Single Species Cultures Research Articles

Glomus rugosae, a new arbuscular mycorrhizal species in Glomeraceae (phylum Glomeromycota) from maritime sand dunes of Poland and an ash pond of Czech Republic

Preliminary morphological analyzes of an isolate producing glomoid spores in culture and comparison of its 45S nuc rDNA sequences (= 18S-ITS-28S) with sequences available in GenBank suggested that this isolate is an undescribed arbuscular mycorrhizal fungus of the genus Glomus in the family Glomeraceae (phylum Glomeromycota). This suggestion was confirmed by phylogenetic analyzes with sequences of 45S and the largest subunit of RNA polymerase II (rpb1) gene that placed this isolate in an autonomous clade sister to that with Glomus macrocarpum, the type species of Glomus and Glomeromycota. In the field, this species, here named G. rugosae sp. nov., was associated with roots of Rosa rugosa, which inhabited the coastal dunes of the Hel Peninsula in northern Poland. In single-species cultures, G. rugosae formed typical vesicular-arbuscular mycorrhiza. Phylogenetic analyzes with 45S sequences of Glomus species and environmental sequences with a similarity of >96% to the 45S sequences of G. rugosae showed that G. rugosae was previously detected in the Czech Republic.

Interspecific competition affects spore germination and gametophore development of mosses

Background Interactions between moss species in their earliest growth stages have received little attention. To what extent interspecific competition or priority effects influence spore germination, protonemal development and gametophore emergence is unknown. We evaluated such effects in pairwise interaction between six common bryophyte species: Atrichum undulatum, Bryum argenteum, Ceratodon purpureus, Funaria hygrometrica, Hypnum cupressiforme, Leptobryum pyriforme Methods Interspecific interactions were assessed in vitro. Spores were sterilized and sown on agar plates in three treatments: 1) as single species cultures (controls), 2) as pairwise species cultures inoculated simultaneously, and 3) with a time lag of 20 days between species. Data on time needed for spore germination, germination rate, the time needed for gametophore differentiation, number of gametophores per germinated spore and average diameter of colonies were collected. We also performed spore germination tests in single-species cultures at the start and end of the study, as well as tests for density-dependency at spore germination and gametophore formation. Results We observed strong pairwise interactive effects when sowing spores of different species simultaneously or with a delay of 20 days. The results indicate that spore germination is often inhibited by interspecific competition. The first species has an advantage as compared to the later colonizing species, i.e., an apparent priority effect. Interspecific interactions were also evident during gametophore development and included both inhibition and facilitation. Conclusion We found pronounced differences in the relative performance of species in interaction with other species during spore germination and gametophore formation. Allelopathic effects are the most probable explanation for these observations. Our results under sterile lab conditions are likely to reflect processes that occur in the wild, governing biotic filtering and bryophyte community assembly during primary and secondary colonization.

Cell pleomorphism and changes in the enzymatic profile of selected Candida albicans strains in interaction with Escherichia coli – pilot study

Interactions between C. albicans and the microbiota play an important role in maintaining the balance between commensal and pathogenic organisms. Although the exact role of bacteria in reducing the pathogenicity of yeast remains poorly understood, a few examples have been documented so far: probiotics administration effectively reduces the formation of biofilm and bacterial metabolites inhibit the formation of hyphae.The aim of the study was to analyze C. albicans virulence levels based on the changes in the morphological structure and enzymatic profile in experimental cultures mixed with Escherichia coli. Viable cell abundance, cell pleomorphism and enzymatic profile were analyzed in single and mixed cultures (C. albicans + E. coli).The microscope analysis showed a large decrease in the number of viable C. albicans cells in mixed cultures with E. coli from 485.3±132.1 immediately after the establishment of the culture to 238.1±71.2 after an hour of incubation and 24.4±5.4 after 24 h. The length of C. albicans cells differed significantly between the single-species cultures and the mixed cultures for 24 h. Our present findings indicate a significant reduction in the secretion of several enzymes by fungi following contact with E. coli, including acid phosphatase, N-acetyl-β-glucosaminidase, naphthol-AS-BI-phosphohydrolase and leucine arylamidase.The interactions between fungi and bacteria appear to be extremely complex. On the one hand, during C. albicans with E. coli co-incubation, the bacteria stimulated the elongation of yeast cells, leading to the formation of a filamentous form; however, the number of yeast cells and their enzymatic activity decreased significantly. Therefore, it can be concluded that while E. coli stimulates some pathogenic properties, e.g. cell elongation, it also inhibits other virulence features, e.g. enzymatic activity of C. albicans.

Modeling interactions of Clostridium cadaveris and Clostridium sporogenes in anaerobic acidogenesis of glucose and peptone

This study focuses on developing a mathematical model to assess interaction among acidogenic bacteria during the anaerobic degradation of two substrates. Clostridium cadaveris and Clostridium sporogenes were cultured in various combinations with glucose and peptone. Parameter estimates are given for both conventional Monod parameters from single substrate-single species cultures and sum kinetics with interaction parameters obtained from dual substrate-single species cultures. The presence of multiple substrates led to both inhibitory and enhancing effects on biodegradation rates for dual substrates compared to single substrate cultures. A new model of interspecies interaction was developed within the framework of Lotka-Volterra incorporating substrate interaction parameters, with a focus on accuracy, realism, simplicity, and biological significance. The model demonstrated competitive interaction for resource sharing and the additional non-linearity parameter eliminated the constraint of the linear relationship between growth rate and population density.

Transcriptional profiling and genetic analysis of a cystic fibrosis airway-relevant model shows asymmetric responses to growth in a polymicrobial community.

Bacterial infections in the lungs of persons with cystic fibrosis are typically composed of multispecies biofilm-like communities, which modulate clinically relevant phenotypes that cannot be explained in the context of a single species culture. Most analyses to date provide a picture of the transcriptional responses of individual pathogens; however, there is relatively little data describing the transcriptional landscape of clinically relevant multispecies communities. Harnessing a previously described cystic fibrosis-relevant, polymicrobial community model consisting of Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica, we performed an RNA-Seq analysis on the biofilm population to elucidate the transcriptional profiles of the community grown in artificial sputum medium (ASM) as compared to growth in monoculture, without mucin, and in fresh medium supplemented with tobramycin. We provide evidence that, although the transcriptional profile of P. aeruginosa is community agnostic, the transcriptomes of S. aureus and S. sanguinis are community aware. Furthermore, P. aeruginosa and P. melaninogenica are transcriptionally sensitive to the presence of mucin in ASM, whereas S. aureus and S. sanguinis largely do not alter their transcriptional profiles in the presence of mucin when grown in a community. Only P. aeruginosa shows a robust response to tobramycin. Genetic studies of mutants altered in community-specific growth provide complementary data regarding how these microbes adapt to a community context. IMPORTANCE Polymicrobial infections constitute the majority of infections in the cystic fibrosis (CF) airway, but their study has largely been neglected in a laboratory setting. Our lab previously reported a polymicrobial community that can help explain clinical outcomes in the lungs of persons with CF. Here, we obtained transcriptional profiles of the community versus monocultures to provide transcriptional information about how this model community responds to CF-related growth conditions and perturbations. Genetic studies provide complementary functional outputs to assess how the microbes adapt to life in a community.

Interspecific competition affects spore germination and gametophore development of mosses.

Interactions between moss species in their earliest growth stages have received little attention. To what extent interspecific competition or priority effects influence spore germination, protonemal development and gametophore emergence is unknown. We evaluated such effects in pairwise interaction between six common bryophyte species: Atrichum undulatum, Bryum argenteum, Ceratodon purpureus, Funaria hygrometrica, Hypnum cupressiforme, Leptobryum pyriforme. Interspecific interactions were assessed in vitro. Spores were sterilized and sown on agar plates in three treatments: 1) as single species cultures (controls), 2) as pairwise species cultures inoculated simultaneously, and 3) with a time lag of 20 days between species. Data on time needed for spore germination, germination rate, the time needed for gametophore differentiation, number of gametophores per germinated spore and average diameter of colonies were collected. We also performed spore germination tests in single-species cultures at the start and end of the study, as well as tests for density-dependency at spore germination and gametophore formation. We observed strong pairwise interactive effects when sowing spores of different species simultaneously or with a delay of 20 days. The results indicate that spore germination is often inhibited by interspecific competition. The first species has an advantage as compared to the later colonizing species, i.e., an apparent priority effect. Interspecific interactions were also evident during gametophore development and included both inhibition and facilitation. We found pronounced differences in the relative performance of species in interaction with other species during spore germination and gametophore formation. Allelopathic effects are the most probable explanation for these observations. Our results under sterile lab conditions are likely to reflect processes that occur in the wild, governing biotic filtering and bryophyte community assembly during primary and secondary colonization.

Epithelial responses and Candida albicans pathogenicity are enhanced in the presence of oral streptococci.

Experimental models that consider host-pathogen interactions are relevant for improving knowledge about oral candidiasis. The aim of this study was to assess the epithelial immune responses, Candida penetration of cell monolayers, and virulence during mixed species culture infections. Single species cultures of Candida albicans and mixed cultures (C. albicans, Streptococcus mutans, and Streptococcus sanguinis) were used to infect monolayers of HaCaT and FaDu ATCC HTB-43 cells for 12 h. After infection, IL-18 and IL-34 gene expression was measured to assess epithelial cell immune responses, and lactate dehydrogenase (LDH) activity was measured as an indicator of cell damage. Microscopy determined C. albicans morphology and penetration of fungal cells through the keratinocyte monolayer. Monolayers devoid of infection served as controls. Data were analyzed by an ANOVA one-way test followed by Tukey's post-hoc test (α = 0.05). The results found that IL-18 and IL-34 gene expression and LDH activity were significantly (p < 0.05) upregulated for both cell lines exposed to mixed species cultures compared with C. albicans alone. Candida albicans yeast and hyphae were evident in C. albicans only infections. In contrast, monolayers infected by C. albicans, S. mutans, and S. sanguinis exhibited higher microbial invasion with several hyphal aggregates detected. The presence of streptococci in C. albicans infection enhances the virulence and pathogenicity of the fungus with associated increased immune responses and tissue damage. Extrapolation of these findings to oral infection would indicate the added potential benefit of managing bacterial components of biofilms during treatment.

303 Investigation of the antibacterial and regenerative properties of a novel AHA dental coating for the treatment of deep caries

OBJECTIVES/GOALS: Our objective is to investigate the antibacterial and regenerative properties of a novel AHA dental coating for the prevention and treatment of deep caries (cavities). Further, we aim to investigate and compare these properties through in vivo murine models and assessment on human saliva samples and human pulpal cells collected from clinical samples. METHODS/STUDY POPULATION: In vitro antibacterial studies were performed by collecting and culturing human salivary bacteria with AHA substrates and quantifying survival of cariogenic Sm (S. mutans). In vivo, C57BL/6 mice were treated with AHA composite fillings, infected with Sm clinical isolates, and fed a high sucrose diet with cavity formation assessment after 6 weeks. To evaluate regeneration, mice were similarly given composite with AHA or MTA (standard of care) upon pulpal exposure with regeneration quantified by microCT and histological analysis of dentin bridge formation, ALP production, and odontoblast migration. In vitro, AHA substrates were cultured with MC3T3-E1 pre-osteoblast cells and dental pulp stem cells obtained from clinical samples over 21 days, with mineralization and ALP assessed indicating osteogenesis. RESULTS/ANTICIPATED RESULTS: In vivo studies have shown the reduction of cavity formation in mice treated with AHA as well as dentin regeneration upon pulpal exposure using microCT and histological image analysis. Coinciding with these findings, AHA substrates eradicated cariogenic Sm in human saliva samples and single species cultures in vitro. Further, preliminary results in vitro have shown increased mineralization of MC3T3-E1 cells when cultured with AHA substrates in comparison to uncoated substrates. We anticipate similar increased mineralization as well as increased ALP production of human pulpal stem cells from clinical samples when cultured with AHA substrates, suggesting osteogenesis. Further, we anticipate increased odontoblast migration and ALP production upon additional analysis of in vivo tissue samples. DISCUSSION/SIGNIFICANCE: This work will elucidate the antibacterial and regenerative properties of AHA dental coatings. These results further support the translation of AHA coatings into the clinic as a novel therapeutic for the prevention and treatment of dental decay, which may overcome the limitations associated with the current treatments.

Nano selenium–alginate edible coating extends hydroponic strawberry shelf life and provides selenium fortification as a micro-nutrient

Although selenium nanoparticles (Nano Se) have gained attention for their anti-cancer and antioxidant activity, this nanomaterial's antimicrobial activity also has great potential. The aim of this study was to synthesize Nano Se edible coating solution through gamma irradiation technique with a bio-stabilizer of sodium alginate (Na-alg.), which is an ingredient commonly used in food preservation. Dynamic light scattering and transmission electron microscopy identified 94-nm-diameter Nano Se, and energy dispersive X-ray spectroscopy and Raman spectroscopy confirmed the formation of Nano Se. Antifungal studies revealed that Na-alg./Nano Se coating solution well inhibited both Neopestalotiopsis rosae and Fusarium oxysporum in single species culture conditions. The effectiveness of edible coatings containing Nano Se and Na-alg. on the physicochemical properties of strawberries at 24 ± 2 °C during 5-day storage period was evaluated. The results showed coated strawberries enhanced firmness by as much as 26% and a significantly less weight loss by as much as three fold compared to the control. The coating application was effective in maintaining a natural appearance and preserving half the amounts of vitamin C and B9 in the strawberries. In addition, the Na-alg./Nano Se coating was utilized as an additive to increase Se nutrient in strawberries. The Se content was supplemented from 0.28 to 0.56 μg per 100 g FW after coating. This study may potentially be used in the bio-preservation and Se-enrichment of various fresh fruits and other foods.

Dual Species Bacterial Challenge of a Biomimetic Nanostructured Surface

AbstractAn ever‐present risk of medical device associated infection has driven a significant body of research toward development of novel anti‐infective materials. Surfaces bearing sharp nanostructures are an emerging technology to address this concern. The in vitro efficacy of antimicrobial nanostructures has previously been verified using single species cultures, but there remains a paucity of data to address the threat of infections containing more than one species. Polymicrobial infections are a concerning threat because they can complicate treatment, promote drug resistance, and harshen patient prognosis. In the present study, dual‐species cultures are employed to challenge the mechano‐bactericidal properties of nanostructured surfaces. Escherichia coli is used with either Staphylococcus aureus or Enterococcus faecalis due to their clinical relevance in implant associated infection. Despite the presence of two mixed species, a high rate of bactericidal activity is found. Interestingly, in the mixed culture containing Escherichia coli with Enterococcus faecalis, the nanostructured surface triggers a shift in species distribution to favor Enterococcus faecalis. Overall, this study highlights the potential for mechano‐bactericidal surfaces to minimize the burden of infections containing more than one species. It also serves as an enticing foundation for further research into more complex biointerfacial interactions.

Connecting environmental and evolutionary microbiology for the development of new agrobiotechnological tools.

Connecting environmental and evolutionary microbiology for the development of new agrobiotechnological tools.

The central role of arginine in Haemophilus influenzae survival in a polymicrobial environment with Streptococcus pneumoniae and Moraxella catarrhalis.

Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis are bacterial species which frequently co-colonise the nasopharynx, but can also transit to the middle ear to cause otitis media. Chronic otitis media is often associated with a polymicrobial infection by these bacteria. However, despite being present in polymicrobial infections, the molecular interactions between these bacterial species remain poorly understood. We have previously reported competitive interactions driven by pH and growth phase between H. influenzae and S. pneumoniae. In this study, we have revealed competitive interactions between the three otopathogens, which resulted in reduction of H. influenzae viability in co-culture with S. pneumoniae and in triple-species culture. Transcriptomic analysis by mRNA sequencing identified a central role of arginine in mediating these interactions. Arginine supplementation was able to increase H. influenzae survival in a dual-species environment with S. pneumoniae, and in a triple-species environment. Arginine was used by H. influenzae for ATP production, and levels of ATP generated in dual- and triple-species co-culture at early stages of growth were significantly higher than the combined ATP levels of single-species cultures. These results indicate a central role for arginine-mediated ATP production by H. influenzae in the polymicrobial community.

Food web and metabolic interactions of the lung inhabitants Streptococcus pneumoniae and Pseudomonas aeruginosa.

Bacteria that successfully adapt to different substrates and environmental niches within the lung and overcome the immune defence can cause serious lung infections. Such infections are generally complex, and recognized as polymicrobial in nature. Both Pseudomonas aeruginosa and Streptococcus pneumoniae can cause chronic lung infections and were both detected in cystic fibrosis (CF) lung at different stages. In this study, single and dual species cultures of Pseudomonas aeruginosa and Streptococcus pneumoniae were studied under well-controlled planktonic growth conditions. Under pH-controlled conditions, both species apparently benefited from the presence of the other. In co-culture with P. aeruginosa, S. pneumoniae grew efficiently under aerobic conditions, whereas in pure S. pneumoniae culture, growth inhibition occurred in bioreactors with dissolved oxygen concentrations above the microaerobic range. Lactic acid and acetoin that are produced by S. pneumoniae were efficiently utilized by P. aeruginosa. In pH-uncontrolled co-cultures, the low pH triggered by S. pneumoniae assimilation of glucose and lactic acid production negatively affected the growth of both strains. Nevertheless, ammonia production improved significantly, and P. aeruginosa growth dominated at later growth stages. This study revealed unreported metabolic interactions of two important pathogenic microorganisms and shed new lights into pathophysiology of bacterial lung infection.

Integrated multi-trophic aquaculture mitigates the effects of ocean acidification: Seaweeds raise system pH and improve growth of juvenile abalone

Integrated multi-trophic aquaculture (IMTA) has the potential to enhance growth, reduce nutrient loads, and mitigate environmental conditions compared to traditional single-species culture techniques. The goal of this project was to develop a land-based system for the integrated culture of seaweeds and shellfish, to test the efficacy of integrated versus non-integrated designs, and to assess the potential for IMTA to mitigate the effects of climate change from ocean acidification on shellfish growth and physiology. We utilized the red abalone (Haliotis rufescens) and the red seaweed dulse (Devaleraea mollis) as our study species and designed integrated tanks at three different recirculation rates (0%, 30%, and 65% recirculation per hour) to test how an integrated design would affect growth rates of the abalone and seaweeds, modify nutrient levels, and change water chemistry. We specifically hypothesized that IMTA designs would raise seawater pH to benefit calcifying species. Our results indicated that juvenile abalone grew significantly faster in weight (22% increase) and shell area (11% increase) in 6 months in tanks with the highest recirculation rates (65%). The 65% recirculation treatment also exhibited a significant increase in mean seawater pH (0.2 pH units higher) due to the biological activity of the seaweed in the connected tanks. We found a significant positive relationship between the mean pH of seawater in the tanks and juvenile abalone growth rates across all treatments. There were no significant differences in the growth of dulse among treatments, but dulse growth did vary seasonally. Seawater phosphate and nitrate concentrations were depleted in the highest recirculation rate treatment, but ammonium concentrations were elevated, likely due to the abalone effluent. Overall, our results indicate that there are benefits to IMTA culture of seaweeds and abalone in terms of improving growth in land-based systems, which will reduce the time to market and buffer commercial abalone operations against the effects of ocean acidification during vulnerable early life stages.

Three new species of arbuscular mycorrhizal fungi of the genus Diversispora from maritime dunes of Poland

ABSTRACT Three new species of arbuscular mycorrhizal fungi of the genus Diversispora (phylum Glomeromycota) were described based on their morphology and molecular phylogeny. The phylogeny was inferred from the analyses of the partial 45S rDNA sequences (18S-ITS-28S) and the largest subunit of RNA polymerase II (rpb1) gene. These species were associated in the field with plants colonizing maritime sand dunes of the Baltic Sea in Poland and formed mycorrhiza in single-species cultures.

Glomus chinense and Dominikia gansuensis, two new Glomeraceae species of arbuscular mycorrhizal fungi from high altitude in the Tibetan Plateau

Two glomoid spore-producing arbuscular mycorrhizal fungi were grown in single-species cultures that were established from soil-borne spores collected from high altitude (2800 m a.s.l.) of Tibet Plateau, China. Phylogenetic analyses were performed with sequences of nuclear rDNA (spanning the partial small subunit, whole internal transcribed spacer, and partial large subunit segment; 18S-ITS-28S) and the largest subunit of RNA polymerase II (RPB1) gene. Morphological and phylogenetic analyses indicated that the two fungi are undescribed species of the genera Glomus and Dominikia. The first fungus, described here as G. chinense sp. nov., forms spores singly or in loose clusters. The spores are orange-yellow to dark brown, globose, and (47–)64(–93) μm diam. Dominikia gansuensis sp. nov. produces glomerocarps with pale yellow to yellow-brown, globose, (20–)47(–86) μm diam spores. The spore wall of both species consists of three layers. Both species differ clearly in morphology and phylogeny from their closest phylogenetic relatives, which are G. atlanticum and G. ibericum, and D. glomerocarpica, respectively.

A Multi–Membrane System to Study the Effects of Physical and Metabolic Interactions in Microbial Co-Cultures and Consortia

Continuous cell-to-cell contact between different species is a general feature of all natural environments. However, almost all research is conducted on single-species cultures, reflecting a biotechnological bias and problems associated with the complexities of reproducibly growing and controlling multispecies systems. Consequently, biotic stress due to the presence of other species remains poorly understood. In this context, understanding the effects of physical contact between species when compared to metabolic contact alone is one of the first steps to unravelling the mechanisms that underpin microbial ecological interactions. The current technologies to study the effects of cell-to-cell contact present disadvantages, such as the inefficient or discontinuous exchange of metabolites when preventing contact between species. This paper presents and characterizes a novel bioreactor system that uses ceramic membranes to create a “multi-membrane” compartmentalized system whereby two or more species can be co-cultured without the mixing of the species, while ensuring the efficient sharing of all of the media components. The system operates continuously, thereby avoiding the discontinuities that characterize other systems, which either have to use hourly backwashes to clean their membranes, or have to change the direction of the flow between compartments. This study evaluates the movement of metabolites across the membrane in co-cultures of yeast, microalgae and bacterial species, and monitors the movement of the metabolites produced during co-culturing. These results show that the multi-membrane system proposed in this study represents an effective system for studying the effects of cell-to-cell contact in microbial consortia. The system can also be adapted for various biotechnological purposes, such as the production of metabolites when more than one species is required for such a process.

The Effects of Nonnutritive Sweeteners on the Cariogenic Potential of Oral Microbiome.

Nonnutritive sweeteners (NNSs) are sugar substitutes widely used to reduce the negative health effects of excessive sugar consumption. Dental caries, one of the most prevalent chronic diseases globally, results from a pathogenic biofilm with microecological imbalance and frequent exposure to sugars. Some research has shown that certain NNSs possess less cariogenic potential than sucrose, indicating their putative effect on oral microbiome. To uncover the alterations of acidogenic pathogens and alkali-generating commensals, as well as the biofilm cariogenic potential under the influence of NNSs, we selected four common NNSs (acesulfame-K, aspartame, saccharin, and sucralose) and established single-, dual-, and multispecies in vitro culture model to assess their effects on Streptococcus mutans (S. mutans) and/or Streptococcus sanguinis (S. sanguinis) compared to sucrose with the same sweetness. The results showed that NNSs significantly suppressed the planktonic growth, acid production, and biofilm formation of S. mutans or S. sanguinis compared with sucrose in single-species cultures. Additionally, decreased S. mutans/S. sanguinis ratio, less EPS generation, and higher pH value were observed in dual-species and saliva-derived multispecies biofilms with supplementary NNSs. Collectively, this study demonstrates that NNSs inhibit the cariogenic potential of biofilms by maintaining microbial equilibrium, thus having a promising prospect as anticaries agents.

Compounds with Distinct Targets Present Diverse Antimicrobial and Antibiofilm Efficacy against Candida albicans and Streptococcus mutans, and Combinations of Compounds Potentiate Their Effect.

Candida albicans and Streptococcus mutans interact synergistically in biofilms associated with a severe form of dental caries. Their synergism is driven by dietary sucrose. Thus, it is necessary to devise strategies to hinder the development of those biofilms and prevent cavities. Six compounds [tt-farnesol (sesquiterpene alcohol that decreases the bacterium acidogenicity and aciduricity and a quorum sensing fungal molecule), myricetin (flavonoid that interferes with S. mutans exopolysaccharides production), two 2’-hydroxychalcones and 4’-hydroxychalcone (intermediate metabolites for flavonoids), compound 1771 (inhibitor of lipoteichoic synthase in Gram-positive bacteria)] with targets in both fungus and bacterium and their products were investigated for their antimicrobial and antibiofilm activities against single-species cultures. The compounds and concentrations effective on single-species biofilms were tested alone and combined with or without fluoride to control initial and pre-formed dual-species biofilms. All the selected treatments eliminated both species on initial biofilms. In contrast, some combinations eliminated the bacterium and others the fungus in pre-formed biofilms. The combinations 4’-hydroxychalcone+tt-farnesol+myricetin, 4’-hydroxychalcone+tt-farnesol+fluoride, and all compounds together with fluoride were effective against both species in pre-formed biofilms. Therefore, combinations of compounds with distinct targets can prevent C. albicans and S. mutans dual-species biofilm build-up in vitro.

Impact of the Peptide WMR-K on Dual-Species Biofilm Candida albicans/Klebsiella pneumoniae and on the Untargeted Metabolomic Profile.

In recent years, the scientific community has focused on the development of new antibiotics to address the difficulties linked to biofilm-forming microorganisms and drug-resistant infections. In this respect, synthetic antimicrobial peptides (AMPs) are particularly regarded for their therapeutic potential against a broad spectrum of pathogens. In this work, the antimicrobial and antibiofilm activities of the peptide WMR-K towards single and dual species cultures of Candida albicans and Klebsiella pneumoniae were investigated. We found minimum inhibitory concentration (MIC) values for WMR-K of 10 µM for K. pneumoniae and of 200 µM for C. albicans. Furthermore, sub-MIC concentrations of peptide showed an in vitro inhibition of biofilm formation of mono and polymicrobial systems and also a good biofilm eradication even if higher concentrations of it are needed. In order to provide additional evidence for the effect of the examined peptide, a study of changes in extracellular metabolites excreted and/or uptaken from the culture medium (metabolomic footprinting) in the poly-microbial association of C. albicans and K. pneumoniae in presence and absence of WMR-K was performed. Comparing to the untreated dual species biofilm culture, the metabolomic profile of the WMR-K treated culture appears significantly altered. The differentially expressed compounds are mainly related to the primary metabolic pathways, including amino acids, trehalose, pyruvic acid, glycerol and vitamin B6.