Quorum sensing architecture network in Escherichia coli virulence and pathogenesis.

Escherichia coli is a Gram-negative commensal bacterium of the normal microbiota of humans and animals. However, several E. coli strains are opportunistic pathogens responsible for severe bacterial infections, including gastrointestinal and urinary tract infections. Due to the emergence of multidrug-resistant serotypes that can cause a wide spectrum of diseases, E. coli is considered one of the most troublesome human pathogens worldwide. Therefore, a more thorough understanding of its virulence control mechanisms is essential for the development of new anti-pathogenic strategies. Numerous bacteria rely on a cell density-dependent communication system known as quorum sensing (QS) to regulate several bacterial functions, including the expression of virulence factors. The QS systems described for E. coli include the orphan SdiA regulator, an autoinducer-2 (AI-2), an autoinducer-3 (AI-3) system, and indole, which allow E. coli to establish different communication processes to sense and respond to the surrounding environment. This review aims to summarise the current knowledge of the global QS network in E. coli and its influence on virulence and pathogenesis. This understanding will help to improve anti-virulence strategies with the E. coli QS network in focus.

Mushroom-shaped structures formed in Acinetobacter baumannii biofilms grown in a roller bioreactor are associated with quorum sensing-dependent Csu-pilus assembly.

There is currently a need to develop simple biofilm models that facilitate investigation of the architecture/biology of mature bacterial biofilms in a consistent/standardized manner given their environmental and clinical importance and the need for new anti-biofilm interventions. This study introduces a novel biofilm culture system termed the rolling biofilm bioreactor (RBB). This easily operated system allows adherent microbial cells to be repeatedly exposed to air/solid/liquid interfaces optimizing biofilm growth. The RBB was exploited to investigate biofilm formation in Acinetobacter baumannii. High levels of A. baumannii biofilm biomass reproducibly accumulate in the RBB and, importantly, undergo a maturation step to form large mushroom-shaped structures that had not been observed in other models. Based on image analysis of biofilm development and genetic manipulation, we show how N-acylhomoserine lactone-dependent quorum sensing (QS) impacts on biofilm differentiation, composition and antibiotic tolerance. Our results indicate that extracellular DNA (eDNA) is a key matrix component in mature Acinetobacter biofilms as the mushroom-like structures consist of dense cellular masses encased in an eDNA mesh. Moreover, this study reveals the contribution of QS to A. baumannii biofilm differentiation through Csu pilus assembly regulation. Understanding the mechanisms of structural development of mature biofilms helps to identify new biofilm eradication and removal strategies.

Use of Quorum Sensing Inhibition Strategies to Control Microfouling.

Interfering with the quorum sensing bacterial communication systems has been proposed as a promising strategy to control bacterial biofilm formation, a key process in biofouling development. Appropriate in vitro biofilm-forming bacteria models are needed to establish screening methods for innovative anti-biofilm and anti-microfouling compounds. Four marine strains, two Pseudoalteromonas spp. and two Vibrio spp., were selected and studied with regard to their biofilm-forming capacity and sensitivity to quorum sensing (QS) inhibitors. Biofilm experiments were performed using two biofilm cultivation and quantification methods: the xCELLigence® system, which allows online monitoring of biofilm formation, and the active attachment model, which allows refreshment of the culture medium to obtain a strong biofilm that can be quantified with standard staining methods. Although all selected strains produced acyl-homoserine-lactone (AHL) QS signals, only the P. flavipulchra biofilm, measured with both quantification systems, was significantly reduced with the addition of the AHL-lactonase Aii20J without a significant effect on planktonic growth. Two-species biofilms containing P. flavipulchra were also affected by the addition of Aii20J, indicating an influence on the target bacterial strain as well as an indirect effect on the co-cultured bacterium. The use of xCELLigence® is proposed as a time-saving method to quantify biofilm formation and search for eco-friendly anti-microfouling compounds based on quorum sensing inhibition (QSI) strategies. The results obtained from these two in vitro biofilm formation methods revealed important differences in the response of biosensor bacteria to culture medium and conditions, indicating that several strains should be used simultaneously for screening purposes and the cultivation conditions should be carefully optimized for each specific purpose.

Biotechnological applications of Bacillus licheniformis.

Bacillus licheniformis is a Gram positive spore-forming bacterial species of high biotechnological interest with numerous present and potential uses, including the production of bioactive compounds that are applied in a wide range of fields, such as aquaculture, agriculture, food, biomedicine, and pharmaceutical industries. Its use as an expression vector for the production of enzymes and other bioproducts is also gaining interest due to the availability of novel genetic manipulation tools. Furthermore, besides its widespread use as a probiotic, other biotechnological applications of B. licheniformis strains include: bioflocculation, biomineralization, biofuel production, bioremediation, and anti-biofilm activity. Although authorities have approved the use of B. licheniformis as a feed additive worldwide due to the absence of toxigenic potential, some probiotics containing this bacterium are considered unsafe due to the possible transference of antibiotic resistance genes. The wide variability in biological activities and genetic characteristics of this species makes it necessary to establish an exact protocol for describing the novel strains, in order to evaluate its biotechnological potential.

Quorum Sensing as a Target for Controlling Surface Associated Motility and Biofilm Formation in Acinetobacter baumannii ATCC® 17978TM.

The important nosocomial pathogen Acinetobacter baumannii presents a quorum sensing (QS) system (abaI/abaR) mediated by acyl-homoserine-lactones (AHLs) and several quorum quenching (QQ) enzymes. However, the roles of this complex network in the control of the expression of important virulence-related phenotypes such as surface-associated motility and biofilm formation is not clear. Therefore, the effect of the mutation of the AHL synthase AbaI, and the exogenous addition of the QQ enzyme Aii20J on surface-associated motility and biofilm formation by A. baumannii ATCC® 17978TM was studied in detail. The effect of the enzyme on biofilm formation by several multidrug-resistant A. baumannii clinical isolates differing in their motility pattern was also tested. We provide evidence that a functional QS system is required for surface-associated motility and robust biofilm formation in A. baumannii ATCC® 17978TM. Important differences were found with the well-studied strain A. nosocomialis M2 regarding the relevance of the QS system depending on environmental conditions The in vitro biofilm-formation capacity of A. baumannii clinical strains was highly variable and was not related to the antibiotic resistance or surface-associated motility profiles. A high variability was also found in the sensitivity of the clinical strains to the action of the QQ enzyme, revealing important differences in virulence regulation between A. baumannii isolates and confirming that studies restricted to a single strain are not representative for the development of novel antimicrobial strategies. Extracellular DNA emerges as a key component of the extracellular matrix in A. baumannii biofilms since the combined action of the QQ enzyme Aii20J and DNase reduced biofilm formation in all tested strains. Results demonstrate that QQ strategies in combination with other enzymatic treatments such as DNase could represent an alternative approach for the prevention of A. baumannii colonization and survival on surfaces and the prevention and treatment of infections caused by this pathogen.

Short-Chain N-Acylhomoserine Lactone Quorum-Sensing Molecules Promote Periodontal Pathogens in In Vitro Oral Biofilms.

Acylhomoserine lactones (AHLs), the quorum-sensing (QS) signals produced by a range of Gram-negative bacteria, are involved in biofilm formation in many pathogenic and environmental bacteria. Nevertheless, the current paradigm excludes a role of AHLs in dental plaque formation, while other QS signals, such as AI-2 and autoinducer peptides, have been demonstrated to play an important role in biofilm formation and virulence-related gene expression in oral pathogens. In the present work, we have explored the effect of externally added AHLs on in vitro oral biofilm models for commensal, cariogenic, and periodontal dental plaque. While little effect on bacterial growth was observed, some AHLs specifically affected the lactic acid production and protease activity of the biofilms. Most importantly, the analysis of bacterial diversity in the biofilms showed that the addition of C6-homoserine lactone (C6-HSL) results in a shift toward a periodontal bacterial composition profile by increasing the relative presence of the orange-complex bacteria Peptostreptococcus and Prevotella These results point to a relevant role of AHL-mediated QS in dental plaque formation and might be involved in the development of dysbiosis, the mechanism of which should be further investigated. This finding potentially opens new opportunities for the prevention or treatment of the periodontal disease.IMPORTANCE Dental plaque is omnipresent in healthy oral cavities and part of our commensal microbial colonization. At the same time, dental plaque is the cause of the most common human diseases, caries and gum disease. Dental plaque consists of billions of microbes attached to the surface of your teeth. Communication among these microbes is pivotal for development of these complex communities yet poorly studied in dental plaque. In the present study, we show that a specific communication molecule induces changes within the community related to the development of gum disease. This finding suggests that interfering with microbial communication may represent an interesting novel strategy to prevent gum disease that should be further investigated.

Immobilization of antimicrobial and anti-quorum sensing enzymes onto GMA-grafted poly(vinyl chloride) catheters.

Catheter-associated infections still represent a challenging thread because of the likelihood of biofilm formation. The aim of this work was the surface modification of catheters to immobilize lysozyme and acylase under mild conditions while preserving antimicrobial and anti-quorum sensing performances. Glycidyl methacrylate (GMA) was grafted onto poly(vinyl chloride) (PVC) catheters by a pre-irradiation method. The effects of monomer concentration, pre-irradiation dose, reaction time, monomer concentration and reaction temperature were investigated. The grafting process was monitored using FTIR-ATR spectroscopy, differential scanning calorimetry, thermogravimetric analysis, and swelling data. Lysozyme was directly immobilized onto PVC-g-GMA maintaining the hydrolytic activity, which hindered Staphylococcus aureus adhesion. For acylase immobilization, the PVC-g-GMA catheters were reacted with ethylenediamine and glutaraldehyde in order to facilitate acylase covalent binding. Free acylase in solution demonstrated notably capability to act as quorum sensing inhibitor, as observed using Chromobacterium violaceum as biosensor, by degrading a wide variety of acylated homoserine lactones (AHLs), including those produced by Pseudomonas aeruginosa and Acinetobacter baumannii. Acylase-immobilized PVC-g-GMA catheters were challenged against degradation of AHLs and the activity monitored using both the biosensor and HPLC-MS. Relevantly, the functionalized catheters completely degraded all tested AHL signals, opening new ways of preventing biofilm formation on medical devices.

Multiple Quorum Quenching Enzymes Are Active in the Nosocomial Pathogen Acinetobacter baumannii ATCC17978.

Acinetobacter baumannii presents a typical luxI/luxR quorum sensing (QS) system (abaI/abaR) but the acyl-homoserine lactone (AHL) signal profile and factors controlling the production of QS signals in this species have not been determined yet. A very complex AHL profile was identified for A. baumannii ATCC17978 as well as for A. nosocomialis M2, but only when cultivated under static conditions, suggesting that surface or cell-to-cell contact is involved in the activation of the QS genes. The analysis of A. baumanni clinical isolates revealed a strain-specific AHL profile that was also affected by nutrient availability. The concentration of OHC12-HSL, the major AHL found in A. baumannii ATCC17978, peaked upon stationary-phase establishment and decreases steeply afterwards. Quorum quenching (QQ) activity was found in the cell extracts of A. baumannii ATCC17978, correlating with the disappearance of the AHLs from the culture media, indicating that AHL concentration may be self-regulated in this pathogen. Since QQ activity was observed in strains in which AidA, a novel α/ß-hydrolase recently identified in A. baumannii, is not present, we have searched for additional QQ enzymes in A. baumannii ATCC17978. Seven putative AHL-lactonase sequences could be identified in the genome and the QQ activity of 3 of them could be confirmed. At least six of these lactonase sequences are also present in all clinical isolates as well as in A. nosocomialis M2. Surface-associated motility and biofilm formation could be blocked by the exogenous addition of the wide spectrum QQ enzyme Aii20J. The differential regulation of the QQ enzymes in A. baumannii ATCC17978 and the full dependence of important virulence factors on the QS system provides a strong evidence of the importance of the AHL-mediated QS/QQ network in this species.

High Prevalence of Quorum-Sensing and Quorum-Quenching Activity among Cultivable Bacteria and Metagenomic Sequences in the Mediterranean Sea.

There is increasing evidence being accumulated regarding the importance of N-acyl homoserine lactones (AHL)-mediated quorum-sensing (QS) and quorum-quenching (QQ) processes in the marine environment, but in most cases, data has been obtained from specific microhabitats, and subsequently little is known regarding these activities in free-living marine bacteria. The QS and QQ activities among 605 bacterial isolates obtained at 90 and 2000 m depths in the Mediterranean Sea were analyzed. Additionally, putative QS and QQ sequences were searched in metagenomic data obtained at different depths (15-2000 m) at the same sampling site. The number of AHL producers was higher in the 90 m sample (37.66%) than in the 2000 m sample (4.01%). However, the presence of QQ enzymatic activity was 1.63-fold higher in the 2000 m sample. The analysis of putative QQ enzymes in the metagenomes supports the relevance of QQ processes in the deepest samples, found in cultivable bacteria. Despite the unavoidable biases in the cultivation methods and biosensor assays and the possible promiscuous activity of the QQ enzymes retrieved in the metagenomic analysis, the results indicate that AHL-related QS and QQ processes could be common activity in the marine environment.

Inhibition of Steptococcus mutans biofilm formation by extracts of Tenacibaculum sp. 20J, a bacterium with wide-spectrum quorum quenching activity.

Background: Previous studies have suggested the quorum sensing signal AI-2 as a potential target to prevent the biofilm formation by Streptococcus mutans, a pathogen involved in tooth decay. Objective: To obtain inhibition of biofilm formation by S. mutans by extracts obtained from the marine bacterium Tenacibaculum sp. 20J interfering with the AI-2 quorum sensing system. Design: The AI-2 inhibitory activity was tested with the biosensors Vibrio harveyi BB170 and JMH597. S. mutans ATCC25175 biofilm formation was monitored using impedance real-time measurements with the xCELLigence system®, confocal laser microscopy, and the crystal violet quantification method. Results: The addition of the cell extract from Tenacibaculum sp. 20J reduced biofilm formation in S. mutans ATCC25175 by 40-50% compared to the control without significantly affecting growth. A decrease of almost 40% was also observed in S. oralis DSM20627 and S. dentisani 7747 biofilms. Conclusions: The ability of Tenacibaculum sp. 20J to interfere with AI-2 and inhibit biofilm formation in S. mutans was demonstrated. The results indicate that the inhibition of quorum sensing processes may constitute a suitable strategy for inhibiting dental plaque formation, although additional experiments using mixed biofilm models would be required.

Quorum sensing network in clinical strains of A. baumannii: AidA is a new quorum quenching enzyme.

Acinetobacter baumannii is an important pathogen that causes nosocomial infections generally associated with high mortality and morbidity in Intensive Care Units (ICUs). Currently, little is known about the Quorum Sensing (QS)/Quorum Quenching (QQ) systems of this pathogen. We analyzed these mechanisms in seven clinical isolates of A. baumannii. Microarray analysis of one of these clinical isolates, Ab1 (A. baumannii ST-2_clon_2010), previously cultured in the presence of 3-oxo-C12-HSL (a QS signalling molecule) revealed a putative QQ enzyme (α/ß hydrolase gene, AidA). This QQ enzyme was present in all non-motile clinical isolates (67% of which were isolated from the respiratory tract) cultured in nutrient depleted LB medium. Interestingly, this gene was not located in the genome of the only motile clinical strain growing in this medium (A. baumannii strain Ab421_GEIH-2010 [Ab7], isolated from a blood sample). The AidA protein expressed in E. coli showed QQ activity. Finally, we observed downregulation of the AidA protein (QQ system attenuation) in the presence of H2O2 (ROS stress). In conclusion, most of the A. baumannii clinical strains were not surface motile (84%) and were of respiratory origin (67%). Only the pilT gene was involved in surface motility and related to the QS system. Finally, a new QQ enzyme (α/ß hydrolase gene, AidA protein) was detected in these strains.

Biofilm Formation and Quorum-Sensing-Molecule Production by Clinical Isolates of Serratia liquefaciens.

Serratia spp. are opportunistic human pathogens responsible for an increasing number of nosocomial infections. However, little is known about the virulence factors and regulatory circuits that may enhance the establishment and long-term survival of Serratia liquefaciens in the hospital environment. In this study, two reporter strains, Chromobacterium violaceum CV026 and VIR24, and high-resolution triple-quadrupole liquid chromatography-mass spectrometry (LC-MS) were used to detect and to quantify N-acyl-homoserine lactone (AHL) quorum-sensing signals in 20 S. liquefaciens strains isolated from clinical samples. Only four of the strains produced sufficient amounts of AHLs to activate the sensors. Investigation of two of the positive strains by high-performance liquid chromatography (HPLC)-MS confirmed the presence of significant amounts of short-acyl-chain AHLs (N-butyryl-l-homoserine lactone [C4-HSL] and N-hexanoyl-l-homoserine lactone [C6-HSL]) in both strains, which exhibited a complex and strain-specific signal profile that included minor amounts of other short-acyl-chain AHLs (N-octanoyl-l-homoserine lactone [C8-HSL] and N-3-oxohexanoyl-l-homoserine lactone [OC6-HSL]) and long-acyl-chain (C10, C12, and C14) AHLs. No correlation between biofilm formation and the production of large amounts of AHLs could be established. Fimbria-like structures were observed by transmission electron microscopy, and the presence of the type 1 fimbrial adhesin gene fimH in all strains was confirmed by PCR. The ability of S. liquefaciens to adhere to abiotic surfaces and to form biofilms likely contributes to its persistence in the hospital environment, increasing the probability of causing nosocomial infections. Therefore, a better understanding of the adherence properties of this species will provide greater insights into the diseases it causes.

In vitro quenching of fish pathogen Edwardsiella tarda AHL production using marine bacterium Tenacibaculum sp. strain 20J cell extracts.

Quorum quenching (QQ) has become an interesting alternative for solving the problem of bacterial antibiotic resistance, especially in the aquaculture industry, since many species of fish-pathogenic bacteria control their virulence factors through quorum sensing (QS) systems mediated by N-acylhomoserine lactones (AHLs). In a screening for bacterial strains with QQ activity in different marine environments, Tenacibaculum sp. strain 20J was identified and selected for its high degradation activity against a wide range of AHLs. In this study, the QQ activity of live cells and crude cell extracts (CCEs) of strain 20J was characterized and the possibilities of the use of CCEs of this strain to quench the production of AHLs in cultures of the fish pathogen Edwardsiella tarda ACC35.1 was explored. E. tarda ACC35.1 produces N-hexanoyl-L-homoserine lactone (C6-HSL) and N-oxohexanoyl-L-homoserine lactone (OC6-HSL). This differs from profiles registered for other E. tarda strains and indicates an important intra-specific variability in AHL production in this species. The CCEs of strain 20J presented a wide-spectrum QQ activity and, unlike Bacillus thuringiensis serovar Berliner ATCC10792 CCEs, were effective in eliminating the AHLs produced in E. tarda ACC35.1 cultures. The fast and wide-spectrum AHL-degradation activity shown by this member of the Cytophaga-Flexibacter-Bacteroidetes group consolidates this strain as a promising candidate for the control of AHL-based QS pathogens, especially in the marine fish farming industry.