The iron stimulon of Xylella fastidiosa includes genes for type IV pilus and colicin V-like bacteriocins

Xylella fastidiosa is the etiologic agent of a wide range of plant diseases, including citrus variegated chlorosis (CVC), a major threat to citrus industry. The genomes of several strains of this phytopathogen were completely sequenced, enabling large-scale functional studies. DNA microarrays representing 2,608 (91.6%) coding sequences (CDS) of X. fastidiosa CVC strain 9a5c were used to investigate transcript levels during growth with different iron availabilities. When treated with the iron chelator 2,2`-dipyridyl, 193 CDS were considered up-regulated and 216 were considered down-regulated. Upon incubation with 100 mu M ferric pyrophosphate, 218 and 256 CDS were considered up- and down-regulated, respectively. Differential expression for a subset of 44 CDS was further evaluated by reverse transcription-quantitative PCR. Several CDS involved with regulatory functions, pathogenicity, and cell structure were modulated under both conditions assayed, suggesting that major changes in cell architecture and metabolism occur when X. fastidiosa cells are exposed to extreme variations in iron concentration. Interestingly, the modulated CDS include those related to colicin V-like bacteriocin synthesis and secretion and to functions of pili/fimbriae. We also investigated the contribution of the ferric uptake regulator Fur to the iron stimulon of X. fastidiosa. The promoter regions of the strain 9a5c genome were screened for putative Fur boxes, and candidates were analyzed by electrophoretic mobility shift assays. Taken together, our data support the hypothesis that Fur is not solely responsible for the modulation of the iron stimulon of X fastidiosa, and they present novel evidence for iron regulation of pathogenicity determinants.

Role of sigma(54) in the regulation of genes involved in type I and type IV pili biogenesis in Xylella fastidiosa

The phytopathogen Xylella fastidiosa produces long type IV pili and short type I pili involved in motility and adhesion. In this work, we have investigated the role of sigma factor sigma(54) (RpoN) in the regulation of fimbrial biogenesis in X. fastidiosa. An rpoN null mutant was constructed from the non-pathogenic citrus strain J1a12, and microarray analyses of global gene expression comparing the wild type and rpoN mutant strains showed few genes exhibiting differential expression. In particular, gene pilA1 (XF2542), which encodes the structural pilin protein of type IV pili, showed decreased expression in the rpoN mutant, whereas two-fold higher expression of an operon encoding proteins of type I pili was detected, as confirmed by quantitative RT-PCR (qRT-PCR) analysis. The transcriptional start site of pilA1 was determined by primer extension, downstream of a sigma(54)-dependent promoter. Microarray and qRT-PCR data demonstrated that expression of only one of the five pilA paralogues, pilA1, was significantly reduced in the rpoN mutant. The rpoN mutant made more biofilm than the wild type strain and presented a cell-cell aggregative phenotype. These results indicate that sigma(54) differentially regulates genes involved in type IV and type I fimbrial biogenesis, and is involved in biofilm formation in X. fastidiosa.

Antimicrobial Particles from Cationic Lipid and Polyelectrolytes

Hybrid nanoparticles from cationic lipid and polymers were prepared and characterized regarding physical properties and antimicrobial activity. Carboxymethylcellulose (CMC) and polydiallyldimethylammonium chloride (PDDA) were sequentially added to cationic bilayer fragments (BF) prepared from ultrasonic dispersion in water of the synthetic and cationic lipid dioctadecyldimethylammonium bromide (DODAB). Particles thus obtained were characterized by dynamic light-scattering for determination of z-average diameter (Dz) and zeta-potential (zeta). Antimicrobial activity of the DODAB BF/CMC/PDDA particles against Pseudomonas aeruginosa or Staphylococcus aureus was determined by plating and CFU counting over a range of particle compositions. DODAB BF/CMC/PDDA particles exhibited sizes and zeta-potentials strictly dependent on DODAB, CM C, and PDDA concentrations. At 0.1 mM DODAB, 0.1 mg/mL CMC, and 0.1 mg/mL PDDA, small cationic particles with Dz = 100 nm and zeta = 30 mV were obtained. At 0.5 mM DODAB, 0.5 mg/mL CMC and 0.5 mg/mL PDDA, large cationic particles with Dz = 470 nm and zeta= 50 mV were obtained. Both particulates were highly reproducible regarding physical properties and yielded 0% of p. aeruginosa viability (10(7) CFU/mL) at 1 or 2 mu g/mL PDDA dissolved in solution or in form of particles, respectively. 99% of S. aureus cells died at 10 mu g/mL PDDA alone or in small or large DODAB BF/CMC/PDDA particles. The antimicrobial effect was dependent on the amount of positive charge on particles and independent of particle size. A high microbicide potency for PDDA over a range of nanomolar concentrations was disclosed. P. aeruginosa was more sensitive to all cationic assemblies than S. aureus.

PILZ Protein Structure and Interactions with PILB and the FIMX EAL Domain: Implications for Control of Type IV Pilus Biogenesis

The PilZ protein was originally identified as necessary for type IV pilus (T4P) biogenesis. Since then, a large and diverse family of bacterial PilZ homology domains have been identified, some of which have been implicated in signaling pathways that control important processes, including motility, virulence and biofilm formation. Furthermore, many PilZ homology domains, though not PilZ itself, have been shown to bind the important bacterial second messenger bis(3`-> 5`)cyclic diGMP (c-diGMP). The crystal structures of the PilZ orthologs from Xanthomonas axonopodis pv Citri (PilZ(XAC1133), this work) and from Xanthomonas campestris pv campestris (XC1028) present significant structural differences to other PilZ homologs that explain its failure to bind c-diGMP. NMR analysis of PilZ(XAC1133) shows that these structural differences are maintained in solution. In spite of their emerging importance in bacterial signaling, the means by which NZ proteins regulate specific processes is not clear. In this study, we show that PilZ(XAC1133) binds to PilB, an ATPase required for TV polymerization, and to the EAL domain of FiMX(XAC2398), which regulates TV biogenesis and localization in other bacterial species. These interactions were confirmed in NMR, two-hybrid and far-Western blot assays and are the first interactions observed between any PilZ domain and a target protein. While we were unable to detect phosphodiesterase activity for FimXX(AC2398) in vitro, we show that it binds c-diGMP both in the presence and in the absence of PilZ(XAC1133). Site-directed mutagenesis studies for conserved and exposed residues suggest that PilZ(XAC1133) interactions with FimX(XAC2398) and PilB(XAC3239) are mediated through a hydrophobic surface and an unstructured C-terminal extension conserved only in PilZ orthologs. The FimX-PilZ-PilB interactions involve a full set of ""degenerate"" GGDEF, EAL and PilZ domains and provide the first evidence of the means by which PilZ orthologs and FimX interact directly with the TP4 machinery. (C) 2009 Elsevier Ltd. All rights reserved.

Chemical Composition and Antimicrobial Activity of the Essential Oil from Rollinia sericea (REFr.) REFr. (Annonaceae) Leaves

The chemical composition of the essential oil of Rollinia sericea (R.E.Fr.) R.E.Fr. leaves was determined by GC and GC/MS analysis. The analysis revealed the presence mainly of sesquiterpenes: beta-elemene (10%), beta-caryophyllene (10.0%), bicyclogermacrene (9.1%), germacrene-D (8.2%), bicycloelemene (6.2%) and (Z)-nerolidol (5.3%). Rollinia sericea oil was able to inhibit the growth of both fungi Aspergillus niger (16404) and Candida albicans (ATCC 10231) as well as the Gram-positive bacterium Staphyloccocus aureus (ATCC 6538) but it was inactive against the Gram-negative bacteria Escherichia coli (ATCC 8739) and Pseudomonas aeruginosa (ATCC 9027).

Identification of an Operon, Pil-Chp, That Controls Twitching Motility and Virulence in Xylella fastidiosa

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases, including Pierce`s disease of grapevines. Disease manifestation by X. fastidiosa is associated with the expression of several factors, including the type IV pili that are required for twitching motility. We provide evidence that an operon, named Pil-Chp, with genes homologous to those found in chemotaxis systems, regulates twitching motility. Transposon insertion into the pilL gene of the operon resulted in loss of twitching motility (pilL is homologous to cheA genes encoding kinases). The X. fastidiosa mutant maintained the type IV pili, indicating that the disrupted pilL or downstream operon genes are involved in pili function, and not biogenesis. The mutated X. fastidiosa produced less biofilm than wild-type cells, indicating that the operon contributes to biofilm formation. Finally, in planta the mutant produced delayed and less severe disease, indicating that the Pil-Chp operon contributes to the virulence of X. fastidiosa, presumably through its role in twitching motility.

Chemical Composition and Antimicrobial Activity of the Essential Oil from Croton heterocalyx Baill. (Euphorbiaceae s.s.) Leaves

The chemical composition and the antimicrobial activity of the essential oil from Croton heterocalyx leaves were evaluated. The oil which was analyzed by GC and GUMS was found to contain germacrene D (12.5%), bicyclogermacrene (11.2%), delta-elemene (9.2%) beta-elemene (8.2%), spathulenol (6.9%), linalool (5.4%) and 1,8-cineole (3.7%) its major components. Croton. heterocalyx oil displayed a high inhibitory activity against the fungi Aspergillus niger (16404) and Candida albicans (ATCC 10231.) as well its the Gram-positive bacterium Staphylococcus aureus (ATCC 6538), hut a very weak activity was observed for the Gram-negative bacteria Escherichia coli (ATCC 8739) and Pseudomonas aeruginosa (ATCC 9027).

Structure and Applications of a Rhamnolipid Surfactant Produced in Soybean Oil Waste

Soybean oil soapstock was utilized as an alternative carbon source for the production of rhamnolipids by Pseudomonas aeruginosa LBI strain. The chemical composition and properties of the rhamnolipid mixture obtained were determined to define its potential applications. The chemical characterization of the rhamnolipid has revealed the presence of ten different homologues. The monorhamnolipid RhaC(10)C(10) and the dirhamnolipid Rha(2)C(10)C(10) were the main components of the mixture that showed predominance of 44% and 29%, respectively, after 144-h of cultivation. The biosurfactant was able to form stable emulsions with several hydrocarbons and showed excellent emulsification for soybean oil and chicken fat (100%). The rhamnolipid removed 67% of crude oil present in sand samples and presented antimicrobial activity against Bacillus cereus and Mucor miehei at 64 mu g/mL and inhibition of Neurospora crassa, Staphylococcus aureus, and Micrococcus luteus at 256 mu g/mL. The results demonstrated that the rhamnolipid produced in soybean oil soapstock can be useful in environmental and food industry applications.

Aspectos microbiológicos da bile de pacientes com suspeita de coledocolilitíase e suas repercussões no tratamento das infecções biliares

A infecção das vias biliares é uma doença freqüente com alta morbidade e mortalidade, que pode variar de 10 a 60% dependendo de sua gravidade. A causa mais comum desta infecção é a presença de cálculos na via biliar principal que propicia o surgimento de bacteriobilia. O profundo conhecimento das características microbiológicas da bile nos casos de coledocolitíase e infecção das vias biliares são fundamentais para o melhor diagnóstico desta infecção e escolha da antibioticoterapia a ser instituída. Assim, o objetivo deste estudo foi de caracterizar os principais aspectos microbiológicos da bile dos pacientes com e sem coledocolitíase e avaliar sua importância na escolha dos antimicrobianos para o tratamento da infecção das vias biliares. Foram analisados 33 pacientes que foram divididos em um grupo de 10 pacientes sem coledocolitíase (grupo controle) no momento da Colangiografia Endoscópica (CPER) e em outro grupo de 23 pacientes com coledocolitíase. A bile de todos os pacientes foi coletada no início do procedimento endoscópico, através de catater introduzido na via biliar. O exame de microscopia direta com coloração de Gram e as culturas da bile foram negativas nos 10 pacientes que não apresentaram coledocolitíase durante a CPER. Dos 23 pacientes com cálculos na via biliar principal, 19 (83%) apresentaram culturas positivas. Desses 19 pacientes com culturas de bile positivas, 18 (94,7%) apresentaram microorganismos detectáveis à microscopia direta com coloração de Gram. Apenas um paciente apresentou crescimento de germe anaeróbio (Bacteroides fragilis). O cultivo de 28 bactérias teve predominância de microorganismos Gram negativos (18 bactérias- 64,3%). Os germes isolados foram E. coli (9, 32,1%), Klebsiella pneumoniae (5, 17,9%), Enterococcus faecalis (5, 17,9%), Streptococcus alfa-haemoliticus (3, 10,7%), Streptococcus viridans (2, 7,1%), Enterobacter cloacae (2, 7,1%), Panteona aglomerans (1, 3,6%) e Pseudomonas aeruginosa (1, 3,6%). Todos os pacientes com microorganismos detectados pela microscopia direta com coloração de Gram tiveram crescimento bacteriano em suas culturas, por outro lado nenhum paciente com cultura negativa apresentou microoorganismos à microsopia direta ( p= 0,0005). Nesses casos, a microsopia direta apresentou uma especificidade de 100% e sensibilidade de 80%. A análise quantitativa das culturas da bile mostrou que das 19 culturas positivas, 12 (63,2%) tiveram pelo menos um germe com contagem superior a 105 ufc/ml. Todas as bactérias Gram positivas isoladas foram sensíveis à ampicilina, da mesma forma que todas as Gram negativas foram sensíveis aos aminoglicosídeos. Os achados deste estudo demonstram uma boa correlação entre a microscopia direta da bile com coloração de Gram e os achados bacteriológicos das culturas da bile coletada por colangiografia endoscópica retrógrada. O esquema terapêutico antimicrobiano tradicionalmente empregado em nosso hospital, que inclui a combinação de ampicilina e gentamicina, parece ser adequado, pois apresenta eficácia terapêutica contra os principais microorganismos responsáveis pela infecção das vias biliares.

Codisposição de resíduos sólidos de serviços de saúde com resíduos sólidos urbanos

O objetivo deste trabalho é estudar a codisposição de resíduos sólidos de serviços de saúde (RSSS) com resíduos sólidos urbanos (RSU), dando ênfase aos microrganismos normalmente encontrados nos RSSS. O trabalho foi desenvolvido em três etapas, julgadas relevantes para o entendimento da codisposição de RSSS com RSU, denominadas de forma geral: Codisposição, Recirculação e Inoculação. A Codisposição foi implementada a partir da montagem de seis células de 70 m3, portanto em escala real, escavadas sobre a Célula 4 do Aterro Sanitário Zona Norte, na cidade de Porto Alegre. As células experimentais foram dispostas lado a lado, apresentando base inferior 2 x 3 m, base superior 7 x 8 m e profundidade 2,5 m. Cada célula recebeu, nesta ordem, cobertura de BIDIM 400, PEAD 2 mm e BIDIM 180. As duas coberturas de BIDIM funcionaram como membranas protetoras da manta de PEAD (impermeabilizante), visando minorar possíveis danos mecânicos determinados principalmente por elementos pérfuro-cortantes. As drenagens dos efluentes líquidos e gasosos de cada célula foram realizadas através de tubos hidráulicos de DN 40 mm, possuindo estes comunicação entre si no interior de cada célula, mas não entre células. Os tubos de saída de gás e lixiviado foram furados em sua superfície para garantir a remoção dos efluentes do interior das células, sendo forrados com BIDIM 400 nas regiões perfuradas para impedir a obstrução dos furos pelo resíduo. As células foram denominadas C1, C2, C3, C4, C5 e C6, diferindo entre si devido às relações de RSSS/RSU que foram utilizadas em seu preenchimento. De C1 a C6, as percentagens de RSU e RSSS foram, respectivamente, (100% e 0%), (95% e 5%), (75% e 25%), (50% e 50%), (25% e 75%) e (0% e 100%). Depois de preenchidas, as células receberam uma camada de argila compactável de 60 cm. Foram analisadas diversas variáveis físicas, químicas e microbiológicas de controle. Na etapa de Recirculação, estudou-se a influência da reposição de todo o lixiviado gerado em uma célula sobre a argila de recobrimento desta mesma célula. Foi estudado ainda o Poder Germinativo de quatro espécies vegetais das quais uma delas viria a ser utilizada para remoção de poluentes do lixiviado recirculado, sendo plantada sobre a superfície superior das células. Foram elas ervilhaca (Vicia sativa), aveia preta (Avena strigosa), alfafa (Medicago sativa) e pensacola (Paspalum notatum), testadas na presença e ausência de lixiviado;. Nesta etapa, foi testada a influência da espécie vegetal ervilhaca (Vicia sativa) – leguminosa de inverno –, plantada na argila, como agente de depuração do lixiviado recirculado. Verificou-se que a utilização desta espécie vegetal não teve influência significativa na remoção de poluentes do lixiviado, nas condições do experimento. A redução das concentrações de poluentes se deveu, portanto, à operação de recirculação. Na etapa denominada Inoculação, foram estudadas as curvas de crescimento de três espécies de microrganismos em lixiviado bruto e esterilizado. Foram utilizados reatores de 1,5 L preenchidos com 1 L de lixiviado esterilizado previamente, sendo inoculadas concentrações conhecidas dos microrganismos Escherichia coli, Pseudomonas aeruginosa e Staphylococcus aureus, separadamente. Para cada microrganismo, foram montados doze reatores: quatro com pH 5, quatro com pH 7 e quatro com pH 9. De cada conjunto de reatores de mesmo pH, três foram inoculados com igual concentração de microrganismos, e um, considerado reator de controle, recebeu somente água deionizada e esterilizada. Desenvolveram-se, como esperado, as curvas de crescimento para as espécies estudadas. A espécie Pseudomonas aeruginosa se adaptou melhor (maior tempo de sobrevivência e concentrações mais elevadas) no pH neutro, enquanto que a espécie Escherichia coli se desenvolveu melhor no pH ácido. No pH 9, ambas as espécies foram inviabilizadas desde a primeira contagem. Para Staphylococcus aureus a concentração reduziu-se drasticamente nas primeiras 24 h para todos os valores de pH. Comparando-se os pH, essa espécie se adaptou melhor no pH neutro e depois no pH ácido. A partir dos estudos desenvolvidos, a codisposição de RSSS com RSU se mostrou uma técnica aceitável.

Pneumonia adquirida na comunidade em adultos hospitalizados : estudo da etiologia, epidemiologia e fatores prognósticos

OBJETIVO: Determinar a etiologia, epidemiologia e os fatores prognósticos de pneumonia adquirida na comunidade (PAC) em adultos imunocompetentes hospitalizados. MÉTODOS: Durante um período de 3 anos, foram estudados, prospectivamente, 110 pacientes consecutivos com diagnóstico de PAC. RESULTADOS: Sessenta e seis (60%) pacientes eram homens, a idade média foi de 54 anos, 42 (38,2%) eram maiores de 65 anos, 81 (73,6%) apresentavam comorbidades, 70 (63%) pertenciam às classes IV e V de Fine e 24 (21,8%) pacientes foram admitidos em unidade de terapia intensiva (UTI). Um agente etiológico foi identificado em 60 (54,5%) casos, incluindo Streptococcus pneumoniae em 23 (20,9%) casos, Staphylococcus aureus em 14 (12,7%), Pseudomonas aeruginosa em 7 (6,4%), Haemophilus influenzae em 5 (4,6%) e Legionella pneumophila em 5 (4,6%) casos, como os patógenos mais freqüentemente isolados. O uso de antimicrobianos previamente à admissão hospitalar ocorreu em 33,6% dos casos e foi significativamente associado com etiologia desconhecida. Houve 15 (13,6%) óbitos e três variáveis foram estatisticamente associadas ao desfecho: idade > 65 anos, índice de Fine V e IV e internação em UTI. Alterações no tratamento empírico inicial foram realizadas em 43 (39%) casos devido à obtenção do diagnóstico etiológico. CONCLUSÕES: Em nosso estudo, S. pneumoniae foi o principal agente etiológico de PAC, seguido de S. aureus e P. aeruginosa, que apresentaram freqüência elevada em indivíduos com pneumonia grave e/ou fatores de risco conhecidos. A determinação do agente etiológico serviu para otimizar o tratamento proposto pelos consensos e estimar a prevalência local dos patógenos.

Superfícies de titânio modificadas termoquimicamente por plasma : avaliação da resposta biológica

This laboratory study involves the participation of a group with professionals from different areas that had contributed to the construction of a multidisciplinary knowledge, about biological response of titanium surfaces modified through thermochemical treatment by plasma. Thus, the crystalline phase was previously characterized in relation to the topography, roughness, molhability and nitrogen concentration in the samples surface. It s indispensable that materials implanted can influence in a good cellular response as well as promotes a bacteria action. Surfaces modified by plasma were exposed to different cultures such as: cellular (human osteoblastic) and bacteria (Staphylococcus epidermidis ATCC35984 and Pseudomonas aeruginosa ATCC 27853) in order to evaluate the biological response. It was evaluated the adhesion, proliferation, morphology and cellular preference of human ostheoblastic cells (HOST), as well as the formation of a biofilm and bacteria proliferation. It was still analyzed the bacteria selectivity ability in relation to the surfaces. The software Image Pro Plus was used to the counting of cells and bacteria adhered to the surface of disks. The results were submitted to the variance analysis (ANOVA), and then, by the Kruskal-Wallis test, using GraphPad Instat ® software, version 3.5 to Windows. The nitrided samples in spite of show a higher roughness and molhability showed a smaller bacteria growing and higher cellular proliferation, when compared to non treated samples, indicating that the treated material present a high efficiency to biomedical implants

Avaliação do sistema conservante em formulação com extrato hidroalcóolico de schinus terebinthifolius raddi nacardiaceae

Schinus terebinthifolius Raddi is used in the treatment of skin and mucosal injuries, infections of respiratory, digestive and genitourinary systems. Currently one of the biggest problems faced for the industry of phytopharmaceuticals with regard to the quality of raw materials is the microbial contamination. The aim this study was to evaluate the antimicrobial action of the hidroalcoholic extract of aroeira, beyond testing the effectiveness of the preservative system in hidrogel to the base of this extract. The extracts were prepared by maceration in the ratio of 1:10 of solvent plant/with alcohol 40%. The methods for microbial count were pour plate and test for specific microorganisms, analyzing in third copy each one of the samples. The antimicrobial activity of aroeira extracts was performed using an agar diffusion method, using strains of S. aureus, P. aeruginosa, E. coli, B. subtilis, C. albicans, C. tropicalis, C. krusei, C. guilliermondii, T. rubrum, M. gypseum, A. flavus and A. niger. The formula with aroeira was evaluated by the challenge test. This method consisted of artificial contamination the sample with separate inóculos of A. niger, C. albicans, E. coli e S. aureus aeruginosa and determinations of survivors for the method of counting for pour plate , during times 0, 24h, 48h, 7 days, 14 days, 21 days and 28 days. How much to the results, one verified that the extract of aroeira in the 13,5 concentration mg/mL presented antimicrobial activity for cepas of E. coli, B. subtilis, P. aeruginosa e S. aureus, producing inhibition zone, on average with 13 mm of diameter. However it did not present no fungi activity. The formula with aroeira containig both methylparaben and propylparaben showed a good efficacy in challenge test front to strains of A. niger, C. albicans, E. coli, S. aureus. The A criteria of European Pharmacopoeia, adopted in this work, was verified that this product revealed the good preservative efficacy for the challenge test, time interval of the 28 days. However, it is interesting to extend this study, in order to carry through the sped up stability and the test of shelf, to establish the validity of this formularization

Rhamnolipid produced from agroindustrial wastes enhances hydrocarbon biodegradation in contaminated soil

A crude biosurfactant solution was produced by Pseudomonas aeruginosa growing on agroindustrial wastes as the substrate and used to study its effect on hydrocarbon biodegradation by the indigenous soil microflora under laboratory conditions. Two concentrations were studied at first and 1 mg of biosurfactant/g of soil showed to be the most efficient for the total petroleum hydrocarbon reduction, which reached 85% at the first 20 days in soil microcosms. Respirometric and microbial analyses showed that the biosurfactant added did not have toxic effects over the microbial population. The use of a biosurfactant for bioremediation has been limited because of its high cost production. Biosurfactants produced from cost-free by-products combines waste minimization with economic potential bioremediation process.