Search Results (219)

Antibiotic misuse accelerates resistance dissemination via plasmid conjugation, but quorum sensing (QS) regulatory mechanisms remain undefined. Using Escherichia coli (E. coli) MG1655 conjugation models (RP4-7/EC600 plasmids), we demonstrate that long-chain acyl-homoserine lactones (C10/C12-HSL) enhance transfer frequency by up to 7.7-fold (200 μM C12-HSL; p < 0.001), while quorum-quenching by sub-inhibitory vanillin suppressed this effect by 95% (p < 0.0001). C12-HSL compromised membrane integrity via ompF upregulation (4-fold; p < 0.01) and conjugative pore assembly (trbBp upregulated by 1.38-fold; p < 0.05), coinciding with ROS accumulation (1.5-fold; p < 0.0001) and SOS response activation (recA upregulated by 1.68-fold; p < 0.001). Crucially, rpoS and rmf deletion mutants reduced conjugation by 65.5% and 55.8%, respectively (p < 0.001), exhibiting attenuated membrane permeability (≤65.5% reduced NPN influx; p < 0.0001), suppressed ROS (≤54% downregulated; p < 0.0001), and abolished transcriptional induction of conjugation/stress genes. Reciprocal RpoS–RMF (ribosomal hibernation factor) crosstalk was essential for AHL responsiveness, with deletions mutually suppressing expression (≤65.9% downregulated; p < 0.05). We establish a hierarchical mechanism wherein long-chain AHLs drive resistance dissemination through integrated membrane restructuring, stress adaptation, and RpoS–RMF-mediated genetic plasticity, positioning QS signaling as a viable target for curbing resistance spread. Full article

Quorum quenching (QQ) is of interest for potential application as a sustainable strategy for bacterial disease control via communication interruption. The QQ enzyme can be used as a good alternative antagonist to combat antibiotic abuse and bacterial resistance. Here, genomic DNA sequencing was performed on N-acyl homoserine lactonase from the deep-sea strain Bacillus velezensis DH82 with Cluster of Orthologous Groups of proteins (COGs) annotation. The homologous sequences with β-lactamase domain-containing protein were predicted to be potential QQ enzymes and were cloned and expressed to study their quorum quenching properties by comparing them with the reported enzyme AiiA_3DHB. The experimental results of enzyme activity analysis and steady-state kinetics, as well as enzyme structure and substrate docking simulations and predictions, all consistently demonstrated that YtnP_DH82 presented superior enzyme structural stability and higher degradation efficiency of N-acyl homoserine lactones than AiiA_DH82 under the effects of pH, and temperature, and performed better on short -chain and 3-O-substituted AHSLs. The findings revealed the structural and biochemical characterization of YtnP_DH82 from the deep sea, which provide the capacity for further application in sustainable aquaculture as an alternative to antibiotics. Full article

Biofilm-associated infections caused by Gram-negative bacteria, especially multidrug-resistant strains, frequently occur in intensive care units and represent a major therapeutic challenge. The economic burden of biofilm-associated infections is considerable, making the search for new treatment approaches a focal point for policymakers and scientific funding bodies. Biofilm formation is regulated by quorum sensing (QS), a population density-dependent communication mechanism between cells mediated by small diffusible signaling molecules. QS modulates various intracellular processes, and some features of QS are common to all Gram-negative bacteria. While there are differences in the QS regulatory networks of different Gram-negative bacterial species, a common feature of most Gram-negative bacteria is the ability of N-acylhomoserine lactones (AHL) as inducers to diffuse across the bacterial membrane and interact with receptors located either in the cytoplasm or on the inner membrane. Targeting QS by inhibiting the synthesis, transport, or perception of signaling molecules using small molecules, quorum quenching enzymes, antibodies, combinatorial therapies, or nanoparticles is a promising strategy to combat virulence. In-depth knowledge of biofilm biology, antibiotic susceptibility, and penetration mechanisms, as well as a deep understanding of anti-QS agents, will contribute to the development of antimicrobial therapies to combat biofilm infections. Advancing antimicrobial therapies against biofilm infections requires a deep understanding of biofilm biology, antibiotic susceptibility, penetration mechanisms, and anti-QS strategies. This can be achieved through in vivo and clinical studies, supported by state-of-the-art tools such as machine learning and artificial intelligence. Full article

This review presents information obtained over the past 10 years on the methods to control the widespread worldwide phytopathogen Pectobacterium carotovorum subsp. carotovorum (Pcc). This bacterium is among the ten most dangerous phytopathogens; it affects a wide range of cultivated plants: vegetables, ornamental and medicinal crops, both during vegetation and during the storage of fruits. Symptoms of Pcc damage include the wilting of plants, blackening of vessels on leaves, stems and petioles. At the flowering stage, the stem core gradually wilts and, starting from the root, the stem breaks and the plant dies. Pcc is a rod-shaped, non-capsule and endospore-forming facultative anaerobic Gram-negative bacterium with peritrichous flagellation. Pcc synthesizes bacteriocins—carocins. The main virulence factors of Pcc are the synthesis of N-acyl-homoserine lactone (AHL) and plant cell wall-degrading enzymes (PCWDEs) (pectinases, polygalacturonases, cellulases, and proteases). Diagnostic methods for this phytopathogen include polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), multilocus genotyping of strain-specific genes and detection of unique volatile organic compounds (VOCs). The main methods to control this microorganism include the use of various chemicals (acids, phenols, esters, salts, gases), plant extracts (from grasses, shrubs, trees, and algae), antagonistic bacteria (Bacillus, Pseudomonas, Streptomyces, and lactic acid bacteria), viruses (including a mixture of bacteriophages), and nanomaterials based on metals and chitosan. Full article

Background/Objectives: Quorum Sensing (QS) refers to the communication mechanism in bacterial cells, which occurs through the production and detection of small signaling molecules to coordinate activities and monitor population size. In Gram-negative bacteria, QS is typically mediated by N-acyl-homoserine lactones (HSLs) and 2-alkyl-4(1H)-quinolone metabolites (AQ). The present study aims to develop and validate an LC-MS/MS method for detecting QS molecules and apply it to the analysis of plasma samples from burn patients with septic shock caused by Acinetobacter baumannii. Methods: The LC-MS/MS method was developed and fully validated for the quantitative, simultaneous determination of five HSLs and four AQ molecules, ultimately derived from the plasma of three patients with septic shock, with samples collected over three consecutive days. Results: The developed method proved to be both specific and selective, demonstrating a good fit and linearity over the entire range of interest. Trueness and accuracy were satisfactory. The method showed excellent intra-assay precision (CV% was lower than 15%) and limits of quantification (LOQ) ranging from 0.02 to 0.79 ng/mL. In the patients’ samples, the concentration of 3-OH-C12-HSL peaked at 1.5 ng/mL on the first day, and C7-PQS, C9-PQS, HHQ, and HQNO ranged from 0.5 to 1.5 ng/mL, peaking at 5 ng/mL in one patient on the third day. Conclusions: A method for the simultaneous determination of nine QS molecules by LC-MS/MS was developed and validated. When applied, it showed good performance for the analysis of plasma samples and could be a useful tool for an improvement in the diagnosis, prognosis, or treatment monitoring of infections in burn patients caused by Acinetobacter baumannii. Full article

This study investigated the mechanism by which N-acyl-homoserine lactone (AHL) signaling molecules influence ammonia-oxidizing microorganisms (AOMs) under inhibitory conditions. In laboratory-scale sequential batch reactors (SBRs), the effects of different AHLs (C6-HSL and C8-HSL) on the metabolic activity, microbial community structure, and quorum sensing (QS) system response of AOMs were examined. Caffeic acid, 1-octyne, and allylthiourea were used as ammoxidation inhibitors. The results indicated that under inhibitory conditions, AHLs effectively reduced the loss of ammonia oxidation activity and enhanced the resistance of AOMs to unfavorable environments. Additionally, AHLs enriched AOMs in the microbial community, wherein C6-HSL significantly increased the abundance of amoA genes in AOMs. Furthermore, AHLs maintained the activity of QS-related genes and preserved the communication ability between microorganisms. Correlation analysis revealed a positive relationship between AOMs and QS functional bacteria, suggesting that AHLs can effectively regulate the ammonia oxidation process. Overall, exogenous AHLs can improve the metabolic activity and competitive survival of AOMs under inhibitory conditions. Full article

Traditional anaerobic digestion (AD) technology continues to have severe limitations in terms of complicated substrate degradation efficiency and methane production. This study optimizes the AD system using corn straw and cattle manure as substrates by introducing an exogenous N-Hexanoyl-L-Homoserine lactone (C6-HSL) signaling molecule in concert with an applied external voltage of 0.8 V, systematically investigating its impact on methanogenic performance and microbial community dynamics. The results show that the combined regulation significantly increased methane production (by 29.74%) and substrate utilization rate (by 74.73%) while preventing acid inhibition and ammonia nitrogen inhibition. Mechanistic analysis revealed that the external voltage enhanced the system’s electrocatalytic activity, while the C6-HSL signaling molecule further facilitated the electron transfer efficiency of the biofilm on the electrode. The combined regulation notably enriched hydrogenotrophic methanogens (with Methanobacterium predominating on the cathode and Methanobrevibacter in the digestate), establishing a stable metabolic cooperative network on both the electrode and in the digestate, optimizing the hydrogenotrophic methanogenesis pathway, and enhancing the synergistic effects among microbial communities and system robustness. This study uncovers the synergistic enhancement mechanism of C6-HSL and external voltage, providing new technological pathways and theoretical support for the efficient conversion of low-quality biomass resources and the production of clean energy. Full article

This study aimed to isolate AHL-degrading bacteria from the intestine of Penaeus vannamei and evaluate their ability to control pathogenic Vibrio harveyi in P. vannamei larvae. Twenty-seven isolates were obtained from the digestive tract of healthy Pacific white shrimp juveniles (P. vannamei) after six cycles of pasteurization at 70 °C, but only three isolates (E₁LP₂, E₂LP₁, and E₂LP₂) could degrade AHL. The 16S sequence results gave a high identity (>95%) with Bacillus sp. The isolates exhibited quorum-quenching abilities by degrading AHLs, thereby disrupting Vibrio quorum sensing and virulence. In Zoea and Mysis, the challenged larvae plus the administration of E₁LP₂ resulted in the lowest survival compared to the other groups. Isolates degrading N-acyl homoserine lactone improved the survival of shrimp Zoea and Mysis larvae when challenged with pathogenic V. harveyi. This is the first report on the use of quorum-sensing disrupter bacteria in P. vannamei larval shrimp culture. Our findings suggest that these Bacillus spp. strains have potential as biocontrol agents for sustainable shrimp aquaculture, reducing the reliance on antibiotics while mitigating vibriosis outbreaks. Full article

Quorum quenchers are emerging as an alternative to conventional antimicrobials, since they hinder the development of virulence or resistance mechanisms but without killing the microorganisms, thus, reducing the risk of antimicrobial resistance. Many quorum quenchers are analogs of the natural quorum-sensing signaling molecules or autoinducers. Thus, different analogs of natural N-acylhomoserine lactones (AHLs) have been reported for controlling virulence or reducing the production of biofilms in Gram-negative pathogens. Herein we report the preparation of AHL analogs with a variety of N-substituents in just two steps from readily available N-substituted hydroxyproline esters. The substrates underwent an oxidative radical scission of the pyrrolidine ring. The resulting N-substituted β-aminoaldehyde underwent reduction and in situ cyclization to give a variety of homoserine lactones, with N- and N,N-substituted amino derivatives and with high optical purity. The libraries were screened for the inhibition of violacein production in Chromobacterium violaceum, a Gram-negative pathogen. For the first time, N,N-disubstituted AHL analogs were studied. Several N-sulfonyl derivatives, one carbamoyl, and one N-alkyl-N-sulfonyl homoserine lactone displayed a promising inhibitory activity. Moreover, they did not display microbicide action against S. aureus, C. jejuni, S. enterica, P. aeruginosa, and C. albicans, confirming a pure QQ activity. The determination of structure–activity relationships and in silico ADME studies are also reported, which are valuable for the design of next generations QQ agents. Full article

Quorum sensing (QS) is a cell-to-cell communication mechanism through which microorganisms can sense their population density and adjust their physiology by producing and detecting small signaling molecules called autoinducers (AIs). QS influences various aspects of microbial physiology, including virulence and pathogenesis by bacterial pathogens, biofilm formation, sporulation, antimicrobial resistance, etc. Lactic acid bacteria (LAB) have been used for centuries in food fermentation to improve sensory and nutritional profiles and preserve against spoilage and pathogenic microflora. This study investigated the potential of foodborne LAB of various genera, including Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, and Enterococcus, to interfere with the QS system of bacterial pathogens. For this, cell-free supernatants (CFSs) of 89 LAB foodborne isolates were collected by centrifugation following a 20 h culture (at 30 °C) in quarter-strength Brain Heart Infusion (BHI) broth. The pH of all CFSs was adjusted to 6.5 and sterilized by filtration. The anti-QS activity of the sterilized and neutralized CFSs was initially screened using the biosensor strains Chromobacterium violaceum 026 and Agrobacterium tumefaciens NTL4 (pZLR4) through an agar well diffusion assay that can detect the inhibition of the QS system that is based on acylated homoserine lactones (AHLs), which are used as AIs by Gram-negative bacteria. Additionally, all the CFSs were also screened for interference with the autoinducer 2 (AI-2) QS system that is mostly used for interspecies communication by both Gram-positive and Gram-negative bacteria. This was assessed using a luminescence bioassay with the Vibrio harveyi BAA-1117 biosensor strain. The results indicate that none of the LAB CFSs could inhibit AHL-based QS. However, 61.8% (55/89) of the CFSs induced luminescence in V. harveyi BAA-1117, while the remaining 38.2% (34/89) of the samples were capable of inhibiting AI-2-based QS. In the next steps, the most representative of these latter AI-2 interfering LAB isolates will be investigated for possible inhibition of biofilm formation by some important foodborne bacterial pathogens. Full article

Pseudomonas aeruginosa is a metabolically versatile opportunistic pathogen capable of surviving in a range of environments. The major contribution to these abilities relies on virulence factor production, e.g., exotoxins, phenazines, and rhamnolipids, regulated through a hierarchical system of communication, named quorum sensing (QS). QS involves the production, release, and recognition of two classes of diffusible signal molecules: N-acyl-homoserine lactones and alkyl-quinolones. These present a central role during P. aeruginosa infection, regulating bacterial virulence and the modulation of the host immune system. The influence of this arsenal of virulence factors on bacterial–host interaction makes P. aeruginosa a highly potential platform for the development of biopharmaceuticals. Here, we comprehensively reviewed the therapeutical applications of P. aeruginosa virulence factors and quorum sensing signaling molecules on pathological conditions, ranging from infections and inflammation to cancer disease. Full article

Biofilms are complex, highly organized structures formed by microorganisms, with functional cell arrangements that allow for intricate communication. Severe clinical challenges occur when anaerobic bacterial species establish long-lasting infections, especially those involving biofilms. These infections can occur in device-related settings (e.g., implants) as well as in non-device-related conditions (e.g., inflammatory bowel disease). Within biofilms, bacterial cells communicate by producing and detecting extracellular signals, particularly through specific small signaling molecules known as autoinducers. These quorum-sensing signals are crucial in all steps of biofilm formation: initial adhesion, maturation, and dispersion, triggering gene expression that coordinates bacterial virulence factors, stimulates immune responses in host tissues, and contributes to antibiotic resistance development. Within anaerobic biofilms, bacteria communicate via quorum-sensing molecules such as N-Acyl homoserine lactones (AHLs), autoinducer-2 (AI-2), and antimicrobial molecules (autoinducing peptides, AIPs). To effectively combat pathogenic biofilms, understanding biofilm formation mechanisms and bacterial interactions is essential. The strategy to disrupt quorum sensing, termed quorum quenching, involves methods like inactivating or enzymatically degrading signaling molecules, competing with signaling molecules for binding sites, or noncompetitively binding to receptors, and blocking signal transduction pathways. In this review, we comprehensively analyzed the fundamental molecular mechanisms of quorum sensing in biofilms formed by anaerobic bacteria. We also highlight quorum quenching as a promising strategy to manage bacterial infections associated with anaerobic bacterial biofilms. Full article

Leaf rust (Puccinia triticina) is a common disease that causes significant yield losses in wheat. The most frequently used methods to control leaf rust are the application of fungicides and the cultivation of resistant genotypes. However, high genetic diversity and associated adaptability of pathogen populations hamper achieving durable resistance in wheat. Emerging alternatives, such as microbial priming, may represent an effective measure to stimulate plant defense mechanisms and could serve as a means of controlling a broad range of pathogens. In this study, 175 wheat genotypes were inoculated with two bacterial strains: Ensifer meliloti strain expR⁺ch (producing N-acyl homoserine lactone (AHL)) or transformed E. meliloti carrying the lactonase gene attM (control). In total, 21 genotypes indicated higher resistance upon bacterial AHL priming. Subsequently, the phenotypic data of 175 genotypes combined with 9917 single-nucleotide polymorphisms (SNPs) in a genome-wide association study to identify quantitative trait loci (QTLs) and associated markers for relative infection under attM and expR⁺ch conditions and priming efficiency using the Genome Association and Prediction Integrated Tool (GAPIT). In total, 15 QTLs for relative infection under both conditions and priming efficiency were identified on chromosomes 1A, 1B, 2A, 3A, 3B, 3D, 6A, and 6B, which may represent targets for wheat breeding for priming and leaf-rust resistance. Full article

Recently, studies suggest that the protective effects of Lactobacillus within the female reproductive tract may be partly due to their ability to form biofilms. This study aims to explore the possibility that Lactobacillus can produce key mediators to further bolster the survival of biofilms in human vaginal microbiomes. Three bacterial species, namely, Lactobacillus gasseri, L. crispatus, and L. jensenii, sourced from human female subjects were used to carry out experiments examining the growth of biofilms using a microfermenter system. The bacteria were used to inoculate a glass rod spatula which was subsequently transferred to the microfermenter system. The resulting biofilm growing on the glass spatula was harvested in media and stored in a −80 °C freezer for gas chromatography–mass spectroscopy analysis. We found that quorum sensing compounds, acyl homoserine lactones (AHLs), were detected in the biofilm of L. crispatus and L. jensenii, but none were detected in L. gasseri. The biofilm produced by L. crispatus and L. jensenii was much higher in quantity than the biofilm produced by L. gasseri. Aside from oligopeptides quorum sensing, lactobacilli were found to also have AHL compounds that may help them produce more biofilms and improve the survival and growth of their bacterial communities in the female genital area. Full article

Dasania marina (isolate SD1D, with 98.5% sequence similarity to Dasania marina DMS 21967 KOPRI 20902) is a marine bacterium that was isolated from ballast tank fluids as part of a biofilm study in 2014. Our previous work indicated that although this strain produced no detectable biofilm, it was the only isolate to produce N-acyl homoserine lactones (AHLs) in assays using the broad-range reporter strain, Agrobacterium tumefaciens KYC55. The goal of the current study was to determine the types of AHL molecules produced by the D. marina isolate using gas chromatography–mass spectroscopy (GCMS) and C4- to C14-AHL as standards. A time course assay indicated that the D. marina strain produced the highest level of AHLs at 20 h of growth. When extracts were subjected to GCMS, detectable levels of C8- and C10-AHL and higher levels of C12-AHL were observed. Interestingly, several biofilm-forming isolates obtained from the same source also produced detectable amounts of several AHLs. Of the isolates tested, a strain designated SD5, with 99.83% sequence similarity to Alteromonas tagae BCRC 17571, produced unstable biofilms, yet detectable levels of C6-, C8-, C10- and C12-AHL, and isolate SD8, an Alteromonas oceani S35 strain (98.85% sequence similarity), produced robust and stable biofilms accompanied by detectable levels of C8- and C12-AHL. All isolates tested produced C12-AHL at higher levels than the other AHLs. Results from this study suggest that quorum sensing and biofilm formation are uncoupled in D. marina. Whether the suite of AHLs produced by this isolate could modulate biofilm formation in other strains requires further study. Full article