• Journal of Microbiology and Biotechnology
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• v.25 no.11
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• pp.1908-1919
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• 2015

This work investigated the potential of curcumin (CCM) and (-)-epigallocatechin gallate (EGCG) to inhibit N-acyl homoserine lactone (AHL)-mediated biofilm formation in gram-negative bacteria from membrane bioreactor (MBR) activated sludge. The minimum inhibitory concentrations (MICs) of CCM alone against all the tested bacteria were 200-350 μg/ml, whereas those for EGCG were 300-600 μg/ml. Biofilm formation at one-half MICs indicated that CCM and EGCG alone respectively inhibited 52-68% and 59-78% of biofilm formation among all the tested bacteria. However, their combination resulted in 95-99% of biofilm reduction. Quorum sensing inhibition (QSI) assay with known biosensor strains demonstrated that CCM inhibited the expression of C4 and C6 homoserine lactones (HSLs)-mediated phenotypes, whereas EGCG inhibited C4, C6, and C10 HSLs-based phenotypes. The Center for Disease Control biofilm reactor containing a multispecies culture of nine bacteria with one-half MIC of CCM (150 μg/ml) and EGCG (275 μg/ml) showed 17 and 14 μg/cm² of extracellular polymeric substances (EPS) on polyvinylidene fluoride membrane surface, whereas their combination (100 μg/ml of each) exhibited much lower EPS content (3 μg/cm²). Confocal laser scanning microscopy observations also illustrated that the combination of compounds tremendously reduced the biofilm thickness. The combined effect of CCM with EGCG clearly reveals for the first time the enhanced inhibition of AHL-mediated biofilm formation in bacteria from activated sludge. Thus, such combined natural QSI approach could be used for the inhibition of membrane biofouling in MBRs treating wastewaters.

• ALGAE
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• v.30 no.2
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• pp.147-161
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• 2015

Brown algal extracts have long been used as feed supplements to promote health of farm animals. Here, we show new molecular insights in to the mechanism of action of a fucose containing polymer (FCP) rich fraction from the brown seaweed Ascophyllum nodosum using the Caenorhabditis elegans-Pseudomonas aeruginosa PA14 infection model. FCP enhanced survival of C. elegans against pathogen stress, correlated with up-regulation of key immune response genes such as: lipases, lysozyme (lys-1), saponin-like protein (spp-1), thaumatin-like protein (tlp-1), matridin SK domain protein (msk-1), antibacterial protein (abf-1), and lectin family protein (lfp). Further, FCP caused down regulation of P. aeruginosa quorum sensing genes: (lasI, lasR, rhlI, and rhlR), secreted virulence factors (lipase, proteases, and elastases) and toxic metabolites (pyocyanin, hydrogen cyanide, and siderophore). Biofilm formation and motility of pathogenic bacteria were also greatly attenuated when the culture media were treated with FCP. Interestingly, FCP failed to mitigate the pathogen stress in skn-1, daf-2, and pmk-1 mutants of C. elegans. This indicated that, FCP treatment acted on the regulation of fundamental innate immune pathways, which are conserved across the majority of organisms including humans. This study suggests the possible use of FCP, a seaweed component, as a functional food source for healthy living.

• Journal of Dairy Science and Biotechnology
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• v.33 no.1
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• pp.59-67
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• 2015

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is well-characterized as an important food-borne pathogen worldwide and causes human diseases such as diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS) by producing shiga-like toxin (Stx). It has been reported that a number of dairy foods, including cheese, can act as the source of EHEC O157:H7 infections. In addition to the toxicity of Stx, recently it has been indicated that EHEC O157:H7 possesses virulence factors related to attachment, quorum sensing, and biofilms. Moreover, these novel virulence factors might become critical points to be considered in the future production of food derived from animals. Here, we review the evidences that support these insights on new virulence factors and discuss the potential mechanisms mediating the pathogenesis of EHEC O157:H7 in the dairy food industry.

• Molecules and Cells
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• v.37 no.6
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• pp.480-486
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• 2014

Growth restriction by antibiotics is a common feature that pathogenic bacteria must overcome for survival. The struggle of bacteria to escape from growth restriction eventually results in development of antibiotic-resistance through the expression of a set of genes. Here we found that some physiologically important transcriptional regulators of Pseudomonas aeruginosa including QscR, a quorum sensing (QS) receptor, SoxR, a superoxide sensor-regulator, and AntR, a regulator of anthranilate-related secondary metabolism, are activated by various growth-restricted conditions. We generated the growth-restricted conditions by various methods, such as overexpression of PA2537 and treatment with antibiotics or disinfectants. The overexpression of PA2537, encoding an acyltransferase homologue, tightly restricted the growth of P. aeruginosa and significantly activated QscR during the growth restriction. Similarly, treatments with gentamycin, tetracycline, and ethanol also activated QscR near their minimal inhibitory concentrations (MICs). Some non-QS regulators, such as AntR and SoxR, were also activated near the MICs in the same conditions. However, LasR and PqsR, other QS receptors of P. aeruginosa, were not activated, suggesting that only a specific set of transcriptional regulators is activated by growth restriction. Since paraquat, a superoxide generator, significantly activated QscR and AntR, we suggest that the oxidative stress generated by growth restriction may be partly involved in this phenomenon.

• Journal of Microbiology and Biotechnology
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• v.25 no.8
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• pp.1299-1306
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• 2015

This study aims to investigate the mechanism of action of the cinnamon bark essential oil (CB), when used singly and also in combination with piperacillin, for its antimicrobial and synergistic activity against beta-lactamase TEM-1 plasmid-conferred Escherichia coli J53 R1. Viable count of bacteria for this combination of essential oil and antibiotic showed a complete killing profile at 20 h and further confirmed its synergistic effect by reducing the bacteria cell numbers. Analysis on the stability of treated cultures for cell membrane permeability by CB when tested against sodium dodecyl sulfate revealed that the bacterial cell membrane was disrupted by the essential oil. Scanning electron microscopy observation and bacterial surface charge measurement also revealed that CB causes irreversible membrane damage and reduces the bacterial surface charge. In addition, bioluminescence expression of Escherichia coli [pSB1075] and E. coli [pSB401] by CB showed reduction, indicating the possibility of the presence of quorum sensing (QS) inhibitors. Gas-chromatography and mass spectrometry of the essential oil of Cinnamomum verum showed that trans-cinnamaldehyde (72.81%), benzyl alcohol (12.5%), and eugenol (6.57%) were the major components in the essential oil. From this study, CB has the potential to reverse E. coli J53 R1 resistance to piperacillin through two pathways; modification in the permeability of the outer membrane or bacterial QS inhibition.

• Animal Bioscience
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• v.37 no.2_spc
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• pp.337-345
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• 2024

Ruminants possess a specialized four-compartment forestomach, consisting of the reticulum, rumen, omasum, and abomasum. The rumen, the primary fermentative chamber, harbours a dynamic ecosystem comprising bacteria, protozoa, fungi, archaea, and bacteriophages. These microorganisms engage in diverse ecological interactions within the rumen microbiome, primarily benefiting the host animal by deriving energy from plant material breakdown. These interactions encompass symbiosis, such as mutualism and commensalism, as well as parasitism, predation, and competition. These ecological interactions are dependent on many factors, including the production of diverse molecules, such as those involved in quorum sensing (QS). QS is a density-dependent signalling mechanism involving the release of autoinducer (AIs) compounds, when cell density increases AIs bind to receptors causing the altered expression of certain genes. These AIs are classified as mainly being N-acyl-homoserine lactones (AHL; commonly used by Gram-negative bacteria) or autoinducer-2 based systems (AI-2; used by Gram-positive and Gram-negative bacteria); although other less common AI systems exist. Most of our understanding of QS at a gene-level comes from pure culture in vitro studies using bacterial pathogens, with much being unknown on a commensal bacterial and ecosystem level, especially in the context of the rumen microbiome. A small number of studies have explored QS in the rumen using 'omic' technologies, revealing a prevalence of AI-2 QS systems among rumen bacteria. Nevertheless, the implications of these signalling systems on gene regulation, rumen ecology, and ruminant characteristics are largely uncharted territory. Metatranscriptome data tracking the colonization of perennial ryegrass by rumen microbes suggest that these chemicals may influence transitions in bacterial diversity during colonization. The likelihood of undiscovered chemicals within the rumen microbial arsenal is high, with the identified chemicals representing only the tip of the iceberg. A comprehensive grasp of rumen microbial chemical signalling is crucial for addressing the challenges of food security and climate targets.

• Microbiology and Biotechnology Letters
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• v.37 no.3
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• pp.248-257
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• 2009

The inhibitors against Vibrio harveyi quorum sensing (QS) signaling were developed by modifying the molecular structure of the major signal, N-3-hydroxybutanoyl-L-homoserine lactone (3-OH-$C_4$-HSL). A series of structural derivatives, N-(3-hydroxysulfonyl)-L-homoserine lactones (HSHLs) were synthesized by the solid-phase organic synthesis method. The in vivo QS inhibition by these compounds was measured by a bioassay system using the V. harveyi bioluminescence, and all showed significant inhibitory effects. To analyze the interaction between these compounds and LuxN, a 3-OH-$C_4$-HSL receptor protein of V. harveyi, we tentatively determined the putative signal binding domain of LuxN based on the sequence homology with other acyl-HSL binding proteins, and predicted the partial 3-D structure of the putative signal binding domain of LuxN by using ORCHESTRA program, and further estimated the binding poses and energies (docking scores) of 3-OH-$C_4$-HSL and HSHLs within the domain. In comparison of the result from this modeling study with that of in vivo bioassay, we suggest that the in silica interpretation of the interaction between ligands and their receptor proteins can be a valuable way to develop better competitive inhibitors, especially in the case that the structural information of the protein is limited.

• Korean Journal of Microbiology
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• v.48 no.3
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• pp.180-186
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• 2012

Pseudomonas aeruginosa, a Gram-negative bacterium, is an ubiquitous and opportunistic human pathogen, which expresses many virulence factors through quorum sensing (QS) regulation. QscR, one of the QS signal receptors of P. aeruginosa, has unique features that make it possible to distinguish QscR from other QS receptors. In the present study, we focused on amino acid residues responsible for such a broad signal specificity of QscR. Thus we constructed mutant QscRs: $QscR_{T72I}$, $QscR_{R132M}$, and $QscR_{T140I}$ by substituting $72^{nd}$ threonine, $132^{nd}$ arginine, and $140^{th}$ threonine residues with isoleucine, methionine, and isoleucine, respectively by site-directed mutagenesis. When we examined the activity of these mutant QscRs, $QscR_{R132M}$ failed to respond to N-3-oxododecanoyl homoserine lactone (3OC12-HSL), but $QscR_{T72I}$ and $QscR_{T140I}$ remained the ability to respond to 3OC12-HSL despite much reduction of the sensitivity. When we treated a variety of acyl-HSLs with different structure, $QscR_{T72I}$ and $QscR_{T140I}$ showed better responsiveness to N-decanoyl HSL (C10-HSL) or N-dodecanoyl HSL (C12-HSL) that has no oxo-moiety at $3^{rd}$ carbon of acyl group than to 3OC12-HSL, and $QscR_{R132M}$ showed no responsiveness to any acyl-HSLs tested here. In addition, $QscR_{T72I}$ and $QscR_{T140I}$ were inhibited by 5f, a QscR inhibitor as similarly as wild type QscR was. These results suggest that while the $130^{th}$ arginine is crucial in both activity and acyl-HSL binding of QscR, the $72^{nd}$ and $140^{th}$ threonines are important in the activity, but they are little responsible for the discrimination of acyl-HSLs or competitive inhibitor.

• Journal of Life Science
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• v.27 no.10
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• pp.1161-1167
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• 2017

In order to find a new antimicrobial bacterium, we performed screening for antimicrobial activity of bacteria isolated from the eggs of a sea hare. The newly identified strain was designated as Phaeobacter inhibens KJ-2, based on the biochemical characterization and 16S rRNA gene sequence analysis. A colony of P. inhibens KJ-2 showed a circular and ruler-like smooth form at the edge, and a brown color. However, when maintained with a longer incubation time, its coloring was transformed into dark brown. From the result of SEM, P. inhibens KJ-2 is a bacillus which has a length of $0.8{\sim}1.0{\mu}m$ and a width of $0.4{\sim}0.6{\mu}m$. The optimal growth and antimicrobial activity were observed by shaking the culture for 24 hr at $20^{\circ}C$, which showed potent activity against pathogenic bacteria including Vibrio logei, Vibrio campbellii, Vibrio mimicus, Vibrio vulnificus, and Vibrio salmonicida. The antimicrobial activity was proportional to the amount of produced acylated homoserine lactones (AHLs). Therefore, we suggest that production of antimicrobial materials from P. inhibens KJ-2 is regulated by Quorum sensing (QS).

• Korean Journal of Microbiology
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• v.46 no.3
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• pp.240-247
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• 2010

Pseudomonas aeruginosa is a Gram (-) opportunistic human pathogen causing a wide variety of infections on lung, urinary tract, eyes, and burn wound sites and quorum sensing (QS), a cell density-sensing mechanism plays an essential role in Pseudomonas pathogenesis. In order to investigate the importance of QS in the Pseudomonas infections of Korean patients, we isolated 189 clinical strains of P. aeruginosa from the patients in Pusan Paik Hospital, Busan, South Korea. The QS signal production of these clinical isolates was measured by signal diffusion assay on solid media using reporter strains. While most clinical strains (79.4%) produced the QS signals as similar level as a wild type strain, PAO1 did, where LasR, the initial QS signal sensor-regulator was fully activated, a minority of them (4.2%) produced much less QS signals at the level to which LasR failed to respond. Similarly, while 72.5% of the clinical isolates produced QS signals enough to activate QscR, an another QS signal sensor-regulator, some few of them (9%) produced the QS signals at much lower level where QscR was not activated. For further analysis, we selected 74 clinical strains that were obtained from the patients under suspicion of Pseudomonas infection and investigated the total protease activity that is considered important for virulence. Interestingly, significant portion of them showed very low protease activity (44.6%) or no detectable protease activity (12.2%). When the biofilm-forming ability that is considered very important in chronic infection was examined, most isolates showed lower biofilm-forming activity than PAO1. Similarly, significant portion of clinical isolates showed reduced motility (reduced swarming activity in 51.4% and reduced twitching activity in 41.9%), or non-detectable motility (swarming-negative in 28.4% and twitching-negative in 28.4%). Our result showed that the clinical isolates that produced QS signals at the similar level to wild type could have significantly reduced activities in the protease production, biofilm formation, and motility, and some clinical isolates had unique patterns of motility, biofilm formation, and protease production that are not correlated to their QS activity.