Linoleic acid inhibits Pseudomonas aeruginosa biofilm formation by activating diffusible signal factor-mediated quorum sensing.

Bacterial biofilm formation causes serious problems in various fields of medical, clinical, and industrial settings. Antibiotics and biocide treatments are typical methods used to remove bacterial biofilms, but biofilms are difficult to remove effectively from surfaces due to their increased resistance. An alternative approach to treatment with antimicrobial agents is using biofilm inhibitors that regulate biofilm development without inhibiting bacterial growth. In the present study, we found that linoleic acid (LA), a plant unsaturated fatty acid, inhibits biofilm formation under static and continuous conditions without inhibiting the growth of Pseudomonas aeruginosa. LA also influenced the bacterial motility, extracellular polymeric substance production, and biofilm dispersion by decreasing the intracellular cyclic diguanylate concentration through increased phosphodiesterase activity. Furthermore, quantitative gene expression analysis demonstrated that LA induced the expression of genes associated with diffusible signaling factor-mediated quorum sensing that can inhibit or induce the dispersion of P. aeruginosa biofilms. These results suggest that LA is functionally and structurally similar to a P. aeruginosa diffusible signaling factor (cis-2-decenoic acid) and, in turn, act as an agonist molecule in biofilm dispersion.

Antibacterial application of covalently immobilized photosensitizers on a surface.

Singlet oxygen produced by irradiating photosensitizers (PSs) can be used to kill pathogens during water treatment. Chemical immobilization of the PSs on surfaces can maintain their disinfection function long-term. In this study, two model PSs (rose bengal (RB) and hematoporphyrin (HP)) were immobilized on a glass surface using a silane coupling agent with an epoxide group, and their antibacterial properties were analyzed. Fourier transform infrared spectroscopy demonstrated that a covalent bond formed between the epoxide group and hydroxyl group in the PSs. A large proportion of the immobilized PSs (approximately 50%) was active in singlet oxygen production, which was evidenced by a comparative analysis with free PSs. RB was more effective at producing singlet oxygen than HP. The immobilized PSs were durable in terms of repeated use. On the other hand, singlet oxygen produced by the PSs was effective at killing bacteria, mostly for Gram-positive bacteria (> 90% death for 2 h of irradiation), by damaging the cell membrane. The preferable antibacterial property against Gram-positive bacteria compared with that against Gram-negative bacteria suggested efficient penetrability of singlet oxygen across the cell membrane, which led to cell death. Taken together, it was concluded that immobilization of PSs on surfaces using the silane coupling agent proposed in this study was effective at killing Gram-positive bacteria by forming singlet oxygen.

Design, synthesis and biological evaluation of 4-(alkyloxy)-6-methyl-2H-pyran-2-one derivatives as quorum sensing inhibitors.

Novel pyrone-derived quorum sensing (QS) ligands to inhibit the binding of OdDHL to the LasR of Pseudomonas aeruginosa were designed, synthesized and evaluated. Among the analogs, the most potent compound 8 exhibited strong in vitro inhibitory activities against biofilm formation and down-regulated OdDHL/LasR-associated genes by 35-67%. The binding mode of 8 in silico was highly similar to that of the crystal ligand OdDHL in the active site of LasR.

Inhibition of biofouling by modification of forward osmosis membrane using quaternary ammonium cation.

In the operation of the forward osmosis (FO) process, biofouling of the membrane is a potentially serious problem. Development of an FO membrane with antibacterial properties could contribute to a reduction in biofouling. In this study, quaternary ammonium cation (QAC), a widely used biocidal material, was conjugated with a silane coupling agent (3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride) and used to modify an FO membrane to confer antibacterial properties. Fourier transform infrared spectroscopy (FT-IR) demonstrated that the conjugated QAC was successfully immobilized on the FO membrane via covalent bonding. Bacterial viability on the QAC-modified membrane was confirmed via colony count method and visualized via bacterial viability assay. The QAC membrane decreased the viability of Escherichia coli to 62% and Staphylococcus aureus to 77% versus the control membrane. Inhibition of biofilm formation on the QAC modified membrane was confirmed via anti-biofilm tests using the drip-flow reactor and FO unit, resulting in 64% and 68% inhibition in the QAC-modified membrane against the control membrane, respectively. The results demonstrate the effectiveness of the modified membrane in reducing bacterial viability and inhibiting biofilm formation, indicating the potential of QAC-modified membranes to decrease operation costs incurred by biofouling.

Repercussion of extracellular polymeric removal by low-temperature calcium peroxide pretreatment on bacterial fragmentation for enhancing biohydrogen production.

This study envisioned attaining the percipience of effective biohydrogen production from paper mill waste-activated sludge through low-temperature calcium peroxide-mediated bacterial pretreatment (TCP-BP). Floc dissociation with limited cell destruction was attained at a calcium peroxide dosage of 0.05 g/g suspended solids (SS) at 70 °C temperature. This TCP-BP method improves bacterial fragmentation, and very high SS solubilization was achieved at 42 h, with the solubilization and solid reduction of 18.6% and 14.1%, respectively. BP-only pretreatment shows lower solubilization efficiency of 9.4% than TCP-BP pretreatment due to the presence of flocs, which inhibit the enzymatic action during bacterial fragmentation. A biohydrogen test shows a high biohydrogen potential of 94.1 mL H2/gCOD for the TCP-BP sample, which is higher than that of the BP-only and control samples. According to the findings, low-temperature calcium peroxide-mediated bacterial fragmentation is validated to be an efficient process for sludge degradation and biohydrogen production.

Rapid and sensitive detection of gram-negative bacteria using surface-immobilized polymyxin B.

Although detection of gram-negative bacteria (GNB) in body fluids is important for clinical purpose, traditional gram staining and other recently developed methods have inherent limitations in terms of accuracy, sensitivity, and convenience. To overcome the weakness, this study proposed a method detecting GNB based on specific binding of polymyxin B (PMB) to lipopolysaccharides (LPS) of GNB. Fluorescent microscopy demonstrated that surface immobilized PMB using a silane coupling agent was possible to detect fluorescent signal produced by a single Escherichia coli (a model GNB) cell. Furthermore, the signal was selective enough to differentiate between GNB and gram-positive bacteria. The proposed method could detect three cells per ml within one hour, indicating the method was very sensitive and the sensing was rapid. These results suggest that highly multifold PMB binding on each GNB cell occurred, as millions of LPS are present on cell wall of a GNB cell. Importantly, the principle used in this study was realized in a microfluidic chip for a sample containing E. coli cells suspended in porcine plasma, demonstrating its potential application to practical uses. In conclusion, the proposed method was accurate, sensitive, and convenient for detecting GNB, and could be applied clinically.

Inhibiting bacterial biofilm formation by stimulating c-di-GMP regulation using citrus peel extract from Jeju Island.

Biofilms consist of single or multiple species of bacteria embedded in extracellular polymeric substances (EPSs), which affect the increase in antibiotic resistance by restricting the transport of antibiotics to the bacterial cells. An alternative approach to treatment with antimicrobial agents is using biofilm inhibitors that regulate biofilm development without inhibiting bacterial growth. In this study, we found that citrus peel extract from Jeju Island (CPEJ) can inhibit bacterial biofilm formation. According to the results, CPEJ concentration-dependently reduces biofilm formation without affecting bacterial growth. Additionally, CPEJ decreased the production of extracellular polymeric substances but increased bacterial swarming motility. These results led to the hypothesis that CPEJ can reduce intracellular bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) concentration. The results showed that CPEJ significantly reduced the c-di-GMP level through increased phosphodiesterase activity. Altogether, these findings suggest that CPEJ as a biofilm inhibitor has new potential for pharmacological (e.g. drug and medication) and industrial applications (e.g. ship hulls, water pipes, and membrane processes biofouling control).

Preparation of single-walled carbon nanotubes/polyvinylchloride membrane and its antibacterial property.

Polyvinylchloride (PVC) ultrafiltration membranes were modified by blending with single-walled carbon nanotubes (SWCNTs) to improve the membranes' antibacterial property. Both modified and control samples were characterized for pore structure, roughness, hydrophilicity, permeability and mechanical properties. The membranes' antibacterial property was accessed with Escherichia coli as the model microbes by several methods. It was found that, after being blended with SWCNTs, the surface roughness of the modified membrane increased. Also, the surface hydrophilicity was improved. The membrane flux increased accordingly. But the membrane elongation decreased obviously with the SWCNTs addition. The modified membranes did not show the antibacterial property as expected in this research. There was no bacterial inhibition circle around the SWCNTs/PVC membrane coupons in the culture plates. There were no morphological differences of the cells on the control and the modified membranes. Hoechst 33342/propidium iodide stain test showed that there were more than 90% living bacterial cells which could grow on the SWCNTs/PVC membranes. This study suggests that the polymer wrapping may reduce the SWCNTs' antibacterial property greatly.

Raffinose Inhibits Streptococcus mutans Biofilm Formation by Targeting Glucosyltransferase.

Streptococcus mutans is a representative biofilm-forming bacterium that causes dental caries through glucosyltransferase (GTF) activity. Glucans are synthesized from sucrose by GTFs and provide binding sites for S. mutans to adhere tightly to the tooth enamel. Therefore, if a novel compound that interferes with GTF function is developed, biofilm formation control in S. mutans would be possible. We discovered that raffinose, an oligosaccharide from natural products, strongly inhibited biofilm formation, GTF-related gene expression, and glucan production. Furthermore, biofilm inhibition on saliva-coated hydroxyapatite discs through the reduction of bacterial adhesion indicated the applicability of raffinose in oral health. These effects of raffinose appear to be due to its ability to modulate GTF activity in S. mutans. Hence, raffinose may be considered an antibiofilm agent for use as a substance for oral supplies and dental materials to prevent dental caries. IMPORTANCE Dental caries is the most prevalent infectious disease and is expensive to manage. Dental biofilms can be eliminated via mechanical treatment or inhibited using antibiotics. However, bacteria that are not entirely removed or are resistant to antibiotics can still form biofilms. In this study, we found that raffinose inhibited biofilm formation by S. mutans, a causative agent of dental caries, possibly through binding to GtfC. Our findings support the notion that biofilm inhibition by raffinose can be exerted by interference with GTF function, compensating for the shortcomings of existing commercialized antibiofilm methods. Furthermore, raffinose is an ingredient derived from natural products and can be safely utilized in humans; it has no smell and tastes sweet. Therefore, raffinose, which can control S. mutans biofilm formation, has been suggested as a substance for oral supplies and dental materials to prevent dental caries.

Role of DegQ in differential stability of flagellin subunits in Vibrio vulnificus.

Biofilm formation of Vibrio vulnificus is initiated by adherence of flagellated cells to surfaces, and then flagellum-driven motility is not necessary during biofilm maturation. Once matured biofilms are constructed, cells become flagellated and swim to disperse from biofilms. As a consequence, timely regulations of the flagellar components' expression are crucial to complete a biofilm life-cycle. In this study, we demonstrated that flagellins' production is regulated in a biofilm stage-specific manner, via activities of a protease DegQ and a chaperone FlaJ. Among four flagellin subunits for V. vulnificus filament, FlaC had the highest affinities to hook-associated proteins, and is critical for maturating flagellum, showed the least susceptibility to DegQ due to the presence of methionine residues in its DegQ-sensitive domains, ND1 and CD0. Therefore, differential regulation by DegQ and FlaJ controls the cytoplasmic stability of flagellins, which further determines the motility-dependent, stage-specific development of biofilms.

Investigation of critical sludge characteristics for membrane fouling in a submerged membrane bioreactor: Role of soluble microbial products and extracted extracellular polymeric substances.

Membrane bioreactors (MBRs) are considered a promising tool for resource recovery in wastewater treatment. Nevertheless, membrane fouling is an inevitable phenomenon that deteriorates the MBR performance. Although many studies have attempted to elucidate the effect of sludge characteristics on MBR fouling, they posed certain limitations. Most of the previous studies focused on the initial sludge or employ the results of short-term batch tests without long-term transmembrane pressure (TMP) profiles in the interpretation of fouling behaviors. This study was conducted considering these limitations to determine the sludge characteristics most closely related to long-term TMP profiles and to identify their role in fouling behaviors. In long-term TMP profiles, critical time (tc; time to TMP jump) and fouling rates (the increase in the TMP slope) were used as fouling indexes, which were used to correlate with average values of sludge characteristics before and after experiments. According to the results, the concentration of the total soluble microbial product (SMP) and extracted extracellular polymeric substance (eEPS) in sludge significantly increased by 1.9 times and up to 28 times after experiment. The increase in the SMP and eEPS caused early TMP jumps and resulted in low-fouling rates by increasing particle size. Owing to the increase in the SMP and eEPS concentration, the origin of fouling potential was shifted from suspended solids to colloids and soluble materials. Fouling resistance caused by soluble material increased by up to 11.38 times.

Combined Treatment of 6-Gingerol Analog and Tobramycin for Inhibiting Pseudomonas aeruginosa Infections.

Pseudomonas aeruginosa is a ubiquitous human pathogen that causes severe infections. Although antibiotics, such as tobramycin, are currently used for infection therapy, their antibacterial activity has resulted in the emergence of multiple antibiotic-resistant bacteria. The 6-gingerol analog, a structural derivative of the main component of ginger, is a quorum sensing (QS) inhibitor. However, it has a lower biofilm inhibitory activity than antibiotics and the possibility to cause toxicity in humans. Therefore, novel and more effective approaches for decreasing dosing concentration and increasing biofilm inhibitory activity are required to alleviate P. aeruginosa infections. In this study, a 6-gingerol analog was combined with tobramycin to treat P. aeruginosa infections. The combined treatment of 6-gingerol analog and tobramycin showed strong inhibitory activities on biofilm formation and the production of QS-related virulence factors of P. aeruginosa compared to single treatments. Furthermore, the combined treatment alleviated the infectivity of P. aeruginosa in an insect model using Tenebrio molitor larvae without inducing any cytotoxic effects in human lung epithelial cells. The 6-gingerol analog showed these inhibitory activities at much lower concentrations when used in combination with tobramycin. Adjuvant effects were observed through increased QS-disrupting processes rather than through antibacterial action. In particular, improved RhlR inactivation by this combination is a possible target for therapeutic development in LasR-independent chronic infections. Therefore, the combined treatment of 6-gingerol analog and tobramycin may be considered an effective method for treating P. aeruginosa infections. IMPORTANCE Pseudomonas aeruginosa is a pathogen that causes various infectious diseases through quorum-sensing regulation. Although antibiotics are mainly used to treat P. aeruginosa infections, they cause the emergence of resistant bacteria in humans. To compensate for the disadvantages of antibiotics and increase their effectiveness, natural products were used in combination with antibiotics in this study. We discovered that combined treatment with 6-gingerol analog from naturally-derived ginger substances and tobramycin resulted in more effective reductions of biofilm formation and virulence factor production in P. aeruginosa than single treatments. Our findings support the notion that when 6-gingerol analog is combined with tobramycin, the effects of the analog can be exerted at much lower concentrations. Furthermore, its improved LasR-independent RhlR inactivation may serve as a key target for therapeutic development in chronic infections. Therefore, the combined treatment of 6-gingerol analog and tobramycin is suggested as a novel alternative for treating P. aeruginosa infections.

Development of a new method to evaluate critical flux and system reliability based on particle properties in a membrane bioreactor.

Membrane fouling occurs when the operating flux exceeds a certain point (i.e., critical flux). Critical flux has therefore been widely adopted to determine the initial operating flux in membrane bioreactor (MBR) processes. The flux steeping method currently used to measure the critical flux is time-consuming and uneconomical. This study was conducted to develop a novel approach for the evaluation of critical flux. Given that particle fouling is dominant during the initial fouling stage, we hypothesized that particle properties may be closely related to critical flux. A critical flux prediction model with an R2 of 0.9 was therefore derived, which indicates that particle properties regulate critical flux. The results imply that most of the fouling potential during the early stages of operation is caused by SS, and that the formation of cakes that comprise large particles is the dominant fouling mechanism. The new method proposed in this study reduced the measurement cost and time to evaluate critical flux by 3.5-and 8 times, respectively, compared to the flux-stepping method. In terms of practical application, the applicability of the model equation was identified by system reliability analysis, which indicates that the system failure increases significantly as the standard deviation of the variables increases. This study demonstrated that the prediction of critical flux and system reliability can be achieved through particle characteristic measurement. A similar approach is expected to be employed in real MBR plants as an economical and convenient fouling control strategy to solve problems involving resource shortages.

Synergistic control of membrane biofouling using linoleic acid and sodium hypochlorite.

Biofouling is a major operational problem in the reverse osmosis (RO) process, affecting the membrane performance. Although sodium hypochlorite (NaOCl) is used to chemically clean the biofouled membranes, high concentrations of NaOCl cause morphological and chemical damage to the RO membrane. The objective of this study is to enhance chemical cleaning efficiency by combining with a dispersion agent (linoleic acid, LA) that does not harm the RO membrane, to overcome the disadvantages of NaOCl. Biofilm cells were initially dispersed with LA treatment and biofouled layers were subsequently cleaned using NaOCl at low concentration. The optimized combination resulted in 3.9-4.4 times higher flux recovery efficiency than that with individual treatments. Furthermore, the combination decreased the volume and thickness of the biofilm as well as the amount of extracellular polymeric substances. Taken together, the combined treatment of LA and NaOCl significantly improves RO biofouling control.

Effect of broad-spectrum biofilm inhibitor raffinose, a plant galactoside, on the inhibition of co-culture biofilm on the microfiltration membrane.

A membrane bioreactor (MBR) integrates process such as membrane filtration and biological treatment of activated sludge. However, organic, inorganic and biological matters cause membrane fouling, which seriously affects membrane performance. The goal of this study was to evaluate the biofouling inhibition capacity of raffinose during the MBR process. The results showed that 0-1,000 µM raffinose significantly reduced the formation of the P. aeruginosa and S. aureus co-culture biofilm by about 25-52 % in a concentration-dependent manner. In addition, the effect of raffinose on the microfiltration membrane biofilm was tested in a flow reactor and lab-scale MBR unit. The results showed that the co-culture biofilm and transmembrane pressure were decreased by raffinose treatment compared to those by furanone C-30 treatment. These results clearly demonstrated that raffinose, broad-spectrum biofilm inhibitor, inhibits biofilm formation in mixed cultures and could be used to mitigate biofouling in MBR processes.

Vertically Aligned Carbon Nanotube Membranes: Water Purification and Beyond.

Vertically aligned carbon nanotube (VACNT) membranes have attracted significant attention for water purification owing to their ultra-high water permeability and antibacterial properties. In this paper, we critically review the recent progresses in the synthesis of VACNT arrays and fabrication of VACNT membrane methods, with particular emphasis on improving water permeability and anti-biofouling properties. Furthermore, potential applications of VACNT membranes other than water purification (e.g., conductive membranes, electrodes in proton exchange membrane fuel cells, and solar electricity-water generators) have been introduced. Finally, future outlooks are provided to overcome the limitations of commercialization and desalination currently faced by VACNT membranes. This review will be useful to researchers in the broader scientific community as it discusses current and new trends regarding the development of VACNT membranes as well as their potential applications.

Discovery and Characterization of Pure RhlR Antagonists against Pseudomonas aeruginosa Infections.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic human pathogen that forms biofilms and produces virulence factors via quorum sensing (QS). Blocking the QS system in P. aeruginosa is an excellent strategy to reduce biofilm formation and the production of virulence factors. RhlR plays an essential role in the QS system of P. aeruginosa. We synthesized 55 analogues based on the chemical structure of 4-gingerol and evaluated their RhlR inhibitory activities using the cell-based reporter strain assay. Comprehensive structure-activity relationship studies identified the alkynyl ketone 30 as the most potent RhlR antagonist. This compound displayed selective RhlR antagonism over LasR and PqsR, strong inhibition of biofilm formation, and reduced production of virulence factors in P. aeruginosa. Furthermore, the survival rate of Tenebrio molitor larvae treated with 30 in vivo greatly improved. Therefore, compound 30, a pure RhlR antagonist, can be utilized for developing QS-modulating molecules in the control of P. aeruginosa infections.

Granular activated carbon supplementation alters the metabolic flux of Clostridium butyricum for enhanced biohydrogen production.

In this work, the effects of granular activated carbon (GAC) supplementation on hydrogen fermentation were investigated in Clostridium butyricum. Maximum hydrogen production rate and ultimate hydrogen volume increased up to 1.7 and 1.9 fold, respectively, with 1 g/L GAC supplementation. Indicators of stable hydrogen production, such as acetate and butyrate formation increased with increasing GAC concentration. To identify the factors for hydrogen production enhancement, transcriptome analysis was conducted. Functional genes related to hydrogen production increased by GAC supplementation (1.75 fold for pfor and 1.48 fold for oxct). On the other hand, functional genes related to hydrogen consumption decreased (1.78 fold for ldh, 0.67 fold for adh, 1.64 fold for hbd, 2.4 fold for crt, and 0.71 fold for buk). Considered together, these results suggested that GAC supplementation increased biohydrogen production by changing the metabolic flux associated with hydrogen production and consumption pathways.

Mitigation of membrane biofouling by a quorum quenching bacterium for membrane bioreactors.

In this study, a quorum-quenching (QQ) bacterium named HEMM-1 was isolated at a membrane bioreactor (MBR) plant. HEMM-1 has diplococcal morphology and 99% sequence identity to Enterococcus species. The HEMM-1 cell-free supernatant (CFS) showed higher QQ activities than the CFS of other QQ bacteria, mostly by degrading N-acyl homoserine lactones (AHLs) with short acyl chains. Instrumental analyses revealed that HEMM-1 CFS degraded AHLs via lactonase activity. Under static, flow, and shear conditions, the HEMM-1 CFS was effective in reducing bacterial and activated-sludge biofilms formed on membrane surfaces. In conclusion, the HEMM-1 isolate is a QQ bacterium applicable to the control of biofouling in MBRs via inhibition of biofilm formation on membrane surfaces.

Structure-Activity Relationships of 6- and 8-Gingerol Analogs as Anti-Biofilm Agents.

Pseudomonas aeruginosa is a causative agent of chronic infections in immunocompromised patients. Disruption of quorum sensing circuits is an attractive strategy for treating diseases associated with P. aeruginosa infection. In this study, we designed and synthesized a series of gingerol analogs targeting LasR, a master regulator of quorum sensing networks in P. aeruginosa. Structure-activity relationship studies showed that a hydrogen-bonding interaction in the head section, stereochemistry and rotational rigidity in the middle section, and optimal alkyl chain length in the tail section are important factors for the enhancement of LasR-binding affinity and for the inhibition of biofilm formation. The most potent compound 41, an analog of (R)-8-gingerol with restricted rotation, showed stronger LasR-binding affinity and inhibition of biofilm formation than the known LasR antagonist (S)-6-gingerol. This new LasR antagonist can be used as an early lead compound for the development of anti-biofilm agents to treat P. aeruginosa infections.