Gene complementation of airway epithelium in the cystic fibrosis mouse is necessary and sufficient to correct the pathogen clearance and inflammatory abnormalities

Increasingly, cystic fibrosis (CF) is regarded as an inflammatory disorder where the response of the lung to Pseudomonas aeruginosa is exaggerated as a consequence of processes mediated by the product of the CF gene, CFTR. Of importance to any gene-replacement strategy for treatment of CF is the identification of the cell type(s) within the lung milieu that need to be corrected and an indication whether this is sufficient to restore a normal inflammatory response and bacterial clearance. We generated G551D CF mice transgenically expressing the human CFTR gene in two tissue compartments previously demonstrated to mediate a CFTR-dependent inflammatory response: lung epithelium and alveolar macrophages. Following chronic pulmonary infection with P. aeruginosa, CF mice with epithelial-expressed but not macrophage-specific CFTR showed an improvement in pathogen clearance and inflammatory markers compared with control CF animals. Additionally, these data indicate the general role for epithelial cell-mediated events in the response of the lung to bacterial pathogens and the importance of CFTR in mediating these processes.

Evaluation of potential biocontrol agents against Claviceps africana in vitro and in vivo

Undiluted culture filtrates of two commercial products of Trichoderma spp., Trichopel and Trichoflow, and two isolates of Penicillium citrinum completely inhibited the conidial germination of macroconidia of Claviceps africana , the cause of ergot or sugary disease of sorghum (Sorghum bicolor) in vitro . Similarly, Pseudomonas aeruginosa and Burkholderia cepacia completely inhibited macroconidial germination, with the former being more effective at high dilutions. In contrast, these bacterial isolates failed to inhibit infection in vivo in glasshouse tests with ergot-inoculated sorghum, but all fungal biocontrol agents (including an isolate of Epicoccum nigrum) reduced the severity of disease (percentage of infected spikelets per panicle), in some cases completely inhibiting the development of ergot. In a second glasshouse trial, optimum control was achieved when the biocontrol agents were applied 3-7 days before inoculation with conidia of C. africana .

A survey of bacterial diversity in ticks, lice and fleas from Australia

We isolated bacteria from ticks, lice and fleas. Partial small subunit rRNA sequences were obtained for each isolate and the closest matches in the FastA database were determined. These bacteria were mostly Gram-positive (Firmicutes), although representatives from the Proteobacteria (alpha, beta, gamma subdivisions) and CFB group were also isolated. Most of the isolates we found were from genera that were present in most of the ectoparasites studied, but a few genera were restricted to one species of ectoparasite. The most commonly isolated genera were Stenotrophomonas, Staphylococcus, Pseudomonas, Acinetobacter and Bacillus. Species of Bacillus and Proteus, which have biopesticide potential, were found in some of these ectoparasites. Overall, the communities of bacteria were similar to those found in other studies of parasitic arthropods.

The use of 16s rDNA restriction fragment length polymorphism analysis for the identification of non-fermenting gram negative bacilli in a cystic fibrosis microbiology referral service

The utility of 16s rDNA restriction fragment length polymorphism (RFLP) analysis for the partial genomovar differentiation of Burkholderia cepacia complex bacterium is well documented. We compared the 16s rDNA RFLP signatures for a number of non-fermenting gram negative bacilli (NF GNB) LMG control strains and clinical isolates pertaining to the genera Burkholderia, Pseudomonas, Achromobacter (Alcaligenes), Ralstonia, Stenotrophomonas and Pandoraea. A collection of 24 control strain (LMG) and 25 clinical isolates were included in the study. Using conventional PCR, a 1.2 kbp 16s rDNA fragment was generated for each organism. Following restriction digestion and electrophoresis, each clinical isolate RFLP signature was compared to those of the control strain panel. Nineteen different RFLP signatures were detected from the 28 control strains included in the study. TwentyoneyTwenty- five of the clinical isolates could be classified by RFLP analysis into a single genus and species when compared to the patterns produced by the control strain panel. Four clinical B. pseudomallei isolates produced RFLP signatures which were indistinguishable from B. cepacia genomovars I, III and VIII. The identity of these four isolates were confirmed using B. pseudomallei specific PCR. 16s rDNA RFLP analysis can be a useful identification strategy when applied to NF GNB, particularly for those which exhibit colistin sulfate resistance. The use of this molecular based methodology has proved very useful in the setting of a CF referral laboratory particularly when utilised in conjunction with B. cepacia complex and genomovar specific PCR techniques. Species specific PCR or sequence analysis should be considered for selected isolates; especially where discrepancies between epidemiology, phenotypic and genotypic characteristics occur.

A Sco homologue plays a role in defence against oxidative stress in pathogenic Neisseria

Sco proteins are found in mitochondria and in a variety of oxidase positive bacteria. Although Sco is required for the formation of the Cu-A centre in a cytochrome oxidase of the aa(3) type, it was observed that oxidases with a Cu-A centre are not present in many bacteria that contain a Sco homologue. Two bacteria of this type are the pathogens Neisseria meningitidis and Neisseria gonorrhoeae. The sco genes of N. gonorrhoeae strain 1291 and N. meningitidis strain MC58 were cloned, inactivated by inserting a kanamycin resistance cassette and used to make knockout mutants by allelic exchange. Both N. gonorrhoeae and N. meningitidis sco mutants were highly sensitive to oxidative killing by paraquat, indicating that Sco is involved in protection against oxidative stress in these bacteria. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

The genetics of glycosylation in Gram-negative bacteria

In recent years there has been a dramatic increase in reports of glycosylation of proteins in various Gram-negative systems including Neisseria meningitidis, Neisseria gonorrhoeae, Campylobacter jejuni, Pseudomonas aeruginosa, Escherichia coli, Caulobacter crescentus, Aeromonas caviae and Helicobacter pylori. Although this growing list contains many important pathogens (reviewed by Benz and Schmidt [Mol. Microbiol. 45 (2002) 267-276]) and the glycosylations are found on proteins important in pathogenesis such as pili, adhesins and flagella the precise role(s) of the glycosylation of these proteins remains to be determined. Furthermore, the details of the glycosylation biosynthetic process have not been determined in any of these systems. The definition of the precise role of glycosylation and the mechanism of biosynthesis will be facilitated by a detailed understanding of the genes involved. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Purification of post-translationally modified proteins from bacteria: homologous expression and purification of histidine-tagged pilin from Neisseria meningitidis

Until recently, glycosylation of proteins in prokaryotes was regarded as uncommon and thought to be limited to special cases such as S-layer proteins and some archeal outer membrane proteins. Now, there are an increasing number of reports of bacterial proteins that are glycosylated. Pilin of pathogenic Neisseria is one of the best characterised post-translation ally modified bacterial proteins, with four different types of modifications reported, including a novel glycosylation. Pilin monomers assemble to form pilus fibres, which are long protein filaments that protrude from the surface of bacterial cells and are key virulence factors. To aid in the investigation of these modifications, pure pilin is required. A number of pilin purification methods have been published, but none are appropriate for the routine purification of pilin from many different isolates. This study describes a novel, rapid, and simple method of pilin purification from Neisseria meningitidis C311#3, which facilitates the production of consistent quantities of pure, native pilin. A 6 x histidine tag was fused to the C-terminus of the pilin subunit structural gene, pilE, via homologous recombination placing the 6 x histidine-tagged allele in the chromosome of N. meningitidis C311#3. Pilin was purified under non-denaturing conditions via a two-step process using immobilised metal affinity chromatography (IMAC), followed by dye affinity chromatography. Analysis of the purified pilin confirmed that it retained both of the post-translational modifications examined. This novel approach may prove to be a generally applicable method for purification and analysis of post-translationally modified proteins in bacteria. (C) 2003 Elsevier Science (USA). All rights reserved.

Identification and characterization of pptA: a gene involved in the phase-variable expression of phosphorylcholine on pili of Neisseria meningitidis

Pili of pathogenic Neisseria are major virulence factors associated with adhesion, cytotoxicity, twitching motility, autoaggregation, and DNA transformation. Pili are modified posttranslationally by the addition of phosphorylcholine. However, no genes involved in either the biosynthesis or the transfer of phosphorylcholine in Neisseria meningitidis have been identified. In this study, we identified five candidate open reading frames (ORFs) potentially involved in the biosynthesis or transfer of phosphorylcholine to pilin in N. meningitidis. Insertional mutants were constructed for each ORF in N. meningitidis strain C311#3 to determine their effect on phosphorylcholine expression. The effect of the mutant ORFs on the modification by phosphorylcholine was analyzed by Western analysis with phosphorylcholine-specific monoclonal antibody TEPC-15. Analysis of the mutants showed that ORF NMB0415, now defined as pptA (pilin phosphorylcholine transferase A), is involved in the addition of phosphorylcholine to pilin in N. meningitidis. Additionally, the phase variation (high frequency on-off switching of expression) of phosphorylcholine on pilin is due to changes in a homopolymeric guanosine tract in pptA.

Pericondrite por piercing: relato de casos e revis��o da literatura

A pericondrite �� uma infec����o que envolve o peric��ndrio do pavilh��o auricular. Desencadeada pelo piercing, �� secund��ria a uma rea����o al��rgica ao n��quel. O diagn��stico �� essencialmente cl��nico, por��m a realiza����o de cultura e antibiograma �� fundamental. Os pat��genos freq��entemente envolvidos s��o germes Gram negativos (Pseudomonas aeruginosa). Os autores apresentam 3 casos de pericondrite por piercing atendidos no ambulat��rio de otorrinolaringologia e realizam uma revis��o da literatura.

Bacteriologia do aspirado do meato m��dio em pacientes com fibrose c��stica

A combina����o de fatores como viscosidade das secre����es dos seios paranasais, diminui����o da drenagem sinusal e comprometimento do transporte mucociliar podem ser respons��veis pela cria����o de um ambiente prop��cio e adequado para a coloniza����o de bact��rias nos seios paranasais de pacientes com fibrose c��stica. OBJETIVO: Analisar a bacteriologia do aspirado do meato m��dio de pacientes portadores de fibrose c��stica. MATERIAL E M��TODO: Atrav��s de um estudo prospectivo de delineamento transversal, avaliou-se uma amostra composta de 23 pacientes, avaliados durante 2 anos. Realizaram-se rela����es entre a cultura do meato m��dio e a avalia����o radiol��gica do seio maxilar e a avalia����o cl��nica. Secundariamente, estudou-se a rela����o da bacteriologia do aspirado do meato m��dio e a do escarro. RESULTADOS: No total foram realizadas 42 aspira����es do meato m��dio. Em 17 (73,91%) dos 23 pacientes, as culturas foram negativas e, em 6 (26,08%), positivas. Das 42 aspira����es, 31 (73,8%) foram negativas e 11 (26,2%), positivas. A presen��a de Pseudomonas aeruginosa foi observada em 18,18% das culturas positivas e o Staphylococcus aerus em 27,28%. CONCLUS��O: A maioria das culturas do aspirado do meato m��dio de pacientes com fibrose c��stica foi negativa.

Atividade antimicrobiana in vitro de produtos vegetais em otite externa aguda

Otite externa aguda �� a inflama����o do conduto auditivo externo, e plantas medicinais podem ser utilizadas, na cultura popular, para seu tratamento. OBJETIVO: Avaliar atividade antimicrobiana in vitro de Aleolanthus suaveolens, Caryophyllus aromaticus, Cymbopogon citratus, Matricaria chamomila, Pithecellobium avaremotemo, Plectranthus amboinicus e Ruta graveolens sobre agentes etiol��gicos de otite externa. CASU��STICA E M��TODOS: A concentra����o inibit��ria m��nima de extratos e ��leos destas plantas foi obtida em amostras de otite externa. RESULTADOS: Staphylococcus aureus em 10 culturas, Pseudomonas aeruginosa em 8, Pseudomonas aeruginosa e Staphylococcus aureus, em associa����o, em 5 culturas e Candida albicans e Candida krusei em 4 culturas. P. aeruginosa foi resistente a todos os extratos e ��leos essenciais testados; os extratos de A. suaveolens, P. avaremotemo e de R. graveolens foram inativos, o ��leo essencial de C. aromaticus e M. chamomila foram ativos contra 3 cepas de S. aureus e as cepas de Candida; Sete das cepas de S. aureus foram sens��veis ao extrato de P. amboinicus, mas o ��leo n��o mostrou atividade, 4 cepas de S.aureus e as cepas de Candida foram sens��veis ao ��leo essencial de R. graveolens. CONCLUS��O: Algumas plantas apresentaram resultados satisfat��rios, dependendo do agente etiol��gico, por��m se faz necess��rio estudos mais detalhados, para melhorar o aproveitamento destas plantas.

Achado de bact��rias selecionadas em crian��as de Trinidad com doen��a amigdaliana cr��nica

Faringoamigdalite na popula����o pedi��trica �� largamente tratada com antibi��ticos. OBJETIVO: Estudar a microflora presente na superf��cie e no n��cleo de am��gdalas ap��s adenoamigdalectomia eletiva em crian��as. M��TODO: Am��gdalas de 102 crian��as de Trinidad foram prospectivamente estudadas por meio de culturas e identifica����es bacteriol��gicas feitas a partir de amostras das superf��cies e n��cleos de suas am��gdalas entre 2005-2006. RESULTADOS: A partir de 360 am��gdalas, foram isolados Streptococcus spp. (51,3%), Staphylococcus spp. (42,3%) e Gram-Negativos (6,4%). A identifica����o de estafilococos e estreptococos tanto na superf��cie quanto no n��cleo foi semelhante (p>0,05). Encontramos mais (p<0,001) Streptococcus spp. nas superf��cies (82,2%) do que nos n��cleos (63,3%); a preval��ncia de estreptococos alfa-hemol��ticos foi maior (p<0,001) do que aquela de estreptococos beta-hemol��ticos nas superf��cies (74,4% vs. 18,6%) do que nos n��cleos (58,9% vs. 13,7%). N��o houve concord��ncia entre superf��cies e n��cleos com rela����o a estreptococos (p<0,0004) e estreptococos alfa-hemol��ticos (p<0,007). Estreptococos beta-hemol��ticos foram mais identificados (p<0,05) em crian��as dentre 6-16 anos do que naquelas entre 1-5 anos de idade (31% e 23,8% vs 12,5% e 8%). A preval��ncia de S. pyogenes na superf��cie e no n��cleo foi de (84,6% vs 70%) e (50,0% vs 25,0%) em crian��as de maior faixa et��ria e crian��as mais novas, respectivamente. Klebsiella spp. (6,6%, 2,2%), Proteus (4,4%, 4,4%) e Pseudomonas (4,4 %, 1,1%) cresceram nas superf��cies e n��cleos, respectivamente. CONCLUS��O: As superf��cies amigdalianas tinham mais estreptococos e estreptococos hemol��ticos do que seus n��cleos. Crian��as mais velhas tiveram mais estreptococos beta-hemol��ticos, e s��o altamente colonizadoras de S. pyogenes. Sugerimos estudos que investiguem os mecanismos de ader��ncia estreptoc��cica em crian��as de Trinidad.

Pericondrite p��s-piercing

O uso de piercing tem se tornado uma pr��tica muito freq��ente entre os jovens. O procedimento na maioria vezes realizado por profissionais n��o-qualificados n��o �� isento de riscos. O manuseio de material contaminado ou a higiene impr��pria predisp��em �� pericondrite e �� celulite. A pericondrite caracteriza-se pelo eritema do pavilh��o auricular, dor intensa e febre. Sem tratamento, desenvolve-se um edema generalizado do pavilh��o com forma����o de abscesso subpericondrial, podendo evoluir para necrose isqu��mica da cartilagem e a tem��vel deformidade est��tica conhecida como "orelha em couve flor". O agente respons��vel mais encontrado �� o Pseudomonas aeruginosa. No est��gio inicial da doen��a o tratamento pode ser feito com antibi��ticos de amplo espectro. Nos casos em que o abscesso est�� presente, a incis��o e drenagem cir��rgica s��o obrigat��rios acompanhado de antibioticoterapia guiado pela cultura e antibiograma. OBJETIVO: O objetivo deste relato de caso �� realizar uma revis��o bibliogr��fica dos ��ltimos 10 anos abordando os aspectos anat��micos do pavilh��o auricular, a hist��ria do uso de piercing e suas mais conhecidas complica����es. M��TODO: Relato de um caso de pericondrite p��s-piercing transcartilaginoso onde houve a necessidade de tratamento cir��rgico com praticamente nenhuma deformidade est��tica. RESULTADO: Aquisi����o de experi��ncia te��rico-pr��tica atrav��s de revis��o bibliogr��fica e relato de um caso de evolu����o favor��vel para a paciente. CONCLUS��O: Incid��ncia crescente das complica����es de pericondrites na popula����o jovem deve levar �� preven����o prim��ria mais elaborada.

Composi����o qu��mica dos gr��os de milho em resposta �� aduba����o mineral e inocula����o com rizobact��rias

Para o aumento da produtividade agr��cola no Pa��s, s��o necess��rios estudos para melhor aproveitamento da aduba����o mineral, a fim de uma produ����o de gr��os com melhor qualidade nutricional. Este trabalho teve como objetivo avaliar o teor e o ac��mulo de prote��na, de cinzas e de nutrientes em gr��os de milho provenientes de plantas submetidas �� inocula����o com Pseudomonas fluorescens e cultivadas em diferentes n��veis de aduba����o NPK no solo. Foram testados seis tratamentos, em delineamento experimental de blocos casualizados, no esquema fatorial 3 x 2, sendo tr��s n��veis de aduba����o qu��mica com NPK (0,125 e 250 kg ha-1) e dois n��veis de inoculante �� base de P. fluorescens (com e sem), com quatro repeti����es, sendo instalado em Latossolo Vermelho eutrof��rrico, utilizando o cultivar de milho h��brido 30F35. Determinaram-se os teores e ac��mulo de N, P, K, Ca, Mg, Zn, Cu, Fe, Mn, prote��na e cinzas dos gr��os. Os dados foram submetidos �� an��lise de vari��ncia e as m��dias, comparadas pelo teste de Tukey (p < 0,05). A aplica����o de P. fluorescens via inocula����o incrementou os teores de P e K dos gr��os de milho, independentemente dos n��veis de aduba����o. Os teores dos nutrientes, de prote��na e de cinzas dos gr��os de milho n��o foram influenciados pelos n��veis de aduba����o mineral, com exce����o do Cu, que aumentou com a eleva����o dos n��veis de aduba����o.