N-acetylcysteine (NAC) attenuates quorum sensing regulated phenotypes in Pseudomonas aeruginosa PAO1.

The expression of many virulence genes in bacteria is regulated by quorum sensing (QS), and the inhibition of this mechanism has been intensely investigated. N-acetylcysteine (NAC) has good antibacterial activity and is able to interfere with biofilm-related respiratory infections, but little is known whether this compound has an effect on bacterial QS communication. This work aimed to evaluate the potential of NAC as a QS inhibitor (QSI) in Pseudomonas aeruginosa PAO1 through in silico and in vitro analyses, as well as in combination with the antibiotic tobramycin. Initially, a molecular docking analysis was performed between the QS regulatory proteins, LasR and RhlR, of P. aeruginosa with NAC, 3-oxo-C12-HSL, C4-HSL, and furanone C30. The NAC sub-inhibitory concentration was determined by growth curves. Then, we performed in vitro tests using the QS reporter strains P. aeruginosa lasB-gfp and rhlA-gfp, as well as the expression of QS-related phenotypes. Finally, the synergistic effect of NAC with the antibiotic tobramycin was calculated by fractional inhibitory concentrations index (FICi) and investigated against bacterial growth, pigment production, and biofilm formation. In the molecular docking study, NAC bound to LasR and RhlR proteins in a similar manner to the AHL cognate, suggesting that it may be able to bind to QS receptor proteins in vivo. In the biosensor assay, the GFP signal was turned down in the presence of NAC at 1000, 500, 250, and 125 µM for lasB-gfp and rhlA-gfp (p < 0.05), suggesting a QS inhibitory effect. Pyocyanin and rhamnolipids decreased (p < 0.05) up to 34 and 37%, respectively, in the presence of NAC at 125 µM. Swarming and swimming motilities were inhibited (p < 0.05) by NAC at 250 to 10000 µM. Additionally, 2500 and 10000 µM of NAC reduced biofilm formation. NAC-tobramycin combination showed synergistic effect with FICi of 0.8, and the best combination was 2500-1.07 µM, inhibiting biofilm formation up to 60%, besides reducing pyocyanin and pyoverdine production. Confocal microscopy images revealed a stronger, dense, and compact biofilm of P. aeruginosa PAO1 control, while the biofilm treated with NAC-tobramycin became thinner and more dispersed. Overall, NAC at low concentrations showed promising anti-QS properties against P. aeruginosa PAO1, adding to its already known effect as an antibacterial and antibiofilm agent.

N-acetylcysteine (NAC) attenuates quorum sensing regulated phenotypes in Pseudomonas aeruginosa PAO1

The expression of many virulence genes in bacteria is regulated by quorum sensing (QS), and the inhibition of this mechanism has been intensely investigated. N-acetylcysteine (NAC) has good antibacterial activity and is able to interfere with biofilm-related respiratory infections, but little is known whether this compound has an effect on bacterial QS communication. This work aimed to evaluate the potential of NAC as a QS inhibitor (QSI) in Pseudomonas aeruginosa PAO1 through in silico and in vitro analyses, as well as in combination with the antibiotic tobramycin. Initially, a molecular docking analysis was performed between the QS regulatory proteins, LasR and RhlR, of P. aeruginosa with NAC, 3-oxo-C12-HSL, C4-HSL, and furanone C30. The NAC sub-inhibitory concentration was determined by growth curves. Then, we performed in vitro tests using the QS reporter strains P. aeruginosa lasB-gfp and rhlA-gfp, as well as the expression of QS-related phenotypes. Finally, the synergistic effect of NAC with the antibiotic tobramycin was calculated by fractional inhibitory concentrations index (FICi) and investigated against bacterial growth, pigment production, and biofilm formation. In the molecular docking study, NAC bound to LasR and RhlR proteins in a similar manner to the AHL cognate, suggesting that it may be able to bind to QS receptor proteins in vivo. In the biosensor assay, the GFP signal was turned down in the presence of NAC at 1000, 500, 250, and 125 μM for lasB-gfp and rhlA-gfp (p < 0.05), suggesting a QS inhibitory effect. Pyocyanin and rhamnolipids decreased (p < 0.05) up to 34 and 37%, respectively, in the presence of NAC at 125 μM. Swarming and swimming motilities were inhibited (p < 0.05) by NAC at 250 to 10000 μM. Additionally, 2500 and 10000 μM of NAC reduced biofilm formation. NAC-tobramycin combination showed synergistic effect with FICi of 0.8, and the best combination was 2500–1.07 μM, inhibiting biofilm formation up to 60%, besides reducing pyocyanin and pyoverdine production. Confocal microscopy images revealed a stronger, dense, and compact biofilm of P. aeruginosa PAO1 control, while the biofilm treated with NAC-tobramycin became thinner and more dispersed. Overall, NAC at low concentrations showed promising anti-QS properties against P. aeruginosa PAO1, adding to its already known effect as an antibacterial and antibiofilm agent.

Outer Membrane Vesicles (OMVs) Produced by Gram-Negative Bacteria: Structure, Functions, Biogenesis, and Vaccine Application.

Gram-negative bacteria produce outer membrane vesicles (OMVs) with 10 to 300 nm of diameter. The contribution of OMVs to bacterial pathogenesis is a topic of great interest, and their capacity to be combined with antigens impact in the future to the development of vaccines.

Outer membrane vesicles (OMVs) produced by gram-negative bacteria: structure, functions, biogenesis, and vaccine application

Gram-negative bacteria produce outer membrane vesicles (OMVs) with 10 to 300 nm of diameter. The contribution of OMVs to bacterial pathogenesis is a topic of great interest, and their capacity to be combined with antigens impact in the future to the development of vaccines.

Novel biotechnological approaches for monitoring and immunization against resistant to antibiotics Escherichia coli and other pathogenic bacteria

The application of next-generation molecular, biochemical and immunological methods for developing new vaccines, antimicrobial compounds, probiotics and prebiotics for zoonotic infection control has been fundamental to the understanding and preservation of the symbiotic relationship between animals and humans. With increasing rates of antibiotic use, resistant bacterial infections have become more difficult to diagnose, treat, and eradicate, thereby elevating the importance of surveillance and prevention programs. Effective surveillance relies on the availability of rapid, cost-effective methods to monitor pathogenic bacterial isolates. In this opinion article, we summarize the results of some research program initiatives for the improvement of live vaccines against avian enterotoxigenic Escherichia coli using virulence factor gene deletion and engineered vaccine vectors based on probiotics. We also describe methods for the detection of pathogenic bacterial strains in eco-environmental headspace and aerosols, as well as samples of animal and human breath, based on the composition of volatile organic compounds and fatty acid methyl esters. We explain how the introduction of these low-cost biotechnologies and protocols will provide the opportunity to enhance co-operation between networks of resistance surveillance programs and integrated routine workflows of veterinary and clinical public health microbiology laboratories.

Effect of Capsicum Frutescens Extract, Capsaicin, and Luteolin on Quorum Sensing Regulated Phenotypes.

Capsicum peppers have not been investigated as sources of quorum sensing (QS) inhibitors. This study aimed to identify compounds in pimenta-malagueta (Capsicum frutescens) and red pepper (Capsicum annuum) extracts and to evaluate their effect on violacein production in Chromobacterium violaceum ATCC 12472 and C. violaceum CV026, as well as biofilm formation (BF) in Pseudomonas aeruginosa PAO1 and Serratia marcescens MG1. Among the extracts, pimenta-malagueta methanolic extract (PMME) was chosen because it contained capsaicin, dihydrocapsaicin, and luteolin in greater amount than the other extracts. In general, PMME partially inhibited bacterial growth at 2.5 and 5.0 mg/mL, as well as capsaicin at 100 µg/mL and luteolin at 62.5, 125, and 250 µg/mL. At lower concentrations, PMME and luteolin reduced violacein production in C. violaceum ATCC 12472 without affecting growth, a result that was not observed with capsaicin. We show that violacein inhibition by PMME is likely due to luteolin. In silico docking evaluation showed that luteolin binds to the CviR QS regulator. Crystal violet staining and confocal microscopy revealed that BF was increased by PMME and capsaicin, being remarkably superior for P. aeruginosa PAO1 at 30 °C. Capsaicin is not an effective QS inhibitor, while luteolin should be further investigated for its potential effects in QS regulated phenotypes. PRACTICAL APPLICATION: Quorum sensing (QS) is a form of bacterial communication targeted for studies aiming to inhibit bacterial virulence. QS regulates phenotypes that influence microbial activities across many areas, including Food Science. Capsicum frutescens is a type of chili pepper consumed in Brazil, rich in bioactive compounds such as capsaicin (which gives its pungency) and luteolin (a phenolic compound). We show that C. frutescens extract and luteolin inhibit QS in a model bacterium, along with the possible molecular mechanism of inhibition. Capsaicin did not inhibit QS neither biofilm formation. Luteolin should be further investigated for its QS inhibition properties and biotechnological applications.

Effect of Capsicum frutescens extract, capsaicin, and luteolin on quorum sensing regulated phenotypes

Capsicum peppers have not been investigated as sources of quorum sensing (QS) inhibitors. This study aimed to identify compounds in pimenta-malagueta (Capsicum frutescens) and red pepper (Capsicum annuum) extracts and to evaluate their effect on violacein production in Chromobacterium violaceum ATCC 12472 and C. violaceum CV026, as well as biofilm formation (BF) in Pseudomonas aeruginosa PAO1 and Serratia marcescens MG1. Among the extracts, pimenta-malagueta methanolic extract (PMME) was chosen because it contained capsaicin, dihydrocapsaicin, and luteolin in greater amount than the other extracts. In general, PMME partially inhibited bacterial growth at 2.5 and 5.0 mg/mL, as well as capsaicin at 100 µg/mL and luteolin at 62.5, 125, and 250 µg/mL. At lower concentrations, PMME and luteolin reduced violacein production in C. violaceum ATCC 12472 without affecting growth, a result that was not observed with capsaicin. We show that violacein inhibition by PMME is likely due to luteolin. In silico docking evaluation showed that luteolin binds to the CviR QS regulator. Crystal violet staining and confocal microscopy revealed that BF was increased by PMME and capsaicin, being remarkably superior for P. aeruginosa PAO1 at 30 °C. Capsaicin is not an effective QS inhibitor, while luteolin should be further investigated for its potential effects in QS regulated phenotypes.

Effect of Capsicum frutescens extract, capsaicin, and luteolin on quorum sensing regulated phenotypes

Capsicum peppers have not been investigated as sources of quorum sensing (QS) inhibitors. This study aimed to identify compounds in pimenta-malagueta (Capsicum frutescens) and red pepper (Capsicum annuum) extracts and to evaluate their effect on violacein production in Chromobacterium violaceum ATCC 12472 and C. violaceum CV026, as well as biofilm formation (BF) in Pseudomonas aeruginosa PAO1 and Serratia marcescens MG1. Among the extracts, pimenta-malagueta methanolic extract (PMME) was chosen because it contained capsaicin, dihydrocapsaicin, and luteolin in greater amount than the other extracts. In general, PMME partially inhibited bacterial growth at 2.5 and 5.0 mg/mL, as well as capsaicin at 100 µg/mL and luteolin at 62.5, 125, and 250 µg/mL. At lower concentrations, PMME and luteolin reduced violacein production in C. violaceum ATCC 12472 without affecting growth, a result that was not observed with capsaicin. We show that violacein inhibition by PMME is likely due to luteolin. In silico docking evaluation showed that luteolin binds to the CviR QS regulator. Crystal violet staining and confocal microscopy revealed that BF was increased by PMME and capsaicin, being remarkably superior for P. aeruginosa PAO1 at 30 °C. Capsaicin is not an effective QS inhibitor, while luteolin should be further investigated for its potential effects in QS regulated phenotypes.

Role of SdiA on biofilm formation by atypical enteropathogenic Escherichia coli

Atypical enteropathogenic Escherichia coli are capable to form biofilm on biotic and abiotic surfaces, regardless of the adherence pattern displayed. Several E. coli mechanisms are regulated by Quorum sensing (QS), including virulence factors and biofilm formation. Quorum sensing is a signaling system that confers bacteria with the ability to respond to chemical molecules known as autoinducers. Suppressor of division inhibitor (SdiA) is a QS receptor present in atypical enteropathogenic E. coli (aEPEC) that detects acyl homoserine lactone (AHL) type autoinducers. However, these bacteria do not encode an AHL synthase, but they are capable of sensing AHL molecules produced by other species, establishing an inter-species bacterial communication. In this study, we performed experiments to evaluate pellicle, ring-like structure and biofilm formation on wild type, sdiA mutants and complemented strains. We also evaluated the transcription of genes involved in different stages of biofilm formation, such as bcsA, csgA, csgD, fliC and fimA. The sdiA mutants were capable of forming thicker biofilm structures and showed increased motility when compared to wild type and complemented strains. Moreover, they also showed denser pellicles and ring-like structures. Quantitative real-time PCR (qRT-PCR) analysis demonstrated increased csgA, csgD and fliC transcription on mutant strains. Biofilm formation, as well as csgD, csgA and fimA transcription decreased on wild type strains by the addition of AHL. These results indicate that SdiA participates on the regulation of these phenotypes in aEPEC and that AHL addition enhances the repressor effect of this receptor on the transcription of biofilm and motility related genes.

Role of SdiA on biofilm formation by atypical enteropathogenic Escherichia coli

Atypical enteropathogenic Escherichia coli are capable to form biofilm on biotic and abiotic surfaces, regardless of the adherence pattern displayed. Several E. coli mechanisms are regulated by Quorum sensing (QS), including virulence factors and biofilm formation. Quorum sensing is a signaling system that confers bacteria with the ability to respond to chemical molecules known as autoinducers. Suppressor of division inhibitor (SdiA) is a QS receptor present in atypical enteropathogenic E. coli (aEPEC) that detects acyl homoserine lactone (AHL) type autoinducers. However, these bacteria do not encode an AHL synthase, but they are capable of sensing AHL molecules produced by other species, establishing an inter-species bacterial communication. In this study, we performed experiments to evaluate pellicle, ring-like structure and biofilm formation on wild type, sdiA mutants and complemented strains. We also evaluated the transcription of genes involved in different stages of biofilm formation, such as bcsA, csgA, csgD, fliC and fimA. The sdiA mutants were capable of forming thicker biofilm structures and showed increased motility when compared to wild type and complemented strains. Moreover, they also showed denser pellicles and ring-like structures. Quantitative real-time PCR (qRT-PCR) analysis demonstrated increased csgA, csgD and fliC transcription on mutant strains. Biofilm formation, as well as csgD, csgA and fimA transcription decreased on wild type strains by the addition of AHL. These results indicate that SdiA participates on the regulation of these phenotypes in aEPEC and that AHL addition enhances the repressor effect of this receptor on the transcription of biofilm and motility related genes.

Integration host factor is important for biofilm formation by Salmonella enterica Enteritidis.

Salmonella enterica Enteritidis forms biofilms and survives in agricultural environments, infecting poultry and eggs. Bacteria in biofilms are difficult to eradicate compared to planktonic cells, causing serious problems in industry and public health. In this study, we evaluated the role of ihfA and ihfB in biofilm formation by S. enterica Enteritidis by employing different microbiology techniques. Our data indicate that ihf mutant strains are impaired in biofilm formation, showing a reduction in matrix formation and a decrease in viability and metabolic activity. Phenotypic analysis also showed that deletion of ihf causes a deficiency in curli fimbriae expression, cellulose production and pellicle formation. These results show that integration host factor has an important regulatory role in biofilm formation by S. enterica Enteritidis.

Metodologias de identificação e estudo de Escherichia coli diarreiogênica

A familia das endobactérias constitui um importante grupo bacteriano devido a sua importância médica e econômica, pois abrigam muitos dos mais importantes patógenos para o homem e os animais. São bacterias Gram-negativas e aeróbias facultativas e possuem diversos fatores de virulência, como os antigenos K, que fazem parte da capsula de algumas espécies desse grupo e protegem os patógenos da ação de fagócitos e anticorpos. As enterobacterias causam, além de infecções no trato intestinal, outras infecções localizadas, sendo as mais comuns nas vias urinárias, nos pulmões, no sistema nervoso central, na pele e no tecido celular subcultâneo. Essas infecções localizadas podem evoluir para infecções sistemicas, ou seja, generalizadas, o que pode levar a morte do organismo infectado. Elas também podem ocorrer devido à translocação de enterobactérias dos intestinos para a corrente sanguínea. Escherichia coli, espécie da família enterobacteriacea, é um dos organismos vivos mais bem estudados atualmente e isso se deve a sua grande diversidade patogênica, podendo ser a causadora de infecções intestinais por vários mecanismos diferentes, infecções extra-intestinais e bacteremias, além de ser parte da microbiota normal do ser humano e demais mamíferos. As categorias de E. coli causadoras de infecção intestinal, também são chamadas de E. coli diarreiogênicas, são divididas em cinco: Escherichia coli Enteropatogênica (EPEC), Escherichia coli Enterohemorrágica (EHEC), Escherichia coli Enteroagregativa (EAEC), Escherichia coli Enterotoxigênica (ETEC) e Escherichia coli Enteroinvasora (EIEC) e Escherichia coli de aderência difusa (DAEC), de acordo com suas características específicas, como produção de toxinas, padrão de adesão em testes in vidro, etc. Este trabalho busca reunir e explicar as principais metodologias para identificação e estudo das E. coli diarreiogênicas, desde os métodos de cultivo e isolamento até os testes genotípicos e fenotípicos, e servir como um manual para o início dos estudos desse grupo de bactérias que, embora já sejam bastante estudadas, ainda necessitam da elucidação de muitos de seus processos, assim como o desenvolvimento de novos tratamentos para as infecções ocasionadas por elas.

Multilocus sequence typing analyses of Clostridium perfringens type A strains harboring tpeL and netB genes

Clostridium perfringens is an anaerobic bacterium ubiquitous in various environments, especially in soil and the gastrointestinal tract of healthy humans and animals. In this study, multilocus sequence typing protocol was used to investigate genotypic relationships among 40 C. perfringens strains isolated from humans and broiler chicken with necrotic enteritis [NE]. The results indicated a few clonal populations, mainly observed in human strains, with 32.5% of all strains associated with one of three clonal complexes and 30 sequences types. The CC-1 cluster showed an interesting and unexpected result because it contained seven strains [six from animals and one of human origin]. Detection assays for toxin genes tpeL and netB were also performed. The netB gene was only observed in 7.5% of the strains from healthy human. The toxin gene tpeL was detected in 22.5% of the C perfringens strains isolated from three individuals and in six broilers with NE. Our study describes the role of some C perfringens strains of human origin acting as reservoirs of virulence genes and sources of infection. In addition, the strains of human and animal origin were found to be genetically distinct but phylogenetically close, and the human strains showed more diversity than the animal strains.

Integration host factor is important for biofilm formation by Salmonella enterica Enteritidis

Salmonella enterica Enteritidis forms biofilms and survives in agricultural environments, infecting poultry and eggs. Bacteria in biofilms are difficult to eradicate compared to planktonic cells, causing serious problems in industry and public health. In this study, we evaluated the role of ihfA and ihfB in biofilm formation by S. enterica Enteritidis by employing different microbiology techniques. Our data indicate that ihf mutant strains are impaired in biofilm formation, showing a reduction in matrix formation and a decrease in viability and metabolic activity. Phenotypic analysis also showed that deletion of ihf causes a deficiency in curli fimbriae expression, cellulose production and pellicle formation. These results show that integration host factor has an important regulatory role in biofilm formation by S. enterica Enteritidis.

Antimelanoma effect of Salmonella Typhimurium integration host factor mutant in murine model

Aim: This study aimed to evaluate an attenuated Salmonella ihfA-null mutant strain as therapeutic agent to control tumor growth. Materials & methods: After bacterial toxicity evaluation, C57BL/6JUnib mice were inoculated with B16F10 cells and treated with two Salmonella strains (LGBM 1.1 and LGBM 1.41). Results: LGBM 1.1 can reduce tumor mass, but it exerts some toxic effects. Although LGBM 1.41 is less toxic than LGBM 1.1, it does not reduce tumor mass significantly. Indeed, animals treated with LGBM 1.41 present only slightly initial delay in tumor progression and increased survival rate as compared with the control. Conclusion: The null-mutants of ihfA gene of Salmonella Typhimurium could be a promising candidate for melanoma treatment