Sub-inhibitory concentrations of ceftazidime and tobramycin reduce the quorum sensing signals of Pseudomonas aeruginosa

Aim: Concentrations of antimicrobials below minimum inhibitory concentration (subMIC) may reduce the production by Pseudomonas aeruginosa of virulence factors such as elastase. We sought to determine whether the reduction in elastase production may be mediated by a reduction in acyl-homoserine lactones. Methods: Pseudomonas aeruginosa in broth was exposed to three conditions for ceftazidime and tobramycin: control, 6% MIC and 25% MIC. Elastase was assayed using elastin congo red. N-(3-Oxododecanoyl)-homoserine lactone (C12-HSL) and N-butyryl-homoserine lactone (C4-HSL) were assayed using biosensor Escherichia coli. Results: Elastase was unchanged with ceftazidime. Elastase was reduced by 16% at 6% MIC tobramycin and reduced by 70% at 25% MIC tobramycin (P

Pseudomonas aeruginosa fimL regulates multiple virulence functions by intersecting with Vfr-modulated pathways

Virulence of Pseudomonas aeruginosa involves the co-ordinate expression of a range of factors including type IV pili (tfp), the type III secretion system (TTSS) and quorum sensing. Tfp are required for twitching motility, efficient biofilm formation, and for adhesion and type III secretion (TTS)-mediated damage to mammalian cells. We describe a novel gene (fimL) that is required for tfp biogenesis and function, for TTS and for normal biofilm development in P. aeruginosa. The predicted product of fimL is homologous to the N-terminal domain of ChpA, except that its putative histidine and threonine phosphotransfer sites have been replaced with glutamine. fimL mutants resemble vfr mutants in many aspects including increased autolysis, reduced levels of surface-assembled tfp and diminished production of type III secreted effectors. Expression of vfr in trans can complement fimL mutants. vfr transcription and production is reduced in fimL mutants whereas cAMP levels are unaffected. Deletion and insertion mutants of fimL frequently revert to wild-type phenotypes suggesting that an extragenic suppressor mutation is able to overcome the loss of fimL. vfr transcription and production, as well as cAMP levels, are elevated in these revertants, while Pseudomonas quinolone signal (PQS) production is reduced. These results suggest that the site(s) of spontaneous mutation is in a gene(s) which lies upstream of vfr transcription, cAMP, production, and PQS synthesis. Our studies indicate that Vfr and FimL are components of intersecting pathways that control twitching motility, TTSS and autolysis in P. aeruginosa.

Aerosol dispersal of the fish pathogen, Amyloodinium ocellatum

Amyloodinium ocellatum, a frequently encountered parasite in marine aquaculture, was investigated to determine if infective dinospore stages could be transported in aerosol droplets. We used an in vivo model incorporating static and dynamic airflow systems and found dinospores of A. ocellatum could travel in aerosol droplets (up to 440 turn in a static system and up to 3 m in a dynamic one). This is the first record of this transmission pathway for a marine protozoan parasite. It is possible that other marine protozoans can transfer via the aerobiological pathway. Management of A. ocellatum infections in aquaculture facilities could be affected, particularly where tanks and ponds are situated in close proximity. (c) 2006 Elsevier B.V. All rights reserved.

Flagellin of Pseudomonas aeruginosa inhibits Na+ transport in airway epithelia

Pseudomonas aeruginosa causes severe life-threatening airway infections that are a frequent cause for hospitalization of cystic fibrosis (CF) patients. These Gram-negative pathogens possess flagella that contain the protein flagellin as a major structural component. Flagellin binds to the host cell glycolipid asialoGM1 (ASGM1), which appears enriched in luminal membranes of respiratory epithelial cells. We demonstrate that in mouse airways, luminal exposure to flagellin leads to inhibition of Na+ absorption by the epithelial Na+ channel ENaC, but does not directly induce a secretory response. Inhibition of ENaC was observed in tracheas of wild-type mice and was attenuated in mice homozygous for the frequent cystic fibrosis conductance regulator (CFTR) mutation G551D. Similar to flagellin, anti-ASGM1 antibody also inhibited ENaC. The inhibitory effects of flagellin on ENaC were attenuated by blockers of the purinergic signaling pathway, although an increase in the intracellular Ca2+ concentration by recombinant or purified flagellin or whole flagella was not observed. Because an inhibitor of the mitogen-activated protein kinase (MAPK) pathway also attenuated the effects of flagellin on Na+ absorption, we conclude that flagellin exclusively inhibits ENaC, probably due to release of ATP and activation of purinergic receptors of the P2Y subtype. Stimulation of these receptors activates the MAPK pathway, thereby leading to inhibition of ENaC. Thus, P. aeruginosa reduces Na+ absorption, which could enhance local mucociliary clearance, a mechanism that seem to be attenuated in CF.

In vitro antibacterial activity of Australian native herb extracts against food-related bacteria

The antibacterial activities of water, ethanol and hexane extracts of five Australian herbs (Backhousia citriodora, Anetholea anisata, Eucalyptus staigerana, Eu. olida and Prostanthera incisa) against seven food-related bacteria (Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella Enteritidis, Sal. Typhimurium and Staphylococcus aureus) were determined by the microtitre broth microdilution assay. The water extracts of all the herbs displayed no or low antimicrobial activity against all of the bacteria tested with the exception of S. aureus. Relatively high levels of activity (minimum inhibitory concentrations of 125-15.6 mu g ml(-1)) against this pathogen were present in water extracts from all herbs except P. incisa. The ethanol and hexane extracts of all herbs displayed some activity against a number of the bacteria tested, with no one particular herb displaying an obviously higher level or range of activity. Staphylococcus aureus proved to be the most sensitive of the bacteria tested against the solvent extracts with all extracts displaying activity ranging from 125 to 7.8 mu g ml(-1), while E. coli and L. monocytogenes, on the other hand, proved the least sensitive with only five of 15 herb/extract combinations displaying any activity against these pathogens. The extracts of the Australian native herbs examined in this study have potential for application in foods to increase shelf-life or promote safety. (c) 2005 Elsevier Ltd. All rights reserved.

Molecular characterization of the Escherichia coli asymptomatic bacteriuria strain 83972: The taming of a pathogen

Escherichia coli 83972 is a clinical asymptomatia bacteriuric isolate that is able to colonize the human urinary bladder without inducing an immune response. Here we demonstrate that one of the mechanisms by which this strain has become attenuated is through the mutation of its genes encoding type I and P fimbriae.

Salicylic acid mediates resistance to the vascular wilt pathogen Fusarium oxysporum in the model host Arabidopsis thaliana

Fusarium oxysporum is a soilborne fungal pathogen that causes major economic losses by inducing necrosis and wilting symptoms in many crop plants. In this study, the interaction between F. oxysporum and the model plant Arabidopsis thaliana has been investigated to better understand the nature of host defences that are effective against the Fusarium wilt pathogen. The expression of salicylate- and jasmonate-responsive defence genes in F. oxysporum-challenged roots of A. thaliana plants as well as in the roots of plants whose leaves were treated with salicylate or jasmonate was analysed. Unexpectedly, genes (e.g. PR1, PDF1.2, and CHIB) encoding proteins with defensive functions or transcription factors (e.g. ERF1, AtERF2, AtERF4 and AtMYC2) known to positively or negatively regulate defences against F. oxysporum were not activated in F. oxysporum-inoculated roots. In contrast, the jasmonate-responsive defence gene PDF1.2 was induced in the leaves of plants whose roots were challenged with F. oxysporum, but the salicylate- responsive PR1 gene was not induced in the leaves of inoculated plants. Exogenous salicylic acid treatment prior to inoculation, however, activated PR1 and BGL2 defence gene expression in leaves and provided increased F. oxysporum resistance as evidenced by reduced foliar necrosis and plant death. Exogenous salicylic acid treatment of the foliar tissue did not activate defence gene expression in the roots of plants. This suggests that salicylate- dependent defences may function in foliar tissue to reduce the development of pathogen-induced wilting and necrosis. Despite the induction of defence gene expression in the leaves by jasmonate, this treatment did not lead to increased resistance to F. oxysporum. Overall, the results presented here suggest that the genetic manipulation of plant defence signalling pathways is a useful strategy to provide increased Fusarium wilt resistance.

Protease IV production in Pseudomonas aeruginosa from the lungs of adults with cystic fibrosis

Protease IV is important in the pathogenesis of Pseudomonas aeruginosa-induced microbial keratitis, but little is known of its role in cystic fibrosis (CF) lung infection. In this study protease IV production was examined in 43 P. aeruginosa isolates (24 non-clonal and 19 clonal) from the lungs of chronically infected adult patients attending the Royal Prince Alfred Hospital CF Clinic, Sydney, Australia. Overall, 32/43 (74 %) isolates were positive for protease IV protein by Western blotting and 22/43 (51 %) had evidence of active protease IV on gelatin zymography. Clonal strains were 1.6 times more likely than non-clonal strains to produce protease IV [18/19 (95 %) versus 14/24 (58 %), RR=1.6, CI 1.1–2.3, P=0.007] and 3 times more likely to secrete the protein [16/19 (84 %) versus 6/24 (25 %), RR=3.4, CI 1.6–6.9, P

Methyl jasmonate induced gene expression in wheat delays symptom development by the crown rot pathogen Fusarium pseudograminearum

The necrotrophic fungal pathogen Fusarium pseudograminearum (F. pseudograminearum) causes crown rot disease (CR) in wheat. This host-pathogen interaction has not been studied previously at the molecular level. In this study. using real-time quantitative PCR, the expression of 26 selected wheat genes was examined 1, 2 and 4 days after inoculation of wheat seedlings of the CR susceptible cultivar Kennedy and the partially field-resistant cultivar Sunco. Reproducible induction of eight defence genes consisting of PR1.1, PR2 (beta,1-3 glucanase), PR3 (chitinase), PR4 (wheativin), PR5 (thaumatin-like protein). TaPERO (peroxidase), PR10 and TaGLP2a (germin-like) was observed. These genes were induced in both cultivars, however. some genes were induced more rapidly in Sunco than in Kennedy. MJ treatment also induced the above pathogen responsive defence genes in both cultivars while benzo(1,2,3)thiadiazole-7-carbothionic acid S-methyl ester (BTH) treatment weakly induced them in Kennedy only. Similarly. treatment with MJ before inoculation significantly delayed the development of necrotic symptoms for 2 weeks in both wheat cultivars, while BTH pre-treatments delayed symptom development in Kennedy only. The chemically induced protection, therefore, correlated with induction of the F. pseudograminearum-responsive genes. These results support the emerging role of jasmonate signalling in defence against necrotrophic fungal pathogens in monocots and future manipulation of this pathway may improve CR resistance in wheat. (c) 2006 Elsevier Ltd. All rights reserved.

Effect of site-specific mutations in different phosphotransfer domains of the chemosensory protein ChpA on Pseudomonas aeruginosa motility

The virulence of Pseudomonas aeruginosa and other surface pathogens involves the coordinate expression of a wide range of virulence determinants, including type IV pili. These surface filaments are important for the colonization of host epithelial tissues and mediate bacterial attachment to, and translocation across, surfaces by a process known as twitching motility. This process is controlled in part by a complex signal transduction system whose central component, ChpA, possesses nine potential sites of phosphorylation, including six histidine-containing phosphotransfer (HPt) domains, one serine-containing phosphotransfer domain, one threonine-containing phosphotransfer domain, and one CheY-like receiver domain. Here, using site-directed mutagenesis, we show that normal twitching motility is entirely dependent on the CheY-like receiver domain and partially dependent on two of the HPt domains. Moreover, under different assay conditions, point mutations in several of the phosphotransfer domains of ChpA give rise to unusual "swarming" phenotypes, possibly reflecting more subtle perturbations in the control of P. aeruginosa motility that are not evident from the conventional twitching stab assay. Together, these results suggest that ChpA plays a central role in the complex regulation of type IV pilus-mediated motility in P. aeruginosa