Zusammenfassung Ribosomale FISH(Fluoreszenz-in-situ-Hybridisierung)-Analysen zeigen, dass die bakterielle Vaginose (BV) ein komplexes klinisches Syndrom ist, das mehrere, nicht miteinander verwandte Infektionsprozesse mit ähnlicher Symptomatik umfasst. Die Biofilm-Vaginose ist die häufigste Ursache des BV-Syndroms, gefolgt von der kohäsiven Lactobacillus-iners -Sludge-Vaginose. Zum BV-Syndrom gehören darüber hinaus bisher nicht eindeutig charakterisierte Dysbiosen, wobei nicht ausgeschlossen ist, dass diese später in weitere nosologisch klar definierte Gruppen unterteilt werden. Derzeit lassen sich pathogene Konsortien nur mittels ribosomaler FISH bestimmen. Weder Lichtmikroskopie noch Multiplex-PCR (Polymerasekettenreaktion) und NGS („next generation sequencing“) sind hierfür hilfreich, da sie lediglich beteiligte Morphotypen bzw. Spezies, nicht aber ihre Wechselbeziehungen aufzeigen. Da alle polymikrobiellen Gemeinschaften auf spezielle Gene angewiesen sind, die für den Zusammenhalt der beteiligten Mikroorganismen notwendig sind, ist es nur eine Frage der Zeit, bis diese DNA-Sequenzen aufgedeckt und ihr Nachweis für die klinische Diagnostik und Therapie zugänglich sind.

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.

A standard management soil priming effect is short term. A current soil plowing, irrigation and waste utilization soil priming action shortcomings elimination is needed. The Biogeosystem Technique (BGT*) priming action system was developed for soil management to provide a higher-level priming effect. The BGT* is focused on a soil biogeochemical cycle activation similar to the biodegradation in a colon of a live organism as an efficient benchmark bioreactor. Fluorescence in situ hybridization showed a cooperation of bacteria within polymicrobial biofilms and a probability of the polymicrobial biofilms biodegradation process acceleration and soil organic matter (SOM) regime improvement. The soil priming actions the BGT* technological management is capable of providing are based on the BGT* constituents, whose essence, applications and corresponding detailed data are presented in available studies. The main BGT* components are as follows. The illuvial horizon intra-soil milling artificial soil aggregation (a form of crumbling and mixing) priming action provides the soil structuring, a SOM content increase and an annual yield increment direct priming effects. A long-term secondary priming effect is a technology life cycle with higher profitability compared to the standard moldboard, chiseling and three-tier plowing. The intra-soil pulse sequential-discrete watering priming action is performed through water injection into the soil in sequence. A next water spreading provides the soil aggregate stability, the seedling length and weight increment direct priming effect and a water-saving secondary priming effect. The intra-soil dispersed liquid, paste-like and loose granular nutrition and structuring matter intra-soil milling application priming action ensures soil structuring direct priming effects, heavy metals passivation and long-term yield increment secondary priming effects. The BGT* methodology could be useful for soil management improvement.

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.

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.

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.

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.

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.

The air pollution with fine particulate matter (PM) of dimension 2.5 µm or less (PM2.5) causes lung and other diseases. The problem of prevention of water and terrestrial systems pollution with PM dry deposits is multifaceted. The ionized O2 is capable to intensify the atmosphere turbulence, PM2.5 сoalescence, and increasing the PM dry deposition velocity. Unfortunately, outdated environmental technologies are incapable to secure this. The quality of air is linked to the pedosphere and plant kingdom. Addressing the problem of environmental quality, including the PM2.5 content reduction in the atmosphere, the Biogeosystem Technique (BGT*) transcendental (nonstandard and not a direct imitation of Nature) methodology has been developed. The BGT* focus is an enrichment of the Earth's biogeochemical cycle. The heuristic approach to land use and air cleaning is a new niche for development to improve the soil system and ensure a high rate of air cleaning. BGT* ingredients are the intra-soil processing, which provides the soil multilevel architecture; intra-soil pulse continuously discrete watering for optimal soil water regime and freshwater saving up to 10-20 times; intra-soil dispersed environmentally safe recycling of the PM sediments and other pollutants; controlled microbial community and biofilm-mediated interactions in the soil. BGT* enriches the biogeochemical cycle, provides a better function of the humic substances, biological preparation and microbial biofilms as a soil-biological starter, priority plant and trees nutrition. BGT* methodology is capable to increase plant resistance to phytopathogens. BGT* provides the formation of higher underground and aboveground biological products, thus increasing reversible C biological sequestration from the atmosphere in the form of additional aboveground biomass and soil organic matter. BGT* provides a higher rate photosynthetic production of light O2 ions, a coalescence of PM2.5, PM0.1 to the PM10 and larger particles, sedimentation of the PM, and a soil transformation of PM sediments into the nutrients. BGT* allows sustainability of the biosphere, enables a high quality of the atmosphere, stabilizes the climate system of the Earth, and is capable to promote a green circular economy. The research was supported by the Strategic Academic Leadership Program of the Southern Federal University ("Priority 2030").

Currently, antibiotics are the mainstay of therapy for bacterial vaginosis (BV). However, the rate of treatment failure in patients with recurrent BV is about 50 %. It has been disputed whether Metronidazole (MDZ) resistance might be a crucial factor for therapy failure in BV, particularly in patients with recurrent BV. We investigated if and how easily the keystone pathogen of BV, Gardnerella, can develop MDZ resistance and at the same time we present a novel Gardnerella biofilm disrupting agent with a low probability to induce resistance, the endolysin PM-477.

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 Ca2+, 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.

Soil organic matter biodegradation is an agent of the soil fertility and passivation of the hazardous substances including heavy metals. Bacteria within specific habitats, be it the mouth, tonsils, intestines, gut, vagina, or soil are not a faceless mixture of the once acquired participants, but the structurally strictly ordered polymicrobial communities where each participant takes its specific functional place. The conditions for polymicrobial biofilms in the soil are important. The aim was tracking down the structural organization and adherence to soil particles of the polymicrobial communities and biofilms, responsible for biodegradation. Polymicrobial communities and biofilms can be used as a starter, indicator, and control tools for the targeted soil and landscape improvements. Multiple skills in identification, characterizing and monitoring of functional activity of polymicrobial biofilms in the human body and gut were developed in the laboratory of polymicrobial infections and biofilms of the Charité hospital over the past 30 years. The biofilms do not occur in all systems and at any time in relevant amounts. The biochemical activity of the microorganisms till now is investigated solely in pure cultures. As soon as more than three different taxa are involved, the cultivation of the target microorganisms got problematic. The mapping of biofilms by the FISH method is promising for the following objectives in the soil system: - identification of the structured polymicrobial biofilms for optimal composting, soil fertility, and a healthy environment; - revealing modelling the polymicrobial starter of soil fertility; - polymicrobial biofilms activity ensuring via control of the soil architecture, soil moisture and aeration; - aerobe/anaerobe conditioning, pH, humic acids, and organic and mineral fertilizers, amelioration and remediation additives; - testing of the substrate-bound polymicrobial biofilms as a starter for the shaping of different lands and agricultures. Development of the soil-microbiological theoretical and technical fundamentals for the long-term soil improvement and environmentally safe organic wastes recycling and heavy metal passivation into the synthesized soil multilevel aggregate system under minimal intra-soil moistening and appropriate intra-soil mineral and organic matter, and waste application using Biogeosystem Technique (BGT*) transcendental environmental services. The soil-microbiological theoretical and technical fundamentals are useful for long-term soil improvement and environmentally safe and eсonomically efficient organic wastes recycling into the synthesized soil aggregate system. The transcendental intra-soil aggregate system construction, the pulse intra-soil continuously-discrete watering, dispersed intra-soil matter application are decisive for higher soil microbial activity and target polymicrobial infections and biofilms transformation into the environmentally safe fertile substances. Comparative characterization of the polymicrobial community dynamics in colon and soils will help to promote the function of polymicrobial biofilms in the soil as a specific starter. The BGT* methodology is capable to ensure the soil fertility, improve the soil polymicrobial biofilms resistance, and provide the soil and human health. The research was financially supported by the RFBR, projects no. 18-29-25071 and 19-29-05265.

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.

Background Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome in which the normally predominant lactobacilli are replaced by other bacterial species. Initiated by strains of the bacterium Gardnerella , a characteristic biofilm forms on the vaginal epithelium, explaining the typical presence of clue cells. This biofilm contributes to the resilience of the bacteria to antibiotic treatment, which may explain the frequent recurrence of BV. Objective In this study, we investigate whether a therapy based on bacteriophage endolysins which specifically lyse Gardnerella , in particular the drug candidate PM-477, might be a promising alternative to broad-spectrum antibiotics and antiseptics. Study design To identify Gardnerella specific endolysins, we searched for endolysin-encoding sequences in regions of Gardnerella genomes that are of prophage origin. This search identified fourteen homologous genes predicted to encode 1,4-beta-N-acetylmuramidase-type endolysins. When expressed in Escherichia coli and purified, the recombinant proteins demonstrated strong bactericidal activity against four different Gardnerella species. By shuffling the N-terminal catalytic domains and C-terminal cell wall-binding domains between the homologues, we produced 81 chimeric endolysins. These endolysins were tested for their activity and specificity in vitro and ex-vivo on vaginal samples from fifteen BV positive patients. Fluorescence in situ hybridization was used for visualization. Results Several engineered endolysins were 10-fold more active than the most active wild-type enzymes. 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 Lactobacillus strains or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested on vaginal samples from fifteen patients with either first time or recurring bacterial vaginosis. In fourteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. Conclusion 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 drug candidate and an alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.

Organic biodegradation is a microbial controlled process that significantly influences soil fertility. The microorganisms involved are polymicrobial and organized in communities. Beyond this general statement, there are no reliable data available on the occurrence, structure and composition of polymicrobial biofilms in soil. The few published data are based on sequence analysis of unsystematically raised soil samples and provide no information to the involved biofilms, their structural organization or adherence to particles, which they are biodegrading. The objective of the following proposal is tracking down polymicrobial communities and biofilms, which are responsible for biodegradation and which in turn, can be used as starter, indicator, and control tools for the targeted soil- and landscaping. Over the last 30 years, the laboratory of polymicrobial infections and biofilms of the Charité hospital has developed multiple skills in identification, characterizing and monitoring of functional activity of polymicrobial biofilms in human body and gut specifically. One of the most striking results of these studies was the assessment, that bacteria within specific habitats of the mouth, tonsils, vagina or gut are not a faceless mixture of the once acquired participants, but structurally strictly ordered polymicrobial communities in which each participant takes its specific functional place. Since the biofilms do not occur in all systems and at any time in relevant amounts, the mapping of biofilms is unavoidable and intentional. The assessment of polymicrobial communities on the FISH methods basis provides the biofilms mapping for the following objectives: - identification of structured polymicrobial biofilms responsible for optimal composting, maximal soil fecundity, and reduction of environmental soil burden; - modeling of soil fecundity based on polymicrobial starter and defined factors controlling their activity such as water supply, aerobe/anaerobe conditioning, pH, humic acids additives and other; - testing of substrate bound polymicrobial biofilms as starter for the shaping of different lands and agricultures; - development of soil-microbiological theoretical and technical fundamentals for the long-term soil improvement and efficient environmentally safe organic wastes recycling into the synthesized soil aggregate system under minimal sufficient intra-soil moistening and appropriate intra-soil mineral and organic matter, and waste load (Biogeosystem Technique – BGT*). The biochemical activity of the microorganisms till now is investigated solely in pure cultures. As soon as more than three different taxa are involved, the cultivation of microorganisms got problematic. The main objective is the development of soil-microbiological theoretical and technical fundamentals for the long-term soil improvement and efficient environmentally safe organic wastes recycling in the synthesized soil aggregate system, for which microbial activity is decisive for polymicrobial infections and biofilms transformation into safe fertile substances. Till now nothing is known about homology or interactions in arrangement and functioning of polymicrobial communities of colon and soil, and a new knowledge to fill this is needed. Objectives of the study: to comparatively describe polymicrobial community dynamics in colon and soils; using BGT* methodology, to promote the function of polymicrobial biofilms in soil as a specific starter to insure the soil fertility, and to improve the human and soil health.

In the insightful publication by Swidsinski et al,1Swidsinski A. Guschin A. Tang Q. et al.Vulvovaginal candidiasis: histologic lesions are primarily polymicrobial and invasive and do not contain biofilms.Am J Obstet Gynecol. 2019; 220: 91.e1-91.e8Abstract Full Text Full Text PDF Scopus (31) Google Scholar the authors address important questions regarding the presence of vaginal biofilm in cases of vulvovaginal candidiasis (VVC). They conclude following evaluation of vaginal biopsies that lesions are primarily polymicrobial and do not contain biofilm. In a related study by our group, demonstrating that Candida albicans actively forms biofilm on the vaginal mucosa in the mouse model of VVC,2Harriott M.M. Lilly E.A. Rodriguez T.E. et al.Candida albicans forms biofilms on the vaginal mucosa: implications for the pathogenesis of vaginal candidiasis.Microbiology. 2010; 156: 3635-3644Crossref PubMed Scopus (188) Google Scholar similar questions regarding clinical VVC were raised. While we applaud the authors for designing a clinical study that addressed the issue through vaginal biopsies, we believe it important to point out some limitations to the study that stress caution in the interpretation of the results. First, because the biopsies were all taken from the middle side wall, and without colposcopy, areas of discontinuous biofilm formation might not have been sampled. Second, although infected areas did appear to be sampled based on the level of invasion and presence of mycelium/hyphae, surface biofilm could have been disrupted or dislodged with biopsy removal and sample preparation. Third, the biopsy sections were not evaluated for fungal extracellular matrix, the hallmark of Candida biofilm formation, or assessed by confocal microscopy for high-resolution serial sectioning that provides 3-dimensional reconstruction of thick microbial biofilm structures.2Harriott M.M. Lilly E.A. Rodriguez T.E. et al.Candida albicans forms biofilms on the vaginal mucosa: implications for the pathogenesis of vaginal candidiasis.Microbiology. 2010; 156: 3635-3644Crossref PubMed Scopus (188) Google Scholar As an alternative argument in agreement of the interpretations/conclusions made, there is evidence for less frequent antifungal resistance in recurrent VVC (RVVC) than in other forms of recurrent candidiasis.3Lynch M.E. Sobel J.D. Fidel Jr., P.L. The role of antifungal drug resistance in the pathogenesis of recurrent vulvovaginal candidiasis.J Med Vet Mycol. 1996; 34: 337-339Crossref PubMed Scopus (58) Google Scholar Less antifungal resistance may reflect reduced biofilm presence. In addition, erythema (redness) is a common documented sign of VVC/RVVC.4Fidel Jr., P.L. Candida albicans: from commensal to pathogen.in: Tannock G. Medical importance of the normal microflora. Kluwer Academic Publishers, London1999: 441-476.80Crossref Google Scholar This sign of infection is synonymous to the erythematous form of oral candidiasis and not the pseudomembranous form that presents as white plaques that visually reflect the presence of biofilm.4Fidel Jr., P.L. Candida albicans: from commensal to pathogen.in: Tannock G. Medical importance of the normal microflora. Kluwer Academic Publishers, London1999: 441-476.80Crossref Google Scholar Conversely, another common sign of VVC/RVVC is a cottage cheese–like discharge that is considered a mix of Candida, dead epithelial cells/leukocytes, and vaginal secretions.4Fidel Jr., P.L. Candida albicans: from commensal to pathogen.in: Tannock G. Medical importance of the normal microflora. Kluwer Academic Publishers, London1999: 441-476.80Crossref Google Scholar As such, parts of the discharge could represent biofilm sloughed from the vaginal wall. Swidsinski et al1Swidsinski A. Guschin A. Tang Q. et al.Vulvovaginal candidiasis: histologic lesions are primarily polymicrobial and invasive and do not contain biofilms.Am J Obstet Gynecol. 2019; 220: 91.e1-91.e8Abstract Full Text Full Text PDF Scopus (31) Google Scholar did not evaluate discharge in their study. In conclusion, while the interpretations made by the authors regarding the histopathology of VVC were correct based on the study design and results obtained, the limitations of the study and inability of the methods to specifically detect biofilm elements, stress caution relative to data overinterpretation. Hence, important questions remain regarding the presence of biofilm or role of biofilm formation in VVC. Vulvovaginal candidiasis: histologic lesions are primarily polymicrobial and invasive and do not contain biofilmsAmerican Journal of Obstetrics & GynecologyVol. 220Issue 1PreviewThe recent demonstration of a vaginal biofilm in bacterial vaginosis and its postulated importance in the pathogenesis of recurrent bacterial vaginosis, including relative resistance to therapy, has led to the hypothesis that biofilms are crucial for the development of vulvovaginal candidiasis. The histopathology and microbial architecture of vulvovaginal candidiasis have not been previously defined; neither has Candida, containing biofilm been reported in situ. The present study aimed at clarifying the histopathology of vulvovaginal candidiasis including the presence or absence of vaginal biofilm. Full-Text PDF ReplyAmerican Journal of Obstetrics & GynecologyVol. 221Issue 2PreviewWe appreciate the thoughtful comments of Noverr and Fidel as to the findings in our recent publication evaluating the presence of Candida-associated biofilm in women with acute vulvovaginal candidiasis (VVC).1 The study produced 2 conclusions: (1) the extensive and previously unreported human vaginal mucosal invasion and infiltration by Candida microorganisms as well as the clinical consequences of tissue invasion; and (2) failure to document using a well-described and widely used technique of fluorescent in situ hybridization with ribosomal gene-based probes, the presence of microscopic biofilm, containing Candida species microorganisms. Full-Text PDF