L-form bacteria cohabitants in human blood: significance for health and diseases.

From a historical perspective, intriguing assumptions about unknown "live units" in human blood have attracted the attention of researchers, reflecting their desire to define a new class of microorganisms. Thus, the concept of "blood microbiota" brings about many questions about the nature, origin, and biological significance of the "unusual microbial cohabitants" in human blood. In contrast to current views that bloodstream in healthy humans is sterile, the hypothesis about the existence of microbes as L-forms (cell wall deficient bacteria) in human blood has evolved on the basis of known facts about their unique biology, as observed in our studies and those of other authors. Recently, we reported that bacterial L-forms persist in the human blood and that filterable, self-replicating bodies with a virus-like size of 100 nm are able to cross the maternal-fetal barrier by vertically transmitted pathway, then enter fetus blood circulation and colonize newborns. Subjects discussed here include the following: Is the existence of L-form bacteria in human blood a natural phenomenon? Are L-form bacteria commensal cohabitants in the human body? Since blood is an unfavorable compartment for the classical bacteria and their propagation, how do L-forms survive in blood circulation? How does L-form microbiota in blood influence the host immune system and contribute to systemic inflammatory, autoimmune, and tumor diseases? The current commentary presents the topic of "human microbiota and L-form bacteria" in its microcosm. It contains details of the hypothesis, supporting evidence and important implications.

Mycobacterial L-forms are found in cord blood: A potential vertical transmission of BCG from vaccinated mothers.

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.

Presence of mycobacterial L-forms in human blood: Challenge of BCG vaccination.

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.

Stress-induced L-forms of Mycobacterium bovis: a challenge to survivability.

This study addressed the ability of Mycobacterium bovis to produce unusual extreme morphologic forms (cell wall-deficient or L-forms) under stress conditions. Models using nutrient starvation and cryogenic stress treatments of Mycobacterium bovis, as well as the filtration technique followed by cultivation in semisolid medium, were used for isolation of L-form variants. Morphological transformations and developmental stages, typical for the bacterial L-cycle were observed by electron microscopy. Of special interest was the formation of giant filaments and common extremely thick membranous structures enveloping the entire L-form population. Following collapse of giant filamentous structures small viable cell elements, mainly granules and coccobacilli, were released and proved able to grow into large bodies or multiply by fission or budding. Derivation of viable filterable forms from L-form cultures and parental strain and their identification as Mycobacterium bovis based on specific IS6110 PCR was noteworthy. We suggest that formation of giant filaments and thick common membranous envelopes, observed under stress conditions, may serve a twofold purpose - protection against an unfavourable environment, and a role in reproduction of Mycobacterium bovis L-forms. The observed L-form conversion phenomenon in Mycobacterium bovis seems to be associated with an adaptive strategy of this pathogen for survival and reproduction in an unfavorable environment.

Unique biological properties of Mycobacterium tuberculosis L-form variants: impact for survival under stress.

Bacteria can, under certain conditions, enter into a cell-less state known as L-form conversion. This phenomenon is universal, but also recognized with difficultly by microbiologists. The current study addresses several aspects concerning the ability of tubercle bacilli to use L-form conversion as a unique adaptive strategy to survive and reproduce under unfavorable conditions. Nutrient starvation of M. tuberculosis in vitro followed by passages in Middlebrook 7H9 semisolid medium was used for stress induction and the selective isolation of mycobacterial L-form variants. Light and electron microscopy images evidence the peculiar characteristics of mycobacterial L-forms. For example, mycobacterial L-forms were observed to lose their acid-fastness and change their morphology. In addition, wide morphological variability, the presence of large and elementary bodies, coccoids and small granular forms, as well as the appearance of unusual modes of irregular cell division were observed. Unlike classical tubercle bacilli, L-form variants grew and developed typical "fried-egg" colonies faster. L-forms were verified as M. tuberculosis by spoligotyping. The results provide insights into the nature of L-form phenomena in M. tuberculosis and link them to the mechanisms allowing mycobacterial survival under stress.

Filterable forms and L-forms of Mycobacterium bovis BCG: impact for live vaccine features.

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.

Bacterial L-forms.

L-forms are "cell wall-deficient" bacteria which are able to grow as spheroplasts or protoplasts. They can be differentiated into four types depending on their ability to revert to the parental, cell-walled form and to the extent of their cell-wall modification. L-forms are significant in modern science because of their contributions to an improved understanding of principal questions and their interactions with eukaryotes. This review particularly focuses on research using stable protoplast-type L-forms which have contributed to a better understanding of the structural and functional organisation of the cytoplasmic membrane and of cell division. These L-forms, which have only a single surrounding bilayer membrane, also represent a unique expression system for production of recombinant proteins. A large proportion of L-form publications concern their putative role in human disease and its therapy, a topic which is discussed briefly. L-forms have also been used to form intracellular associations with plant cells and have been shown to elicit induced disease resistance offering a novel method for plant protection. The recent decline in active research on L-forms is a concern as knowledge and experience, as well as unique L-form strains which have been maintained for decades, are being lost.

Persistence of Staphylococcus aureus L-form during experimental lung infection in rats.

The course of pulmonary infection in rats infected by intranasal inoculation with a Staphylococcus aureus stable protoplast L-form was studied. Blood and bronchoalveolar samples were taken on days 3, 7, 14 and 30 after challenge and were investigated by microbiological, electron-microscopic, cytochemical and cytometric methods. The electron microscopic data and isolation of L-form cultures from bronchoalveolar samples at all experimental times demonstrated the ability of S. aureus L-form cells to internalize, replicate and persist in the lungs of infected rats to the end of the observation period, in contrast to the S. aureus parental form. It was found that persisting L-form evoked ineffectual phagocytose by alveolar macrophages and low but long-lasting inflammatory reaction in rats. The experimental model of pulmonary infection with S. aureus L-form suggests that the cell-wall-deficient bacterial forms may be involved in the pathogenesis of chronic and latent lung infections.

[Salmonella typhimurium population in water environment under the influence of temperature]. / Sostoianie pipuliatsii Salmonella typhimurium v vodnoi srede pod vliianiem temperatury.

The strategy of the adaptation of S. typhimurium population to water environment under the influence of temperature factor was studied by scanning electron microscopy. Salmonellae were found to adhere to the surface of the Daphnia chitin covering. The study revealed that S. typhimurium population existed in water in the form of covered microcolonies as well as in the form of spheroplast-type cells and small cells in the L-form, joined with bands. The viability of salmonellae in water environment was studied without interaction and following interaction with Daphnia.

Induction of L-form-like cell shape change of Bacillus subtilis under microculture conditions.

A remarkable cell shape change was observed in Bacillus subtilis strain 168 under microculture conditions on CI agar medium (Spizizen's minimal medium supplemented with a trace amount of yeast extract and Casamino acids). Cells cultured under a cover glass changed in form from rod-shaped to spherical, large and irregular shapes that closely resembled L-form cells. The cell shape change was observed only with CI medium, not with Spizizen's minimum medium alone or other rich media. The whole-cell protein profile of cells grown under cover glass and cells grown on CI agar plates differed in several respects. Tandem mass analysis of nine gel bands which differed in protein expression between the two conditions showed that proteins related to nitrate respiration and fermentation were expressed in the shape-changed cells grown under cover glass. The cell shape change of CI cultures was repressed when excess KNO3 was added to the medium. Whole-cell protein analysis of the normal rod-shaped cells grown with 0.1% KNO3 and the shape-changed cells grown without KNO3 revealed that the expression of the branched-chain alpha-keto acid dehydrogenase complex (coded by the bfmB gene locus) was elevated in the shape-changed cells. Inactivation of the bfmB locus resulted in the repression of cell shape change, and cells in which bfmB expression was induced by IPTG did show changes in shape. Transmission electron microscopy of ultrathin sections demonstrated that the shape-changed cells had thin walls, and plasmolysis of cells fixed with a solution including 0.1 M sucrose was observed. Clarifying the mechanism of thinning of the cell wall may lead to the development of a new type of cell wall biosynthetic inhibitor.

[Formation of giant and ultramicroscopic forms of Nostoc muscorum CALU 304 during cocultivation with Rauwolfia tissues]. / Obrazovanie gigantskikh i ultramikroform Nostoc muscorum CALU 304 pri vzaimodeistvii s kul'tiviruemymi tkaniami rauvol'fii.

The study of heteromorphic Nostoc muscorum CALU 304 cells, whose formation was induced by 6- to 7-week cocultivation with the Rauwolfia callus tissues under unfavorable conditions, revealed the occurrence of giant cell forms (GCFs) with a volume which was 35-210 times greater than that of standard cyanobacterial cells. Some GCFs had an impaired structure of the murein layer of the cell wall, which resulted in the degree of impairment of the cell wall ranging from the mere loss of its rigidity to its profound degeneration with the retention of only small peptidoglycan fragments. An analysis of thin sections showed that all GCFs had enlarged nucleoids. The photosynthetic membranes of spheroplast-like GCFs formed vesicles with the contents analogous to that of nucleoids (DNA strands and ribosomes). About 60% of the vesicles had a size exceeding 300 nm. With the degradation of GCFs, the vesicles appeared in the intercellular slimy matrix. It is suggested that the vesicles are analogous to elementary bodies, which are the minimal and likely primary reproductive elements of L-forms. The data obtained in this study indicate that such L-forms may be produced in the populations of the cyanobionts of natural and model syncyanoses. Along with the other known cyanobacterial forms induced by macrosymbionts, L-forms may represent specific adaptive cell forms generated in response to the action of plant symbionts.

Novel bacterial membrane surface display system using cell wall-less L-forms of Proteus mirabilis and Escherichia coli.

We describe a novel membrane surface display system that allows the anchoring of foreign proteins in the cytoplasmic membrane (CM) of stable, cell wall-less L-form cells of Escherichia coli and Proteus mirabilis. The reporter protein, staphylokinase (Sak), was fused to transmembrane domains of integral membrane proteins from E. coli (lactose permease LacY, preprotein translocase SecY) and P. mirabilis (curved cell morphology protein CcmA). Both L-form strains overexpressed fusion proteins in amounts of 1 to 100 microg ml(-1), with higher expression for those with homologous anchor motifs. Various experimental approaches, e.g., cell fractionation, Percoll gradient purification, and solubilization of the CM, demonstrated that the fusion proteins are tightly bound to the CM and do not form aggregates. Trypsin digestion, as well as electron microscopy of immunogold-labeled replicas, confirmed that the protein was localized on the outside surface. The displayed Sak showed functional activity, indicating correct folding. This membrane surface display system features endotoxin-poor organisms and can provide a novel platform for numerous applications.

Cell wall-deficient forms (L-forms) of Listeria monocytogenes in experimentally infected rats.

Experimental infections were induced with different bacterial forms of Listeria monocytogenes: parental (S-forms), protoplastic (L-forms) and combined inoculum of both forms by i.p. injection of rats. The parental bacterial forms (S-forms) were isolated up to 7 days after challenge from the peritoneal cavity and the liver, while the L-forms were isolated up to 60 days from the peritoneal cavity. Continuous adhesion of L-forms on the peritoneal macrophage surface was found by scanning-electron microscopy. Erythrocyte and leucocyte count as well as some clinical chemistry parameters were measured during infections. They showed different dynamics in the three experimental groups. Histomorphological changes in the liver (microabscesses and mononuclear cellular granulomas) of infected animals were observed. They were less intensive and appeared later in rats infected with L-forms. The experiments demonstrated that infections caused by parental bacterial forms and by combined inoculum took an acute course, while the infection caused by L-forms could be distinguished as a prolonged and persistent one.

Bacterial persistence and expression of disease.

A considerable body of experimental and clinical evidence supports the concept that difficult-to-culture and dormant bacteria are involved in latency of infection and that these persistent bacteria may be pathogenic. This review includes details on the diverse forms and functions of individual bacteria and attempts to make this information relevant to the care of patients. A series of experimental studies involving host-bacterium interactions illustrates the probability that most bacteria exposed to a deleterious host environment can assume a form quite different from that of a free-living bacterium. A hypothesis is offered for a kind of reproductive cycle of morphologically aberrant bacteria as a means to relate their diverse tissue forms to each other. Data on the basic biology of persistent bacteria are correlated with expression of disease and particularly the mechanisms of both latency and chronicity that typify certain infections. For example, in certain streptococcal and nocardial infections, it has been clearly established that wall-defective forms can be induced in a suitable host. These organisms can survive and persist in a latent state within the host, and they can cause pathologic responses compatible with disease. A series of cases illustrating idiopathic conditions in which cryptic bacteria have been implicated in the expression of disease is presented. These conditions include nephritis, rheumatic fever, aphthous stomatitis, idiopathic hematuria, Crohn's disease, and mycobacterial infections. By utilizing PCR, previously nonculturable bacilli have been identified in patients with Whipple's disease and bacillary angiomatosis. Koch's postulates may have to be redefined in terms of molecular data when dormant and nonculturable bacteria are implicated as causative agents of mysterious diseases.

Effect of Escherichia coli L-form cytoplasmic membranes on the interaction between macrophages and Lewis lung carcinoma cells: scanning electron microscopy.

Scanning electron microscopy (SEM) investigations on the interactions between peritoneal macrophages from Lewis lung carcinoma (LLC)-bearing mice and LLC tumour cells during 21 days after tumour implantation were carried out. The action of lipopolysaccharide (LPS)-containing cytoplasmic membranes (CM), from the stable protoplast type L-form of Escherichia coli, on the activity of in vitro phagocytosis was studied; CM induced a continuous increase in macrophage numbers. Activation of macrophage surfaces in healthy and tumour-bearing mice was established. Lamelipods, pseudopods and migration fringes 14 days after CM application were seen. Crater-like cavities deeply in the macrophage cells as well as adherent or prominent engulfed tumour cells within macrophages were observed during in vitro interaction with LLC cells. Macrophages from tumour-bearing mice without CM treatment showed less activation evaluated by SEM during earlier stages of tumour growth. The SEM investigation proved the temporary stimulating effect of E. coli L-form CM on the cell surface activation of peritoneal macrophages in healthy and LLC-bearing mice.

[A clinical and experimental study of L-form bacteria in 66 cases].

In order to study the relationship between the time for establishing the diagnosis of infectious diseases and L-form bacteria, a series of clinical specimens taken from 321 cases of patients suspected to have infection were collected. Besides routine bacterial culture, special culture for L-form bacteria was also performed. The results were as follows: the rate of positive routine bacterial culture was 10.90% (35/321); the rate of negative routine bacterial culture but positive L-form bacterial culture was 20.56% (66/321). In this study, L-form bacteria infection was treated with sensitive antibiotics and a satisfactory result was obtained. It is shown that L-form bacterial culture is very useful in detection of pathogenic bacteria and helpful to the therapy of infectious diseases. Ultrastructure organization of these bacteria was studied by using transmission electron microscope.

[The mechanisms of the pathogenicity of bacteria in different infections]. / Mekhanizmy patogennosti bakterii pri razlichnykh infektsiiakh.

The following ultrastructural formations are found in the bacteria of various infections: fibrillar and drop-like microcapsules, an increase of nucleotide size and number, micropyles. The dynamics of staphylococcus L-form formation in sepsis as well as the phenomenon of incomplete phagocytosis and endocytobiosis were studied. The latter is observed in mixed infection: dysentery bacteria lamblia, gonococci and trichomonas. These alterations indicate increased bacterial pathogenicity and seem to reflect the evolution of the bacteria adaptive mechanisms under the conditions of antibiotic therapy.

[The variability of Yersinia pestis in the body of the flea]. / Izmenchivost' Yersinia pestis v organizme blokhi.

Changes in the morphology and ultrastructure of Y.pestis cells at different periods of their stay in the body of fleas (Citellophilus tesquorum altaicus) have been studied. The study, carried out by means of optical and electron microscopy, as well as with the use of a culture medium for the isolation of L-forms, has revealed that in the body of fleas Y.pestis cells undergo the effect of processes leading to their L-transformation. As the result of L-transformation, the infective agent may take altered forms, including L-like variants. Such forms are retained in hungry insects and are capable of prolonged survival in the body of the carrier.

Serum inhibits penicillin-induced L-form growth in Staphylococcus aureus: a note of caution on the use of serum in cultivation of bacterial L-forms.

Heat-inactivated horse serum inhibited penicillin-induced L-form colony formation in Staphylococcus aureus when included in an osmotically stabilized culture medium. Most, perhaps all, L-form colonies that appeared with low frequencies on the serum-supplemented medium were of the penicillin-independent, stable type. This relationship must be taken into account when use of serum is considered for L-form cultivation.

[Tinctorial properties of mycobacteria using standard and nontraditional staining technics]. / Tinktorialnye osobennosti mikobakterii pri ispolzovanii standartnykh i netraditsionnykh metodov okraski.

Tinctural specificity was studied for various mycobacterial species (M. tuberculosis Erdman TMC 107, M. tuberculosis Academia, M. Bovis Bovinus-8, M. Bovis BCG Russian, M. Avium Avium-53) subjected to staining according to Ziehl-Neelsen, Boy (fluorescent staining), Gram and Romanovsky-Gimze. The cultures were studied on Pavlovsky's continuous medium under natural ageing. The authors tested adequacy of diverse bacterioscopic methods for registration of all the cells in the mycobacterial population. Mycobacterial morphological and tinctural features are shown to be dependent on the culture duration and species. Non-conventional Gram's and Romanovsky-Gimze techniques were able to identify L-form granular balls and non-acid-fast coccus-like forms. The presence and the number of mycobacterial changed forms are species-specific and related to the culture duration.