Stenosis triggers spread of helical Pseudomonas biofilms in cylindrical flow systems

Biofilms are multicellular bacterial structures that adhere to surfaces and often endow the bacterial population with tolerance to antibiotics and other environmental insults. Biofilms frequently colonize the tubing of medical devices through mechanisms that are poorly understood. Here we studied the helicoidal spread of Pseudomonas putida biofilms through cylindrical conduits of varied diameters in slow laminar flow regimes. Numerical simulations of such flows reveal vortical motion at stenoses and junctions, which enhances bacterial adhesion and fosters formation of filamentous structures. Formation of long, downstream-flowing bacterial threads that stem from narrowings and connections was detected experimentally, as predicted by our model. Accumulation of bacterial biomass makes the resulting filaments undergo a helical instability. These incipient helices then coarsened until constrained by the tubing walls, and spread along the whole tube length without obstructing the flow. A three-dimensional discrete filament model supports this coarsening mechanism and yields simulations of helix dynamics in accordance with our experimental observations. These findings describe an unanticipated mechanism for bacterial spreading in tubing networks which might be involved in some hospital-acquired infections and bacterial contamination of catheters.

Polyporus squamosus (Huds.) Fr from different origins: chemical characterization, screening of the bioactive properties and specific antimicrobial effects against Pseudomonas aeruginosa

Wild mushrooms are mainly collected during the rainy season and valued as a nutritious food and sources of natural medicines and nutraceuticals. The aim of this study was to determine the chemical composition and bioactive properties (antioxidant, antimicrobial and cytotoxicity) of Polyporus squamosus from two different origins, Portugal and Serbia. The sample from Portugal showed higher contents of as protein (17.14 g/100 g), fat (2.69 g/100 g), ash (3.15 g/100 g) and carbohydrates (77.02 g/100 g); the same sample gave the highest antioxidant activity: highest reducing power, DPPH radical scavenging activity, and lipid peroxidation inhibition in both β-carotene/linoleate and TBARS assay. These results could be related to its higher content in total tocopherols (1968.65 μg/100 g) and phenolic compounds (1.29 mg/100 g). Both extracts exhibited antibacterial activity against all the tested organisms. The samples from Serbia gave higher overall antibacterial activity and showed excellent antibiofilm activity (88.30 %). Overall, P. squamosus methanolic extracts possessed antioxidant, antimicrobial, antibiofilm and anti-quorum sensing activity, and without toxicity for liver cells. This investigation highlights alternatives to be explored for the treatment of bacterial infections, in particular against Pseudomonas aeruginosa. This study provides important results for the chemical and bioactive properties, especially antimicrobial activity of the mushroom P. squamosus. Moreover, to the authors’ knowledge this is the first report on sugars, organic acids, and individual phenolic compounds in P. squamosus.

Pseudomonas fluorescentes provenientes da rizosfera de solanáceas no controle de Ralstonia solanacearum em tomateiro

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Fitopatologia, Programa de Pós-Graduação em Fitopatologia, 2015.

PAMP-triggered immunity against Pseudomonas syringae involves microRNA-mediated regulation of several uncharacterized R genes

Two main types of noncoding small RNA molecules have been found in plants: microRNAs (miRNAs) and small interfering RNAs (siRNAs). They differ in their biogenesis and mode of action, but share similar sizes (20-24 nt). Their precursors are processed by Dicer-Like RNase III (dcl) proteins present in Arabidopsis thaliana, and in their mature form can act as negative regulators of gene expression, being involved in a vast array of plant processes, including plant development, genomic integrity or response to stress. Small-RNA mediated regulation can occurs at transcriptional level (TGS) or at post-transcriptional level (PTGS). In recent years, the role of gene silencing in the regulation of expression of genes related to plant defence responses against bacterial pathogens is becoming clearer. Comparisons carried out in our lab between the expression profiles of different mutants affected in gene silencing, and plants challenged with Pseudomonas syringae pathovar tomato DC3000, led us to identify a set of uncharacterized R genes, belonging to the TIR-NBS-LRR gene family, differentially expressed in these conditions. Through the use of bioinformatics tools, we found a miRNA* of 22 nt putatively responsible for down-regulating expression of these R genes through the generation of siRNAs. We have also found that the corresponding pri-miRNA is down-regulated after PAMP-perception in a SA-dependent manner. We also demonstrate that plants with altered levels of miRNA* (knockdown lines or overexpression lines) exhibit altered PTI-associated phenotypes, suggesting a role for this miRNA* in this defence response against bacteria. In addition we identify one of the target genes as a negative regulator of defence response against Pseudomonas syringae.

Mutational and acquired carbapenem resistance mechanisms in multidrug resistant Pseudomonas aeruginosa clinical isolates from Recife, Brazil

An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem- resistant Pseudomonas aeruginosa isolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding β-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosa isolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXYOprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and β-lactamase production were responsible for the multidrug resistance in the isolates analysed.

Ferrous iron sensing and responding in Pseudomonas aeruginosa

Controlling iron distribution is important for all organisms, and is key in bacterial pathogenesis. It has long been understood that cystic fibrosis (CF) patient sputum contains elevated iron concentrations. However, anaerobic bacteria have been isolated from CF sputum and hypoxic zones in sputum have been measured. Because ferrous iron [Fe(II)] is stable in reducing, acidic conditions, it could exist in the CF lung. I show that a two-component system, BqsRS, specifically responds to Fe(II) in the CF pathogen, Pseudomonas aeruginosa. Concurrently, a clinical study found that Fe(II) is present in CF sputum at all stages of lung function decline. Fe(II), not Fe(III) correlates with patients in the most severe disease state. Furthermore, transcripts of the newly identified BqsRS were detected in sputum. Two component systems are the main method bacteria interact with their extracellular environment. A typical two-component system contains a sensor histidine kinase, which upon activation phosphorylates a response regulator that then acts as a transcription factor to elicit a cellular response to stimuli. To explore the mechanism of BqsRS, I describe the Fe(II)-sensing RExxE motif in the sensor BqsS and determine the consensus DNA sequence BqsR binds. With the BqsR binding sequence, I identify novel regulon members through bioinformatic and molecular biology techniques. From the predicted function of new BqsR regulon members, I find that Fe(II) elicits a response that globally protects the cells against cationic stressors, including clinically relevant antibiotics. Subsequently, I use BqsR as a case study to determine if promoter outputs can accurately be predicted based only on a deep understanding of a transcriptional activator’s operator or if a broader regulatory context is required for accurate predictions at all genomic loci. This work highlights the importance of Fe(II) as a (micro)environmental factor, even in conditions typically thought of as aerobic. Since the presence of Fe(II) can alter P. aeruginosa’s antibiotic susceptibility, combining the current strategy of targeting Fe(III) with a new approach targeting Fe(II) may help eradicate infections in the CF lung in the future.

Bioorthogonal Noncanonical Amino Acid Tagging for Selective Analysis of the Pseudomonas aeruginosa Proteome

In natural environments, bacterial physiology is frequently characterized by slow metabolic rates and complex cellular heterogeneities. The opportunistic pathogen Pseudomonas aeruginosa provides one such example; P. aeruginosa forms untreatable chronic biofilm infections of the cystic fibrosis lung, where oxygen limitation can lead to states of metabolic dormancy. To better understand the biology of these states, in vitro experiments must be adapted to better recapitulate natural settings. However, low rates of protein turnover and cellular or phenotypic complexity make these systems difficult to study using established methods. Here we adapt the bioorthogonal noncanonical amino acid tagging (BONCAT) method for time- and cell-selective proteomic analysis to the study of P. aeruginosa. Analysis of proteins synthesized in an anoxic dormancy state led to the discovery of a new type of transcriptional regulator which we designated SutA. We performed detailed analyses of SutA’s role in transcription under slow growth states and we elucidated the structural basis for its regulatory behavior. Additionally, we used cell-selective targeting of BONCAT labeling to measure the dynamic proteomic response of an antibiotic-tolerant biofilm subpopulation. Overall this work shows the utility of selective proteomics as applied to bacterial physiology and describes the broad biological insight obtained from that application.

Avaliação da segurança de uso de pseudomonas como biorremediador em áreas degradadas de pastagens.

A integração agricultura pecuária consiste em uma alternativa promissora de produção, pelo aumento da eficiência de utilização de recursos naturais e a preservação do meio ambiente, além de cooperar com a segurança alimentar. Porém, para se obter sucesso em seu uso, a pastagem não deve estar em estádio avançado de degradação. Em áreas já degradadas, pode-se utilizar as Pseudomonas que são rizobactérias promotoras de crescimento. Neste trabalho, foram avaliados os efeitos decorrentes da exposição a P. putida em mamíferos e um invertebrado aquático como sistema teste para avaliação de risco da introdução desses agentes microbiológicos nessas áreas. Não foram encontrados sinais de patogenicidade e infectividade desses agentes microbianos utilizados como biorremediadores. Apesar disso, seria interessante a realização de testes adicionais a fim de garantir a inocuidade dos agentes bem como a sua segurança. Os protocolos empregados fornecem subsídios técnicos para gerenciar os possíveis riscos envolvidos na liberação e/ou uso do produto. Os resultados obtidos, além de sua aplicação na identificação de efeitos prejudiciais à saúde ambiental, poderão subsidiar e orientar avaliações P. putida por agências reguladoras, quanto ao seu uso comercial para fins de biorremediação.

Impacto da utilização de ácido gálhico, ácido ferúlico e eugenol em modo contínuo na suscetibilidade de Pseudomonas aeruginosa

Dissertação de Mestrado, Biologia Molecular e Microbiana, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2016

Influence of Pseudomonas putida AF7 inoculation on soil enzymes.

There has been some concern about the environmental impact of microbial agents. Pseudomonas may be used as bioremediator and as biopesticide. In this study, we report the use of soil enzyme assays as biological indicator of possible negative effects in soil functioning after the P. putida AF7 inoculation. For that, P. putida AF7 was originally isolated from the rizosphere of rice and was inoculated on three soil types: Rhodic Hapludox (RH), Typic Hapludox (TH); and Arenic Hapludult (AH). The acid phosphatase, b-glucosidase and protease enzymes activities were measured for three period of evaluation (7, 14 and 21 days). In general, the enzymatic activities pre- sented variation among the tested soils. The highest activities of b-glucosidase and acid phosphatase were observed in the RH and AH soils, while the protease activity was higher in the TH soil. Also, the soil charac- teristics were measured for each plot. The activity of enzymes from the carbon cycle was positively correlated with the N and the P and the enzyme from the nitrogen cycle was negatively correlated with N and C.org. The presented data indicate that soil biochemical properties can be an useful tool for use as an indicator of soil perturba- tions by microbial inoculation in a risk assessment.

Assessing the safety of Pseudomonas putida introduction in the environment: an overview of ecotoxicological tests.

Risk assessment guidelines for the environmental release of microbial agents are performed in a tiered sequence which includes evaluation of exposure effects on non target organisms. However, it becomes important to verify whether environmental risk assessment from temperate studies is applicable to tropical countries, as Brazil. Pseudomonas putida is a bacteria showing potential to be used for environmental applications as bioremediation and plant disease control. This study investigates the effects of this bacteria exposure on rodents and aquatic organisms (Daphnia similes) that are recommended to be used as non-target organism in environmental risk assessments. Also, the microbial activity in three different soils under P. putida exposure was evaluated. Rats did not show clinical alterations, although the agent was recovered 16 h after the exposure in lung homogenates. The bacteria did not reduce significantly the reproduction and survival of D. similis. The soil enzymatic activities presented fluctuating values after inoculation with bacteria. The measurement of perturbations in soil biochemical characteristics is presented as an alternative way of monitoring the overall effects of the microbial agent to be introduced even in first stage (Tier I) of the risk assessment in tropical ecosystems.

Efeito de Pseudomonas chlororaphis sobre a podridão radicular (Pythium aphanidermatum)e o crescimento vegetal em pimentão hidropônico.

A podridão radicular causada por Pythium spp. é uma das principais doenças em cultivos hidropônicos. O objetivo do trabalho foi avaliar o efeito de Pseudomonas chlororaphis (63-28) sobre a podridão radicular e o crescimento vegetal em pimentão hidropônico. Plantas de pimentão cv. 35-206 RZ foram cultivadas em unidades hidropônicas, e infestadas com P. chlororaphis sete dias antes da inoculação com o patógeno. Os tratamentos foram: testemunha, testemunha inoculada com o patógeno, P. chlororaphis, P. chlororaphis com a adição do patógeno. As variáveis avaliadas foram: severidade da doença, expansão foliar, clorofila foliar e incidência do patógeno. A severidade da doença decresceu em 51% nas plantas inoculadas com a bactéria aos 12 dias após a inoculação, em comparação com a testemunha inoculada. O conteúdo de clorofila decresceu em 12% nas plantas inoculadas com Pythium, em comparação com a testemunha. Plantas tratadas com P. chlororaphis sem a presença do patógeno tiveram maior taxa de expansão foliar. Conclui-se que P. chlororaphis é um promissor agente de controle biológico da podridão radicular e promotor de crescimento de pimentão hidropônico.

Formulação e vida de prateleira de Pseudomonas chlororaphis 63- 28 em fibra de coco, talco e turfa.

2009

Pseudomonas Aeruginosa AmpR Transcriptional Regulatory Network

In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. Previous studies showed that in addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, the transcriptional profiles generated using DNA microarrays and RNA-Seq of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion mutant, PAO∆ampR were analyzed. Transcriptome analysis demonstrates that the AmpR regulon is much more extensive than previously thought influencing the differential expression of over 500 genes. In addition to regulating resistance to β-lactam antibiotics via AmpC, AmpR also regulates non-β-lactam antibiotic resistance by modulating the MexEF-OprN efflux pump. Virulence mechanisms including biofilm formation, QS-regulated acute virulence, and diverse physiological processes such as oxidative stress response, heat-shock response and iron uptake are AmpR-regulated. Real-time PCR and phenotypic assays confirmed the transcriptome data. Further, Caenorhabditis elegans model demonstrates that a functional AmpR is required for full pathogenicity of P. aeruginosa. AmpR, a member of the core genome, also regulates genes in the regions of genome plasticity that are acquired by horizontal gene transfer. The extensive AmpR regulon included other transcriptional regulators and sigma factors, accounting for the extensive AmpR regulon. Gene expression studies demonstrate AmpR-dependent expression of the QS master regulator LasR that controls expression of many virulence factors. Using a chromosomally tagged AmpR, ChIP-Seq studies show direct AmpR binding to the lasR promoter. The data demonstrates that AmpR functions as a global regulator in P. aeruginosa and is a positive regulator of acute virulence while negatively regulating chronic infection phenotypes. In summary, my dissertation sheds light on the complex regulatory circuit in P. aeruginosa to provide a better understanding of the bacterial response to antibiotics and how the organism coordinately regulates a myriad of virulence factors.

Characterization of the poxAB operon encoding a class D carbapenemase in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a dreaded opportunistic pathogen that causes severe and often intractable infections in immunocompromised and critically ill patients. This bacterium is also the primary cause of fatal lung infections in patients with cystic fibrosis and a leading nosocomial pathogen responsible for nearly 10% of all hospital-acquired infections. P. aeruginosa is intrinsically recalcitrant to most classes of antibiotics and has the ability to acquire additional resistance during treatment. In particular, resistance to the widely used β-lactam antibiotics is frequently mediated by the expression of AmpC, a chromosomally encoded β-lactamase that is ubiquitously found in P. aeruginosa strains. This dissertation delved into the role of a recently reported chromosomal β-lactamase in P. aeruginosa called PoxB. To date, no detailed studies have addressed the regulation of poxB expression and its contribution to β-lactam resistance in P. aeruginosa. In an effort to better understand the role of this β-lactamase, poxB was deleted from the chromosome and expressed in trans from an IPTG-inducible promoter. The loss of poxB did not affect susceptibility. However, expression in trans in the absence of ampC rendered strains more resistant to the carbapenem β-lactams. The carbapenem-hydrolyzing phenotype was enhanced, reaching intermediate and resistant clinical breakpoints, in the absence of the carbapenem-specific outer membrane porin OprD. As observed for most class D β-lactamases, PoxB was only weakly inhibited by the currently available β-lactamase inhibitors. Moreover, poxB was shown to form an operon with the upstream located poxA, whose expression in trans decreased pox promoter (Ppox) activity suggesting autoregulation. The transcriptional regulator AmpR negatively controlled Ppox activity, however no direct interaction could be demonstrated. A mariner transposon library identified genes involved in the transport of polyamines as potential regulators of pox expression. Unexpectedly, polyamines themselves were able induce resistance to carbapenems. In summary, P. aeruginosa carries a chromosomal-encoded β-lactamase PoxB that can provide resistance against the clinically relevant carbapenems despite its narrow spectrum of hydrolysis and whose activity in vivo may be regulated by polyamines.^