Gama de hospedeiros e reação de genótipos de tomateiro a Pseudomonas cichorii

Em 2005, foi constatada em dois campos comerciais de tomate no Estado de São Paulo, a ocorrência da queima bacteriana, causada por Pseudomonas cichorii. em vista disso, foram desenvolvidos estudos visando a determinação da gama de hospedeiros de isolados de Pseudomonas cichorii (IBSBF 2309 e IBSBF 2323), obtidos de tomateiro, provenientes de campos comerciais localizados nos municípios de Bragança Paulista e Mogi Guaçú, SP. Plantas de abobrinha, alface, beldroega, berinjela, beterraba, cenoura, couvebrócolo, datura, fumo, girassol, jiló, melão, pepino, petúnia, pimentão, rabanete, repolho, rúcula, salsa e tomateiro foram inoculadas por pulverização, separadamente, com os dois isolados de P. cichorii de tomateiro e um isolado de girassol (GIR-1). Os isolados IBSBF 2309 e IBSBF 2323 foram patogênicos à beldroega, datura, girassol, pimentão e tomate; GIR-1 foi patogênico apenas à beldroega, datura e girassol, não sendo patogênico ao pimentão e ao tomateiro. No Brasil não se conhecem fontes de resistência dentro do gênero Lycopersicon ou a reação de cultivares de tomateiros a esta bactéria. Vinte e oito genótipos de tomateiro provenientes do Banco de Germoplasma da empresa Sakata Seed Sudamerica Ltda., foram avaliados quanto a reação aos isolados IBSBF 2309 e IBSBF 2323 de P. cichorii, pelo método de inoculação nas folhas. Os maiores níveis de resistência foram observados em AF 11768, AF 2521, AF 11766, AF 11772, AF 229, AF 5719-1 e AF 8162. O genótipo AF 5719-1, que possui o gene Pto, que confere resistência a P. syringae pv. tomato, apresentou um bom nível de resistência a P. cichorii. A identificação de genótipos que apresentem bons níveis de resistência a este patógeno é importante para utilização em programas de melhoramento genético do tomateiro, visando a incorporação de genes de resistência a P. cichorii.

Cough-generated aerosols of Pseudomonas aeruginosa and other Gram Negative Bacteria from cystic fibrosis patients.

Background: Pseudomonas aeruginosa is the most common bacterial pathogen in cystic fibrosis (CF) patients. Current infection control guidelines aim to prevent transmission via contact and respiratory droplet routes and do not consider the possibility of airborne transmission. We hypothesized that with coughing, CF subjects produce viable, respirable bacterial aerosols. Methods: Cross-sectional study of 15 children and 13 adults with CF, 26 chronically infected with P. aeruginosa. A cough aerosol sampling system enabled fractioning of respiratory particles of different size, and culture of viable Gram negative non-fermentative bacteria. We collected cough aerosols during 5 minutes voluntary coughing and during a sputum induction procedure when tolerated. Standardized quantitative culture and genotyping techniques were used. Results: P. aeruginosa was isolated in cough aerosols of 25 (89%) subjects of whom 22 produced sputum samples. P. aeruginosa from sputum and paired cough aerosols were indistinguishable by molecular typing. In 4 cases the same genotype was isolated from ambient room air. Approximately 70% of viable aerosols collected during voluntary coughing were of particles ≤ 3.3 microns aerodynamic diameter. P. aeruginosa, Burkholderia cenocepacia Stenotrophomonas maltophilia and Achromobacter xylosoxidans were cultivated from respiratory particles in this size range. Positive room air samples were associated with high total counts in cough aerosols (P=0.003). The magnitude of cough aerosols were associated with higher FEV1 (r=0.45, P=0.02) and higher quantitative sputum culture results (r=0.58, P=0.008). Conclusion: During coughing, CF patients produce viable aerosols of P. aeruginosa and other Gram negative bacteria of respirable size range, suggesting the potential for airborne transmission.

High resolution melt analysis : a novel method for studying the genetic relatedness of Pseudomonas aeruginosa isolates from clinical and environmental sources

A novel method was developed for studying the genetic relatedness of Pseudomonas aeruginosa isolates from clinical and environmental sources. This bacterium is ubiquitous in the natural environment and is an important pathogen known to infect Cystic Fibrosis (CF) patients. The transmission route of strains has not yet been defined; current theories include acquisition from an environmental source or through patient-to-patient spread. A highly discriminatory, bioinformatics based, DNA typing method was developed to investigate the relatedness of clinical and environmental isolates. This study found a similarity between the environmental and several CF clonal strains and also highlighted occurrence of environmental P. aeruginosa strains in CF infections.

Identification and characterization of a virulence-associated protease from a pathogenic Pseudomonas fluorescens strain

Pseudomonas fluorescens is an aquaculture pathogen that can infect a number of fish species. The virulence mechanisms of aquatic P. fluorescens remain largely unknown. Many P. fluorescens strains are able to secrete an extracellular protease called AprX, yet no AprX-like proteins have been identified in pathogenic P. fluorescens associated with aquaculture. In this study, a gene encoding an AprX homologue was cloned from TSS, a pathogenic A fluorescens strain isolated from diseased fish. In TSS, AprX is secreted into the extracellular milieu, and the production of AprX is controlled by growth phase and calcium. Mutation of aprX has multiple effects, which include impaired abilities in interaction with cultured host cells, adherence to host mucus, modulation of host immune response, and dissemination and survival in host tissues and blood. Purified recombinant AprX exhibits apparent proteolytic activity, which is optimal at pH 8.0 and 50 degrees C. The protease activity of recombinant AprX is enhanced by Ca2+ and Zn2+ and reduced by Co2+. Cytotoxicity analyses showed that purified recombinant AprX has profound toxic effect on cultured fish cells. These results demonstrate that AprX is an extracellular metalloprotease that is involved in bacterial virulence. (C) 2009 Elsevier B.V. All rights reserved.

Pseudomonas aeruginosa vesicles associate with and are internalized by human lung epithelial cells.

BACKGROUND: Pseudomonas aeruginosa is the major pathogen associated with chronic and ultimately fatal lung infections in patients with cystic fibrosis (CF). To investigate how P. aeruginosa-derived vesicles may contribute to lung disease, we explored their ability to associate with human lung cells. RESULTS: Purified vesicles associated with lung cells and were internalized in a time- and dose-dependent manner. Vesicles from a CF isolate exhibited a 3- to 4-fold greater association with lung cells than vesicles from the lab strain PAO1. Vesicle internalization was temperature-dependent and was inhibited by hypertonic sucrose and cyclodextrins. Surface-bound vesicles rarely colocalized with clathrin. Internalized vesicles colocalized with the endoplasmic reticulum (ER) marker, TRAPalpha, as well as with ER-localized pools of cholera toxin and transferrin. CF isolates of P. aeruginosa abundantly secrete PaAP (PA2939), an aminopeptidase that associates with the surface of vesicles. Vesicles from a PaAP knockout strain exhibited a 40% decrease in cell association. Likewise, vesicles from PAO1 overexpressing PaAP displayed a significant increase in cell association. CONCLUSION: These data reveal that PaAP promotes the association of vesicles with lung cells. Taken together, these results suggest that P. aeruginosa vesicles can interact with and be internalized by lung epithelial cells and contribute to the inflammatory response during infection.

Comparison of the binding specificity of two bacterial metalloproteases, LasB of Pseudomonas aeruginosa and ZapA of Proteus mirabilis, using N-alpha mercaptoamide template-based inhibitor analogues

The metalloproteases ZapA of Proteus mirabilis and LasB of Pseudomonas aeruginosa are known to be virulence factors their respective opportunistic bacterial pathogens, and are members of the structurally related serralysin and thermolysin families of bacterial metalloproteases respectively. Secreted at the site of infection, these proteases play a key role in the infection process, contributing to tissue destruction and processing of components of the host immune system. Inhibition of these virulence factors may therefore represent an antimicrobial strategy, attenuating the virulence of the infecting pathogen. Previously we have screened a library of N-alpha mercaptoamide dipeptide inhibitors against both ZapA and LasB, with the aim of mapping the S1' binding site of the enzymes, revealing both striking similarities and important differences in their binding preferences. Here we report the design, synthesis, and screening of several inhibitor analogues, based on two parent inhibitors from the original library. The results have allowed for further characterization of the ZapA and LasB active site binding pockets, and have highlighted the possibility for development of broad-spectrum bacterial protease inhibitors, effective against enzymes of the thermolysin and serralysin metalloprotease families.

Tobramycin Inhalation Powder™:a novel drug delivery system for treating chronic Pseudomonas aeruginosa infection in cystic fibrosis

Lung disease in cystic fibrosis (CF) is typified by the development of chronic airways infection culminating in bronchiectasis and progression to end-stage respiratory disease. Pseudomonas aeruginosa, a ubiquitous gram-negative bacteria, is the archetypical CF pathogen and is associated with an accelerated clinical decline. The development and widespread use of chronic suppressive aerosolized antibacterial therapies, in particular Tobramycin Inhalation Solution (TIS), in CF has contributed to reduced lung function decline and improved survival. However, the requirement for the aerosolization of these agents through nebulizers has been associated with increased treatment burden, reduced quality of life and remain a barrier to broader uptake. Tobramycin Inhalation Powder (TIP™) has been developed by Novartis with the express purpose of delivering the same benefits as TIS in a time-effective manner. Administered via the T-326™ (Novartis) Inhaler in four individual 28-mg capsules, TIP can be administered in a quarter of the time of traditional nebulizers and is inherently portable. In clinical studies, TIP has been shown to be safe, result in equivalent or superior reductions in P. aeruginosa sputum density and produce similar improvements in pulmonary function. TIP offers significant advantages in time saving, portability and convenience over traditional nebulized TIS with comparable clinical outcomes for individuals with CF.

Developing an international Pseudomonas aeruginosa reference panel

Pseudomonas aeruginosa is a major opportunistic pathogen in cystic fibrosis (CF) patients and causes a wide range of infections among other susceptible populations. Its inherent resistance to many antimicrobials also makes it difficult to treat infections with this pathogen. Recent evidence has highlighted the diversity of this species, yet despite this, the majority of studies on virulence and pathogenesis focus on a small number of strains. There is a pressing need for a P. aeruginosa reference panel to harmonize and coordinate the collective efforts of the P. aeruginosa research community. We have collated a panel of 43 P. aeruginosa strains that reflects the organism's diversity. In addition to the commonly studied clones, this panel includes transmissible strains, sequential CF isolates, strains with specific virulence characteristics, and strains that represent serotype, genotype or geographic diversity. This focussed panel of P. aeruginosa isolates will help accelerate and consolidate the discovery of virulence determinants, improve our understanding of the pathogenesis of infections caused by this pathogen, and provide the community with a valuable resource for the testing of novel therapeutic agents.

Pseudomonas aeruginosa exhibits frequent recombination, but only a limited association between genotype and ecological setting

Pseudomonas aeruginosa is an opportunistic pathogen and an important cause of infection, particularly amongst cystic fibrosis (CF) patients. While specific strains capable of patient-to-patient transmission are known, many infections appear to be caused by unique and unrelated strains. There is a need to understand the relationship between strains capable of colonising the CF lung and the broader set of P. aeruginosa isolates found in natural environments. Here we report the results of a multilocus sequence typing (MLST)-based study designed to understand the genetic diversity and population structure of an extensive regional sample of P. aeruginosa isolates from South East Queensland, Australia. The analysis is based on 501 P. aeruginosa isolates obtained from environmental, animal and human (CF and non-CF) sources with particular emphasis on isolates from the Lower Brisbane River and isolates from CF patients obtained from the same geographical region. Overall, MLST identified 274 different sequence types, of which 53 were shared between one or more ecological settings. Our analysis revealed a limited association between genotype and environment and evidence of frequent recombination. We also found that genetic diversity of P. aeruginosa in Queensland, Australia was indistinguishable from that of the global P. aeruginosa population. Several CF strains were encountered frequently in multiple ecological settings; however, the most frequently encountered CF strains were confined to CF patients. Overall, our data confirm a non-clonal epidemic structure and indicate that most CF strains are a random sample of the broader P. aeruginosa population. The increased abundance of some CF strains in different geographical regions is a likely product of chance colonisation events followed by adaptation to the CF lung and horizontal transmission among patients.

Cough-generated aerosols of Pseudomonas aeruginosa and other Gram-negative bacteria from patients with cystic fibrosis

BACKGROUND: Pseudomonas aeruginosa is the most common bacterial pathogen in patients with cystic fibrosis (CF). Current infection control guidelines aim to prevent transmission via contact and respiratory droplet routes and do not consider the possibility of airborne transmission. It was hypothesised that subjects with CF produce viable respirable bacterial aerosols with coughing.

METHODS: A cross-sectional study was undertaken of 15 children and 13 adults with CF, 26 chronically infected with P aeruginosa. A cough aerosol sampling system enabled fractioning of respiratory particles of different sizes and culture of viable Gram-negative non-fermentative bacteria. Cough aerosols were collected during 5 min of voluntary coughing and during a sputum induction procedure when tolerated. Standardised quantitative culture and genotyping techniques were used.

RESULTS: P aeruginosa was isolated in cough aerosols of 25 subjects (89%), 22 of whom produced sputum samples. P aeruginosa from sputum and paired cough aerosols were indistinguishable by molecular typing. In four cases the same genotype was isolated from ambient room air. Approximately 70% of viable aerosols collected during voluntary coughing were of particles <or=3.3 microm aerodynamic diameter. P aeruginosa, Burkholderia cenocepacia, Stenotrophomonas maltophilia and Achromobacter xylosoxidans were cultivated from respiratory particles in this size range. Positive room air samples were associated with high total counts in cough aerosols (p = 0.003). The magnitude of cough aerosols was associated with higher forced expiratory volume in 1 s (r = 0.45, p = 0.02) and higher quantitative sputum culture results (r = 0.58, p = 0.008).

CONCLUSION: During coughing, patients with CF produce viable aerosols of P aeruginosa and other Gram-negative bacteria of respirable size range, suggesting the potential for airborne transmission.

Bacteria microarrays as sensitive tools for exploring pathogen surface epitopes and recognition by host receptors

We describe a protocol for the generation and validation of bacteria microarrays and their application to the study of specific features of the pathogen's surface and interactions with host receptors. Bacteria were directly printed on nitrocellulose-coated glass slides, using either manual or robotic arrayers, and printing quality, immobilization efficiency and stability of the arrays were rigorously controlled by incorporating a fluorescent dye into the bacteria. A panel of wild type and mutant strains of the human pathogen Klebsiella pneumoniae, responsible for nosocomial and community-acquired infections, was selected as model bacteria, and SYTO-13 was used as dye. Fluorescence signals of the printed bacteria were found to exhibit a linear concentration-dependence in the range of 1 x 10(8) to 1 x 10(9) bacteria per ml. Similar results were obtained with Pseudomonas aeruginosa and Acinetobacter baumannii, two other human pathogens. Successful validation of the quality and applicability of the established microarrays was accomplished by testing the capacity of the bacteria array to detect recognition by anti-Klebsiella antibodies and by the complement subcomponent C1q, which binds K. pneumoniae in an antibody-independent manner. The biotin/AlexaFluor-647-streptavidin system was used for monitoring binding, yielding strain-and dose-dependent signals, distinctive for each protein. Furthermore, the potential of the bacteria microarray for investigating specific features, e.g. glycosylation patterns, of the cell surface was confirmed by examining the binding behaviour of a panel of plant lectins with diverse carbohydrate-binding specificities. This and other possible applications of the newly developed arrays, as e.g. screening/evaluation of compounds to identify inhibitors of host-pathogen interactions, make bacteria microarrays a useful and sensitive tool for both basic and applied research in microbiology, biomedicine and biotechnology.

Phenotypic characterization of an international Pseudomonas aeruginosa reference panel: strains of cystic fibrosis (CF) origin show less in vivo virulence than non-CF strains

Pseudomonas aeruginosa causes chronic lung infections in people with cystic fibrosis (CF) and acute opportunistic infections in people without CF. Forty two P. aeruginosa strains from a range of clinical and environmental sources were collated into a single reference strain panel to harmonise research on this diverse opportunistic pathogen. To facilitate further harmonized and comparable research on P. aeruginosa, we characterised the panel strains for growth rates, motility, virulence in the Galleria mellonella infection model, pyocyanin and alginate production, mucoid phenotype, lipopolysaccharide (LPS) pattern, biofilm formation, urease activity, antimicrobial and phage susceptibilities. Phenotypic diversity across the P. aeruginosa panel was apparent for all phenotypes examined agreeing with the marked variability seen in this species. However, except for growth rate, the phenotypic diversity among strains from CF versus non-CF sources was comparable. CF strains were less virulent in the G. mellonella model than non-CF strains (p=0.037). Transmissible CF strains generally lacked O antigen, produced less pyocyanin, and had low virulence in G. mellonella. Further, in the three sets of sequential CF strains, virulence, O-antigen expression and pyocyanin production were higher in the earlier isolate compared to the isolate obtained later in infection. Overall, full phenotypic characterization of the defined panel of P. aeruginosa strains increases our understanding of the virulence and pathogenesis of P. aeruginosa and may provide a valuable resource for the testing of novel therapies against this problematic pathogen.

Environmental risk factors for the initial acquisition of Pseudomonas aeruginosa in young children with cystic fibrosis

Thesis (Ph.D.)--University of Washington, 2013

Pathogen-induced hatching and population-specific life-history response to water-borne cues in brown trout (Salmo trutta)

Hatching is an important niche shift, and embryos in a wide range of taxa can either accelerate or delay this life-history switch in order to avoid stage-specific risks. Such behavior can occur in response to stress itself and to chemical cues that allow anticipation of stress. We studied the genetic organization of this phenotypic plasticity and tested whether there are differences among populations and across environments in order to learn more about the evolutionary potential of stress-induced hatching. As a study species, we chose the brown trout (Salmo trutta; Salmonidae). Gametes were collected from five natural populations (within one river network) and used for full-factorial in vitro fertilizations. The resulting embryos were either directly infected with Pseudomonas fluorescens or were exposed to waterborne cues from P. fluorescens-infected conspecifics. We found that direct inoculation with P. fluorescens increased embryonic mortality and induced hatching in all host populations. Exposure to waterborne cues revealed population-specific responses. We found significant additive genetic variation for hatching time, and genetic variation in trait plasticity. In conclusion, hatching is induced in response to infection and can be affected by waterborne cues of infection, but populations and families differ in their reaction to the latter.

An unusual postharvest spotting disease of the comercial mushroom, Agaricus bisporus, caused by a novel pathovar of Pseudomonas tolaasii

An unusual postharvest spotting disease of the commercial mushroom, Agaricus bisporus, which was observed on a commercial mushroom farm in Ontario, was found to be caused by a novel pathovar of Pseudomonas tolaasii. Isolations from the discoloured lesions, on the mushroom pilei, revealed the presence of several different bacterial and fungal genera. The most frequently isolated genus being Pseudomonas bacteria. The most frequently isolated fungal genus was Penicillium. Of the bacteria and fungi assayed for pathogenicity to mushrooms, only Pseudomonas tolaasii was able to reproduce the postharvest spotting symptom. This symptom was typically reproduced 1 to 7 days postharvest, when mushroom pilei were inoculated with 101 to 105 cfu. Of the fungi tested for pathogenicity only a Penicillium sp. and Verticillium fungicola were shown to be pathogenic, however, neither produced the postharvest spotting symptom. The Pseudomonas tolaasii strain isolated from the postharvest lesions differed from a type culture (Pseudomonas tolaasii ATCC 33618) in the symptoms it produced on Agaricus bisporus pilei under the same conditions and at the same inoculum concentration. It was therefore designated a pathovar. This strain also differed from the type culture in its cellular protein profile. Neither the type culture, nor the mushroom pathogen was found to contain plasmid DNA. The presence of plasmid DNA is therefore not responsible for the difference in pathogenicity between the two strains.