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1.
Arch Microbiol ; 206(7): 324, 2024 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-38913239

RESUMO

Among the ESKAPE pathogens, Pseudomonas aeruginosa is an extensively notorious superbug that causes difficult-to-treat infections. Since quorum sensing (QS) directly promotes pseudomonal virulence, targeting QS circuits is a promising approach for disarming phenotypic virulence. Hence, this study scrutinizes the anti-QS, antivirulence, and anti-biofilm potential of citral (CiT; phytochemical) and triclosan (TcN; disinfectant), alone and in combination, against P. aeruginosa PAO1/PA14. The findings confirmed synergism between CiT and TcN and revealed their quorum quenching (QQ) potential. At sub-inhibitory levels, CiT-TcN combination significantly impeded pyocyanin, total bacterial protease, hemolysin, and pyochelin production alongside inhibiting biofilm formation in P. aeruginosa. Moreover, the QQ and antivirulence potential of CiT and TcN was positively correlated by molecular docking studies that predicted strong associations of the drugs with QS receptors of P. aeruginosa. Collectively, the study identifies CiT-TcN as an effective drug combination that harbors QQ, antivirulence, and anti-biofilm prospects against P. aeruginosa.


Assuntos
Monoterpenos Acíclicos , Antibacterianos , Biofilmes , Sinergismo Farmacológico , Simulação de Acoplamento Molecular , Pseudomonas aeruginosa , Percepção de Quorum , Triclosan , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Percepção de Quorum/efeitos dos fármacos , Triclosan/farmacologia , Biofilmes/efeitos dos fármacos , Monoterpenos Acíclicos/farmacologia , Antibacterianos/farmacologia , Virulência/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Piocianina/metabolismo
2.
Microb Cell Fact ; 23(1): 174, 2024 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-38867319

RESUMO

BACKGROUND: The objectives of the current study were to extract pyocyanin from Pseudomonas aeruginosa clinical isolates, characterize its chemical nature, and assess its biological activity against different bacteria and cancer cells. Due to its diverse bioactive properties, pyocyanin, being one of the virulence factors of P. aeruginosa, holds a promising, safe, and available therapeutic potential. METHODS: 30 clinical P. aeruginosa isolates were collected from different sources of infections and identified by routine methods, the VITEK 2 compact system, and 16 S rRNA. The phenazine-modifying genes (phzM, phzS) were identified using polymerase chain reaction (PCR). Pyocyanin chemical characterization included UV-Vis spectrophotometry, Fourier Transform Infra-Red spectroscopy (FTIR), Gas Chromatography-Mass Spectrometry (GC-MS), and Liquid Chromatography-Mass Spectrometry (LC-MS). The biological activity of pyocyanin was explored by determining the MIC values against different clinical bacterial strains and assessing its anticancer activity against A549, MDA-MB-231, and Caco-2 cancer cell lines using cytotoxicity, wound healing and colony forming assays. RESULTS: All identified isolates harboured at least one of the phzM or phzS genes. The co-presence of both genes was demonstrated in 13 isolates. The UV-VIS absorbance peaks were maxima at 215, 265, 385, and 520 nm. FTIR could identify the characteristic pyocyanin functional groups, whereas both GC-MS and LC-MS elucidated the chemical formula C11H18N2O2, with a molecular weight 210. The quadri-technical analytical approaches confirmed the chemical nature of the extracted pyocyanin. The extract showed broad-spectrum antibacterial activity, with the greatest activity against Bacillus, Staphylococcus, and Streptococcus species (MICs 31.25-125 µg/mL), followed by E. coli isolates (MICs 250-1000 µg/mL). Regarding the anticancer activity, the pyocyanin extract showed IC50 values against A549, MDA-MB-231, and Caco-2 cancer cell lines of 130, 105, and 187.9 µg/mL, respectively. Furthermore, pyocyanin has markedly suppressed colony formation and migratory abilities in these cells. CONCLUSIONS: The extracted pyocyanin has demonstrated to be a potentially effective candidate against various bacterial infections and cancers. Hence, the current findings could contribute to producing this natural compound easily through an affordable method. Nonetheless, future studies are required to investigate pyocyanin's effects in vivo and analyse the results of combining it with other traditional antibiotics or anticancer drugs.


Assuntos
Antibacterianos , Antineoplásicos , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa , Piocianina , Piocianina/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/isolamento & purificação , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Células CACO-2
3.
Biosens Bioelectron ; 261: 116521, 2024 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-38917514

RESUMO

Oceanic facilities and equipment corrosion present considerable economic and safety concerns, predominantly due to microbial corrosion. Early detection of corrosive microbes is pivotal for effective monitoring and prevention. Yet, traditional detection methods often lack specificity, require extensive processing time, and yield inaccurate results. Hence, the need for an efficient real-time corrosive microbe monitoring technology is evident. Pseudomonas aeruginosa, a widely distributed microorganism in aquatic environments, utilizes its production of quinone-like compounds, specifically pyocyanin (PYO), to corrode metals. Here, we report a novel fiber optic surface plasmon resonance (SPR) sensor modified by the C-terminal of BrlR protein (BrlR-C), which is a specific receptor of PYO molecule, to detect P. aeruginosa in aquatic environments. The results showed that the sensor had a good ability to recognize PYO in the concentration range of 0-1 µg/mL, and showed excellent sensing performance in real-time monitoring the growth status of P. aeruginosa. With a strong selectivity of PYO, the sensor could clearly detect P. aeruginosa against other bacteria in seawater environment, and exhibited excellent anti-interference ability against variations in pH, temperature and pressure and other interfering substances. This study provides a useful tool for monitoring corrosive P. aeruginosa biofilm in aquatic environments, which is a first of its kind example that serves as a laboratory model for the application of fiber optic technology in real-world scenarios to monitoring biofilms in microbial corrosion and biofouling.


Assuntos
Biofilmes , Técnicas Biossensoriais , Tecnologia de Fibra Óptica , Pseudomonas aeruginosa , Piocianina , Ressonância de Plasmônio de Superfície , Pseudomonas aeruginosa/isolamento & purificação , Ressonância de Plasmônio de Superfície/métodos , Piocianina/análise , Piocianina/química , Técnicas Biossensoriais/métodos , Corrosão , Fibras Ópticas , Água do Mar/microbiologia , Água do Mar/química , Desenho de Equipamento
4.
Front Cell Infect Microbiol ; 14: 1375872, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38846355

RESUMO

Introduction: Pseudomonas aeruginosa is notorious for its multidrug resistance and its involvement in hospital-acquired infections. In this study, 20 bacterial strains isolated from soil samples near the Hindan River in Ghaziabad, India, were investigated for their biochemical and morphological characteristics, with a focus on identifying strains with exceptional drug resistance and pyocyanin production. Methods: The isolated bacterial strains were subjected to biochemical and morphological analyses to characterize their properties, with a particular emphasis on exopolysaccharide production. Strain GZB16/CEES1, exhibiting remarkable drug resistance and pyocyanin production. Biochemical and molecular analyses, including sequencing of its 16S rRNA gene (accession number LN735036.1), plasmid-curing assays, and estimation of plasmid size, were conducted to elucidate its drug resistance mechanisms and further pyocynin based target the Candida albicans Strain GZB16/CEES1 demonstrated 100% resistance to various antibiotics used in the investigation, with plasmid-curing assays, suggesting plasmid-based resistance gene transmission. The plasmid in GZB16/CEES1 was estimated to be approximately 24 kb in size. The study focused on P. aeruginosa's pyocyanin production, revealing its association with anticandidal activity. The minimum inhibitory concentration (MIC) of the bacterial extract against Candida albicans was 50 µg/ml, with a slightly lower pyocyanin-based MIC of 38.5 µg/ml. Scanning electron microscopy illustrated direct interactions between P. aeruginosa strains and Candida albicans cells, leading to the destruction of the latter. Discussion: These findings underscore the potential of P. aeruginosa in understanding microbial interactions and developing strategies to combat fungal infections. The study highlights the importance of investigating bacterial-fungal interactions and the role of pyocyanin in antimicrobial activity. Further research in this area could lead to the development of novel therapeutic approaches for combating multidrug-resistant infections.


Assuntos
Antifúngicos , Candida albicans , Farmacorresistência Bacteriana Múltipla , Testes de Sensibilidade Microbiana , Plasmídeos , Pseudomonas aeruginosa , Piocianina , RNA Ribossômico 16S , Microbiologia do Solo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Piocianina/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , RNA Ribossômico 16S/genética , Índia , Plasmídeos/genética , Antibacterianos/farmacologia , Antibiose
5.
mSphere ; 9(5): e0021024, 2024 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-38712943

RESUMO

Metallothioneins (MTs) are small cysteine-rich proteins that play important roles in homeostasis and protection against heavy metal toxicity and oxidative stress. The opportunistic pathogen, Pseudomonas aeruginosa, expresses a bacterial MT known as PmtA. Utilizing genetically modified P. aeruginosa PAO1 strains (a human clinical wound isolate), we show that inducing pmtA increases levels of pyocyanin and biofilm compared to other PAO1 isogenic strains, supporting previous results that pmtA is important for pyocyanin and biofilm production. We also show that overexpression of pmtA in vitro provides protection for cells exposed to oxidants, which is a characteristic of inflammation, indicating a role for PmtA as an antioxidant in inflammation. We found that a pmtA clean deletion mutant is phagocytized faster than other PAO1 isogenic strains in THP-1 human macrophage cells, indicating that PmtA provides protection from the phagocytic attack. Interestingly, we observed that monoclonal anti-PmtA antibody binds to PmtA, which is accessible on the surface of PAO1 strains using both flow cytometry and enzyme-linked immunosorbent assay techniques. Finally, we investigated intracellular persistence of these PAO1 strains within THP-1 macrophages cells and found that the phagocytic endurance of PAO1 strains is affected by pmtA expression. These data show for the first time that a bacterial MT (pmtA) can play a role in the phagocytic process and can be found on the outer surface of PAO1. Our results suggest that PmtA plays a role both in protection from oxidative stress and in the resistance to the host's innate immune response, identifying PmtA as a potential therapeutic target in P. aeruginosa infection. IMPORTANCE: The pathogen Pseudomonas aeruginosa is a highly problematic multidrug-resistant (MDR) pathogen with complex virulence networks. MDR P. aeruginosa infections have been associated with increased clinical visits, very poor healthcare outcomes, and these infections are ranked as critical on priority lists of both the Centers for Disease Control and Prevention and the World Health Organization. Known P. aeruginosa virulence factors have been extensively studied and are implicated in counteracting host defenses, causing direct damage to the host tissues, and increased microbial competitiveness. Targeting virulence factors has emerged as a new line of defense in the battle against MDR P. aeruginosa strains. Bacterial metallothionein is a newly recognized virulence factor that enables evasion of the host immune response. The studies described here identify mechanisms in which bacterial metallothionein (PmtA) plays a part in P. aeruginosa pathogenicity and identifies PmtA as a potential therapeutic target.


Assuntos
Proteínas de Bactérias , Biofilmes , Macrófagos , Metalotioneína , Estresse Oxidativo , Fagocitose , Pseudomonas aeruginosa , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Pseudomonas aeruginosa/metabolismo , Humanos , Metalotioneína/genética , Metalotioneína/metabolismo , Macrófagos/microbiologia , Macrófagos/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Células THP-1 , Piocianina/metabolismo
6.
J Antibiot (Tokyo) ; 77(7): 454-465, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38724627

RESUMO

Antibiotic resistance is a major health problem worldwide. Pseudomonas aeruginosa is a Gram-negative pathogen with an arsenal of virulence factors and elevated antimicrobial resistance. It is a leading cause of nosocomial infections with high morbidity and mortality. The significant time and effort required to develop new antibiotics can be circumvented using alternative therapeutic strategies, including anti-virulence targets. This study aimed to investigate the anti-virulence activity of the FDA-approved drugs miconazole and phenothiazine against P. aeruginosa. The phenotypic effect of sub-inhibitory concentrations of miconazole and phenothiazine on biofilm, pyocyanin, protease, rhamnolipid and hemolysin activities in PAO1 strain was examined. qRT-PCR was used to assess the effect of drugs on quorum-sensing genes that regulate virulence. Further, the anti-virulence potential of miconazole and phenothiazine was evaluated in silico and in vivo. Miconazole showed significant inhibition of Pseudomonas virulence by reducing biofilm-formation approximately 45-48%, hemolytic-activity by 59%, pyocyanin-production by 47-49%, rhamnolipid-activity by approximately 42-47% and protease activity by 36-40%. While, phenothiazine showed lower anti-virulence activity, it inhibited biofilm (31-35%), pyocyanin (37-39%), protease (32-40%), rhamnolipid (35-40%) and hemolytic activity (47-56%). Similarly, there was significantly reduced expression of RhlR, PqsR, LasI and LasR following treatment with miconazole, but less so with phenothiazine. In-silico analysis revealed that miconazole had higher binding affinity than phenothiazine to LasR, RhlR, and PqsR QS-proteins. Furthermore, there was 100% survival in mice injected with PAO1 treated with miconazole. In conclusion, miconazole and phenothiazine are promising anti-virulence agents for P. aeruginosa.


Assuntos
Antibacterianos , Biofilmes , Miconazol , Fenotiazinas , Pseudomonas aeruginosa , Percepção de Quorum , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/patogenicidade , Pseudomonas aeruginosa/genética , Percepção de Quorum/efeitos dos fármacos , Miconazol/farmacologia , Fenotiazinas/farmacologia , Biofilmes/efeitos dos fármacos , Virulência/efeitos dos fármacos , Antibacterianos/farmacologia , Animais , Testes de Sensibilidade Microbiana , Piocianina/biossíntese , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Fatores de Virulência/genética , Camundongos , Simulação de Acoplamento Molecular , Glicolipídeos
7.
J Appl Microbiol ; 135(5)2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38587815

RESUMO

AIMS: Drug repurposing is an attractive strategy to control biofilm-related infectious diseases. In this study, two drugs (montelukast and cefoperazone) with well-established therapeutic applications were tested on Pseudomonas aeruginosa quorum sensing (QS) inhibition and biofilm control. METHODS AND RESULTS: The activity of montelukast and cefoperazone was evaluated for Pqs signal inhibition, pyocyanin synthesis, and prevention and eradication of Ps. aeruginosa biofilms. Cefoperazone inhibited the Pqs system by hindering the production of the autoinducer molecules 2-heptyl-4-hydroxyquinoline (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone (the Pseudomonas quinolone signal or PQS), corroborating in silico results. Pseudomonas aeruginosa pyocyanin production was reduced by 50%. The combination of the antibiotics cefoperazone and ciprofloxacin was synergistic for Ps. aeruginosa biofilm control. On the other hand, montelukast had no relevant effects on the inhibition of the Pqs system and against Ps. aeruginosa biofilm. CONCLUSION: This study provides for the first time strong evidence that cefoperazone interacts with the Pqs system, hindering the formation of the autoinducer molecules HHQ and PQS, reducing Ps. aeruginosa pathogenicity and virulence. Cefoperazone demonstrated a potential to be used in combination with less effective antibiotics (e.g. ciprofloxacin) to potentiate the biofilm control action.


Assuntos
Acetatos , Antibacterianos , Biofilmes , Cefoperazona , Ciclopropanos , Pseudomonas aeruginosa , Quinolinas , Percepção de Quorum , Sulfetos , Pseudomonas aeruginosa/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Sulfetos/farmacologia , Percepção de Quorum/efeitos dos fármacos , Antibacterianos/farmacologia , Acetatos/farmacologia , Quinolinas/farmacologia , Ciclopropanos/farmacologia , Cefoperazona/farmacologia , Testes de Sensibilidade Microbiana , Piocianina/metabolismo , Ciprofloxacina/farmacologia , Quinolonas/farmacologia
8.
Microbiology (Reading) ; 170(3)2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38568202

RESUMO

Understanding the evolution of antibiotic resistance is important for combating drug-resistant bacteria. In this work, we investigated the adaptive response of Pseudomonas aeruginosa to ciprofloxacin. Ciprofloxacin-susceptible P. aeruginosa ATCC 9027, CIP-E1 (P. aeruginosa ATCC 9027 exposed to ciprofloxacin for 14 days) and CIP-E2 (CIP-E1 cultured in antibiotic-free broth for 10 days) were compared. Phenotypic responses including cell morphology, antibiotic susceptibility, and production of pyoverdine, pyocyanin and rhamnolipid were assessed. Proteomic responses were evaluated using comparative iTRAQ labelling LC-MS/MS to identify differentially expressed proteins (DEPs). Expression of associated genes coding for notable DEPs and their related regulatory genes were checked using quantitative reverse transcriptase PCR. CIP-E1 displayed a heterogeneous morphology, featuring both filamentous cells and cells with reduced length and width. By contrast, although filaments were not present, CIP-E2 still exhibited size reduction. Considering the MIC values, ciprofloxacin-exposed strains developed resistance to fluoroquinolone antibiotics but maintained susceptibility to other antibiotic classes, except for carbapenems. Pyoverdine and pyocyanin production showed insignificant decreases, whereas there was a significant decrease in rhamnolipid production. A total of 1039 proteins were identified, of which approximately 25 % were DEPs. In general, there were more downregulated proteins than upregulated proteins. Noted changes included decreased OprD and PilP, and increased MexEF-OprN, MvaT and Vfr, as well as proteins of ribosome machinery and metabolism clusters. Gene expression analysis confirmed the proteomic data and indicated the downregulation of rpoB and rpoS. In summary, the response to CIP involved approximately a quarter of the proteome, primarily associated with ribosome machinery and metabolic processes. Potential targets for bacterial interference encompassed outer membrane proteins and global regulators, such as MvaT.


Assuntos
Ciprofloxacina , Infecções por Pseudomonas , Humanos , Ciprofloxacina/farmacologia , Pseudomonas aeruginosa/genética , Cromatografia Líquida , Proteômica , Piocianina , Espectrometria de Massas em Tandem , Antibacterianos/farmacologia
9.
Bioprocess Biosyst Eng ; 47(6): 903-917, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38630261

RESUMO

In the present study, the potential of Pseudomonas citronellolis 620C strain was evaluated, for the first time, to generate electricity in a standard, double chamber microbial fuel cell (MFC), with oily wastewater (OW) being the fuel at 43.625 mg/L initial chemical oxygen demand (COD). Both electrochemical and physicochemical results suggested that this P. citronellolis strain utilized efficiently the OW substrate and generated electricity in the MFC setup reaching 0.05 mW/m2 maximum power. COD removal was remarkable reaching 83.6 ± 0.1%, while qualitative and quantitative gas chromatography/mass spectrometry (GC/MS) analysis of the OW total petroleum and polycyclic aromatic hydrocarbons, and fatty acids revealed high degradation capacity. It was also determined that P. citronellolis 620C produced pyocyanin as electron shuttle in the anodic MFC chamber. To the authors' best knowledge, this is the first study showing (phenazine-based) pyocyanin production from a species other than P. aeruginosa and, also, the first time that P. citronellolis 620C has been shown to produce electricity in a MFC. The production of pyocyanin, in combination with the formation of biofilm in the MFC anode, as observed with scanning electron microscopy (SEM) analysis, makes this P. citronellolis strain an attractive and promising candidate for wider MFC applications.


Assuntos
Fontes de Energia Bioelétrica , Pseudomonas , Piocianina , Águas Residuárias , Fontes de Energia Bioelétrica/microbiologia , Piocianina/biossíntese , Piocianina/metabolismo , Águas Residuárias/microbiologia , Pseudomonas/metabolismo , Eletricidade
10.
World J Microbiol Biotechnol ; 40(6): 184, 2024 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-38683406

RESUMO

The use of engineered nanoparticles against pathogenic bacteria has gained attention. In this study, zinc oxide nanoparticles conjugated with rutin were synthesized and their antivirulence properties against Pseudomonas aeruginosa and Staphylococcus aureus. The physicochemical characteristics of ZnO-Rutin NPs were investigated using SEM, FT-IR, XRD, DLS, EDS, and zeta potential analyses. Antimicrobial properties were evaluated by well diffusion, microdilution, growth curve, and hemolytic activity assays. The expression of quorum sensing (QS) genes including the lasI and rhlI in P. aeruginosa and agrA in S. aureus was assessed using real-time PCR. Swimming, swarming, twitching, and pyocyanin production by P. aeruginosa were evaluated. The NPs were amorphous, 14-100 nm in diameter, surface charge of -34.3 mV, and an average hydrodynamic size of 161.7 nm. Regarding the antibacterial activity, ZnO-Rutin NPs were more potent than ZnO NPs and rutin, and stronger inhibitory effects were observed on S. aureus than on P. aeruginosa. ZnO-Rutin NPs inhibited the hemolytic activity of P. aeruginosa and S. aureus by 93.4 and 92.2%, respectively, which was more efficient than bare ZnO NPs and rutin. ZnO-Rutin NPs reduced the expression of the lasI and rhlI in P. aeruginosa by 0.17-0.43 and 0.37-0.70 folds, respectively while the expression of the agrA gene in S. aureus was decreased by 0.46-0.56 folds. Furthermore, ZnO-Rutin NPs significantly reduced the swimming and twitching motility and pyocyanin production of P. aeruginosa. This study demonstrates the antivirulence features of ZnO-Rutin NPs against pathogenic bacteria which can be associated with their QS inhibitory effects.


Assuntos
Antibacterianos , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa , Percepção de Quorum , Rutina , Staphylococcus aureus , Óxido de Zinco , Óxido de Zinco/farmacologia , Óxido de Zinco/química , Rutina/farmacologia , Rutina/química , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/farmacologia , Antibacterianos/química , Percepção de Quorum/efeitos dos fármacos , Nanopartículas/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Nanopartículas Metálicas/química , Hemólise/efeitos dos fármacos , Virulência/efeitos dos fármacos , Tamanho da Partícula , Piocianina/metabolismo
11.
Microb Pathog ; 191: 106664, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38679245

RESUMO

Pseudomonas aeruginosa causes life-threatening diseases and is resistant to almost all conventional antibiotics. The quorum sensing (QS) system of P. aeruginosa contributes to many pathogenic factors some of which are pigment production, motility, and biofilm. The disruption of quorum sensing system may be an impactful strategy to deal with infections. The present study investigates the anti-quorum sensing property of a bioactive molecule extracted from marine epibiotic bacteria present on the surface of seaweeds. Among all the isolates tested against monitor strain Chromobacterium violaceum (MTCC 2656), the one with the highest activity was identified as Bacillus zhangzhouensis SK4. The culture supernatant was extracted with chloroform which was then partially purified by TLC and column chromatography. The probable anti-QS compound was identified as 1,2-benzenedicarboxylic acid, bis (2-methylpropyl ester) by GC-MS and NMR analysis. The treatment of P. aeruginosa MCC 3457 with the lead compound resulted in the reduced production of pyocyanin, rhamnolipids, exopolysaccharide, biofilm, and motility. The observations of light and scanning electron microscopy also supported the biofilm inhibition. The lead compound showed synergism with the meropenem antibiotic and significantly reduced MIC. The molecular docking and pharmacokinetics study predicted 1, 2-benzenedicarboxylic acid, bis (2-methylpropyl ester), a phthalate derivative as a good drug candidate. The molecular dynamics study was also performed to check the stability of the lead compound and LasR complex. Further, lead compounds did not exhibit any cytotoxicity when tested on human embryonic kidney cells. As per our knowledge, this is the first report on the anti-QS activity of B. zhangzhouensis SK4, indicating that epibiotic bacteria can be a possible source of novel compounds to deal with the multidrug resistance phenomenon.


Assuntos
Antibacterianos , Bacillus , Biofilmes , Simulação de Acoplamento Molecular , Pseudomonas aeruginosa , Percepção de Quorum , Fatores de Virulência , Percepção de Quorum/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Fatores de Virulência/metabolismo , Humanos , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/isolamento & purificação , Bacillus/efeitos dos fármacos , Bacillus/química , Bacillus/metabolismo , Chromobacterium/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Piocianina/metabolismo , Proteínas de Bactérias/metabolismo , Glicolipídeos/farmacologia , Glicolipídeos/química , Polissacarídeos Bacterianos/farmacologia , Polissacarídeos Bacterianos/isolamento & purificação , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/metabolismo
12.
Microb Pathog ; 191: 106663, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38679246

RESUMO

Quorum sensing (QS) has a central role in biofilm lifestyle and antimicrobial resistance, and disrupting these signaling pathways is a promising strategy to control bacterial pathogenicity and virulence. In this study, the efficacy of three structurally related benzaldehydes (4-hydroxybenzaldehyde, 4-hydroxy-3-methoxybenzaldehyde (vanillin) and 4-hydroxy-3,5-dimethoxybenzaldehyde (syringaldehyde)) in disrupting the las and pqs systems of Pseudomonas aeruginosa was investigated using bioreporter strains and computational simulations. Additionally, these benzaldehydes were combined with tobramycin and ciprofloxacin antibiotics to evaluate their ability to increase antibiotic efficacy in preventing and eradicating P. aeruginosa biofilms. To this end, the total biomass, metabolic activity and culturability of the biofilm cells were determined. In vitro assays results indicated that the aromatic aldehydes have potential to inhibit the las and pqs systems by > 80 %. Molecular docking studies supported these findings, revealing the aldehydes binding in the same pocket as the natural ligands or receptor proteins (LasR, PQSA, PQSE, PQSR). Benzaldehydes were shown to act as virulence factor attenuators, with vanillin achieving a 48 % reduction in pyocyanin production. The benzaldehyde-tobramycin combination led not only to a 60 % reduction in biomass production but also to a 90 % reduction in the metabolic activity of established biofilms. A similar result was observed when benzaldehydes were combined with ciprofloxacin. 4-Hydroxybenzaldehyde demonstrated relevant action in increasing biofilm susceptibility to ciprofloxacin, resulting in a 65 % reduction in biomass. This study discloses, for the first time, that the benzaldehydes studied are potent QS inhibitors and also enhancers of antibiotics antibiofilm activity against P. aeruginosa.


Assuntos
Antibacterianos , Proteínas de Bactérias , Benzaldeídos , Biofilmes , Ciprofloxacina , Simulação de Acoplamento Molecular , Pseudomonas aeruginosa , Percepção de Quorum , Tobramicina , Biofilmes/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Benzaldeídos/farmacologia , Antibacterianos/farmacologia , Antibacterianos/química , Tobramicina/farmacologia , Ciprofloxacina/farmacologia , Proteínas de Bactérias/metabolismo , Fatores de Virulência/metabolismo , Testes de Sensibilidade Microbiana , Sinergismo Farmacológico , Piocianina/metabolismo , Transativadores/metabolismo , Transativadores/antagonistas & inibidores
13.
Antimicrob Agents Chemother ; 68(5): e0011824, 2024 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-38526048

RESUMO

Quorum sensing is a type of cell-cell communication that modulates various biological activities of bacteria. Previous studies indicate that quorum sensing contributes to the evolution of bacterial resistance to antibiotics, but the underlying mechanisms are not fully understood. In this study, we grew Pseudomonas aeruginosa in the presence of sub-lethal concentrations of ciprofloxacin, resulting in a large increase in ciprofloxacin minimal inhibitory concentration. We discovered that quorum sensing-mediated phenazine biosynthesis was significantly enhanced in the resistant isolates, where the quinolone circuit was the predominant contributor to this phenomenon. We found that production of pyocyanin changed carbon flux and showed that the effect can be partially inhibited by the addition of pyruvate to cultures. This study illustrates the role of quorum sensing-mediated phenotypic resistance and suggests a strategy for its prevention.


Assuntos
Antibacterianos , Ciprofloxacina , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana , Fenazinas , Pseudomonas aeruginosa , Piocianina , Percepção de Quorum , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Ciprofloxacina/farmacologia , Percepção de Quorum/efeitos dos fármacos , Fenazinas/farmacologia , Fenazinas/metabolismo , Antibacterianos/farmacologia , Piocianina/biossíntese , Farmacorresistência Bacteriana/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Quinolonas/farmacologia
14.
mSystems ; 9(4): e0116523, 2024 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-38530056

RESUMO

To establish infections in human hosts, Pseudomonas aeruginosa must overcome innate immune-generated oxidative stress, such as the hypochlorous acid (HOCl) produced by neutrophils. We set out to find specific biomarkers of oxidative stress through the development of a protocol for the metabolic profiling of P. aeruginosa cultures grown in the presence of different oxidants using a novel ionization technique for mass spectrometry, laser desorption rapid evaporative ionization mass spectrometry (LD-REIMS). We demonstrated the ability of LD-REIMS to classify samples as untreated or treated with a specific oxidant with 100% accuracy and identified a panel of 54 metabolites with significantly altered concentrations after exposure to one or more of the oxidants. Key metabolic changes were conserved in P. aeruginosa clinical strains isolated from patients with cystic fibrosis lung infections. These data demonstrated that HOCl stress impacted the Pseudomonas quinolone signal (PQS) quorum sensing system. Ten 2-alkyl-4-quinolones (AHQs) associated with the PQS system were significantly lower in concentration in HOCl-stressed P. aeruginosa cultures, including 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), the most active signal molecule of the PQS system. The PQS system regulates the production of virulence factors, including pyocyanin and elastase, and their levels were markedly affected by HOCl stress. No pyocyanin was detectable and elastase concentrations were reduced by more than 75% in cultures grown with sub-lethal concentrations of HOCl, suggesting that this neutrophil-derived oxidant may disrupt the ability of P. aeruginosa to establish infections through interference with production of PQS-associated virulence factors. IMPORTANCE: This work demonstrates that a high-throughput ambient ionization mass spectrometry method can be used successfully to study a bacterial stress response. Its application to the opportunistic pathogen Pseudomonas aeruginosa led to the identification of specific oxidative stress biomarkers, and demonstrated that hypochlorous acid, an oxidant specifically produced by human neutrophils during infection, affects quorum sensing and reduces production of the virulence factors pyocyanin and elastase. No pyocyanin was detectable and elastase levels were reduced by more than 75% in bacteria grown in the presence of hypochlorous acid. This approach has the potential to be widely applicable to the characterization of the stress responses of bacteria.


Assuntos
Quinolonas , Percepção de Quorum , Humanos , Pseudomonas aeruginosa , Ácido Hipocloroso/metabolismo , Piocianina/metabolismo , Quinolonas/análise , Fatores de Virulência/metabolismo , Espectrometria de Massas , Oxidantes/metabolismo , Elastase Pancreática/metabolismo , Biomarcadores/metabolismo , Lasers
15.
Folia Med (Plovdiv) ; 66(1): 88-96, 2024 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-38426470

RESUMO

AIM: Due to the importance of exotoxin A and pyocyanin in the pathogenicity of this bacterium, we decided to evaluate the prevalence of genes encoding these virulence factors in clinical isolates of P.aeruginosa.


Assuntos
Infecções por Pseudomonas , Piocianina , Humanos , Pseudomonas aeruginosa/genética , Proteínas de Bactérias/genética , Exotoxinas/genética , Fatores de Virulência/genética , Infecções por Pseudomonas/epidemiologia , Infecções por Pseudomonas/microbiologia
16.
Appl Microbiol Biotechnol ; 108(1): 271, 2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38517512

RESUMO

Various virulence determinants in Pseudomonas aeruginosa are regulated by the quorum sensing (QS) network producing and releasing signalling molecules. Two of these virulence determinants are the pyocyanin and pyoverdine, which interfere with multiple cellular functions during infection. The application of QS-inhibiting agents, such as cyclodextrins (CDs), appears to be a promising approach. Further to method development, this research tested in large-volume test systems the effect of α- and ß-CD (ACD, BCD) at 1, 5, and 10 mM concentrations on the production of pyocyanin in the P. aeruginosa model system. The concentration and time-dependent quorum quenching effect of native CDs and their derivatives on pyoverdine production was tested in a small-volume high-throughput system. In the large-volume system, both ACD and BCD significantly inhibited pyocyanin production, but ACD to a greater extent. 10 mM ACD resulted in 58% inhibition, while BCD only ~40%. Similarly, ACD was more effective in the inhibition of pyoverdine production; nevertheless, the results of RMANOVA demonstrated the significant efficiency of both ACD and BCD, as well as their derivatives. Both the contact time and the cyclodextrin treatments significantly influenced pyoverdine production. In this case, the inhibitory effect of ACD after 48 h at 12.5 mM was 57%, while the inhibitory effect of BCD and its derivatives was lower than 40%. The high-level significant inhibition of both pyocyanin and pyoverdine production by ACD was detectable. Consequently, the potential value of CDs as QS inhibitors and the antivirulence strategy should be considered. KEYPOINTS: • Applicability of a simplified method for quantification of pyocyanin production was demonstrated. • The cyclodextrins significantly affected the pyocyanin and pyoverdine production. • The native ACD exhibited the highest attenuation in pyoverdine production.


Assuntos
Oligopeptídeos , Infecções por Pseudomonas , Percepção de Quorum , Humanos , Pseudomonas aeruginosa , Virulência , Piocianina , Fatores de Virulência , Antibacterianos/farmacologia , Biofilmes
17.
Anal Sci ; 40(5): 891-905, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38472735

RESUMO

Combating Pseudomonas aeruginosa infection is challenging. It secretes pyocyanin (PCN) pigment that contributes to its virulence. Neutralizing PCN via reaction with thiol-containing compounds may represent a potential therapeutic option. This study investigates the neutralization reaction between PCN and N-acetyl cysteine (NAC) for bacterial inhibition and explores its mechanism of action. The neutralization adduct (PCN-NAC) was synthesized by reacting the purified PCN and NAC. The adduct was analyzed and its structure was elucidated. LC-MS/MS method was developed for the determination of PCN-NAC in P. aeruginosa cultures post-treatment with NAC (0-5 mg/mL). The corresponding anti-bacterial potential was estimated and compared to nanoparticles (NPs) alone and under stress conditions. In silico studies were performed to support explaining the mechanism of action. Results revealed that PCN-NAC was exclusively detected in NAC-treated cultures in a concentration-dependent manner. PCN-NAC concentration (230-915 µg/mL) was directly proportional to the reduction in the bacterial viable count (28.3% ± 7.1-87.5% ± 5.9) and outperformed all tested NPs, where chitosan NPs induced 56.9% ± 7.9 inhibition, followed by zinc NPs (49.4% ± 0.9) and gold NPs (17.8% ± 7.5) even post-exposure to different stress conditions. A concomitant reduction in PCN concentration was detected. In silico studies revealed possible interactions between key bacterial proteins and PCN-NAC rather than the NAC itself. These results pose NAC as a potential choice for the management of P. aeruginosa infection, where it neutralizes PCN via the formation of PCN-NAC adduct.


Assuntos
Acetilcisteína , Pseudomonas aeruginosa , Piocianina , Fatores de Virulência , Acetilcisteína/química , Acetilcisteína/farmacologia , Antibacterianos/farmacologia , Antibacterianos/química , Espectrometria de Massa com Cromatografia Líquida , Pseudomonas aeruginosa/efeitos dos fármacos , Piocianina/metabolismo , Piocianina/antagonistas & inibidores , Piocianina/análise , Piocianina/química , Fatores de Virulência/antagonistas & inibidores , Fatores de Virulência/metabolismo
18.
J Biol Chem ; 300(3): 105741, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38340793

RESUMO

Type VI secretion systems (T6SS) are bacterial macromolecular complexes that secrete effectors into target cells or the extracellular environment, leading to the demise of adjacent cells and providing a survival advantage. Although studies have shown that the T6SS in Pseudomonas aeruginosa is regulated by the Quorum Sensing system and second messenger c-di-GMP, the underlying molecular mechanism remains largely unknown. In this study, we discovered that the c-di-GMP-binding adaptor protein PA0012 has a repressive effect on the expression of the T6SS HSI-I genes in P. aeruginosa PAO1. To probe the mechanism by which PA0012 (renamed TssZ, Type Six Secretion System -associated PilZ protein) regulates the expression of HSI-I genes, we conducted yeast two-hybrid screening and identified HinK, a LasR-type transcriptional regulator, as the binding partner of TssZ. The protein-protein interaction between HinK and TssZ was confirmed through co-immunoprecipitation assays. Further analysis suggested that the HinK-TssZ interaction was weakened at high c-di-GMP concentrations, contrary to the current paradigm wherein c-di-GMP enhances the interaction between PilZ proteins and their partners. Electrophoretic mobility shift assays revealed that the non-c-di-GMP-binding mutant TssZR5A/R9A interacts directly with HinK and prevents it from binding to the promoter of the quorum-sensing regulator pqsR. The functional connection between TssZ and HinK is further supported by observations that TssZ and HinK impact the swarming motility, pyocyanin production, and T6SS-mediated bacterial killing activity of P. aeruginosa in a PqsR-dependent manner. Together, these results unveil a novel regulatory mechanism wherein TssZ functions as an inhibitor that interacts with HinK to control gene expression.


Assuntos
Proteínas de Bactérias , Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa , Transcrição Gênica , Sistemas de Secreção Tipo VI , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Imunoprecipitação , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Piocianina/metabolismo , Percepção de Quorum , Sistemas do Segundo Mensageiro , Técnicas do Sistema de Duplo-Híbrido , Sistemas de Secreção Tipo VI/genética , Sistemas de Secreção Tipo VI/metabolismo
19.
Diagn Microbiol Infect Dis ; 109(1): 116212, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38387214

RESUMO

Pseudomonas aeruginosa, one of the most notorious organisms, causes fatal diseases like-, meningitis, pneumonia as well as worsens the prognosis of cystic fibrosis patients. It is also multi-drug resistant and resists a wide range of antibiotics. Attempts have been made to reduce its virulence/pathogenic potential using a number of organic compounds. For this purpose, the Quorum sensing (QS) system of P. aeruginosa was targeted, which regulates its virulence. Pseudomonas Quinolone System (PQS), one of the four quorum sensing systems, producing pyocyanin pigment was chosen. 2-heptyl-3-hydroxy-4-quinolone (HHQ) is a ligand which binds to PQS protein is responsible for pyocyanin pigment production. Attempts were made to find a compound analogous to HHQ which could bind to PQS active site and inhibit the pigment formation. In-silico analysis was performed to estimate possible interactions and to find/predict the possible PQS inhibitors.


Assuntos
Infecções por Pseudomonas , Quinolonas , Humanos , Percepção de Quorum/fisiologia , Pseudomonas aeruginosa/metabolismo , Pseudomonas/metabolismo , Piocianina/metabolismo , Quinolonas/farmacologia , Infecções por Pseudomonas/tratamento farmacológico , Proteínas de Bactérias/metabolismo
20.
World J Microbiol Biotechnol ; 40(3): 90, 2024 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-38341389

RESUMO

Pyocyanin is a bioactive pigment produced by Pseudomonas aeruginosa. It is an important virulence factor that plays a critical role in P. aeruginosa infections as a redox-active secondary metabolite and a quorum sensing (QS) signaling molecule. Pyocyanin production from chorismic acid requires the involvement of two homologous operons, phz1 and phz2, which are activated by QS regulatory proteins. Pyocyanin inhibits the proliferation of bacterial, fungal, and mammalian cells by inducing oxidative stress due to which it acts as a potent antibacterial, antifungal, and anticancer agent. Its potential role as a neuroprotectant needs further exploration. However, pyocyanin exacerbates the damaging effects of nosocomial infections caused by P. aeruginosa in immunocompromised individuals. Further, cystic fibrosis (CF) patients are highly susceptible to persistent P. aeruginosa infections in the respiratory system. The bacterial cells form colonies and three interconnected QS networks-pqs, las, and rhl-get activated, thus stimulating the cells to produce pyocyanin which exacerbates pulmonary complications. As an opportunistic pathogen, P. aeruginosa produces pyocyanin to impede the recovery of injuries like burn wounds through its anti-proliferative activity. Moreover, pyocyanin plays a vital role in compounding P. aeruginosa infections by promoting biofilm formation. This review begins with a brief description of the characteristics of pyocyanin, its activity, and the different aspects of its production including its biosynthesis, the role of QS, and the effect of environmental factors. It then goes on to explore the potential applications of pyocyanin as a biotherapeutic molecule while also highlighting the biomedical challenges and limitations that it presents.


Assuntos
Infecções por Pseudomonas , Piocianina , Animais , Humanos , Biofilmes , Pseudomonas aeruginosa , Proteínas de Bactérias/metabolismo , Percepção de Quorum , Fatores de Virulência/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Infecções por Pseudomonas/microbiologia , Mamíferos/metabolismo
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