Expression Of lasR Research Articles

Fatty acid synthesis promoted by PA1895-1897 operon delays quorum sensing activation in Pseudomonas aeruginosa

PA1895-1897 is a quorum sensing (QS) operon regulated by the anti-activator LuxR homologue QscR in Pseudomonas aeruginosa. We aimed to investigate its impact on bacterial metabolism, and whether it contributes to the delayed QS activation. We performed liquid chromatograph-mass spectrometer-based metabolomics using wildtype PAO1, PA1895-1897-knockout mutant, and mutant with pJN105.PA1895-1897 overexpression vector. The impact of metabolites on QS signaling molecule (3OC12-HSL and C4-HSL) concentrations, pyocyanin production, and QS gene (lasR, lasI, rhlR, and rhlI) expression was examined. Metabolomics analysis found that fatty acid biosynthesis had the highest fold enrichment among all metabolic pathways in PA1895-1897-overexpressed mutants. Among these enriched fatty acids, palmitoleic acid and acetic acid were the predominantly abundant ones that significantly affected by PA1895-1897 operon. When different doses of exogenous palmitoleic acid or acetic acid were added to the cultures of PA1895-1897 knockout mutants, their levels of 3OC12-HSL, C4-HSL, and pyocyanin were decreased in a dose-dependent manner. High doses of palmitoleic acid and acetic acid suppressed the mRNA expression of lasR, rhlR, and rhlI. Inhibition of fatty acid biosynthesis increased the production of 3OC12-HSL, C4-HSL, and pyocyanin in PA1895-1897-overexpressed cultures. Our data suggest that fatty acid synthesis is promoted by PA1895-1897 operon, and contributes the delayed expression of QS phenotypes, furthering the understanding about the regulation of bacterial QS activation.

Roles of transcriptional factor PsrA in the regulation of quorum sensing in Pseudomonas aeruginosa PAO1.

The transcription factor PsrA regulates fatty acid metabolism, the type III secretion system, and quinolone signaling quorum sensing system in Pseudomonas aeruginosa. To explore additional roles of PsrA in P. aeruginosa, this study engineered a P. aeruginosa PAO1 strain to carry a recombinant plasmid with the psrA gene (pMMBpsrA) and examined the impact of elevated psrA expression to the bacterium. Transcriptomic analysis revealed that PsrA significantly downregulated genes encoding the master quorum-sensing regulators, RhlR and LasR, and influenced many quorum-sensing-associated genes. The role of PsrA in quorum sensing was further corroborated by testing autoinducer synthesis in PAO1 [pMMBpsrA] using two reporter bacteria strains Chromobacterium violaceum CV026 and Escherichia coli [pSB1075], which respond to short- and long-chain acyl homoserine lactones, respectively. Phenotypic comparisons of isogenic ΔpsrA, ΔlasR, and ΔpsrAΔlasR mutants revealed that the reduced elastase, caseinase, and swarming activity in PAO1 [pMMBpsrA] were likely mediated through LasR. Additionally, electrophoretic mobility shift assays demonstrated that recombinant PsrA could bind to the lasR promoter at a 5'-AAACGTTTGCTT-3' sequence, which displays moderate similarity to the previously reported consensus PsrA binding motif. Furthermore, the PsrA effector molecule oleic acid inhibited PsrA binding to the lasR promoter and restored several quorum sensing-related phenotypes to wild-type levels. These findings suggest that PsrA regulates certain quorum-sensing phenotypes by negatively regulating lasR expression, with oleic acid acting as a crucial signaling molecule.

Exoprotease exploitation and social cheating in a Pseudomonas aeruginosa environmental lysogenic strain with a noncanonical quorum sensing system.

Social cheating is the exploitation of public goods that are costly metabolites, like exoproteases. Exoprotease exploitation in Pseudomonas aeruginosa has been studied in reference strains. Experimental evolution with reference strains during continuous growth in casein has demonstrated that nonexoprotease producers that are lasR mutants are selected while they behave as social cheaters. However, noncanonical quorum-sensing systems exist in P. aeruginosa strains, which are diverse. In this work, the exploitation of exoproteases in the environmental strain ID4365 was evaluated; ID4365 has a nonsense mutation that precludes expression of LasR. ID4365 produces exoproteases under the control of RhlR, and harbors an inducible prophage. As expected, rhlR mutants of ID4365 behave as social cheaters, and exoprotease-deficient individuals accumulate upon continuous growth in casein. Moreover, in all continuous cultures, population collapses occur. However, this also sometimes happens before cheaters dominate. Interestingly, during growth in casein, ID4565's native prophage is induced, suggesting that the metabolic costs imposed by social cheating may increase its induction, promoting population collapses. Accordingly, lysogenization of the PAO1 lasR mutant with this prophage accelerated its collapse. These findings highlight the influence of temperate phages in social cheating.

Inhibition of Adherence and Biofilm Formation of Pseudomonas aeruginosa by Immobilized ZnO Nanoparticles on Silicone Urinary Catheter Grafted by Gamma Irradiation.

Nosocomial infections caused by microbial biofilm formation on biomaterial surfaces such as urinary catheters are complicated by antibiotic resistance, representing a common problem in hospitalized patients. Therefore, we aimed to modify silicone catheters to resist microbial adherence and biofilm formation by the tested microorganisms. This study used a simple direct method to graft poly-acrylic acid onto silicone rubber films using gamma irradiation to endow the silicone surface with hydrophilic carboxylic acid functional groups. This modification allowed the silicone to immobilize ZnO nanoparticles (ZnO NPs) as an anti-biofilm. The modified silicone films were characterized by FT-IR, SEM, and TGA. The anti-adherence ability of the modified silicone films was evidenced by the inhibition of biofilm formation by otherwise strong biofilm-producing Gram-positive, Gram-negative, and yeast clinical isolates. The modified ZnO NPs grafted silicone showed good cytocompatibility with the human epithelial cell line. Moreover, studying the molecular basis of the inhibitory effect of the modified silicone surface on biofilm-associated genes in a selected Pseudomonas aeruginosa isolate showed that anti-adherence activity might be due to the significant downregulation of the expression of lasR, lasI, and lecB genes by 2, 2, and 3.3-fold, respectively. In conclusion, the modified silicone catheters were low-cost, offering broad-spectrum anti-biofilm activity with possible future applications in hospital settings.

Effects of Poly-N-isopropylacrylamide Microgels Containing Antibiofilm Substances on Pseudomonas aeruginosa Isolated from Chronic Wounds

Biofilm formation helps Pseudomonas aeruginosa (P. aeruginosa) survive in various environments. Microgels can be effective in treatment of bacterial infections. The major aim of this study was to investigate effects of poly-N-isopropylacrylamide microgels (PNIPAM) on P. aeruginosa. Totally, 100 P. aeruginosa strains were isolated from chronic wound infections. Quantitative assessments of biofilm formation and antibiotic susceptibility were carried out. Furthermore, algD, lasR, and PA2714 genes were amplified to investigate gene frequencies and expression rates. Significant decreases were seen in lasR expression in EDTA-treated samples. Significant decreases were observed in expression of algD and lasR treated with xylitol. Decreased expression of PA2714 was seen in samples treated with xylitol with no significance. The PNIPAM containing xylitol or EDTA could penetrate biofilms of P. aeruginosa and significantly decrease expression of lasR and algD. This can be a novel strategy in the management of chronic wounds.

Expression of LasR and LasI genes in quorum sensing system under the influence of iron oxide nanoparticles modified with silver surface structure in the clinical strain of Pseudomonas aeruginosa

This study was an attempt to examine expression of LasR and LasI genes in quorum sensing system under the influence of iron oxide nanoparticles modified with silver surface structure in the clinical strain of Pseudomonas aeruginosa. The participants of this study 51 samples of Pseudomonas aeruginosa bacteria (samples from different sources) such as Wounds, blood, urine and sputum (from Milad Hospital) in January 2019 to May 2020. The steady development of antibiotic resistance in various bacteria poses a high-risk threat to the health of the global community. Activation of quorum sensing signaling and biofilm formation leads to antimicrobial resistance of pathogens, thus increasing the difficulty in treating bacterial diseases. In this study, 50 clinical samples of patients were prepared in Milad Hospital, Tehran. By confirmatory and differential tests, these samples were confirmed as strains of Pseudomonas aeruginosa. After obtaining the MIC of iron oxide nanoparticles, this concentration (MIC = 0.512) of the nanoparticles was applied to the bacteria. The effect of iron-oxide surface nanoparticles on bacterial pathogens and genes affecting it (LasI and LasR) was investigated. Therefore, in this study, the expression of LasR and LasI genes in quorum sensing system under the influence of iron oxide nanoparticles modified surface structure with silver was investigated in the clinical strain of Pseudomonas aeruginosa.The analysis of data revealed that In microbiological (phenotypic) studies, the effect of iron oxide nanoparticles on surface structure modified with silver led to the lack of growth of Pseudomonas aeruginosa. The expression of LasR and LasI genes was significantly reduced under the influence of iron oxide nanoparticles modified surface structure with silver.In this study, the fidings that iron oxide nanoparticles modified by surface structure can be effective on preventing pathogens by Pseudomonas aeruginosa. Also, surface iron oxide modified silver nanoparticles have antimicrobial properties in inhibiting bacterial growth there are various that are applied through different mechanisms. Decreased expression of LasR and LasI genes causes our nanoparticles to inhibit the quorum sensing system. Therefore, this study proposes to investigate the synergistic effect of iron oxide nanoparticles with antibiotics and other genes from the quorum sensing system.

Quorum quenching activity of Andrographis paniculata (Burm f.) Nees andrographolide compounds on metallo-β-lactamase-producing clinical isolates of Pseudomonas aeruginosa PA22 and PA247 and their effect on lasR gene expression

Andrographis paniculata (Burm f.) Nees is a tropical plant native to Southeast Asia that has been used as an effective remedy for a wide variety of illnesses in traditional Chinese and Ayurvedic medicine. The antimicrobial activity of its crude extract had been shown to be due to its quorum quenching activity. The study determined the effect of purified extracted compounds from the leaf of A. paniculata, namely: andrographolide, 14-deoxyandrographolide, 14-deoxy-12-hydroxyandrographolide and neoandrographolide on quorum sensing-mediated virulence mechanisms in clinical isolates of metallo-β-lactamase (MβL)-producing Pseudomonas aeruginosa. Their effect on the expression of the lasR gene, which codes for LasR, a transcription activator protein of the quorum sensing system in P. aeruginosa was also determined using RT-qPCR. All the pure compounds significantly decreased the biofilm formation, protease production and swarming motility of the P. aeruginosa isolates compared to the untreated controls (p < 0.05). Results of the RT-qPCR assay showed that all compounds significantly downregulated the expression of lasR compared to the untreated control (p < 0.05), supporting the position that the lower virulence activities of the treated group were due to quorum quenching activity of the pure compounds. Multiple comparisons using Tukey's HSD analysis revealed that the means of the relative expression of lasR of the isolates treated with the different compounds were not significantly different from each other (p > 0.05), suggesting equal potencies. Results show the potential of the isolated pure compounds from A. paniculata for use as antimicrobial agents as a result of their quorum quenching activities.

The inhibitory effects of Staphylococcus aureus on the antibiotic susceptibility and virulence factors of Pseudomonas aeruginosa: A549 cell line model

Pseudomonas aeruginosa and Staphylococcus aureus often lead to serious lung infections. This study aimed to investigate the role of S. aureus in the expression of the β-lactamase enzymes and virulence factors of P. aeruginosa in the polymicrobial infections of the respiratory tract. Biofilm and planktonic co-culture of P. aeruginosa and S. aureus were performed in the A549 cell line. Then, antibiotic resistance and virulence factors of P. aeruginosa were examined, and the expression of lasR, lasI, algD, mexR, and KPC genes were determined using qPCR. S.aureus decreased β-lactam resistance but increased resistance to tobramycin in the biofilm condition. Furthermore, S.aureus showed a positive effect on reducing resistance to meropenem, doripenem, and tobramycin (except PA-2). Altough it was demonstrated that S.aureus reduced the viability of P. aeruginosa, particularly in the biofilm state, the pathogenicity of the recovered strains of P.aeruginosa increased. Moreover, the gene expression levels for lasR/I and algD were increased in biofilm conditions. The levels of lasI were more prominent in the virulent strain than the β-lactamase producing strain. Furthermore, the expression of KPC was increased in all strains of P. aeruginosa. According to the findings of this study, S. aureus has an inhibitory effect in polymicrobial infections by suppressing the β-lactamase genes and viability of P. aeruginosa. Also, it cooperates with the biofilm-producing P. aeruginosa strains to increase pathogenicity and resistance to tobramycin.

Analysis of the organization and expression patterns of the convergent Pseudomonas aeruginosa lasR/rsaL gene pair uncovers mutual influence.

The two adjacent genes encoding the major Pseudomonas aeruginosa quorum-sensing regulator, LasR, and its opponent, RsaL, overlap in their coding 3' ends and produce mRNA transcripts with long untranslated 3' ends that overlap with the sense transcripts of the gene on the opposing DNA strand. In this study, we evaluated whether the overlapping genes are involved in mutual regulatory events and studied interference by natural antisense transcripts. We introduced various gene expression constructs into a P. aeruginosa PA14 lasR/rsaL double deletion mutant, and found that although complementary RNA is produced, this does not interfere with the sense gene expression levels of lasR and rsaL and does not have functional consequences on down-stream gene regulation. Nevertheless, expression of lasR, but not of rsaL, was shown to be enhanced if transcription was terminated at the end of the respective gene so that no overlapping transcription was allowed. Our data indicate that the natural organization with a partial overlap at the 3' ends of the lasR/rsaL genes gives rise to a system of checks and balances to prevent dominant and unilateral control by LasR over the RsaL transcriptional regulator of opposing function.

LasR/rhlR Expression Linked to Quorum Sensing-Mediated Biofilm Formation in Pseudomonas aeruginosa Using Gold Nanoparticles Synthesized with Ethnobotanical Extracts

The rapid emergence of multidrug-resistant bacteria endangers the efficacy of antibiotics, which leads to the search for novel anti-infective measures that do not rely on antibiotics. Quorum sensing inhibition (QSI) presents potential in making straightforward approach in disrupting cell-to-cell communication in bacteria, thereby decreasing virulence factors such as biofilm formation. To assess QSI of gold nanoparticles conjugated with ethnobotanical extracts (AuNPs + CEs) against Pseudomonas aeruginosa, antibacterial activity was assessed as a preliminary test and AuNPs + CEs that exhibited antibacterial activity were not used in the biofilm formation assay to rule out antimicrobial decrease in biofilm formation. RNA of bacterial sample treated with AuNPs + CEs with biofilm formation inhibitory activity was subjected to gene expression analysis. The results revealed that all AuNPs + CEs used in the microtiter biofilm formation assay showed a significant decrease in biofilm formation in P. aeruginosa. Gene expression analysis of lasR and rhlR revealed that AuNPs + CEs that exhibited decrease in biofilm formation also have downregulated expression of lasR and rhlR, an indication of quorum sensing–mediated decrease in virulence. Furthermore, samples treated with AuNPs + CEs, showed lower expression of lasR compared to samples treated with only CEs. This result presents the role of gold nanoparticles in enhancing the delivery of bioactive compounds present in the ethnobotanicals, with QSI activity against P. aeruginosa. Hence, the result offers the potential of AuNPs + CEs in QSI of disease-causing human pathogens.

Detection of biofilm formation and assessment of biofilm genes expression in different Pseudomonas aeruginosa clinical isolates

IntroductionBiofilm formation is an important antibiotic resistance mechanism among different bacterial species. AimThis study aimed to determine the ability of different P. aeruginosa clinical isolates to produce biofilm and its association with las R, lec A and pel A genes expression and antibiotic resistance. MethodsIn this study, 100 isolates of P. aeruginosa were obtained from patients with wound infections at Minia University Hospitals. The antibiotic sensitivity of the isolated strains was done using the disc diffusion method. The isolated bacteria were tested for their ability to form biofilm using a microtiter plate assay. The expression of lasR, lecA and pel A genes in isolated strains was detected using real-time rt-PCR. Results: 27 out of 100 P. aeruginosa isolates (27%) were positive biofilm producers: 14% were strong biofilm producers, 7% were moderate biofilm producers and 6% were weak biofilm producers. Resistance to all used antibiotics and multidrug resistance was higher among biofilm producing than non-biofilm producing strains, but the difference was statistically non-significant. The expression of lasR, lecA and pel A genes were detected in 20 (74.1%), 26(96.3%) and 18(66.7%) isolates out of 27 biofilm-producing strains respectively, compared to 23(31.5%), 26(35.6%) and 24(24%) out of 73 non-biofilm producing strains respectively. ConclusionBiofilm producing strains demonstrated higher expression level of lasR, lecA, and pel A genes with a mean fold change of 357.02 ± 248.5, 26.7 ± 23.2 and 420.2 ± 271.3 respectively, therefore las R, lec A, and pel A genes have a significant role in biofilm formation.

The interference of nonylphenol with bacterial cell-to-cell communication

The interference of nonylphenol (NP) with humans and animals, especially in hormone systems, has been well-studied. There is rarely any record of its effect on bacteria, which dominate in various environments. In our study, we employed Pseudomonas aeruginosa PAO1 as a model microorganism and took its common lifestyle biofilm, mainly regulated by quorum sensing (QS), as a cut-in point to investigate the effect of NP (1, 5, 10 mg L−1) on bacteria. The results showed that more than 5 mg L−1 of NP did interfere with biofilm formation and affected bacterial QS. In detail, the LasI/R circuit, but not the RhlI/R circuit, was considerably obstructed. The decrease in lasI and lasR expression resulted in a significant reduction in N-3-oxo-dodecanoyl homoserine lactone (3OC12-HSL) signals and the downstream production of elastases. Docking results indicated the binding of NP with LasR protein, simulating the binding of 3OC12-HSL with LasR protein, which explained the obstruction of the LasIR circuit. We concluded that NP competed with 3OC12-HSL and blocked 3OC12-HSL binding with the LasR protein, resulting in a direct interference in bacterial biofilm formation. This is the first report of NP interference with bacterial signaling, which is not only helpful to understand the effect of NP on various ecosystems, but is also beneficial to enrich our knowledge of inter-kingdom communication.

LasR Might Act as an Intermediate in Overproduction of Phenazines in the Absence of RpoS in Pseudomonas aeruginosa.

As an opportunistic bacterial pathogen, Pseudomonas aeruginosa PAO1 contains two phenazineproducing gene operons, phzA1B1C1D1E1F1G1 (phz1) and phzA2B2C2D2E2F2G2 (phz2), each of which is independently capable of encoding all enzymes for biosynthesizing phenazines, including phenazine-1-carboxylic acid and its derivatives. Other previous study reported that the RpoS-deficient mutant SS24 overproduced pyocyanin, a derivative of phenazine-1- carboxylic acid. However, it is not known how RpoS mediates the expression of two phz operons and regulates pyocyanin biosynthesis in detail. In this study, with deletion of the rpoS gene in the PAΔphz1 mutant and the PAΔphz2 mutant respectively, we demonstrated that RpoS exerted opposite regulatory roles on the expression of the phz1and phz2 operons. We also confirmed that the phz1 operon played a critical role and especially biosynthesized much more phenazines than the phz2 operon when the rpoS gene was knocked out in P. aeruginosa. By constructing the translational reporter fusion vector lasR'-'lacZ and the chromosomal fusion mutant PAΔlasR::lacZ, we verified that RpoS deficiency caused increased expression of lasR, a transcription regulator gene in a first quorum sensing system (las) that activates overexpression of the phz1 operon, suggesting that in the absence of RpoS, LasR might act as an intermediate in overproduction of phenazine biosynthesis mediated by the phz1 operon in P. aeruginosa.

Virulence of Pseudomonas aeruginosa exposed to carvacrol: alterations of the Quorum sensing at enzymatic and gene levels.

The main goal of this study was to evaluate the inhibition of Pseudomonas aeruginosa virulence factors and Quorum Sensing during exposure to carvacrol. P. aeruginosa (ATCC 10154) was exposed to carvacrol determining changes in biofilm development, motility, acyl-homoserine lactones (AHL) synthesis and relative expression of lasI/lasR. Docking analysis was used to determinate interactions between carvacrol with LasI and LasR proteins. P. aeruginosa produced 60% lower AHLs when exposed to carvacrol (1.9mM) compared to control, without affecting cellular viability, indicating a reduction on the LasI synthase activity. AHL-C12, C6, and C4 were detected and related to biofilm development, motility, and pyocyanin production, respectively. The presence of carvacrol reduced the expression of lasR, without affecting lasI gen. Moreover, computational docking showed interactions of carvacrol with amino acids in the active site pocket of LasI (-5.6kcalmol-1) and within the binding pocket of LasR (-6.7kcalmol-1) of P. aeruginosa. These results demonstrated that virulence of P. aeruginosa was reduced by carvacrol, by inhibiting LasI activity with the concomitant reduction on the expression of lasR, biofilm and swarming motility. This study provides relevant information about the effect of carvacrol against quorum sensing to inhibit virulence factors of P. aeruginosa at enzymatic and gene levels. These findings can contribute to the development of natural anti-QS products, which can affect pathogenesis.

A complex hierarchical quorum-sensing circuitry modulates phenazine gene expression in Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) modulates the expression of a myriad of virulence factors via two complicated hierarchical quorum-sensing (QS) cascade. This study shed light on the interrelation between P. aeruginosa QS systems and pyocyanin production. Transcription analysis of lasR, rhlR, rhlI and phz genes using quantitative real time-reverse transcriptase PCR (qRT-PCR) assay, followed by sequencing of the autoinducer synthase (lasI gene) were applied for 15 P. aeruginosa strains recovered from diverse animal clinical sources. Expression studies revealed that most P. aeruginosa strains demonstrated statistically significant differences (p < 0.05) with a very wide range of transcript levels of QS and phz genes in comparison to P. aeruginosa ATCC 27853. We have identified significant positive correlations (r ≥ 0.3) between the expressions of QS and phz genes in eleven analyzed strains, whereas pyocyanin production positively correlated with the expression of lasR only in three strains (r ≥ 0.6). We further found that there was a negative correlation between the transcript levels of QS and phz genes in one bacterial strain. Analysis of lasI sequences showed point mutations explaining the alterations in pyocyanin expression. The deficiencies of lasI, lasR and rhlI with rhlR-dependent expression of phz in one strain were also recorded. These results provided new insights to the pivotal role of QS signal molecules on pyocyanin production presenting the las system as the dominant regulator.

Chitosan extracted from Aspergillus flavus shows synergistic effect, eases quorum sensing mediated virulence factors and biofilm against nosocomial pathogen Pseudomonas aeruginosa

The biological methods for extraction of chitosan were used as alternative procedures for chemical methods In biological methods, the chitosan was extracted from A. flavus by using of Lactobacillus paracasei for demineralization and Bacillus subtilis for deproteinization. The yield of extracted chitosan reached to 53.8%, pH was 7.8 and complete solubility in 1% acitic acid. Purified chitosan had the ability to reduce the biofilm forming capacity of P. aeruginosa clearly visible in light microscopic staining and scanning electron microscopy. The QS dependent phenotype and QS regulated gene expression was significantly reduced in the influence of chitosan. A significant decrease in biofilm formation was seen in the presence of chitosan. The chitosan treatment showed a decrease in the expression of lasR and rhlR genes. Same time production of pyocyanin and proteases was also inhibited in dose dependent manner. Chitosan led to increasing antimicrobial activity of antibiotics and had synergism effect, thus chitosan may be a useful adjuvant agent for the treatment of many bacterial infections in combination with antibiotics.

Inhibition of quorum sensing-controlled biofilm formation in Pseudomonas aeruginosa by quorum-sensing inhibitors

Antimicrobial therapy against extensively drug-resistant (XDR) P. aeruginosa biofilms is less efficient compared to the treatment of equal bacterial counts of free-floating planktonic cells, which has become a serious threat in hospital environment. P. aeruginosa regulate their cooperative activities and physiological processes through a cell to cell chemical communication process called Quorum sensing (QS). This attracted our interest to synthesize, and to chemically characterize two anti-QS compounds, N-(4-{4-fluoroanilno} butanoyl) -l-homoserine lactone (FABHL) and N-(4-{4-chlororoanilno} butanoyl) -l-homoserine lactone (CABHL) to inhibit biofilm formation via disabling the QS circuits. Structural and morphological properties of these compounds were characterized by 1H Nuclear Magnetic Resonance (NMR), 13C NMR and High-resolution mass spectrometry (HRMS). Two biofilm forming XDR P. aeruginosa isolates were included in this study. Anti-biofilm property of FABHL or CABHL was confirmed by biofilm formation assay and it was shown to occur without affecting the bacterial growth. Anti-QS property of FABHL or CABHL was determined by evaluating the expression levels of QS genes (lasR and rhlR) by quantitative real time PCR (qRT-PCR). Although, FABHL and CABHL downregulates the expression levels of QS genes, lasR expression was significantly reduced. Molecular modeling studies revealed that the binding energy of FABHL and CABHL with LasR protein was −4.27 and −4.51, respectively. Hence, the synthesized compounds have the potential to serve as a potent anti-biofilm agent via disabling the QS systems. Lethality of FABHL and CABHL against PBMCs was assessed by 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphynyl tetrazolium bromide (MTT) assay. Cell viability was observed for both the compounds.

Hyperoside inhibits biofilm formation of Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) is a common pathogen in hospital-acquired infection and is readily able to form biofilms. Due to its high antibiotic resistance, traditional antibacterial treatments exert a limited effect on P. aeruginosa biofilm infections. It has been indicated that hyperoside inhibits P. aeruginosa PAO1 (PAO1) biofilm formation without affecting growth. Therefore, the current study examined the biofilm formation and quorum sensing (QS) system of PAO1 in the presence of hyperoside. Confocal laser scanning microscopy analysis demonstrated that hyperoside significantly inhibited biofilm formation. It was also observed that hyperoside inhibited twitching motility in addition to adhesion. Data from reverse transcription-quantitative polymerase chain reaction indicated that hyperoside inhibited the expression of lasR, lasI, rhlR and rhlI genes. These results suggest that the QS-inhibiting effect of hyperoside may lead to a reduction in biofilm formation. However, the precise mechanism of hyperoside on P. aeruginosa pathogenicity remains unclear and requires elucidation in additional studies.

A new quorum-sensing inhibitor attenuates virulence and decreases antibiotic resistance in Pseudomonas aeruginosa

Quorum sensing (QS) has been a novel target for the treatment of infectious diseases. Here structural analogs of Pseudomonas aeruginosa autoinducer N-acyl homoserine lactone (AHL) were investigated for QS inhibitor (QSI) activity and a novel QSI was discovered, N-decanoyl-L-homoserine benzyl ester (C2). Virulence assays showed that C2 down-regulated total protease and elastase activities, as well as the production of rhamnolipid, that are controlled by QS in P. aeruginosa wild-type strain PAO1 without affecting growth. C2 was also shown to inhibit swarming motility of PAO1. Using a microdilution checkerboard method, we identified synergistic interactions between C2 and several antibiotics, tobramycin, gentamycin, cefepime, and meropenem. Data from real-time RT-PCR suggested that C2 inhibited the expression of lasR (29.67%), lasI (21.57%), rhlR (28.20%), and rhlI (29.03%).

Manuka honey is bactericidal against Pseudomonas aeruginosa and results in differential expression of oprF and algD

The presence of Pseudomonas aeruginosa in cutaneous wounds is of clinical significance and can lead to persistent infections. Manuka honey has gained ground in clinical settings due to its effective therapeutic action and broad spectrum of antibacterial activity. In this study, the effect of manuka honey on P. aeruginosa was investigated using MIC, MBC, growth kinetics, confocal microscopy, atomic force microscopy and real-time PCR. A bactericidal mode of action for manuka honey against P. aeruginosa was deduced (12 %, w/v, MIC; 16 %, w/v, MBC) and confirmed by confocal and atomic force microscopy, which showed extensive cell lysis after 60 min exposure to inhibitory concentrations of manuka honey. The inability of honey-treated cells to form microcolonies was demonstrated and investigated using Q-PCR for three key microcolony-forming genes: algD, lasR and oprF. The expression of algD increased 16-fold whereas oprF expression decreased 10-fold following honey treatment; lasR expression remained unaltered. These findings confirm that manuka honey is effective at inducing cell lysis and identify two targets, at the genetic level, that might be involved in this process.