Interaction between hyaluronate and the cell surface of Pseudomonas aeruginosa

Abstract Treatment of Pseudomonas aeruginosa cells with the non-metabolizable polysaccharide hyaluronate led to a strong increase in extracellular lipase activity. Alteration of the cell surface either by treatment with the chelator EDTA or by selecting for phage-resistant mutants significantly altered the bacterial response to hyaluronate. Binding of ¹⁴C-labeled hyaluronate to the bacteria was shown to depend on polysaccharide concentration and on cell number. Cell-free exolipase interacted with chemically cross-linked hyaluronate. The results suggested an interaction between hyaluronate and the cell surface of P. aeruginosa as a prerequisite for the polysaccharide to be effective.     

Leaching of Pseudomonas aeruginosa, and transconjugants containing pR68.45 through unsaturated, intact soil columns

Abstract Leaching of genetically engineered microbes (GEMs) through soil is a significant concern related to groundwater quality. The objective of this study was to examine the leaching, survival and gene transfer of a genetically engineered microbe and indigenous recipients of pR68.45 in nonsterile, undisturbed soil columns. Pseudomonas aeruginosa PAO25, containing the plasmid R68.45, was added to the surface of undisturbed soil columns (10 cm diameter × 80 cm length). Unsaturated flow conditions were maintained by 100 ml daily additions of 2 mM CaCl₂ for a period of 70 days. The population of the GEM exhibited a significant ( P = 0.05) linear decline with time. The GEM leached only to a depth of 30–40 cm in 70 days. Transfer of pR68.45 was shown to occur from P. aeruginosa into the indigenous bacterial population although relatively low numbers of transconjugants were observed (log 2 cfu g⁻¹ dry soil). The number of transconjugants also decreased with depth and time. Leaching of transconjugants, however, occured more readily than that of the GEM, probably as a result of plasmid transfer into smaller, more mobile bacteria. At 70 days incubation, no GEMs were detected in the columns, while transconjugants were observed at several depths. These results demonstrate the importance of examining both the survival and movement of GEMs and transconjugants in soil.     

Genome analysis of Pseudomonas aeruginosa by field inversion gel electrophoresis

Abstract The genome of Pseudomonas aeruginosa was analysed by digestion with rare-cutting restriction endonucleases and subsequent field inversion gel electrophoresis (FIGE). P. aeruginosa strain PAO and the 17 IATS strains were investigated. Each strain displayed a unique pattern of restriction fragments. Digestion with Dra I and Ssp I yielded, respectively 7–11 and 2–5 fragments of more than 130 kb in size, indicating the non-random occurrence of AT-rich sequences in the P. aeruginosa genome. The genome size of P. aeruginosa PAO was estimated to be (2.2 ± 0.3) × 10⁶ bp. The applications of DNA fingerprinting for gene cloning, construction of a physical chromosome map, and epidemiological studies, are discussed.     

Rapid distinction between Leptonema and Leptospira by PCR amplification of 16S-23S ribosomal DNA spacer

Abstract The protein-D2 porin of Pseudomonas aeruginosa is lacking in carbapenem or fluoroquinolone-resistant strains and hence was thought to facilitate the diffusion of these antibiotics. We examined the effect of several antibiotics on the single channel conductivity of protein-D2 in planar lipid bilayers and found that fluoroquinolones and carbapenems at concentrations of around 1 mM caused closure of the protein-D2 channel. Tetracycline, ampicillin, piperacillin, and latamoxef did not exert any detectable effect on the protein-D2 channel activity.     

Iron uptake in Pseudomonas aeruginosa mediated by N-(2,3-dihydroxybenzoyl)-L-serine and 2,3-dihydroxybenzoic acid

Abstract Pseudomonas aeruginosa is known to have an inducible uptake system for the enterobacterial siderophore enterobactin. In this work we have examined iron transport mediated by the biosynthetic precursor 2,3-dihydroxybenzoic acid and N -(2,3-dihydroxybenzoyl)- l -serine, a breakdown product of enterobactin. Iron complexed with 2,3-dihydroxybenzoyl-L-serine was transported into P. aeruginosa IA1 via a transport system which is energy-dependent and iron-repressible. The rate of transport was not altered by growing the cells in the presence of either pyoverdin or pyochelin, which have been shown previously to induce transport via that system. Growth of the cells in the presence of enterobactin did cause an increase in the rate of transport, indicating that the complex can be transported by the inducible enterobactin uptake system, but also that a separate system must exist. In contrast, transport of iron complexed with 2,3-dihydroxybenzoic acid was neither iron-repressible nor strongly energy-dependent, from which we conclude that there must be a novel mode of transport not characteristic of iron-siderophore transport systems.     

Pseudomonas aeruginosa cholinesterase and phosphorylcholine phosphatase: two enzymes contributing to corneal infection

Choline, acetylcholine and betaine used as the sole carbon, nitrogen or carbon and nitrogen source increase cholinesterase activity in addition to phosphorylcholine phosphatase and phospholipase C activities in Pseudomonas aeruginosa. The cholinesterase activity catalyses the hydrolysis of acetylthiocholine (Km approx. 0.13 mM) and propionylthiocholine (Km approx. 0.26 mM), but not butyrylthiocholine, which is a pure competitive inhibitor (Ki 0.05 mM). Increasing choline concentrations in the assay mixture decreased the affinity of cholinesterase for acetylthiocholine, but in all cases prevented inhibition raised by high substrate concentrations. Considering the properties of these enzymes, and the fact that in the corneal epithelium there exists a high acetylcholine concentration and that Pseudomonas aeruginosa produces corneal infection, it is proposed that these enzymes acting coordinately might contribute to the breakdown of the corneal epithelial membrane.     

Exploiting the antivirulence efficacy of an ajoene-ciprofloxacin combination against Pseudomonas aeruginosa biofilm associated murine acute pyelonephritis

The study investigated the in vitro, ex vivo and in vivo efficacy of ajoene and ciprofloxacin (CIP) alone and in combination against Pseudomonas aeruginosa biofilms and biofilm-associated murine acute pyelonephritis. The ajoene–CIP combination exhibited significant greater (p < 0.05) antimotility and biofilm inhibitory effects than those obtained when they were applied individually. The combined action of the agents resulted in a significant increase in serum sensitivity and phagocytic uptake and killing of P. aeruginosa (p < 0.001) compared to the untreated control. Mice groups treated with an ajoene (25 mg kg^?1) and CIP (30 mg kg^?1 or 15 mg kg^?1) combination showed a significantly (p < 0.001) reduced bacterial load in the kidney and bladder as compared to that of infected controls and mice treated with solo agents on the fifth day post-infection. The decreased levels of biomarkers and photomicrographs of the kidney tissue of the treated mice showed a reduced severity of damage. Hence, the study highlights the antivirulent and therapeutic efficacy of the ajoene-CIP combination at the minimal dosage of CIP.