<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Cystic Fibrosis isolates of similar RAPD genotype exhibit diversity in biofilm forming ability <Emphasis Type="Italic">in vitro</Emphasis>

Background  

Pseudomonas aeruginosa is considered to grow in a biofilm in cystic fibrosis (CF) chronic lung infections. Bacterial cell motility is one of the main factors that have been connected with P. aeruginosa adherence to both biotic and abiotic surfaces. In this investigation, we employed molecular and microscopic methods to determine the presence or absence of motility in P. aeruginosa CF isolates, and statistically correlated this with their biofilm forming ability in vitro.     

Cloning,expression, purification,and characterization of recombinant flagellin isolated from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Pseudomonas aeruginosa as an opportunistic pathogen causes lethal infections in immunocompromised individuals. This bacterium possesses a polar flagellum made up of flagellin subunits. Flagella have important roles in motility, chemotaxis, and establishment of P. aeruginosa in acute phase of infections. Isolation, cloning, and expression of flagellin were aimed at in this study. Flagellin gene (fliC) of P. aeruginosa strain 8821M was isolated by PCR and cloned into a pET expression vector. The recombinant flagellin (46 kDa) was overexpressed as inclusion bodies (IBs). IBs were solubilized in guanidine hydrochloride (GuHCl) followed by affinity-purification and renatured using Ni²⁺-Sepharose resin. Recombinant flagellins reacted with the serum from a rabbit previously immunized with native flagellin. In addition, polyclonal antiserum raised against the recombinant flagellin was shown to significantly inhibit the cell motility of P. aeruginosa strain 8821M in vitro.     

Iron dictates the virulence of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> in urinary tract infections

Iron-limiting conditions have been reported to be prevalent in the milieu of urinary tract. In the present investigation, effect of iron on virulence of uropathogenic Pseudomonas aeruginosa in planktonic and biofilm cell mode was studied. Significant enhancement in elaboration of all the virulence traits along with increased adherence to uroepithelial cells and decreased phagocytosis of P. aeruginosa was observed following growth in iron-deplete medium. On the contrary, decrease in all these parameters except phagocytosis was observed when P. aeruginosa was grown in iron-rich medium. In vivo, P. aeruginosa grown in iron-deplete medium showed increased renal bacterial load and tissue pathology in a mouse model of ascending urinary tract infection compared with organisms grown in iron-replete medium. The results of the present study may help in understanding host–parasite interaction and in developing alternative preventive approach against P. aeruginosa induced urinary tract infections.     

Rhamnolipid-producing thermophilic bacteria of species <Emphasis Type="Italic">Thermus</Emphasis> and <Emphasis Type="Italic">Meiothermus</Emphasis>

Novel rhamnolipid-producing strains of three thermophilic bacteria, Thermus sp., T. aquaticus and Meiothermus ruber were identified that have not been previously described as rhamnolipid producers. Rhamnolipids were extracted from supernatant and further purified by thin-layer chromatography. Mass spectrometry with negative electrospray ionization revealed 77 rhamnolipid homologues varying in chain length and unsaturation. Tandem mass spectrometry identified mono-rhamnolipid and di-rhamnolipid homologues containing one or two 3-hydroxy-fatty acids, saturated, monounsaturated or diunsaturated, even- or odd-chain, up to unusual long chains with 24 carbon atoms. The stereochemistry of rhamnose was L and that of 3-hydroxy-fatty acids was R, the position of double bonds in monoenoic acids was cis ω-9. All three strains produced a rhamnolipid that differs in structure from Pseudomonas aeruginosa rhamnolipids and exhibits excellent surfactant properties. Importantly, in comparison to P. aeruginosa both strains, i.e., Thermus and Meiothermus, are Biosafety level 1 microorganisms and are not pathogenic to humans.     

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.     

A three-phase <Emphasis Type="Italic">in-vitro</Emphasis> system for studying <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> adhesion and biofilm formation upon hydrogel contact lenses

Background  

Pseudomonas aeruginosa is commonly associated with contact lens (CL) -related eye infections, for which bacterial adhesion and biofilm formation upon hydrogel CLs is a specific risk factor. Whilst P. aeruginosa has been widely used as a model organism for initial biofilm formation on CLs, in-vitro models that closely reproduce in-vivo conditions have rarely been presented.     

<Emphasis Type="Italic">Staphylococcus aureus</Emphasis> sigma B-dependent emergence of small-colony variants and biofilm production following exposure to <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> 4-hydroxy-2-heptylquinoline-<Emphasis Type="Italic">N-</Emphasis> oxide

Background  

Staphylococcus aureus and Pseudomonas aeruginosa are often found together in the airways of cystic fibrosis (CF) patients. It was previously shown that the P. aeruginosa exoproduct 4-hydroxy-2-heptylquinoline-N-oxide (HQNO) suppresses the growth of S. aureus and provokes the emergence of small-colony variants (SCVs). The presence of S. aureus SCVs as well as biofilms have both been associated with chronic infections in CF.     

Synthesis and biotransformation of 2-alkyl-4(1<Emphasis Type="Italic">H</Emphasis>)-quinolones by recombinant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> KT2440

2-Alkyl-4(1H)-quinolones (AQs) and related derivatives, which exhibit a variety of biological properties, are secondary metabolites produced by, e.g., Pseudomonas and Burkholderia spp. Due to their main role as signaling molecules in the quorum sensing system of Pseudomonas aeruginosa, 2-heptyl-4(1H)-quinolone (HHQ) and its 3-hydroxy derivative, termed the “Pseudomonas quinolone signal” (PQS), have received considerable attention. Since chemical synthesis of different AQs is complex, we assessed the applicability of recombinant P. putida KT2440 strains for the biosynthetic production of AQs. In mineral salts medium supplemented with octanoate and anthranilate, batch cultures of P. putida KT2440 [pBBR-pqsABCD] produced about 45 μM HHQ, 30% and 70% of which were localized in the culture supernatant and methanolic cell extract, respectively. 2,4-Dihydroxyquinoline and minor amounts of C₃- to C₁₃-saturated and C_7:1 to C_13:1 monounsaturated AQs were formed as by-products. Mass spectrometry and nuclear magnetic resonance analyses spectroscopy indicated that unsaturated AQs having the same molecular mass are cis and trans isomers rather than position isomers, with the double bond located between the α and β carbon of the alkyl chain. Supplementing the cultures with hexanoate instead of octanoate shifted the AQ profile towards increased formation of C₅-AQ. Individual AQs can be prepared from concentrated methanolic extracts by preparative high-performance liquid chromatography (HPLC). Regioselective hydroxylation of HHQ to PQS can be achieved in >90% yield by biotransformation with P. putida KT2440 [pBBR-pqsH]. PQS can be isolated from methanolic cell extracts by HPLC, or be precipitated as Fe(III)-PQS complex. Preparation of a library of AQs will facilitate studies on the biological functions of these compounds.     

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.     

Widespread Emergence of Multidrug Resistant <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Isolated from CSF Samples

Bacterial infections of the central nervous system, especially acute infections such as bacterial meningitis require immediate, invariably empiric antibiotic therapy due to the widespread emergence of resistance among bacterial species. Nosocomial infections by Pseudomonas aeruginosa have been described with an increasing trend towards multidrug resistance. P. aeruginosa isolates n = 53 (66%) isolated from the cerebrospinal fluid (CSF) were used for this study. Antibiotic resistance in 53 P. aeruginosa clinical isolates from 80 CSF samples were evaluated. Of these, n = 42 (80%) of the isolates showed multidrug resistance to more than eight antibiotics and n = 17 (32%) isolates were found to be imipenem resistant P. aeruginosa (IMPR-Pa). Genotypical examination by ERIC based PCR revealed minor genetic variations. Polymicrobial infections are common in the CSF samples. However, high prevalence of P. aeruginosa as an opportunistic pathogen has been developing with increased resistance to antimicrobial agents and thus becoming a significant threat.     

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.     

Co-inoculation of Urea and DAP Tolerant <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> and <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> as Integrated Approach for Growth Enhancement of <Emphasis Type="Italic">Brassica juncea</Emphasis>

Two plant growth promoting rhizobacteria––Sinorhizobium meliloti RMP1 and Pseudomonas aeruginosa GRC₂ were studied for integrated nutrient management to obtain improved yield of Brassica juncea. Low concentrations of urea and diammonium phosphate (DAP) stimulated the growth of both S. meliloti RMP1 and P. aeruginosa GRC₂. 1 M of urea and 0.35 M of DAP was found lethal for RMP1, while 1.3 M and 0.37 M concentrations of urea and DAP proved to be toxic for GRC₂. Lc₅₀ was observed as 0.49 M of urea and 0.15 M of DAP for RMP1, and 0.66 M urea and 0.18 M of DAP for GRC₂. Urea and DAP adaptive variants of RMP1 and GRC₂ was isolated. Adaptive bacterial variants had better growth rates at sub-lethal (Lc₅₀) concentrations of urea and DAP as compared to non-adaptive variants. They also retained plant growth promoting attributes similar to non adaptive variants. GRC₂ and RMP1 did not affect the growth of each other and were chemotactically active for DAP, urea as well as root exudates of B. juncea. Both the isolates colonized well in the rhizosphere of B. juncea, as their populations were recorded ≈5 log₁₀ cfu g⁻¹ after 120 days. Interestingly, the colonization ability was found even better when both strains were co-inoculated, as their population was recorded in the range of ≈6 log₁₀ cfu g⁻¹ after 120 days. In field trials, application of RMP1 and GRC₂ resulted in significant increase in biomass and yield of B. juncea as compared to control. However, yield was better with application of half dose and full dose of recommended fertilizers. Interestingly, the biomass as well as yield improved further when both isolates were applied together along with half dose of recommended fertilizers.     

Combined field efficacy of <Emphasis Type="Italic">Paranosema locustae</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> var. <Emphasis Type="Italic">acridum</Emphasis> for the control of sahelian grasshoppers

Field trials were conducted to evaluate the efficacy of wheat bran bait formulations of Paranosema locustae and Metarhizium anisopliae for controlling grasshoppers in southeast Niger. Treatments consisted of wheat bran baits mixed with M. anisopliae, P. locustae + M. anisopliae or with P. locustae spores and P. locustae + sugar. Oedaleus senegalensis, Pyrgomorpha cognata and Acrotylus blondeli were the predominant species at the time of application representing ca. 94% of the total population. Bran application was done when O. senegalensis (ca. 75% of the population) was at its early developmental stages, with first, second and third instars accounting for 64–85%. Grasshopper population reduction, P. locustae prevalence and level of infections in the predominant species were monitored. Manual application of P. locustae and M. anisopliae formulated in wheat bran has proven to induce consistent pathogen infection in grasshopper populations. Population density over the three weeks monitoring, typically decreased by 44.7 ± 6.9%, 52.8 ± 8.4%, 73.7 ± 5.5% and 89.1 ± 1.8% in P. locustae, P. locustae + sugar, M. anisopliae and P. locustae + M. anisopliae treated plots respectively. Paranosema locustae prevalence in surviving adult grasshoppers at 28 after application was 48.1 ± 2.3%, 28.9 ± 4.8% and 27.4 ± 3.7%, with infection level of 6.2 ± 0.8 × 10⁶, 2.3 ± 0.3 × 10⁴ and 2.1 ± 0.3 × 10³ spores mg⁻¹ host weight in O. senegalensis, A blondeli and P. cognate respectively. Other species that each accounted for <2% of the community, namely Aiolopus thalassinus, A. simulatrix, Acorypha glaucopsis, Acrotylus patruelis, Anacridium melanorhodon, Diabolocatantops axillaris, Kraussaria angulifera and Schistocerca gregaria were found to show sign of infection. The results from this study suggest that wheat bran application of M. anisopliae and P. locustae alone or in combination, targeting early instars grasshopper could be a valuable option in grasshopper control programs.     

Anti-cyanobacterial activity of <Emphasis Type="Italic">Moringa oleifera</Emphasis> seeds

Filtrates from crushed Moringa oleifera seeds were tested for their effects on growth and Photosystem II efficiency of the common bloom-forming cyanobacterium Microcystis aeruginosa. M. aeruginosa populations exhibited good growth in controls and treatments with 4- and 8-mg crushed Moringa seeds per liter, having similar growth rates of 0.50 (±0.01) per day. In exposures of 20- to 160-mg crushed Moringa seeds L⁻¹, growth rates were negative and on average −0.23 (±0.05) .day⁻¹. Presumably, in the higher doses of 20- to 160-mg crushed seeds per liter, the cyanobacteria died, which was supported by a rapid drop in the Photosystem II efficiency (Φ_PSII), while the Φ_PSII was high and unaffected in 0, 4, and 8 mg L⁻¹. High-density populations of M. aeruginosa (chlorophyll-a concentrations of ∼270 μg L⁻¹) were reduced to very low levels within 2 weeks of exposure to ≥80-mg crushed seeds per liter. At the highest dosage of 160 mg L⁻¹, the Φ_PSII dropped to zero rapidly and remained nil during the course of the experiment (14 days). Hence, under laboratory conditions, a complete wipeout of the bloom could be achieved. This is the first study that yielded evidence for cyanobactericidal activity of filtrate from crushed Moringa seeds, suggesting that Moringa seed extracts might have a potential as an effect-oriented measure lessening cyanobacterial nuisance.     

Phosphorus cycling between the colonial cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> and attached bacteria, <Emphasis Type="Italic">Pseudomonas</Emphasis>

Phosphorus (P) transfer between Microcystis aeruginosa and the attached bacterium Pseudomonas was studied using radioactive P (³²P) and green fluorescence protein-labeled Pseudomonas. M. aeruginosa in P-starved condition took up most ³²P (70%) in water and about 50% of ³²P in ³²P-saturated bacteria in individual experiments. However, only 26% of ³²P in the ³²P-saturated M. aeruginosa was transferred to P-starved bacteria. The P-starved M. aeruginosa had an advantage to take up P over the bacteria and its growth rates and abundance were higher in combined cultures, with bacteria as the biotic P source. The rate of P transfer from bacteria to the cyanobacteria was slow. P cycles predominantly between M. aeruginosa and Pseudomonas with little variation in the water. This ability is very useful for the colony-forming M. aeruginosa, especially if phosphate concentrations in water are low during water bloom periods.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of a high value medicinal plant: <Emphasis Type="Italic">Asparagus racemosus</Emphasis> Willd.

Asparagus racemosus Willd. is an important medicinal plant of tropical and subtropical India. Its medicinal usage has been reported in the Indian and British Pharmacopoeias and in traditional systems of medicine such as Ayurveda, Unani, and Siddha. The multiple uses of this species have increased its commercial demand, resulting in over-exploitation. Because of destructive harvesting, the natural population of A. racemosus is rapidly disappearing, and it is recognized as ‘vulnerable’ (Warner et al., Some important medicinal plants of the Western Ghats, India: a profile. International Development Research Centre, Artstock, New Delhi, India, 15 pp, 2001). The development of an efficient micropropagation protocol will play a significant role in meeting the requirements for commercial cultivation, thereby conserving the species in its natural habitat. In the present study, in vitro shoot proliferation was obtained by culturing single node segments in Murashige and Skoog’s (MS) medium supplemented with 3.69 μM 2-isopentyl adenine and 3% sucrose with a multiplication rate of 3.5. For proper root formation, the in vitro-formed shoot clusters were cultured on half strength (major salts reduced to half) MS medium with 1.61 μM 1-naphthalene acetic acid, 0.46 μM kinetin, 98.91 μM adenine sulfate, 500 mg/l malt extract, 198.25 μM phloroglucinol, and 3% sucrose. On this medium, 85% rooting was observed within 20 d. Following a simple hardening procedure involving sequential transfer of plants to a greenhouse, polyhouse, and shade net, the tissue-cultured plants were transferred to the field where the survival rate was 100%.     

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.