Polymicrobial Biofilms Research Articles

Recent Advances and Opportunities in the Study of Candida albicans Polymicrobial Biofilms.

It is well known that the opportunistic pathogenic yeast, Candida albicans, can form polymicrobial biofilms with a variety of bacteria, both in vitro and in vivo, and that these polymicrobial biofilms can impact the course and management of disease. Although specific interactions are often described as either synergistic or antagonistic, this may be an oversimplification. Polymicrobial biofilms are complex two-way interacting communities, regulated by inter-domain (inter-kingdom) signaling and various molecular mechanisms. This review article will highlight advances over the last six years (2016-2021) regarding the unique biology of polymicrobial biofilms formed by C. albicans and bacteria, including regulation of their formation. In addition, some of the consequences of these interactions, such as the influence of co-existence on antimicrobial susceptibility and virulence, will be discussed. Since the aim of this knowledge is to inform possible alternative treatment options, recent studies on the discovery of novel anti-biofilm compounds will also be included. Throughout, an attempt will be made to identify ongoing challenges in this area.

Inactivation of Polymicrobial Biofilms of Foodborne Pathogens Using Epsilon Poly-L-Lysin Conjugated Chitosan Nanoparticles.

A mixed culture (polymicrobial) biofilm provides a favorable environment for pathogens to persist in the food processing environment and to contaminate food products. Inactivation and eradication of such biofilms from food processing environments are achieved by using harsh disinfectants, but their toxicity and environmentally hostile characteristics are unsustainable. This study aims to use food-grade natural nanoparticulated antimicrobials to control mixed-culture biofilms. Chitosan, a natural broad-spectrum antimicrobial biopolymer (polysaccharide) from crustaceans, was derivatized to produce chitosan nanoparticles (ChNP) as a carrier for another broad-spectrum antimicrobial agent, ε-poly-L-lysine (PL), to synthesize ChNP-PL conjugate. The antimicrobial activity of ChNP and ChNP-PL was tested against mixed-culture biofilms. ChNP-PL (~100 nm) exhibited a synergistic antimicrobial and anti-biofilm effect against mono or mixed-culture biofilms of five foodborne pathogens, including Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica serovar Enteritidis, Escherichia coli O157:H7, and Pseudomonas aeruginosa. ChNP-PL treatment prevented biofilm formation by mono or mixed cultures of L. monocytogenes, P. aeruginosa, and E. coli O157:H7, and bacterial counts were either below the detection limit or caused 3.5–5 log reduction. ChNP-PL also inactivated preformed biofilms. In monoculture biofilm, ChNP-PL treatment reduced L. monocytogenes counts by 4.5 logs, S. Enteritidis by 2 logs, E. coli by 2 logs, and S. aureus by 0.5 logs, while ChNP-PL had no inhibitory effect on P. aeruginosa. In vitro mammalian cell-based cytotoxicity analysis confirmed ChNP-PL to have no deleterious effect on intestinal HCT-8 cell line. In conclusion, our results show ChNP-PL has strong potential to prevent the formation or inactivation of preformed polymicrobial biofilms of foodborne pathogens.

Development of a Highly Specific, Selective, and Sensitive Fluorescent Probe for Detection of Mycobacteria in Human Tissues.

Tuberculosis (TB), including extrapulmonary TB, is responsible for more than one million deaths in a year worldwide. Existing methods of mycobacteria detection have poor sensitivity, selectivity, and specificity, especially in human tissues. Herein, the synthesis of a cholic acid-derived fluorescent probe (P4) that can specifically stain the mycobacterium species is presented. It is shown that P4 probe specifically binds with mycobacterial lipids, trehalose monomycolate, and phosphatidylinositol mannoside 6. P4 probe can detect mycobacteria in polymicrobial planktonic cultures and biofilms with high specificity, selectivity, and sensitivity. Moreover, it can detect a single mycobacterium in the presence of 10000 other bacilli. Unlike the probes that depend on active mycobacterial enzymes, the membrane-specific P4 probe can detect mycobacteria even in formalin-fixed paraffin-embedded mice and human tissue sections. Therefore, the ability of the P4 probe to detect mycobacteria in different biological milieu makes it a potential candidate for diagnostic and prognostic applications in clinical settings.

An In Vitro Evaluation of Denture Cleansing Regimens against a Polymicrobial Denture Biofilm Model.

Denture stomatitis (DS) is an inflammatory disease resulting from a polymicrobial biofilm perturbation at the denture surface–palatal mucosa interface. Recommendations made by dental health care professionals often lack clarity for appropriate denture cleaning. This study investigated the efficacy of brushing with off-the-shelf denture cleanser (DC) tablets (Poligrip®) vs. two toothpastes (Colgate® and Crest®) in alleviating the viable microorganisms (bacteria and fungi) in an in vitro denture biofilm model. Biofilms were grown on poly(methyl)methacrylate (PMMA) discs, then treated daily for 7 days with mechanical disruption (brushing), plus Poligrip® DC, Colgate® or Crest® toothpastes. Weekly treatment with Poligrip® DC on day 7 only was compared to daily modalities. All treatment parameters were processed to determine viable colony forming units for bacteria and fungi using the Miles and Misra technique, and imaged by confocal laser scanning microscopy (CLSM). Brushing with daily DC therapy was the most effective treatment in reducing the viable biofilm over 7 days of treatment. Brushing only was ineffective in controlling the viable bioburden, which was confirmed by CLSM imaging. This data indicates that regular cleansing of PMMA with DC was best for polymicrobial biofilms.

Bacterial vaginosis: controversial issues

The purpose of the review. Consideration of the most controversial issues regarding the possibility of sexual transmission of BV-associated microorganisms (bacterias) in women and men.
 Basic provisions. Bacterial vaginosis (BV) is a common disease associated with an increased risk of contracting sexually transmitted infections (including human papillomavirus and human immunodeficiency virus) in women and their male sexual partners. BV is characterized by polymicrobial transformations caused by Gardnerella vaginalis, which is the main etiological microorganism of this disease. G. vaginalis has a proven ability to form microbial biofilms on the mucosal surface. As a rule, from 10 to 12 different G. vaginalis genotypes can simultaneously reside in one biofilm, which provides it with a longer lifespan and viability. It has been shown that microorganisms in the biofilm acquire properties that reduce sensitivity to standard etiotropic therapy even at high doses of antibiotics. It was found that the cause of BV is a polymicrobial gardnerella biofilm, all components of which are transferred as a whole (for example, with the help of key cells), including during sexual contact. In this regard, the article discusses the possibility of using a new term biofilm gardnerellosis, which more accurately reflects the essence of this problem. Microbial biofilms organized by G. vaginalis are found in a significant number of women with BV and their sexual partners.

Formation, Development, and Cross-Species Interactions in Biofilms.

Biofilms, which are essential vectors of bacterial survival, protect microbes from antibiotics and host immune attack and are one of the leading causes that maintain drug-resistant chronic infections. In nature, compared with monomicrobial biofilms, polymicrobial biofilms composed of multispecies bacteria predominate, which means that it is significant to explore the interactions between microorganisms from different kingdoms, species, and strains. Cross-microbial interactions exist during biofilm development, either synergistically or antagonistically. Although research into cross-species biofilms remains at an early stage, in this review, the important mechanisms that are involved in biofilm formation are delineated. Then, recent studies that investigated cross-species cooperation or synergy, competition or antagonism in biofilms, and various components that mediate those interactions will be elaborated. To determine approaches that minimize the harmful effects of biofilms, it is important to understand the interactions between microbial species. The knowledge gained from these investigations has the potential to guide studies into microbial sociality in natural settings and to help in the design of new medicines and therapies to treat bacterial infections.

Efficacy of bakuchiol-garlic combination against virulent genes of Candida albicans.

BackgroundPolymicrobial biofilms are notorious for causing intraoral tissue destruction. Streptococcus sanguinis and Streptococcus mitis, commensals of oral cavities, have been found co-existing with C. albicans in resistant oral infections. There is an urgent need to find alternative treatment options. This study aims to assess the efficacy of garlic (G) and bakuchiol (Bk) combination against candida virulent genes and their subsequently secreted proteins.MethodsIn vitro single species biofilms of C. albicans, and mixed species biofilms formed in combination with streptococci were exposed to bakuchiol and garlic extract (Bk+G). Gene expression of agglutinin-like sequence (ALS1), (ALS3), adhesin-like wall proteins (HWP1) and aspartyl proteinases (SAP5) were determined using qPCR and their subsequent proteins were assessed through Western blotting.ResultsVirulent genes were significantly downregulated in single species biofilms when they were treated with Bk+G combination. However, Bk+G did not have significant effect on ALS1 and HWP1 gene in polymicrobial biofilms. ALS3 and SAP5 were significantly downregulated in Bk+G treated polymicrobial biofilm. Similar results were portrayed in Western blotting.ConclusionBk+G combination exhibited antimicrobial effects against single and mixed species biofilms. The findings might provide insights for treating resistant candida infections. This combination could potentially serve as an herbal alternative to traditional antifungals following further research.

Effectiveness of polyhexamethylene biguanide, chlorhexidine, and calcium hydroxide intracanal medicament against intraradicular mature polymicrobial biofilm: A microbiological study.

To compare the effectiveness of different intracanal medicaments against polymicrobial biofilm formed by Enterococcus faecalis, Staphylococcus aureus, and Candida albicans. Eighty mature human roots with intraradicular polymicrobial biofilm were randomly assigned into four groups (n = 20). Intracanal medicaments 0.2% polyhexamethylene biguanide (PHMB), 2% chlorhexidine (CHX), and calcium hydroxide (CH) were applied into the root canals. Collected dentine samples were tested at 7th, 15th, and 30th day for microbial growth, and the colony-forming units per ml (CFU/ml) were determined. The CFU/ml data were analyzed using unpaired t-test and one-way ANOVA-F comparison test. All medicaments resulted in a significant reduction (P < 0.05) in microbial growth at all time intervals compared to the control group. CHX and PHMB showed a similar reduction in CFU/ml at 7th and 15th day but significantly more than CH at all time intervals. At 30th day, PHMB caused a significantly more reduction in CFU/ml than CHX. All the three tested intracanal medicaments such as CH, CHX, and PHMB Gel were effective in reducing the microbial count. CH has a limited antimicrobial effect against the polymicrobial biofilm found inside the root canal. 2% CHX gel has a time-dependent antimicrobial effect. PHMB has a superior antimicrobial effect in comparison with CHX and CH.

Recent Updates on Microbial Biofilms in Periodontitis: An Analysis of In Vitro Biofilm Models.

The development of oral biofilm models has been extremely important to study the specific role of most microbial species at the early stages of periodontitis. The current knowledge on monospecies or multispecies biofilms originates mainly from the observation of in vitro dynamic or static biofilm model systems, which were engineered to mimic clinical oral conditions. In the last few decades, mounting evidence has confirmed that biofilms are the major form of bacterial lifestyle, and more importantly, that microorganisms dwelling in sessile mixed-species aggregates display completely different phenotypes and physiological characteristics than when living in planktonic pure cultures. Interspecies interactions within these communities, mediated by chemical communication systems, have been shown to affect biofilm physiology and increase antimicrobial resistance by up to 1000 fold. These aspects reinforce the importance of developing multispecies biofilm models to better understand and control biofilms. Literature reports demonstrate that while monospecies models are still most commonly used in caries research, authors have used different multispecies models to study periodontal diseases. Periodontitis is a polymicrobial biofilm-dependent disease mainly associated with Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. Interestingly, these species hardly adhere to substrates commonly used for biofilm formation, which makes multispecies models essential for an accurate analysis of periodontitis-related biofilms. The multispecies models currently available are generally composed of 6-10 species, but a more recent 34-species model was developed to better examine the dynamics within oral biofilms. The complexity of such polymicrobial biofilm models mimics more consistently the oral microbiome and different aspects of the oral environment. Collectively, the evidence on multispecies biofilm models described herein may support future studies on the use of antimicrobials for biofilm control as well as provide research opportunities to expand the current knowledge on interspecies interactions. The present manuscript reviews the most recent updates on in vitro biofilm model systems for periodontitis.

Evaluation of biofilm formation on acrylic resin surfaces coated with silicon dioxide: an in situ study.

Biofilm on acrylic resin dental prostheses may cause gingival inflammation. This study evaluated the influence of a silicon dioxide coating layer applied onto acrylic resin on the adhesion of microorganisms. Blocks (5 x 5 x 3 mm) of acrylic resin were evaluated for surface roughness and divided into two groups: control (CG) and coated with silicon dioxide (LG group). The specimens were evaluated by scanning electron microscopy (n = 1) and by contact angle analysis (n = 3). For the in situ study, 20 volunteers wore acrylic palatal devices containing three samples from each group (n = 60) for 2 days. The biofilm formed was quantified by metabolic activity and total biomass using the crystal violet assay. The results were subjected to Bartlett's normality test and Gamma model with random effect for the response variable (α = 5%). The mean contact angle of the coated group was significantly lower than that of the uncoated group (p < 0.05). The metabolic activity of microorganisms in the biofilm on the blocks treated with coating was significantly lower than that of control blocks (p = 0.02). Regarding the amount of extracellular matrix produced by the microorganisms, there was no difference between the CG and LG group (p = 0.05). The application of a silicon dioxide coating on acrylic resin reduced the activity of the polymicrobial biofilm formed in situ. This coating may be advantageous for patients with conventional complete dentures or implants made of acrylic resin and who have motor difficulties that prevent them from cleaning their prostheses properly.

Antibacterial efficacy of antibiotic pastes versus calcium hydroxide intracanal dressing: A systematic review and meta-analysis of ex vivo studies.

Conflicting findings on the potency of antibiotic pastes versus calcium hydroxide (CH) have been evident in the literature. To compare the antibacterial efficacy of single antibiotic paste (SAP), double antibiotic paste (DAP), triple antibiotic paste (TAP), and modified TAP (mTAP) with CH on bacterial biofilms. PubMed, Scopus, and Embase were comprehensively searched until August 23, 2021. The study protocol was registered in the PROSPERO. Ex vivo studies performed on Enterococcus faecalis or polymicrobial biofilms incubated on human/bovine dentin were selected. The quality of the studies was assessed using a customized quality assessment tool. Standardized mean difference (SMD) with a 95% confidence interval (CI) was calculated for the meta-analysis. Meta-regression models were used to identify the sources of heterogeneity and to compare the efficacy of pastes. The qualitative and quantitative synthesis included 40 and 23 papers, respectively, out of 1421 search results. TAP (SMD = -3.82; CI, -5.44 to -2.21; P < 0.001) and SAPs (SMD = -2.38; CI, -2.81 to - 1.94; P < 0.001) had significantly higher antibacterial efficacy compared to the CH on E. faecalis biofilm. However, no significant difference was found between the efficacy of DAP (SMD = -2.74; CI, -5.56-0.07; P = 0.06) or mTAP (SMD = -0.28; CI, -0.82-0.26; P = 0.31) and CH. Meta-regression model on E. faecalis showed that SAPs have similar efficacy compared to TAP and significantly better efficacy than DAP. On dual-species (SMD = 0.15; CI, -1.00-1.29; P = 0.80) or multi-species (SMD = 0.23; CI, -0.08-0.55; P = 0.15) biofilms, DAP and CH had similar efficacy. Ex vivo evidence showed that antibiotic pastes were either superior or equal to CH. The studied SAPs had considerably higher or similar antibacterial effectiveness compared to DAP, CH, and TAP. Hence, combined antibiotic therapy was not necessarily required for root canal disinfection ex vivo.

Psl-Dependent Cooperation Contributes to Drug Resistance of Pseudomonas aeruginosa in Dual-Species Biofilms with Acinetobacter baumannii.

Co-infection of Pseudomonas aeruginosa (Pa) and Acinetobacter baumannii (Ab) is frequently observed in intensive care unit (ICU) patients but difficult to eliminate. Current clinical practice based on microbial population characterization and single-species-based antibiotic resistance profiling has ignored the potential interspecies interactions, which might lead to novel drug-resistance phenotypes. Here, we investigated the impacts of interspecies interactions on antibiotic therapies by establishing a Pa and Ab dual-species biofilm model. Our data showed that antibiotic exposure would reshape the community compositions of dual-species biofilms, and those of the extracellular polymeric substance (EPS) matrix of Pa, Psl exopolysaccharide in particular, promoted its interactions with Ab against imipenem stress. We further found other EPS structural fiber-eDNA contributed to the Psl-dependent dual-species biofilm stability under antibiotic treatment. Thus, targeting the EPS structural fibers such as Psl and extracellular DNA (eDNA) is a potent strategy for controlling polymicrobial biofilm related infections.

Using Genomics to Shape the Definition of the Agglutinin-Like Sequence (ALS) Family in the Saccharomycetales.

The Candida albicans agglutinin-like sequence (ALS) family is studied because of its contribution to cell adhesion, fungal colonization, and polymicrobial biofilm formation. The goal of this work was to derive an accurate census and sequence for ALS genes in pathogenic yeasts and other closely related species, while probing the boundaries of the ALS family within the Order Saccharomycetales. Bioinformatic methods were combined with laboratory experimentation to characterize 47 novel ALS loci from 8 fungal species. AlphaFold predictions suggested the presence of a conserved N-terminal adhesive domain (NT-Als) structure in all Als proteins reported to date, as well as in S. cerevisiae alpha-agglutinin (Sag1). Lodderomyces elongisporus, Meyerozyma guilliermondii, and Scheffersomyces stipitis were notable because each species had genes with C. albicans ALS features, as well as at least one that encoded a Sag1-like protein. Detection of recombination events between the ALS family and gene families encoding other cell-surface proteins such as Iff/Hyr and Flo suggest widespread domain swapping with the potential to create cell-surface diversity among yeast species. Results from the analysis also revealed subtelomeric ALS genes, ALS pseudogenes, and the potential for yeast species to secrete their own soluble adhesion inhibitors. Information presented here supports the inclusion of SAG1 in the ALS family and yields many experimental hypotheses to pursue to further reveal the nature of the ALS family.

Metabolic Adaptations During Staphylococcus aureus and Candida albicans Co-Infection.

Successful pathogens require metabolic flexibility to adapt to diverse host niches. The presence of co-infecting or commensal microorganisms at a given infection site can further influence the metabolic processes required for a pathogen to cause disease. The Gram-positive bacterium Staphylococcus aureus and the polymorphic fungus Candida albicans are microorganisms that asymptomatically colonize healthy individuals but can also cause superficial infections or severe invasive disease. Due to many shared host niches, S. aureus and C. albicans are frequently co-isolated from mixed fungal-bacterial infections. S. aureus and C. albicans co-infection alters microbial metabolism relative to infection with either organism alone. Metabolic changes during co-infection regulate virulence, such as enhancing toxin production in S. aureus or contributing to morphogenesis and cell wall remodeling in C. albicans. C. albicans and S. aureus also form polymicrobial biofilms, which have greater biomass and reduced susceptibility to antimicrobials relative to mono-microbial biofilms. The S. aureus and C. albicans metabolic programs induced during co-infection impact interactions with host immune cells, resulting in greater microbial survival and immune evasion. Conversely, innate immune cell sensing of S. aureus and C. albicans triggers metabolic changes in the host cells that result in an altered immune response to secondary infections. In this review article, we discuss the metabolic programs that govern host-pathogen interactions during S. aureus and C. albicans co-infection. Understanding C. albicans-S. aureus interactions may highlight more general principles of how polymicrobial interactions, particularly fungal-bacterial interactions, shape the outcome of infectious disease. We focus on how co-infection alters microbial metabolism to enhance virulence and how infection-induced changes to host cell metabolism can impact a secondary infection.

Mucoadhesive controlled-release formulations containing morin for the control of oral biofilms

This study aimed to evaluate the antimicrobial and anti-biofilm activity of morin on polymicrobial biofilms and its cytotoxicity in controlled-release films and tablets based on gellan gum. Polymicrobial biofilms were formed from saliva for 48 h under an intermittent exposure regime to 1% sucrose and in contact with films or tablets of gellan gum containing 2 mg of morin each. Acidogenicity, bacterial viability, dry weight and insoluble extracellular polysaccharides from biofilms were evaluated. The cytotoxicity of morin was evaluated in oral keratinocytes. Morin released from the systems reduced the viability of all the microbial groups evaluated, as well as the dry weight and insoluble polysaccharide concentration in the matrix and promoted the control of acidogenicity when compared with the control group without the substance. Morin was cytotoxic only at the highest concentration evaluated. In conclusion, morin is an effective agent and shows antimicrobial and anti-biofilm activity against polymicrobial biofilms.

A nano phototheranostic approach of toluidine blue conjugated gold silver core shells mediated photodynamic therapy to treat diabetic foot ulcer

Diabetic foot infection caused by multidrug-resistant bacteria, is becoming serious problem. Moreover, polymicrobial biofilms contribute significantly to the persistent infections. In the present study, we investigated the effectiveness of novel toluidine blue conjugated chitosan coated gold–silver core–shell nanoparticles (TBO–chit–Au–AgNPs) mediated photodynamic therapy and demonstrate their use as a nontoxic antibacterial therapy to combat diabetic foot ulcer (DFU) caused by multi-drug resistant strains both in monomicrobial and polymicrobial state of infection. In vitro efficacy of TBO–chit–Au–AgNPs mediated photodynamic therapy (PDT) against polymicrobial biofilms was determined using standard plate count method and compared with that of monomicrobial biofilms of each species. Different anti-biofilm assays and microscopic studies were performed to check the efficacy of TBO–chit–Au–AgNPs mediated PDT, displayed significant decrease in the formation of biofilm. Finally, its therapeutic potential was validated in vivo type-2DFU. Cytokines level was found reduced, using nano-phototheranostic approach, indicating infection control. Expression profile of growth factors confirmed both the pathogenesis and healing of DFU. Hence, we conclude that TBO–chit–Au–AgNPs mediated PDT is a promising anti-bacterial therapeutic approach which leads to a synergistic healing of DFU caused by MDR bacterial strains.

A New PNA-FISH Probe Targeting Fannyhessea vaginae.

Bacterial vaginosis (BV) is the most common vaginal infection in women of reproductive age and has been associated with serious health complications, mainly in pregnant women. It is characterized by a decrease in the number of Lactobacillus species in the healthy vaginal microbiota and an overgrowth of strict and facultative anaerobic bacteria that develop a polymicrobial biofilm. Despite over 60 years of research investigating BV, its etiology is not fully understood. Gardnerella spp. is a crucial microorganism that contributes to the formation of the biofilm and the development of BV, but the role of other BV-associated bacteria is not clear. Nevertheless, Fannyhessea vaginae (previously known as Atopobium vaginae) is a highly specific species for BV, and co-colonization with Gardnerella is thought to be a very specific diagnostic marker. The diagnosis of BV still presents some limitations, since currently used methods often fail to accurately detect BV. This work aims to develop a novel peptide nucleic acid (PNA) probe targeting F. vaginae. This probe was further validated in a multiplex assay, which included a Gardnerella-specific PNA probe, as a possible method for diagnosis of BV, and was compared with quantification by qPCR. The new PNA probe showed excellent sensitivity and specificity and could discriminate F. vaginae-Gardnerella biofilms, confirming the potential to be used for the detection of BV-associated pathogens.

Efficacy and safety of biofilm dispersal by glycoside hydrolases in wounds

Novel anti-biofilm and dispersal agents are currently being investigated in an attempt to combat biofilm-associated wound infections. Glycoside hydrolases (GHs) are enzymes that hydrolyze the glycosidic bonds between sugars, such as those found within the exopolysaccharides of the biofilm matrix. Previous studies have shown that GHs can weaken the matrix, inducing bacterial dispersal, and improving antibiotic clearance. Yet, the number of GH enzymes that have been examined for potential therapeutic effects is limited. In this study, we screened sixteen GHs for their ability to disperse mono-microbial and polymicrobial biofilms grown in different environments. Six GHs, α-amylase (source: A. oryzae), alginate lyase (source: various algae), pectinase (source: Rhizopus sp.), amyloglucosidase (source: A. niger), inulinase (source: A. niger), and xylanase (source: A. oryzae), exhibited the highest dispersal efficacy in vitro. Two GHs, α-amylase (source: Bacillus sp.) and cellulase (source: A. niger), used in conjunction with meropenem demonstrated infection clearing ability in a mouse wound model. GHs were also effective in improving antibiotic clearance in diabetic mice. To examine their safety, we screened the GHs for toxicity in cell culture. Overall, there was an inverse relationship between enzyme exposure time and cellular toxicity, with twelve out of sixteen GHs demonstrating some level of toxicity in cell culture. However, only one GH exhibited harmful effects in mice. These results further support the ability of GHs to improve antibiotic clearance of biofilm-associated infections and help lay a foundation for establishing GHs as therapeutic agents for chronic wound infections.

Gardnerella Vaginalis Dominates Multi-Species Biofilms in both Pre-Conditioned and Competitive In Vitro Biofilm Formation Models.

Bacterial vaginosis (BV) is one of the most common bacterial vaginal infections worldwide. Despite its high prevalence, BV etiology is still unknown. Nevertheless, a hallmark of BV is the presence of a highly structured polymicrobial biofilm on the vaginal epithelium, formed primarily by Gardnerella spp. and other anaerobic species, of which co-colonization with Fannyhessea vaginae is considered an important diagnostic marker. We previously developed an in vitro biofilm model wherein Gardnerella was first allowed to establish an early biofilm that served as a scaffold for other species to adhere to. To better understand ecological interactions between BV-associated bacteria, we compared triple-species biofilms formed using two distinct models: a pre-conditioned (wherein Gardnerella vaginalis formed the early biofilm) model and a competitive (wherein all three bacteria were co-incubated together) model. Interestingly, synergistic growth interactions were more significant in the competitive model. Furthermore, the biofilm structure and species-specific distribution, as assessed by confocal laser scanning microscopy and using peptide nucleic acid fluorescence in situ hybridization method, revealed two very different triple-species morphotypes, suggesting that different interactions occur in the different models. Interestingly, independent of the model or triple-species consortium tested, we observed that G. vaginalis represented most of the biofilm bacterial composition, further highlighting the relevance of this taxon in BV.

Mechanisms underlying interactions between two abundant oral commensal bacteria

Complex polymicrobial biofilm communities are abundant in nature particularly in the human oral cavity where their composition and fitness can affect health. While the study of these communities during disease is essential and prevalent, little is known about interactions within the healthy plaque community. Here we describe interactions between two of the most abundant species in this healthy microbiome, Haemophilus parainfluenzae and Streptococcus mitis. We discovered that H. parainfluenzae typically exists adjacent to mitis group streptococci in vivo with which it is also positively correlated based on microbiome data. By comparing in vitro coculture data to ex vivo microscopy we revealed that this co-occurrence is density dependent and further influenced by H2O2 production. We discovered that H. parainfluenzae utilizes a more redundant, multifactorial response to H2O2 than related microorganisms and that this system’s integrity enhances streptococcal fitness. Our results indicate that mitis group streptococci are likely the in vivo source of NAD for H. parainfluenzae and also evoke patterns of carbon utilization in vitro for H. parainfluenzae similar to those observed in vivo. Our findings describe mechanistic interactions between two of the most abundant and prevalent members of healthy supragingival plaque that contribute to their in vivo survival.