Genes lasR Research Articles

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.

Evolution of biofilm-adapted gene expression profiles in lasR-deficient clinical Pseudomonas aeruginosa isolates

The overall success of a pathogenic microbe depends on its ability to efficiently adapt to challenging conditions in the human host. Long-term evolution experiments track and predict adaptive trajectories and have contributed significantly to our understanding of the driving forces of bacterial adaptation. In this study, we conducted a cross-sectional study instead of long-term longitudinal evolution experiments. We analyzed the transcriptional profiles as well as genomic sequence variations of a large number of clinical Pseudomonas aeruginosa isolates that have been recovered from different infected human sites. Convergent changes in gene expression patterns were found in different groups of clinical isolates. The majority of repeatedly observed expression patterns could be attributed to a defective lasR gene, which encodes the major quorum-sensing regulator LasR. Strikingly, the gene expression pattern of the lasR-defective strains appeared to reflect a transcriptional response that evolves in a direction consistent with growth within a biofilm. In a process of genetic assimilation, lasR-deficient P. aeruginosa isolates appear to constitutively express a biofilm-adapted transcriptional profile and no longer require a respective environmental trigger. Our results demonstrate that profiling the functional consequences of pathoadaptive mutations in clinical isolates reveals long-term evolutionary pathways and may explain the success of lasR mutants in the opportunistic pathogen P. aeruginosa in a clinical context.

Occurrence of Pseudomonas spp. in Raw Vegetables: Molecular and Phenotypical Analysis of Their Antimicrobial Resistance and Virulence-Related Traits.

Pseudomonas is characterized by its great capacity to colonize different ecological niches, but also by its antimicrobial resistance and pathogenicity, causing human, animal, or plant diseases. Raw and undercooked food is a potential carrier of foodborne disease. The aim of this study was to determine the occurrence of Pseudomonas spp. among raw vegetables, analysing their antimicrobial resistance, virulence, and molecular typing. A total of 163 Pseudomonas spp. isolates (12 different species) were recovered from 77 of the 145 analysed samples (53.1%) and were classified into 139 different pulsed-field gel electrophoresis patterns. Low antimicrobial resistance levels, but one multidrug-resistant isolate, were found. Among the 37 recovered P. aeruginosa strains, 28 sequence-types and nine serotypes were detected. Eleven OprD patterns and an insertion sequence (ISPa1635) truncating the oprD gene of one imipenem-resistant strain were found. Ten virulotypes were observed, including four exoU-positive and thirty-one exoS-positive strains. The lasR gene was absent in three ST155 strains and was truncated by different insertion sequences (ISPre2, IS1411, and ISPst7) in other three strains. High biofilm, motility, pigment, elastase, and rhamnolipid production were detected. Our study demonstrated a low occurrence of P. aeruginosa (18%) and low antimicrobial resistance, but a high number of virulence-related traits in these P. aeruginosa strains, highlighting their pathological importance.

Antimicrobial Resistance and Genomic Characterization of Six New Sequence Types in Multidrug-Resistant Pseudomonas aeruginosa Clinical Isolates from Pakistan.

Pseudomonas aeruginosa (P. aeruginosa) is a major bacterial pathogen associated with a variety of infections with high mortality rates. Most of the clinical P. aeruginosa isolates belong to a limited number of genetic subgroups characterized by multiple housekeeping genes’ sequences (usually 5–7) through the Multi-Locus Sequence Typing (MLST) scheme. The emergence and dissemination of novel multidrug-resistant (MDR) sequence types (ST) in P. aeruginosa pose serious clinical concerns. We performed whole-genome sequencing on a cohort (n = 160) of MDR P. aeruginosa isolates collected from a tertiary care hospital lab in Pakistan and found six isolates belonging to six unique MLST allelic profiles. The genomes were submitted to the PubMLST database and new ST numbers (ST3493, ST3494, ST3472, ST3489, ST3491, and ST3492) were assigned to the respective allele combinations. MLST and core-genome-based phylogenetic analysis confirmed the divergence of these isolates and positioned them in separate branches. Analysis of the resistome of the new STs isolates revealed the presence of genes blaOXA-50, blaPAO, blaPDC, blaVIM-2, aph(3′)-IIb, aac(6′)-II, aac(3)-Id, fosA, catB7, dfrB2, crpP, merP and a number of missense and frame-shift mutations in chromosomal genes conferring resistance to various antipseudomonal antibiotics. The exoS, exoT, pvdE, rhlI, rhlR, lasA, lasB, lasI, and lasR genes were the most prevalent virulence-related genes among the new ST isolates. The different genotypic features revealed the adaptation of these new clones to a variety of infections by various mutations in genes affecting antimicrobial resistance, quorum sensing and biofilm formation. Close monitoring of these antibiotic-resistant pathogens and surveillance mechanisms needs to be adopted to reduce their spread to the healthcare facilities of Pakistan. We believe that these strains can be used as reference strains for future comparative analysis of isolates belonging to the same STs.

A new filamentous bulking control strategy: The role of N-acyl homoserine lactone (AHL)-mediated quorum sensing in filamentous bacteria proliferation within activated sludge

The microcosmic mechanisms underlying filamentous bulking are still unclear. Quorum sensing (QS) is an important mechanism by which bacteria control self-reproduction and growth in response to changes in population density. The potential role of QS in filamentous bulking and the feasibility of using a Quorum quenching (QQ) strategy to inhibit filamentous bulking were investigated in this study. The results indicated that the rate of increase in the population density and the abundance of filamentous bacteria in the serious filamentous bulking stage (250 mL/g < SVI) was 1.46 and 2.76 times greater, respectively, than that of the limited filamentous bulking stage (150 mL/g < SVI < 250 mL/g). The proliferation of filamentous bacteria was clearly population density-dependent. The concentration of C6-HSL increased from 31.39 ± 3.52 ng/g VSS to 125.29 ± 6.70 ng/g VSS with an increase in the abundance of filamentous bacteria. Filamentous bulking could be promoted by exogenous N-acyl homoserines (AHLs), especially C6-HSL. The hdtS gene related to the synthesis of AHLs and the lasR and cciR genes related to receptors showed significantly increased expressions during filamentous bulking. SDHA, ND1, and CDK2 genes related to proliferation showed significantly increased expression during filamentous bulking. The QS-AHL system was triggered during filamentous bulking, which promoted the proliferation of filamentous bacteria. As expected, filamentous bulking was significantly inhibited by the addition of vanillin, a quorum sensing inhibitor. Therefore, QQ is a potential strategy for the prevention and control of filamentous bulking. This study provides new information regarding the microcosmic mechanisms of filamentous bulking.

Virulence gene expression of Pseudomonas aeruginosa isolated from children with nephrotic syndrome

Pseudomonas aeruginosa is Gram-negative, considered as the most common opportunistic pathogens which lead to high rate of mortality and morbidity when subject suffered from compromised immune systems. This study was aimed to estimate the lasI and lasR genes expression of P. aeruginosa isolated from children with nephrotic syndrome. Nineteen isolates from urine samples of children with nephropathy syndrome are included in this study and confirmed by VITEK test as a P. aeruginosa strains. The result showed that the LasI and LasR genes showed a high level of gene expression in strains that grown with ceftriaxone than the strains that grown alone, but the gene Las I had a higher level of expression than the Las R gene, also a regression analysis were done to reveal the effect of Las I on the Las R gene which showed a positive effect which mean any increment in the gene expression level of Las I gene will combined with an increment in the Las R gene, (R= 0.899 and P= 0.001).

Effects of Gentamicin-Loaded Chitosan-ZnO Nanocomposite on Quorum-Sensing Regulation of Pseudomonas Aeruginosa.

Cell density-based intercellular signaling mechanism is known as Quorum sensing (QS); it serves a significant role in regulating the pathogenic factors. The objective of the present study was to assess the influence of chitosan-zinc oxide nanocomposite (CH-ZnO nanocomposite), alone and in combination with gentamicin, on the sensitivity to hydrogen peroxide (H2O2), the production of pathogenic factors and QS-regulated genes of Pseudomonas aeruginosa. The efficacy of the minimum inhibitory concentration (MIC) and 1/4 MIC of the CH-ZnO nanocomposite, alone and in combination with gentamicin, on the sensitivity to H2O2, pyocyanin secretion, swarming and twitching motilities was evaluated. In addition, the expression of some QS-regulated genes including rhlI, rhlR, lasI and lasR genes was measured by Real-time quantitative PCR (RT-qPCR) following exposure to the nanocomposite. The results demonstrated that at MIC concentrations, the gentamicin-loaded CH-ZnO nanocomposite significantly inhibited QS-regulated phenotypes such as pyocyanin secretion (82.4%), swarming (76%) and twitching (73.6%) motilities; further it increased the inhibition growth zone (134.5%), as well as, at 1/4 MIC concentration decreased the expression of lasI (72%), lasR (78%), rhlI (76%) and rhlR (82%) genes; as compared to untreated P. aeruginosa PAO1 (P < 0.05). Our results also demonstrated that the CH-ZnO nanocomposite combined with gentamicin could be a potential innovative candidate, which could be broadly applied in the treatment of P. aeruginosa infections.

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.

Elastase Activity From Pseudomonas aeruginosa Respiratory Isolates and ICU Mortality

Pseudomonas aeruginosa (PA) is a common cause of respiratory infection and morbidity. Pseudomonas elastase is an important virulence factor regulated by the lasR gene. Whether PA elastase activity is associated with worse clinical outcomes in ICU patients is unknown. Is there an association between PA elastase activity and worse host outcomes in a cohort of ICU patients? PA respiratory isolates from 238 unique ICU patients from two tertiary-care centers within the University of Pittsburgh Medical Center health system were prospectively collected and screened for total protease and elastase activity, biofilm production, antimicrobial resistance, and polymicrobial status. The association between pathogen characteristics and 30-day and 90-day mortality was calculated using logistic regression. For subgroup analysis, two patterns of early (≤72 h) and late sample (>72 h) collection from the index ICU admission were distinguished using a finite mixture model. Lung inflammation and injury was evaluated in a mouse model using a PA high elastase vslow elastase producer. PA elastase activity was common in ICU respiratory isolates representing 75%of samples and was associated with increased 30-day mortality (adjusted OR [95% CI]: 1.39 [1.05-1.83]). Subgroup analysis demonstrated that elastase activity was a risk factor for 30- and 90-day mortality in the early sample group, whereas antimicrobial resistance was a risk factor for 90-day mortality in the late sample group. Whole genome sequencing of high and low elastase producers showed that predicted loss-of-function lasR genotypes were less common among high elastase producers. Mice infected with a high elastase producer showed increased lung bacterial burden and inflammatory profile compared with mice infected with a low elastase producer. Elastase activity is associated with 30-day ICU mortality. A high elastase producing clinical isolate confers increased lung tissue inflammation compared with a low elastase producer invivo.

Anti-Pseudomonas aeruginosa Activity of Metal Schiff Base Complex and Probiotics Against Planktonic- and Biofilm-Growing Cells

Introduction: The biofilm formation by Pseudomonas aeruginosa seems to protect the bacteria from antibiotics since these entities are highly resistant to such antimicrobial agents. The aim of this study was to investigate the role of Lactobacillus salivarus, Lactobacillus plantarum supernatants and CuII Schiff base complex in eliminating planktonic cells and biofilm of P. aeruginosa. Methods: One hundred specimens of blood, urine, cerebrospinal fluid, respiratory samples, and wound swabs were collected from patients attending three hospitals in Mashhad. All specimens were identified by biochemical tests. The susceptibility of the isolates to the conventional antibiotics was assessed using disk diffusion method. The biofilm formation ability of P. aeruginosa isolates was evaluated by crystal violet assay and confirmed using PCR. The anti-planktonic and antibiofilm ability of L. salivarus, L. plantarum supernatants and CuII Schiff base complex was evaluated separately in P. aeruginosa isolates. Results: The highest and lowest resistance rates were detected in Cefazoline (95%) and cefepime (23%), respectively. The thickest biofilm was produced by 8% of P. aeruginosa isolates, 9% and 83% of the isolates were considered as moderate and weak biofilm producers, respectively. The rhlR and lasR genes were reported in 100% of the isolates, but the algD gene existed in 92% of them. Conclusion: Particularly, the CuII Schiff base complex could affect both planktonic and biofilm cells by the lowest concentration in comparison of probiotic supernatants. L. plantarum supernatant inhibited planktonic cells at a lower concentration than L. salivarius. Also, L. salivarius showed better antibiofilm activity than another probiotic in lower doses of supernatant. Unlike that, these compounds have not completely eliminated biofilm cells, but only reduced the biofilm formation. : Metal Schiff base complex and Lactobacillus supernatants is a potent antimicrobial agent against Pseudomonas aeruginosa biofilm cells.

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.

Study of quorum-sensing LasR and RhlR genes and their dependent virulence factors in Pseudomonas aeruginosa isolates from infected burn wounds

BackgroundPseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen responsible for burn-wound infection. High incidence, infection severity and increasing resistance characterize P. aeruginosa -induced burn infection.PurposeTo estimate quorum-sensing (QS)-dependent virulence factors of P. aeruginosa isolates from burn wounds and correlate it to the presence of QS genes.MethodsA cross-sectional descriptive study included 50 P . aeruginosa isolates from burn patients in Mansoura University Plastic and Burn Hospital, Egypt. Antibiotic sensitivity tests were done. All isolates were tested for their ability to produce biofilm using a micro-titration assay method. Protease, pyocyanin and rhamnolipid virulence factors were determined using skimmed milk agar, King’s A medium and CTAB agar test, respectively. The identity of QS lasR and rhlR genes was confirmed using PCR.Results In total, 86 % of isolates had proteolytic activity. Production of pyocyanin pigment was manifested in 66 % of isolates. Altogether, 76 % of isolates were rhamnolipid producers. Biofilm formation was detected in 96 % of isolates. QS lasR and rhlR genes were harboured by nearly all isolates except three isolates were negative for both lasR and rhlR genes and two isolates were positive for lasR gene and negative for rhlR gene. Forty-nine isolates were considered as extremely QS-proficient strains as they produced QS-dependent virulence factors. In contrast, one isolate was a QS deficient strain.ConclusionsQS affects P. aeruginosa virulence-factor production and biofilm in burn wounds. Isolates containing lasR and rhlR seem to be a crucial regulator of virulence factors and biofilm formation in P. aeruginosa whereas the lasR gene positively regulates biofilm formation, proteolytic activity, pyocyanin production and rhamnolipid biosurfactant synthesis. The QS regulatory RhlR gene affects protease and rhamnolipid production positively.

Antibiotic Resistance, Biofilm Formation, and Presence of Genes Encoding Virulence Factors in Strains Isolated from the Pharmaceutical Production Environment.

The spread of bacterial resistance to antibiotics affects various areas of life. The aim of this study was to assess the occurrence of Pseudomonas aeruginosa, and other bacteria mainly from orders Enterobacterales and Staphylococcus in the pharmaceutical production sites, and to characterize isolated strains in the aspects of antibiotic resistance, biofilm formation, and presence of genes encoding virulence factors. Genes encoding selected virulence factors were detected using PCR techniques. Antimicrobial susceptibility testing was applied in accordance with the EUCAST recommendations. A total of 46 P. aeruginosa strains were isolated and 85% strains showed a strong biofilm-forming ability. The qualitative identification of genes taking part in Quorum Sensing system demonstrated that over 89% of strains contained lasR and rhlI genes. An antimicrobial susceptibility testing revealed nine strains resistant to at least one antibiotic, and two isolates were the metallo-β-lactamase producers. Moreover, the majority of P. aeruginosa strains contained genes encoding various virulence factors. Presence of even low level of pathogenic microorganisms or higher level of opportunistic pathogens and their toxic metabolites might result in the production inefficiency. Therefore, the prevention of microbial contamination, effectiveness of sanitary and hygienic applied protocols, and constant microbiological monitoring of the environment are of great importance.

Proteomic Analysis of Vesicle-Producing Pseudomonas aeruginosa PAO1 Exposed to X-Ray Irradiation.

Ionizing irradiation kills pathogens by destroying nucleic acids without protein structure destruction. However, how pathogens respond to irradiation stress has not yet been fully elucidated. Here, we observed that Pseudomonas aeruginosa PAO1 could release nucleic acids into the extracellular environment under X-ray irradiation. Using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), X-ray irradiation was observed to induce outer membrane vesicle (OMV) formation in P. aeruginosa PAO1. The size distribution of the OMVs of the irradiated PAO1 was similar to that of the OMVs of the non-irradiated PAO1 according to nanoparticle tracking analysis (NTA). The pyocin-related proteins are involved in OMV production in P. aeruginosa PAO1 under X-ray irradiation conditions, and that this is regulated by the key SOS gene recA. The OMV production was significantly impaired in the irradiated PAO1 Δlys mutant, suggesting that Lys endolysin is associated with OMV production in P. aeruginosa PAO1 upon irradiation stress. Meanwhile, no significant difference in OMV production was observed between PAO1 lacking the pqsR, lasR, or rhlR genes and the parent strain, demonstrating that the irradiation-induced OMV biosynthesis of P. aeruginosa was independent of the Pseudomonas quinolone signal (PQS).

Through quorum sensing, Pseudomonas aeruginosa resists noble metal-based nanomaterials toxicity.

Noble metal-based nanomaterials (NMNs), such as platinum nanoparticles (Pt@NPs) and palladium nanoparticles (Pd@NPs), are increasingly being used as antibacterial agents. However, little information is available on bacterial resistance to NMNs. In this study, owing to their oxidase-like and peroxidase-like properties, both Pt@NPs and Pd@NPs induce reactive oxygen species (ROS) and manifest antibacterial activities: 6.25μg/mL of either Pt@NPs or Pd@NPs killed >50% of Staphylococcus aureus strain ATCC29213. However, Pseudomonas aeruginosa strain PAO1 completely resisted 12.5μg/mL of Pt@NPs and 6.25μg/mL of Pd@NPs. Compared to the non-NMN groups, these NMNs promoted 2-3-fold upregulation of the quorum sensing (QS) gene lasR in strain PAO1. In fact, the lasR gene upregulation induced a 1.5-fold reduction in ROS production and increased biofilm formation by 11% (Pt@NPs) and 27% (Pd@NPs) in strain PAO1. The ΔlasR mutants (lasR gene knock out in strain PAO1), became sensitive to NMNs. The survival rates of ΔlasR mutants at 12.5μg/mL Pt@NPs and Pd@NPs treatments were only 77% and 58%, respectively. This is the first report indicating that bacteria can resist NMNs through QS. Based on these results, evaluation of the ecological risks of using NMNs as antibacterial agents is necessary.

Intraspecies Signaling between Common Variants of Pseudomonas aeruginosa Increases Production of Quorum-Sensing-Controlled Virulence Factors.

The opportunistic pathogen Pseudomonas aeruginosa damages hosts through the production of diverse secreted products, many of which are regulated by quorum sensing (QS). The lasR gene, which encodes a central QS regulator, is frequently mutated in clinical isolates from chronic infections, and loss of LasR function (LasR-) generally impairs the activity of downstream QS regulators RhlR and PqsR. We found that in cocultures containing LasR+ and LasR- strains, LasR- strains hyperproduce the RhlR/RhlI-regulated antagonistic factors pyocyanin and rhamnolipids in diverse models and media and in different strain backgrounds. Diffusible QS autoinducers produced by the wild type were not required for this effect. Using transcriptomics, genetics, and biochemical approaches, we uncovered a reciprocal interaction between wild-type and lasR mutant pairs wherein the iron-scavenging siderophore pyochelin produced by the lasR mutant induced citrate release and cross-feeding from the wild type. Citrate, a metabolite often secreted in low iron environments, stimulated RhlR signaling and RhlI levels in LasR-but not in LasR+ strains. These studies reveal the potential for complex interactions between recently diverged, genetically distinct isolates within populations from single chronic infections.IMPORTANCE Coculture interactions between lasR loss-of-function and LasR+ Pseudomonas aeruginosa strains may explain the worse outcomes associated with the presence of LasR- strains. More broadly, this report illustrates how interactions within a genotypically diverse population, similar to those that frequently develop in natural settings, can promote unpredictably high virulence factor production.

Inhibitory activity of metal-curcumin complexes on quorum sensing related virulence factors of Pseudomonas aeruginosa PAO1

The use of metal complexes to reduce or inhibit virulence factors of Pseudomonas aeruginosa is a promising strategy for the management and control of infections caused by this multidrug-resistant pathogen. The present study aimed to investigate the anti-quorum sensing activity of sub-minimum inhibitory concentrations (sub-MIC) of copper(II) sulfate pentahydrate-curcumin complex (Cu-CUR), iron(III) nitrate nonahydrate -curcumin complex (Fe-CUR), zinc(II) chloride-curcumin complex (Zn-CUR) and free curcumin (free-CUR) against P. aeruginosa PAO1. Metal-CUR complexes were synthesized and characterized by spectroscopic methods. The effect of sub-MIC (1/4 and 1/16 MIC) concentrations of metal-CUR complexes and free-CUR on cell growth, biofilm formation, motility, alginate and pyocyanin production, H2O2 susceptibility and expression of lasI and lasR genes in PAO1 was determined. MIC of metal-CUR complexes and free-CUR was determined as 62.5 and 125 µg/ml, respectively. Metal-CUR complexes at concentration of 62.5 µg/ml significantly reduced the cell growth to 1.5%–3.3%. Although we did not measure the anti-QS activity of metal-CUR complexes directly against PAO1, they indicated anti-QS activity in C. violaceum CV026. Copper-CUR complex at the concentration of 1/4 MIC showed the greatest inhibitory effect on swarming and twitching motilities, biofilm formation, alginate and pyocyanin production, sensitivity to H2O2 and reduction in the expression levels of lasI and lasR genes (P < 0.001). Considering the biological effects of Cu-CUR complex and its inhibitory activity on virulence factors, it may be used as an effective compound for treatment and control of infections caused by P. aeruginosa.

Synthesis, characterization and\xa0assessment of anti-quorum sensing activity of copper(II)-ciprofloxacin complex against Pseudomonas aeruginosa PAO1

Quorum sensing (QS) inhibition by metal-antibiotic complexes is a promising strategy for the management and control of multidrug resistant Pseudomonas aeruginosa infections. We investigated the anti-quorum sensing activity of sub-minimum inhibitory concentration (sub-MIC) of copper(II) sulfate pentahydrate-ciprofloxacin (Cu-CIP) complex and free ciprofloxacin (free-CIP) against P. aeruginosa PAO1. Copper-CIP complex was synthesized and its characterization was assessed using spectroscopic methods and single crystal X-ray analysis. The effect of sub-MIC (1/4 and 1/16 MIC) concentrations of Cu-CIP and free-CIP on cell growth, biofilm formation, motility, alginate and pyocyanin production, H2O2 susceptibility and expression of QS circuit genes lasI and lasR in PAO1 was determined. Minimum inhibitory concentration of Cu-CIP complex and free-CIP was determined as 0.125 µg/ml. Copper-CIP complex did not show significant effect on the cell growth at concentrations of 1/4 and 1/16 MIC. However, sub-MIC concentrations (1/4 and 1/16 MIC) of Cu-CIP showed the significant reduction in violacein production, motility, biofilm formation, alginate and pyocyanin production and sensitivity to H2O2 in a concentration dependent manner (P < 0.001). Copper-CIP at the concentration of 1/4 MIC showed the greatest reduction in lasI and lasR transcriptional expression (89.5% and 96.2% respectively). Considering the biological effects of Cu-CIP complex and its inhibitory activity on QS related virulence traits at low concentrations (0.03 and 0.007 µg/ml), it may be used as an effective approach in the management of infections caused by P. aeruginosa.

The Study of Mutation in LasR and PqsR Genes in Extremely Drug Resistant and Multidrug Resistant Strains of Pseudomonas aeruginosa from Burn Wound Infection

Background: Pseudomonas aeruginosa is among the top 10 resistant bacteria worldwide. Its extraordinary resistance is principally due to the function of quorum sensing genes, such as lasR and pqsR, which are mainly involved in antibiotic resistance. Objectives: This study aimed to examine possible mutations in lasR and pqsR in resistant clinical isolates of P. aeruginosa. Methods: We obtained 120 suspected isolates of P. aeruginosa from burn patients. The isolates were identified by biochemical tests and toxA gene analysis. The PCR products of LasR and PqsR genes were analyzed in seven resistant isolates. Then, the sequences were compared with a reference strain. Results: We verified Ninety-six isolates as absolute P. aeruginosa. According to antibiograms, 95.8% of the isolates were considered as multidrug-resistant, of which 87.5% were extensively drug-resistant. Based on DNA and protein sequences, only one missense mutation was observed, including R180Q in lasR and A314V in pqsR. While R180Q decreased the stability of LasR and had a deleterious impact on protein function, A314V had a neutral impact on the protein and increased PqsR protein stability. Also, two nonsense mutations in position E259 were observed in the PqsR protein. Conclusions: The lasR and pqsR genes possibly can play a key role in antibiotic resistance, but they are not the only factors. Hence, studying mutations helps design a promising antibiotic to overcome antibiotic resistance as much as possible.

Regulation of Pseudomonas aeruginosa-Mediated Neutrophil Extracellular Traps.

Pseudomonas aeruginosa is the most prevalent opportunistic pathogen in the airways of cystic fibrosis (CF) patients. The pulmonary disorder is characterized by recurrent microbial infections and an exaggerated host inflammatory immune response led primarily by influx of neutrophils. Under these conditions, chronic colonization with P. aeruginosa is associated with diminished pulmonary function and increased morbidity and mortality. P. aeruginosa has a wide array of genetic mechanisms that facilitate its persistent colonization of the airway despite extensive innate host immune responses. Loss of function mutations in the quorum sensing regulatory gene lasR have been shown to confer survival advantage and a more pathogenic character to P. aeruginosa in CF patients. However, the strategies used by LasR-deficient P. aeruginosa to modulate neutrophil-mediated bactericidal functions are unknown. We sought to understand the role of LasR in P. aeruginosa-mediated neutrophil extracellular trap (NET) formation, an important anti-microbial mechanism deployed by neutrophils, the first-line responder in the infected airway. We observe mechanistic and phenotypic differences between NETs triggered by LasR-sufficient and LasR-deficient P. aeruginosa strains. We uncover that LasR-deficient P. aeruginosa strains fail to induce robust NET formation in both human and murine neutrophils, independently of bacterial motility or LPS expression. LasR does not mediate NET release via downstream quorum sensing signaling pathways but rather via transcriptional regulation of virulence factors, including, but not restricted to, LasB elastase and LasA protease. Finally, our studies uncover the differential requirements for NADPH oxidase in NET formation triggered by different P. aeruginosa strains.