Antibacterial effect of Moringa oleifera on Staphylococcus aureus and Pseudomonas aeruginosa isolated from raw milk and some dairy products with special reference to biofilm gene expression.

Background: Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) are well defined as food poisoning pathogens that are highly resistant and need continuous studies. Aim: The purpose of the work was to examine phenotypic and genotypic characteristics of both P. aeruginosa and S. aureus, and treatment trials with medicinal plants. Methods: Samples were examined for isolation of P. aeruginosa and S. aureus on selective media followed by biochemical confirmation, biofilm formation, genes detection, and expression of P. aeruginosa pslA biofilm gene was performed by quantitative real-time polymerase chain reaction after treatment with 0.312 mg/ml Moringa oleifera aqueous extract as a minimum inhibitory concentration. Results: The highest isolation rate of P. aeruginosa was 20% from both raw milk and Kariesh cheese, followed by 16% and 12% from ice cream and processed cheese, respectively, while the highest isolation rate of S. aureus was 36% from raw milk followed by 28% in ice cream and 16% in both Kariesh cheese and processed cheese. 30% of P. aeruginosa isolates were biofilm producers, while only 21% of S. aureus isolates were able to produce biofilm. The P. aeruginosa isolates harbor virulence-associated genes nan1, exoS, toxA, and pslA at 100%, 80%, 40%, and 40%, respectively. Staphylococcus aureus SEs genes were examined in S. aureus strains, where SEA and SEB genes were detected with 60%, but no isolate harbored SEC, SED, or SEE. The significant fold change of P. aeruginosa pslA expression was 0.40332 after treatment with M. oleifera aqueous extract. Conclusion: Pseudomonas aeruginosa and S. aureus harbor dangerous virulence genes that cause food poisoning, but M. oleifera extract could minimize their action.

Performance evaluation of rhamnolipid biosurfactant produced by Pseudomonas aeruginosa and its effect on marine oil-spill remediation.

This study aimed to reveal that the effect of biosurfactant on the dispersion and degradation of crude oil. Whole genome analysis showed that Pseudomonas aeruginosa GB-3 contained abundant genes involved in biosurfactant synthesis and metabolic processes and had the potential to degrade oil. The biosurfactant produced by strain GB-3 was screened by various methods. The results showed that the surface tension reduction activity was 28.6 mN·m-1 and emulsification stability was exhibited at different pH, salinity and temperature. The biosurfactant was identified as rhamnolipid by LC-MS and FTIR. The fermentation conditions of strain GB-3 were optimized by response surface methodology, finally the optimal system (carbon source: glucose, nitrogen source: ammonium sulfate, C/N ratio:16:1, pH: 7, temperature: 30-35 °C) was determined. Compared with the initial fermentation, the yield of biosurfactant increased by 4.4 times after optimization. In addition, rhamnolipid biosurfactant as a dispersant could make the dispersion of crude oil reach 38% within seven days, which enhanced the bioavailability of crude oil. As a biostimulant, it could also improve the activity of indigenous microorganism and increase the degradation rate of crude oil by 10-15%. This study suggested that rhamnolipid biosurfactant had application prospect in bioremediation of marine oil-spill.

Discovery of psoralen as a quorum sensing inhibitor suppresses Pseudomonas aeruginosa virulence.

Pseudomonas aeruginosa is a common opportunistic pathogen with growing resistance and presents heightened treatment challenges. Quorum sensing (QS) is a cell-to-cell communication system that contributes to the production of a variety of virulence factors and is also related to biofilm formation of P. aeruginosa. Compared to traditional antibiotics which kill bacteria directly, the anti-virulence strategy by targeting QS is a promising strategy for combating pseudomonal infections. In this study, the QS inhibition potential of the compounds derived from the Traditional Chinese Medicines was evaluated by using in silico, in vitro, and in vivo analyses. The results showed that psoralen, a natural furocoumarin compound derived from Psoralea corylifolia L., was capable of simultaneously inhibiting the three main QS regulators, LasR, RhlR, and PqsR of P. aeruginosa. Psoralen had no bactericidal activity but could widely inhibit the production of extracellular proteases, pyocyanin, and biofilm, and the cell motilities of the model and clinical P. aeruginosa strains. RNA-sequencing and quantitative PCR analyses further demonstrated that a majority of QS-activated genes in P. aeruginosa were suppressed by psoralen. The supplementation of psoralen could protect Caenorhabditis elegans from P. aeruginosa challenge, especially for the hypervirulent strain PA14. Moreover, psoralen showed synergistic antibacterial effects with polymyxin B, levofloxacin, and kanamycin. In conclusions, this study identifies the anti-QS and antibiofilm effects of psoralen against P. aeruginosa strains and sheds light on the discovery of anti-pseudomonal drugs among Traditional Chinese Medicines. KEY POINTS: • Psoralen derived from Psoralea corylifolia L. inhibits the virulence-related phenotypes of P. aeruginosa. • Psoralen simultaneously targets the three core regulators of P. aeruginosa QS system and inhibits the expression of a large part of downstream genes. • Psoralen protects C. elegans from P. aeruginosa challenge and enhances the susceptibility of P. aeruginosa to antibiotics.

Emergence of cefiderocol resistance during ceftazidime/avibactam treatment caused by a large genomic deletion, including ampD and piuCD genes, in Pseudomonas aeruginosa.

We report the emergence of cefiderocol resistance during the treatment of a ST312 Pseudomonas aeruginosa respiratory infection with ceftazidime/avibactam. whole genome sequencing (WGS) revealed that resistance was caused by a large genomic deletion, including PiuDC (iron transport system) and AmpD (ampC negative regulator), driven by the integration of phage DNA. Thus, our findings alert that this type of deletion could be an efficient (two mechanisms in one step) specific cefiderocol resistance mechanism that might occur nonspecifically upon treatment with ß-lactams that select for AmpC overexpression.

Diesel degradation capability and environmental robustness of strain Pseudomonas aeruginosa WS02.

Petroleum hydrocarbon (PHC) degrading bacteria have been frequently discovered. However, in practical application, a single species of PHC degrading bacterium with weak competitiveness may face environmental pressure and competitive exclusion due to the interspecific competition between petroleum-degrading bacteria as well as indigenous microbiota in soil, leading to a reduced efficacy or even malfunction. In this study, the diesel degradation ability and environmental robustness of an endophytic strain Pseudomonas aeruginosa WS02, were investigated. The results show that the cell membrane surface of WS02 was highly hydrophobic, and the strain secreted glycolipid surfactants. Genetic analysis results revealed that WS02 contained multiple metabolic systems and PHC degradation-related genes, indicating that this strain theoretically possesses the capability of oxidizing both alkanes and aromatic hydrocarbons. Gene annotation also showed many targets which coded for heavy metal resistant and metal transporter proteins. The gene annotation-based inference was confirmed by the experimental results: GC-MS analysis revealed that short chain PHCs (C10-C14) were completely degraded, and the degradation of PHCs ranging from C15-C22 were above 90% after 14 d in diesel-exposed culture; Heavy metal (Mn2+, Pb2+ and Zn2+) exposure was found to affect the growth of WS02 to some extent, but not its ability to degrade diesel, and the degradation efficiency was still maintained at 39-59%. WS02 also showed a environmental robustness along with PHC-degradation performance in the co-culture system with other bacterial strains as well as in the co-cultured system with the indigenous microbiota in soil fluid extracted from a PHC-contaminated site. It can be concluded that the broad-spectrum diesel degradation efficacy and great environmental robustness give P. aeruginosa WS02 great potential for application in the remediation of PHC-contaminated soil.

Differential expression of urease genes and ureolytic activity of uropathogenic Escherichia coli and Pseudomonas aeruginosa isolates in different nutritional conditions.

Uropathogens have adaptation strategies to survive in the host urinary tract by efficiently utilizing and tolerating the urinary metabolites. Many uropathogens harbour the enzyme urease for the breakdown of urea and the enzymatic breakdown of urea increases the pH and facilitate the struvite crystallization. In this study, the differential urease activity of uropathogenic Escherichia coli and Pseudomonas aeruginosa strains was investigated under different nutritional conditions. The experiments included measurement of growth, pH, urease activity, NH4-N generation and urease gene (ureC) expression among the bacterial strains under different conditions. Further, the implications of urea breakdown on the struvite crystallization in vitro and biofilm formation were also assessed. The study included urease positive isolates and for comparison urease negative isolates were included. Compared to the urease negative strains the urease positive strains formed higher biofilms and motility. The urease positive P. aeruginosa showed significantly higher (p < 0.01) pH and urease activity (A557-A630) compared to E. coli under experimental conditions. Further, supplementation of glucose to the growth media significantly increased the urease activity in P. aeruginosa and in contrast, it was significantly lower in E. coli. The expression profile of urease gene (ureC) was significantly higher (p < 0.001) in P. aeruginosa compared to E. coli and was consistent with the biochemical results of the urease activity under the nutritional conditions. The differential urease activity under two nutritional conditions influenced the biogenic struvite crystallization. It correlated with the urease activity showing higher crystallization rate in P. aeruginosa compared to E. coli. The results highlight the differential urease activity in two common uropathogens under different nutritional conditions that may have significant role on the regulation of virulence, pathogenicity and in the kidney stone disease.

Suppressor mutants demonstrate the metabolic plasticity of unsaturated fatty acid synthesis in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa PAO1 has two aerobic pathways for synthesis of unsaturated fatty acids (UFAs), DesA and DesB plus the oxygen independent FabAB pathway. The DesA desaturase acts on saturated acyl chains of membrane phospholipid bilayers whereas the substrates of the DesB desaturase are thought to be long chain saturated acyl-CoA thioesters derived from exogeneous saturated fatty acids that are required to support DesB-dependent growth. Under suitable aerobic conditions either of these membrane-bound desaturates can support growth of P. aeruginosa ∆fabA strains lacking the oxygen independent FabAB pathway. We previously studied function of the desA desaturase of P. putida in a P. aeruginosa ∆fabA ∆desA strain that required supplementation with a UFA for growth and noted bypass suppression of the P. aeruginosa ∆fabA ∆desA strain that restored UFA synthesis. We report three genes encoding lipid metabolism proteins that give rise to suppressor strains that bypass loss of the DesA and oxygen independent FabAB pathways.

Characterization of Pseudomonas aeruginosa resistance to ceftolozane-tazobactam due to ampC and/or ampD mutations observed during treatment using semi-mechanistic PKPD modeling.

A double ampC (AmpCG183D) and ampD (AmpDH157Y) genes mutations have been identified by whole genome sequencing in a Pseudomonas aeruginosa (PaS) that became resistant (PaR) in a patient treated by ceftolozane/tazobactam (C/T). To precisely characterize the respective contributions of these mutations on the decreased susceptibility to C/T and on the parallel increased susceptibility to imipenem (IMI), mutants were generated by homologous recombination in PAO1 reference strain (PAO1- AmpCG183D, PAO1-AmpDH157Y, PAO1-AmpCG183D/AmpDH157Y) and in PaR (PaR-AmpCPaS/AmpDPaS). Sequential time-kill curve experiments were conducted on all strains and analyzed by semi-mechanistic PKPD modeling. A PKPD model with adaptation successfully described the data, allowing discrimination between initial and time-related (adaptive resistance) effects of mutations. With PAO1 and mutant-derived strains, initial EC50 values increased by 1.4, 4.1, and 29-fold after AmpCG183D , AmpDH157Y and AmpCG183D/AmpDH157Y mutations, respectively. EC50 values were increased by 320, 12.4, and 55-fold at the end of the 2 nd experiment. EC50 of PAO1-AmpCG183D/AmpDH157Y was higher than that of single mutants at any time of the experiments. Within the PaR clinical background, reversal of AmpCG183D, and AmpDH157Y mutations led to an important decrease of EC50 value, from 80.5 mg/L to 6.77 mg/L for PaR and PaR-AmpCPaS/AmpDPaS, respectively. The effect of mutations on IMI susceptibility mainly showed that the AmpCG183D mutation prevented the emergence of adaptive resistance. The model successfully described the separate and combined effect of AmpCG183D and AmpDH157Y mutations against C/T and IMI, allowing discrimination and quantification of the initial and time-related effects of mutations. This method could be reproduced in clinical strains to decipher complex resistance mechanisms.

Bacterial respiratory inhibition triggers dispersal of Pseudomonas aeruginosa biofilms.

Pseudomonas aeruginosa grows as a biofilm under many environmental conditions, and the bacterium can disperse from biofilms via highly regulated, dynamic processes. However, physiologic triggers of biofilm dispersal remain poorly understood. Based on prior literature describing dispersal triggered by forms of starvation, we tested bacterial respiratory inhibitors for biofilm dispersal in two models resembling chronic airway infections. Our underlying hypothesis was that respiratory inhibitors could serve as a model for the downstream effects of starvation. We used two experimental conditions. In the first condition, biofilms were grown and dispersed from the surface of airway epithelial cells, and the second condition was a model where biofilms were grown on glass in cell culture media supplemented with host-relevant iron sources. In both biofilm models, the respiratory inhibitors potassium cyanide and sodium azide each triggered biofilm dispersal. We hypothesized that cyanide-induced dispersal was due to respiratory inhibition rather than signaling via an alternative mechanism, and, indeed, if respiration was supported by overexpression of cyanide-insensitive oxidase, dispersal was prevented. Dispersal required the activity of the cyclic-di-GMP regulated protease LapG, reinforcing the role of matrix degradation in dispersal. Finally, we examined the roles of individual phosphodiesterases, previously implicated in dispersal to specific triggers, and found signaling to be highly redundant. Combined deletion of the phosphodiesterases dipA, bifA, and rbdA was required to attenuate the dispersal phenotype. In summary, this work adds insight into the physiology of biofilm dispersal under environmental conditions in which bacterial respiration is abruptly limited. IMPORTANCE The bacterium Pseudomonas aeruginosa grows in biofilm communities that are very difficult to treat in human infections. Growing as a biofilm can protect bacteria from antibiotics and the immune system. Bacteria can leave a biofilm through a process called "dispersal." Dispersed bacteria seed new growth areas and are more susceptible to killing by antibiotics. The triggers for biofilm dispersal are not well understood, and if we understood dispersal better it might lead to the development of new treatments for infection. In this paper, we find that inhibiting P. aeurginosa's ability to respire (generate energy) can trigger dispersal from a biofilm grown in association with human respiratory epithelial cells in culture. The dispersal process requires a protease which is previously known to degrade the biofilm matrix. These findings give us a better understanding of how the biofilm dispersal process works so that future research can discover better ways of clearing bacteria growing in biofilms.

Case Commentary: When Voldemort Meets Sauron: Treatment Considerations for Emerging Dual-Carbapenemase-Producing Extensively Drug-Resistant Pseudomonas aeruginosa.

Infections caused by extensively drug-resistant Pseudomonas aeruginosa are difficult to treat due to limited effective treatment options. In this issue, a patient with a corneal infection caused by a Verona integron-encoded metallo-ß-lactamase (VIM)- and Guiana extended-spectrum ß-lactamase (GES)-coproducing P. aeruginosa strain associated with the recent artificial tears-related outbreak in the United States is described. This resistance genotype/phenotype further compromises therapeutic options, and this report provides insights into diagnostic and treatment approaches for clinicians dealing with infections due to this highly resistant P. aeruginosa.

Investigating and Treating a Corneal Ulcer Due to Extensively Drug-Resistant Pseudomonas aeruginosa.

Resistant Gram-negative bacteria are a growing concern in the United States, leading to significant morbidity and mortality. We identified a 72-year-old female patient who presented with unilateral vision loss. She was found to have a large corneal ulcer with hypopyon. Culture of corneal scrapings grew extensively drug-resistant Pseudomonas aeruginosa. Treatment involved a combination of systemic and topical antibiotics. Whole genome sequencing revealed the presence of blaVIM-80, blaGES-9, and other resistance determinants. This distinctive organism was linked to an over-the-counter artificial tears product.

Analysis of the Plasmid-Based ts-Mutant ΔfabA/pTS-fabA Reveals Its Lethality under Aerobic Growth Conditions That Is Suppressed by Mild Overexpression of desA at a Restrictive Temperature in Pseudomonas aeruginosa.

It is uncertain whether PA1610|fabA is essential or dispensable for growth on LB-agar plates under aerobic conditions in Pseudomonas aeruginosa PAO1. To examine its essentiality, we disrupted fabA in the presence of a native promoter-controlled complementary copy on ts-plasmid. In this analysis, we showed that the plasmid-based ts-mutant ΔfabA/pTS-fabA failed to grow at a restrictive temperature, consistent with the observation by Hoang and Schweizer (T. T. Hoang, H. P. Schweizer, J Bacteriol 179:5326-5332, 1997, https://doi.org/10.1128/jb.179.17.5326-5332.1997), and expanded on this by showing that ΔfabA exhibited curved cell morphology. On the other hand, strong induction of fabA-OE or PA3645|fabZ-OE impeded the growth of cells displaying oval morphology. Suppressor analysis revealed a mutant sup gene that suppressed a growth defect but not cell morphology of ΔfabA. Genome resequencing and transcriptomic profiling of sup identified PA0286|desA, whose promoter carried a single-nucleotide polymorphism (SNP), and transcription was significantly upregulated (level increase of >2-fold, P < 0.05). By integration of the SNP-bearing promoter-controlled desA gene into the chromosome of ΔfabA/pTS-fabA, we showed that the SNP is sufficient for ΔfabA to phenocopy the sup mutant. Furthermore, mild induction of the araC-PBAD-controlled desA gene but not desB rescued ΔfabA. These results validated that mild overexpression of desA fully suppressed the lethality but not the curved cell morphology of ΔfabA. Similarly, Zhu et al. (Zhu K, Choi K-H, Schweizer HP, Rock CO, Zhang Y-M, Mol Microbiol 60:260-273, 2006, https://doi.org/10.1111/j.1365-2958.2006.05088.x) showed that multicopy desA partially alleviated the slow growth phenotype of ΔfabA, the difference in which was that ΔfabA was viable. Taken together, our results demonstrate that fabA is essential for aerobic growth. We propose that the plasmid-based ts-allele is useful for exploring the genetic suppression interaction of essential genes of interest in P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen whose multidrug resistance demands new drug development. Fatty acids are essential for viability, and essential genes are ideal drug targets. However, the growth defect of essential gene mutants can be suppressed. Suppressors tend to be accumulated during the construction of essential gene deletion mutants, hampering the genetic analysis. To circumvent this issue, we constructed a deletion allele of fabA in the presence of a native promoter-controlled complementary copy in the ts-plasmid. In this analysis, we showed that ΔfabA/pTS-fabA failed to grow at a restrictive temperature, supporting its essentiality. Suppressor analysis revealed desA, whose promoter carried a SNP and whose transcription was upregulated. We validated that both the SNP-bearing promoter-controlled and regulable PBAD promoter-controlled desA suppressed the lethality of ΔfabA. Together, our results demonstrate that fabA is essential for aerobic growth. We propose that plasmid-based ts-alleles are suitable for genetic analysis of essential genes of interest.

Quorum sensing activities and genomic insights of plant growth-promoting rhizobacteria isolated from Assam tea.

Secretion of quorum sensing (QS) molecules is important for the effective colonization of host plants by plant growth-promoting rhizobacteria. The current study aims at the isolation and characterization of tea rhizo bacteria, which produce the QS molecules, acyl homoserine lactone (AHLs), along with multiple plant growth-promoting (PGP) activities. Thirty-one isolates were isolated from the tea rhizosphere, and screening for PGP activities resulted in the selection of isolates RTE1 and RTE4 with multiple PGP traits, inhibiting the growth of tea fungal pathogens. Both isolates also showed production of AHL molecules when screened using two biosensor strains, Chromobacterium violaceum CV026 and Escherichia coli MT 102(jb132). The isolates identified as Burkholderia cepacia RTE1 and Pseudomonas aeruginosa RTE4 based on genome-based analysis like phylogeny, dDDH, and fastANI calculation. Detailed characterization of AHLs produced by the isolates using reverse-phase TLC, fluorometry, and LC-MS indicated that the isolate RTE1 produced a short chain, C8, and a long chain C12 AHL, while RTE4 produced short-chain AHLs C4 and C6. Confocal microscopy revealed the formation of thick biofilm by RTE1 and RTE4 (18 and 23 µm, respectively). Additionally, we found several genes involved in QS, and PGP, inducing systemic resistance (ISR) activities such as lasI/R, qscR, pqq, pvd, aldH, acdS, phz, Sod, rml, and Pch, and biosynthetic gene clusters like N-acyl homoserine lactone synthase, terpenes, pyochelin, and pyocyanin. Based on the functional traits like PGP, biofilm formation and production of AHL molecules, and genetic potential of the isolates B. cepacia RTE1 and P. aeruginosa RTE4 appear promising candidates to improve the health and growth of tea plantations.

Simultaneous and divergent evolution of resistance to cephalosporin/ß-lactamase inhibitor combinations and imipenem/relebactam following ceftazidime/avibactam treatment of MDR Pseudomonas aeruginosa infections.

OBJECTIVES: To describe and characterize the emergence of resistance to ceftolozane/tazobactam, ceftazidime/avibactam and imipenem/relebactam in a patient receiving ceftazidime/avibactam treatment for an MDR Pseudomonas aeruginosa CNS infection. METHODS: One baseline (PA1) and two post-exposure (PA2 and PA3) isolates obtained before and during treatment of a nosocomial P. aeruginosa meningoventriculitis were evaluated. MICs were determined by broth microdilution. Mutational changes were investigated through WGS. The impact on ß-lactam resistance of mutations in blaPDC and mexR was determined through cloning experiments and complementation assays. RESULTS: Isolate PA1 showed baseline resistance mutations in DacB (I354A) and OprD (N142fs) conferring resistance to conventional antipseudomonals but susceptibility to ceftazidime/avibactam, ceftolozane/tazobactam and imipenem/relebactam. Post-exposure isolates showed two divergent ceftazidime/avibactam-resistant phenotypes associated with distinctive mutations affecting the intrinsic P PDC ß-lactamase (S254Ins) (PA2: ceftolozane/tazobactam and ceftazidime/avibactam-resistant) or MexAB-OprM negative regulator MexR in combination with modification of PBP3 (PA3: ceftazidime/avibactam and imipenem/relebactam-relebactam-resistant). Cloning experiments demonstrated the role of PDC modification in resistance to ceftolozane/tazobactam and ceftazidime/avibactam. Complementation with a functional copy of the mexR gene in isolate PA3 restored imipenem/relebactam susceptibility. CONCLUSIONS: We demonstrated how P. aeruginosa may simultaneously develop resistance and compromise the activity of new ß-lactam/ß-lactamase inhibitor combinations when exposed to ceftazidime/avibactam through selection of mutations leading to PDC modification and up-regulation of MexAB-OprM-mediated efflux.

Exploration of the inhibitory effect of Cassia fistula on quorum sensing mediated virulence factor production and biofilm activity in Pseudomonas aeruginosa: an in vivo study in model organism Caenorhabditis elegans.

Introduction. Resistance to antibiotics is leading to challenges in the treatment of microbial diseases. One amongst the various approaches to control these pathogens is quorum sensing (QS), which is used to rectify resistance issues. Blocking the bacterial QS circuit is the most reliable anti-virulence therapy to control pathogenicity-associated genes. Pseudomonas aeruginosa is a contagious bacterium that proliferates in the host by using signalling molecules like acyl-homoserine lactones; these molecules generate and disseminate toxins and virulence factors for increasing host infection.Hypothesis. The herb Cassia fistula is known to have antimicrobial, antidiabetic, anti-inflammatory, antitumor medicinal properties amongst others. We hypothesize that its crude extracts will inhibit the QS circuit of Pseudomonas aeruginosa (P. aeruginosa).Aim. The research work was aimed at evaluating anti-quorum sensing and anti-biofilm activity of various crude extracts from Cassia fistula against P. aeruginosa.Methodology. Various extraction methods and solvents were availed for maximum separation, and the extracts were screened for anti-quorum sensing activity. The most potent Fruit Ethyl acetate (FEE) extract at non-inhibitory concentrations was found to interrupt both short-chain (RhlI/R) and long-chain (LasI/R) QS circuits and other virulence factors (P<0.05) such as elastase, protease, rhamnolipids and pyocyanin levels in P. aeruginosa. Biofilm inhibitory properties of FEE were demonstrated using atomic force microscopy, scanning electron microscope and confocal laser microscope. Caenorhabditis elegans infection model (Paralytic assay) was developed to determine the protective role of FEE by reducing the pathogenicity of P. aeruginosa.Results. The study results suggest that hot crude FEE extract interfered in the QS circuit, leading to comprehensive debilitation of QS-controlled virulence factors. The extract reduced virulence factor production in P. aeruginosa at 4 mg ml-1 concentration whilst paradoxically promoting biofilm formation. Possibly, higher sugar content in the extract promoted clump formation of biofilm architecture by increasing exopolysaccharide production. Moreover, in vivo analysis of bacterial pathogenesis on Caenorhabditis elegans reveals a drastic increase in survival rates in FEE treated worms compared to untreated control.Conclusions. FEE showed promising QS inhibitory activity against P. aeruginosa. In the future, additional purification of crude FEE is required to remove carbohydrates, and pure isolated phytochemicals from FEE could be used as therapeutic agents to control QS-mediated infections in P. aeruginosa.

Genome-scale model of Pseudomonas aeruginosa metabolism unveils virulence and drug potentiation.

Pseudomonas aeruginosa is one of the leading causes of hospital-acquired infections. To decipher the metabolic mechanisms associated with virulence and antibiotic resistance, we have developed an updated genome-scale model (GEM) of P. aeruginosa. The model (iSD1509) is an extensively curated, three-compartment, and mass-and-charge balanced BiGG model containing 1509 genes, the largest gene content for any P. aeruginosa GEM to date. It is the most accurate with prediction accuracies as high as 92.4% (gene essentiality) and 93.5% (substrate utilization). In iSD1509, we newly added a recently discovered pathway for ubiquinone-9 biosynthesis which is required for anaerobic growth. We used a modified iSD1509 to demonstrate the role of virulence factor (phenazines) in the pathogen survival within biofilm/oxygen-limited condition. Further, the model can mechanistically explain the overproduction of a drug susceptibility biomarker in the P. aeruginosa mutants. Finally, we use iSD1509 to demonstrate the drug potentiation by metabolite supplementation, and elucidate the mechanisms behind the phenotype, which agree with experimental results.

Scavenging of reactive oxygen species effectively reduces Pseudomonas aeruginosa biofilms through disrupting policing.

Biofilm formation is likely to contribute greatly to antibiotic resistance in bacteria and therefore the efficient removal of bacterial biofilms needs addressing urgently. Here, we reported that the supplement of non-inhibitory concentration of N-acetyl-L-cysteine (NAC), a common reactive oxygen species (ROS) scavenger, can significantly reduce the biomass of mature Pseudomonas aeruginosa biofilms (corroborated by crystal violet assay and laser scanning confocal microscopy). 1 mM NAC increased the cheater (ΔlasR mutant) frequency to 89.4 ± 1.5% in the evolved PAO1 after the 15-day treatment. Scavenging of ROS by NAC induced the collapse of P. aeruginosa biofilms, but it did not alter quorum sensing-regulated genes expression (e.g., hcnC and cioAB) and hydrogen cyanide production. The replenishment of public good protease contributed to the recovery of biofilm biomass, indicating the role of disrupting policing in biofilm inhibition. Furthermore, 7 typical ROS scavengers (e.g., superoxide dismutase, catalase and peroxidase, etc.) also effectively inhibited mature P. aeruginosa biofilms. This study demonstrates that scavenging of ROS can promote the selective control of P. aeruginosa biofilms through policing disruption as a targeted biofilm control strategy in complex water environments.

Investigation of antimicrobial activities and molecular characterization of the species belong to Origanum, Thymus and Thymbra genera by ISSR.

BACKGROUND: The aim of this study is to investigate the antimicrobial activities of the species belonging to the genera Origanum L., Thymus L., and Thymbra L. in the Lamiaceae family and molecular characterization using ISSR markers and to determine the correlations between anti-microbial activities of the plant extracts and ISSR loci. METHODS AND RESULTS: Anti-microbial active extracts were obtained after 24-hours extraction using either of the three different solvents (ethanol, hexane, and chloroform) from the plants using the Soxhlet device. The effects of extracts on the bacterial strains (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis) were determined using the disc-diffusion method. The species Thymbra spicata var. spicata L., Thymus vulgaris L., Thymus citriodorus, Thymus cilicicus, Origanum syriacum L., and Origanum vulgare L. subsp. hirtum displayed significant anti-microbial activities, while the Origanum minutiflorum, Origanum onites L., Origanum saccatum and Origanum vulgare L. ssp. gracile displayed less activities on the bacterial strains. The plant species under study had a high level of genetic diversity. Significant correlations were determined between the anti-microbial activities of the plant species and the ISSR loci. CONCLUSION: Staphylococcus aureus was the most sensitive and Pseudomonas aeruginosa was the least sensitive strain. The ethanol and chloroform extracts were the most effective solvents. ISSR markers were successful for determining high levels of genetic diversity and clustering the species belonging to the genera Origanum, Thymus, and Thymbra. Conducting molecular marker analyses facilitated in distinguishing the species correctly for molecular breeding studies. The studies identified the antimicrobial activities of the plants against the bacteria used in the study and suggested their potential role in the pharmaceutical industry.

Characterization of petroleum degrading bacteria and its optimization conditions on effective utilization of petroleum hydrocarbons.

Hydrocarbon contamination is continuing to be a serious environmental problem because of their toxicity. Hydrocarbon components have been known to be carcinogens and neurotoxic organic pollutants. The physical and chemical methods of petroleum removal have become ineffective and also are very costly. Therefore, bioremediation is considered the promising technology for the treatment of these contaminated sites since it is cost-effective and will lead to complete mineralization.The current study also concentrates on bioremediation of petroleum products by bacterium isolated from petroleum hydrocarbon contaminated soil. The current work shows that bacterial strains obtained from a petroleum hydrocarbon contaminated environment may degrade petroleum compounds. Two strains Bacillus licheniformis ARMP2 and Pseudomonas aeruginosa ARMP8 were identified as petroleum-degrading bacteria of the isolated bacterial colonies. The best growth conditions for the ARMP2 strain were determined to be pH 9, temperature 29 °C with sodium nitrate as its nitrogen source, whereas for the ARMP8 strain the optimal growth was found at pH 7, temperature 39 °C, and ammonium chloride as the nitrogen source. Both strains were shown to be effective at degrading petroleum chemicals confirmed by GCMS. Overall petroleum product degradation efficiency of the strains ARMP2 and ARMP8 was about 88 % and 73 % respectively in 48 h.The strains Bacillus licheniformis ARMP2 and Pseudomonas aeruginosa ARMP8 were shown to be effective at degrading petroleum compounds in the current study. Even greater results might be obtained if the organisms were utilised in consortia or the degradation time period was extended.

A Novel FadL Homolog, AltL, Mediates Transport of Long-Chain Alkanes and Fatty Acids in Acinetobacter venetianus RAG-1.

Due to the barrier effect of lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria, transporters are required for hydrophobic alkane uptake. However, there are few reports on long-chain alkane transporters. In this study, a potential long-chain alkane transporter (AltL) was screened in Acinetobacter venetianus RAG-1 by comparative transcriptome analysis. Growth and degradation experiments showed that altL deletion led to the loss of n-octacosane utilization capacity of RAG-1. To identify the function of AltL, we measured the existence and accumulation of alkanes in cells through the constructed alkane detection system and isotope transport experiment, which proved its long-chain alkane transport function. Growth experiments using different chain-length n-alkanes and fatty acids as substrates showed that AltL was responsible for the transport of (very) long-chain n-alkanes (C20 to C38) and fatty acids (C18A to C28A) and was also involved in the uptake of medium-chain n-alkanes (C16 to C18). Subsequently, we analyzed the distribution of AltL in bacteria, and found that AltL homologs are widespread in Gamma-, Beta-, and Deltaproteobacteria. An AltL homolog in Pseudomonas aeruginosa was also identified to participate in long-chain alkane transport by a gene deletion and growth assay. We also found that overexpression of altL in Pseudomonas aeruginosa enhanced the degradation of C16 to C32 n-alkanes. In addition, structure analysis showed that AltL has longer extracellular loops than other FadL family members, which may be involved in the binding of alkanes. These results showed that AltL is a novel transporter and that it is mainly responsible for the transport of long-chain n-alkanes and (very) long-chain fatty acids and has broad application potential. IMPORTANCE Petroleum pollution has caused great harm to the natural environment, and alkanes are the main components of petroleum. Many Gram-negative bacteria can use alkanes as carbon and energy sources, which is an important strategy for oil pollution remediation. Alkane uptake is the first step for its utilization. Hence, the characterization of transport proteins is of great significance for the recovery of oil pollution and other potential applications in industrial engineering bacteria. At present, some short- and medium-chain alkane transporters have been identified, but stronger hydrophobic long-chain alkane transporters have received little attention. In this study, the broad-spectrum transporter AltL, identified in RAG-1, makes up for the lack of research on the transport of long-chain alkanes and (very) long-chain fatty acids. Meanwhile, the structural features of longer extracellular loops might be related to its unique transport function on more hydrophobic and larger substrates, indicating it is a novel type alkane transporter.