Monospecies Biofilms Research Articles

Combined Analysis of Transcriptomes and Metabolomes Reveals Key Genes and Substances That Affect the Formation of a Multi-Species Biofilm by Nine Gut Bacteria

Biofilms are one of the ways microorganisms exist in natural environments. In recent years, research has gradually shifted its focus to exploring the complexity and interactions of multi-species biofilms. A study showed that nine gut bacteria can form a multi-species biofilm on wheat fibers (M9 biofilm). However, the previous study did not clarify the reasons why M9 exhibited a better biofilm formation ability than the mono-species biofilms. In this study, the gene expression levels and metabolic accumulation of the M9 multi-species biofilm and biofilms of each individual bacterium were analyzed using transcriptomes and metabolomes. The differentially expressed genes (DEGs) showed that there were 740 common DEGs that existed in all of the nine groups, and they could regulate five pathways related to bacterial motility, cellular communication, and signal transduction. The metabolome results revealed that many peptides/amino acids and derivatives were produced in the M9 biofilm. Furthermore, purine metabolism was significantly enhanced in the M9 biofilm. L-arginine, l-serine, guanosine, and hypoxanthine were the common differentially accumulated metabolites (DAMs). The combined analysis of the transcriptomes and metabolomes showed that there were 26 common DEGs highly correlated with the four common DAMs, and they were involved in five metabolic pathways related to amino acids and purines. These results indicate that M9 can regulate multi-species biofilm formation by modulating genes related to bacterial motility, cellular communication, signal transduction, and the metabolism of amino acids and purines. This study provides insights into the interactions of microbial biofilms.

Antimicrobial and anti-biofilm activity of a mucoadhesive hydrogel functionalized with aminochalcone on titanium surfaces and in Galleria mellonella model: in vitro and in vivo study.

Peri-implantitis associated with dental implants shares characteristics with destructive periodontal diseases. Both conditions are multifactorial and strongly correlated with the presence of microorganisms surrounding the prostheses or natural dentition. This study aimed to evaluate the antimicrobial activity and toxicity of a mucoadhesive hydrogel functionalized with aminochalcone (HAM-15) against Aggregatibacter actinomycetemcomitans, Fusobacterium periodonticum, Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia, and Candida albicans. Various experiments were conducted to determine the minimum inhibitory concentrations (MIC) and minimum bactericidal/fungicidal concentrations (MBC/MFC), as well as the antibiofilm potential and toxicity in human gingival fibroblasts and a G. mellonella animal model. Infection and treatment studies were also performed in G. mellonella. The results demonstrated that both aminochalcone (AM-15) and the aminochalcone-functionalized hydrogel (HAM-15) exhibited antimicrobial activity, with MICs ranging from 7.8 to 31.2 μg/mL for the tested strains. Treatment with HAM-15 at 300 μg/mL reduced the monospecies biofilm of C. albicans and P. gingivalis by 7 log10 and 6 log10, respectively, and the mixed-species biofilm of these microorganisms by 7 log10 and 8 log10, respectively. Regarding toxicity, HAM-15 showed cytotoxic effects on human gingival fibroblasts at high concentrations, but in the G. mellonella model, survival was 70% at a dose of 1 mg/mL. Additionally, AM-15, when administered after larval infection, protected 90% of the animals (p < 0.05). These results suggest that AM-15 is a promising candidate for the prevention and treatment of anaerobic infections and yeasts, demonstrating significant antimicrobial efficacy and an acceptable safety profile in experimental models.

High release of Candida albicans eDNA as protection for the scaffolding of polymicrobial biofilm formed with Staphylococcus aureus and Streptococcus mutans against the enzymatic activity of DNase I.

This study aimed to determine the protective role of the high release of C. albicans extracellular DNA (eDNA) in a polymicrobial biofilm formed by S. aureus and S. mutans in the course of DNase I treatment. A tube-flow biofilm bioreactor was developed to mimic biofilm formation in the oral cavity. eDNA release was quantified by real-time PCR (qPCR) and confocal microscopy analysis were used to determine the concentration and distribution of eDNA and intracellular DNA (iDNA). The mean amount of eDNA released by each species in the polymicrobial was higher than that in monospecies biofilms (S. aureus: 3.1 × 10-2ng/μl polymicrobial versus 5.1 × 10-4ng/μl monospecies; S. mutans: 3 × 10-1ng/μl polymicrobial versus 2.97 × 10-2ng/μl monospecies; C. albicans: 8.35ng/μl polymicrobial versus 4.85ng/μl monospecies). The large amounts of eDNA released by C. albicans (96%) in polymicrobial biofilms protects the S. aureus and S. mutans cells against the degradation by DNase I and dampens the effect of clindamycin.

The effect of soaking heat-polymerized acrylic resin denture base in avocado seed extract (Persea americana Mill.) on the inhibition of denture-plaque microorganisms biofilm growth

Background Heat polymerized acrylic (HPA) resins are known to have high porosity that contributes to increased surface roughness and microcrack formation in stress areas. This facilitates the attachment and growth of polymicrobial biofilms contributing to increased antimicrobial resistance. Many research had been carried out on avocado seeds, but no research that studies avocado seeds effect on denture-plaque microorganism biofilm on HPA resin has been found. Methods This study used 144 samples (n=144), namely HPA resin discs covered with mono-species and polymicrobial biofilms. The discs were soaked for 8 hours in the 5%, 10%, 15%, 20% avocado seed extract, positive control (alkaline peroxide), and negative control (aquadest). Each disc was shaken with a vortex mixer for 1 minute, and 100 μL was added into 96-well microplates with three times repetition and incubated for 24 hours. A microtiter plate biofilm formation assay was then conducted The inhibition values were determined from the percentage inhibition value formula which required absorption values from a microplate reader (595 nm). The research data were analyzed using a univariant test, and a one-way ANOVA test, accompanied by Welch ANOVA on non-homogenous data. Results In this research, it was found that the MBIC50 of avocado seed extract against the mono-species of C. albicans (5%), C. glabrata (5%), A. odontolyticus (15%), S. gordonii (15%), S. aureus (10%), while against polymicrobial was 20%. There was a significant effect of soaking HPA resin in avocado seed extract on the inhibition of mono-species and polymicrobial biofilms with a value of p&lt;0.001 (p&lt;0.05). Conclusion The MBIC50 of avocado seed extract in polymicrobial biofilm group was higher than that in the mono-species biofilm groups. Although alkaline peroxide showed higher inhibition value than that of the MBIC50 in polymicrobial biofilm group, 20% avocado seed extract was concluded effective as it inhibited &gt;50% polymicrobial biofilm.

Klebsiella pneumoniae AI-2 transporters mediate interspecies interactions and composition in a three-species biofilm community

Biofilms in nature often exist as communities. In this study, an experimental mixed-species community consisting of Pseudomonas aeruginosa, Pseudomonas protegens and Klebsiella pneumoniae was used to investigate how AI-2 transporters affect interspecies interactions and composition. The K. pneumoniae lsrB/lsrD deletion mutants had a 10–25-fold higher concentration of extracellular AI-2 compared to the wild-type. Although these deletion mutants produced monospecies biofilms of similar biomass, the substitution of these mutants for the parental strain significantly altered composition. Dual-species biofilm assays demonstrated that the changes in composition were due to the cumulative effect of pairwise interactions. It was further revealed that K. pneumoniae being present physically in the consortium was important in AI-2 mediating composition in the consortium, and that AI-2 transporters were crucial in achieving maximum biomass in the community. In conclusion, these findings demonstrate that AI-2 transporters mediate interspecies interactions and is important in maintaining the compositional equilibrium of the community.

Effect of exposure to sub-inhibitory concentrations of biocides on the susceptibility to antibiotics of sessile cells of Listeria monocytogenes and Salmonellaenterica

The effect of sub-inhibitory concentrations of several disinfectants on the antibiotic resistance of sessile cells of Listeria monocytogenes and Salmonella enterica was investigated. Biofilms were formed in the presence of biocides at MIC/2. Five compounds were used: peracetic acid (PAA), benzalkonium chloride (BZK), sodium hypochlorite (SHY), polyvinyl pyrrolidone iodine (PVI) and bis(3-aminopropyl)amine (BAPA). Two strains of L. monocytogenes (LM30 and LM28) and S. enterica (S. Typhimurium -ST- and S. Enteritidis -SE-) were used. Mono-species biofilms (MSB) and mixed biofilms (MB, LM30 + ST and LM28 + SE) were tested. LM30 went from susceptible to resistant to tetracycline and erythromycin after exposure to BAPA in MB. LM28 went from susceptible to resistant to tetracycline in the presence of PVI in MSB, from susceptible to intermediate to chloramphenicol after exposure to PAA in both MSB and MB, from susceptible to resistant to erythromycin after exposure to BAPA in MB, and from susceptible to resistant to ampicillin after exposure to SHY and BAPA in MB. ST went from intermediate to resistant to gentamicin after exposure to SHY, PVI and BAPA (MSB), or to PVI and BAPA (MB). This strain went from susceptible to intermediate to cefoxitin after exposure to BZK and PVI (MSB) or to BZK, PVI and BAPA (MB). For fosfomycin, ST went from susceptible to intermediate after exposure to PAA, PVI and BAPA (MSB) or to SHY, BZK, PVI and BAPA (MB). In the case of cephalothin, ST moved from intermediate to resistant category in MSB exposed to BAPA. SE moved from susceptible to intermediate to cefoxitin after contact with PAA in both MSB and MB. By contrast, in several occasions, strains increased their susceptibility to antibiotics after exposure to biocides. In some additional cases, contact with disinfectants caused significative modifications in the MIC of various antibiotics without the strains changing from one category to another. These findings show that contact with low concentrations of biocides can modify the antibiotic resistance of sessile cells of L. monocytogenes and S. enterica. The need to use adequate doses of disinfectants in food facilities in order to avoid changes in bacterial resistance is highlighted.

The effect of soaking heat-polymerized acrylic resin denture base in avocado seed extract (Persea americana Mill.) on the inhibition of denture-plaque microorganisms biofilm growth

Background Heat polymerized acrylic (HPA) resins are known to have high porosity that contributes to increased surface roughness and microcrack formation in stress areas. This facilitates the attachment and growth of polymicrobial biofilms contributing to increased antimicrobial resistance. Many research had been carried out on avocado seeds, but no research that studies the effect of avocado seeds on denture-plaque microorganism biofilm on HPA resin has been found. Methods This study used 144 samples (n=144), namely HPA resin discs covered with mono-species and polymicrobial biofilms consisting of Candida albicans, Candida glabrata, Actinomyces odontolyticus, Streptococcus gordonii, and Staphylococcus aureus. The discs were soaked for 8 hours in the 5%, 10%, 15%, 20% avocado seed extract, positive control (alkaline peroxide), and negative control (aquadest). Each disc was shaken with a vortex mixer for 1 minute, and 100 μL was added into 96-well microplates with three times repetition and incubated for 24 hours. The inhibition values were determined from the percentage inhibition value formula which required absorption values from a microplate reader (595 nm). Results In this research, it was found that the MBIC50 of avocado seed extract against the mono-species of C. albicans (5%), C. glabrata (5%), A. odontolyticus (15%), S. gordonii (15%), S. aureus (10%), while against the biofilm was 20%. There was a significant effect of soaking HPA resin in avocado seed extract of 5%, 10%, 15%, 20% on the inhibition of mono-species and polymicrobial biofilms of denture-plaque microorganisms with a value of p&lt;0.001 (p&lt;0.05). Conclusion The MBIC50 of avocado seed extract in polymicrobial biofilm group was higher than that in the mono-species biofilm groups. Although alkaline peroxide showed higher inhibition value than that of the MBIC50 in polymicrobial biofilm group, 20% avocado seed extract was effective in inhibiting polymicrobial biofilm because it was able to inhibit more than 50% polymicrobial biofilm.

Antimicrobial Potential of Strontium-Functionalized Titanium Against Bacteria Associated With Peri-Implantitis.

To explore the antimicrobial potential of strontium (Sr)-functionalized wafers against multiple bacteria associated with per-implant infections, in both mono- and multispecies biofilms. The bactericidal and bacteriostatic effect of silicon wafers functionalized with a strontium titanium oxygen coating (Sr-Ti-O) or covered only with Ti (controls) against several bacteria, either grown as a mono-species or multispecies biofilms, was assessed using a bacterial viability assay and a plate counting method. Mono-species biofilms were assessed after 2 and 24 h, while the antimicrobial effect on multispecies biofilms was assessed at Days 1, 3, and 6. The impact of Sr functionalization on the total percentage ofPorphyromonas gingivalis in the multispecies biofilm, using qPCR, and gingipain activity was also assessed. Sr-functionalized wafers, compared to controls, were associated with statistically significant less viable cells in both mono- and multispecies tests. The number of colony forming units (CFUs) within the biofilm was significantly less in Sr-functionalized wafers, compared to control wafers, for Staphylococcus aureus at all time points of evaluation and forEscherichia coli at Day 1. Gingipain activity was less in Sr-functionalized wafers, compared to control wafers, and the qPCR showed that P. gingivalis remained below detection levels at Sr-functionalized wafers, while it consisted of 15% of the total biofilm on control wafers at Day 6. Sr functionalization displayed promising antimicrobial potential, possessing bactericidal and bacteriostatic ability against bacteria associated with peri-implantitis grown either as mono-species or mixed in a multispecies consortium with several common oral microorganisms.

Antimicrobial resistance induction potential of grapefruit seed extract on multi-species biofilm of E. coli in food industry

Biofilm formation in natural environments involving complex multi-structural arrangements hinders challenges in antimicrobial resistance. This study investigated the antimicrobial resistance potential of grapefruit seed extract (GSE) by examining the formation of mono-, dual-, and multi-species biofilms. We also explored the counterintuitive effect in response to GSE at various concentrations, including minimum inhibitory concentration (MIC) and sub-MIC (1/2 and 1/4 MIC). The results of the swimming and swarming motility tests revealed increased motility at the sub-MIC of GSE. The crystal violet assay demonstrated increased biofilm formation in multi-species biofilms, highlighting the synergistic effect of Escherichia coli, Salmonella Typhimurium, and Listeria monocytogenes. At the MIC concentration of GSE, field emission scanning electron microscopy (FE-SEM) revealed cell morphology damage, while sub-MIC increased biofilm formation and architectural complexity. Multi-species biofilms demonstrated greater biofilm-forming ability and antimicrobial resistance than mono-species biofilms, indicating synergistic interactions and enhanced resilience. These findings highlight the importance of understanding biofilm dynamics and antimicrobial resistance to ensure environmental safety.

Microbial interactions in mixed-species biofilms on the surfaces of Baijiu brewing environments

Environmental microorganisms commonly inhabit dense multispecies biofilms, fostering mutualistic relationships and co-evolution. However, the mechanisms underlying biofilm formation and microbial interactions within the Baijiu fermentation microecosystem remain poorly understood. Hence, the objective of this study was to investigate the composition, structure, and interactions of microorganisms residing in biofilms on environmental surfaces in Baijiu production. The results revealed a shift in the bacteria-fungi interaction network following fermentation, transitioning from a cooperative/symbiotic relationship to a competitive/antagonistic dynamic. Core microbiota within the biofilms comprised lactic acid bacteria (LAB), yeast, and filamentous fungi. From the environmental surface samples, we isolated two strains of LAB (Lactiplantibacillus pentosus EB27 and Pediococcus pentosaceus EB35) and one strain of yeast (Pichia kudriavzevii EF8), all displaying remarkable biofilm formation and fermentation potential. Co-culturing LAB and yeast demonstrated a superior capacity for dual-species biofilm formation compared to mono-species biofilms. The dual-species biofilm displayed a two-layer structure, with LAB in the lower layer and serving as the foundation for the yeast community in the upper layer. The upper layer exhibited a dense distribution of yeast, enhancing aerobic respiration. Metabolic activities in the dual-species biofilm, such as ABC transporter, oxidative phosphorylation, citric acid cycle, sulfur metabolism, glycine, serine, threonine metabolism, lysine degradation, and cysteine and methionine metabolism, showed significant alterations compared to LAB mono-species biofilms. Moreover, bacterial chemotaxis, starch, and sucrose metabolism in the dual-species biofilm exhibited distinct patterns from those observed in the yeast mono-species biofilm. This study demonstrated that a core microbiota with fermentation potential may exist in the form of a biofilm on the surface of a Baijiu brewing environment. These findings provide a novel strategy for employing synthetic stable microbiotas in the intelligent brewing of Baijiu.

The interplay of Pseudomonas aeruginosa and Staphylococcus aureus in dual-species biofilms impacts development, antibiotic resistance and virulence of biofilms in in vitro wound infection models.

Due to high tolerance to antibiotics and pronounced virulence, bacterial biofilms are considered a key factor and major clinical challenge in persistent wound infections. They are typically composed of multiple species, whose interactions determine the biofilm's structural development, functional properties and thus the progression of wound infections. However, most attempts to study bacterial biofilms in vitro solely rely on mono-species populations, since cultivating multi-species biofilms, especially for prolonged periods of time, poses significant challenges. To address this, the present study examined the influence of bacterial composition on structural biofilm development, morphology and spatial organization, as well as antibiotic tolerance and virulence on human skin cells in the context of persistent wound infections. By creating a wound-mimetic microenvironment, the successful cultivation of dual-species biofilms of two of the most prevalent wound pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, was realized over a period of 72 h. Combining quantitative analysis with electron microscopy and label-free imaging enabled a comprehensive evaluation of the dynamics of biofilm formation and matrix secretion, revealing a twofold increased maturation of dual-species biofilms. Antibiotic tolerance was comparable for both mono-species cultures, however, dual-species communities showed a 50% increase in tolerance, mediated by a significantly reduced penetration of the applied antibiotic into the biofilm matrix. Further synergistic effects were observed, where dual-species biofilms exacerbated wound healing beyond the effects observed from either Pseudomonas or Staphylococcus. Consequently, predicting biofilm development, antimicrobial tolerance and virulence for multi-species biofilms based solely on the results from mono-species biofilms is unreliable. This study underscores the substantial impact of a multi-species composition on biofilm functional properties and emphasizes the need to tailor future studies reflecting the bacterial composition of the respective in vivo situation, leading to a more comprehensive understanding of microbial communities in the context of basic microbiology and the development of effective treatments.

Antimicrobial and antibiofilm activities of Brazilian organic honey against oral microorganisms.

To evaluate the antimicrobial activity of Brazilian honeys against oral microorganisms. Organic honeys (OH-1 to OH-8) were diluted (%-w/v) and sterilized by filtration. Antimicrobial activity was defined by determining MIC and CBM against oral Streptococcus. The component responsible for the antimicrobial action was defined by a catalase assay. Antibiofilm activity was evaluated against the monospecies biofilm of Streptococcus mutans(ATCC 700610). OHs showed antimicrobial activity principally OH-1, OH-2, OH-3, and OH-7 with MIC values ​​ranging between 10 and 25%. The mechanism of action occurs mainly by hydrogen peroxide produced by honey enzymes. OH-1, OH-2, and OH-7 showed total biofilm destruction at low concentrations. Brazilian honeys have promising antimicrobial and antibiofilm activity with the potential to control oral microbiota.

Effect of hydroxyapatite nanoparticles coating of titanium surface on biofilm adhesion: An in vitro study

AimTo evaluate the adhesion of mono and duospecies biofilm on a commercially available dental implant surface coated with hydroxyapatite nanoparticles (nanoHA). Material and methodsTitanium discs were divided into two groups: double acid-etched (AE) and AE coated with nanoHA (NanoHA). Surface characteristics evaluated were morphology, topography, and wettability. Mono and duospecies biofilms of Streptococcus sanguinis (S. sanguinis) and Candida albicans (C. albicans) were formed. Discs were exposed to fetal bovine serum (FBS) to form the pellicle. Biofilm was growth in RPMI1640 medium with 10% FBS and 10% BHI medium for 6 h. Microbial viability was evaluated using colony-forming unit and metabolic activity by a colorimetric assay of the tetrazolium salt XTT. Biofilm architecture and organization were evaluated by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). ResultsAE surface had more pores, while NanoHA had even nanoHA crystals distribution. Roughness was similar (AE: 0.59 ± 0.07 µm, NanoHA: 0.69 ± 0.18 µm), but wettability was different (AE: Θw= 81.79 ± 8.55°, NanoHA: Θw= 53.26 ± 11.86°; P = 0.01). NanoHA had lower S. sanguinis viability in monospecies biofilm (P = 0.007). Metabolic activity was similar among all biofilms. In SEM both surfaces on C. albicans biofilm show a similar distribution of hyphae in mono and duospecies biofilms. AE surface has more S. sanguinis than the NanoHA surface in the duospecies biofilm. CLSM showed a large proportion of live cells in all groups. ConclusionsThe nanoHA surface reduced the adhesion of S. sanguinis biofilm but did not alter the adhesion of C. albicans or the biofilm formed by both species.

Identification of microbial communities and multi-species biofilms contamination in seafood processing environments with different hygiene conditions

Biofilms formed by spoilage and pathogenic bacteria increase microbial persistence, causing an adverse influence on the quality of seafood. The mono-species biofilms are widely reported, however, the contamination of multi-species biofilms and their matrix in food environments are still not fully understood. Here, we assessed the contamination of multi-species biofilms in three seafood processing environments with different hygiene levels by detecting bacterial number and three biofilm matrix components (carbohydrates, extracellular DNA (eDNA), and proteins). Samples comprising seven food matrix surfaces and eight food processing equipment surfaces were collected from two seafood processing plants (XY and XC) and one seafood market (CC). The results showed that the bacterial counts ranged from 1.89 to 4.91 CFU/cm2 and 5.68 to 9.15 BCE/cm2 in these surfaces by cultivation and real-time PCR, respectively. Six biofilm hotspots were identified, including four in CC and two in XY. Among the three processing environments, the amplicon sequence variants (ASVs) of Proteobacteria, Bacteroidetes, and Actinobacteria decreased with improved processing hygiene, while Firmicutes showed a decrease in the four most abundant phyla. The most prevalent bacteria belonged to genera Psychrobacter, Acinetobacter, and Pseudomonas, demonstrating the significant differences and alteration in bacterial community composition during different environments. From the biofilm hotspots, 15 isolates with strong biofilm forming ability were identified, including 7 Pseudomonas, 7 Acinetobacter, and 1 Psychrobacter. The Pseudomonas isolates exhibited the highest production of EPS components and three strong motilities, whose characteristics were positively correlated. Thus, this study verified the presence of multi-species biofilms in seafood processing environments, offering preliminary insights into the diversity of microbial communities during processing. It highlights potential contamination sources and emphasizes the importance of understanding biofilms composition to control biofilms formation in seafood processing environments.

Probiotic Effect of Streptococcus dentisani on Oral Pathogens: An In Vitro Study.

Probiotics, including Streptococcus dentisani, have been proposed as an alternative to re-establish the ecology of the oral cavity and inhibit the formation of pathogenic biofilms. The main objective of this work was to assess the probiotic ability of S. dentisani against Streptococcus mutans, Streptococcus mitis, and Candida albicans biofilms. The ability of the strains to form a monospecies biofilm and the probiotic potential of S. dentisani using the competition, exclusion, and displacement strategies were determined. All strains were moderate biofilm producers. The ability of S. dentisani to compete with and exclude S. mutans and S. mitis during biofilm formation was not significant. However, S. dentisani significantly reduced pathologic streptococcal biofilms using the displacement strategy. Also S. dentisani reduced the formation of the C. albicans biofilm mainly through competition and displacement. In vitro, S. dentisani exhibited probiotic potential to reduce the formation of potentially pathogenic biofilms. Further investigation is required to understand the biofilm-inhibiting mechanisms exhibited by this probiotic strain.

A gain-of-function mutation in zinc cluster transcription factor Rob1 drives Candida albicans adaptive growth in the cystic fibrosis lung environment.

Candida albicans chronically colonizes the respiratory tract of patients with Cystic Fibrosis (CF). It competes with CF-associated pathogens (e.g. Pseudomonas aeruginosa) and contributes to disease severity. We hypothesize that C. albicans undergoes specific adaptation mechanisms that explain its persistence in the CF lung environment. To identify the underlying genetic and phenotypic determinants, we serially recovered 146 C. albicans clinical isolates over a period of 30 months from the sputum of 25 antifungal-naive CF patients. Multilocus sequence typing analyses revealed that most patients were individually colonized with genetically close strains, facilitating comparative analyses between serial isolates. We strikingly observed differential ability to filament and form monospecies and dual-species biofilms with P. aeruginosa among 18 serial isolates sharing the same diploid sequence type, recovered within one year from a pediatric patient. Whole genome sequencing revealed that their genomes were highly heterozygous and similar to each other, displaying a highly clonal subpopulation structure. Data mining identified 34 non-synonymous heterozygous SNPs in 19 open reading frames differentiating the hyperfilamentous and strong biofilm-former strains from the remaining isolates. Among these, we detected a glycine-to-glutamate substitution at position 299 (G299E) in the deduced amino acid sequence of the zinc cluster transcription factor ROB1 (ROB1G299E), encoding a major regulator of filamentous growth and biofilm formation. Introduction of the G299E heterozygous mutation in a co-isolated weak biofilm-former CF strain was sufficient to confer hyperfilamentous growth, increased expression of hyphal-specific genes, increased monospecies biofilm formation and increased survival in dual-species biofilms formed with P. aeruginosa, indicating that ROB1G299E is a gain-of-function mutation. Disruption of ROB1 in a hyperfilamentous isolate carrying the ROB1G299E allele abolished hyperfilamentation and biofilm formation. Our study links a single heterozygous mutation to the ability of C. albicans to better survive during the interaction with other CF-associated microbes and illuminates how adaptive traits emerge in microbial pathogens to persistently colonize and/or infect the CF-patient airways.

How xylitol, gluten, and lactose change human gut microbiota Escherichia coli and Lactobacillus rhamnosus GG biofilm

The human gut microbiota is composed of many viruses, bacteria, and fungi. Escherichia coli representatives are facultative anaerobic bacteria in the colon that play a crucial role in the metabolism of lactose, vitamin synthesis, and immune system modulation. E. coli forms a biofilm on the epithelial cell surface of the intestine that can be modified by diet compounds, i.e., gluten, xylitol, lactose, and probiotics. In the present study, the impact of probiotic-derived Lactobacillus rhamnosus GG strain on non-pathogenic E. coli biofilm was examined. The mono- and multispecies biofilm was also treated with gluten, xylitol, and lactose. We used 96-well plates to obtain biofilm growth. Biofilm was stained using crystal violet. To evaluate the type of interaction in mono- and multispecies biofilm, a new formula was introduced: biofilm interaction ratio index (BIRI). To describe the impact of nutrients on biofilm formation, the biofilm formation impact ratio (BFIR) was calculated. The biofilms formed by both examined species are stronger than in monocultures. All the BIRI values were above 3.0. It was found that the monospecies biofilm of L. rhamnosus is strongly inhibited by gluten (84.5%) and the monospecies biofilm of E. coli by xylitol (85.5%). The mixed biofilm is inhibited by lactose (78.8%) and gluten (90.6%). The relations between bacteria in the mixed biofilm led to changes in biofilm formation by E. coli and L. rhamnosus GG. Probiotics might be helpful in rebuilding the gut microbiota after broad spectrum antibiotic therapy, but only if gluten and lactose are excluded from diet.

Potential of Indonesian Plants as Polymicrobial Anti-Biofilm

Biofilm infection occurs in 80% of chronic infections caused by 60% of biofilms from plankton cells and polymicrobial biofilms. Due to synergistic interactions between species, infections caused by polymicrobial biofilms are more virulent than monospecies biofilm infections. New anti-biofilm candidates are constantly being developed by tracing the content of active compounds from medicinal plants native to Indonesia. The need to find new plant sources that have the potential as anti-biofilms is increasingly needed along with increasing microbial resistance. Various studies show that active compounds that have anti-biofilm potential are polyphenols, quercetin, curcumin, gallic acid, and ferulic acid. The mechanism of action of anti-biofilms is through the prevention of attachment and formation of biofilms, inhibition of quorum sensing, and inhibition of gene expression in microbes.

Surface topography and chemistry of food contact substances, and microbial nutrition affect pathogen persistence and symbiosis in cocktail Listeria monocytogenes biofilms

The foodborne pathogen Listeria monocytogenes (L. monocytogenes) can form very persistent biofilms on food contact substances, either in monospecies, or in conjunction with background microflora. Although some studies have investigated how bacterial symbiosis facilitates L. monocytogenes survival in complex cocktail biofilms, very little is known about how this core symbiotic relationship is affected by environmental factors. Here, we investigate how L. monocytogenes persistence is affected by symbiosis with Escherichia coli O157:H7, Pseudomonas fluorescens, and Ralstonia insidiosa, and how these relationships are impacted by environmental factors, including food contact surface topography and chemistry, and microbial nutrition. We find that symbiosis can significantly enhance L. monocytogenes persistence in four-species cocktail biofilms (5.71 ± 0.03 Log CFU/cm2) by up to 1.9 Log CFU/cm2 compared to monospecies biofilms (3.81 ± 0.08 Log CFU/cm2). We also report that surface topography and chemistry, microbial nutrition, and symbiont species significantly impact the symbiotic relationships involving L. monocytogenes, revealing the adaptability of bacterial symbiosis to changing environmental conditions and the complex nature of cocktail pathogen biofilms. Antagonistic and synergistic interactions involving pathogens in cocktail biofilms, and factors affecting those interactions, are elucidated and can establish a foundation to study symbiosis and its role in mitigating pathogen persistence in food systems, and helping identify unanticipated increased food safety risks of pathogens due to enhanced persistence.

L-lysine as a Potential Agent for Controlling Biofilm Formation Using Fusobacterium nucleatum and Porphyromonas gingivalis

Background Periodontitis, a globally-pervasive chronic inflammatory condition, is precipitated by forming microbial biofilms. A key player in this process is Fusobacterium nucleatum (F. nucleatum), an anaerobic bacterium exhibiting invasive and pro-inflammatory characteristics primarily associated with the onset of periodontitis. Consequently, developing novel biofilm control strategies specifically targeting F. nucleatum is essential. The objective of this research is to examine the suppressive impact of L-lysine on biofilm formation using F. nucleatum. Methods Fusobacterium nucleatum(F. nucleatum) and Porphyromonas gingivalis (P. gingivalis) were cultured in a liquid medium containing L-lysine of different concentrations. The antimicrobial activity of L-lysine was evaluated using the broth microdilution method. The effect of L-lysine on the mono-species and dual-species F. nucleatum biofilms was assessed using techniques such as crystal violet staining. The synthesis of extracellular polysaccharide substances (EPSs) from biofilms was measured using phenol-sulfuric acid. The expression level of virulence factors (FN1420, FN0669, FN0634, FN1162) and the biofilm-formation-related gene radD were quantified using real-time PCR. Results L-lysine exhibited the antibacterial and antibiofilm activity of F. nucleatum and F. nucleatum/ P. gingivalis double strains, which was concentration-dependent. The MIC and MBC values were 10 mg/ml and 20 mg/ml in F. nucleatum single strains, while those in F. nucleatum/ P. gingivalis double strains were 20 mg/ml and 40 mg/ml. L-lysine with a concentration ≥20 mg/ml could reduce the EPS production in F. nucleatum and F. nucleatum/ P. gingivalis dual-species biofilms. L-lysine also inhibited the expression of genes encoding the F. nucleatum virulence factor (FN1420, FN0669, FN0634, FN1162) and the adhesive factor radD. Conclusion These findings corroborate the potential of L-lysine as an agent impeding the development of both mono-species biofilms of F. nucleatum and dual-species biofilms of F. nucleatum and P. gingivalis.