Genes lasI Research Articles

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).

Heavy Vacuum Gas Oil Upregulates the Rhamnosyltransferases and Quorum Sensing Cascades of Rhamnolipids Biosynthesis in Pseudomonas sp. AK6U.

We followed a comparative approach to investigate how heavy vacuum gas oil (HVGO) affects the expression of genes involved in biosurfactants biosynthesis and the composition of the rhamnolipid congeners in Pseudomonas sp. AK6U. HVGO stimulated biosurfactants production as indicated by the lower surface tension (26 mN/m) and higher yield (7.8 g/L) compared to a glucose culture (49.7 mN/m, 0.305 g/L). Quantitative real-time PCR showed that the biosurfactants production genes rhlA and rhlB were strongly upregulated in the HVGO culture during the early and late exponential growth phases. To the contrary, the rhamnose biosynthesis genes algC, rmlA and rmlC were downregulated in the HVGO culture. Genes of the quorum sensing systems which regulate biosurfactants biosynthesis exhibited a hierarchical expression profile. The lasI gene was strongly upregulated (20-fold) in the HVGO culture during the early log phase, whereas both rhlI and pqsE were upregulated during the late log phase. Rhamnolipid congener analysis using high-performance liquid chromatography-mass spectrometry revealed a much higher proportion (up to 69%) of the high-molecularweight homologue Rha–Rha–C10–C10 in the HVGO culture. The results shed light on the temporal and carbon source-mediated shifts in rhamonlipids’ composition and regulation of biosynthesis which can be potentially exploited to produce different rhamnolipid formulations tailored for specific applications.

Quorum sensing inhibitory effect of bergamot oil and aspidosperma extract against Chromobacterium violaceum and Pseudomonas aeruginosa.

Quorum sensing (QS) represents a major target for reducing bacterial pathogenicity and antibiotic resistance. This study identifies bergamot and aspidosperma as new potential sources of anti-QS agents. We investigated the anti-QS activity of plant materials on both Chromobacterium violaceum and Pseudomonas aeruginosa. Initially, we determined the minimum inhibitory concentrations (MICs) of plant materials using a broth microdilution method. Subsequently, we tested the effect of sub-MIC concentrations on QS-regulated traits and virulence factors production in test bacteria. Results revealed that bergamot and aspidosperma inhibited the ability of C. violaceum to produce violacein. Other QS-controlled phenotypes of C. violaceum, namely chitinolytic activity, motility, and biofilm formation, were also reduced by both plant materials. Moreover, QS-linked traits of P. aeruginosa were also reduced. Bergamot inhibited swarming but not swimming motility, while aspidosperma diminished both motility types in P. aeruginosa. Both plant materials also demonstrated antibiofilm activity and inhibited the production of protease and pyocyanin in P. aeruginosa. Furthermore, we tested the anti-QS effect of plant materials on the transcriptional level using RT-qPCR. Bergamot dramatically downregulated the C. violaceum autoinducer synthase gene cviI and the vioB gene involved in violacein biosynthesis, confirming the phenotypic observation on its anti-QS activity. Aspidosperma also reduced the expression of cviI and vioB but less drastically than bergamot. In P. aeruginosa, downregulation in the transcripts of the QS genes lasI, lasR, rhlI, and rhlR was also achieved by bergamot and aspidosperma. Therefore, data in the present study suggest the usefulness of bergamot and aspidosperma as sources of antivirulence agents.

Anaerobic biosynthesis of rhamnolipids by Pseudomonas aeruginosa: performance, mechanism and its application potential for enhanced oil recovery

BackgroundPseudomonas aeruginosa, the rhamnolipids-producer, is one of dominant bacteria in oil reservoirs. Although P. aeruginosa strains are facultative bacteria, the anaerobic biosynthesis mechanism of rhamnolipids is unclear. Considering the oxygen scarcity within oil reservoirs, revealing the anaerobic biosynthesis mechanism of rhamnolipids are significant for improving the in-situ production of rhamnolipids in oil reservoirs to enhance oil recovery.ResultsPseudomonasaeruginosa SG anaerobically produced rhamnolipids using glycerol rather than glucose as carbon sources. Two possible hypotheses on anaerobic biosynthesis of rhamnolipids were proposed, the new anaerobic biosynthetic pathway (hypothesis 1) and the highly anaerobic expression of key genes (hypothesis 2). Knockout strain SGΔrmlB failed to anaerobically produce rhamnolipids using glycerol. Comparative transcriptomics analysis results revealed that glucose inhibited the anaerobic expression of genes rmlBDAC, fabABG, rhlABRI, rhlC and lasI. Using glycerol as carbon source, the anaerobic expression of key genes in P. aeruginosa SG was significantly up-regulated. The anaerobic biosynthetic pathway of rhamnolipids in P. aeruginosa SG were confirmed, involving the gluconeogenesis from glycerol, the biosynthesis of dTDP-l-rhamnose and β-hydroxy fatty acids, and the rhamnosyl transfer process. The engineered strain P. aeruginosa PrhlAB constructed in previous work enhanced 9.67% of oil recovery higher than the wild-type strain P. aeruginosa SG enhancing 8.33% of oil recovery.ConclusionThe highly anaerobic expression of key genes enables P. aeruginosa SG to anaerobically biosynthesize rhamnolipids. The genes, rmlBDAC, fabABG, rhlABRI, rhlC and lasI, are key genes for anaerobic biosynthesis of rhamnolipid by P. aeruginosa. Improving the anaerobic production of rhamnolipids better enhanced oil recovery in core flooding test. This study fills the gaps in the anaerobic biosynthesis mechanism of rhamnolipids. Results are significant for the metabolic engineering of P. aeruginosa to enhance anaerobic production of rhamnolipids.

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.

VIRULENCE OF ISOLATED PSEUDOMONAS AERUGINOSA INFECTING DUCKLING AND ANTIBIOTIC RESISTANCE WITH AN EXPERIMENTAL TREATMENT TRIAL

ABSTRACT The present study was conducted in Kafrelsheikh governorate to isolate P.aeruginosa from 120 ducks, sampling from cloacal swab, internal organs and tracheal swab of freshly dead, healthy and diseased ducklings at different ages which showed depression, emaciation, ocular and or nasal discharges, diarrhea and sometimes enlargement of hock joint and had lesions of airsacculitis, congested liver, catarrhal enteritis and sometimes presence of gelatinous material in the hock joints with rate of isolation of 16.66% (20 samples were positive). Serological identification of suspected isolatesof P. aeruginosa showed that 16 isolates of polyvalent I group Iand 4 isolates of polyvalent II group J. Susceptibility of the isolatedP. aeruginosa for 11 antibiotics demonstrated that the highest resistance was noted against Erythromycin, Oxytetracycline, Ampicillin and Amoxicillin (95%). The highest sensitivity of P. aeruginosa was observed againstFlorphenicol(80%) and it used for treatment in experimental design and give good results.Multiplex PCR used to identify virulencerepresented bytoxA gene, lasI gene and oprL gene ofP. aeruginosa show that all isolates where positive for the 3 examined virulence genes and these indicate that all isolates were highly virulent strains and antibiotic resistance genesrepresented by qnrS gene, blaCTX gene and mexR gene ofP. aeruginosa showed that all isolates where positive for the 3 examined AB resistanceexept isolate No 4 were negative forblaCTX and these indicate that all isolates were highly AB resistanceexept isolate No 4 wasnot haveblaCTXresistance gene.Experimental infection was done to study the Pathogenicity of P. aeruginosa which isolated from diseased ducklings on newly hatched ducklings with dose of infection of 0.2ml of 1x107 cfu/ml s/c and use the potent antibiotic florphenicol for treatment of experimentally infected ducks. Our study proved that virulence genes owned by the P. aeruginosa confirming its pathogenicity for ducks, especially in the presence of oprL gene which plays a great role in antimicrobial resistance, so biosafety was recommended for hatcheries and farms, the hygiene, cleaning and disinfection will reduce P. aeruginosa spreading in the farms.

New Sequence Type ST3449 in Multidrug-Resistant Pseudomonas aeruginosa Isolates from a Cystic Fibrosis Patient.

Pseudomonas aeruginosa is one of the most critical bacterial pathogens associated with chronic infections in cystic fibrosis patients. Here we show the phenotypic and genotypic characterization of five consecutive multidrug-resistant isolates of P. aeruginosa collected during a month from a CF patient with end-stage lung disease and fatal outcome. The isolates exhibited distinct colony morphologies and pigmentation and differences in their capacity to produce biofilm and virulence potential evaluated in larvae of Galleria mellonella. Whole genome-sequencing showed that isolates belonged to a novel sequence type ST3449 and serotype O6. Analysis of their resistome demonstrated the presence of genes blaOXA-396, blaPAO, aph(3’)-IIb, catB, crpP and fosA and new mutations in chromosomal genes conferring resistance to different antipseudomonal antibiotics. Genes exoS, exoT, exoY, toxA, lasI, rhlI and tse1 were among the 220 virulence genes detected. The different phenotypic and genotypic features found reveal the adaptation of clone ST3449 to the CF lung environment by a number of mutations affecting genes related with biofilm formation, quorum sensing and antimicrobial resistance. Most of these mutations are commonly found in CF isolates, which may give us important clues for future development of new drug targets to combat P. aeruginosa chronic infections.

The anti-biofilm capability of nano-emodin-mediated sonodynamic therapy on multi-species biofilms produced by burn wound bacterial strains

BackgroundManagement of burn wound infections (BWIs) is difficult due to the emergence of multidrug-resistant microorganisms. This study aimed to explore the anti-biofilm efficacy of sonodynamic therapy (SDT) using nano-emodin (N-EMO) against multi-species bacterial biofilms containing Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii. MethodsFollowing synthesis and confirmation of N-EMO as a sonosensitizer, the anti-biofilm efficacy of SDT against multi-species bacterial biofilms was determined using minimum inhibitory concentrations (MIC), minimum biofilm inhibitory concentration (MBIC), and minimal biofilm eradication concentration (MBEC) of N-EMO. The reduction of multi-species bacterial biofilms was then evaluated following the treatments using Log reduction and crystal violet (CV) assays. In addition, the expression profiling of abaI, agrA, and lasI genes using SDT with sub-MIC, sub-MBIC, and sub-MBEC of N-EMO was assessed. ResultsSuccessful synthesis of N-EMO was confirmed through several characterization tests. As the results demonstrated, the MIC value of N-EMO for the multi-species bacterial suspension was 0.15 × 10–4 g/L, as well as, the MBEC value of N-EMO was 2.5 × 10–4 g/L, approximately 4-fold higher than that of MBIC (0.62 × 10–4 g/L). According to the CV assay, there were 57.8 %, 71.0 %, and 81.5 % reduction in the biofilm of multi-species bacterial growth following SDT using 1/128 MBEC, 1/16 MBIC, and 1/2 MIC of N-EMO, respectively. Log reductions analysis demonstrated that 1/2 MIC of N-EMO was more potent in inhibiting the biofilm growth of multi-species test bacteria by 5.725 ± 0.12 (99.9993 %). In this study, N-EMO-mediated SDT could obviously downregulate the gene expression of virulence factors (P < 0.05). The gene expression of lasI, agrA, and abaI were downregulated about 2.5-, 3.6-, and 5.5-fold; and 3.0-, 5.2-, and 7.4-fold following SDT with sub-MBIC and sub-MBEC of N-EMO, respectively. ConclusionThese results highlight the potential of N-EMO-mediated SDT in inhibition of biofilm formation, degradation of formed biofilms, and reduction of virulence factor associated with biofilms of multi-species bacterial biofilms in BWIs.

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.

An innovative role for luteolin as a natural quorum sensing inhibitor in Pseudomonas aeruginosa

AimsThe emergence of antibiotic tolerance was a tricky problem in the treatment of chronic Pseudomonas aeruginosa-infected cystic fibrosis and burn victims. The quorum sensing (QS) inhibitor may serve as a new tactic for the bacterial resistance by inhibiting the biofilm formation and the production of virulence factors. This study explored the potential of luteolin as a QS inhibitor against P. aeruginosa and the molecular mechanism involved. Main methodsCrystal violet staining, CLSM observation, and SEM analysis were carried out to assess the effect of luteolin on biofilm formation. The motility assays and the production of virulence factors were determined to evaluate the QS-inhibitory activity of luteolin. Acyl-homoserine lactone, RT-PCR, and molecular docking assays were conducted to explain its anti-QS mechanisms. Key findingsThe biofilm formation, the production of virulence factors, and the motility of P. aeruginosa could be efficiently inhibited by luteolin. Luteolin could also attenuate the accumulation of the QS-signaling molecules N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) and N-butanoyl-L-homoserine lactone (BHL) (P < 0.01) and downregulate the transcription levels of QS genes (lasR, lasI, rhlR, and rhlI) (P < 0.01). Molecular docking analysis indicated that luteolin had a greater docking affinity with LasR regulator protein compared with OdDHL. SignificanceThis study is important as it reports the molecular mechanisms involved in the anti-biofilm formation activity of luteolin against P. aeruginosa. This study also indicated that luteolin could be helpful when used for the treatment of clinical drug-resistant infections of P. aeruginosa.

Effect of azithromycin and phenylalanine-arginine beta-naphthylamide on quorum sensing and virulence factors in clinical isolates of Pseudomonas aeruginosa

Background and Objectives:Pseudomonas aeruginosa is a problematic opportunistic pathogen causing several types of nosocomial infections with a high resistance rate to antibiotics. Production of many virulence factors in P. aeruginosa is regulated by quorum sensing (QS), a cell-to-cell communication mechanism. In this study, we aimed to assess and compare the inhibitory effect of azithromycin (AZM) and EPI-PAβN (efflux pump inhibitor-Phenylalanine-Arginine Beta-Naphthylamide) on QS system and QS-dependent virulence factors in P. aeruginosa clinical isolates.Materials and Methods:A total of 50 P. aeruginosa isolates were obtained from different types of clinical specimens. Isolates were investigated for detection of QS system molecules by AHL cross-feeding bioassay and QS-dependent virulence factors; this was also confirmed by detection of QS genes (lasR, lasI, rhlR, and rhlI) using PCR assay. The inhibitory effect of sub-MIC AZM and EPI PAβN on these virulence factors was assessed.Results:All the P. aeruginosa, producing QS signals C4HSL, failed to produce C4HSL in the presence of sub-MIC AZM, In the presence of EPI PAβN (20 μg/ml) only 14 isolates were affected, there was a significant reduction in QS-dependent virulence factors production (protease, biofilm, rhamnolipid and pyocyanin) in the presence of either 20 μg/ml EPI or sub-MIC of AZM with the inhibitory effect of AZM was more observed than PAβN.Conclusion:Anti-QS agents like AZM and EPI (PAβN) are useful therapeutic options for P. aeruginosa due to its inhibitory effect on QS-dependent virulence factors production without selective pressure on bacteria growth, so resistance to these agents is less likely to develop.

New insights into the antibacterial and quorum sensing inhibition mechanism of Artemisia argyi leaf extracts towards Pseudomonas aeruginosa PAO1.

The online version contains supplementary material available at 10.1007/s13205-021-02663-5.

Attenuation of Pseudomonas aeruginosa biofilm by thymoquinone: an individual and combinatorial study with tetrazine-capped silver nanoparticles and tryptophan.

Microbial biofilm indicates a cluster of microorganisms having the capability to display drug resistance property, thereby increasing its proficiency in spreading diseases. In the present study, the antibiofilm potential of thymoquinone, a black seed-producing natural molecule, was contemplated against the biofilm formation by Pseudomonas aeruginosa. Substantial antimicrobial activity was exhibited by thymoquinone against the test organism wherein the minimum inhibitory concentration of the compound was found to be 20 μg/mL. Thereafter, an array of experiments (crystal violet staining, protein count, and microscopic observation, etc.) were carried out by considering the sub-MIC doses of thymoquinone (5 and 10 μg/mL), each of which confirmed the biofilm attenuating capacity of thymoquinone. However, these concentrations did not show any antimicrobial activity. Further explorations on understanding the underlying mechanism of the same revealed that thymoquinone accumulated reactive oxygen species (ROS) and also inhibited the expression of the quorum sensing gene (lasI) in Pseudomonas aeruginosa. Furthermore, by taking up a combinatorial approach with two other reported antibiofilm agents (tetrazine-capped silver nanoparticles and tryptophan), the antibiofilm efficiency of thymoquinone was expanded. In this regard, the highest antibiofilm activity was observed when thymoquinone, tryptophan, and tetrazine-capped silver nanoparticles were applied together against Pseudomonas aeruginosa. These combinatorial applications of antibiofilm molecules were found to accumulate ROS in cells that resulted in the inhibition of biofilm formation. Thus, the combinatorial study of these antibiofilm molecules could be applied to control biofilm threats as the tested antibiofilm molecules alone or in combinations showed negligible or very little cytotoxicity.

The Effect of Qourum Sensing genes (lasI, rhlI) in Some Virulence Factors of Pseudomonas aeruginosa Isolated From Different Clinical Sources

Background and Objective: Quorum sensing (QS) is a chemical language of bacteria, and use of N-acylhomoserine lactone (AHL) signals is one of the most known mechanisms by which bacteria can communicatewith each other to regulate the gene expression based on bacterial density. Previous studies suggested that QShas an important role in pathogenicity of P.aeruginosa, so that the present study investigation the correlationof QS genes in local isolate of P.aeruginosa with production of virulence factors.Materials and Method: The study included collection of 126 samples from various clinical sources and,these samples included wounds, burns, ear, cystic fibrosis and urinary tract infections (UTI). Pyocyaninand biofilm production were determined spectrophotometrically, QS genes were detected by PCR based onspecific sequences for lasI and rhlI genes.Results: Final diagnosis showed that 51 isolates belong to target bacteria and, the phenotypic detectionof some virulence factors showed that 46 isolates (90%) showed variance in the strength of Pyocyaninproduction, and 44 (86%) of the isolates showed a differences in their ability to biofilm formation and,screed QS genes showed that 48 (94%) of isolates were positive for lasI gene, while 44 (86%) of isolateshave rhlI gene, and this study pointed that 9 isolates have one of QS genes lasI or rhlI, and also have one ormore of virulence factors, as well as the results showed that 1 isolate out of 51 negative for both QS genesand weakly in production of virulence factors.Conclusion: Most of local isolates were positive for QS genes and, QS plays an important role in thepathogenesis, not all of the virulence factors controlled by QS. The study indicated that the isolates whichhave both QS genes were more virulence than isolates that have single gene.

Effects of disinfectants and ciprofloxacin on quorum sensing genes and biofilm of clinical Pseudomonas aeruginosa isolates

BackgroundPseudomonas aeruginosa is one of the most virulent bacteria and quorum sensing (QS) genes have an importance on virulence factors such as biofilm that provide resistance against disinfectants and antibiotics. ObjectiveThis study aimed to determine the minimum inhibitory concentrations of the disinfectants, to investigate the effects of disinfectants and ciprofloxacin on biofilm production mature biofilm of clinical P. aeruginosa isolates, and it was aimed to investigate the effects of the agents on the expression levels of several QS-related genes in the isolates. MethodsMinimum inhibitory concentration (MIC) levels of polyhexamethylene biguanide (PHMB), chlorhexidine (CHX), quaternary ammonium compounds (QAC), glutaraldehyde (GLU) and ciprofloxacin (CIP) against clinical P. aeruginosa isolates were evaluated by microdilution method. Effects of the agents on the biofilm producing capacities of clonally unrelated nine strains were investigated by spectrophotometric method. Alterations in the expression of QS-related genes (lasI, lasR, rhlI and rhlR) were investigated by qPCR in three isolates that were CIP-susceptible and strong biofilm producer. ResultsAccording to microdilution method results, three isolates were found as resistant, one isolate was found as intermediate susceptible and five isolates were found as susceptible to CIP, and CHX (7.81–31.25 μg/mL) had the lowest MIC against P. aeruginosa. CHX inhibited biofilm production levels of eight of nine isolates, and GLU and CIP inhibited six of nine isolates in the presence of agents at MIC levels. GLU inhibited the mature biofilm levels of three of nine isolates at MIC and MIC/4 levels and four of nine isolates at MIC/2 levels. Expression levels of QS-related genes were reduced or induced in the presence of different disinfectants. ConclusionsMore efforts are required to decrease the risk of ineffective and low-dose application of disinfectants and antimicrobials against bacteria. Targeting of QS-related genes may be a reasonable strategy for the inhibition of virulence factors in P. aeruginosa.

Investigation of Pseudomonas aeruginosa Biofilm Formation and Quorum Sensing Genes in Piperacillin/Tazobactam and Ciprofloxacin Sub-minimal Inhibitory Concentrations

Pseudomonas aeruginosa is a non-fermentative, oxidase-positive, motile gram-negative bacillus widespread in nature. The virulence factors of P.aeruginosa including the ability to grow under minimal growth conditions, the widespread presence in nature, and the ability to form biofilms make P.aeruginosa a highly important bacterium along with its resistance mechanisms against many antibiotics. The ability to form biofilms increases the symptom severity in diseases caused by P.aeruginosa and causes difficulties in the treatment. The aim of this study was to investigate the effects of sub-minimal inhibitory concentrations (sub-MIC) of piperacillin/tazobactam (TZP) and ciprofloxacin (CIP) which are used for the treatment of P.aeruginosa infections on biofilm formation and to investigate the relationship between the severity of biofilm formation and Quorum Sensing (QS) genes. The study included 24 P.aeruginosa isolates from the culture collection of Medical Microbiology Laboratory of Gazi University Faculty of Medicine. MIC values of TZP and CIP antibiotics were determined by the microdilution method. The biofilm layers in the antibiotic-free medium and in the sub-MIC (MIC/2, MIC/4 ve MIC/8) concentrations of antibiotics were visualized by using a scanning electron microscope (SEM). The QS genes (lasI, lasR, rhlI, and rhlR) of the 24 isolates with known biofilm characteristics were identified via the amplification of chromosomal DNA by using PCR method. In the study, it was foundthat both antibiotics reduced biofilm formation in a dose-dependent manner in sub-MIC concentrations compared to the antibiotic-free condition and that MIC/2 was the concentration, which reduced the biofilm formation most. These results were further confirmed by viewing the SEM images. The QS genes lasI, lasR, and rhlI were detected in a total of 19 isolates with moderately strong and strong biofilm formation, the rhlR gene was detected in six of the strong biofilm-forming isolates, in four of the moderately strong biofilm-forming isolates, and in three of the weak biofilm-forming isolates, respectively. The investigation of the effects of sub-MIC concentrations of antimicrobials, used for the treatment of P.aeruginosa infections, on the biofilm formation of P.aeruginosa and the investigation and better understanding of the QS systems associated with biofilm production will allow for finding out new treatment approaches and offer different options in combating infections with high morbidity and mortality.

Effect of Sub-Minimum Inhibitory Concentrations of Tyrosol and EDTA on Quorum Sensing and Virulence of Pseudomonas aeruginosa.

IntroductionPseudomonas aeruginosa is considered a dangerous pathogen, as it causes many human diseases, besides that it is resistant to almost all types of antibacterial agents. So, new strategies to overcome P. aeruginosa infection have evolved to attenuate its virulence factors and inhibit its quorum-sensing (QS) activity.PurposeThis study investigated the effect of tyrosol and EDTA as anti-quorum-sensing and antivirulence agents against P. aeruginosa PAO1.MethodsAnti-quorum activity of sub-minimum inhibitory concentrations (sub-MICs) of tyrosol and EDTA was tested using Chromobacterium violaceum (CV 12,472) biosensor bioassay. Miller assay was used to assess the inhibition of QS signal molecules by β-galactosidase activity determination. Also, their effects on the production of protease, lipase, lecithinase, and motility were tested. The inhibitory effects of these molecules on QS regulatory genes and exotoxins genes expression were evaluated by real-time PCR.ResultsTyrosol and EDTA at sub-MICs inhibited the production of violacein pigment. Both compounds inhibited QS molecules production and their associated virulence factors (protease, lipase, lecithinase, and motility) (P≤ 0.05). Besides, the expression levels of QS regulatory genes (lasI, lasR, rhƖI, rhIR, pqsA, and pqsR) and exotoxins genes (exoS and exoY) were significantly reduced (P≤ 0.05).ConclusionBoth tyrosol and EDTA can be used to fight P. aeruginosa infection as anti-quorum-sensing and antivirulence agents at their sub-MICs.

In silico Screening Unveil the Great Potential of Ruminal Bacteria Synthesizing Lasso Peptides.

Studies of rumen microbial ecology suggest that the capacity to produce antimicrobial peptides could be a useful trait in species competing for ecological niches in the ruminal ecosystem. However, little is known about the synthesis of lasso peptides by ruminal microorganisms. Here we analyzed the distribution and diversity of lasso peptide gene clusters in 425 bacterial genomes from the rumen ecosystem. Genome mining was performed using antiSMASH 5, BAGEL4, and a database of well-known precursor sequences. The genomic context of the biosynthetic clusters was investigated to identify putative lasA genes and protein sequences from enzymes of the biosynthetic machinery were evaluated to identify conserved motifs. Metatranscriptome analysis evaluated the expression of the biosynthetic genes in the rumen microbiome. Several incomplete (n = 23) and complete (n = 11) putative lasso peptide clusters were detected in the genomes of ruminal bacteria. The complete gene clusters were exclusively found within the phylum Firmicutes, mainly (48%) in strains of the genus Butyrivibrio. The analysis of the genetic organization of complete putative lasso peptide clusters revealed the presence of co-occurring genes, including kinases (85%), transcriptional regulators (49%), and glycosyltransferases (36%). Moreover, a conserved pattern of cluster organization was detected between strains of the same genus/species. The maturation enzymes LasB, LasC, and LasD showed regions highly conserved, including the presence of a transglutaminase core in LasB, an asparagine synthetase domain in LasC, and an ABC-type transporter system in LasD. Phylogenetic trees of the essential biosynthetic proteins revealed that sequences split into monophyletic groups according to their shared single common ancestor. Metatranscriptome analyses indicated the expression of the lasso peptides biosynthetic genes within the active rumen microbiota. Overall, our in silico screening allowed the discovery of novel biosynthetic gene clusters in the genomes of ruminal bacteria and revealed several strains with the genetic potential to synthesize lasso peptides, suggesting that the ruminal microbiota represents a potential source of these promising peptides.

Hypertonic glucose inhibits growth and attenuates virulence factors of multidrug-resistant Pseudomonas aeruginosa

BackgroundPseudomonas aeruginosa is the most common Gram-negative pathogen responsible for chronic wound infections, such as diabetic foot infections, and further exacerbates the treatment options and cost of such conditions. Hypertonic glucose, a commonly used prolotherapy solution, can accelerate the proliferation of granulation tissue and improve microcirculation in wounds. However, the action of hypertonic glucose on bacterial pathogens that infect wounds is unclear. In this study, we investigated the inhibitory effects of hypertonic glucose on multidrug-resistant P. aeruginosa strains isolated from diabetic foot infections. Hypertonic glucose represents a novel approach to control chronic wound infections caused by P. aeruginosa.ResultsFour multidrug-resistant P. aeruginosa clinical strains isolated from diabetic foot ulcers from a tertiary hospital in China and the reference P. aeruginosa PAO1 strain were studied. Hypertonic glucose significantly inhibited the growth, biofilm formation, and swimming motility of P. aeruginosa clinical strains and PAO1. Furthermore, hypertonic glucose significantly reduced the production of pyocyanin and elastase virulence factors in P. aeruginosa. The expression of major quorum sensing genes (lasI, lasR, rhlI, and rhlR) in P. aeruginosa were all downregulated in response to hypertonic glucose treatment. In a Galleria mellonella larvae infection model, the administration of hypertonic glucose was shown to increase the survival rates of larvae infected by P. aeruginosa strains (3/5).ConclusionsHypertonic glucose inhibited the growth, biofilm formation, and swimming motility of P. aeruginosa, as well as reduced the production of virulence factors and quorum sensing gene expression. Further studies that investigate hypertonic glucose therapy should be considered in treating chronic wound infections.

Natural products and polysorbates: Potential Inhibitors of biofilm formation in Pseudomonas aeruginosa.

With all the challenges super bugs are imposing, biofilm formation opens the door against various more complicated challenges. Such issue may be highlighted with the ability of the latter to render the antibiotics hardly accessible to bacterial cells and sheds the light on the importance of finding antibiofilm formers. Therefore, we assessed the inhibitory effect of natural product extracts (ginger, wild blueberry) and polysorbates (PS20, PS80) on biofilm formation at the molecular level. Growth inhibition assay was performed to test the effect of ginger (Zingiber Officinale), wild blueberry (Vaccinium Angustifolium), and polysorbates on Pseudomonas aeruginosa (PAN14) growth. Transcription levels of biofilm exopolysaccharides encoding genes (ndvB, pelC, algC) and quorum sensing genes (lasI, lasR, rhlI, rhlR) for LasI/LasR and RhlI/ RhlR systems were evaluated by RT qPCR. The polysorbates and the extracts of both ginger and wild blueberry had no effect on the growth of P. aeruginosa. Biofilms' examination has unraveled the effectiveness of treatments used in reducing its formation. Moreover, a significant reduction in the expression of all genes tested for biofilm exopolysaccharides and its quorum sensing system was observed. The decrease in the relative gene expression of the exopolysaccharides and quorum sensing encoding genes sheds the light on the mechanism of action of ginger and wild blueberry's constituents as well as polysorbates 20 and 80 on P. aeruginosa biofilm formation. Future studies need to assess the antibiofilm effect of each fraction of herbal extracts separately.