Polymicrobial consortia in the pathogenesis of biofilm vaginosis visualized by FISH. Historic review outlining the basic principles of the polymicrobial infection theory.

The manuscript disputes the exclusive mono-infectious way of thinking, which presumes that for every infection only one pathogen is responsible and sufficient, when infectious vectors, close contact and reduced immunity meet. In situations involving heavily colonized anatomical sites such an approach often ends in insoluble contradictions. Upon critical reflection and evaluation of 20 years research on spatial organization of vaginal microbiota it is apparent, that in some situations, pathogens may act and operate in permanent, structurally organized consortia, whereas its individual components may be innocuous and innocent, failing to express any pathogenic effect. In these cases, consortia are the true pathogens responsible for many infectious conditions, which usually remain unrecognized as long as improperly diagnosed. The structure of such consortia can be unraveled using ribosomal fluorescence in situ hybridization (FISH). FISH methodology, that not only offers an ex vivo opportunity to recognize bacterial species, but provides unique physical insight into their specific role in the pathogenesis of polymicrobial infections. Ribosomal FISH technique applied to both, women with bacterial vaginosis (BV) and their male partners, has added significantly to our understanding of the pathogenesis of this condition and contributed to appreciating the mechanisms of polymicrobial, community-based infection, potentially leading to therapeutic advances.

Ketogenic Diet Has Moderate Effects on the Fecal Microbiota of Wild-Type Mice.

The ketogenic diet (KD) is a high-fat, low-carbohydrate diet that has been reported to have neuroprotective effects. The health effects of KD might be linked to an altered gut microbiome, which plays a major role in host health, leading to neuroprotective effects via the gut-brain axis. However, results from different studies, most often based on the 16S rRNA gene and metagenome sequencing, have been inconsistent. In this study, we assessed the effect of a 4-week KD compared to a western diet (WD) on the colonic microbiome of female C57Bl/6J mice by analyzing fecal samples using fluorescence in situ hybridization. Our results showed distinct changes in the total number of gut bacteria following the 4-week KD, in addition to changes in the composition of the microbiome. KD-fed mice showed higher absolute numbers of Actinobacteria (especially Bifidobacteria spp.) and lower absolute levels of Proteobacteria, often linked to gut inflammation, in comparison with WD-fed mice. Furthermore, an increased abundance of the typically rare genus Atopobium was observed. These changes may indicate the possible anti-inflammatory effects of the KD. However, since the overall changes in the microbiota seem low, the KD effects might be linked to the differential abundance of only a few key genera in mice.

New approach to soil management focusing on soil health and air quality: one earth one life (critical review).

Soil plays a key role in ecosphere and air quality regulation. Obsolete environmental technologies lead to soil quality loss, air, water, and land systems pollution. Pedosphere and plants are intertwined with the air quality. Ionized O2 is capable to intensify atmosphere turbulence, providing particulate matter (PM2.5) coalescence and dry deposition. Addressing environmental quality, a Biogeosystem Technique (BGT*) heuristic transcendental (nonstandard and not direct imitation of nature) methodology has been developed. A BGT* main focus is an enrichment of Earth's biogeochemical cycles through land use and air cleaning. An intra-soil processing, which provides the soil multilevel architecture, is one of the BGT* ingredients. A next BGT* implementation is intra-soil pulse continuously discrete watering for optimal soil water regime and freshwater saving up to 10-20 times. The BGT* comprises intra-soil dispersed environmentally safe recycling of the PM sediments, heavy metals (HMs) and other pollutants, controlling biofilm-mediated microbial community interactions in the soil. This provides abundant biogeochemical cycle formation and better functioning of the humic substances, biological preparation, and microbial biofilms as a soil-biological starter, ensuring priority plants and trees nutrition, growth and resistance to phytopathogens. A higher underground and aboveground soil biological product increases a reversible C biological sequestration from the atmosphere. An additional light O2 ions photosynthetic production ensures a PM2.5 and PM0.1 coalescence and strengthens an intra-soil transformation of PM sediments into nutrients and improves atmosphere quality. The BGT* provides PM and HMs intra-soil passivation, increases soil biological productivity, stabilizes a climate system of the earth and promotes a green circular economy.

Bacterial Vaginosis-Vaginal Polymicrobial Biofilms and Dysbiosis.

BACKGROUND: Bacterial vaginosis (BV) is the most common genital disease worldwide in women of sexually active age, with a prevalence of 23-29%. Its traditional definition as dysbiosis, i.e., a disruption of the normal balance of the vaginal microbiota, with a massive increase of facultative and obligate anaerobic bacteria (mainly Gardnerella spp.) and a loss of lactobacilli, accurately describes the change in the vaginal microbiota, but does not explain the underlying pathophysiology. METHODS: This review is based on information in pertinent articles retrieved by a selective literature search and on the authors' own research findings. RESULTS: Fluorescent in situ hybridization (FISH) has revealed Gardnerella spp.-dominated polymicrobial vaginal biofilm as a cause of ascending gynecologic and pregnancy-related infections, preterm birth, and infertility in patients with BV. The biofilm-induced disturbance of epithelial homeostasis favors co-infection with pathogens of sexually transmitted infection (STI). Standard antibiotic therapy is ineffective against biofilms, and there is thus a recurrence rate above 50%. The characteristic biofilm can be followed as a diagnostic marker and is considered evidence of sexual transmission when heterosexual couples and ejaculate samples are examined. FISH studies have shown that, in addition to biofilm-related vaginosis, there are other dysbiotic changes in the vaginal microbiota that have not yet been characterized in detail. It is therefore justified to speak of a "bacterial vaginosis syndrome." CONCLUSION: The simplistic view of BV as dysbiosis, characterizable by microscopic reference methods, has so far led to inadequate therapeutic success. An evaluation of molecular genetic testing methods that would be suitable for routine use and the development of therapeutic agents that are effective against biofilms are urgently needed if the "bacterial vaginosis syndrome" is to be effectively treated.

Inflammatory invasion on human vaginal mucosa correlated with combined drug treatment and recurrence in recurrent vulvovaginal candidiasis.

AIM: Recurrent vulvovaginal candidiasis (RVVC) is a chronic, difficult to treat vaginal infection, caused by Candida species, which affects women of all ages and ethnic and social background. Most RVVC studies use animal models, and there is still a lack of observation on human tissue samples and effective therapy to reduce recurrence. MATERIALS AND METHODS: We observed CD163+ macrophages and NLRP3 expression by immunohistochemistry, also investigated bacteria and fungi co-invasion by fluorescence in situ hybridization from 144 human vaginal biopsy tissues (48 RVVC, 48 VVC, 48 healthy volunteers), and we also explored the effect of combining metronidazole in the treatment of RVVC. RESULTS: A large number of neutrophils, lymphocytes and plasma cells infiltrated the mucosa, basement membrane and submucosa, accompanied by significantly overexpressed NLRP3 inflammasome. While CD163+ macrophages often infiltrated under the basement membrane in patients with RVVC, 29.2% of cases were found Gardnerella and fungi jointly invaded the vaginal mucosas. RVVC vaginal mucosal histopathology revealed mucosal inflammatory responses dominated by neutrophils, which may involve activation of NLRP3 and immune tolerance of M2 macrophages (CD163+ ). Fluconazole combined with metronidazole can achieve higher efficiency (95.8% vs. 70.8%) and reduce the recurrence rate more (8.3% vs. 37.5%) at 6-month follow-up. CONCLUSION: Inflammatory invasion on human vaginal mucosa correlated with combined drug treatment and recurrence in RVVC. The combined medication will need to further evaluate in future.

Clue Cells and Pseudo Clue Cells in Different Morphotypes of Bacterial Vaginosis.

Introduction: Clue cells (epithelial cells heavily covered with adherent bacteria) are an accepted clue to the diagnosis of bacterial vaginosis. However, the exact morphologic criteria of clue cells and bacterial adherence were never elaborated. Materials and Methods: We investigated adhesive and cohesive patterns of main microbiota groups in vaginal discharge using fluorescence in situ hybridization (FISH). Samples from 500 women diagnosed with bacterial vaginosis and positive for clue cells with classic microscopy were collected from 42 gynecologic practices in Berlin and reexamined in our FISH laboratory for the spatial distribution of Bifidobacteriaceae, Gardnerella, Fannyhessea vaginae (Atopobium); low G+C (guanine+cytosine) bacteria, lactobacilli, Lactobacillus iners; Lactobacillus crispatus, Gamma-Proteobacteria; and Enterobacteriaceae, Prevotella-Bacteroides, Veillonella, and Coriobacterium groups. Results: Bacterial taxa present in vaginal smears were not accidentally assembled according to their relative abundance but were built in group-specific distribution patterns, which can be well described by two features: cohesiveness to each other and adherence to epithelial cells. Accordingly, four patterns can be distinguished: dispersed (non-adherent bacteria), dispersed adherent bacteria, cohesive (non-adherent) bacteria, and cohesive adherent bacteria. Direct cohesive adherence to the epithelial cells representing true clue cells was unique for Gardnerella species and observed only in 56% of the investigated samples. In the remaining vaginal samples, the epithelial cells were mechanically entrapped in bacterial masses, and the composition was unrelated to the epithelial cell surface, building non-adherent pseudo clue cells. The proportion of women with true clue cells in their samples from different gynecologic practices varied from 19% to 80%. Discussion: Taxon indifferent imaging is inadequate for the exact analysis of the microbial layer adjacent to the vaginal epithelial cells. Morphologically seen bacterial vaginosis is a mix of at least two different conditions: biofilm vaginosis and bacterial excess vaginosis.

Antimicrobial Susceptibility of Microbiota in Bacterial Vaginosis Using Fluorescence In Situ Hybridization.

BACKGROUND: Testing of antibiotic resistance of intact vaginal microbiota in pure culture is not feasible. METHODS: Metronidazole, antiseptic octenisept®, antimycotic ciclopirox, bacterial probiotic Lactobacillus crispatus, yeast probiotic Saccharomyces boulardii, Gardnerella-phage-endolysin named phagolysin and phagolysin in combination with probiotics were tested for bacteriolytic activity. Included were vaginal swabs from 38 random women with Amsel-confirmed bacterial vaginosis (BV). Test aliquots were incubated by 37° for 2 and 24 h. Gardnerella, low G+C, Atopobium, lactobacilli, Lactobacillus iners and crispatus, Prevotella-Bacteroides, and Gammaproteobacteria microbial groups were quantified using fluorescence in situ hybridization (FISH). RESULTS: The probiotic strain Lactobacillus crispatus demonstrated the weakest bacteriolytical effects, followed by metronidazole. Both had no impact on Gardnerella species, instead lysing Prevotella-Bacteroides, Enterobacteriaceae (by L.crispatus) or LGC, Atopobium and Prevotella-Bacteroides (by metronidazole) groups of the microbiota. Cytolytic activity on Gardnerella was highly pronounced and increased from octenisept to ciclopirox, phagolysin, phagolysin with L.crispatus, being best in the combination of phagolysin with S.boulardii. Universally active ciclopirox and octenisept® suppressed nearly all microbial groups including those which are regarded as beneficial. Phagolysin had no effect on naturally occurring Lactobacillus crispatus. Conclusions: FISH susceptibility testing allows unique efficacy evaluation of individually adjusted topical therapy without microbial isolation facilitating optimal therapy choice.

Engineered Phage Endolysin Eliminates Gardnerella Biofilm without Damaging Beneficial Bacteria in Bacterial Vaginosis Ex Vivo.

Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria, and is frequently refractory to antibiotic treatment. We investigated endolysins of the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages as an alternative treatment. When recombinantly expressed, these proteins demonstrated strong bactericidal activity against four different Gardnerella species. By domain shuffling, we generated several engineered endolysins with 10-fold higher bactericidal activity than any wild-type enzyme. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13-8 µg/mL. PM-477 had no effect on beneficial lactobacilli or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested by fluorescence in situ hybridization on vaginal samples of fifteen patients with either first time or recurring bacterial vaginosis. In thirteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. The high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.

Thermodynamic mathematical model of the Kastanozem complex and new principles of sustainable semiarid protective silviculture management.

The Kastanozem complex in the dry steppe of southern Russia underlies an artificially-constructed forest strips. Deep ploughing to a depth of 45 cm was used to process the soil prior to planting. Between 20 and 40 cm depth, soil density was high, 1.57 t m-3. Soil hardness was also high, 440 psi. Soil aggregates greater than 5 cm in size were impermeable to tree roots. The content of such aggregates was high, comprising 35%. The number of tree roots with diameters greater than 0.5 cm that cross the soil profile was as low as 0.15 to 0.3 pcs cm-2. The soil matric potential signifying water availability was low in the vegetation period -0.9 MPa to a depth of 1.0 m. According to modelling experiments, the main salt components in the soil solution drive the transfer of soil organic matter (SOM) and heavy metals (HM). The composition of the soil solution determined by the calcium carbonate equilibrium (CCE) and the association and complexation of ions. ION-3 software was used to calculate the ion equilibrium in the soil solution. Macro-ions Cа2+, Mg2+, SO42-, and CO32- partly bonded as ion pairs. Oversaturation of the soil solution with CaCO3 was calculated according to the analytical content of macro-ion, which was high up to 1000 units, and its value decreased in response to ionic strength, activity, association, complexation, and thermodynamic equilibrium of macro-ions in the soil solution. Oversaturation calculated for Salic Solonetz and Gleyic Solonetz soil solutions was small considering the SOM content. Calculations indicate the profile and lateral loss of C from the soil to the vadose zone. The content of Pb in the soil solution was calculated sirca 75%-80%. The calculated coefficient of Pb2+ association was as high as 52.0. The probability of Pb passivation by SOM in the Kastanozem complex was significant. The probability of uncontrolled transfer and accumulation of HM in the soil and vadose zone was high. Biogeosystem Technique (BGT*) transcendental methodology, an innovative methodology created for stable geomorphological system formation to achieve sustainable agriculture and silviculture, was applied. The BGT* elements were: intra-soil milling of the 30-60 cm soil layer for geophysical conditioning; intra-soil continuously-discrete pulse watering for plants and trees to improve the hydrologic regime. The BGT* methodology reduced HM mobility, controlled biodegradation, enriched nutrient biogeochemical cycling, increased C content, increased soil productivity, and reversible carbon sequester in biological form.