Enhancement of pyocyanin production by subinhibitory concentration of royal jelly in Pseudomonas aeruginosa

Background: Pseudomonas aeruginosa, a multidrug-resistant Gram-negative bacterium, produces pyocyanin, a virulence factor associated with antibiotic tolerance. High concentrations of royal jelly have an antibacterial effect, which may potentially overcome antibacterial resistance. However, in some cases, antibiotic tolerance can occur due to prolonged stress of low-dose antibacterial agents. This study aimed to investigate the effect of subinhibitory concentrations of royal jelly on bacterial growth, pyocyanin production, and biofilm formation of P. aeruginosa. Methods: Pseudomonas aeruginosa ATCC 10145 and clinical isolates were cultured in a royal jelly-containing medium to test the antibacterial activity. Pyocyanin production was observed by measuring the absorbance at 690 nm after 36 h culture and determined using extinction coefficient 4310 M-1 cm-1. Static microtiter plate biofilm assay performed to detect the biofilm formation, followed by scanning electron microscopy. Results: Royal jelly effectively inhibited the viability of both strains from a concentration of 25%. The highest production of pyocyanin was observed in the subinhibitory concentration group 6.25%, which gradually decreased along with the decrease of royal jelly concentration. Results of one-way ANOVA tests differed significantly in pyocyanin production of the two strains between the royal jelly groups. Tukey HSD test showed concentrations of 12.5%, 6.25%, and 3.125% significantly increased pyocyanin production of ATCC 10145, and the concentrations of 12.5% and 6.25% significantly increased production of the clinical isolates. Concentrations of 12.5% and 6.125% significantly induced biofilm formation of P. aeruginosa ATCC 10145, in line with the results of the SEM analysis. Conclusions: The royal jelly concentration of 25% or higher inhibits bacterial growth; however, the subinhibitory concentration increases pyocyanin production and biofilm formation in P. aeruginosa. It is advisable to determine the appropriate concentration of royal jelly to obtain beneficial virulence inhibiting activity.

Enhancement of pyocyanin production by subinhibitory concentration of royal jelly in Pseudomonas aeruginosa

Background: Pseudomonas aeruginosa, a multidrug-resistant Gram-negative bacterium, produces pyocyanin, a virulence factor associated with antibiotic tolerance. High concentrations of royal jelly have an antibacterial effect, which may potentially overcome antibacterial resistance. However, in some cases, antibiotic tolerance can occur due to prolonged stress of low-dose antibacterial agents. This study aimed to investigate the effect of subinhibitory concentrations of royal jelly on bacterial growth, pyocyanin production, and biofilm formation of P. aeruginosa. Methods: Pseudomonas aeruginosa ATCC 10145 and clinical isolates were cultured in a royal jelly-containing medium to test the antibacterial activity. Pyocyanin production was observed by measuring the absorbance at 690 nm after 36 h culture and determined using extinction coefficient 4310 M-1 cm-1. Static microtiter plate biofilm assay performed to detect the biofilm formation, followed by scanning electron microscopy. Results: Royal jelly effectively inhibited the viability of both strains from a concentration of 25%. The highest production of pyocyanin was observed in the subinhibitory concentration group 6.25%, which gradually decreased along with the decrease of royal jelly concentration. Results of one-way ANOVA tests differed significantly in pyocyanin production of the two strains between the royal jelly groups. Tukey HSD test showed concentrations of 12.5%, 6.25%, and 3.125% significantly increased pyocyanin production of ATCC 10145, and the concentrations of 12.5% and 6.25% significantly increased production of the clinical isolates. Concentrations of 12.5% and 6.125% significantly induced biofilm formation of P. aeruginosa ATCC 10145, in line with the results of the SEM analysis. Conclusions: The royal jelly concentration of 25% or higher inhibits bacterial growth; however, the subinhibitory concentration increases pyocyanin production and biofilm formation in P. aeruginosa. It is advisable to determine the appropriate concentration of royal jelly to obtain beneficial virulence inhibiting activity.

Disruption of the Pseudomonas aeruginosa Tat system perturbs PQS-dependent quorum sensing and biofilm maturation through lack of the Rieske cytochrome bc1 sub-unit

Extracellular DNA (eDNA) is a major constituent of the extracellular matrix of Pseudomonas aeruginosa biofilms and its release is regulated via pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening a P. aeruginosa transposon library to identify factors required for DNA release, mutants with insertions in the twin-arginine translocation (Tat) pathway were identified as exhibiting reduced eDNA release, and defective biofilm architecture with enhanced susceptibility to tobramycin. P. aeruginosa tat mutants showed substantial reductions in pyocyanin, rhamnolipid and membrane vesicle (MV) production consistent with perturbation of PQS-dependent QS as demonstrated by changes in pqsA expression and 2-alkyl-4-quinolone (AQ) production. Provision of exogenous PQS to the tat mutants did not return pqsA, rhlA or phzA1 expression or pyocyanin production to wild type levels. However, transformation of the tat mutants with the AQ-independent pqs effector pqsE restored phzA1 expression and pyocyanin production. Since mutation or inhibition of Tat prevented PQS-driven auto-induction, we sought to identify the Tat substrate(s) responsible. A pqsA::lux fusion was introduced into each of 34 validated P. aeruginosa Tat substrate deletion mutants. Analysis of each mutant for reduced bioluminescence revealed that the primary signalling defect was associated with the Rieske iron-sulfur subunit of the cytochrome bc1 complex. In common with the parent strain, a Rieske mutant exhibited defective PQS signalling, AQ production, rhlA expression and eDNA release that could be restored by genetic complementation. This defect was also phenocopied by deletion of cytB or cytC1. Thus, either lack of the Rieske sub-unit or mutation of cytochrome bc1 genes results in the perturbation of PQS-dependent autoinduction resulting in eDNA deficient biofilms, reduced antibiotic tolerance and compromised virulence factor production.

Enhancement of pyocyanin production by subinhibitory concentration of royal jelly in Pseudomonas aeruginosa

Background: Pseudomonas aeruginosa, a multidrug-resistant Gram-negative bacterium, produces pyocyanin, a virulence factor associated with antibiotic tolerance. High concentrations of royal jelly have an antibacterial effect, which may potentially overcome antibacterial resistance. However, in some cases, antibiotic tolerance can occur due to prolonged stress of low-dose antibacterial agents. This study aimed to investigate the effect of subinhibitory concentrations of royal jelly on bacterial growth, pyocyanin production, and biofilm formation of P. aeruginosa. Methods: Pseudomonas aeruginosa ATCC 10145 and clinical isolates were cultured in a royal jelly-containing medium to test the antibacterial activity. Pyocyanin production was observed by measuring the absorbance at 690 nm after 36 h culture and determined using extinction coefficient 4310 M-1 cm-1. Static microtiter plate biofilm assay performed to detect the biofilm formation, followed by scanning electron microscopy. Results: Royal jelly effectively inhibited the viability of both strains from a concentration of 25%. The highest production of pyocyanin was observed in the subinhibitory concentration group 6.25%, which gradually decreased along with the decrease of royal jelly concentration. Results of one-way ANOVA tests differed significantly in pyocyanin production of the two strains between the royal jelly groups. Tukey HSD test showed concentrations of 12.5%, 6.25%, and 3.125% significantly increased pyocyanin production of ATCC 10145, and the concentrations of 12.5% and 6.25% significantly increased production of the clinical isolates. Concentrations of 12.5% and 6.125% significantly induced biofilm formation of P. aeruginosa ATCC 10145, in line with the results of the SEM analysis. Conclusions: Royal jelly concentrations of 25% or higher can inhibit bacterial growth; however, subinhibitory concentrations could increase pyocyanin production and biofilm formation in P. aeruginosa. It is advisable to determine the appropriate concentration of royal jelly to obtain beneficial virulence inhibiting activity.

High pyocyanin production and non-motility of Pseudomonas aeruginosa isolates are correlated with septic shock or death in bacteremic patients

Studies of the outcome of Pseudomonas aeruginosa bacteremia (Pab) have focused mainly on antibiotic appropriateness. However, P. aeruginosa possesses many virulence factors whose roles in outcomes have not been examined in humans, except for the type III secretion system (T3SS) toxins. The purpose of this study was to examine the role of virulence factors other than the T3SS toxins. Bacterial isolates were collected from 75 patients who suffered from Pa blood stream infections. Host factors such as neutropenia, immunosuppression, comorbidities, time to effective antibiotics, source of bacteremia, and presence of multidrug resistant (MDR) isolate were studied. The isolates were analyzed for the presence of toxin genes, proteolytic activity, swimming and twitching motility, and pyocyanin production. The data were analyzed to ascertain which virulence factors correlated with poor outcomes defined as septic shock or death (SS) within 7 days. Septic shock or death occurred in 25/75 patients. Univariate analysis identified age as a host factor that exerted a significant effect on these outcomes. Ineffective antibiotics administered during the first 24 hours of treatment or MDR P. aeruginosa did not influence the frequency of SS, nor did the presence of lasB, exoA, exoS exoU, plcH genes and proteolytic activity. However, 6/8 patients infected with non-motile isolates, developed SS, p = 0.014 and 5/6 isolates that produced large amounts of pyocyanin (>18ug/ml), were associated with SS, p = 0.014. Multivariate analysis indicated that the odds ratio (OR) for development of SS with a non-motile isolate was 6.8, with a 95% confidence interval (CI) (1.37, 51.5), p = 0.030 and with high pyocyanin producing isolates, an OR of 16.9, 95% CI = (2.27, 360), p = .017. This study evaluating the role of microbial factors that significantly effect outcomes following Pa bloodstream infection suggests that P. aeruginosa strains showing high pyocyanin production and the lack of motility independently increase the risk of SS.

Possible antivirulent activity of some agents against clinical isolates of Pseudomonas aeruginosa

Background: Pseudomonas aeruginosa has developed different mechanisms of resistance against antibiotics and became one of the most life-threatening pathogens. Fighting against its virulence Factors are an alternative therapeutic target. Objective: This study was directed towards the investigation of anti-quorum sensing activity and inhibitory action on virulence factors of different agents including antibacterial agents to which Pseudomonas aeruginosa isolates are resistant and non-antibacterial agents. Methodology: Anti-quorum sensing activity of ceftriaxone, ceftazidime (CAZ), cefepime (FEP), vancomycin (VA), paracetamol (PA), and pheniramine maleate (PHE) investigated as well as their ability to reduce other virulence factors including protease, hemolysin, and pyocyanin production. Results: This study showed that 3rd and 4th generations cephalosporins could be used as anti-quorum sensing agents effectively in the treatment of Pseudomonas aeruginosa infections, however, vancomycin, paracetamol, and pheniramine maleate had no effect on inhibiting the studied virulence factors. Conclusion: From our study we conclude that although cephalosporins at the used concentrations did not show anti-pseudomonal activity they were effective as anti virulent agents that could be utilized in therapeutically in controlling Pseudomonas aeruginosa infections.

Disruption of the Pseudomonas aeruginosa Tat system perturbs PQS-dependent quorum sensing and biofilm maturation through loss of the Rieske cytochrome bc1 sub-unit

Extracellular DNA (eDNA) is a major constituent of the extracellular matrix of P seudomonas aeruginosa biofilms and its release is regulated via the pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening a P. aeruginosa transposon library to identify factors required for DNA release, mutants with insertions in the twin-arginine translocation (Tat) pathway were identified as exhibiting reduced eDNA release, and defective biofilm architecture with enhanced susceptibility to tobramycin. P. aeruginosa tat mutants showed substantial reductions in pyocyanin, rhamnolipid and membrane vesicle (MV) production consistent with perturbation of 2-heptyl-3-hydroxy-4-quinolone (PQS) dependent QS as demonstrated by changes in pqsA expression and 2-alkyl-4-quinolone (AQ) production. Provision of exogenous PQS to the tat mutants did not return pqsA , rhlA or phzA1 expression or pyocyanin production to wild type levels. However, transformation of the tat mutants with the AQ-independent pqs effector pqsE restored phzA1 expression and pyocyanin production. Since mutation or inhibition of Tat prevented PQS-driven auto-induction, we sought to identify the Tat secretion substrate responsible. A pqsA::lux fusion was introduced into each of 34 validated P. aeruginosa Tat substrate deletion mutants. Analysis of each mutant for reduced bioluminescence revealed that the signalling defect was associated with the Rieske iron-sulfur subunit of the cytochrome bc 1 complex. In common with the parent strain, a Rieske mutant exhibited defective PQS signalling, AQ production, rhlA expression and eDNA release that could be restored by genetic complementation. Thus, lack of the Rieske sub-unit export is clearly responsible for the Tat-mediated perturbation of PQS-dependent QS, the loss of virulence factor production, biofilm eDNA and the tobramycin tolerance of P. aeruginosa biofilms.

The Effect of Silver Nanoparticles on Pyocyanin Production of Pseudomonas aeruginosa Isolated From Clinical Specimens

Background: Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen causing a wide range of human infections. The organism is resistant to a wide range of antibiotics. The purpose of this study was to investigate the effect of AgNPs on pyocyanin pigment production of P. aeruginosa bacteria isolated from clinical specimens. Methods: In this study, 15 clinical isolates of P. aeruginosa were collected from different specimens of hospitalized patients. P. aeruginosa was detected by biochemical and molecular (detection of pbo1 gene by colony PCR method) methods and the MIC and MBC of AgNPs were determined by agar dilution method. Inhibition of P. aeruginosa pyocyanin production at AgNPs concentrations of 0, 0.3, 0.5, 1 and 1.5 mg/ml of was studied with OD of 520 nm. Results: The mean MIC and MBC of AgNPs were 1.229 and 1.687 mg/ml, respectively. Pyocyanin production was investigated for all isolates at different concentrations of nanoparticles, and their comparison showed that with increasing nanoparticle concentration, pyocyanin production significantly decreased (p<0.05). Conclusion: According to the results of this study, AgNPs had an inhibitory effect on P. aeruginosa and its pigment production and with increasing nanoparticles concentration, pigment production decreased; therefore, it seems that the nanoparticles can be used to treat and prevent diseases caused by P. aeruginosa.

The three NADH dehydrogenases of Pseudomonas aeruginosa: Their roles in energy metabolism and links to virulence

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen which relies on a highly adaptable metabolism to achieve broad pathogenesis. In one example of this flexibility, to catalyze the NADH:quinone oxidoreductase step of the respiratory chain, P. aeruginosa has three different enzymes: NUO, NQR and NDH2, all of which carry out the same redox function but have different energy conservation and ion transport properties. In order to better understand the roles of these enzymes, we constructed two series of mutants: (i) three single deletion mutants, each of which lacks one NADH dehydrogenase and (ii) three double deletion mutants, each of which retains only one of the three enzymes. All of the mutants grew approximately as well as wild type, when tested in rich and minimal medium and in a range of pH and [Na+] conditions, except that the strain with only NUO (ΔnqrFΔndh) has an extended lag phase. During exponential phase, the NADH dehydrogenases contribute to total wild-type activity in the following order: NQR > NDH2 > NUO. Some mutants, including the strain without NQR (ΔnqrF) had increased biofilm formation, pyocyanin production, and killed more efficiently in both macrophage and mouse infection models. Consistent with this, ΔnqrF showed increased transcription of genes involved in pyocyanin production.