Eubiotic vs. dysbiotic human blood microbiota: the phenomenon of cell wall deficiency and disease-trigger potential of bacterial and fungal L-forms.

The current review provides data and focuses on blood as a niche for the presence of cell wall-deficient microbes (L-forms). The hypothesis for the existence of L-form microbiota in humans was tested by us using an innovative methodology for the isolation of L-form cultures from human blood. Criteria were conceived for the individual assessment of blood microbiota and recognition of two types of states -- "eubiotic" and "dysbiotic" blood microbiota. Cell wall-deficient microbes (CWD) that inhabit blood in healthy people are in natural balance with the host homeostasis, which corresponds to the "eubiotic" state. When interacting with a host, CWD bacteria or fungi employ a strategy distinctive for a latent lifestyle. In contrast to "eubiotic," "dysbiotic" blood microbiota manifests when the balance is disrupted and there is an excess of L-form variants of opportunistic microbes that invade from the external microbiota, i.e., from all body sites in contact with the external environment. Our case studies on people with multiple sclerosis (MS), Parkinson's disease, psoriasis, thyroid cancer, and diabetes revealed the appearance of "dysbiotic" blood microbiota that outlined the disease-trigger potential of opportunistic bacteria and fungi existing in blood as CWD variants. Blood microbiota assessment could be of diagnostic and prognostic importance for the pathological processes occurring within the body, as well as for understanding the microbial pathogenesis.

Cell-wall deficient L. monocytogenes L-forms feature abrogated pathogenicity.

Stable L-forms are cell wall-deficient bacteria which are able to multiply and propagate indefinitely, despite the absence of a rigid peptidoglycan cell wall. We investigated whether L-forms of the intracellular pathogen L. monocytogenes possibly retain pathogenicity, and if they could trigger an innate immune response. While phagocytosis of L. monocytogenes L-forms by non-activated macrophages sometimes resulted in an unexpected persistence of the bacteria in the phagocytes, they were effectively eliminated by IFN-γ preactivated or bone marrow-derived macrophages (BMM). These findings were in line with the observed down-regulation of virulence factors in the cell-wall deficient L. monocytogenes. Absence of Interferon-ß (IFN-ß) triggering indicated inability of L-forms to escape from the phagosome into the cytosol. Moreover, abrogated cytokine response in MyD88-deficient dendritic cells (DC) challenged with L. monocytogenes L-forms suggested an exclusive TLR-dependent host response. Taken together, our data demonstrate a strong attenuation of Listeria monocytogenes L-form pathogenicity, due to diminished expression of virulence factors and innate immunity recognition, eventually resulting in elimination of L-form bacteria from phagocytes.

[Correlation between bacterial L-form infection, expression of HIF-1α/MMP-9 and vasculogenic mimicry in epithelial ovarian cancer].

The aim of the present study is to explore whether vasculogenic mimicry (VM) and bacterial L-form infection exist in human epithelial ovarian cancer (EOC) or not and to elucidate the correlation of L-form infection, expression of hypoxia inducible factor 1α (HIF-1α)/MMP-9 and VM. In 87 specimens of EOC and 20 specimens of ovarian benign epithelial tumor tissues, L-form infection was detected by Gram's staining, expression of HIF-1α/MMP-9 and VM were detected by immunohistochemical and histochemical staining. The results showed that the positive rates of HIF-1α and MMP-9 protein in EOC were 52.9% and 66.7%, while in benign epithelial tumor tissues, the positive rates were 10.0% and 10.0% respectively, and there were significant differences between them (P < 0.05). In EOC and benign epithelial tumor tissues, L-form infections ratios were 24.1% and 0, respectively, and the difference was also significant (P < 0.01). Expression of VM, HIF-1α and MMP-9 in EOC was significantly related to differentiation, abdominal implantation and lymph node metastasis and FIGO stage (P < 0.01). L-form infection had relationship with abdominal implantation, lymph node metastasis and FIGO stage (P < 0.01 or 0.05). The expression of HIF-1α had positive relationship with expression of MMP-9 and VM (r = 0.505, 0.585, respectively, P < 0.01); there was also a positive relationship between MMP-9 expression and VM (r = 0.625, P < 0.01). Overexpression of VM, HIF-1α and MMP-9 were related to poor prognosis: the survival rates were significantly lower in positive patients than those in negative patients (P < 0.05). And the group with L-form infection also had poor prognosis: the survival rates were lower than those in group without infection (P < 0.05). FIGO stage, expression of VM, HIF-1α and MMP-9 were independent prognosis factors of EOC (P < 0.05). The results suggest that L-form infection, the expression of HIF-1α, MMP-9 and VM in EOC are related to differentiation, lymph node metastasis, clinical stage and prognosis. Combined detection of these indexes has an important role in predicting the progression and prognosis of EOC.

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.

Cell wall-deficient (CWD) bacterial pathogens: could amylotrophic lateral sclerosis (ALS) be due to one?

Recently, a number of diseases that had been thought previously to be caused by something other than an infectious agent are now known to be caused by bacteria. It now appears that it is not uncommon that bacteria, viruses, or fungi can cause diseases even when these organisms have not been detected or cultured. The most recent, well-publicized case is that of stomach ulcers; these are largely due to Helicobacter pylori infections. Here, the possibility is explored that amylotrophic lateral sclerosis (ALS) is caused by a cell wall-deficient microorganism.

[The antilysozyme activity of Mycobacterium tuberculosis L forms]. / Antilizotsimnaia aktivnost' L-form Mycobacterium tuberculosis.

The method for the detection of antilysozyme activity (ALA) in M. tuberculosis L forms was developed. The level of ALA in M. tuberculosis L forms isolated from patients with different clinical forms of the disease varied within 1-5 micrograms. M. tuberculosis L forms with the ALA level > 4 micrograms were isolated from patients with the progressing course of the disease. The method for the prognostication of the course of the tuberculous process in the lungs by the results of the antilysozyme test was proposed.

[Study on pathogenicity of sputum from cavity of sputum negative patients with pulmonary tuberculosis after short course chemotherapy].

OBJECTIVE: To find out whether or not the sputa of pulmonary tuberculosis(PTB) patients with cavitation but sputum negative have pathogenicity after short course chemotherapy. METHODS: Guinea pigs were inoculated with sputa of PTB patients who were with cavitation but sputum negative after having finished short course chemotherapy. Then their body weight, enlargement of local lymph nodes and other ordinary symptom were observed. Six weeks later, pathological changes of TB in the internal organs were examined by dissecting these guinea pigs. Culture and drug resistance test of tubercle bacillus were also conducted. All of which were with negative and positive controls. RESULTS: Of the 63 cases included, 3(5%) patients' sputa resulted in tuberculous nodulation varying in amount in lung, liver and spleen of these guinea pigs, and the culture for tubercle bacillus of these sputa was positive too. CONCLUSIONS: 5% of sputum collected from PTB patients with cavitation but sputum negative still show pathogenicity after short course chemotherapy. For the cases with drug-resistant PTB and slow sputum negative conversion, the treatment should be prolonged and tubercle bacilli in their sputa should be monitored.

[Microorganism persistence in hematopoietic tissue: means for detecting it and its significance]. / Persistentsiia mirkoorganizmov v krovetvornoi tkani: puti vyiavleniia i znachimost'.

Many microorganisms are capable of prolonged persistence in the marrow. In this study, carried out by the method of negative selection based on the treatment of mouse marrow cells with specific antimicrobial sera and complement, Mycoplasma arthritidis and L-forms of group B streptococci were found to be capable of persisting in the marrow in close association with the late category of clonogenic precursor cells, CFU-7, as well as, to a lesser extent, with late erythroid precursors, CFUe. Early colony-forming cells, CFUs-12 and PFUe, as well as granulocyto-macrophagal precursors, CFUgm, did not practically express antigens to the given infective agents on their surface.

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.

[The pathomorphology and pathogenesis of glanders in laboratory animals]. / Patomorfologiia i patogenez sapa u laboratornykh zhivotnykh.

The pathomorphology and cell-mediated response of the body to Burkholderia mallei in laboratory animals, highly sensitive and resistant to these bacteria. In the comparative study of the pathomorphology and pathogenesis of glanders in golden hamsters and white rats quantitative and qualitative differences in the histogenesis of response reaction and the morphology of immunocompetent organs were established. Cell-mediated reactions play a greater role in the limitation of the early spread of B. mallei in the host body than antigen-mediated mechanisms.

[An experimental study of the possibility for the preservation of the causative agent of plague in the nest substrate of the long-tailed suslik]. / Eksperimental'noe izuchenie vozmozhnosti sokhraneniia vozbuditelia chumy v substrate gnezda dlinnokhvostogo suslika.

How long Yersinia pestis can preserve in the nest substrate of Spermophililus undulatus and whether it can infect the animals contacting with the substrate infected with flea feces and carcasses were studied in a Tuva natural focus. The infected material was kept in special niches of a bunkering laboratory, which imitated a souslik nest. Biological, serological, and bacteriological studies were conducted after different storage periods. The experiment used 25 sousliks, 56 albino mice, and 3256 fleas. Two hundred and ninety flea carcasses were individually explored. Y.pestis was ascertained to survive in the nest substrate infected with flea feces and carcasses. The pathogen may be preserved as a mutant in the flea carcasses, as evidenced by the isolation of 6 Y. pestis cultures from flea carcasses as a L-form. There is evidence for the fact that healthy animals may be infected on their contact with the contaminated substrate.

Experimental otitis media induced by coagulase negative staphylococcus and its L-forms.

In a previous study, we found 15% L-forms of bacteria (predominately coagulase negative staphylococci (CNS)) in ears which gave negative cultures by conventional methods. In this study, we used an animal model to test whether CNS and its L-forms can be pathogenic and whether L-forms have a crucial role in the tendency to secretory otitis media (SOM). We inoculated the tympanic bullas of guinea pigs, in 2 groups, with CNS and its L-forms (revertant forms). We observed that both CNS and its L-forms had the capability of causing infection. However, it was milder for the L-forms than CNS. We clearly noticed that on day 30 60% of the ears inoculated with L-forms had effusion and/or retraction of the tympanic membrane. These ears were histopathologically characterized by hypertrophied pseudostratified epithelium or stratified squamous epithelial metaplasia. The ears inoculated with the original form of CNS had only 16.66% effusion. On day 60 we observed similar findings. Thus, it might be proposed that L-forms could be responsible for chronic irritation to middle ear mucosa leading to SOM.

[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 biomedical aspects of the persistence of bacteria]. / Biomeditsinskie aspekty persistentsii bakterii.

A correlation between the structure and function of bacteria was analyzed in the process of their persistence in the host body. A variety of persistent forms of bacteria was shown to be based on the isolation of their morphological substrate, peptidoglycane, which a cell "masked" (screening by surface bacterial structures, antigenic mimicry), "lost" (L-forms of bacteria, mycoplasmas) or protected against the system of host immunity by secreted factors. A new group of secreted bacterial (antilysozyme, anti-interferon, anti-immunoglobulin, anticomplement) factors, permitting microbial persistence in the host body, was described. Different methodological approaches to their determination were developed on the basis of the principle of "delayed antagonism". Applied aspects of the problem of persistence of bacteria were reviewed. The efficacy of new methods developed for the isolation and identification of a causative agent under the control of persistence markers was demonstrated on facultative microflora in different surgical, obstetrical, gynecological, urological diseases and diseases of internal organs. The facts concerning the use of the factors of bacterial persistence were presented for the solution of therapeutic (selection of means for controlling cell parasites), prognostic (development of carrier state in convalescents) and ecological (microbiological monitoring of the environment) problems.

[An immunological analysis of bacterial persistence in the bone marrow]. / Immunologicheskii analiz bakterial'noi persistentsii v kostnom mozge.

A method for the evaluation of bacterial persistence in the bone marrow in association with particular clonogenic target cells was developed. The method was based on the negative selection of cells expressing microbial antigens after treatment with hyperimmune antiserum specific to a given infective agent and the subsequent quantitation of target cells thus eliminated in appropriate assays. Using this approach, we demonstrated that Mycoplasma arthritidis and L-forms of Streptococcus strain L-406 were capable of persisting in murine bone marrow in close association with CFUs-7 (a subpopulation of hematopoietic stem cells) for at least several months after experimental infection. Francisella tularensis was also found to be capable to express on the CFUs-7 membranes. Persisting microorganisms enhanced both proliferation and migration of CFUs-7.

[The results of a 15-year study of the persistence of Yersinia pestis L-forms]. / Itogi 15-letnego izucheniia persistentsii L-form Yersinia pestis.

New diagnostic preparations for the detection of L-forms, immunoglobulin erythrocyte diagnosticum and luminescent antimembrane immunoglobulins, are described. Proofs indicating that Y.pestis L-forms are constantly isolated from wild rodents and their ectoparasites in the natural foci of plague are given. To control the effectiveness of the sanitation of such natural foci, the use of the radioimmunoassay and the enzyme immunoassay, known to be the most sensitive techniques, are recommended. The facts indicating that Y.pestis contaminating ticks and fleas are converted into L-forms, capable of persisting for several years, are presented. After their reversion to back the initial rod-shaped form the transmission of infection is possible. A suggestion is put forward that the persisting forms of Y.pestis play an important role in the preservation of this infective agent in the natural focus of infection.

[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.