Decreased Biofilm Formation Research Articles

The Alterations of Biofilm Formation and EPS Characteristics of a Diatom by a Sponge-Associated Bacterium Psychrobacter sp.

A sponge-associated bacterium, which was identified as Psychrobacter sp. in this study, was found with high activity against biofilm formation of benthic diatoms, including Amphora sp., Nitzschia closterium, Nitzschia frustulum, and Stauroneis sp. The activity against diatom biofilm formation by the tested strain was confirmed mostly in the culture supernatant and could be extracted using organic solvents. Treatment with its supernatant crude extract significantly reduced the cells of Stauroneis sp. forming biofilm and slightly increased the cells floating in the culture medium, which results in the ratio of biofilm cell/floating cell altering from 0.736 in control to 0.414 in treatment. Use of the supernatant crude extract led to increased production of extracellular polymeric substances (EPSs) by diatom Stauroneis sp. from 16.66 to 41.59 (g/g cell dry weight). The increase in EPS production was mainly contributed by soluble EPS (SL-EPS) and followed by the EPS that was tightly bound to biofilm cells (BF-TB-EPS). In addition, the supernatant crude extract caused significant changes in the monosaccharides composition of the EPS of Stauroneis sp. Specifically, glucuronic acid (Glc-A) and N-acetyl-D-glucosamine (Glc-NAc) in BF-TB-EPS were 55% fold decreased and 1219% fold increased, respectively. Based on our findings, we proposed that these changes in monosaccharides composition might lead to a decreased biofilm formation efficiency of diatom.

Herring Oil and Omega Fatty Acids Inhibit Staphylococcus aureus Biofilm Formation and Virulence.

Staphylococcus aureus is notorious for its ability to become resistant to antibiotics and biofilms play a critical role in antibiotic tolerance. S. aureus is also capable of secreting several exotoxins associated with the pathogenesis of sepsis and pneumonia. Thus, the objectives of the study were to examine S. aureus biofilm formation in vitro, and the effects of herring oil and its main components, omega fatty acids [cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) and cis-5,8,11,14,17-eicosapentaenoic acid (EPA)], on virulence factor production and transcriptional changes in S. aureus. Herring oil decreased biofilm formation by two S. aureus strains. GC-MS analysis revealed the presence of several polyunsaturated fatty acids in herring oil, and of these, two omega-3 fatty acids, DHA and EPA, significantly inhibited S. aureus biofilm formation. In addition, herring oil, DHA, and EPA at 20 μg/ml significantly decreased the hemolytic effect of S. aureus on human red blood cells, and when pre-treated to S. aureus, the bacterium was more easily killed by human whole blood. Transcriptional analysis showed that herring oil, DHA, and EPA repressed the expression of the α-hemolysin hla gene. Furthermore, in a Caenorhabditis elegans nematode model, all three prolonged nematode survival in the presence of S. aureus. These findings suggest that herring oil, DHA, and EPA are potentially useful for controlling persistent S. aureus infection.

Alkaline Phosphatase Activity of Staphylococcus aureus Grown in Biofilm and Suspension Cultures.

Staphylococcus aureus is known for its resistance to antibiotic treatment as well as the ability to form biofilms. Biofilm formation has been seen in S. aureus infections, yet, the mechanism of biofilm formation is not completely understood. Many molecules, such as DNA and polysaccharides, have been identified in the biofilm microenvironment, but little is known about the enzymes involved in the process. In this paper, alkaline phosphatase (ALP) activity was investigated in S. aureus grown either in biofilm or suspension cultures, achieved using DNase I. A significant increase of ALP activity was observed in S. aureus biofilm culture compared to its suspension counterpart. Treatment of sodium orthovanadate, an ALP inhibitor, significantly decreased biofilm formation. Its inhibition was on par with DNase I treatment at specific doses. Thus, ALP may play an important role in the biofilm formation. Likewise, ALP inhibition may be a novel target for anti-biofilm therapeutics.

A Novel Regulator Modulates Glucan Production, Cell Aggregation and Biofilm Formation in Streptococcus sanguinis SK36.

Streptococcus sanguinis is an early colonizer of tooth surfaces and a key player in plaque biofilm development. However, the mechanism of biofilm formation of S. sanguinis is still unclear. Here, we showed that deletion of a transcription factor, brpL, promotes cell aggregation and biofilm formation in S. sanguinis SK36. Glucan, a polysaccharide synthesized from sucrose, was over-produced and aggregated in the biofilm of ΔbrpL, which was necessary for better biofilm formation ability of ΔbrpL. Quantitative RT-PCR demonstrated that gtfP was significantly up-regulated in ΔbrpL, which increased the productions of water-insoluble and water-soluble glucans. The ΔbrpLΔgtfP double mutant decreased biofilm formation ability of ΔbrpL to a level similar like that of ΔgtfP. Interestingly, the biofilm of ΔbrpL had an increased tolerance to ampicillin treatment, which might be due to better biofilm formation ability through the mechanisms of cellular and glucan aggregation. RNA sequencing and quantitative RT-PCR revealed the modulation of a group of genes in ΔbrpL was mediated by activating the expression of ciaR, another gtfP-related biofilm formation regulator. Double deletion of brpL and ciaR decreased biofilm formation ability to the phenotype of a ΔciaR mutant. Additionally, RNA sequencing elucidated a broad range of genes, related to carbohydrate metabolism and uptake, were activated in ΔbrpL. SSA_0222, a gene involved in the phosphotransferase system, was dramatically up-regulated in ΔbrpL and essential for S. sanguinis survival under our experimental conditions. In summary, brpL modulates glucan production, cell aggregation and biofilm formation by regulating the expression of ciaR in S. sanguinis SK36.

Dichromatic and monochromatic laser radiation effects on antibiotic resistance, biofilm formation, and division rate of Pantoea agglomerans

Since infection is a common cause of delayed wound healing, it is important to understand the effect of low-level laser therapy (LLLT) in bacterial mechanisms. In this study we evaluated the effects of LLLT on antibiotic resistance, division rate, and biofilm formation of Pantoea agglomerans. P. agglomerans samples were isolated from human pressure injuries in humans and cultures were exposed to low-level monochromatic and simultaneous dichromatic laser radiation to study the susceptibility of an antimicrobial to ampicillin and piperacillin + tazobactam, quantification of areas of bacterial colonies, and biofilm formation of bacterial cells. Fluence, wavelength, and emission mode were used in the therapeutic protocols for wound healing. The data showed no changes in the areas of the colonies, but dichromatic laser radiation decreased biofilm formation, while a monochromatic red laser at low dose increased biofilm formation and infrared at high dose decreased antibiotic resistance to ampicillin. LLLT modulates antibiotic resistance and biofilm formation of P. agglomerans, but these depend on the laser irradiation parameters, since dichromatic laser radiation induces biological effects that differ from those induced by monochromatic laser radiation. Thus, simultaneous dichromatic low-level red and infrared lasers could be a new option for the treatment of infected wounds, reducing biofilm formation, without altering antibiotic resistance and the division rate of P. agglomerans cultures.

Transcriptional and Post‐Transcriptional Regulation of mtlA in Vibrio cholerae

Vibrio cholerae, the bacterium that causes cholera, performs a complex regulation of genes encoding carbon‐source transporters in order to survive in both aquatic reservoirs and the human small intestine. One such transporter is MtlA, which operates within the phosphotransferase system (PTS), a highly‐conserved system for carbohydrate uptake across bacteria. MtlA concomitantly phosphorylates and transports mannitol, the six‐carbon sugar alcohol produced by algae, into the cell. In addition to mtlA, the mtl locus contains mtlS and mtlR, which encode two major regulators of MtlA. mtlS encodes a 120‐nucleotide small regulatory RNA (sRNA) that posttranscriptionally regulates MtlA synthesis by binding to the mtlA mRNA 5′ untranslated region. Our objective is to identify regulators of mtlS expression. We have constructed a V. cholerae strain harboring a chromosomal mtlS‐lacZ transcriptional fusion. Using a V. cholerae genomic library, we identified putative regulators of mtlS expression. The second regulator of mtlA, encoded by mtlR, showed repressive effects in Escherichia coli, but had not been well characterized in V. cholerae. We studied how MtlR regulates MtlA activity in V. cholerae grown in a variety of carbon sources. Biofilm assays demonstrated that mtlR overexpression results in a greater than 50% decrease in biofilm formation (p<0.001 by unpaired two‐tailed t‐test), an activity known to be positively affected by MtlA. qRT‐PCR showed that a ΔmtlR mutant has higher mtlA RNA levels than a wild‐type control when grown in glucose‐supplemented minimal medium (p<0.0001 by unpaired two‐tailed t‐test), and even higher mtlA RNA levels when grown in maltose‐ and mannose‐ supplemented minimal medium (p<0.01 by unpaired two‐tailed t‐test). Western blotting analysis reflected these increases; MtlA protein levels were more elevated in ΔmtlR compared to wild‐type when grown in glucose, maltose, and mannose. The ΔmtlR mutant was partially complemented, as demonstrated by qRT‐PCR (p<0.01 by unpaired two‐tailed ttest) and western blotting analysis. This shows that the observed increases in MtlA were a direct effect of the gene product of mtlR. These results contribute to a model in which MtlR acts as a transcriptional repressor of mtlA in non‐mannitol conditions, working synergistically with MtlS to regulate mtlA.Support or Funding InformationNIH R15 AI090606This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

An N-Terminal Retention Module Anchors the Giant Adhesin LapA of Pseudomonas fluorescens at the Cell Surface: a Novel Subfamily of Type I Secretion Systems.

LapA of Pseudomonas fluorescens Pf0-1 belongs to a diverse family of cell surface-associated bacterial adhesins that are secreted via the type I secretion system (T1SS). We previously reported that the periplasmic protease LapG cleaves the N terminus of LapA at a canonical dialanine motif to release the adhesin from the cell surface under conditions unfavorable to biofilm formation, thus decreasing biofilm formation. Here, we characterize LapA as the first type I secreted substrate that does not follow the "one-step" rule of T1SS. Rather, a novel N-terminal element, called the retention module (RM), localizes LapA at the cell surface as a secretion intermediate. Our genetic, biochemical, and molecular modeling analyses support a model wherein LapA is tethered to the cell surface through its T1SS outer membrane TolC-like pore, LapE, until LapG cleaves LapA in the periplasm. We further demonstrate that this unusual retention strategy is likely conserved among LapA-like proteins, and it reveals a new subclass of T1SS ABC transporters involved in transporting this group of surface-associated LapA-like adhesins. These studies demonstrate a novel cell surface retention strategy used throughout the Proteobacteria and highlight a previously unappreciated flexibility of function for T1SS.IMPORTANCE Bacteria have evolved multiple secretion strategies to interact with their environment. For many bacteria, the secretion of cell surface-associated adhesins is key for initiating contact with a preferred substratum to facilitate biofilm formation. Our work demonstrates that P. fluorescens uses a previously unrecognized secretion strategy to retain the giant adhesin LapA at its cell surface. Further, we identify likely LapA-like adhesins in various pathogenic and commensal proteobacteria and provide phylogenetic evidence that these adhesins are secreted by a new subclass of T1SS ABC transporters.

The role of TonB-dependent copper receptor in virulence of Acinetobacter baumannii

Acinetobacter baumannii is an opportunistic gram negative pathogen that can adhere to different surfaces and cause different nosocomial infections. To investigate the role of TonB-dependent copper receptor, an outer membrane protein, in virulence of A. baumannii, we deleted this receptor from A. baumannii chromosome. There was a significant decrease in biofilm formation by copper receptor deficient mutant strain. Similarly, the adherence to human epithelial cell and the hydrophobicity were declined. The survival rate of the mutant strain in human sera was reduced while no change was observed in motility of strains. In murine pneumonia model, the bacterial lethal dose 0 (LD0), LD50 and LD100 were increased for mutant strain. Moreover, in vivo and in vitro experiments revealed changes in growth rate and dissemination of mutant strain; so that the bacterial load of the mutant was significantly reduced in the spleen and lung. The findings suggest a critical role for TonB-dependent copper receptor in virulence of A. baumannii.

Pneumonia infection in mice reveals the involvement of the feoA gene in the pathogenesis of Acinetobacter baumannii

ABSTRACT Acinetobacter baumannii has emerged in the last decade as an important nosocomial pathogen. To identify genes involved in the course of a pneumonia infection, gene expression profiles were obtained from A. baumannii ATCC 17978 grown in mouse infected lungs and in culture medium. Gene expression analysis allowed us to determine a gene, the A1S_0242 gene (feoA), over-expressed during the pneumonia infection. In the present work, we evaluate the role of this gene, involved in iron uptake. The inactivation of the A1S_0242 gene resulted in an increase susceptibility to oxidative stress and a decrease in biofilm formation, in adherence to A549 cells and in fitness. In addition, infection of G. mellonella and pneumonia in mice showed that the virulence of the Δ0242 mutant was significantly attenuated. Data presented in this work indicated that the A1S_0242 gene from A. baumannii ATCC 17978 strain plays a role in fitness, adhesion, biofilm formation, growth, and, definitively, in virulence. Taken together, these observations show the implication of the feoA gene plays in the pathogenesis of A. baumannii and highlight its value as a potential therapeutic target.

AmrZ is a major determinant of c-di-GMP levels in Pseudomonas fluorescens F113

The transcriptional regulator AmrZ is a global regulatory protein conserved within the pseudomonads. AmrZ can act both as a positive and a negative regulator of gene expression, controlling many genes implicated in environmental adaption. Regulated traits include motility, iron homeostasis, exopolysaccharides production and the ability to form biofilms. In Pseudomonas fluorescens F113, an amrZ mutant presents a pleiotropic phenotype, showing increased swimming motility, decreased biofilm formation and very limited ability for competitive colonization of rhizosphere, its natural habitat. It also shows different colony morphology and binding of the dye Congo Red. The amrZ mutant presents severely reduced levels of the messenger molecule cyclic-di-GMP (c-di-GMP), which is consistent with the motility and biofilm formation phenotypes. Most of the genes encoding proteins with diguanylate cyclase (DGCs) or phosphodiesterase (PDEs) domains, implicated in c-di-GMP turnover in this bacterium, appear to be regulated by AmrZ. Phenotypic analysis of eight mutants in genes shown to be directly regulated by AmrZ and encoding c-di-GMP related enzymes, showed that seven of them were altered in motility and/or biofilm formation. The results presented here show that in P. fluorescens, AmrZ determines c-di-GMP levels through the regulation of a complex network of genes encoding DGCs and PDEs.

Silver-pig skin nanocomposites and mesenchymal stem cells: suitable antibiofilm cellular dressings for wound healing

BackgroundTreatment of severe or chronic skin wounds is an important challenge facing medicine and a significant health care burden. Proper wound healing is often affected by bacterial infection; where biofilm formation is one of the main risks and particularly problematic because it confers protection to microorganisms against antibiotics. One avenue to prevent bacterial colonization of wounds is the use of silver nanoparticles (AgNPs); which have proved to be effective against non-multidrug-resistant and multidrug-resistant bacteria. In addition, the use of mesenchymal stem cells (MSC) is an excellent option to improve wound healing due to their capability for differentiation and release of relevant growth factors. Finally, radiosterilized pig skin (RPS) is a biomatrix successfully used as wound dressing to avoid massive water loss, which represents an excellent carrier to deliver MSC into wound beds. Together, AgNPs, RPS and MSC represent a potential dressing to control massive water loss, prevent bacterial infection and enhance skin regeneration; three essential processes for appropriate wound healing with minimum scaring.ResultsWe synthesized stable 10 nm-diameter spherical AgNPs that showed 21- and 16-fold increase in bacteria growth inhibition (in comparison to antibiotics) against clinical strains Staphylococcus aureus and Stenotrophomonas maltophilia, respectively. RPS samples were impregnated with different AgNPs suspensions to develop RPS-AgNPs nanocomposites with different AgNPs concentrations. Nanocomposites showed inhibition zones, in Kirby–Bauer assay, against both clinical bacteria tested. Nanocomposites also displayed antibiofilm properties against S. aureus and S. maltophilia from RPS samples impregnated with 250 and 1000 ppm AgNPs suspensions, respectively. MSC were isolated from adipose tissue and seeded on nanocomposites; cells survived on nanocomposites impregnated with up to 250 ppm AgNPs suspensions, showing 35% reduction in cell viability, in comparison to cells on RPS. Cells on nanocomposites proliferated with culture days, although the number of MSC on nanocomposites at 24 h of culture was lower than that on RPS.ConclusionsAgNPs with better bactericide activity than antibiotics were synthesized. RPS-AgNPs nanocomposites impregnated with 125 and 250 ppm AgNPs suspensions decreased bacterial growth, decreased biofilm formation and were permissive for survival and proliferation of MSC; constituting promising multi-functional dressings for successful treatment of skin wounds.

Anti-biofilm and Anti-Virulence Efficacy of Celastrol Against Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is a multi-drug resistant opportunistic pathogen that causes nosocomial infections in immunocompromised patients. This pathogen is difficult to treat owing to its intrinsic multidrug resistance and ability to form antimicrobial-tolerant biofilms. In the present study, we aimed to assess the potential use of celastrol as a novel anti-biofilm and/or anti-virulence agent against S. maltophilia. Results showed that celastrol at its sub-inhibitory doses decreased biofilm formation and disrupt the established biofilms produced by S. maltophilia. Celastrol-induced decrease in biofilm formation was dose-dependent based on the results of the microtiter plate biofilm assays and confocal laser scanning microscopy. In addition, our data validated the anti-virulence efficacy of celastrol, wherein it significantly interfered with the production of protease and motility of S. maltophilia. To support these phenotypic results, transcriptional analysis revealed that celastrol down-regulated the expression of biofilm- and virulence- associated genes (smeYZ, fsnR, and bfmAK) in S. maltophilia. Interestingly, celastrol significantly inhibited the expression of smeYZ gene, which encodes the resistance-nodulation-division (RND)-type efflux pump, SmeYZ. Overall, our findings suggested that celastrol might be a promising bioactive agent for treatment of biofilm- and virulence-related infections caused by the multi-drug resistant S. maltophilia.

Antifungal Activity of Oleylphosphocholine on In Vitro and In Vivo Candida albicans Biofilms

In this study, we investigated the potential antifungal activity of the alkylphospholipid oleylphosphocholine (OlPC), a structural analogue of miltefosine, on in vitro and in vivoCandida albicans biofilm formation. The effect of OlPC on in vitro and in vivoC. albicans biofilms inside triple-lumen polyurethane catheters was studied. In vivo biofilms were developed subcutaneously after catheter implantation on the lower back of Sprague-Dawley rats. Animals were treated orally with OlPC (20 mg/kg of body weight/day) for 7 days. The effect of OlPC on biofilms that developed on the mucosal surface was studied in an ex vivo model of oral candidiasis. The role of OlPC in C. albicans morphogenesis was investigated by using hypha-inducing media, namely, Lee, Spider, and RPMI 1640 media. OlPC displayed activity against both planktonic cells and in vitroC. albicans biofilms. To completely abolish preformed, 24-h-old biofilms, higher concentrations (8, 10, and 13 mg/liter) were needed. Moreover, OlPC was able to reduce C. albicans biofilms formed by caspofungin-resistant clinical isolates and acted synergistically when combined with caspofungin. The daily oral administration of OlPC significantly reduced in vivoC. albicans biofilms that developed subcutaneously. In addition, OlPC decreased biofilm formation on mucosal surfaces. Interestingly, the application of subinhibitory concentrations of OlPC already inhibited the yeast-to-hypha transition, a crucial virulence factor of C. albicans We document, for the first time, the effects of OlPC on C. albicans cells and suggest the potential use of OlPC for the treatment of C. albicans biofilm-associated infections.

Rethinking Pyogenic Flexor Tenosynovitis: Biofilm Formation Treated in a Cadaveric Model.

Introduction Pyogenic flexor tenosynovitis (PFT) of the hand remains a challenging problem that often requires surgical irrigation and parenteral or oral antibiotics. The authors hypothesize that the pathophysiology and microenvironment of PFT can be likened to that of periprosthetic joint infections (PJIs), in which bacteria thrive in a closed synovial space with limited blood supply. As such, they postulate that PFT is also facilitated by bacterial attachment and biofilm formation rendering standard treatments less effective. In this study, they evaluate infected tendons for the presence of biofilm and explore new treatment strategies. Methods Fresh human cadaveric hand tendons were harvested and divided into 0.5-cm segments. Samples were sterilized and inoculated with 1 × 10 4 CFU/mL green fluorescent Staphylococcus aureus (GFP-SA) for 48 hours at 37°C. After saline washing to remove plank tonic bacteria, samples were treated for 24 hours with (1) saline irrigation, (2) antibiotics (vancomycin), (3) corticosteroids, or (4) antibiotics/corticosteroid combined. Samples were visualized using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Results Following bacterial challenge, CLSM revealed heterogeneous green fluorescence representing bacterial attachment with dense biofilm formation. SEM at > 3,000X, also demonstrated bacterial colonization in grape-like clusters consisted with a thick matrix characteristic of biofilm. Bacterial load by direct colony counting decreased by 18.5% with saline irrigation alone, 42.6% with steroids, 54.4% with antibiotics, and 77.3% with antibiotics/steroids combined ( p < 0.05). Conclusion Staphylococcus aureus readily formed thick biofilm on human cadaveric tendons. The addition of both local antibiotics and corticosteroids resulted in greater decreases in biofilm formation on flexor tendons than the traditional treatment of saline irrigation alone. We suggest rethinking the current treatment of PFT and recommend considering a strategy more analogous to PJI management with the adjunctive use of local antibiotics, corticosteroids, and mechanical agitation.

Đánh giá tác dụng kháng sâu răng của nước súc miệng chứa α-mangostin

The mouthwash containing 150 mM α-mangostin concentration was created which had inhibiting activities against the acid secretion and biofilm formation by bacterial S. mutans as well as commercial X mouthwash. The final pH value of cultivation medium after using this mouthwash liquid was approximate 4.9, higher than the control sample using ethanol solvent. Our mouthwash product decreases biofilm formation to 32% in comparison with the control sample. The S. mutans inhibiting activity of the mouthwash product remained unchanged after 8 months storage at room temperature.

Clonal Expansion of Biofilm-Forming Salmonella Typhimurium ST34 with Multidrug-Resistance Phenotype in the Southern Coastal Region of China.

To disclose the antibiotics susceptibility and wide adaptability of commonly occurring genotypes of Salmonella Typhimurium, the antibiotic resistance and biofilm formation of different multi-locus sequence typing (MLST) types of a collection of 240 S. Typhimurium isolates (33 food and 207 clinical ones) during 2010–2014 in Shenzhen were analyzed. Among these strains, 167 was ST34 (69.58%), and 57 was ST19 (23.75%), respectively. A total of 159 (95.21%) ST34 strains displayed the multidrug resistant phenotype (≥ three classes of antibiotic), whereas only 23 (40.35%) ST19 ones did (P < 0.01). Moreover, a relative high proportion (72.46%) of ST34 isolates was classified as moderate to strong biofilm-producers, while only 15.79% of ST19 (P < 0.01) was. Among the food isolates, more than half (51.52%) were from livestock products, among which 41.18% classified as moderate to strong biofilm-producers. In summary, this study highlights the expansion of S. Typhimurium ST34 of strong biofilm-forming ability and multidrug resistance in the southern coastal region of China. Therefore, monitoring the occurrence of ST34 S. Typhimurium in food sources, especially in livestock products, and taking appropriate measures to control Salmonella spp. infections via decreasing biofilm formation should be addressed.

Sigma factor RpoN employs a dual transcriptional regulation for controlling twitching motility and biofilm formation in Lysobacter enzymogenes OH11.

Lysobacter is a Gram-negative genus comprising a group of environmental bacteria with abilities to produce abundant novel antibiotics, as well as adopting a unique type IV pilus (T4P)-mediated twitching motility (TM) that remains poorly understood. Here, we employ L. enzymogenes OH11 exhibiting significant antifungal activity as a working model to address this issue. Via mutating the 28 potential sigma factors in strain OH11, we have identified one protein RpoNOH11 (sigma 54) that is indispensable for T4P formation and TM. We further showed that RpoNOH11 not only regulates the transcription of pilA, but also another crucial gene chpA that encodes a hybrid two-component transduction system. The L. enzymogenes RpoNOH11 was found to directly bind to the promoter of chpA to control its transcription, which is found to be essential for the T4P-mediated TM. To our knowledge, such a transcriptional regulation performed by RpoN in control of bacterial TM has never been reported. Finally, we showed that L. enzymogenes OH11 could also produce biofilm that is likely employed by this strain to infect fungal pathogens. Mutation of rpoN OH11, pilA and chpA all led to a significant decrease in biofilm formation, suggesting that the dual transcriptional regulation of pilA and chpA by RpoNOH11 plays a key role for RpoNOH11 to modulate the biofilm formation in L. enzymogenes. Overall, this study identified chpA as a new target of RpoN for controlling the T4P-mediated twitching motility and biofilm formation in L. enzymogenes OH11.

Biocontrol of Bacterial Fruit Blotch by Bacillus subtilis 9407 via Surfactin-Mediated Antibacterial Activity and Colonization.

In this study, Bacillus subtilis 9407 showed a strong antibacterial activity against Acidovorax citrulli in vitro and 61.7% biocontrol efficacy on melon seedlings 4 days post inoculation under greenhouse conditions. To understand the biocontrol mechanism of B. subtilis 9407, identify the primary antibacterial compound and determine its role in controlling bacterial fruit blotch (BFB), a srfAB deletion mutant (ΔsrfAB) was constructed. The ΔsrfAB which was deficient in production of surfactin, not only showed almost no ability to inhibit growth of A. citrulli but also decreased biofilm formation and reduced swarming motility. Colonization assay demonstrated that B. subtilis 9407 could conlonize on melon roots and leaves in a large population, while ΔsrfAB showed a four- to ten-fold reduction in colonization of melon roots and leaves. Furthermore, a biocontrol assay showed that ΔsrfAB lost the biocontrol efficacy. In summary, our results indicated that surfactin, which consists of C13- to C16-surfactin A was the primary antibacterial compound of B. subtilis 9407, and it played a major role in biofilm formation, swarming motility, colonization and suppressing BFB. We propose that the biocontrol activity of B. subtilis 9407 is the results of the coordinated action of surfactin-mediated antibacterial activity and colonization. This study reveals for the first time that the use of a B. subtilis strain as a potential biological control agent could efficiently control BFB by producing surfactin.

Repurposing metformin as a quorum sensing inhibitor in Pseudomonas aeruginosa.

Quorum sensing is a mechanism of intercellular communication that controls the production of virulence factors in Pseudomonas aeruginosa. Inhibition of quorum sensing can disarm the virulence factors without exerting stress on bacterial growth that leads to emergence of antibiotic resistance. Finding a new quorum sensing inhibitor and determining its inhibitory activities against virulence factors of Pseudomonas aeruginosa PAO1 strain. Quorum sensing was evaluated by estimation of violacein production by Chromobacterium violaceum CV026. Molecular docking was used to investigate the possible binding of metformin to LasR and rhlR receptors. The inhibition of pyocyanin, hemolysin, protease, elastase in addition to swimming and twitching motilities, biofilm formation and resistance to oxidative stress by metformin was also assessed. Metformin significantly reduced the production of violacein pigment. Significant inhibition of pyocyanin, hemolysin, protease and elastase was achieved. Metformin markedly decreased biofilm formation, swimming and twitching motilities and increased the sensitivity to oxidative stress. In the molecular docking study, metformin could bind to LasR by hydrogen bonding and electrostatic interaction and to rhlR by hydrogen bonding only. Metformin can act as a quorum sensing inhibitor and virulence inhibiting agent that may be useful in the treatment of Pseudomonas aeruginosa infection.

Chitosan extracted from Aspergillus flavus shows synergistic effect, eases quorum sensing mediated virulence factors and biofilm against nosocomial pathogen Pseudomonas aeruginosa

The biological methods for extraction of chitosan were used as alternative procedures for chemical methods In biological methods, the chitosan was extracted from A. flavus by using of Lactobacillus paracasei for demineralization and Bacillus subtilis for deproteinization. The yield of extracted chitosan reached to 53.8%, pH was 7.8 and complete solubility in 1% acitic acid. Purified chitosan had the ability to reduce the biofilm forming capacity of P. aeruginosa clearly visible in light microscopic staining and scanning electron microscopy. The QS dependent phenotype and QS regulated gene expression was significantly reduced in the influence of chitosan. A significant decrease in biofilm formation was seen in the presence of chitosan. The chitosan treatment showed a decrease in the expression of lasR and rhlR genes. Same time production of pyocyanin and proteases was also inhibited in dose dependent manner. Chitosan led to increasing antimicrobial activity of antibiotics and had synergism effect, thus chitosan may be a useful adjuvant agent for the treatment of many bacterial infections in combination with antibiotics.