Biosurfactant Production by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> from Renewable Resources

This study deals with production and characterization of biosurfactant from renewable resources by Pseudomonas aeruginosa. Biosurfactant production was carried out in 3L fermentor using waste motor lubricant oil and peanut oil cake. Maximum biomass (11.6 mg/ml) and biosurfactant production (8.6 mg/ml) occurred with peanut oil cake at 120 and 132 h respectively. Characterization of the biosurfactant revealed that, it is a lipopeptide with chemical composition of protein (50.2%) and lipid (49.8%). The biosurfactant (1 mg/ml) was able to emulsify waste motor lubricant oil, crude oil, peanut oil, kerosene, diesel, xylene, naphthalene and anthracene, comparatively the emulsification activity was higher than the activity found with Triton X-100 (1 mg/ml). Results obtained in the present study showed the possibility of biosurfactant production using renewable, relatively inexpensive and easily available resources. Emulsification activity found with the biosurfactant against different hydrocarbons showed its possible application in bioremediation of environments polluted with various hydrocarbons.     

<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> inhibits <Emphasis Type="Italic">in-vitro Candida</Emphasis> biofilm development

Background  

Elucidation of the communal behavior of microbes in mixed species biofilms may have a major impact on understanding infectious diseases and for the therapeutics. Although, the structure and the properties of monospecies biofilms and their role in disease have been extensively studied during the last decade, the interactions within mixed biofilms consisting of bacteria and fungi such as Candida spp. have not been illustrated in depth. Hence, the aim of this study was to evaluate the interspecies interactions of Pseudomonas aeruginosa and six different species of Candida comprising C. albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis, and C. dubliniensis in dual species biofilm development.     

Biofilm dispersion in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

In recent decades, many researchers have written numerous articles about microbial biofilms. Biofilm is a complex community of microorganisms and an example of bacterial group behavior. Biofilm is usually considered a sessile mode of life derived from the attached growth of microbes to surfaces, and most biofilms are embedded in self-produced extracellular matrix composed of extracellular polymeric substances (EPSs), such as polysaccharides, extracellular DNAs (eDNA), and proteins. Dispersal, a mode of biofilm detachment indicates active mechanisms that cause individual cells to separate from the biofilm and return to planktonic life. Since biofilm cells are cemented and surrounded by EPSs, dispersal is not simple to do and many researchers are now paying more attention to this active detachment process. Unlike other modes of biofilm detachment such as erosion or sloughing, which are generally considered passive processes, dispersal occurs as a result of complex spatial differentiation and molecular events in biofilm cells in response to various environmental cues, and there are many biological reasons that force bacterial cells to disperse from the biofilms. In this review, we mainly focus on the spatial differentiation of biofilm that is a prerequisite for dispersal, as well as environmental cues and molecular events related to the biofilm dispersal. More specifically, we discuss the dispersal-related phenomena and mechanisms observed in Pseudomonas aeruginosa, an important opportunistic human pathogen and representative model organism for biofilm study.     

Biodegradation of 4-chlorobenzoic acid by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PA01 NC

A bacterial consortium capable of degrading chloroaromatic compounds was isolated from pulp and paper mill effluents by selective enrichment on 4-chlorobenzoic acid as sole source of carbon and energy. The four different bacterial isolates obtained from bacterial consortium were identified as Pseudomonas aeruginosa AY792969 (A), P. aeruginosa PA01 NC (B), Pseudomonas sp. ZZ5 DQ113452 (C) and Pseudomonas sp. AY762360 (D) based on their morphological and biochemical characteristics and by phylogenetic analysis based on 16S rRNA gene sequences. These bacterial isolates were found to be versatile in degrading a variety of chloroaromatic compounds including fluoro- and iodobenzoic acids. P. aeruginosa PA01 NC utilized 4-chlorobenzoic acid at 2 g/l as growth substrate. Biodegradation studies have revealed that this organism degraded 4-chlorobenzoic acid through 4-chlorocatechol which was further metabolized by ortho-cleavage pathway and the dechlorination occurred after the ring-cleavage.     

N-acetylcysteine inhibit biofilms produced by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Background  

Pseudomonas aeruginosa is a common pathogen in chronic respiratory tract infections. It typically makes a biofilm, which makes treatment of these infections difficult. In this study, we investigated the inhibitory effects of N-acetylcysteine (NAC) on biofilms produced by P. aeruginosa.     

<Emphasis Type="Italic">Drosophila melanogaster</Emphasis> as a polymicrobial infection model for <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Non-mammalian infection models have been developed over the last two decades, which is a historic milestone to understand the molecular basis of bacterial pathogenesis. They also provide small-scale research platforms for identification of virulence factors, screening for antibacterial hits, and evaluation of antibacterial efficacy. The fruit fly, Drosophila melanogaster is one of the model hosts for a variety of bacterial pathogens, in that the innate immunity pathways and tissue physiology are highly similar to those in mammals. We here present a relatively simple protocol to assess the key aspects of the polymicrobial interaction in vivo between the human opportunistic pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, which is based on the systemic infection by needle pricking at the dorsal thorax of the flies. After infection, fly survival and bacteremia over time for both P. aeruginosa and S. aureus within the infected flies can be monitored as a measure of polymicrobial virulence potential. The infection takes ~24 h including bacterial cultivation. Fly survival and bacteremia are assessed using the infected flies that are monitored up to ~60 h post-infection. These methods can be used to identify presumable as well as unexpected phenotypes during polymicrobial interaction between P. aeruginosa and S. aureus mutants, regarding bacterial pathogenesis and host immunity.     

UDP-N-acetylglucosamine enolpyruvyl transferase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Multi-drug resistant Pseudomonas aeruginosa (MDRPA) are emerging as a major threat in the hospitals as they have become resistant to current antibiotics. There is an immediate requirement of drugs with novel mechanisms as the pipeline of investigational drugs against these organisms is lean. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) enzyme that catalyzes the first committed step of bacterial cell wall biosynthesis is an ideal target for the discovery of novel antibiotics against Gram negative pathogens as they have only one copy of murA gene in its genome. We have performed biochemical characterization and comparative kinetic analysis of MurA from E. coli and P. aeruginosa. Both enzymes were active at broad range of pH with temperature optima of 37°C. Metal ions did not enhance the activity of both enzymes. These enzymes had an apparent affinity constant (K _m ) for its substrate UDP-N-acetylglucosamine 36 ± 5.2 and 17.8 ± 2.5 μM and for phosphoenolpyruvate 0.84 ± 0.13 μM and 0.45 ± 0.07 μM for E. coli and P. aeruginosa enzymes respectively. Both the enzymes showed 5–7 fold shift in IC₅₀ for the known inhibitor fosfomycin upon pre-incubation with the substrate UDP-N-acetylglucosamine. This observation was used to develop a novel rapid sensitive high throughput assay for the screening of MurA inhibitors.     

Transferable Carbenicillin Resistance in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

IN 1969, after carbenicillin had been in use for three years in this unit, highly resistant strains of Pseudomonas aeruginosa were isolated for the first time¹. Because these resistant strains included, from their first appearance, representatives of two unrelated types, it seemed likely that the resistance was transferable; this hypothesis was supported by experiments showing the transfer of carbenicillin resistance between Ps. aeruginosa and Escherichia coli K12 in vitro and in vivo^2–4;. The resistant Ps. aeruginosa produced a penicillinase (β lactamase) similar to that normally produced by some strains of Enterobacteria and different from that normally produced by Ps. aeruginosa^2,3, so it seemed likely that the Ps. aeruginosa had initially acquired resistance by the transfer of an R factor from a carbenicillin-resistant member of the Enterobacteriaceae colonizing the same burn. This hypothesis is now supported by a study on strains of Enterobacteria and Ps. aeruginosa isolated in a number of hospitals. We have also found evidence suggesting that Ps. aeruginosa which has acquired this R factor may not show resistance until it has been exposed repeatedly to carbenicillin.     

Antibiotic synergy against biofilm-forming <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Eight antibiotics (aztreonam, ceftazidim, cefoperazon, cefepim, netilmicin, amikacin, ofloxacin and ciprofloxacin) exhibited antimicrobial activity individually and/or in combinations against 20 wild-type biofilm-forming strains of Pseudomonas aeruginosa. The strains were less susceptible in biofilm; in 10 strains antibiotic synergy was observed for the combination of aztreonam and ciprofloxacin. Synergy was also demonstrated in the case of β-lactams and aminoglycosides, β-lactams and fluoroquinolones, aminoglycosides and fluoroquinolones, and for monobactams and β-lactams although the strains were resistant to the individual antibiotics. Synergism or partial synergism was found with one or more antibiotic combinations against 32.4% of isolates.     

Assessing biodegradation of furfuryl alcohol using <Emphasis Type="Italic">Pseudomonas putida</Emphasis> MTCC 1194 and <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> MTCC 1034 and its kinetics

The biodegradation of furfuryl alcohol (FA) in shake flask experiments using a pure culture of Pseudomonas putida (MTCC 1194) and Pseudomonas aeruginosa (MTCC 1034) was studied at 30 °C and pH 7.0. Experiments were performed at different FA concentrations ranging from 50 to 500 mg/l. Before carrying out the biodegradation studies, the bacterial strains were acclimatized to the concentration of 500 mg/l of FA by gradually raising 100 mg/l of FA in each step. The well acclimatized culture of P. putida and P. aeruginosa degraded about 80 and 66% of 50 mg/l FA, respectively. At higher concentration of FA, the percentage of FA degradation decreased. The purpose of this study was to determine the kinetics of biodegradation of FA by measuring biomass growth rates and concentration of FA as a function of time. Substrate inhibition was calculated from experimental growth parameters using the Haldane equation. Data for P. putida were determined as µ _max ?=?0.23 h^?1, K _s ?=?23.93 mg/l and K _i ?=?217.1 mg/l and for P. aeruginosa were determined as µ _max ?=?0.13 h^?1, K _s ?=?21.3 mg/l and K _i ?=?284.9 mg/l. The experimental data were fitted in Haldane, Aiba and Edwards inhibition models.     

Prevalence and analysis of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> in chinchillas

Background  

Chinchillas (Chinchilla laniger) are popular as pets and are often used as laboratory animals for various studies. Pseudomonas aeruginosa is a major infectious agent that causes otitis media, pneumonia, septicaemia enteritis, and sudden death in chinchillas. This bacterium is also a leading cause of nosocomial infections in humans. To prevent propagation of P. aeruginosa infection among humans and animals, detailed characteristics of the isolates, including antibiotic susceptibility and genetic features, are needed. In this study, we surveyed P. aeruginosa distribution in chinchillas bred as pets or laboratory animals. We also characterized the isolates from these chinchillas by testing for antibiotic susceptibility and by gene analysis.     

Production of 7,10-dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic acid from olive oil by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3

Microbial modification of naturally occurring materials is one of the efficient ways to add new values to them. Hydroxylation of free unsaturated fatty acids by microorganism is a good example of those modifications. Among microbial strains studied for that purpose, a new bacterial isolate Pseudomonas aeruginosa PR3 has been well studied to produce several hydroxy fatty acids from different unsaturated fatty acids. Of those hydroxy fatty acids, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was efficiently produced from oleic acid by strain PR3. However, it was highly plausible to use vegetable oil containing oleic acid rather than free oleic acid as a substrate for DOD production by strain PR3. In this study, we firstly tried to use olive oil containing high content of oleic acid as a substrate for DOD production. DOD production from olive oil was confirmed by structural determination with GC, TLC, and GC/MS analysis. DOD production yield from olive oil was 53.5%. Several important environmental factors were also tested. Galactose and glutamine were optimal carbon and nitrogen sources, and magnesium ion was critically required for DOD production from olive oil. Results from this study demonstrated that natural vegetable oils containing oleic acid could be used as efficient substrate for the production of DOD by strain PR3.     

Changes in <Emphasis Type="Italic">Galleria mellonella</Emphasis> lysozyme level and activity during <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> infection

The level of lysozyme in fat body, hemocytes and cell-free hemolymph from Galleria mellonella larvae infected with Pseudomonas aeruginosa was determined and evaluated. In the samples of fat body and hemocytes, an increase in lysozyme content was detected 1 d after infection and then a significant decrease was observed after a prolonged infection time. In the case of cell-free hemolymph, an increase in the lysozyme level was noticeable during the first 30 h post injection and stayed at a similar level for 42 h. The smaller decrease of the lysozyme level after 42 h might be associated with the development of bacteremia of P. aeruginosa in insects. In addition, the gradual increase in the content of lysozyme correlated with the increase of its activity in the hemolymph of the infected larvae as a response to injection with P. aeruginosa. The G. mellonella lysozyme appeared to be insensitive to extracellular proteinases produced in vivo by P. aeruginosa.     

Urovirulence of<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>: Planktonic cells<Emphasis Type="Italic">vs.</Emphasis> biofilm cells

Planktonic and biofilm cells of a clinical urinary isolate of P. aeruginosa were compared in vitro for their ability to adhere to uroepithelial cells, interaction with macrophages, and for production of virulence factors like extracellular proteinase, elastase, hemolysin, phospholipase C and pyochelin. Biofilm cells showed increased adherence to UECs, which was coupled with reduced uptake and intracellular killing by macrophages. Overall there was a decrease in production of extracellular products by biofilm cells. Comparing the two cell forms for their ability to establish infection in an ascending model of acute pyelonephritis, significant enhancement of renal bacterial load, as well as more pronounced renal pathology developed with biofilm cells.     

Enhanced rhamnolipid production by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> under phosphate limitation

Rhamnolipid biosurfactants are effective antimicrobial agents and provide a promising alternative to synthetic medicine. Rhamnolipid accumulation by Pseudomonas aeruginosa ATCC 9027, and associated antimicrobial activity, was quantified during phosphate limited culture. The onset of rhamnolipid production occurred below 0.35 mg phosphate/l. Thereafter rhamnolipid accumulated during phosphate exhaustion where nitrogen remained above 0.9 g/l. A maximum 4.261 g rhamnolipid/l (measured as 1.333 g rhamnose/l) was attained at a productivity of 0.013 g rhamnose/l/h. Rhamnolipid accumulation under conditions of phosphate exhaustion and nitrogen excess suggests a non-specificity of the limiting nutrient, and that rhamnolipids will be synthesised provided carbon is in excess of the metabolic capacity. Antimicrobial activity was demonstrated against Mycobacterium aurum, a surrogate for M. tuberculosis, the causal agent of most forms of tuberculosis, by a 45 mm zone of M. aurum inhibition around a well of supernatant containing 3.954 g rhamnolipid/l.     

Antibiofilm activity of <Emphasis Type="Italic">Andrographis paniculata</Emphasis> against cystic fibrosis clinical isolate <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Respiratory tract and device associated infections caused by biofilm forming Pseudomonas aeruginosa play a primary role in the pathogenesis and prognosis of cystic fibrosis (CF) diseases. The biofilm formed by these pathogens attributes to the antibiotic resistance and protection from host immune response. Once established, the pathogens respond poorly to therapeutic agents. Recently medicinal plants are largely explored as potential source of bioactive agents. In this context the present study reports the antibiofilm activity of the folkloric medicinal plant Andrographis paniculata against biofilm forming CF causative Pseudomonas aeruginosa isolated from CF sputum. P. aeruginosa was also assessed for their growth and development of the biofilm, phylogenetic relationship and antibiotic susceptibility. Antibiogram of the strains indicated that they were resistant to more than one antibiotic. Six extracts of A. paniculata showed significant antibiofilm activity. P. aeruginosa strains, KMS P03 and KMS P05, were found to be maximally inhibited by the methanol extract to an extent of 88.6 and 87.5% respectively. This is the first report on antibiofilm activity of A. paniculata extracts, and our results indicate scope for development of complementary medicine for biofilm associated infections.     

Extracellular expression of keratinase Ker P from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> in <Emphasis Type="Italic">E. coli</Emphasis>

Keratinase from Pseudomonas aeruginosa KS-1 was expressed constitutively as an extracellular protein in Escherichia coli with high specific activity of 3.7 kU/mg. It was purified fourfold as a 33 kDa monomeric protein by Q-Sepharose ion exchange chromatography with a recovery of 95%. It is a serine protease with optimal activity at pH 9 and 50°C. It was stable from pH 4 to 12 for 1 h with a t_1/2 of 12 min at 70°C. It hydrolyzed haemoglobin > fibrin > feather keratin > azo-casein > casein > meat protein > gelatin. Among synthetic substrates, it efficiently hydrolyzed N-Suc-ala-ala-pro-phe-pNA, N-Suc-ala-ala-ala-pNA, N-Suc-ala-ala-pro-leu-pNA and also plasmin substrate, d-Val-Leu-Lys-pNA     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Expression and Functional Characterization of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Recombinant <Emphasis Type="SmallCaps">l</Emphasis>.Asparaginase

Recombinant _l.asparaginase, L.ASNase, from Pseudomonas aeruginosa was purified using nickel affinity chromatography. The affinity purified L.ASNase exhibited a protein band with a molecular weight of 72.4 kDa on a native polyacrylamide gel and 36.276 kDa using SDS–PAGE. The activity of the purified L.ASNase was enhanced by Mg²⁺ and inhibited by Zn²⁺ at a concentration of 5 mM. The specificity of the recombinant L.ASNase towards different substrates was examined, and it was found that the enzyme showed the highest activity towards l.asparagine. Moreover, the enzyme showed lower activity towards other substrates such as _L.glutamine, urea and acrylamide. The in vitro hemolysis assay revealed that the purified L.ASNase did not show hemolysis effect on blood erythrocytes. Serum and trypsin half-life of L.ASNase suggested that the recombinant L.ASNase retained 50% of its initial activity after 90 and 60 min incubation period in serum and trypsin separately.