Development Of Multi-species Biofilms Research Articles

Unraveling the role of inert biomass in membrane aerated biofilm reactors for simultaneous nitrification and denitrification

This study presents an innovative 2D spatio-temporal model that sheds light on the intricate formation of biofilms, incorporating two essential biomass decay pathways: cell lysis and endogenous respiration. The model encompasses heterotrophic bacteria (HB), anaerobic heterotrophic bacteria (AHB), and autotrophic bacteria (AB), offering a comprehensive understanding of multi-species biofilm development. Through meticulous simulations, we explore the primary mechanisms behind inert biomass formation in biofilms, revealing the key roles played by the lysis of HB, AHB, and AB, as well as the endogenous respiration of HB. Moreover, the simulations reveal how species of higher abundance contribute significantly to inert biomass generation, reshaping our understanding of biofilm dynamics. Crucially, this study highlights the indispensability of considering biofilm inert biomass when modeling the nitrification and denitrification behaviors of a membrane aerated biofilm reactor (MABR). The distribution of oxygen and acetate across biofilm thickness is remarkably different when inert biomass is factored in, underscoring the necessity for a more holistic approach to modeling biofilm behavior. With the introduction of the inert biomass inclusive biofilm model, our simulations explore the interactive effects of key process conditions - bulk concentrations of oxygen ($O_{\infty}$), ammonium nitrogen ($N_{1\infty}$), acetate ($A_{\infty}$), and biofilm thickness - on the nitrification and denitrification performance of MABR. A compelling correlation emerges between higher bulk concentrations of oxygen and ammonium nitrogen and optimal nitrification rates, achieving an impressive range of 0.3 to 1.1 g ammonium/$m^{2}/d. Delving into denitrification, we observe that high $O_{\infty}$, low $N_{1\infty}$, and either high or low $A_{\infty}$ levels impede AHB formation and consequently hinder denitrification. Our findings provide a roadmap for achieving simultaneous nitrification and denitrification, contingent on specific conditions: $10[gm^{-3}] < O_{\infty} < 15[gm^{-3}]$, $12[gm^{-3}] < N_{1\infty} < 20[gm^{-3}]$, $A_{\infty}$ ranging from $3[gm^{-3}]$ to $12[gm^{-3}]$, and a biofilm thickness $> 1.4[mm]$. While our study reveals promising avenues for simultaneous nitrification and denitrification, denitrification rates still lag behind nitrification rates under the same conditions. As a result, we advocate for further investigations to devise strategies that can enhance denitrification in MABR systems. In conclusion, this study advances our knowledge of biofilm dynamics by introducing a comprehensive model and illuminating the key factors driving nitrification and denitrification performance in MABRs. These findings pave the way for improved biofilm engineering and wastewater treatment strategies, opening new horizons for sustainable environmental practices.

Inhibition of Biofilm Formation in Cutibacterium acnes, Staphylococcus aureus, and Candida albicans by the Phytopigment Shikonin.

Skin microbiota, such as acne-related Cutibacterium acnes, Staphylococcus aureus, and fungal Candida albicans, can form polymicrobial biofilms with greater antimicrobial tolerance to traditional antimicrobial agents and host immune systems. In this study, the phytopigment shikonin was investigated against single-species and multispecies biofilms under aerobic and anaerobic conditions. Minimum inhibitory concentrations of shikonin were 10 µg/mL against C. acnes, S. aureus, and C. albicans, and at 1-5 µg/mL, shikonin efficiently inhibited single biofilm formation and multispecies biofilm development by these three microbes. Shikonin increased porphyrin production in C. acnes, inhibited cell aggregation and hyphal formation by C. albicans, decreased lipase production, and increased hydrophilicity in S. aureus. In addition, shikonin at 5 or 10 µg/mL repressed the transcription of various biofilm-related genes and virulence-related genes in C. acnes and downregulated the gene expression levels of the quorum-sensing agrA and RNAIII, α-hemolysin hla, and nuclease nuc1 in S. aureus, supporting biofilm inhibition. In addition, shikonin prevented multispecies biofilm development on porcine skin, and the antimicrobial efficacy of shikonin was recapitulated in a mouse infection model, in which it promoted skin regeneration. The study shows that shikonin inhibits multispecies biofilm development by acne-related skin microbes and might be useful for controlling bacterial infections.

Antibiofilm activity of lawsone against polymicrobial enterohemorrhagic Escherichia coli O157:H7 and Candida albicans by suppression of curli production and hyphal growth

Most bacteria and fungi form biofilms that attach to living or abiotic surfaces. These biofilms diminish the efficacy of antimicrobial agents and contribute to chronic infections. Furthermore, multispecies biofilms composed of bacteria and fungi are often found at chronic infection sites. In this study, lawsone (2‑hydroxy-1,4-naphthoquinone) and its parent 1,4-naphthoquinone were studied for antimicrobial and antibiofilm activities against single-species and multispecies biofilms of enterohemorrhagic Escherichia coli O157:H7 (EHEC) and Candida albicans. Biofilm formation assays, biofilm eradication assays, antimicrobial assays, live cell imaging microscopy, confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), extracellular polymeric substances and indole production, cell surface hydrophilicity assay, cell motility, cell aggregation, hyphal growth, dual species biofilm formation, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), and toxicity assays on plant seed germination and nematode model were utilized to investigate how lawsone affect biofilm development. Sub-inhibitory concentrations of lawsone (35µg/ml) significantly inhibited single-and multispecies biofilm development. Lawsone reduced the production of curli and indole, and the swarming motility of EHEC, efficiently inhibited C. albicans cell aggregation and hyphal formation, and increased the cell surface hydrophilicity of C. albicans. Transcriptomic analysis showed that lawsone suppressed the expression of the curli-related genes csgA and csgB in EHEC, and the expression of several hypha- and biofilm-related genes (ALS3, ECE1, HWP1, and UME6) in C. albicans. In addition, lawsone up to 100µg/ml was nontoxic to the nematode Caenorhabditis elegans and to the seed growth of Brassica rapa and Triticum aestivum. These results show that lawsone inhibits dual biofilm development and suggest that it might be useful for controlling bacterial or fungal infections and multispecies biofilms.

NaCl-induced modulation of species distribution in a mixed P. aeruginosa / S. aureus /B.cepacia biofilm

Pseudomonas aeruginosa, Staphylococcus aureus, and Burkholderia cepacia are notorious pathogens known for their ability to form resilient biofilms, particularly within the lung environment of cystic fibrosis (CF) patients. The heightened concentration of NaCl, prevalent in the airway liquid of CF patients' lungs, has been identified as a factor that promotes the growth of osmotolerant bacteria like S. aureus and dampens host antibacterial defenses, thereby fostering favorable conditions for infections.In this study, we aimed to investigate how increased NaCl concentrations impact the development of multi-species biofilms in vitro, using both laboratory strains and clinical isolates of P. aeruginosa, S. aureus, and B. cepacia co-cultures. Employing a low-nutrient culture medium that fosters biofilm growth of the selected species, we quantified biofilm formation through a combination of adherent CFU counts, qPCR analysis, and confocal microscopy observations.Our findings reaffirmed the challenges faced by S. aureus in establishing growth within 1:1 mixed biofilms with P. aeruginosa when cultivated in a minimal medium. Intriguingly, at an elevated NaCl concentration of 145 mM, a symbiotic relationship emerged between S. aureus and P. aeruginosa, enabling their co-existence. Notably, this hyperosmotic environment also exerted an influence on the interplay of these two bacteria with B. cepacia. We demonstrated that elevated NaCl concentrations play a pivotal role in orchestrating the distribution of these three species within the biofilm matrix.Furthermore, our study unveiled the beneficial impact of NaCl on the biofilm growth of clinically relevant mucoid P. aeruginosa strains, as well as two strains of methicillin-sensitive and methicillin-resistant S. aureus. This underscores the crucial role of the microenvironment during the colonization and infection processes. The results suggest that hyperosmotic conditions could hold the key to unlocking a deeper understanding of the genesis and behavior of CF multi-species biofilms.

Process optimization of a moving bed bioreactor undergoing simultaneous nitrification and denitrification for wastewater in the absence of organic carbon

ABSTRACT Moving bed bioreactors are efficient for the removal of nitrogen effluents by simultaneous nitrification and denitrification (SND), which is particularly challenging for effluents with low C/N ratio due to insufficient carbon available for denitrification. The addition of external organic carbon in such cases increases treatment costs. Endogenous degradation of biomass offers a cost-effective and persistent source of carbon. Internal carbon leads to low denitrification rates and optimizing the biomass content leads to a sustainable solution for treating such effluents. The present study aims to optimize the process of SND using endogenous carbon by investigating the effect of operational parameters like dissolved oxygen, influent ammonium (NH4–N) loading, hydraulic retention time (HRT) and carrier filling ratio (CFR). The synthetic wastewater used in the study does not inherently contain any organic carbon, thus indicating the responsible carbon for denitrification to be of internal origin. Using a novel carrier for multispecies biofilm development, initial NH4–N concentration variation showed an insignificant effect on SND efficiency. At higher loading, nitrite accumulation due to incomplete denitrification was observed. The effect of CFR was investigated in a range of 20–35%, beyond which non-homogeneous mixing was caused in the reactor. Under controlled DO and temperature of about 5 mg/L and 28 ± 2°C, respectively, an optimum filling ratio of 35% was finalised, resulting in an SND efficiency of 97.9% at 22 h HRT for high influent NH4–N concentration of 500 mg/L. Bacteriological species identification revealed the coexistence of nitrogen-fixing, aerobic nitrifying and anaerobic denitrifying bacteria in the biofilm growing on the biocarrier. Mechanism of SND occurring in aerobic moving bed bioreactors: Laboratory-scale aerobic Moving Bed Bioreactor setup for a continuous study evaluating the influence of operational parameters on SND Occurrence of aerobic and anoxic zones in biofilm grown on the surface of novel carriers due to diffusional gradient of dissolved oxygen

Quaternary ammonium salt (QAS)-modified thermoresponsive PNIPAM-PDMAEA copolymer for antibiofilm and antimicrobial applications

The development of antimicrobial and antibiofilm materials for treating bacterial pathogens is attracting considerable research attention. Polymers with specific functionalities play important roles in the preparation of antimicrobial materials. In this study, a novel copolymer with dual functions was synthesized. A one-pot copolymerization technique was used to copolymerize N-isopropylacrylamide (NIPAM) and 2-dimethylaminoethylmethacrylate (DMAEA) monomers and quaternized them with alkyl bromide to produce thermoresponsive poly (N-isopropylacrylamide) (PNIPAM) segments. These PNIPAM segments exhibited high-efficient antimicrobial and antibiofilm properties of quaternary ammonium salt (QAS)-based PDMAEMA segments together in a single copolymer system. The synthesized PNIPAM-DMAEMA (CP- 9:1, 8:2, 7:3, 6:4, and 5:5) copolymer-QAS (QAS- C4, C6, and C8) were characterized using a range of techniques. Among them, PNIPAM-DMAEMA-7:3@QAS-C4 copolymer (CP-7:3@QAS-C4) at 100 μg/mL inhibited biofilm formation by fungal Candida albicansvia impeding hyphal formation. In addition, CP-7:3@QAS-C4 and CP-7:3@QAS-C6, at 100 μg/mL markedly inhibited single biofilm formation by uropathogenic Escherichia coli (UPEC) and multispecies biofilm development by UPEC and C. albicans. Overall, a novel synthetic method was developed for producing QAS-based antimicrobial copolymers with potential antibiofilm efficiency that could be applied in biomedical applications.

Drinking-water isolated Delftia acidovorans selectively coaggregates with partner bacteria and facilitates multispecies biofilm development

Coaggregation plays an important role in the development of multispecies biofilms in different environments, often serving as an active bridge between biofilm members and other organisms that, in their absence, would not integrate the sessile structure. The ability of bacteria to coaggregate has been reported for a limited number of species and strains. In this study, 38 bacterial strains isolated from drinking water (DW) were investigated for their ability to coaggregate, in a total of 115 pairs of combinations. Among these isolates, only Delftia acidovorans (strain 005P) showed coaggregating ability. Coaggregation inhibition studies have shown that the interactions mediating D. acidovorans 005P coaggregation were both polysaccharide-protein and protein-protein, depending on the interacting partner bacteria. Dual-species biofilms of D. acidovorans 005P and other DW bacteria were developed to understand the role of coaggregation on biofilm formation. Biofilm formation by Citrobacter freundii and Pseudomonas putida strains highly benefited from the presence of D. acidovorans 005P, apparently due to the production of extracellular molecules/public goods favouring microbial cooperation. This was the first time that the coaggregation capacity of D. acidovorans was demonstrated, highlighting its role in providing a metabolic opportunity for partner bacteria.

Effects of Acylase Treatment Episodes on Multispecies Biofilm Development

Effects of Acylase Treatment Episodes on Multispecies Biofilm Development

Design and evaluation of a novel nano copper/chitosan–starch bio-composite on antimicrobial property and wound-healing efficacy

The development of multi-species biofilms in wounds is a serious health problem that primarily generates strong resistance mechanisms to antimicrobial therapy. Chitosan based composites are interesting bio composites with applications in wound restorations and coatings for artificial implants. However, the lack of antimicrobial activity restricts its further application. Thus, the purpose of this study is to prepare a novel chitosan based metal nanocomposite. Therefore, we report the green synthesis of nontoxic, stable, and small size copper nanoparticle by using chitosan/starch hydrogel with reducing/capping ability without any toxic reducing agents. Starch was used as a reducing agent for the synthesis of Cu NPs that next stabilized by chitosan polymers. The in situ prepared Cu NPs/CS-Starch bio-composite were characterized by advanced physicochemical techniques like Fourier Transformed Infrared spectroscopy (FT-IR), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Energy Dispersive X-ray spectroscopy (EDX), X-ray Diffraction (XRD) study and inductively coupled plasma-optical emission spectroscopy (ICP-OES). It has been established that Cu NPs/CS-Starch bio-composite have a spherical shape with a mean diameter from 10 to 30 nm. The Cu NPs/CS-Starch bio-composite showed promising antimicrobial activity against various pathogens (Gram-negative, Gram-positive and fungus). In vivo design, Cu NPs/CS-Starch bio-composite ointment significantly (p ≤ 0.01) increase the levels of wound contracture, vessel, hydroxyl proline, hexuronic acid, hexosamine, fibrocyte, and fibroblast, and significantly (p ≤ 0.01) decrease the levels of wound area, total cells, neutrophil, and lymphocyte compared to other groups in rats. Moreover, a certain concentration of Cu NPs/CS-Starch bio-composite can improve the adhesion and proliferation of cells, which has also been proved in animal experiments, including gross appearance and histological observations. Together, these results suggest the efficacy of Cu NPs/CS-Starch bio-composite for broad applications associated with wound healing.

Phytopigment Alizarin Inhibits Multispecies Biofilm Development by Cutibacterium acnes, Staphylococcus aureus, and Candida albicans.

Acne vulgaris is a common chronic inflammatory skin disease involving Cutibacterium acnes with other skin commensals such as Staphylococcus aureus and Candida albicans in the anaerobic and lipid-rich conditions of pilosebaceous units. These microbes readily form multispecies biofilms that are tolerant of traditional antibiotics as well as host immune systems. The phytopigment alizarin was previously found to prevent biofilm formation by S. aureus and C. albicans strains under aerobic conditions. Hence, we hypothesized that alizarin might control C. acnes and multispecies biofilm development. We found that under anaerobic conditions, alizarin efficiently inhibited single biofilm formation and multispecies biofilm development by C. acnes, S. aureus, and C. albicans without inhibiting planktonic cell growth. Alizarin increased the hydrophilicities of S. aureus and C. albicans cells, decreased lipase production by S. aureus, diminished agglutination by C. acnes, and inhibited the aggregation of C. albicans cells. Furthermore, the co-administration of alizarin and antibiotics enhanced the antibiofilm efficacies of alizarin against C. acnes. A transcriptomic study showed that alizarin repressed the transcriptions of various biofilm-related genes such as lipase, hyaluronate lyase, adhesin/invasion-related, and virulence-related genes of C. acnes. Furthermore, alizarin at 100 µg/mL prevented C. acnes biofilm development on porcine skin. Our results show that alizarin inhibits multispecies biofilm development by acne-causing microbes and suggest it might be a useful agent for treating or preventing C. acnes-causing skin diseases.

Combined utilization of metabolic inhibitors to prevent synergistic multi-species biofilm formation

Biofilm is ubiquitous in industrial water systems, causing biofouling and leading to heat transfer efficiency decreases. In particular, multi-species living in biofilms could boost biomass production and enhance treatment resistance. In this study, a total of 37 bacterial strains were isolated from a cooling tower biofilm where acetic acid and propionic acid were detected as the main carbon sources. These isolates mainly belonged to Proteobacteria and Firmicutes, which occupied more than 80% of the total strains according to the 16S rRNA gene amplicon sequencing. Four species (Acinetobacter sp. CTS3, Corynebacterium sp. CTS5, Providencia sp. CTS12, and Pseudomonas sp. CTS17) were observed co-existing in the synthetic medium. Quantitative comparison of biofilm biomass from mono- and multi-species showed a synergistic effect towards biofilm formation among these four species. Three metabolic inhibitors (sulfathiazole, 3-bromopyruvic acid, and 3-nitropropionic acid) were employed to prevent biofilm formation based on their inhibitory effect on corresponding metabolic pathways. All of them displayed evident inhibition profiles to biofilm formation. Notably, combining these three inhibitors possessed a remarkable ability to block the multi-species biofilm development with lower concentrations, suggesting an enhanced effect appeared in simultaneous use. This study demonstrates that combined utilization of metabolic inhibitors is an alternative strategy to prevent multi-species biofilm formation.

Development of multi-species biofilm formed by thermophilic bacteria on stainless steel immerged in skimmed milk

Thermophilic bacteria, such as Bacillus licheniformis, Geobacillus stearothermophilus, Bacillus Subtilis and Anoxybacillus flavithermus, are detected frequently in milk powder products. Biofilms of those strains act as a major contamination to milk powder manufactures and pose potential risks in food safety. In this study, we explored the developing process of multi-species biofilm formed by the four thermophilic bacteria on stainless steel immerged in skimmed milk. The results showed that the thermophilic strains possessed strong capacities to decompose proteins and lactose in skimmed milk, and the spoilage effects were superimposed from multiple strains. B. licheniformis was the most predominant species in the mixed-species biofilm after 12-h incubation. From 24 h to 48 h, G. stearothermophilus occupied the highest proportion. Within the multi-species biofilm, competitive relation existed between B. licheniformis and G. stearothermophilus, while synergistic impacts were observed between B. licheniformis and A. flavithermus. The interspecies mutual influences on biofilm development provided important evidences for understanding colonization of the predominant thermophilic bacteria during milk powder processing.

Metabolic plasticity enables lifestyle transitions of Porphyromonas gingivalis

Our understanding of how the oral anaerobe Porphyromonas gingivalis can persist below the gum line, induce ecological changes, and promote polymicrobial infections remains limited. P. gingivalis has long been described as a highly proteolytic and asaccharolytic pathogen that utilizes protein substrates as the main source for energy production and proliferation. Here, we report that P. gingivalis displays a metabolic plasticity that enables the exploitation of non-proteinaceous substrates, specifically the monocarboxylates pyruvate and lactate, as well as human serum components, for colonization and biofilm formation. We show that anabolism of carbohydrates from pyruvate is powered by catabolism of amino acids. Concomitantly, the expression of fimbrial adhesion is upregulated, leading to the enhancement of biofilm formation, stimulation of multispecies biofilm development, and increase of colonization and invasion of the primary gingival epithelial cells by P. gingivalis. These studies provide the first glimpse into the metabolic plasticity of P. gingivalis and its adaptation to the nutritional condition of the host niche. Our findings support the model that in response to specific nutritional parameters, P. gingivalis has the potential to promote host colonization and development of a pathogenic community.

Plant-based oral care product exhibits antibacterial effects on different stages of oral multispecies biofilm development in vitro

BackgroundExcessive biofilm formation on surfaces in the oral cavity is amongst the main reasons for severe infection development like periodontitis and peri-implantitis. Mechanical biofilm removal as well as the use of adjuvant antiseptics supports the prevention of pathogenic biofilm formation. Recently, the antibacterial effect of the oral care product REPHA-OS®, based on medicinal plant extracts and essential oils, has been demonstrated on oral pathogens grown on agar plates. In the present study, the effectiveness of the product on medical relevant oral biofilm development should be demonstrated for the first time.MethodsAn established in vitro oral multispecies biofilm, composed of Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar and Porphyromonas gingivalis, was used to analyze the antibacterial effect of different REPHA-OS® concentrations on planktonic bacteria, biofilm formation and mature biofilms. It was quantified using metabolic activity assays and live/dead fluorescence staining combined with three-dimensional confocal laser-scanning microscopy. Additionally, effects on species distribution inside the biofilm were assessed by means of quantitative real-time PCR.ResultsREPHA-OS® showed statistically significant antimicrobial effects on all stages of biofilm development: a minimal inhibitory concentration of 5% could be detected for both, for planktonic bacteria and for biofilm formation. Interestingly, only a slightly higher concentration of 10% was necessary to completely kill all bacteria in mature biofilms also. In contrast, an influence on the biofilm matrix or the species distribution could not be observed. The effect could be attributed to the herbal ingredients, not to the contained ethanol.ConclusionThe strong antibacterial effect of REPHA-OS® on different stages of oral biofilm development strengthens its application as an alternative adjuvant in oral care therapies.

The Rhodamine Isothiocyanate Analogue as a Quorum Sensing Inhibitor Has the Potential to Control Microbially-Induced Biofouling.

Quorum sensing inhibitors (QSIs) have been proven to be an innovative approach to interfering with biofilm formation, since this process is regulated by QS signals. However, most studies have focused on single-species biofilm formation, whereas studies of the effects of signal interference on the development of multispecies biofilm, especially in the natural environment, are still lacking. Here we develop and evaluate the anti-biofilm capability of a new QSI (rhodamine isothiocyanate analogue, RIA) in natural seawater. During the experiment, biofilm characteristics, microbial communities/functions and network interactions were monitored at 36, 80, and 180 h, respectively. The results showed that the biomass and 3D structure of the biofilm were significantly different in the presence of the QSI. The expression of genes involved in extracellular polysaccharide synthesis was also downregulated in the QSI-treated group. Dramatic differences in microbial composition, β-diversity and functions between the RIA-treated group and the control group were also observed, especially in the early stage of biofilm development. Furthermore, co-occurrence model analysis showed that RIA reduced the complexity of the microbial network. This study demonstrates that rhodamine isothiocyanate analogue is an efficient QS inhibitor and has potential applications in controlling biofouling caused by multispecies biofilm, especially in the early stage of biofouling formation.

Effects of a sub-minimum inhibitory concentration of chlorhexidine gluconate on the development of in vitro multi-species biofilms

Following antimicrobial administrations in oral environments, bacteria become exposed to a sub-minimum inhibitory concentration (sub-MIC), which can induce in vitro single-species biofilms. This study explored the effects of chlorhexidine gluconate (CHG) at a sub-MIC on in vitro multi-species biofilms comprising Streptococcus mutans, Streptococcus oralis and Actinomyces naeslundii. CHG at a sub-MIC was found to induce in vitro biofilm growth, although the bacterial growth was not significantly different from that in the control. The gene transcription related to S. mutans multi-species biofilm formation with CHG at a sub-MIC was significantly higher than that of the control, but this was not found in S. mutans single-species biofilms. The bio-volume of extracellular polysaccharides with CHG at a sub-MIC was significantly higher than that of the control. This suggests that CHG at a sub-MIC may promote the development of multi-species biofilms by affecting the gene transcription related to S. mutans biofilm formation.

Quantitative and Compositional Study of Monospecies Biofilms of Spoilage Microorganisms in the Meat Industry and Their Interaction in the Development of Multispecies Biofilms.

Food spoilage is a serious problem in the food industry, since it leads to significant economic losses. One of its main causes is the cross-contamination of food products from industrial surfaces. Three spoilage bacterial species which are highly present in meat and the gastrointestinal tract of chickens were selected: Pseudomonas fragi, Leuconostoc gasicomitatum, and Lactobacillus reuteri. The dual aim was to determine their ability to form monospecies biofilms and to examine how they interact when they coexist together. To do so, mature monospecies biofilms were produced statically for seven days at a temperature of 30 °C. L. gasicomitatum was also used to investigate the behavior of P. fragi and L. reuteri in the formation of multispecies biofilms. The structure and composition of the monospecies biofilms were evaluated by direct epifluorescence microscopy, and the multispecies biofilms were evaluated by plate counting. Both L. gasicomitatum and L. reuteri were able to form biofilms, with counts of approximately 7 Log CFU/cm2 and a defined structure. However, P. fragi obtained counts to the order of 4 Log CFU/cm2, which is significantly different from the previous species (P < 0.05), and it had no network of cell conglomerates. The content of the L. gasicomitatum and L. reuteri biofilm matrices were 70–80% protein, unlike P. fragi, which presented a higher polysaccharide content (P < 0.05). In the multispecies biofilms, the presence of P. fragi did not affect the growth of L. gasicomitatum, which remained at between 5.76 to 6.1 Log CFU/cm2. However, L. reuteri was able to displace L. gasicomitatum growth after 24 h of coexisting in a mixed biofilm, presenting differences in counts of approximately 2 Log CFU/cm2. The study of the biofilms constructed by food industry resident microbiota can help to understand the ecological relations that exist between species, characterize them, and propose strategies to eliminate them. The name of genes and species should be written in italic.

Multispecies Biofilm Development of Marine Bacteria Implies Complex Relationships Through Competition and Synergy and Modification of Matrix Components.

Microbial communities composition is largely shaped by interspecies competition or cooperation in most environments. Ecosystems are made of various dynamic microhabitats where microbial communities interact with each other establishing metabolically interdependent relationships. Very limited information is available on multispecies biofilms and their microhabitats related to natural environments. The objective of this study is to understand how marine bacteria isolated from biofilms in the Mediterranean Sea interact and compete with each other when cultivated in multispecies biofilms. Four strains (Persicivirga mediterranea TC4, Polaribacter sp. TC5, Shewanella sp. TC10 and TC11) with different phenotypical traits and abilities to form a biofilm have been selected from a previous study. Here, the results show that these strains displayed a different capacity to form a biofilm in static versus dynamic conditions where one strain, TC11, was highly susceptible to the flux. These bacteria appeared to be specialized in the secretion of one or two exopolymers. Only TC5 seemed to secrete inhibitory molecule(s) in its supernatant, with a significant effect on TC10. Most of the strains negatively impacted each other, except TC4 and TC10, which presented a synergetic effect in the two and three species biofilms. Interestingly, these two strains produced a newly secreted compound when grown in dual-species versus mono-species biofilms. TC5, which induced a strong inhibition on two of its partners in dual-species biofilms, outfitted the other bacteria in a four-species biofilm. Therefore, understanding how bacteria respond to interspecific interactions should help comprehending the dynamics of bacterial populations in their ecological niches.

Cell wall associated protein TasA provides an initial binding component to extracellular polysaccharides in dual-species biofilm

Many bacteria in biofilm surround themselves by an extracellular matrix composed mainly of extracellular polysaccharide (EP), proteins such as amyloid-like fibers (ALF) and nucleic acids. While the importance of EP in attachment and acceleration of biofilm by a number of different bacterial species is well established, the contribution of ALF to attachment in multispecies biofilm remains unknown. The study presented here aimed to investigate the role of TasA, a precursor for ALF, in cell-cell interactions in dual-species biofilms of Bacillus subtilis and Streptococcus mutans. Expression of major B. subtilis matrix operons was significantly up-regulated in the presence of S. mutans during different stages of biofilm formation, suggesting that the two species interacted and modulated gene expression in each other. Wild-type B. subtilis expressing TasA adhered strongly to S. mutans biofilm, while a TasA-deficient mutant was less adhesive and consequently less abundant in the dual-species biofilm. Dextran, a biofilm polysaccharide, induced aggregation of B. subtilis and stimulated adhesion to S. mutans biofilms. This effect was only observed in the wild-type strain, suggesting that interactions between TasA and dextran-associated EP plays an important role in inter-species interactions during initial stages of multispecies biofilm development.

Low-abundant species facilitates specific spatial organization that promotes multispecies biofilm formation.

Microorganisms frequently co-exist in matrix-embedded multispecies biofilms. Within biofilms, interspecies interactions influence the spatial organization of member species, which likely play an important role in shaping the development, structure and function of these communities. Here, a reproducible four-species biofilm, composed of Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus, was established to study the importance of individual species spatial organization during multispecies biofilm development. We found that the growth of species that are poor biofilm formers, M. oxydans and P. amylolyticus, were highly enhanced when residing in the four-species biofilm. Interestingly, the presence of the low-abundant M. oxydans (0.5% of biomass volume) was observed to trigger changes in the composition of the four-species community. The other three species were crucially needed for the successful inclusion of M. oxydans in the four-species biofilm, where X. retroflexus was consistently positioned in the top layer of the mature four-species biofilm. These findings suggest that low abundance key species can significantly impact the spatial organization and hereby stabilize the function and composition of complex microbiomes.