Control Biofilm Formation Research Articles

Elucidating the contribution of the Pseudomonas aeruginosa CPX system in biofilm formation

An investigation of Pseudomonas aeruginosa attachment to a diverse range of polymers in a high-throughput microarray format resulted in the discovery of novel biofilm-resistant and biofilm-stimulating materials. These findings raised questions about the nature of the surface interactions involved and in particular the sensory mechanisms use by P. aeruginosa to distinguish between different surface chemistries. To further investigate this, Tn5 mutants of the P. aeruginosa PAO1 Washington sub-line were tested for biofilm formation on the polymer microarrays. This revealed that a Tn5::cpxR mutant had a significant difference in biofilm formation when compared with PAO1-W. In Escherichia coli cpxR forms an operon with cpxA and cpxP. Together they form the CPX two-component signalling system controlling biofilm formation in response to cell envelope stress. To further characterise the P. aeruginosa cpxsystem, ΔcpxR,ΔcpxP and ΔcpxA deletion mutants were constructed. No significant differences were observed in their growth or in the swimming, swarming or twitch motility phenotypes normally required for surface colonization. Unlike E. coli the PAO1-W ΔcpxAmutant was able to invade lung epithelial cells and did not display increased sensitivity to antibiotics. However, the ΔcpxRmutant showed increased biofilm formation on glass and eDNA secretion in both biofilm and liquid modes of growth. This work highlights the relationship between biofilm formation and the CPX system in P. aeruginosa. However, further assays need to be conducted in order to understand the sensory mechanism(s) involved in surface sensing via the CPX system.

Susceptibility to Oral Antiseptics and Virulence Factors Ex Vivo Associated with Candida spp. Isolated from Dental Prostheses.

To isolate Candida spp. from dental prosthesis users' saliva and to evaluate the isolates for the presence of several virulence factors. This research also aimed to investigate the antifungal activity of 3 commercial mouthwashes/oral antiseptic formulations containing 0.12% chlorhexidine, 0.07% cetylpyridinium, or 0.075% cetylpyridinium against planktonic and sessile (biofilm mode) yeast cells. Forty-three Candida yeasts were isolated from 32 of 70 selected patients, and the virulence factors of C. albicans, C. krusei, C. glabrata, C. tropicalis, and C. parapsilosis species were investigated by polymerase chain reaction (PCR) and proteinase in plates. Minimum inhibitory concentration (MIC), and in vitro biofilm assay evaluated the antifungal activity of antiseptics. C. albicans, C. krusei, C. glabrata, C. tropicalis, and C. parapsilosis were detected in mono and mixed cultures. Only C. albicans displayed genes related to adhesion and proteinases (ALS2, ALS3, SAP1, and SAP3). The aspartate proteinase activity was found in 60.46% of isolates. The tested antiseptic formulations exhibited a MIC less than 1.25% toward yeasts in the planktonic mode. According to XTT ((2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay results, most Candida isolates and all mixed cultures formed biofilms within 24 hours. The evaluated antiseptic formulations were also active against biofilms. Most virulence factors investigated here (ALS2, ALS3, SAP1, and SAP3) occurred in the majority of the Candida spp. isolates, especially in C. albicans. The tested mouthwash formulations were effective against all the yeast isolates in both the planktonic and sessile growth modes. Developing alternative therapies that can avoid or control biofilm formation is necessary to prevent oral candidiasis and other Candida spp. infections.

Cross Talk between Chemosensory Pathways That Modulate Chemotaxis and Biofilm Formation.

Complex chemosensory systems control multiple biological functions in bacteria, such as chemotaxis, gene regulation, and cell cycle progression. Many species contain more than one chemosensory system per genome, but little is known about their potential interplay. In this study, we reveal cross talk between two chemosensory pathways that modulate chemotaxis and biofilm formation in Comamonas testosteroni We demonstrate that some chemoreceptors that govern chemotaxis also contribute to biofilm formation and these chemoreceptors can physically interact with components of both pathways. Finally, we show that the chemotaxis histidine kinase CheA can phosphorylate not only its cognate response regulator CheY2 but also one of the response regulators from the pathway mediating biofilm formation, FlmD. The phosphoryl group transfer from CheA to CheY2 is much faster than that from CheA to FlmD, which is consistent with chemotaxis being a fast response and biofilm formation being a much slower developmental process. We propose that cross talk between chemosensory pathways may play a role in coordination of complex behaviors in bacteria.IMPORTANCE In many bacteria, two or more homologous chemosensory pathways control several cellular functions, such as motility and gene regulation, in response to changes in the cell's microenvironment. Cross talk between signal transduction systems is poorly understood; while generally it is considered to be undesired, in some instances it might be beneficial for coregulation of complex behaviors. We demonstrate that several receptors from the pathway controlling motility can physically interact with downstream components of the pathway controlling biofilm formation. We further show that a kinase from the pathway controlling motility can also phosphorylate a response regulator from the pathway controlling biofilm formation. We propose that cross talk between two chemosensory pathways might be involved in coordination of two types of cell behavior-chemotaxis and biofilm formation.

Nanocomposite of Ag-Doped ZnO and AgO Nanocrystals as a Preventive Measure to Control Biofilm Formation in Eggshell and Salmonella spp. Entry Into Eggs.

Salmonella spp. is an important foodborne agent of salmonellosis, whose sources in humans often include products of avian origin. The control of this bacterium is difficult especially when Salmonella spp. is organized into biofilms. We hypothesized that the novel nanocomposites of ZnO nanocrystals doped with silver (Ag) and silver oxide (AgO) nanocrystals (ZnO:Ag-AgO) synthesized by the coprecipitation method could control or prevent the formation of Salmonella Enteritidis (SE) and Salmonella Heidelberg (SH) biofilm and its entry into turkey eggs. The diffraction characteristics of ZnO and AgO showed sizes of 28 and 30 nm, respectively. The Zn to Ag substitution into the ZnO crystalline structure was evidenced by the ionic radius of Ag+2 (1.26 Å), which is greater than Zn+2 (0.74 Å). For the SE analyses post-biofilm formation, the ZnO:Ag-AgO was not able to eliminate the biofilm, but the bacterial load was lower than that of the control group. Additionally, SE was able to infiltrate into the eggs and was found in both albumen and yolk. For the SH analyses applied onto the eggshells before biofilm formation, the ZnO:Ag-AgO treatment prevented biofilm formation, and although the bacterium infiltration into the eggs was observed in all treated groups, it was significantly smaller in ZnO:Ag-AgO pre-treated eggs, and SH could not reach the yolk. There was no difference in pore size between groups; therefore, the inhibition of biofilm formation and the prevention of bacterium entry into the egg were attributable to the use of ZnO:Ag-AgO, which was not influenced by the egg structure. Although the amount of Ag and Zn in the shell of the ZnO:Ag-AgO group was greater in relation to the control, this difference was not detected in the other egg components. In the search for new measures that are effective, safe and viable for controlling microorganisms in poultry farming, the application of a nanocomposite of Ag-doped ZnO and AgO nanocrystals appears as an alternative of great potential to prevent Salmonella sp biofilms in eggshells and other surfaces.

Nitric oxide increases biofilm formation in Saccharomyces cerevisiae by activating the transcriptional factor Mac1p and thereby regulating the transmembrane protein Ctr1

BackgroundBiofilms with immobilized cells encased in extracellular polymeric substance are beneficial for industrial fermentation. Their formation is regulated by various factors, including nitric oxide (NO), which is recognized as a quorum-sensing and signal molecule. The mechanisms by which NO regulates bacterial biofilms have been studied extensively and deeply, but were rarely studied in fungi. In this study, we observed the effects of low concentrations of NO on biofilm formation in Saccharomyces cerevisiae. Transcriptional and proteomic analyses were applied to study the mechanism of this regulation.ResultsAdding low concentrations of NO donors (SNP and NOC-18) enhanced biofilm formation of S. cerevisiae in immobilized carriers and plastics. Transcriptional and proteomic analyses revealed that expression levels of genes regulated by the transcription factor Mac1p was upregulated in biofilm cells under NO treatment. MAC1 promoted yeast biofilm formation which was independent of flocculation gene FLO11. Increased copper and iron contents, both of which were controlled by Mac1p in the NO-treated and MAC1-overexpressing cells, were not responsible for the increased biofilm formation. CTR1, one out of six genes regulated by MAC1, plays an important role in biofilm formation. Moreover, MAC1 and CTR1 contributed to the cells’ resistance to ethanol by enhanced biofilm formation.ConclusionsThese findings suggest that a mechanism for NO-mediated biofilm formation, which involves the regulation of CTR1 expression levels by activating its transcription factor Mac1p, leads to enhanced biofilm formation. The role of CTR1 protein in yeast biofilm formation may be due to the hydrophobic residues in its N-terminal extracellular domain, and further research is needed. This work offers a possible explanation for yeast biofilm formation regulated by NO and provides approaches controlling biofilm formation in industrial immobilized fermentation by manipulating expression of genes involved in biofilm formation.

Two-Component Signal Transduction Systems: A Major Strategy for Connecting Input Stimuli to Biofilm Formation.

Biofilms are multicellular communities of microbes that are encased within an extracellular matrix. Environmental factors induce bacteria to form biofilm. Bacteria have several regulatory mechanisms in response to environmental changes, and the two-component signal transduction system (TCS) is a major strategy in connecting changes in input signals to changes in cellular physiological output. The TCS employs multiple mechanisms such as cross-regulation, to integrate and coordinate various input stimuli to control biofilm formation. In this mini-review, we demonstrate the roles of TCS on biofilm formation, illustrating these input signals and modulation modes, which may be utilized by future investigations in elucidating the regulatory signals and underlying the mechanisms of biofilm formation.

Recombinant protein expression in biofilms.

Biofilm research is usually focused on the prevention or control of biofilm formation. Recently, the significance of the biofilm mode of growth in biotechnological applications received increased attention. Since biofilm reactors show many advantages over suspended cell reactors, especially in their higher biomass density and operational stability, bacterial biofilms have emerged as an interesting approach for the expression of specific proteins. Despite the potential of biofilm systems, recombinant protein production using biofilms has been scarcely investigated for the past 25 years. Our group has demonstrated that E. coli biofilms were able to produce a model recombinant protein, the enhanced green fluorescent protein (eGFP), at much higher levels than their planktonic counterparts. Even without optimization of cultivation conditions, an attractive productivity was obtained, indicating that biofilm cultures can be used as an alternative form of high cell density cultivation (HCDC). E. coli remains one of the favorite hosts for recombinant protein production and it has been successfully used in metabolic engineering for the synthesis of high value products. This review presents the advantages and concerns of using biofilms for the production of recombinant proteins and summarizes the different biofilm systems which have been described for this purpose. The relative advantages and disadvantages of the four microbial hosts tested for recombinant protein production in biofilms (two bacteria and two filamentous fungi) are also discussed.

Controlling biofilm formation with nitroxide functional surfaces

Nitroxide functional polymer coatings with inherent antibiofilm activity are introduced as an avenue to combat bacterial biofilm contamination of medical devices.

Molecular characterization of alkaline protease-coding gene from Bacillus licheniformis MK90 mutants with biofilm inhibitory activity

Background Enzymes are organic materials that accelerate biochemical processes without themselves undergoing change. They can be produced by plant, animal, fungi, and bacteria. Bacterial proteases are much favorable than any other sources, because bacteria can grow quickly and can be easily cultivated in laboratory. Objective To isolate and screen bacteria from soil samples for their ability to produce alkaline protease, and to improve the alkaline protease production followed by evaluation of its antimicrobials and antibiofilm activity. Materials and methods Sample collection was carried out from different locations in Egypt. Isolation of bacteria from soil samples was done using serial dilution method on skim milk agar. All isolated bacteria were screened for their ability to produce protease enzyme. The bacterial isolate showing maximum alkaline protease activity was identified using 16 S rRNA genetic identification. To induce mutations, ultraviolet (UV) irradiation was used. The most active mutant strains were selected for production, purification, and characterization of alkaline protease followed by evaluation of alkaline protease antimicrobial and antibiofilm activity. Results and conclusion Three UV mutants (MT2, MT4, and MT26) out of 48 displayed proteolytic activity more than other mutants and wild type (WT). Bacillus alkaline extracellular protease gene was genetically characterized through isolation of the genomic DNA of Bacillus licheniformis MK90 WT, and the best protease-producing UV mutants were followed by amplification, sequencing, and analyses. WT strain and best protease-producing mutants were compared at proteomic level through sodium dodecyl sulfate polyacrylamide gel electrophoresis for total cellular proteins. Then protease enzyme of WT and mutants was purified and characterized. This study reports that the B. licheniformis protease was active at an alkaline pH and wide range of temperatures (40–60°C), reflecting its potential application in detergent and laundry industries. On the contrary, the antibiofilm activity of the protease enzymes was evaluated toward four pathogenic bacterial strains, i.e., Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Bacillus subtilis, and the results showed that proteases from B. licheniformis MK90 may be useful for controlling biofilm formation by some pathogenic bacteria.

Biofilm Formation of Methicillin-resistant Coagulase-Negative Staphylococci Isolated from Clinical Samples in Northern Thailand.

Background:Methicillin-resistant coagulase-negative staphylococci (MR-CoNS) are multidrug-resistant bacteria that are difficult to treat because of their ability to form biofilms.Objectives:In the present study, we evaluated the antibiotic-resistant phenotypes, biofilm-forming ability, and biofilm associated genes of 55 clinical MR-CoNS isolates obtained from two hospitals in Thailand.Materials and Methods:MALDI-TOF-MS and tuf gene sequencing were performed to determine the species of all isolates. Biofilm production was determined using Congo red agar (CRA) and the microtiter plate (MTP) assay. Biofilm-associated genes were characterized using polymerase chain reaction (PCR).Results:Among the 55 MR-CoNS isolates, five species were identified as Staphylococcus haemolyticus (34.5%), Staphylococcus epidermidis (32.7%), Staphylococcus capitis (18.2%), Staphylococcus cohnii (9.1%), and Staphylococcus hominis (5.5%). The antimicrobial susceptibility pattern of MR-CoNS isolates indicated high resistance to cefoxitin (100%), penicillin (98.2%), erythromycin (96.4%), ciprofloxacin (67.3%), sulfamethoxazole/trimethoprim (67.3%), gentamicin (67.3%), and clindamycin (63.6%). All the isolates were susceptible to vancomycin and linezolid. The biofilm production was detected in 87.3% isolates through the CRA method and in 38.1% isolates through the MTP assay. The prevalence rates of icaAD, bap, fnbA, and cna were 18.2%, 12.7%, 47.3%, and 27.3%, respectively. There were significant differences in the presence of these biofilm-associated genes among the MR-CoNS isolates. Moreover, quantitative biofilm formation was significantly different among MR-CoNS species.Conclusion:The present study revealed that biofilm-associated genes are important for biofilm biomass in MR-CoNS isolates, and the findings of this study are essential for finding new strategies to control biofilm formation and prevent the spread of MR-CoNS infectious diseases.

Two Regulators, PA3898 and PA2100, Modulate the Pseudomonas aeruginosa Multidrug Resistance MexAB-OprM and EmrAB Efflux Pumps and Biofilm Formation.

It is generally believed that the Pseudomonas aeruginosa biofilm matrix itself acts as a molecular sieve or sink that contributes to significant levels of drug resistance, but it is becoming more apparent that multidrug efflux pumps induced during biofilm growth significantly enhance resistance levels. We present here a novel transcriptional regulator, PA3898, which controls biofilm formation and multidrug efflux pumps in P. aeruginosa A mutant of this regulator significantly reduced the ability of P. aeruginosa to produce biofilm in vitro and affected its in vivo fitness and pathogenesis in Drosophila melanogaster and BALB/c mouse lung infection models. Transcriptome analysis revealed that PA3898 modulates essential virulence genes/pathways, including multidrug efflux pumps and phenazine biosynthesis. Chromatin immunoprecipitation sequencing (ChIP-seq) identified its DNA binding sequences and confirmed that PA3898 directly interacts with promoter regions of four genes/operons, two of which are mexAB-oprM and phz2 Coimmunoprecipitation revealed a regulatory partner of PA3898 as PA2100, and both are required for binding to DNA in electrophoretic mobility shift assays. PA3898 and PA2100 were given the names MdrR1 and MdrR2, respectively, as novel repressors of the mexAB-oprM multidrug efflux operon and activators for another multidrug efflux pump, EmrAB. The interaction between MdrR1 and MdrR2 at the promoter regions of their regulons was further characterized via localized surface plasmon resonance and DNA footprinting. These regulators directly repress the mexAB-oprM operon, independent of its well-established MexR regulator. Mutants of mdrR1 and mdrR2 caused increased resistance to multiple antibiotics in P. aeruginosa, validating the significance of these newly discovered regulators.

Nitric oxide inhibits biofilm formation by Vibrio fischeri via the nitric oxide sensor HnoX.

Nitric oxide (NO) is an important defense molecule secreted by the squid Euprymna scolopes and sensed by the bacterial symbiont, Vibrio fischeri, via the NO sensor HnoX. HnoX inhibits colonization through an unknown mechanism. The genomic location of hnoX adjacent to hahK, a recently identified positive regulator of biofilm formation, suggested that HnoX may inhibit colonization by controlling biofilm formation, a key early step in colonization. Indeed, the deletion of hnoX resulted in early biofilm formation in vitro, an effect that was dependent on HahK and its putative phosphotransfer residues. An allele of hnoX that encodes a protein with increased activity severely delayed wrinkled colony formation. Control occurred at the level of transcription of the syp genes, which produce the polysaccharide matrix component. The addition of NO abrogated biofilm formation and diminished syp transcription, effects that required HnoX. Finally, an hnoX mutant formed larger symbiotic biofilms. This work has thus uncovered a host-relevant signal controlling biofilm and a mechanism for the inhibition of biofilm formation by V. fischeri. The study of V. fischeri HnoX permits us to understand not only host-associated biofilm mechanisms, but also the function of HnoX domain proteins as regulators of important bacterial processes.

Overshadow Effect of Psl on Bacterial Response to Physiochemically Distinct Surfaces Through Motility-Based Characterization.

Biofilms of Pseudomonas aeruginosa are ubiquitously found on surfaces of many medical devices, which are the major cause of hospital-acquired infections. A large amount of work has been focused on bacterial attachment on surfaces. However, how bacterial cells evolve on surfaces after their attachment is the key to get better understanding and further control of biofilm formation. In this work, by employing both single-cell- and collective-motility of cells, we characterized the bacterial surface movement on physiochemically distinct surfaces. The measurement of cell surface motility showed consistent results that gold and especially platinum surfaces displayed a stronger capability in microcolony formation than polyvinyl chloride and polycarbonate surfaces. More interestingly, we found that overproduction of Psl led to a narrower variance in cell surface motility among tested surfaces, indicating an overshadow effect of Psl for bacteria by screening the influence of physicochemical properties of solid surfaces. Our results provide insights into how Pseudomonas aeruginosa cells adapt their motion to physiochemically distinct surfaces, and thus would be beneficial for developing new anti-biofouling techniques in biomedical engineering.

Microbiome Analysis of Biofilms of Silver Nanoparticle-Dispersed Silane-Based Coated Carbon Steel Using a Next-Generation Sequencing Technique.

Previously, we demonstrated that silver nanoparticle-dispersed silane-based coating could inhibit biofilm formation in conditions where seawater was used as a bacterial source and circulated in a closed laboratory biofilm reactor. However, it is still unclear whether the microbiome of a biofilm of silver nanoparticle-dispersed silane-based coating samples (Ag) differs from that of a biofilm of non-dispersed silane-based coating samples (Non-Ag). This study aimed to perform a microbiome analysis of the biofilms grown on the aforementioned coatings using a next-generation sequencing (NGS) technique. For this, a biofilm formation test was conducted by allowing seawater to flow through a closed laboratory biofilm reactor; subsequently, DNAs extracted from the biofilms of Ag and Non-Ag were used to prepare 16S rRNA amplicon libraries to analyze the microbiomes by NGS. Results of the operational taxonomy unit indicated that the biofilms of Non-Ag and Ag comprised one and no phyla of archaea, respectively, whereas Proteobacteria was the dominant phylum for both biofilms. Additionally, in both biofilms, Non-Ag and Ag, Marinomonas was the primary bacterial group involved in early stage biofilm formation, whereas Anaerospora was primarily involved in late-stage biofilm formation. These results indicate that silver nanoparticles will be unrelated to the bacterial composition of biofilms on the surface of silane-based coatings, while they control biofilm formation there.

Influence of incubation conditions on the formation of model biofilms by Listeria monocytogenes and Salmonella Typhimurium on abiotic surfaces.

This research aims to develop strongly adherent and mature model biofilms (on a 20cm² polystyrene surface) for two pathogenic species, i.e. Listeria monocytogenes and Salmonella Typhimurium. These model biofilms can be used as standards to study biofilms or to study/compare the influence of different inactivation technologies. Three influencing factors on the formation of biofilms are investigated, i.e. growth medium, incubation temperature and incubation time, which are three easily controllable environmental factors. Optical density measurement and plate counts were used to evaluate the adherence and the maturity of the biofilms, respectively. Confocal laser scanning microscopy was used to verify most important findings obtained with previously mentioned assays. Results indicated that mature and strongly adherent L.monocytogenes biofilms are obtained following 13h of incubation at 30°C with BHI as growth medium. For S.Typhimurium, an incubation period of 19h at 25°C was required with 20-fold diluted TSB as growth medium. Based on previously mentioned assays, a protocol for the formation of reproducible model biofilms was obtained. The developed model biofilms can be applied as a standard to study biofilms (in different research fields) and their subsequent inactivation by different methods. In addition, the results of this study could be used to control biofilm formation (e.g. by setting a maximum allowed surface temperature).

Use of extracellular polysaccharides, secreted by Lactobacillus plantarum and Bacillus spp., as reducing indole production agents to control biofilm formation and efflux pumps inhibitor in Escherichia coli

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.

Quorum sensing LuxS/autoinducer-2 inhibits Enterococcus faecalis biofilm formation ability.

Objective:To investigate the relation between biofilm formation ability and quorum sensing gene LuxS/AI-2.Materials and Methods: Enterococcus faecalis (E. faecalis) standard strain ATCC 29212 was used in the study. Long flanking homology polymerase chain reaction method was used to build the LuxS gene knockout strain. Sequential culture turbidity measurement and CFU counting were used to assess the proliferation ability of E. faecalis after the depletion of LuxS. 96-well plate assay was used to quantify the biofilm formation ability; CLSM was used to observe the attached bacteria areas, while scanning electron microscopy (SEM) was performed to observe biofilm microstructure conditions.Results:LuxS gene knockout strains were successfully constructed and identified. The results showed that proliferation ability of E. faecalis was not affected by the depletion of the luxS gene, and the biofilm formation ability of ΔLuxS 29212 significantly decreased (P<0.05).Conclusions:Collectively, our studies provide the LuxS gene's key role in controlling biofilm formation of E. faecalis, which presented a negative regulation, and furthermore, providing us a possible way to conquer the persistent apical periodontitis.

Vibrio fischeri Biofilm Formation Prevented by a Trio of Regulators.

Biofilms, complex communities of microorganisms surrounded by a self-produced matrix, facilitate attachment and provide protection to bacteria. A natural model used to study biofilm formation is the symbiosis between Vibrio fischeri and its host, the Hawaiian bobtail squid, Euprymna scolopes Host-relevant biofilm formation is a tightly regulated process and is observed in vitro only with strains that have been genetically manipulated to overexpress or disrupt specific regulators, primarily two-component signaling (TCS) regulators. These regulators control biofilm formation by dictating the production of the symbiosis polysaccharide (Syp-PS), the major component of the biofilm matrix. Control occurs both at and below the level of transcription of the syp genes, which are responsible for Syp-PS production. Here, we probed the roles of the two known negative regulators of biofilm formation, BinK and SypE, by generating double mutants. We also mapped and evaluated a point mutation using natural transformation and linkage analysis. We examined traditional biofilm formation phenotypes and established a new assay for evaluating the start of biofilm formation in the form of microscopic aggregates in shaking liquid cultures, in the absence of the known biofilm-inducing signal calcium. We found that wrinkled colony formation is negatively controlled not only by BinK and SypE but also by SypF. SypF is both required for and inhibitory to biofilm formation. Together, these data reveal that these three regulators are sufficient to prevent wild-type V. fischeri from forming biofilms under these conditions.IMPORTANCE Bacterial biofilms promote attachment to a variety of surfaces and protect the constituent bacteria from environmental stresses, including antimicrobials. Understanding the mechanisms by which biofilms form will promote our ability to resolve them when they occur in the context of an infection. In this study, we found that Vibrio fischeri tightly controls biofilm formation using three negative regulators; the presence of a single one of these regulators was sufficient to prevent full biofilm development, while disruption of all three permitted robust biofilm formation. This work increases our understanding of the functions of specific regulators and demonstrates the substantial negative control that one benign microbe exerts over biofilm formation, potentially to ensure that it occurs only under the appropriate conditions.

Control of Biofilm Formation in Healthcare: Recent Advances Exploiting Quorum-Sensing Interference Strategies and Multidrug Efflux Pump Inhibitors.

Biofilm formation in healthcare is an issue of considerable concern, as it results in increased morbidity and mortality, imposing a significant financial burden on the healthcare system. Biofilms are highly resistant to conventional antimicrobial therapies and lead to persistent infections. Hence, there is a high demand for novel strategies other than conventional antibiotic therapies to control biofilm-based infections. There are two approaches which have been employed so far to control biofilm formation in healthcare settings: one is the development of biofilm inhibitors based on the understanding of the molecular mechanism of biofilm formation, and the other is to modify the biomaterials which are used in medical devices to prevent biofilm formation. This review will focus on the recent advances in anti-biofilm approaches by interrupting the quorum-sensing cellular communication system and the multidrug efflux pumps which play an important role in biofilm formation. Research efforts directed towards these promising strategies could eventually lead to the development of better anti-biofilm therapies than the conventional treatments.

Novel Silver Nanoparticles Synthesized from Anthers of Couroupita Guianensis Abul. Control Growth and Biofilm Formation in Human Pathogenic Bacteria

Novel Silver Nanoparticles Synthesized from Anthers of Couroupita Guianensis Abul. Control Growth and Biofilm Formation in Human Pathogenic Bacteria