Linking microbial slime community structure with abiotic factors and antifouling strategy in hydroelectric cooling systems.

Microfouling can have significant economic impacts for hydroelectric power plants. However, knowledge concerning the composition and metabolism of microbial biofilm in cooling systems remains scarce. We examined the metagenome present in a cooling system, comprising a filter (F) and heat exchanger (HE), in the Nova Ponte hydroelectric power plant in Brazil, to identify bacteria and pathways that could be targeted to monitor and control biofilm formation. Our data revealed that the microfouling sample from heat exchanger 1 (HEM1), with porous consistency, presented enriched bacterial members not frequently described as biofilm formers in cooling systems, besides it has been shown to be an autoinducer repression pathway. Furthermore, the microfouling sample from heat exchanger 2 (HEM2), with gelatinous consistency, seemed to be an established biofilm, containing enriched bacterial groups such as Desulfotomaculum and Crenothrix and autoinducers, with biotechnological relevance in industrial biofilms. The results demonstrate that biofilm composition will vary depending on different abiotic conditions and the antifouling strategy used, including type of compound, concentration, and frequency of use. Therefore, all these variables must be evaluated when a power plant is affected by microbial slime in the cooling system. Our findings could help to define strategies for efficient and ecofriendly measures to contain microfouling in power plants.

The use of synthetic agonists of quorum sensing N- acyl homoserine lactone pathway improves the bioleaching ability in Acidithiobacillus and Pseudomonas bacteria.

Metal solubilization from discarded electrical material and electronic devices (e-waste) using the bioleaching capabilities of bacterial cells is highly effective. However, gaps in understanding about the microbiological processes involved in the bioleaching reaction leads to less efficient metal solubilization in large-scale e-waste processing. In this study, bacterial species belonging to the genera Acidithiobacillus and Pseudomonas were used to leach copper and gold from discarded printed circuit boards (PCB). Through modulation of the cell-to-cell communication system in these bacteria, phenotypic traits directly involved in the bioleaching reaction were regulated in order to improve the metal solubilization. Addition of the long chain synthetic autoinducer molecule N-acyl homoserine lactone (AHL) of the quorum sensing pathway to the bioleaching reaction resulted in a significant enhancement of metal extraction from PCB. Factors such as: cell attachment to PCB, biofilm formation and hydrogen cyanide (HCN) production were regulated by the quorum sensing system and could be directly related to the improvement of metal bioleaching. Bioleaching reactions using bacterial quorum sensing modulation could represent a valuable tool in overcoming limitations at the industrial level imposed by microbiological traits that lead to inefficient metal bioleaching from e-waste.

N-acyl-homoserine lactone produced by Rahnella inusitata isolated from the gut of Galleria mellonella influences Salmonella phenotypes.

The most studied mechanism of quorum sensing in Gram-negative bacteria is mediated by autoinducer 1 (AI-1), namely, acyl-homoserine lactone (AHL). This system allows communication among different bacterial species and regulates the expression of virulence genes in many pathogens. Although AHL-producing bacteria have been detected in the intestines of humans and other animals, no report was found about AHL-producing bacteria in the insect gut and the possible effects of these autoinducers on enteropathogenic bacteria. Therefore, this study aimed to identify AHL-producing bacteria in the gut of larvae of Galleria mellonella and to evaluate the influence of this quorum sensing signal on the regulation of adhesion and motility phenotypes in the intestinal pathogen Salmonella. Sequencing of the 16S rRNA gene, 16S rRNA gene-based phylogenetic analyses, and phenotypic characterization of gut isolates was performed. The profile of AHLs produced by the isolates was determined using thin-layer chromatography (TLC) and revealed with the biosensor strain Chromobacterium violaceum CV026. Sequencing, phylogenetic analyses and phenotypic characterization of gut isolates showed that the three AHL-producing strains belong to the species Rahnella inusitata, named GM34, GM56, and GM60. The TLC showed that R. inusitata produces a six-carbon AHL. In the presence of cell-free extract of R. inusitata containing AHL and under anaerobic conditions, Salmonella enterica increased the adhesion to stainless steel coupons and presented swarming motility. Extracts from the culture medium of R. inusitata isolates containing AHL increased the adhesion on stainless steel coupons and swarming motility of Salmonella enterica serovar Enteritidis PT4 under anaerobic conditions. The results suggest the possibility of communication between members of the G. mellonella intestinal microbiota with pathogens such as Salmonella.

Quorum Sensing Regulation as a Target for Antimicrobial Therapy.

Some bacterial species use a cell-to-cell communication mechanism called Quorum Sensing (QS). Bacteria release small diffusible molecules, usually termed signals which allow the activation of beneficial phenotypes that guarantee bacterial survival and the expression of a diversity of virulence genes in response to an increase in population density. The study of the molecular mechanisms that relate signal molecules with bacterial pathogenesis is an area of growing interest due to its use as a possible therapeutic alternative through the development of synthetic analogues of autoinducers as a strategy to regulate bacterial communication as well as the study of bacterial resistance phenomena, the study of these relationships is based on the structural diversity of natural or synthetic autoinducers and their ability to inhibit bacterial QS, which can be approached with a molecular perspective from the following topics: i) Molecular signals and their role in QS regulation; ii) Strategies in the modulation of Quorum Sensing; iii) Analysis of Bacterial QS circuit regulation strategies; iv) Structural evolution of natural and synthetic autoinducers as QS regulators. This mini-review allows a molecular view of the QS systems, showing a perspective on the importance of the molecular diversity of autoinducer analogs as a strategy for the design of new antimicrobial agents.

Salmonella enterica Optimizes Metabolism After Addition of Acyl-Homoserine Lactone Under Anaerobic Conditions.

Acyl-homoserine lactones (AHLs) are quorum sensing (QS) signaling molecules that mediate cell-to-cell communication in Gram-negative bacteria. Salmonella does not produce AHL, however, it can recognize AHLs produced by other species through SdiA protein modulating important cellular functions. In this work, the influence of the N-dodecanoyl-DL-homoserine lactone (C12-HSL) on glucose consumption, metabolic profile, and gene expression of Salmonella throughout the cultivation time in Tryptic Soy Broth (TSB) under anaerobic conditions was evaluated. Analysis of the supernatant culture in high-performance liquid chromatography (HPLC) revealed lower glucose uptake after 4 and 6 h of the addition of C12-HSL. Gas chromatography-mass spectrometry (GC-MS) based analysis of the intracellular metabolites revealed C12-HSL perturbation in the abundance levels of metabolites related to the metabolic pathways of glycerolipids, purines, amino acids, and aminoacyl-tRNA biosynthesis. The real-time quantitative PCR (RT-qPCR) indicated that Salmonella increase expression of genes associated with nucleoside degradation and quantification of metabolites supported the induction of pentose phosphate pathway to ensure growth under lower glucose consumption. The obtained data suggest an important role of C12-HSL in the optimization of metabolism at a situation of high population densities.

Analysis of two Mexican Pectobacterium brasiliense strains reveals an inverted relationship between c-di-GMP levels with exopolysaccharide production and swarming motility.

Pectobacterium is a diverse genus of phytopathogenic species from soil and water that cause infection either to restricted or multiple plant hosts. Phylogenetic analysis and metabolic fingerprinting of large numbers of genomes have expanded classification of Pectobacterium members. Pectobacterium brasiliense sp. nov has been elevated to the species level having detached from P. carotovorum. Here we present two P. brasiliense strains BF20 and BF45 isolated in Mexico from Opuntia and tobacco, respectively, which cluster into two different groups in whole genome comparisons with other Pectobacterium. We found that BF20 and BF45 strains are phenotypically different as BF45 showed more severe and rapid symptoms in comparison to BF20 in the host models celery and broccoli. Both strains produced similar levels of the main autoinducers, but BF45 shows an additional low abundant autoinducer compared to strain BF20. The two strains had different levels of c-di-GMP, which regulates the transition from motile to sessile lifestyle. In contrast to BF45, BF20 had the highest levels of c-di-GMP, was more motile (swarming), non-flocculant and less proficient in biofilm formation and exopolysaccharide production. Genomic comparisons revealed that differences in c-di-GMP accumulation and perhaps the associated phenotypes might be due to unique c-di-GMP metabolic genes in these two strains. Our results improve our understanding of the associations between phenotype and genotype and how this has shaped the physiology of Pectobacterium strains.

Virulence of Pseudomonas aeruginosa exposed to carvacrol: alterations of the Quorum sensing at enzymatic and gene levels.

The main goal of this study was to evaluate the inhibition of Pseudomonas aeruginosa virulence factors and Quorum Sensing during exposure to carvacrol. P. aeruginosa (ATCC 10154) was exposed to carvacrol determining changes in biofilm development, motility, acyl-homoserine lactones (AHL) synthesis and relative expression of lasI/lasR. Docking analysis was used to determinate interactions between carvacrol with LasI and LasR proteins. P. aeruginosa produced 60% lower AHLs when exposed to carvacrol (1.9 mM) compared to control, without affecting cellular viability, indicating a reduction on the LasI synthase activity. AHL-C12, C6, and C4 were detected and related to biofilm development, motility, and pyocyanin production, respectively. The presence of carvacrol reduced the expression of lasR, without affecting lasI gen. Moreover, computational docking showed interactions of carvacrol with amino acids in the active site pocket of LasI (-5.6 kcal mol-1) and within the binding pocket of LasR (-6.7 kcal mol-1) of P. aeruginosa. These results demonstrated that virulence of P. aeruginosa was reduced by carvacrol, by inhibiting LasI activity with the concomitant reduction on the expression of lasR, biofilm and swarming motility. This study provides relevant information about the effect of carvacrol against quorum sensing to inhibit virulence factors of P. aeruginosa at enzymatic and gene levels. These findings can contribute to the development of natural anti-QS products, which can affect pathogenesis.

Acyl homoserine lactone-based quorum sensing stimulates biofilm formation by Salmonella Enteritidis in anaerobic conditions.

Quorum sensing regulates a variety of phenotypes in bacteria including the production of virulence factors. Salmonella spp. have quorum sensing systems mediated by three autoinducers (AI-1, AI-2, and AI-3). The AI-1-mediated system is incomplete in that the bacterium relies on the synthesis of signaling molecules by other microorganisms. This study aimed to evaluate the influence of the AI-1 N-dodecanoyl-DL-homoserine lactone (C12-HSL) on the growth, motility, adhesion, and biofilm formation of Salmonella enterica serovar Enteritidis PT4 578 on a polystyrene surface. Experiments were conducted at 37 °C in anaerobic tryptone soy broth supplemented with C12-HSL and/or a mixture of four synthetic furanones, at the concentration of 50 nM each. The planktonic growth, adhesion, swarming, and twitching motility were not altered in the presence of C12-HSL and/or furanones under anaerobic conditions. However, C12-HSL induced biofilm formation after 36 h of cultivation as determined by quantification of biofilm formation, by enumeration of adhered cells to polystyrene coupons, and finally by imaging the presence of multilayered cells on an epifluorescence microscope. When furanones were present in the medium, an antagonistic effect against C12-HSL on the biofilm development was observed. The results demonstrate an induction of biofilm formation in Salmonella Enteritidis by AI-1 under anaerobic conditions. Considering that Salmonella does not produce AI-1 but respond to it, C12-HSL synthesized by other bacterial species could trigger biofilm formation by this pathogen in conditions that are relevant for its pathogenesis.

Acyl homoserine lactone changes the abundance of proteins and the levels of organic acids associated with stationary phase in Salmonella Enteritidis.

Quorum sensing (QS) is cell-cell communication mechanism mediated by signaling molecules known as autoinducers (AIs) that lead to differential gene expression. Salmonella is unable to synthesize the AI-1 acyl homoserine lactone (AHL), but is able to recognize AHLs produced by other microorganisms through SdiA protein. Our study aimed to evaluate the influence of AI-1 on the abundance of proteins and the levels of organic acids of Salmonella Enteritidis. The presence of N-dodecyl-homoserine lactone (C12-HSL) did not interfere on the growth or the total amount of extracted proteins of Salmonella. However, the abundance of the proteins PheT, HtpG, PtsI, Adi, TalB, PmgI (or GpmI), Eno, and PykF enhanced while the abundance of the proteins RplB, RplE, RpsB, Tsf, OmpA, OmpC, OmpD, and GapA decreased when Salmonella Enteritidis was anaerobically cultivated in the presence of C12-HSL. Additionally, the bacterium produced less succinic, lactic, and acetic acids in the presence of C12-HSL. However, the concentration of extracellular formic acid reached 20.46 mM after 24 h and was not detected when the growth was in the absence of AI-1. Considering the cultivation period for protein extraction, their abundance, process and function, as well as the levels of organic acids, we observed in cells cultivated in presence of C12-HSL a correlation with what is described in the literature as entry into the stationary phase of growth, mainly related to nitrogen and amino acid starvation and acid stress. Further studies are needed in order to determine the specific role of the differentially abundant proteins and extracellular organic acids secreted by Salmonella in the presence of quorum sensing signaling molecules.

Novel insights from molecular docking of SdiA from Salmonella Enteritidis and Escherichia coli with quorum sensing and quorum quenching molecules.

Quorum sensing is a cell-to-cell communication mechanism leading to differential gene expression in response to high population density. The autoinducer-1 (AI-1) type quorum sensing system is incomplete in Escherichia coli and Salmonella due to the lack of the AI-1 synthase (LuxI homolog) responsible for acyl homoserine lactone (AHL) synthesis. However, these bacteria encode the AHL receptor SdiA (a LuxR homolog) leading to gene regulation in response to AI-1 produced by other bacteria. This study aimed to model the SdiA protein of Salmonella enterica serovar Enteritidis PT4 578 based on three crystallized SdiA structures from Enterohemorrhagic E. coli (EHEC) with different ligands. Molecular docking of these predicted structures with AHLs, furanones and 1-octanoyl-rac-glycerol were also performed. The available EHEC SdiA structures provided good prototypes for modeling SdiA from Salmonella. The molecular docking of these proteins showed that residues Y63, W67, Y71, D80 and S134 are common binding sites for different quorum modulating signals, besides being conserved among other LuxR type proteins. We also show that AHLs with twelve carbons presented better binding affinity to SdiA than AHLs with smaller side chains in our docking analysis, regardless of the protein structures used. Interestingly, the conformational changes provided by AHL binding resulted in structural models with increased affinities to brominated furanones. These results suggest that the use of brominated furanones to inhibit phenotypes controlled by quorum sensing in Salmonella and EHEC may present a good strategy since these inhibitors seem to specifically compete with AHLs for binding to SdiA in both pathogens.

Inducers and autoinducers on Salmonella enterica serovar Typhimurium motility, growth and gene expression / Indutores e autoindutores na motilidade, no crescimento e na expressão gênica de Salmonella enterica serovar Typhimurium

Genus Salmonella bacteria are among the major pathogenic microorganisms in food. This bacterium pathogenicity is related to a number of virulence factors, among which its flagella. Flagellum expression is one of the virulence factors modulated by Quorum Sensing. Epinephrine produced by mammals uses the same signaling pathway of the 3 bacteria autoinducer. This study evaluated the effect of molecules inducer (epinephrine) and autoinducers (autoinducer 2 and autoinducer 3) and their association with the motility, growth and expression genes flhC, fliA, fliY, motA, motB e fliC of Salmonella Typhimurium (ST). Initially, ST was inoculated in BHI. Then, motility assays, growth curves and gene expression were performed by testing different concentrations of epinephrine (50, 125, 250, 500µM), conditioned medium (10 and 50%) and a combination of these. ST was exposed to different concentrations of epinephrine, conditioned medium and an association of both. Following, motility assays, bacterial growth and gene expression were performed. The results obtained showed that the combination of 500uM epinephrine with 50% conditioned medium increased ST bacterial motility by increasing the expression of genes involved in flagellum assembly.

Salmonella está entre os principais micro-organismos patogênicos veiculados por alimentos. A patogenicidade dessa bactéria está relacionada a uma série de fatores de virulência e, dentre estes, podemos citar os flagelos. A expressão do flagelo está entre os fatores de virulência modulados por Quorum Sensing. A adrenalina produzida pelos mamíferos utiliza a mesma via de sinalização do autoindutor 3 das bactérias. Nesse sentido, este trabalho teve como objetivo avaliar o efeito de moléculas indutora (adrenalina) e autoindutoras (auto-indutor 2 e auto-indutor 3) e a associação destas na motilidade, no crescimento celular e na expressão dos genes flhC, fliA, fliY, motA, motB e fliC de SalmonellaTyphimurium (ST). Inicialmente, ST foi semeada em caldo BHI. Após, ensaios de motilidade, curvas de crescimento e expressão gênica foram feitos, testando diferentes concentrações de adrenalina (50, 125, 250, 500µM), meio condicionado (10 e 50%) e a associação destes. A partir dos resultados obtidos, observou-se que o tratamento que utilizou 50% de meio condicionado + 500µM de adrenalina aumentou a motilidade de ST, em decorrência do aumento de genes envolvidos com montagem do flagelo.

Inducers and autoinducers on Salmonella enterica serovar Typhimurium motility, growth and gene expression / Indutores e autoindutores na motilidade, no crescimento e na expressão gênica de Salmonella enterica serovar Typhimurium

Genus Salmonella bacteria are among the major pathogenic microorganisms in food. This bacterium pathogenicity is related to a number of virulence factors, among which its flagella. Flagellum expression is one of the virulence factors modulated by Quorum Sensing. Epinephrine produced by mammals uses the same signaling pathway of the 3 bacteria autoinducer. This study evaluated the effect of molecules inducer (epinephrine) and autoinducers (autoinducer 2 and autoinducer 3) and their association with the motility, growth and expression genes flhC, fliA, fliY, motA, motB e fliC of Salmonella Typhimurium (ST). Initially, ST was inoculated in BHI. Then, motility assays, growth curves and gene expression were performed by testing different concentrations of epinephrine (50, 125, 250, 500µM), conditioned medium (10 and 50%) and a combination of these. ST was exposed to different concentrations of epinephrine, conditioned medium and an association of both. Following, motility assays, bacterial growth and gene expression were performed. The results obtained showed that the combination of 500uM epinephrine with 50% conditioned medium increased ST bacterial motility by increasing the expression of genes involved in flagellum assembly.(AU)

Salmonella está entre os principais micro-organismos patogênicos veiculados por alimentos. A patogenicidade dessa bactéria está relacionada a uma série de fatores de virulência e, dentre estes, podemos citar os flagelos. A expressão do flagelo está entre os fatores de virulência modulados por Quorum Sensing. A adrenalina produzida pelos mamíferos utiliza a mesma via de sinalização do autoindutor 3 das bactérias. Nesse sentido, este trabalho teve como objetivo avaliar o efeito de moléculas indutora (adrenalina) e autoindutoras (auto-indutor 2 e auto-indutor 3) e a associação destas na motilidade, no crescimento celular e na expressão dos genes flhC, fliA, fliY, motA, motB e fliC de Salmonella Typhimurium (ST). Inicialmente, ST foi semeada em caldo BHI. Após, ensaios de motilidade, curvas de crescimento e expressão gênica foram feitos, testando diferentes concentrações de adrenalina (50, 125, 250, 500µM), meio condicionado (10 e 50%) e a associação destes. A partir dos resultados obtidos, observou-se que o tratamento que utilizou 50% de meio condicionado + 500µM de adrenalina aumentou a motilidade de ST, em decorrência do aumento de genes envolvidos com montagem do flagelo.(AU)

Production of AI-2 is mediated by the S-ribosylhomocystein lyase gene luxS in Bacteroides fragilis and Bacteroides vulgatus.

Quorum sensing is a cell-cell signaling mechanism based on cell density and that involves the production of hormone-like molecules called autoinducers (AI). One of the most studied AIs has been termed AI-2, and its biosynthesis requires the enzyme encoded by luxS. We have previously described for the first time that Bacteroides species can produce molecules with AI-2 activity. In this study, we focus on the detection of luxS and its activity as the AI-2 synthase in Bacteroides species. The strains Bacteroides fragilis B3b and Bacteroides vulgatus ATCC 8482 were selected based on a positive phenotype for AI-2 production and the presence of a putative luxS in the genome, respectively. In order to identify the luxS gene, cloning and heterologous expression strategies were utilized. We demonstrate that both strains contain functional luxS orthologs that can complement AI-2 production in Escherichia coli.

The use of quorum sensing to improve vaccine immune response.

Enterotoxigenic Escherichia coli (ETEC) infection is an important cause of diarrhea in both newborn and post-weaning pigs, it is also responsible for economic losses on farms worldwide. Vaccines that use ETEC virulence factors have been well documented, and several vaccines containing inactivated bacteria with protective antigens, or purified (isolated) antigens are available on the market. Vaccination of pregnant sows is widely seen as an effective strategy for the control of the disease. Yet these vaccines very often do not lead to efficient protection. In this study, we produced an ETEC bacterin with the use of quorum sensing (QS), and observed a significant expression of F4 adhesin, and heat-labile toxin (LT) in the cultures when compared to the controls. Mice, and pigs vaccinated with the QS bacterin demonstrated higher antibody titers against these antigens when compared with commercial and control bacterin. Our results suggest that the system might bring promising improvements in ETEC bacterin efficacy.

El impacto biológico de los autoinductores bacterianos / The biological impact of bacterial autoinducers

Las bacterias, a pesar de ser organismos unicelulares, presentan una gran complejidad. Durante mucho tiempo fueron consideradas como organismos asociales cuyas funciones principales eran el nutrirse y el reproducirse. Sin embargo, se ha observado que las bacterias son los microorganismos con la mayor capacidad de adaptación a ambientes diversos, además responden a múltiples estímulos, tanto nutricionales como ambientales (pH, disponibilidad de oxígeno, osmolaridad, etc.). En las últimas décadas se ha reportado que tanto las bacterias grampositivas como las gramnegativas son capaces de comunicarse entre si mediante sistemas especializados de comunicación celular. A tales sistemas se les ha denominado “sistemas de señalización” y “autoinductores” a las moléculas señal que desencadenan diferentes respuestas celulares, como la formación de biopelículas, la transformación bacteriana, la producción de bioluminiscencia, la producción de antibióticos o de factores de virulencia, entre otras. En este trabajo se presentan los aspectos más relevantes relacionados a los autoinductores de bacterias grampositivas y gramnegativas, así como su participación en diferentes procesos biológicos.

Bacteria, in spite of being unicellular organisms, present great complexity. During a long time they were considered as asocial organisms whose main functions were feeding and reproducing. Nevertheless, it has been observed that bacteria are the microorganisms with the greatest capacity for adapting to diverse environments, also responding to multiple stimuli, both nutritional and environmental (pH, oxygen availability, osmolarity, etc.). During the last decades it has been reported that bacteria, both gram negative and gram positive, are capable of communicating among them through specialized cell-communication systems. These systems have been called “signaling systems” and the signaling molecules which unchain the various cell responses such as biofilm formation, bacterial transformation, luminescence production, antibiotic production, or virulence factor production, among others, have been called “autoinducers”. This paper presents the most relevant aspects related with gram positive and gram negative bacteria autoinducers, as well as their participation in different biological processes.