Από 3 πρότυπα στελέχη Pseudomonas aeruginosa το Smooth στέλεχος PAC-IR και τα Rough στελέχη PAC-557 και PAC-605, που δώρησε ευγενώς στο Εργαστήριο Μικροβιολογίας του Πανεπιστημίου Πατρών η Dr. ΡΜ Meadow του University Co 1 lege, London παρελήφθησαν με ειδική μεθοδολογία ο λιποπολυσακχαρίτης L.P.S, η εξωκυττάρια ουσία (Slime) και οι εξωτερικές μεμβράνες. Έγιναν ηλεκτροφορήσεις με αποδιατακτικούς παράγοντες (SDS-PAGE) και χρώση των πηκτωμάτων για πρωτεΐνες με Coomassie blue, και για L.P.S με AgNÖ3. Οι ηλεκτροφορητικές εικόνες του Slime, του L.P.S και των εξωτερικών μεμβρανών διαφέρουν μεταξύ τους και στα 3 στελέχη. Ανάλυση των ουδετέρων σακχάρων έδειξε ότι και τα 3 Slime περί έχουν με ποσοτικές διαφορές 6 σάκχαρα, τη ραμνόζη, τη φουκόζη, τη ξυλόζη, τη μαννόζη και τη γαλακτόζη. Αντίθετα μαννόζη και γαλακτόζη δεν περιέχονται στο Smooth L.P.S, ενώ οι Rough L.P.Ss περί έχουν μόνο ραμνόζη και γλυκόζη. Ανάλυση του λιπιδικού στοιχείου των 3 Slime και των 3 L.P.Ss έδειξε ότι ποιοτικά περιέχουν 5 ίδια λιπαρά οξέα. Δωδεκανοικό οξύ (~Cii), 20Η-δωδεκανοικό (-Ci?), δεκατετρανικό οξύ (~Ci 4), δεκαεξανικό οξύ (~Cit), δεκαοκτανικό οξύ (-Ci s) και τα ισομερή του. Μετά από χρωματογραφία μοριακής διήθησης στα 3 Slime ο πολυσακχαρίτης που παραλαμβάνεται από την υδατανθρακική κορυφή δεν περιέχει L πρωτεΐνη και είναι μοριακού βάρους 40.000-100.000 daltons. Τα αποτελέσματα αυτά αποδεικνύουν ότι η παραγωγή του Slime είναι κοινή ιδιότητα Smooth και Rough στελεχών Ρ.aeruginosa και ότι το υδατανθρακικό στοιχείο του Slime είναι αυτοτελής ουσία, ελεύθερη πρωτεϊνών, που δεν αποτελείται από τις πλευρικές αλυσίδες του L.P.S. / Lipopolysaccharide (L.P.S), slime and outer membranes were obtained from a smooth, nonmucoid Pseudomonas aeruginosa strain, PAC-IR and its two rough mutants, PAC~ 557 and PAC-605 (Kindly provided by Dr. PM Meadow, University College, London). Electrophoretic analysis on SDS-polyacrylamide gels and staining with silver nitrate and Coomassie blue showed different profiles between L.P.S, Slime and outer membranes in all three strains. Comparative analysis of the saccharide moiety between L.P.S and Slime of each strain by H.P.L.C demonstrated that the saccharide moiety of slime has different composition from that of L.P.S. Six neutral sugars, rhamnose, fucose, xylose, mannose, galactose and glucose were identified in all three slimes though in different amounts. Mannose and galactose were not found in the smooth L.P.S, whereas only rhamnose and glucose were identified in the L.P.Ss of the rough strains. Comparative analysis of the lipid moiety between L.P.S and Slime in all three strains with Gas Chromatography and Mass Spectroscopy indicated that lipid moiety had no quaiitative differences concerning the lipid acids. Five lipid acids were identified in all three slimes and L.P.Ss dodecanoic acid (~Ci2)> 20H~dodecanoic acid (-C2?), tetradecanoic (-C14), exadecanoic (Ci(,), octadecanoic (- Cis) and its isomeric forms. After gel filtration of all three slimes the polysaccharide obtained from the carbohydrate peak fraction was found to be protein free. By gel filtration the mo 1 ecular size of the polysaccharide was estimated to be about 40000-100000 daltons. Whether the polysaccharide moiety is a glycolipid is not clear at the present. This problem is currently under investigation. Our results suggest that slime production is a common property shared by both Smooth and Hough Ρ.aeruginosa strains. The saccharide moiety of slime does not represent the side chains of L.P.S and it is protein free.

Pseudomonas chlororaphis (aureofaciens) 30-84 is a biocontrol bacterium effective against take-all disease of wheat. Phenazine (PZ) production by strain 30-84 is the primary mechanism responsible for pathogen inhibition and the rhizosphere persistence of 30-84. The PhzR/PhzI system of strain 30-84 directly regulates PZ production and mutations in this QS system are defective in biofilm formation. Genetic complementation or direct addition of AHL signal restored biofilm formation to a phzI mutant. Mutations in PZ biosynthesis were equally defective in biofilm formation. Addition of PZ or genetic complementation of the PZ biosynthetic mutation restored biofilm formation. QS and PZ production also were involved in the establishment of populations on wheat seeds and plant roots. Presence of 10% wild type strain 30-84 in mixtures with QS or PZ mutants restored root colonization. These data demonstrate that QS and specifically PZ production are essential for biofilm formation by strain 30-84. This is a new role for PZs in the rhizosphere community.Strain 30-84 produces primarily phenazine-1-carboxylic acid (PCA) and 2-hydroxy-PCA (2-OH-PCA). We generated derivatives of strain 30-84 that produced the same total amount of PZs as the wild type but produced only PCA, or more efficiently converted PCA to 2-OH-PCA. These derivatives with altered PZ ratios differed from the wild type in initial attachment, biofilm architecture, and dispersal. Increased 2-OH-PCA production increased initial attachment, although both alterations resulted in thicker biofilms and reduced dispersal rates. Loss of 2-OH-PCA production resulted in a significant reduction in pathogen inhibition. My findings indicate that alterations in the endogenous ratios of PZs have wide-ranging effects on the biology of strain 30-84. I initiated studies to understand the mechanisms by which PZs affect surface attachment and biofilm development. Addition of PZs to metabolically inactivated cells improved adhesion compared to the inactive cells alone, suggesting that PZs may improve initial binding to surfaces. Results from whole genome transcription profiles of wild type strain 30-84 to a PZ mutant indicate that genes potentially involved in biofilm formation were up-regulated in the presence of PZs. These results provide initial evidence that PZs may modulate cell adhesion and biofilm formation via multiple mechanisms.

Cystic Fibrosis is one of the most common genetic recessive diseases among Caucasians and is caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene on chromosome 7. There are different classes of CFTR mutation, leading to differences in disease severity among patients. In addition to the CFTR genotype, secondary genetic factors, modifier genes, also influence CF phenotypes. Due to the dysfunction of CFTR protein and production of thickened mucus, bacterial infection in the lungs is favored and can lead to further clinical complications in CF patients. Pseudomonas aeruginosa is one of the most common bacteria detected among patients. The aim of this project was to investigate four candidate modifier genes, Factor B, Complement Factor 3, Toll-like Receptor 4 and Heme oxygenase-1, which might affect the status of Pseudomonas aeruginosa infection. A total of 22 single nucleotide polymorphisms (SNPs) were selected in these four genes and they were tested against five phenotypic traits, including age of diagnosis, FEV1% predicted andstandard deviation value, age of first Pseudomonas aeruginosa infection and Pseudomonas aeruginosa infection status. Among the selected SNPs, both case-control studies and family-based analysis were performed in order to establish any correlation between the genotypes and the phenotypes. In addition, haplotype analysis was performed to determine whether there was interaction between SNPs or whether there were unidentified SNPs in the vicinity of the selected ones that might contribute to the observed phenotypic traits. Among the 22 chosen SNPs, 13 of them were found to be significantly linked to one or more of the tested phenotypes. The three most significant associations were BF_2557 with lung function, HMOX1_9531 with lung function and BF_7202 with age of diagnosis. Several haplotypes were significantly associated with one of the five phenotypes. There was no evidence for the presence of unidentified SNPs or interaction between SNPs. Most of haplotype associations were likely due to the presence of a single SNP which was found to be significantly linked to the phenotype. Conclusively, both SNPs and haplotype analyses suggest that the four candidate genes are modifiers of disease severity in CF.

This thesis is an investigation of Pseudomonas aeruginosa bacterial biofilms. The objective of the first study was to evaluate the biofilm-forming capacity of canine otitis isolates of P. aeruginosa and to compare the minimum inhibitory concentrations (MICs) of antimicrobials for planktonic versus biofilm-embedded bacteria. Biofilm forming ability was assessed using a microtitre plate assay. Broth microdilution was used to assess the MICs of neomycin, polymyxin B, enrofloxacin and gentamicin for the planktonic and biofilm-embedded bacteria of eighty-three isolates. Thirty-three (40%) isolates were biofilm producers and MICs for biofilm-embedded bacteria were significantly higher than their planktonic counterparts for all antimicrobials (all P<0.05). The objective of the second study was to evaluate the impact of Tromethamine edetate disodium dihydrate (Triz-EDTA®) in combination with antimicrobials on antimicrobial susceptibility of P. aeruginosa biofilm-embedded bacteria. MICs of the four antimicrobials for the biofilm embedded bacteria and biofilm-embedded bacteria with added Triz-EDTA® were assessed with broth microdilution for thirty-one biofilm-producing isolates. Addition of Triz-EDTA® significantly reduced MICs for neomycin (P < 0.008) and gentamicin (P < 0.04) but not enrofloxacin (P = 0.7), or polymyxin B (P = 0.5). The objective of the third study was to determine the presence of biofilm-associated genes in biofilm forming and non-biofilm forming isolates. Four genes involved with carbohydrate matrix production (pelA), irreversible attachment (sadB) and quorum sensing (lasB, rhlA) were selected. DNA was extracted and polymerase chain reaction (PCR) was performed for all isolates. All isolates possessed lasB and sadB, 74 (90%) possessed pelA and 74 (90%) possessed rhlA. All thirty-two (100%) isolates that were classified as biofilm producers contained all genes. There was an association between the presence of pelA and rhlA and biofilm production (P < 0.017) and between the presence of rhlA and pelA and the quantity of biofilm produced (both P < 0.001). These results highlight that biofilm formation of Pseudomonas aeruginosa otic isolates does occur and can impact antimicrobial therapy. Certain compounds can also influence antimicrobial susceptibility of biofilm-embedded bacteria. Genetics may also play a role in biofilm formation.

Pseudomonas syringae delivers a plethora of effector proteins into host cells to sabotage host immune responses and physiology to favor infection. We have previously shown that P. syringae pv. tomato DC3000 effector HopF2 suppresses Arabidopsis innate immunity triggered by multiple pathogen-associated molecular patterns (PAMP) at the plasma membrane. We show here that HopF2 possesses distinct mechanisms in the suppression of two branches of PAMP-activated MAP kinase cascades. In contrast to blocking MKK5 in MEKK1-MKK4/5-MPK3/6 cascade, HopF2 targets additional component(s) upstream of MEKK1 in MEKK1-MKK1/2-MPK4 cascade and plasma membrane-localized receptor-like cytoplasmic kinase BIK1 and its homologs. We further show that HopF2 directly targets BAK1, a plasma membrane-localized receptor-like kinase involved in multiple PAMP signaling. The interaction between BAK1 and HopF2 or two additional P. syringae effectors AvrPto and AvrPtoB, was confirmed in vivo and in vitro. Consistent with BAK1 as a physiological target of HopF2, the lethality of overexpression of HopF2 in wild-type Arabidopsis transgenic plants was largely alleviated in bak1 mutant plants. Identification of BAK1 as an additional HopF2 virulence target not only explains HopF2 suppression of multiple PAMP signaling at the plasma membrane, but also supports the notion that pathogen virulence effectors have multiple targets in host cells.

Expression of the mexCD-oprJ multidrug efflux operon is enhanced by the presence of membrane damaging agents [e.g., the biocide chlorhexidine (Chx)] or mutations in the nfxB gene encoding a repressor of efflux gene expression, both dependent on the AlgU envelope stress response sigma factor. Details of mexCD-oprJ regulation are, however, lacking. In examining the mexCD-oprJ locus, a gene, PA4596, encoding a homologue of NfxB (61% identity) was identified downstream of oprJ, a location conserved in all sequenced Pseudomonas aeruginosa isolates and in Pseudomonas putida. Opposite to mexCD-oprJ, PA4596 expression was reduced by Chx exposure, as assessed using RT-PCR; although like mexCD-oprJ, this was AlgU-dependent (i.e., lost in a ΔalgU strain). Deletion of PA4596 had no impact on Chx resistance indicating that it is not required for Chx-inducible mexCD-oprJ expression/ MexCD-OprJ-dependent Chx resistance. In contrast, mexCD-oprJ expression and the attendant multidrug resistance of nfxB deletion mutants were compromised upon deletion of PA4596, indicating that PA4596 plays a positive role in mexCD-oprJ expression in such mutants. Consistent with this, PA4596 expression increased in nfxB deletion and missense mutants in parallel with mexCD-oprJ. Intriguingly, mexCD-oprJ expression and multidrug resistance were observed in a mutant lacking an nfxB mutation (demonstrating an NfxB-like phenotype) and in an nfxB missense mutant and these were not compromised upon deletion of PA4596. Thus, mexCD-oprJ hyperexpression can be both PA4596-dependent and -independent. A bacterial 2-hybrid assay revealed a PA4596-PA4596 interaction, consistent with the protein forming dimers as NfxB has been shown to do. Two-hybrid assays also demonstrated that NfxB and PA4596 interact. While the functional significance of this remains to be elucidated, it is consistent with their common role in regulating mexCD-oprJ expression and is suggestive of a complex and possibly novel regulatory mechanism. These data highlight the complexity of mexCD-oprJ regulation and the apparently multiple pathways to efflux gene expression, suggestive of multiple roles for this efflux system in P. aeruginosa independent of antimicrobial efflux. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2009-08-18 14:25:18.107

Pseudomonas aeruginosa is a common soil organism as well as an opportunistic human pathogen. Treatment of P. aeruginosa infections is often complicated by innate resistance to a variety of antimicrobials mediated by multidrug efflux systems. The MexAB-OprM efflux system is constitutively expressed in wildtype strains and contributes to innate antimicrobial resistance, while hyperexpression of the system results in acquired high levels of resistance. MexAB-OprM is hyperexpressed in nalC mutants containing mutations in the gene encoding NalC, a repressor of a two-gene operon, PA3720-armR. armR encodes a protein modulator of MexR, a repressor of mexAB-oprM expression. Previous reports showed that genes encoding the MexAB-OprM efflux system are upregulated in response to pentachlorophenol (PCP), a phenolic compound that is a common environmental contaminant. Induction of mexAB-oprM and PA3720-armR by PCP was confirmed using RT-PCR, and MexAB-OprM was shown to be involved in PCP resistance. An electromobility shift assay (EMSA) showed that PCP interacts with NalC, interfering with its binding to the PA3720-armR promoter region and thereby promoting PA3720-armR expression. Nonetheless, the increase in ArmR did not drive mexAB-oprM expression suggesting that PCP induction of this efflux operon occurred via a different mechanism. A direct PCP-MexR interaction could not be demonstrated using an EMSA. PCP exposure did, however, reduce expression of nalD, encoding a second repressor of mexAB-oprM, which might explain the PCP-promoted increase in mexAB-oprM expression. PCP is unlikely to be the intended inducer(s)/substrate(s) for this system but probably resembles these. Several compounds related to PCP were tested for an interaction with NalC but all were negative in EMSAs. Plants produce a variety of phenolic compounds, which are often antimicrobial and, so, root extracts of various plants were tested for an ability to interact with NalC and interfere with promoter binding. Extracts from Boehmeria tricuspis, Uncaria tomentosa and Ixiolirion tataricum were shown to interact with NalC, suggesting that plant compounds may be the intended inducers/substrates for NalC/MexAB-OprM. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2010-09-10 10:35:16.271

No description available.

The adhesion of seven meat spoilage bacteria to model meat surfaces (thin fat and tendon slices) was studied in a specially designed flow chamber. In general, adhesion was not influenced by the physiological age of the cells and was not correlated with the cell surface characteristics (electrical charge, hydrophobicity) of the organisms. Also, adhesion data did not corroborate the predictions based on changes in the free energy of adhesion, calculated from contact angles and surface tension measurements. A more detailed study of the adhesion of Pseudomonas fluorescens to tendon was subsequently undertaken. Neither physiological activity nor the presence of flagella was found to be essential for adhesion. Selective chemical alterations of the cell surface pointed to no direct implication of carboxyl or amino groups in an adhesive bond with tendon. These groups may participate in adhesion, however, through electrostatic interactions as suggested from the variations of adhesion with ionic strength.

No description available.