Isolation of L-form Bacteria from Urine using Filtration Method.

Transition of bacteria to the L-form state is thought to play a possible role in immune evasion and bacterial persistence during treatment with cell wall-targeting antibiotics. However, isolation and handling of L-form bacteria is challenging, mainly due to their high sensitivity to changes in osmolarity. Here, we describe detailed protocols for the preparation of L-form medium, isolation of L-forms from urine using a filtration method, detection of L-forms in urine samples by phase contrast microscopy and induction of L-forms in vitro. The exact requirements for survival and growth of L-forms may vary from strain to strain. Therefore, the methods presented here are intended to act as basic guidelines for establishing L-form protocols within individual laboratories, rather than as precise instructions. The filtration method can lead to a reduction in the number of L-forms in a sample and should not be used for quantification. However, it is the only method used so far for effective separation of cell wall-deficient variants from their walled counterparts and for identification of bacterial strains, which are capable of L-form switching in patients with urinary tract infections. The filtration method has the potential to be adapted for the isolation of L-forms from patients with other categories of bacterial infections and from environmental samples.

Dysbiotic microbiota in autistic children and their mothers: persistence of fungal and bacterial wall-deficient L-form variants in blood.

Based on our hypothesis for existing microbiota of wall-deficient variants (L-forms) in human blood, we created an innovative methodology, which allowed for the development of L-form populations from blood of all investigated people. In contrast to healthy controls, blood L-forms from autistic children and their mothers converted under appropriate conditions of cultivation into detectable opportunistic bacteria and fungi, а process demonstrated by light and transmission electron microscopy. It can be distinguished into two types of states - "eubiotic" blood microbiota in healthy individuals, and "dysbiotic" in autistic children and their mothers. Remarkably, the unifying finding for autistic children and their mothers was the presence in blood of wall-free variants from life-cycle of filamentous fungi. Increased specific IgG, IgM and IgA, together with typical mold growth were a decisive argument for proven presence of Aspergillus fumigatus in almost all of the autistic children. As it was demonstrated in our previous study, filterable L-forms can be transmitted by vertical pathway from mother to child before birth. Thus, it can be suggested that autistic children may be born already colonized with fungi, while a "silent aspergillosis" could contribute or even be a leading cause for neurodevelopmental disorders in the early childhood.

Mother-to-newborn transmission of mycobacterial L-forms and Vδ2 T-cell response in placentobiome of BCG-vaccinated pregnant women.

The ability of bacteria to exist as a population of self-replicating forms with defective or entirely missing cell wall (L-forms) is an adaptive mechanism for their survival and reproduction under unfavorable conditions. Bacterial mother-to-fetus transfer is a universal phenomenon in the animal kingdom. However, data about vertical transfer of L bacterial forms are extremely scarce. Bacille Calmette-Guérin is an attenuated strain of M. bovis and the only licensed vaccine used for tuberculosis prevention. We already have shown that filterable L-forms of BCG exist freely in the vaccine and are able to reproduce and to form colonies. The present study was focused on the placental microbiome in the context of mother's BCG vaccination. Here we report an isolation of filterable mycobacterial L-form cultures from gestational tissues and blood of healthy newborns delivered by healthy BCG-vaccinated mothers after normal pregnancy. Of note, vertically transmitted mycobacterial L-forms as a part of placentobiome of the pregnant women didn't influence the number of resident pathogen-reactive Vδ2 cells. Placenta colonization with mycobacterial L-forms occurs by maternal blood-to-decidua transfer very early in gestation. Together, these data showed that BCG L-forms have the capacity to pass trans-placental barrier and that maternal BCG vaccination affects the placentobiome.

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.

Salmonella L-forms: formation in human bile in vitro and isolation culture from patients' gallbladder samples by a non-high osmotic isolation technique.

Bacterial L-forms have always been considered as osmotic-pressure-sensitive cell-wall-deficient bacteria and isolation culture of L-forms must use media with high osmotic pressure. However, isolation culture of stable L-forms formed in humans and animals is very difficult because they have adapted to the physiological osmotic pressure condition of the host. We use a non-high osmotic isolation technique to isolate stable L-forms of Salmonella Typhi and Salmonella Paratyphi A from bile-inducer cultures in vitro and from patients' gallbladder specimens. Multiplex PCR assay for Salmonella-specific genes and nucleotide sequencing are used to identify the Salmonella L-forms in stable L-form isolates. Using this method, we confirmed that Salmonella Paratyphi A and Salmonella Typhi cannot be isolated from bile-inducer cultures cultured for 6 h or 48 h, but the L-forms can be isolated from 1 h to 45 days. In the 524 gallbladder samples, the positive rate for bacterial forms was 19.7% and the positive rate for Salmonella spp. was 0.6% by routine bacteriological methods. The positive rate for bacterial L-forms was 75.4% using non-high osmotic isolation culture. In the L-form isolates, the positive rate of Salmonella invA gene was 3.1%. In these invA-positive L-form isolates, four were positive for the invA and flic-d genes of Salmonella Typhi, and ten were positive for the invA and flic-a genes of Salmonella Paratyphi A.

The Helicobacter pylori L-form: formation and isolation in the human bile cultures in vitro and in the gallbladders of patients with biliary diseases.

BACKGROUND: The Helicobacter pylori is considered the important causative agent causing biliary diseases, but the H. pylori can be isolated from very few gallbladder specimens with diseases. We studied the formation of H. pylori L-forms in bile in vitro and isolated the H. pylori L-forms from gallbladder of patients with biliary diseases. METHODS: We inoculated the H. pylori into the human bile to induce the L-form in vitro. The gallbladder specimens were collected from patients with biliary diseases to isolate the bacterial L-forms by the nonhigh osmotic isolation technique, and the H. pylori L-forms in the L-form isolates were identified by the gene assay for the H. pylori-specific genes 16S rRNA and UreA. RESULTS: The H. Pylori cannot be isolated from the bile-induced cultures, but the H. pylori L-form can be isolated from the H. pylori-negative bile-induced cultures. The L-form isolates of bile-induced cultures showed a positive reaction of the H. pylori-specific genes by PCR, and the coincidence ratio of the nucleotide sequences between the L-forms and the H. pylori is 99%. The isolation rate of bacteria L-form is 93.2% in the gallbladder specimens with bacteria-negative isolation culture by the nonhigh osmotic isolation technique, and the positive rate of the H. pylori-specific genes in the L-form isolates is 7.1% in the bacterial L-form-positive isolation cultures by the PCR. CONCLUSIONS: H. pylori can be rapidly induced into the L-form in the human bile; the L-form, as the latent bacteria, can live in the host gallbladder for a long times, and they made the host became a latent carrier of the H. pylori L-form. The H. pylori L-form can be isolated by the nonhigh osmotic isolation technique, and the variant can be identified by the gene assay for the H. pylori-specific genes 16S rRNA and reA.

L-forms of Staphylococcus epidermidis induced by penicillin.

L-forms of S. epidermidis were induced at 35 degrees C with the use of an L-form medium with penicillin. The aim of this study was to evaluate the frequency of L-form induction and demonstrate whether the origin of the clinical strains affects the frequency of L-forms induction, as well as to study whether the time of action of the antibiotic has an influence on frequency of L-form induction.

[The composition and drug sensitivity of a mycobacterial population in patients with suspected tuberculosis].

By using the diagnostic material (175 sputum samples and 103 bronchoalveolar lavage fluid samples) taken from 39 patients with suspected tuberculous infection during a 2.5-month follow-up, the authors traced the time course of changes in the composition and drug sensitivity of a mycobacterial population to rifampicin. Along with the traditional microbiological studies, the latest molecular biological studies, a TB-BIOCHIP test system (enzyme immunoassay) in particular, were employed to detect the bacterial and L-transformed forms of the causative agent. A molecular biological assay was first developed to detect the drug sensitivity of L-forms of Mycobacterium tuberculosis.

[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.

Molecular detection and beta-glucuronidase expression of gus-marked Bacillus subtilis L-form bacteria in developing Chinese cabbage seedlings.

AIM: To detect L-form bacteria in developing Chinese cabbage seedlings. METHODS AND RESULTS: Stable Bacillus subtilis L-forms were genetically modified to express the gus gene (encoding beta-glucuronidase). Germinated seeds of Chinese cabbage were soaked in mannitol based suspensions of the L-form bacteria or with mannitol alone and after washing were grown in aseptic conditions on plant growth medium. Histochemical staining of beta-glucuronidase activity (X-gluc) and Polymerase Chain Reaction (PCR) detection of the gus gene were achieved in the L-form associated seedlings. beta-Glucuronidase was localized in discrete spots, mainly in the roots with staining, and was also observed in the cotyledons and base of stems. Correlation was observed between PCR detection of the gus gene and histochemical staining with detection in similar tissues. Stable L-form bacteria were non-culturable after their association with plant material. CONCLUSIONS: The gus reporter gene system with its associated histological staining for enzyme activity was used successfully for detecting B. subtilis L-form bacteria in plant material. SIGNIFICANCE AND IMPACT OF THE STUDY: These molecular marked L-forms should provide a specific and sensitive technique for detecting L-form bacteria in planta and offer a method for further understanding the L-form/plant association.

Recovery of cell wall-deficient organisms from blood does not distinguish between patients with sarcoidosis and control subjects.

STUDY OBJECTIVE: To determine if cell wall-deficient forms (CWDF) of mycobacteria can be grown in culture of blood from subjects with sarcoidosis. DESIGN: A special multicenter study of sarcoidosis (A Case Control Etiologic Study of Sarcoidosis), supported by the National Heart, Lung, and Blood Institute. PATIENTS AND CONTROL SUBJECTS: PATIENTS AND CONTROL SUBJECTS were recruited at 10 institutions in the United States. Control subjects (controls) were of the same gender and race, and within 5 years of age as matching patients with sarcoidosis (cases). RESULTS: Cultures were incubated from 347 blood specimens (197 cases, 150 controls). Two investigators trained to recognize CWDF mycobacteria examined material obtained from culture tubes after 3 weeks. Structures thought to be CWDF were seen with equal frequency in cases (38%) and controls (41%). Thirty-nine percent of cases and 37% of controls were read as negative for CWDF. CONCLUSION: This study fails to confirm earlier reports that CWDF mycobacteria can be grown from the blood of patients with sarcoidosis, but not from control subjects.

[Prognostic value of isolation of L-forms of Mycobacterium tuberculosis]. / Prognosticheskoe znachenie vydeleniia L-form mikobakterii tuberkuleza.

A procedure has been developed to detect antilysozymatic activity n L forms of Mycobacterium tuberculosis (MBT). A mode of predicting the course of a tuberculosis process in the lungs by the degree of antilysozymatic activity of MBT is outlined. Thus, when the level is 4 micrograms/ml or higher, progression or exacerbation of tuberculosis is predicted, when that of 0-3 micrograms/ml, a good prognosis is expected.

[Identification of L-forms of Mycobacterium tuberculosis complex by polymerase chain reaction (PCR)]. / Identifikatsiia L-form mikobakterii tuberkuleznogo kompleksa s primeneniem polimeraznoi tsepnoi reaktsii (PTsR).

Polymerase chain reaction (PCR) was used to verify the tuberculous origin of L-forms isolated from clinical non-respiratory samples from patients with extrapulmonary tuberculosis. PCR was made by using cultured L-forms obtained from negative and positive cultures. PCR used a total of 60 cultured L-forms different in the morphology of colonies and the rate of growth. The total count of L-forms yielding positive amplification with M. tuberculosis complex-specific primers was 51 (85%). L-form passages were subjected to PCR analysis. A total of 14 third-generation L-forms were examined. They turned out to be positive. Thus, the fact that L-forms isolated from nonrespiratory clinical samples from patients with tuberculosis are most commonly L-forms of M. tuberculosis was genetically substantiated.

An ELISA for the detection of Bacillus subtilis L-form bacteria confirms their symbiosis in strawberry.

AIMS: To develop an ELISA for the detection of antigens derived from stable Bacillus subtilis L-form bacteria and to detect these in plants injected with L-form bacteria. METHODS AND RESULTS: A sandwich ELISA was developed and its specificity was investigated using L-forms and cell-walled forms of B. subtilis, different Bacillus species and a range of bacteria isolated from glasshouse-grown strawberry plants. The detection limits of the ELISA were approximately 10(3) viable cells ml(-1) for L-forms compared with 10(7) viable cells ml(-1) for cell-walled forms. Results showed that L-forms survived and moved within strawberry tissues injected with L-form bacteria. CONCLUSION: An ELISA that selectively detects B. subtilis L-form bacteria was developed and shown to confirm the presence of L-forms in plants. SIGNIFICANCE AND IMPACT OF THE STUDY: This will be a valuable rapid method to further studies on L-form plant interactions.

[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.

[Detecting mycobacteria and their L-forms in peripheral blood from pulmonary tuberculosis patients by cultivation with hemolyzed-centrifugated blood in liquid medium].

OBJECTIVE: To establish a new method of detecting mycobacteria and their L-forms in blood. METHODS: 65 samples of peripheral blood from patients with pulmonary tuberculosis were cultivated directly or cultivated with hemolyzed-centrifugated blood sediments in 92-3TB and 92-3TBL liquid medium, and the cultures were staining with immunoenzyme technique (ABC method). RESULTS: In 65 specimens, the positive rates of mycobacteria and their L-forms were 15%, 26% respectively with total isolation rate of 32%. In cultivating with hemolyzed-centrifugated blood the positive rate was much higher than direct cultural method (P < 0.05). In sputum the positive rates of acid-fast bacilli and their L-forms were 38% and 20% respectively, with total isolation rate of 52%, higher than that in peripheral blood (P < 0.05), and the total positive rate of combinative detection of blood and sputum salmples was 65%. CONCLUSIONS: Mycobacteria and their L-forms exist in peripheral blood of patients with pulmonary tuberculosis, and mainly in L-forms. Cultivation with hemolyzed-centrifugated blood in liquid medium and staining with immunoenzyme technique are valuable in routine detection of mycobacteria and their L-forms in peripheral blood. The positive rate of bacterial culture of pulmonary tuberculosis could be increased in combination with sputum mycobacteria and their L-forms examination.

[Laboratory diagnostic methods in primary pulmonary tuberculosis in adults]. / Laboratorni metody diahnostyky pervynnoho tuberkul'ozu leheniv u doroslykh.

A comprehensive clinical and roentgenological evaluation was done in 495 patients with primary tuberculosis aged more than eighteen. The clinical pattern of primary forms of the specific process was characterized by predominance of tuberculosis of intrathoracic lymph nodes (tumorous form) infiltrative tuberculosis of pararoot or lower-lobe localization, and exudative pleurisy. Primary tuberculosis was diagnosed in 56.4% of adults. Examination of sputum for changed forms of MBT increases the frequency of verification of diagnosis by 16.0% in comparison with conventional microbiological assays. Primary tuberculosis of the respiratory system in 63.5% of adults runs its course against the background of significant disorders of T- and B-systems of lymphocytes, antituberculous immune defence and bodily metabolic processes. The use of a complex of immunologic studies was found to promote the accuracy of diagnosis of primary tuberculosis from 56.4% up to 79.6%, that of biochemical ones-from 56.4% to 77.5%.