assumed as a potential mechanism for survival of microbes under unfavorable conditions. In this article, we provide evidence of paradoxal survival through L-form conversion of E. coli high cell density population after lethal treatments (boiling or autoclaving). Light and transmission electron microscopy demonstrated conversion from classical rod to polymorphic L-form shape morphology and atypical growths of E. coli. Microcrystal formations observed at this stage were interpreted as being closely linked to the processes of L-form conversion and probably involved in the general phenomenon of protection against lethal environment. Identity of the morphologically modified L-forms as E. coli was verified by species specific DNA-based test. Our study might contribute to a better understanding of the L-form phenomenon and its importance for bacterial survival, as well as provoke reexamination of the traditional view of killing strategies against bacteria.
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.
We suggest that the expression of phenotypic resistance to EMB in M. tuberculosis can be associated with alterations or loss of cell walls in L-form bacteria, respectively, which results in a lack of a specific target for EMB action. In addition, production of capsule-like structures and biofilm matrix by L-forms could contribute to their resistance and survival in the presence of antibacterial agents.
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