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Microbial electrochemical systems (MESs), as a green and sustainable technology, can decompose organics in wastewater to recover bioelectricity. Electroactive biofilms, a microbial community structure encased in a self-produced matrix, play a decisive role in determining the efficiency of MESs. However, as an essential component of the biofilm matrix, the role of exopolysaccharides in electroactive biofilm formation and their influence on extracellular electron transfer (EET) have been rarely studied. Herein, to explore the effects of exopolysaccharides on biofilm formation and EET rate, we first inhibited the key genes responsible for exopolysaccharide biosynthesis (namely, so_3171 , so_3172 , so_3177 , and so_3178 ) by using antisense RNA in Shewanella oneidensis MR-1. Then, to explore the underlying mechanisms why inhibition of exopolysaccharide synthesis could enhance biofilm formation and promote the EET rate, we characterized cell physiology and electrophysiology. The results showed inhibition of exopolysaccharide biosynthesis not only altered cell surface hydrophobicity and promoted intercellular adhesion and aggregation, but also increased biosynthesis of c -type cytochromes and decreased interfacial resistance, thus promoting electroactive biofilm formation and improving the EET rate of S. oneidensis . Lastly, to evaluate and intensify the capability of exopolysaccharide-reduced strains in harvesting electrical energy from actual liquor wastewater, engineered strain Δ3171-as3177 was further constructed to treat an actual thin stillage. The results showed that the output power density reached 380.98 mW m -2 , 11.1-fold higher than that of WT strain, which exhibited excellent capability of harvesting electricity from actual liquor wastewater. This study sheds light on the underlying mechanism of how inhibition of exopolysaccharides impacts electroactive biofilm formation and EET rate, which suggested that regulating exopolysaccharide biosynthesis is a promising avenue for increasing the EET rate.

Pseudomonas aeruginosa is well-known for its antimicrobial resistance and the ability to survive in harsh environmental conditions due to an abundance of resistance mechanisms, including the formation of biofilms and the production of exopolysaccharides. Exopolysaccharides are among the major components of the extracellular matrix in biofilms and aggregates of P. aeruginosa . Although their contribution to antibiotic resistance has been previously shown, their roles in resistance to oxidative stressors remain largely elusive. Here, we studied the function of the exopolysaccharides Psl and Pel in the resistance of P. aeruginosa to the commonly used disinfectants and strong oxidizing agents NaOCl and H 2 O 2 . We observed that the simultaneous inactivation of Psl and Pel in P. aeruginosa PAO1 mutant strain ∆ pslA pelF resulted in a significant increase in susceptibility to both NaOCl and H 2 O 2 . Further analyses revealed that Pel is more important for oxidative stress resistance in P. aeruginosa and that the form of Pel (i.e., cell-associated or cell-free) did not affect NaOCl susceptibility. Additionally, we show that Psl/Pel-negative strains are protected against oxidative stress in co-culture biofilms with P. aeruginosa PAO1 WT. Taken together, our results demonstrate that the EPS matrix and, more specifically, Pel exhibit protective functions against oxidative stressors such as NaOCl and H 2 O 2 in P. aeruginosa ., Importance: Biofilms are microbial communities of cells embedded in a self-produced polymeric matrix composed of polysaccharides, proteins, lipids, and extracellular DNA. Biofilm bacteria have been shown to possess unique characteristics, including increased stress resistance and higher antimicrobial tolerance, leading to failures in bacterial eradication during chronic infections or in technical settings, including drinking and wastewater industries. Previous studies have shown that in addition to conferring structure and stability to biofilms, the polysaccharides Psl and Pel are also involved in antibiotic resistance. This work provides evidence that these biofilm matrix components also contribute to the resistance of Pseudomonas aeruginosa to oxidative stressors including the widely used disinfectant NaOCl. Understanding the mechanisms by which bacteria escape antimicrobial agents, including strong oxidants, is urgently needed in the fight against antimicrobial resistance and will help in developing new strategies to eliminate resistant strains in any environmental, industrial, and clinical setting., Competing Interests: The authors declare no conflict of interest.

Co-aggregation of anaerobic microorganisms into suspended microbial biofilms (aggregates) serves ecological and biotechnological functions. Tightly packed aggregates of metabolically interdependent bacteria and archaea play key roles in cycling of carbon and nitrogen. Additionally, in biotechnological applications, such as wastewater treatment, microbial aggregates provide a complete metabolic network to convert complex organic material. Currently, experimental data explaining the mechanisms behind microbial co-aggregation in anoxic environments is scarce and scattered across the literature. To what extent does this process resemble co-aggregation in aerobic environments? Does the limited availability of terminal electron acceptors drive mutualistic microbial relationships, contrary to the commensal relationships observed in oxygen-rich environments? And do co-aggregating bacteria and archaea, which depend on each other to harvest the bare minimum Gibbs energy from energy-poor substrates, use similar cellular mechanisms as those used by pathogenic bacteria that form biofilms? Here, we provide an overview of the current understanding of why and how mixed anaerobic microbial communities co-aggregate and discuss potential future scientific advancements that could improve the study of anaerobic suspended aggregates. KEY POINTS: • Metabolic dependency promotes aggregation of anaerobic bacteria and archaea • Flagella, pili, and adhesins play a role in the formation of anaerobic aggregates • Cyclic di-GMP/AMP signaling may trigger the polysaccharides production in anaerobes., (© 2024. The Author(s).)

Biofilm formation is an effective survival strategy of plant-associated microorganisms in hostile environments, so the application of biofilm-forming and exopolysaccharide (EPS)-producing beneficial microbes to plants has received more attention in recent years. This study examined the ability of biofilm and EPS production of Bacillus subtilis and Bacillus thuringiensis strains under different NaCl concentrations (0, 50, 100, 200, and 400 mmol/L), pH values (5.5, 6.5, 7.5, and 8.5), and phosphate levels (0, 25, 50, and 100 mmol/L at 0 and 400 mmol/L NaCl). B. subtilis BS2 and B. thuringiensis BS6/BS7 strains significantly increased biofilm formation in a similar pattern to EPS production under salt stress. B. subtilis BS2/BS3 enhanced biofilm production at slightly acidic pH with a lower EPS production but the other strains formed considerably more amount of biofilm and EPS at alkaline pH. Interestingly, higher levels of phosphate substantially decreased biofilm and EPS production at 0 mmol/L NaCl but increased biofilm formation at 400 mmol/L salt concentration. Overall, contrary to phosphate, salt and pH differently influenced biofilm and EPS production by Bacillus strains. EPS production contributed to biofilm formation to some extent under all the conditions tested. Some Bacillus strains produced more abundant biofilm under salt and pH stress, indicating their potential to form in vivo biofilms in rhizosphere and on plants, particularly under unfavorable conditions., (© 2023. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

Biofilms are an intricate community of microbes that colonize solid surfaces, communicating via a quorum-sensing mechanism. These microbial aggregates secrete exopolysaccharides facilitating adhesion and conferring resistance to drugs and antimicrobial agents. The escalating global concern over biofilm-related infections on medical devices underscores the severe threat to human health. Carbon dots (CDs) have emerged as a promising substrate to combat microbes and disrupt biofilm matrices. Their numerous advantages such as facile surface functionalization and specific antimicrobial properties, position them as innovative anti-biofilm agents. Due to their minuscule size, CDs can penetrate microbial cells, inhibiting growth via cytoplasmic leakage, reactive oxygen species (ROS) generation, and genetic material fragmentation. Research has demonstrated the efficacy of CDs in inhibiting biofilms formed by key pathogenic bacteria such as Escherichia coli , Staphylococcus aureus , and Pseudomonas aeruginosa. Consequently, the development of CD-based coatings and hydrogels holds promise for eradicating biofilm formation, thereby enhancing treatment efficacy, reducing clinical expenses, and minimizing the need for implant revision surgeries. This review provides insights into the mechanisms of biofilm formation on implants, surveys major biofilm-forming pathogens and associated infections, and specifically highlights the anti-biofilm properties of CDs emphasizing their potential as coatings on medical implants.

Biofilm formation protects bacteria from antibiotic treatment and host immune responses, making biofilm infections difficult to treat. Within biofilms, bacterial cells are entangled in a self-produced extracellular matrix that typically includes exopolysaccharides. Molecular-level descriptions of biofilm matrix components, especially exopolysaccharides, have been challenging to attain due to their complex nature and lack of solubility and crystallinity. Solid-state nuclear magnetic resonance (NMR) has emerged as a key tool to determine the structure of biofilm matrix exopolysaccharides without degradative sample preparation. In this review, we discuss challenges of studying biofilm matrix exopolysaccharides and opportunities to develop solid-state NMR approaches to study these generally intractable materials. We specifically highlight investigations of the exopolysaccharide called Pel made by the opportunistic pathogen, Pseudomonas aeruginosa. We provide a roadmap for determining exopolysaccharide structure and discuss future opportunities to study such systems using solid-state NMR. The strategies discussed for elucidating biofilm exopolysaccharide structure should be broadly applicable to studying the structures of other glycans., (© 2023 John Wiley & Sons Ltd.)

The ability of most opportunistic bacteria to form biofilms, coupled with antimicrobial resistance, hinder the efforts to control widespread infections, resulting in high risks of negative outcomes and economic costs. Endolysins are promising compounds that efficiently combat bacteria, including multidrug-resistant strains and biofilms, without a low probability of subsequent emergence of stable endolysin-resistant phenotypes. However, the details of antibiofilm effects of these enzymes are poorly understood. To elucidate the interactions of bacteriophage endolysins LysAm24, LysAp22, LysECD7, and LysSi3 with bacterial films formed by Gram-negative species, we estimated their composition and assessed the endolysins' effects on the most abundant exopolymers in vitro. The obtained data suggests a pronounced efficiency of these lysins against biofilms with high (Klebsiella pneumoniae) and low (Acinetobacter baumannii) matrix contents, or dual-species biofilms, resulting in at least a twofold loss of the biomass. These peptidoglycan hydrolases interacted diversely with protective compounds of biofilms such as extracellular DNA and polyanionic carbohydrates, indicating a spectrum of biofilm-disrupting effects for bacteriolytic phage enzymes. Specifically, we detected disruption of acid exopolysaccharides by LysAp22, strong DNA-binding capacity of LysAm24, both of these interactions for LysECD7, and neither of them for LysSi3., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Exopolysaccharides (EPS) are essential constituents of the extracellular matrix within oral biofilms and are significantly influenced by the local microenvironment. This study aimed to investigate the impact of two distinct antimicrobial agents, DJK-5 and chlorhexidine (CHX), on the EPS volume and pH levels in oral biofilms., Methods: Oral biofilms obtained from two donors were cultured on hydroxyapatite discs for durations of 3 days, 1 week, 2 weeks, 3 weeks, and 4 weeks. Subsequently, these biofilms were subjected to treatment with 10 µg/mL DJK-5 or 2% CHX for 3 min. The impact of these antimicrobial treatments on factors such as the proportion of dead bacterial, in situ pH, and EPS volume within the biofilms was assessed using corresponding fluorescent probes. The examination was carried out utilizing confocal laser scanning microscopy, and the resulting images were analyzed with a focus on the upper and lower layers of the biofilm, respectively., Results: DJK-5 exhibited a more potent bactericidal effect compared to CHX across the 3-day to 4-week duration of the biofilm (P < 0.05). The biofilms were acidic, with the upper layer being less acidic than the lower layer (P < 0.05). Both antimicrobial agents increased the pH, but DJK-5 had a greater effect than CHX (P < 0.05). The volume of EPS was significantly lower in DJK-5 treated biofilms compared to that of CHX, regardless of age or layer (P < 0.05)., Conclusion: DJK-5 exhibited superior effectiveness in reducing viable bacteria and EPS volume, as well as in raising extracellular pH, as compared to chlorhexidine., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Bacteria biofilm consists of microorganisms, accounting for 5-35% of the biofilm volume, and of the extracellular matrix (65-95%), made of water (97%), proteins (2%), polysaccharides (1-2%) and nucleic acids (DNA/RNA, both <1%). The physiology of bacteria in the biofilms entails adaptive changes with expression of genes which are different from those translated in the planktonic state. While most of our applied knowledge on bacterial biology stems from the study in the planktonic state, an increasing interest is currently paid to bacterial behaviour as biofilm generators, as it is estimated that 65% of all bacterial infections are associated with bacterial biofilms. Infections of both upper and lower airways, bacterial endocarditis, chronic otitis media, urinary tract infections, periodontitis, ocular infections and chronic wound infections (including diabetic foot ulcer) are all associated with biofilm formation. The role of biofilm is also relevant in case of infections taking place on abiotic surfaces, as in the case of infections occurring on prostheses and several other medical devices. Here, we review current knowledge on biofilm formation and its impact on human infections, discussing recent means for its inhibition, with particular emphasis on an interesting anti-biofilm activity exerted by exopolysaccharides derived from marine strains of Bacillus licheniformis.

Background: Corynebacterium striatum is a microorganism with an excellent capacity for biofilm production and thus has been correlated with nosocomial transmission and invasive infections. However, little is known about the mechanism of biofilm formation of this commensal pathogen. In this study, we aimed to investigate the biofilm formation abilities of multidrug-resistant Corynebacterium striatum clinical isolates and the roles of extracellular proteins, exopolysaccharides and extracellular DNA in mediating more robust biofilm formation by the isolates of C. striatum., Methods: C. striatum isolates were identified using VITEK-2 ANC card, matrix-assisted laser desorption/ionization-time of flight mass spectrometry and 16S rRNA sequencing. The antibiotic susceptibility test was performed using the broth microdilution method. The distribution of spaDEF genes among C. striatum isolates was detected by polymerase chain reaction, and pulsed-field gel electrophoresis typing was employed to analyze the genotypes of the isolates. Crystal violet staining and scanning electron microscopy techniques were used to detect biofilm production by C. striatum isolates. Biofilm degradation assay was performed to observe the effects of extracellular matrix degradative agents (proteinase K, dispersin B, and DNase I) on C. striatum biofilms., Results: Twenty-seven C. striatum isolates were enrolled in the study, and the resistance rates were the highest (100%, 27/27) against penicillin and ceftriaxone. Approximately 96.3% (26/27) C. striatum isolates were resistant to at least three different types of antimicrobial agents tested. All isolates were confirmed to be biofilm producers, and 74.07% (20/27) isolates presented moderate to strong biofilm production abilities. P7 genotype (44.4%, 12/27) was identified to as the predominant genotype, and all of isolates belonging to this genotype were multidrug-resistant and had stronger biofilm-forming abilities. Most C. striatum isolates (74.07%, 20/27) carry spaD, spaE, and spaF genes, which encode spa-type pili. However, the correlation between the expression of spa-type genes and the biofilm production abilities of the C. striatum isolates was not found. The biofilms of 80% (8/10), 90% (9/10), and 100% (10/10) C. striatum isolates with moderate to strong biofilm production abilities were significantly eliminated upon the treatment of dispersin B (20 μg/mL), DNase I (20 μg/mL), and proteinase K (20 μg/mL) (p < 0.05), respectively. For the combination groups with two kinds of biofilm-degradative agents, the combination of 20 μg/mL proteinase K/dispersin B showed the strongest biofilm-eliminating effects, when the biofilms of 90% (9/10) C. striatum isolates degraded more than 50%., Conclusions: The C. striatum isolates that belonged to the predominant genotype showed a multidrug resistance (MDR) phenotype and strong biofilm formation abilities. Extracellular matrix seems to be an essential determinant in mediating biofilm formation of MDR C. striatum, since extracellular matrix degradative agents (proteinase K, dispersin B and DNase I) showed strong biofilm-eliminating effects toward multidrug-resistant C. striatum isolates. The findings of this study highlight new ideas/directions to explore the whole nature of biofilm formation of C. striatum and the function of extracellular matrix in this process., (© 2022. The Author(s).)

Different microbial aggregates show substantial differences in morphology, and extracellular polymer substances have been confirmed to play a key role in the formation of aggregates. In this study, three different microbial aggregates and their exopolysaccharides were compared. The results show that the granular sludge was largest in size and the most compact in shape. Biofilms with a certain thickness had the next greatest density, and flocculent sludge, with the smallest particle size, was the loosest. The extended Derjaguin-Landau-Verwey-Overbeek analysis shows that hydrogen bonding, hydrophobic and electrostatic interactions affect the aggregation of microorganisms. A comparison of exopolysaccharides shows that granular sludge exopolysaccharides show the highest hydrophobicity (38.08%) and lowest surface charge (-20.5 mV), followed by biofilm exopolysaccharides (27.9% and -24.8 mV respectively). The results of Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy show that the contents of hydrophilic and hydrophobic functional groups and charged functional groups of exopolysaccharides affect the above properties of exopolysaccharides, thereby affecting microbial aggregation. In addition, the hydrogen bond content of exopolysaccharides in granular sludge (19.3%), biofilm (19.2%) and activated sludge (18.9%) decreased sequentially. This also affects the cross-linking of microbial exopolysaccharides to form hydrogels. Finally, the results of confocal laser scanning microscopy showed that, different from the other two aggregates, the extracellular α-polysaccharides of granular sludge are mainly distributed in the nucleus, which is more conducive to aggregation. The research results of this thesis provide a new understanding of the differences in the aggregation morphology of different aggregates from the perspective of exopolysaccharides.

Many bacteria form mats at the air-liquid interface of static microcosms. These structures typically involve the secretion of exopolysaccharides, the production of which is often controlled by the secondary messenger c-di-GMP. Mechanisms of mat formation have been particularly well characterized in Pseudomonas fluorescens SBW25; stimuli or mutations that increase c-di-GMP production by diguanylate cyclases (WspR, AwsR, and MwsR) result in the secretion of cellulose and mat formation. Here, we characterize and compare mat formation in two close relatives of SBW25: Pseudomonas simiae PICF7 and P. fluorescens A506. We find that PICF7-the strain more closely related to SBW25-can form mats through mutations affecting the activity of the same three diguanylate cyclases as SBW25. However, instead of cellulose, these mutations activate production of the exopolysaccharide Pel. We also provide evidence for at least two further-as yet uncharacterized-routes to mat formation by PICF7. P. fluorescens A506, while retaining the same mutational routes to mat formation as SBW25 and PICF7, preferentially forms mats by a semi-heritable mechanism that culminates in Psl and Pga over-production. Our results demonstrate a high level of evolutionary flexibility in the molecular and structural routes to mat formation, even among close relatives., (© 2021 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

&lt;b&gt;Background and Objective:&lt;/b&gt; The coastal environment is often polluted by various toxic compounds such as heavy metals. Exposure to these toxic compounds causes coastal bacteria to adapt so that they can be used as bioremediation agents for heavy metals. This study aims for finding and screening the ability of bacteria to produce exopolysaccharide biofilms and then determine the characteristics of bacterial isolates as agents candidates for heavy metal bioremediation in the coastal environment. &lt;b&gt;Materials and Methods:&lt;/b&gt; Samples were collected on submerged seawater substrate from Bungus Coastal, Padang and West Sumatra, on the wet area that was exposed by seawater (on the rocks, on the wood and the ship, the lower out part on the ship that exposed to seawater). Bacterial isolation process using Marine Agar Medium. The isolate discovered then observed and purified. Furthermore, Congo Red Agar was used for bacteria screening for detecting EPS produced by biofilm bacteria. &lt;b&gt;Results:&lt;/b&gt; The results of the isolation, found 9 bacterial isolates attached to the substrate submerged seawater. The screening results showed that isolates K4, K5 and K7 were positive as biofilm-forming bacteria as indicated by the colour change of the bacterial colonies to black on Congo Red Media after 24 hrs incubation. The characteristics of the three bacterial isolates were gram-negative, with cocci and bacilli cells form. &lt;b&gt;Conclusion:&lt;/b&gt; Three isolates of positive exopolysaccharide biofilm bacteria that 1 isolate gram-negative coccus (K4) and the other 2 isolates (K5 and K7) were bacillus. Then, the 3 isolates can be used for remediation of metal contamination research in aquatic.

Dental caries is one of the most prevalent and costly biofilm-associated infectious diseases affecting most of the world's population. In particular, dental caries is driven by dysbiosis of the dental biofilm adherent to the enamel surface. Specific types of acid-producing bacteria, especially Streptococcus mutans , colonize the dental surface and cause damage to the hard tooth structure in the presence of fermentable carbohydrates. Streptococcus mutans has been established as the major cariogenic pathogen responsible for human dental caries, with a high ability to form biofilms. The exopolysaccharide (EPS) matrix, mainly contributed by S. mutans , has been considered as a virulence determinant of cariogenic biofilm. As EPS is an important virulence factor, targeting EPS metabolism could be useful in preventing cariogenic biofilm formation. This review summarizes plausible strategies targeting S. mutans biofilms by degrading EPS structure, inhibiting EPS production, and disturbing the EPS metabolism-related gene expression and regulatory systems.

The treatment of lung infection in the context of cystic fibrosis (CF) is limited by a biofilm mode of growth of pathogenic organisms. When compared to planktonically grown bacteria, bacterial biofilms can survive extremely high levels of antimicrobials. Within the lung, bacterial biofilms are aggregates of microorganisms suspended in a matrix of self-secreted proteins within the sputum. These structures offer both physical protection from antibiotics as well as a heterogeneous population of metabolically and phenotypically distinct bacteria. The bacteria themselves and the components of the extracellular matrix, in addition to the signaling pathways that direct their behaviour, are all potential targets for therapeutic intervention discussed in this review. This review touches on the successes and failures of current anti-biofilm strategies, before looking at emerging therapies and the mechanisms by which it is hoped they will overcome current limitations.

In developing countries, Shigella flexneri is the most common enteric pathogen causing bacillary dysentery. Biofilm formation by S. flexneri can cause the emergence of antibiotic-resistant strains, which poses serious threats to food safety and human health. In this study, the effects of Lactobacillus plantarum 12 exopolysaccharides (L-EPSs) and S. flexneri exopolysaccharides (S-EPSs) on S. flexneri CMCC51574 biofilm formation were investigated. The results showed that L-EPS could decrease polysaccharide production in the extracellular polymeric matrix of S. flexneri and inhibit biofilm formation by S. flexneri L-EPS could decrease the minimum biofilm elimination concentration (MBEC) of antibiotics against S. flexneri biofilm and inhibit S. flexneri adhesion to and invasion into HT-29 cell monolayers, which might be ascribed to S. flexneri biofilm disturbance by L-EPS. In contrast, S-EPS exhibited the opposite effects compared to L-EPS. The monosaccharide composition analysis showed that L-EPS was composed of mannose, glucuronic acid, galactosamine, glucose, galactose, and xylose, with the molar ratio of 32.26:0.99:1.79:5.63:0.05:4.07, while S-EPS was composed of mannose, glucuronic acid, galactosamine, glucose, and galactose, with the molar ratio of 25.43:2.28:7.13:5.35. L-EPS was separated into the neutral polysaccharide L-EPS 1-1 and the acidic polysaccharide L-EPS 2-1 by ion-exchange chromatography and gel chromatography. L-EPS 2-1 exerted higher antibiofilm activity than L-EPS 1-1. The antibiofilm activity of L-EPS might be associated with its structure. IMPORTANCE S. flexneri is a widespread foodborne pathogen causing food contamination and responsible for food poisoning outbreaks related to various foods in developing countries. Not only has biofilm formation by S. flexneri been difficult to eliminate, but it has also increased the drug resistance of the strain. In the present study, it was demonstrated that L-EPSs secreted by Lactobacillus plantrum 12 could inhibit S. flexneri biofilm formation on, adhesion to, and invasion into HT-29 cells. Also, L-EPSs could decrease the minimum biofilm elimination concentration (MBEC) of the antibiotics used against S. flexneri biofilm. Therefore, L-EPSs were shown to be bioactive macromolecules with the potential ability to act against S. flexneri infections., (Copyright © 2020 American Society for Microbiology.)

Introduction: With all the challenges super bugs are imposing, biofilm formation opens the door against various more complicated challenges. Such issue may be highlighted with the ability of the latter to render the antibiotics hardly accessible to bacterial cells and sheds the light on the importance of finding antibiofilm formers. Therefore, we assessed the inhibitory effect of natural product extracts (ginger, wild blueberry) and polysorbates (PS20, PS80) on biofilm formation at the molecular level., Methodology: Growth inhibition assay was performed to test the effect of ginger (Zingiber Officinale), wild blueberry (Vaccinium Angustifolium), and polysorbates on Pseudomonas aeruginosa (PAN14) growth. Transcription levels of biofilm exopolysaccharides encoding genes (ndvB, pelC, algC) and quorum sensing genes (lasI, lasR, rhlI, rhlR) for LasI/LasR and RhlI/ RhlR systems were evaluated by RT qPCR., Results: The polysorbates and the extracts of both ginger and wild blueberry had no effect on the growth of P. aeruginosa. Biofilms' examination has unraveled the effectiveness of treatments used in reducing its formation. Moreover, a significant reduction in the expression of all genes tested for biofilm exopolysaccharides and its quorum sensing system was observed., Conclusion: The decrease in the relative gene expression of the exopolysaccharides and quorum sensing encoding genes sheds the light on the mechanism of action of ginger and wild blueberry's constituents as well as polysorbates 20 and 80 on P. aeruginosa biofilm formation. Future studies need to assess the antibiofilm effect of each fraction of herbal extracts separately., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2020 Mariam Miari, Sari S Rasheed, Nathaline Haidar Ahmad, Dana Itani, Antoine Abou Fayad, Ghassan M Matar.)

Acinetobacter baumannii is an opportunistic nosocomial pathogen, and responsible for high mortality and morbidity. Biofilm formation is one of the resistance determinants, where extracellular polysaccharide (EPS) is an essential component. EPS synthesis and its export is regulated by the bacterial Wza-Wzb-Wzc system. Wzc exhibits auto-phosphorylation protein tyrosine kinase activity, while Wzb is a protein tyrosine phosphatase. Wzb mediates dephosphorylation of Wzc. Dephosphorylated Wzc is required for the export of the EPS through porin Wza-Wzc complex. It shows that the interaction of Wzb with Wzc is critical for the export of EPS. Therefore, if the Wzb-Wzc interaction is inhibited, then it might hinder the EPS transport and diminish the biofilm formation. In this study, we have modelled the Wzb, and Wzc proteins and further validated using PSVS, ProSA, RAMPAGE, and PDBsum. The modelled proteins were used for protein-protein docking. The docked protein-protein complex was minimized by Schrodinger software using OPLS&#95;2005 force field. The binding site of the minimized Wzb-Wzc complex was identified by Sitemap. The high throughput virtual screening identified Labetalol hydrochloride and 4-{1-hydroxy-2-[(1-methyl-3-phenylpropyl) amino] propyl} phenol from FDA-approved drug library based on their interaction at the interface of Wzb-Wzc complex. The inhibitor-protein complex was further undergone molecular mechanics analysis using Generalized Born model and Solvent Accessibility (MMGBSA) to estimate the binding free energies. The lead was also used to generate the pharmacophore model and screening the molecule with antimicrobial scaffold. The identified lead was experimentally validated for its effect on EPS quantity and biofilm formation by A. baumannii. Wzb-Wzc interaction is essential for biofilm and EPS export; hence, the identified lead might be useful to regulate the biofilm formation by A. baumannii., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Dental caries is a diet-biofilm-dependent disease. Streptococcus mutans contributes to cariogenic biofilms by producing an extracellular matrix rich in exopolysaccharides and acids. The study aimed to determine the effect of topical treatments with compound 1771 (modulates lipoteichoic acid (LTA) metabolism) and myricetin (affects the synthesis of exopolysaccharides) on S. mutans biofilms. In vitro S. mutans UA159 biofilms were grown on saliva-coated hydroxyapatite discs, alternating 0.1% sucrose and 0.5% sucrose plus 1% starch. Twice-daily topical treatments were performed with both agents alone and combined with and without fluoride: compound 1771 (2.6 µg/mL), myricetin (500 µg/mL), 1771 + myricetin, fluoride (250 ppm), 1771 + fluoride, myricetin + fluoride, 1771 + myricetin + fluoride, and vehicle. Biofilms were evaluated via microbiological, biochemical, imaging, and gene expression methods. Compound 1771 alone yielded less viable counts, biomass, exopolysaccharides, and extracellular LTA. Moreover, the combination 1771 + myricetin + fluoride decreased three logs of bacterium counts, 60% biomass, >74% exopolysaccharides, and 20% LTA. The effect of treatments on extracellular DNA was not pronounced. The combination strategy affected the size of microcolonies and exopolysaccharides distribution and inhibited the expression of genes linked to insoluble exopolysaccharides synthesis. Therefore, compound 1771 prevented the accumulation of S. mutans biofilm; however, the effect was more pronounced when it was associated with fluoride and myricetin.

Elucidation of biofilm structure formation in the plant growth-promoting rhizobacterium Azospirillum brasilense is necessary to gain a better understanding of the growth of cells within the extracellular matrix and its role in the colonization of plants of agronomic importance. We used immunofluorescence microscopy and confocal laser scanning microscopy to study spatio-temporal biofilm formation on an abiotic surface. Observations facilitated by fluorescence microscopy revealed the presence of polar flagellin, exopolysaccharides, outer major membrane protein (OmaA) and extracellular DNA in the Azospirillum biofilm matrix. In static culture conditions, the polar flagellum disaggregated after 3 days of biofilm growth, but exopolysaccharides were increasing. These findings suggest that the first step in biofilm formation may be attachment, in which the bacterium first makes contact with a surface through its polar flagellum. After attaching to the surface, the long flagella and OmaA intertwine the cells to form a network. These bacterial aggregates initiate biofilm development. The underlying mechanisms dictating how the biofilm matrix components of A. brasilense direct the overall morphology of the biofilm are not well known. The methods developed here might be useful in further studies that analyze the differential spatial regulation of genes encoding matrix components that drive biofilm construction., (© FEMS 2020.)

The genomes of several Acinetobacter species possess three distinct polysaccharide-producing operons [two poly-N-acetyl glucosamine (PNAG) and one K-locus]. Using a microfluidic device, an increased amount of polysaccharides and enhanced biofilm formation were observed following continuous exposure to H 2 O 2 and removal of the H 2 O 2 -sensing key regulator, OxyR, in Acinetobacter oleivorans DR1 cells. Gene expression analysis revealed that genes located in PNAG1, but not those in PNAG2, were induced and that genes in the K-locus were expressed in the presence of H 2 O 2 . Interestingly, the expression of the K-locus gene was enhanced in the PNAG1 mutant and vice versa. The absence of either OxyR or PNAG1 resulted in enhanced biofilm formation, higher surface hydrophobicity, and increased motility, implying that K-locus-driven polysaccharide production in both the oxyR and PNAG1 deletion mutants may be related to these phenotypes. Both the oxyR and K-locus deletion mutants were more sensitive to H 2 O 2 compared with the wildtype and PNAG1 mutant strains. Purified OxyR binds to the promoter regions of both polysaccharide operons with a higher affinity toward the K-locus promoter. Although oxidized OxyR could bind to both promoter regions, the addition of dithiothreitol further enhanced the binding efficiency of OxyR, suggesting that OxyR might function as a repressor for controlling these polysaccharide operons.

The pathogenic bacterium Vibrio vulnificus exhibits the ability to form biofilm, for which initiation is dependent upon swimming motility by virtue of a polar flagellum. The filament of its flagellum is composed of multiple flagellin subunits, FlaA, -B, -C, and -D. In V. vulnificus genomes, however, open reading frames (ORFs) annotated by FlaE and -F are also present. Although neither FlaE nor FlaF is involved in filament formation and cellular motility, they are well expressed and secreted to the extracellular milieu through the secretion apparatus for flagellar assembly. In the extrapolymeric matrix of V. vulnificus biofilm, significant levels of FlaEF were detected. Mutants defective in both flaE and flaF formed significantly decreased biofilms compared to the wild-type biofilm. Thus, the potential role of FlaEF during the biofilm-forming process was investigated by exogenous addition of recombinant FlaEF (rFlaEF) to the biofilm assays. The added rFlaE and rFlaF were predominantly incorporated into the biofilm matrix formed by the wild type. However, biofilms formed by a mutant defective in exopolysaccharide (EPS) biosynthesis were not affected by added FlaEF. These results raised a possibility that FlaEF specifically interact with EPS within the biofilm matrix. In vitro pulldown assays using His-tagged rFlaEF or rFlaC revealed the specific binding of EPS to rFlaEF but not to rFlaC. Taken together, our results demonstrate that V. vulnificus FlaEF, flagellin-homologous proteins (FHPs), are crucial for biofilm formation by directly interacting with the essential determinant for biofilm maturation, EPS. Further analyses performed with other pathogenic Vibrio species demonstrated both the presence of FHPs and their important role in biofilm formation. IMPORTANCE Flagellar filaments of the pathogenic Vibrio species, including V. vulnificus , V. parahaemolyticus , and V. cholerae , are composed of multiple flagellin subunits. In their genomes, however, there are higher numbers of the ORFs encoding flagellin-like proteins than the numbers of flagellin subunits required for filament assembly. Since these flagellin-homologous proteins (FHPs) are well expressed and excreted to environments via a flagellin transport channel, their extracellular role in the pathogenic Vibrio has been enigmatic. Their biological significance, which is not related with flagellar functions, has been revealed to be in maturation of biofilm structures. Among various components of the extracellular polymeric matrix produced in the V. vulnificus biofilms, the exopolysaccharides (EPS) are dominant constituents and crucial in maturation of biofilms. The enhancing role of the V. vulnificus FHPs in biofilm formation requires the presence of EPS, as indicated by highly specific interactions among two FHPs and three EPS., (Copyright © 2019 Jung et al.)

This article reviews modern applications of mathematical descriptions of biofilm formation. The focus is on theoretically obtained results which have implications for areas including the medical sector, food industry and wastewater treatment. Examples are given as to how models have contributed to the overall knowledge on biofilms and how they are used to predict biofilm behaviour. We conclude that the use of mathematical models of biofilms has demonstrated over the years the ability to significantly contribute to the vast field of biofilm research. Among other things, they have been used to test various hypotheses on the nature of interspecies interactions, viability of biofilm treatment methods or forces behind observed biofilm pattern formations. Mathematical models can also play a key role in future biofilm research. Many models nowadays are analysed through computer simulations and continue to improve along with computational capabilities. We predict that models will keep on providing answers to important challenges involving biofilm formation. However, further strengthening of the ties between various disciplines is necessary to fully use the tools of collective knowledge in tackling the biofilm phenomenon.

Adaptation to osmotic stress is crucial for bacterial growth and survival in changing environments. Although a large number of osmotic stress response genes have been identified in various bacterial species, how osmotic changes affect bacterial motility, biofilm formation, and colonization of host niches remains largely unknown. In this study, we report that the LrhA regulator is an osmoregulated transcription factor that directly binds to the promoters of the flhDC , eps , and opgGH operons and differentially regulates their expression, thus inhibiting motility and promoting exopolysaccharide (EPS) production, synthesis of osmoregulated periplasmic glucans (OPGs), biofilm formation, and root colonization of the plant growth-promoting bacterium Pantoea alhagi LTYR-11Z. Further, we observed that the LrhA-regulated OPGs control RcsCD-RcsB activation in a concentration-dependent manner, and a high concentration of OPGs induced by increased medium osmolarity is maintained to achieve the high level of activation of the Rcs phosphorelay, which results in enhanced EPS synthesis and decreased motility in P. alhagi Moreover, we showed that the osmosensing regulator OmpR directly binds to the promoter of lrhA and promotes its expression, while lrhA expression is feedback inhibited by the activated Rcs phosphorelay system. Overall, our data support a model whereby P. alhagi senses environmental osmolarity changes through the EnvZ-OmpR two-component system and LrhA to regulate the synthesis of OPGs, EPS production, and flagellum-dependent motility, thereby employing a hierarchical signaling cascade to control the transition between a motile lifestyle and a biofilm lifestyle. IMPORTANCE Many motile bacterial populations form surface-attached biofilms in response to specific environmental cues, including osmotic stress in a range of natural and host-related systems. However, cross talk between bacterial osmosensing, swimming, and biofilm formation regulatory networks is not fully understood. Here, we report that the pleiotropic regulator LrhA in Pantoea alhagi is involved in the regulation of flagellar motility, biofilm formation, and host colonization and responds to osmotic upshift. We further show that this sensing relies on the EnvZ-OmpR two-component system that was known to detect changes in external osmotic stress. The EnvZ-OmpR-LrhA osmosensing signal transduction cascade is proposed to increase bacterial fitness under hyperosmotic conditions inside the host. Our work proposes a novel regulatory mechanism that links osmosensing and motile-sessile lifestyle transitions, which may provide new approaches to prevent or promote the formation of biofilms and host colonization in P. alhagi and other bacteria possessing a similar osmoregulatory mechanism., (Copyright © 2019 American Society for Microbiology.)

The overuse of antibiotics and biofilm formation ability has led to the emergence of bacterial resistant strains. The combined use of several antibiotics has been found as an efficient strategy to overcome this resistance. In this study, two exopolysaccharides (EPS) obtained from Lactobacillus plantarum (EPS-Lp) and Bacillus spp. (EPS-B), isolated from a traditional Tunisian food "ricotta cheese" and hypersaline environment respectively, were used to counteract the biofilm formation and efflux pumps activities in Escherichia coli ATCC35218. The obtained results revealed that the tested EPSs can be effective against E. coli at a concentration > 1 mg/ml and were able to modulate biofilm formation by 50%. Moreover, at a concentration of 512 μg/ml, the tested EPSs inhibit the EtBr efflux in the tested bacteria and no significant difference was shown compared to cells treated with reserpine (P > 0.05). The positive effect of the tested EPSs may be due to the decrease of Indole production level proposed as a signal involved in quorum sensing and through the significant reduction of the hydrophobicity percentage between the treated and untreated cells. Overall, EPS-Lp and EPS-B, when used at appropriate concentration, may inhibit biofilm formation and reduce efflux pumps implicated in bacterial adhesion and antimicrobial resistance. These results make them an interesting candidate in the design of a new strategies to control bacterial biofilm-associated infections., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Biofilms are robust multicellular aggregates of bacteria that are encased in an extracellular matrix. Different bacterial species have been shown to use a range of biopolymers to build their matrices. Pseudomonas aeruginosa is a model organism for the laboratory study of biofilms, and past work has suggested that exopolysaccharides are a required matrix component. However, we found that expression of the matrix protein CdrA, in the absence of biofilm exopolysaccharides, allowed biofilm formation through the production of a CdrA-rich proteinaceous matrix. This represents a novel function for CdrA. Similar observations have been made for other species such as Escherichia coli and Staphylococcus aureus , which can utilize protein-dominant biofilm matrices. However, we found that these CdrA-containing matrices were susceptible to both exogenous and self-produced proteases. We previously reported that CdrA directly binds the biofilm matrix exopolysaccharide Psl. Now we have found that when CdrA bound to Psl, it was protected from proteolysis. Together, these results support the idea of the importance of multibiomolecular components in matrix stability and led us to propose a model in which CdrA-CdrA interactions can enhance cell-cell packing in an aggregate that is resistant to physical shear, while Psl-CdrA interactions enhance aggregate integrity in the presence of self-produced and exogenous proteases. IMPORTANCE forms multicellular aggregates or biofilms using both exopolysaccharides and the CdrA matrix adhesin. We showed for the first time that Pseudomonas aeruginosa can use CdrA to build biofilms that do not require known matrix exopolysaccharides. It is appreciated that biofilm growth is protective against environmental assaults. However, little is known about how the interactions between individual matrix components aid in this protection. We found that interactions between CdrA and the exopolysaccharide Psl fortify the matrix by preventing CdrA proteolysis. When both components-CdrA and Psl-are part of the matrix, robust aggregates form that are tightly packed and protease resistant. These findings provide insight into how biofilms persist in protease-rich host environments.P. aeruginosa can use CdrA to build biofilms that do not require known matrix exopolysaccharides. It is appreciated that biofilm growth is protective against environmental assaults. However, little is known about how the interactions between individual matrix components aid in this protection. We found that interactions between CdrA and the exopolysaccharide Psl fortify the matrix by preventing CdrA proteolysis. When both components-CdrA and Psl-are part of the matrix, robust aggregates form that are tightly packed and protease resistant. These findings provide insight into how biofilms persist in protease-rich host environments., (Copyright © 2018 Reichhardt et al.)

Bacterial biofilms remain a persistent threat to human healthcare due to their role in the development of antimicrobial resistance. To combat multi-drug resistant pathogens, it is crucial to enhance our understanding of not only the regulation of biofilm formation, but also its contribution to bacterial virulence. Iron acquisition lies at the crux of these two subjects. In this review, we discuss the role of iron acquisition in biofilm formation and how hosts impede this mechanism to defend against pathogens. We also discuss recent findings that suggest that biofilm formation can also have the reciprocal effect, influencing siderophore production and iron sequestration.

Corals are hotspots of ocean microbial diversity and imbalance in the composition of coral associated microbes has been mostly correlated with the emergence of climate change driven diseases which affect the overall stability of the reef ecosystem. Coral sampling was performed by SCUBA diving at Palk Bay (latitude 9.271580, longitude 79.132203) south Indian coast. Among the 54 bacterial isolates, an isolate MGL-D26 showed comparatively high biofilm formation and was identified as Staphylococcus sciuri based on phylogenetic analysis. The production of exopolysaccharide (EPS) confirmed the formation of a slimy EPS matrix associated with the biofilm. The biofilm formation in S. sciuri D26 was induced significantly by UV exposure followed by other stress factors including pollution, agitation, and salinity. The strain inhibited innate immune factors of corals such as melanin synthesis and phenoloxidase. Challenge experiments in a model organism Aiptasia sp. showed pathogenicity of S. sciuri. Histopathological analysis revealed tissue invasion by S. sciuri which was a predisposing factor leading to mortality in challenged Aiptasia sp. However, specific disease condition of corals infected by S. sciuri requires continuous field monitoring and further investigation. Based on the findings, S. sciuri was a first reported multi-host opportunistic pathogen which has emerged in corals under environmental stress., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Bacterial colonization and biofilm formation on surfaces are typically mediated by the deposition of exopolysaccharides and conditioning protein layers. Pseudomonas aeruginosa is a nosocomial opportunistic pathogen that utilizes strain-specific exopolysaccharides such as Psl, Pel or alginate for both initial surface attachment and biofilm formation. To generate surfaces that resist P. aeruginosa colonization, we covalently bound a Psl-specific glycoside hydrolase (PslG h ) to several, chemically-distinct surfaces using amine functionalization (APTMS) and glutaraldehyde (GDA) linking. In situ quartz crystal microbalance (QCM) experiments and fluorescence microscopy demonstrated a complete lack of Psl adsorption on the PslG h -bound surfaces. Covalently-bound PslG h was also found to significantly reduce P. aeruginosa surface attachment and biofilm formation over extended growth periods (8 days). The PslG h surfaces showed a ∼99.9% (∼3-log) reduction in surface associated bacteria compared to control (untreated) surfaces, or those treated with inactive enzyme. This work demonstrates a non-eluting 'bioactive' surface that specifically targets a mechanism of cell adhesion, and that surface-bound glycoside hydrolase can significantly reduce surface colonization of bacteria through local, continuous enzymatic degradation of exopolysaccharide (Psl). These results have significant implications for the surface design of medical devices to keep bacteria in a planktonic state, and therefore susceptible to antibiotics and antimicrobials., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Caries etiology is biofilm-diet-dependent. Biofilms are highly dynamic and structured microbial communities enmeshed in a three-dimensional extracellular matrix. The study evaluated the expression dynamics of Streptococcus mutans genes associated with exopolysaccharides (EPS) (gtfBCD, gbpB, dexA), lipoteichoic acids (LTA) (dltABCD, SMU&#95;775c) and extracellular DNA (eDNA) (lytST, lrgAB, ccpA) during matrix development within a mixed-species biofilm of S. mutans, Actinomyces naeslundii and Streptococcus gordonii. Mixed-species biofilms using S. mutans strains UA159 or ΔgtfB formed on saliva-coated hydroxyapatite discs were submitted to a nutritional challenge (providing an abundance of sucrose and starch). Biofilms were removed at eight developmental stages for gene expression analysis by quantitative polymerase chain reaction. The pH of spent culture media remained acidic throughout the experimental periods, being lower after sucrose and starch exposure. All genes were expressed at all biofilm developmental phases. EPS- and LTA-associated genes had a similar expression profile for both biofilms, presenting lower levels of expression at 67, 91 and 115 hours and a peak of expression at 55 hours, but having distinct expression magnitudes, with lower values for ΔgtfB (eg, fold-difference of ~382 for gtfC and ~16 for dltB at 43 hours). The eDNA-associated genes presented different dynamics of expression between both strains. In UA159 biofilms lrgA and lrgB genes were highly expressed at 29 hours (which were ~13 and ~5.4 times vs ΔgtfB, respectively), whereas in ΔgtfB biofilms an inverse relationship between lytS and lrgA and lrgB expression was detected. Therefore, the deletion of gtfB influences dynamics and magnitude of expression of genes associated with matrix main components., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Streptococcus mutans-derived exopolysaccharides are virulence determinants in the matrix of biofilms that cause caries. Extracellular DNA (eDNA) and lipoteichoic acid (LTA) are found in cariogenic biofilms, but their functions are unclear. Therefore, strains of S. mutans carrying single deletions that would modulate matrix components were used: eDNA - ∆lytS and ∆lytT; LTA - ∆dltA and ∆dltD; and insoluble exopolysaccharide - ΔgtfB. Single-species (parental strain S. mutans UA159 or individual mutant strains) and mixed-species (UA159 or mutant strain, Actinomyces naeslundii and Streptococcus gordonii) biofilms were evaluated. Distinct amounts of matrix components were detected, depending on the inactivated gene. eDNA was found to be cooperative with exopolysaccharide in early phases, while LTA played a larger role in the later phases of biofilm development. The architecture of mutant strains biofilms was distinct (vs UA159), demonstrating that eDNA and LTA influence exopolysaccharide distribution and microcolony organization. Thus, eDNA and LTA may shape exopolysaccharide structure, affecting strategies for controlling pathogenic biofilms.

Biofilm formation is a universal virulence strategy in which bacteria grow in dense microbial communities enmeshed within a polymeric extracellular matrix that protects them from antibiotic exposure and the immune system. Pseudomonas aeruginosa is an archetypal biofilm-forming organism that utilizes a biofilm growth strategy to cause chronic lung infections in cystic fibrosis (CF) patients. The extracellular matrix of P. aeruginosa biofilms is comprised mainly of exopolysaccharides (EPS) and DNA. Both mucoid and nonmucoid isolates of P. aeruginosa produce the Pel and Psl EPS, each of which have important roles in antibiotic resistance, biofilm formation, and immune evasion. Given the central importance of the EPS for biofilms, they are attractive targets for novel anti-infective compounds. In this study, we used a high-throughput gene expression screen to identify compounds that repress expression of the pel genes. The pel repressors demonstrated antibiofilm activity against microplate and flow chamber biofilms formed by wild-type and hyperbiofilm-forming strains. To determine the potential role of EPS in virulence, pel / psl mutants were shown to have reduced virulence in feeding behavior and slow killing virulence assays in Caenorhabditis elegans The antibiofilm molecules also reduced P. aeruginosa PAO1 virulence in the nematode slow killing model. Importantly, the combination of antibiotics and antibiofilm compounds increased killing of P. aeruginosa biofilms. These small molecules represent a novel anti-infective strategy for the possible treatment of chronic P. aeruginosa infections., (Copyright © 2017 American Society for Microbiology.)

A novel approach to the quantification of extracellular polysaccharides in miniaturized biofilms presenting a wide variety of extracellular matrices was developed. The assay used the periodic acid-Schiff reagent and was first calibrated on dextran and alginate solutions. Then it was implemented on 24-h and 48-h biofilms from three strains known to produce different exopolymeric substances (Pseudomonas aeruginosa, Bacillus licheniformis, Weissella confusa). The assay allowed quantification of the total exopolysaccharides, taking into account possible interferences due to cells or other main expolymers of the matrix (eDNA, proteins)., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Bacteria often colonize in-dwelling medical devices and grow as complex biofilm communities of cells embedded in a self-produced extracellular polymeric matrix, which increases their resistance to antibiotics and the host immune system. During biofilm growth, bacterial cells cooperate through specific quorum-sensing (QS) signals. Taking advantage of this mechanism of biofilm formation, we hypothesized that interrupting the communication among bacteria and simultaneously degrading the extracellular matrix would inhibit biofilm growth. To this end, coatings composed of the enzymes acylase and α-amylase, able to degrade bacterial QS molecules and polysaccharides, respectively, were built on silicone urinary catheters using a layer-by-layer deposition technique. Multilayer coatings of either acylase or amylase alone suppressed the biofilm formation of corresponding Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus. Further assembly of both enzymes in hybrid nanocoatings resulted in stronger biofilm inhibition as a function of acylase or amylase position in the layers. Hybrid coatings, with the QS-signal-degrading acylase as outermost layer, demonstrated 30% higher antibiofilm efficiency against medically relevant Gram-negative bacteria compared to that of the other assemblies. These nanocoatings significantly reduced the occurrence of single-species (P. aeruginosa) and mixed-species (P. aeruginosa and Escherichia coli) biofilms on silicone catheters under both static and dynamic conditions. Moreover, in an in vivo animal model, the quorum quenching and matrix degrading enzyme assemblies delayed the biofilm growth up to 7 days.

Development of effective therapies to control oral biofilms is challenging, as topically introduced agents must avoid rapid clearance from biofilm-tooth interfaces while targeting biofilm microenvironments. Additionally, exopolysaccharides-matrix and acidification of biofilm microenvironments are associated with cariogenic (caries-producing) biofilm virulence. Thus, nanoparticle carriers capable of binding to hydroxyapatite (HA), saliva-coated HA (sHA), and exopolysaccharides with enhanced drug release at acidic pH were developed. Nanoparticles are formed from diblock copolymers composed of 2-(dimethylamino)ethyl methacrylate (DMAEMA), butyl methacrylate (BMA), and 2-propylacrylic acid (PAA) (p(DMAEMA)-b-p(DMAEMA-co-BMA-co-PAA)) that self-assemble into ∼21 nm cationic nanoparticles. Nanoparticles exhibit outstanding adsorption affinities (∼244 L-mmol(-1)) to negatively charged HA, sHA, and exopolysaccharide-coated sHA due to strong electrostatic interactions via multivalent tertiary amines of p(DMAEMA). Owing to hydrophobic cores, nanoparticles load farnesol, a hydrophobic antibacterial drug, at ∼22 wt %. Farnesol release is pH-dependent with t1/2 = 7 and 15 h for release at pH 4.5 and 7.2, as nanoparticles undergo core destabilization at acidic pH, characteristic of cariogenic biofilm microenvironments. Importantly, topical applications of farnesol-loaded nanoparticles disrupted Streptococcus mutans biofilms 4-fold more effectively than free farnesol. Mechanical stability of biofilms treated with drug-loaded nanoparticles was compromised, resulting in >2-fold enhancement in biofilm removal under shear stress compared to free farnesol and controls. Farnesol-loaded nanoparticles effectively attenuated biofilm virulence in vivo using a clinically relevant topical treatment regimen (2×/day) in a rodent dental caries disease model. Strikingly, treatment with farnesol-loaded nanoparticles reduced both the number and severity of carious lesions, while free farnesol had no effect. Nanoparticle carriers have great potential to enhance the efficacy of antibiofilm agents through multitargeted binding and pH-responsive drug release due to microenvironmental triggers.

Biofilms are highly structured microbial communities that are enmeshed in a self-produced extracellular matrix. Within the complex oral microbiome, Streptococcus mutans is a major producer of extracellular polymeric substances including exopolysaccharides (EPS), eDNA, and lipoteichoic acid (LTA). EPS produced by S. mutans-derived exoenzymes promote local accumulation of microbes on the teeth, while forming a spatially heterogeneous and diffusion-limiting matrix that protects embedded bacteria. The EPS-rich matrix provides mechanical stability/cohesiveness and facilitates the creation of highly acidic microenvironments, which are critical for the pathogenesis of dental caries. In parallel, S. mutans also releases eDNA and LTA, which can contribute with matrix development. eDNA enhances EPS (glucan) synthesis locally, increasing the adhesion of S. mutans to saliva-coated apatitic surfaces and the assembly of highly cohesive biofilms. eDNA and other extracellular substances, acting in concert with EPS, may impact the functional properties of the matrix and the virulence of cariogenic biofilms. Enhanced understanding about the assembly principles of the matrix may lead to efficacious approaches to control biofilm-related diseases.

The ubiquitous bacterial cyclic di-guanosine monophosphate (c-di-GMP) emerges as an important messenger for the control of many bacterial cellular functions including virulence, motility, bioluminescence, cellulose biosynthesis, adhesion, secretion, community behaviour, biofilm formation and cell differentiation. The synthesis of this cyclic nucleotide arises from external stimuli on various signalling domains within the N-terminal region of a dimeric diguanylate cyclase. This initiates the condensation of two molecules of guanosine triphosphate juxtaposed to each other within the C-terminal region of the enzyme. The biofilm from pathogenic microbes is highly resistant to antimicrobial agents suggesting that diguanylate cyclase and its product - c-di-GMP - are key biomedical targets for the inhibition of biofilm development. Furthermore the formation and long-term stability of the aerobic granule, a superior biofilm for biological wastewater treatment, can be controlled by stimulation of c-di-GMP. Any modulation of the synthetic pathways for c-di-GMP is clearly advantageous in terms of medical, industrial and/or environmental bioremediation implications. This review discusses the structure and reaction of individual diguanylate cyclase enzymes with a focus on new directions in c-di-GMP research. Specific attention is made on the molecular mechanisms that control bacterial exopolysaccharide biofilm formation and aerobic granules., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Pseudomonas aeruginosa, an opportunistic pathogen frequently associated with nosocomial infections, is emerging as a serious threat due to its resistance to broad spectrum antimicrobials. The biofilm mode of growth confers resistance to antibiotics and novel anti-biofilm agents are urgently needed. Nanoparticle based treatments and therapies have been of recent interest because of their versatile applications. This study investigates the anti-biofilm activity of copper nanoparticles (CuNPs) synthesized by the one pot method against P. aeruginosa. Standard physical techniques including UV-visible and Fourier transform infrared spectroscopy, X-ray diffraction and transmission electron microscopy were used to characterize the synthesized CuNPs. CuNP treatments at 100 ng ml(-1) resulted in a 94, 89 and 92% reduction in biofilm, cell surface hydrophobicity and exopolysaccharides respectively, without bactericidal activity. Evidence of biofilm inhibition was also seen with light and confocal microscope analysis. This study highlights the anti-biofilm potential of CuNPs, which could be utilized as coating agents on surgical devices and medical implants to manage biofilm associated infections.

In the food industry, successful bacterial pathogen colonization and persistence begin with their adhesion to a surface, followed by the spatial development of mature biofilm of public health concerns. Compromising bacterial settlement with natural inhibitors is a promising alternative to conventional anti-fouling treatments typically based on chemical biocides that contribute to the growing burden of antimicrobial resistance. In this study, three extracellular polymeric substance (EPS) fractions extracted from microalgae biofilms of Cylindrotheca closterium (fraction C) and Tetraselmis suecica (fraction Ta rich in insoluble scale structure and fraction Tb rich in soluble EPS) were screened for their anti-adhesive properties, against eight human food-borne pathogens belonging to Escherichia coli, Staphylococcus aureus, Salmonella enterica subsp. enterica, and Listeria monocytogenes species. The results showed that the fraction Ta was the most effective inducing statistically significant reduction for three strains of E. coli, S. aureus, and L. monocytogenes. Overall, EPSs coating on polystyrene surfaces of the different fractions increased the hydrophilic character of the support. Differences in bacterial adhesion on the different coated surfaces could be explained by several dissimilarities in the structural and physicochemical EPS compositions, according to HPLC and ATR-FTIR analysis. Interestingly, while fractions Ta and Tb were extracted from the same microalgal culture, distinct adhesion patterns were observed, highlighting the importance of the extraction process. Overall, the findings showed that EPS extracted from microalgal photosynthetic biofilms can exhibit anti-adhesive effects against food-borne pathogens and could help develop sustainable and non-toxic anti-adhesive surfaces for the food industry. Key points: •EPSs from a biofilm-based culture of C. closterium/T. suecica were characterized. •Microalgal EPS extracted from T. suecica biofilms showed bacterial anti-adhesive effects. •The anti-adhesive effect is strain-specific and affects both Gram − and Gram + bacteria. [ABSTRACT FROM AUTHOR]

Bacterial biofilms are formed by communities, which are encased in a matrix of extracellular polymeric substances (EPS). Notably, bacteria in biofilms display a set of 'emergent properties' that vary considerably from free-living bacterial cells. Biofilms help bacteria to survive under multiple stressful conditions such as providing immunity against antibiotics. Apart from the provision of multi-layered defense for enabling poor antibiotic absorption and adaptive persistor cells, biofilms utilize their extracellular components, e.g., extracellular DNA (eDNA), chemical-like catalase, various genes and their regulators to combat antibiotics. The response of biofilms depends on the type of antibiotic that comes into contact with biofilms. For example, excessive production of eDNA exerts resistance against cell wall and DNA targeting antibiotics and the release of antagonist chemicals neutralizes cell membrane inhibitors, whereas the induction of protein and folic acid antibiotics inside cells is lowered by mutating genes and their regulators. Here, we review the current state of knowledge of biofilm-based resistance to various antibiotic classes in bacteria and genes responsible for biofilm development, and the key role of quorum sensing in developing biofilms and antibiotic resistance is also discussed. In this review, we also highlight new and modified techniques such as CRISPR/Cas, nanotechnology and bacteriophage therapy. These technologies might be useful to eliminate pathogens residing in biofilms by combating biofilm-induced antibiotic resistance and making this world free of antibiotic resistance. [ABSTRACT FROM AUTHOR]

Abstract Background Exopolysaccharides (EPS) secreted by beneficial lactobacilli exert a plethora of positive activities, but little is known about their effects on biofilms of opportunistic vaginal pathogens and especially on biofilms of lactobacilli themselves. Here, the EPS produced by six vaginal lactobacilli, belonging to Lactobacillus crispatus (BC1, BC4, BC5) and Lactobacillus gasseri (BC9, BC12, BC14) species were isolated from cultural supernatants and lyophilized. Results Lactobacillus EPS were chemically characterized in terms of monosaccharide composition by liquid chromatography (LC) analysis coupled to UV and mass spectrometry (MS) detection. Moreover, the ability of EPS (0.1, 0.5, 1 mg/mL) to stimulate the biofilm formation of lactobacilli and to inhibit the formation of pathogens’ biofilms was evaluated by crystal violet (CV) staining and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Isolated EPS (yields 133–426 mg/L) were heteropolysaccharides mainly composed of d-mannose (40–52%) and d-glucose (11–30%). For the first time we demonstrated that Lactobacillus EPS were able to stimulate in a dose-dependent manner (p

Bacterial cells dwelling in the Polycyclic Aromatic Hydrocarbons (PAH) contaminated ecosystem occur as an eco-community or biofilms having biosurfactants and exopolymeric substances (EPS) producing capacity. Bacteria have developed several mechanisms to utilize the low accessible PAH compounds by modifying their structural and physiological process. EPS provides an adsorption site for PAH binding and acts as an emulsifier, enhancing PAH uptake in bacterial cells. Biosurfactants aid in the solubilization of the low-bioavailable carbon sources by reducing the interfacial surface tension between the aqueous phase and PAH-sorbent matrix, solubilizing PAHs thus making them bioavailable. Mining of exopolysaccharides synthesizing key genes (priming Glycosyltransferase) and biosurfactant producing genes (synthetases) in PAH degrading bacteriomes established their concomitant involvement in PAH solubilization and uptake. The transcriptional and translational regulators (secondary messenger cyclic-di-GMP, quorum sensing molecules, small ribosomal RNAs, two-component signaling molecules) control the synthesis of these 'bioavailability enhancers' towards PAH utilization and have been elucidated explicitly in the current review. [ABSTRACT FROM AUTHOR]

Objectives: The study aimed to evaluate the role of Streptococcus mutans (S. mutans) dexA gene on biofilm structure and microecological distribution in multispecies biofilms. Materials and Methods: A multispecies biofilm model consisting of S. mutans and its dexA mutants, Streptococcus gordonii (S. gordonii) and Streptococcus sanguinis (S. sanguinis) was constructed, and bacterial growth, biofilm architecture and microbiota composition were determined to study the effect of the S. mutans dexA on multispecies biofilms. Results: Our results showed that either deletion or overexpression of S. mutans dexA had no effect on the planktonic growth of bacterium, while S. mutans dominated in the multispecies biofilms to form cariogenic biofilms. Furthermore, we revealed that the SmudexA+ group showed structural abnormality in the form of more fractures and blank areas. The morphology of the SmudexA group was sparser and more porous, with reduced and less agglomerated exopolysaccharides scaffold. Interestingly, the microbiota composition analysis provided new insights that the inhibition of S. gordonii and S. sanguinis was alleviated in the SmudexA group compared to the significantly suppressed condition in the other groups. Conclusion: In conclusion, deletion of S. mutans dexA gene re‐modules biofilm structure and microbiota composition, thereby leading to decreased cariogenicity. Thus, the S. mutans dexA may be an important target for regulating the cariogenicity of dental plaque biofilms, expecting to be a probiotic for caries control. [ABSTRACT FROM AUTHOR]

Penicillin-binding proteins (PBPs) are considered essential for bacterial peptidoglycan biosynthesis and cell wall assembly. Clavibacter michiganensis is a representative Gram-positive bacterial species that causes bacterial canker in tomato. pbpC plays a significant role in maintaining cell morphological characteristics and stress responses in C. michiganensis. The current study demonstrated that the deletion of pbpC commonly enhances bacterial pathogenicity in C. michiganensis and revealed the mechanisms through which this occurs. The expression of interrelated virulence genes, including celA, xysA, xysB, and pelA, were significantly upregulated in △pbpC mutants. Compared with those in wild-type strains, exoenzyme activities, the formation of biofilm, and the production of exopolysaccharides (EPS) were significantly increased in △pbpC mutants. It is noteworthy that EPS were responsible for the enhancement in bacterial pathogenicity, with the degree of necrotic tomato stem cankers intensifying with the injection of a gradient of EPS from C. michiganensis. These findings highlight new insights into the role of pbpC affecting bacterial pathogenicity, with an emphasis on EPS, advancing the current understanding of phytopathogenic infection strategies for Gram-positive bacteria. [ABSTRACT FROM AUTHOR]

Background: Exopolysaccharides (EPS) secreted by beneficial lactobacilli exert a plethora of positive activities, but little is known about their effects on biofilms of opportunistic vaginal pathogens and especially on biofilms of lactobacilli themselves. Here, the EPS produced by six vaginal lactobacilli, belonging to Lactobacillus crispatus (BC1, BC4, BC5) and Lactobacillus gasseri (BC9, BC12, BC14) species were isolated from cultural supernatants and lyophilized. Results: Lactobacillus EPS were chemically characterized in terms of monosaccharide composition by liquid chromatography (LC) analysis coupled to UV and mass spectrometry (MS) detection. Moreover, the ability of EPS (0.1, 0.5, 1 mg/mL) to stimulate the biofilm formation of lactobacilli and to inhibit the formation of pathogens' biofilms was evaluated by crystal violet (CV) staining and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Isolated EPS (yields 133–426 mg/L) were heteropolysaccharides mainly composed of d-mannose (40–52%) and d-glucose (11–30%). For the first time we demonstrated that Lactobacillus EPS were able to stimulate in a dose-dependent manner (p < 0.05) the formation of biofilms of ten strains belonging to L. crispatus, L. gasseri and Limosilactobacillus vaginalis species, in terms of cell viability (84–282% increase at 1 mg/mL) and especially biofilm biomass (40–195% increase at 1 mg/mL), quantified with MTT assay and CV staining, respectively. EPS released from L. crispatus and L. gasseri were found to better stimulate the biofilms of the same producer species rather than that of other species, including producing strains themselves and other strains. Conversely, the biofilm formation of bacterial (Escherichia coli, Staphylococcus spp., Enterococcus spp. and Streptococcus agalactiae) and fungal (Candida spp.) pathogens was inhibited. The anti-biofilm activity was dose-dependent and was more marked for L. gasseri-derived EPS (inhibition up to 86%, 70%, and 58% at 1 mg/mL, 0.5 mg/mL, and 0.1 mg/mL, respectively), whilst L. crispatus-derived EPS resulted overall less efficient (inhibition up to 58% at 1 mg/mL and 40% at 0.5 mg/mL) (p < 0.05). Conclusions: Lactobacilli-derived EPS favour the biofilm formation of lactobacilli preventing, at the same time, that of opportunistic pathogens. These results support the possible employment of EPS as postbiotics in medicine as a therapeutic/preventive strategy to counteract vaginal infections. [ABSTRACT FROM AUTHOR]

Background: Streptococcus mutans (S. mutans) is a pivotal cariogenic pathogen contributing to its multiple virulence factors, one of which is synthesizing exopolysaccharides (EPS). VicK, a sensor histidine kinase, plays a major role in regulating genes associated with EPS synthesis and adhesion. Here we first identified an antisense vicK RNA (ASvicK) bound with vicK into double-stranded RNA (dsRNA). Objective: This study aims to investigate the effect and mechanism of ASvicK in the EPS metabolism and cariogenesis of S. mutans. Methods: The phenotypes of biofilm were detected by scanning electron microscopy (SEM), gas chromatography-mass spectrometery (GC-MS), gel permeation chromatography (GPC), transcriptome analysis and Western blot. Co-immunoprecipitation (Co-ip) assay and enzyme activity experiment were adopted to investigate the mechanism of ASvicK regulation. Caries animal models were developed to study the relationship between ASvicK and cariogenicity of S. mutans. Results: Overexpression of ASvicK can inhibit the growth of biofilm, reduce the production of EPS and alter genes and protein related to EPS metabolism. ASvicK can adsorb RNase III to regulate vicK and affect the cariogenicity of S. mutans. Conclusions: ASvicK regulates vicK at the transcriptional and post-transcriptional levels, effectively inhibits EPS synthesis and biofilm formation and reduces its cariogenicity in vivo. [ABSTRACT FROM AUTHOR]

Bacterial biofilms are formed by communities, which are encased in a matrix of extracellular polymeric substances (EPS). Notably, bacteria in biofilms display a set of ‘emergent properties’ that vary considerably from free-living bacterial cells. Biofilms help bacteria to survive under multiple stressful conditions such as providing immunity against antibiotics. Apart from the provision of multi-layered defense for enabling poor antibiotic absorption and adaptive persistor cells, biofilms utilize their extracellular components, e.g., extracellular DNA (eDNA), chemical-like catalase, various genes and their regulators to combat antibiotics. The response of biofilms depends on the type of antibiotic that comes into contact with biofilms. For example, excessive production of eDNA exerts resistance against cell wall and DNA targeting antibiotics and the release of antagonist chemicals neutralizes cell membrane inhibitors, whereas the induction of protein and folic acid antibiotics inside cells is lowered by mutating genes and their regulators. Here, we review the current state of knowledge of biofilm-based resistance to various antibiotic classes in bacteria and genes responsible for biofilm development, and the key role of quorum sensing in developing biofilms and antibiotic resistance is also discussed. In this review, we also highlight new and modified techniques such as CRISPR/Cas, nanotechnology and bacteriophage therapy. These technologies might be useful to eliminate pathogens residing in biofilms by combating biofilm-induced antibiotic resistance and making this world free of antibiotic resistance.

• A Synechocystis mutant with reduced exopolysaccharides generates four-fold higher photocurrent than wild-type cells. • Exopolysaccharide deprivation strengthens biofilms on electrodes. • Exopolysaccharides can inhibit diffusion and/or entry of some molecules to the cells. • Indirect extracellular electron transfer is the bioelectrochemical mechanism of exoelectrogenesis in Synechocystis. Photosynthetic microorganisms, including algae and cyanobacteria, export electrons in a light-stimulated phenomenon called 'exoelectrogenesis'. However, the route(s) by which electrons reach an external electrode from the cell remain(s) unclear. For the model cyanobacterium Synechocystis sp. PCC 6803, it has been established that electron transfer does not depend on direct extracellular electron transfer by type IV pili. However, the role of the exopolysaccharide matrix in which cells are embedded has not been investigated. We show that a Synechocystis mutant with substantially reduced exopolysaccharide production has a four-fold greater photocurrent than wild-type cells. This increase is due in part to increased adhesion of exopolysaccharide-deficient cells to electrodes. Stirred system experiments reveal that a substantial portion of the photocurrent depends on an endogenous diffusible electron mediator, supporting indirect extracellular electron transfer as the bioelectrochemical mechanism of exoelectrogenesis. These findings will be important in harnessing exoelectrogenesis for sustainable electricity generation in biophotovoltaic devices. [Display omitted] [ABSTRACT FROM AUTHOR]

Extracellular DNA (eDNA) is a critical component in the extracellular matrix (ECM) of bacterial biofilms, while little is known about the mechanisms underlying how eDNA integrates into the ECM through potential macromolecular interactions. Myxococcus xanthus biofilm was employed as a suitable model for the investigation due to the co-distribution of eDNA and exopolysaccharides (EPS) owing to their direct interactions in the ECM. DNA is able to combine with M. xanthus EPS to form a macromolecular conjugate, which is dominated by the electrostatic forces participating in the polymer-polymer interactions. Without intercalation binding, DNA-EPS interactions exhibit a certain degree of reversibility. Acting as a strong extracellular framework during biofilm formation process, the eDNA-EPS complex not only facilitates the initial cell adhesion and subsequent establishment of ECM architecture, but also renders cells within biofilms stress resistances that are relevant to the survival of M. xanthus in some hostile environments. Furthermore, the EPS protects the conjugated DNA from the degradation by nucleic acid hydrolases, which leads to the continuous and stable existence of eDNA in the native ECM of M. xanthus biofilms. These results will shed light on developing prevention and treatment strategies against biofilm-related risks. [ABSTRACT FROM AUTHOR]