Molecular Mechanisms Involved in Regulating the Mucoid-to-Nonmucoid Conversion of Pseudomonas aeruginosa Associated with Cystic Fibrosis Patients

Pseudomonas aeruginosa is an opportunistic pathogen that has received attention because of its close association with cystic fibrosis (CF). Chronic pulmonary infection with the mucoid P. aeruginosa is the leading cause of mortality in CF patients. This bacterium has the ability to sense and adapt to the harsh environment in the CF lung by converting from a nonmucoid to a mucoid state. The mucoid phenotype is caused by overproduction of a polysaccharide called alginate. Alginate production is regulated by the algT/U operon containing five genes, algT/U-mucA-mucB-mucC-mucD. Alginate overproduction in CF isolates has been partially attributed to a loss-of-function mutation in mucA that results in the overexpression of algT. This mucoid phenotype is unstable, reverting to the nonmucoid form when the isolates are cultured outside of the CF lung. This study was undertaken to determine the mechanisms involved in the conversion from the mucoid to the nonmucoid form. Thirty-six spontaneous nonmucoid variants of a known mucoid isolate with a mucA mutation were analyzed. Ten of these isolates were complemented in trans by plasmids containing the algT operon and the algT gene. Chromosomal DNA was extracted and the mucA and algT genes were amplified by the polymerase chain reaction. Sequence analysis of the genes showed that these mutants retained the original mucA mutation but acquired secondary mutations in the algT gene.

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.

Nanovecteurs pour cibler pseudomonas aeruginosa dans la fibrose kystique.

La production excessive de mucus visqueaux dans les poumons des patients atteints de la fibrose kystique (FK) gêne la diffusion des médicaments et entraîne des infections bactériennes. En effet, l’infection pulmonaire par Pseudomonas aeruginosa (PA) est la principale cause de mortalité. Les travaux effectués dans cette thèse avaient pour but de développer des nouvelles formulations de nanoparticules (NP) et de liposomes (LP) chargées avec des antibiotiques pour erradiquer le PA chez les patients atteints de KF. Tout d’abord, les polymères PEG-g-PLA et PLA-OH ont été synthétisés et caractérisés. Ensuite, l'efficacité d'encapsulation (EE) de la tobramycine, du sulfate de colistine et de la lévofloxacine (lévo) a été testée dans des NP de PEG-g-PLA et / ou PLA-OH. Les premiers essais d'optimisation ont montré que les NP chargées avec la lévo présentaient une augmentation de l’EE. La lévo reste alors le médicament de choix. Cependant, la meilleure charge de médicament obtenue était de 0,02% m/m. Pour cette raison, nous avons décidé d'évaluer l'encapsulation de la lévo dans les LP. En fait, des LP chargés de lévo ont présenté une EE d’environ 8% m/m. De plus, la taille et la charge de ces LP étaient appropriées pour la pénétration du vecteur dans le mucus. Le test de biofilm n'est pas reproductible, mais le test standard a montré que la souche mucoïde de PA était susceptible à la lévo. Ainsi, nous avons comparé les activités des LP fraîchement préparées (vides et chargés ) et de la lévo libre sous la forme planctonique de PA. Les résultats ont montré que des LP vides ne gênent pas la croissance bactérienne. Pour la souche mucoïde (Susceptible à la lévo) les LP chargés et le médicament libre ont présenté la même concentration minimale inhibitrice (CMI). Toutefois, les souches non mucoïdes (résistant à la lévo) ont présenté une CMI deux fois plus faible que celle pour le médicament libre. Finalement, les LP se sont avérés plus appropriés pour encapsuler des médicaments hydrophiles que les NP de PEG-g-PLA. En outre, les LP semblent améliorer le traitement contre la souche résistante de PA. Toutefois, des études complémentaires doivent être effectuées afin d'assurer la capacité des liposomes èa traiter la fibrose kystique.

Repair of lamellar scleral lesions in dogs with preserved equine renal capsule: short report

OBJETIVO: Avaliar o uso da cápsula renal de eqüino preservada em glicerina 98% no reparo de lesões lamelares esclerais em cães. MÉTODOS: Foram utilizados 12 cães, machos e fêmeas, com peso médio de 12kg. Foram realizadas avaliações clínica e morfológica aos 1, 3, 7, 15, 30 e 60 dias de pós-operatório. Após anestesia geral e procedimentos padrões de preparo do campo operatório, foi realizada cantotomia temporal, seguida de incisão conjutival e escleral com área de 0,5x0,5 cm na posição de 1hora, próxima ao limbo. em seguida, um fragmento de mesma dimensão de cápsula renal de eqüino preservada em glicerina, previamente hidratado em solução salina, foi aplicado ao defeito escleral criado sendo fixado com pontos simples isolados com vicryl 7-0®. RESULTADOS: A avaliação clínica revelou blefaroespasmo/fotofobia até o sétimo dia de pós-operatório. Foi observado edema conjuntival até o quinto dia, acompanhado de secreção ocular mucóide, que persistiu até o décimo dia de pós-operatório. Não foram observados sinais clínicos de rejeição do enxerto em todos os animais, em todos os períodos avaliados. Os segmentos anterior e posterior do bulbo ocular não apresentaram sinais de inflamação. A análise morfológica revelou exsudação inflamatória aguda nos períodos precoces e intermediários da avaliação e inflamação crônica nos períodos tardios da observação. Houve incorporação do enxerto ao leito receptor. CONCLUSÃO: Os resultados sugerem que a cápsula renal de eqüino preservada pode ser mais uma alternativa de membrana biológica para o reparo de lesões esclerais lamelares em cães e no homem.

Conservative Treatment of Odontogenic Myxoma

Odontogenic myxomas (OMs) are nonencapsulated rare benign tumors that can occur in gnathic bones. They are locally invasive and have a high recurrence rate. Radiologically, OMs show a multilocular (in the majority of cases) or unilocular radiolucency, with either distinct or poorly defined margins. Histopathologically, OMs are characterized by spindle-, wedge-, or stellate-shaped cells loosely arranged in an abundant mucoid background. Myxomas are mainly asymptomatic. Radical surgery, excision, and enucleation followed by curettage of the surrounding bony tissue have all been advocated as treatment options. This study presents a successful case of conservative treatment of OMs with a 5-year follow-up.

Caracterização das áreas hemófagas da placenta bovina

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Intoxicação por veneno de sapo em um canino

O sapo do gênero Bufo possui nas suas glândulas paratóides uma secreção mucóide contendo toxinas como bufaginas e Bufotoxinas, que são esteróides cardiogênicos. Os cães podem atacar os sapos, entrando em contato com o veneno por meio das mucosas. Um canino, da raça Bulldog Francês, foi encaminhado ao Setor de Patologia Veterinária da Universidade Federal do Rio Grande do Sul (UFRGS) para a necropsia com histórico de provável intoxicação por veneno de sapo. Na necropsia o canino apresentava pulmões aumentados de volume, avermelhados e com edema, e rins de coloração vermelho-escura. As alterações microscópicas indicaram congestão, hemorragia e edema pulmonar. Nos rins, no baço e nos linfonodos foi observada congestão. As análises toxicológicas para os venenos de rotina foram negativas. Porém, a investigação do veneno de sapo a partir de cromatografia por camada delgada e gasosa demonstrou resultado positivo, revelando ser esta a causa da morte do canino.

Natural latex graft in lamellar and penetrating sclerectomies in rabbits

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Avaliação prognóstica e características anatomoclínicas do carcinoma mucinoso da mama

Pós-graduação em Ginecologia, Obstetrícia e Mastologia - FMB

Aspectos clínicos e epidemiológicos da infecção por pseudomonas aeruginosa em pacientes portadores de fibrose cística no Estado do Pará

Pseudomonas aeruginosa é um bacilo gram-negativo, importante patógeno para pacientes neutropênicos, queimados e em condições de ventilação artificial em Unidades de Tratamento Intensivo, onde causam infecção nosocomial. Nestas condições, a infecção pode ser séria e muitas vezes letal. Em pacientes com fibrose cística, o curso da patologia por P. aeruginosa evolui como uma infecção pulmonar crônica severa, pois a bactéria produz diversas toxinas e outros fatores de virulência responsáveis pelo estabelecimento da colonização persistente do trato respiratório destes pacientes. A apresentação característica da persistente infecção por P. aeruginosa é a produção de alginato mucóide e a formação de microcolônias, que é considerada a estratégia de sobrevivência da bactéria no meio ambiente, P. aeruginosa crescendo em biofilm é altamente resistente a antibióticos, estando usualmente associada com progressiva perda da função pulmonar. Esta pesquisa realizou uma avaliação epidemiológica e clínica de portadores de fibrose cística, colonizados por P. aeruginosa, atendidos no Hospital Universitário João de Barros Barreto, na cidade de Belém, Pará no ano de 2003. Foi feito coleta de escarro dos pacientes expectoradores e swab de orofaringe nos demais para estudo microbiológico realizado no laboratório microbiologia deste hospital. Foram avaliados 32 pacientes fibrocísticos, distribuídos em três grupos, conforme: ausência de infecção por P. aeruginosa (G1), infecção pela bactéria sem colonização (G2) e colonização crônica (G3). Pacientes pertencentes a G3 apresentaram complicações respiratórias mais frequëntes e mais graves que os demais. A ocorrência de cepas mucóidaes de P. aeruginosa foi significativamente mais prevalente neste grupo, onde a doença respiratória se apresentou de forma mais severa. Cepas não-mucóides foram identificadas de forma similar nos grupos G2 e G3. Os sintomas respiratórios foram os mais frequëntes ao diagnóstico. A idade média dos pacientes ao diagnóstico foi de 7 anos. Condições sócio-econômicas adversas, diagnóstico tardio, desnutrição e mutação genética parecem ter favorecido a colonização e contribuído para ocorrência de óbito no grupo G3.

The effects of particulate ambient air pollution on the murine umbilical cord and its vessels: A quantitative morphological and immunohistochemical study

Previous studies have shown that particulate matter (PM) compromise birth weight and placental morphology. We hypothesized that exposing mice to ambient PM would affect umbilical cord (UC) morphology. To test this, mice were kept in paired open-top exposure chambers at the same location and ambient conditions but, in one chamber, the air was filtered (F) and, in the other, it was not (NF). UCs were analysed stereologically and by immunohistochemistry to localize isoprostane and endothelin receptors. The cords of mice from NF chambers were smaller in volume due to loss of mucoid connective tissue and decrease in volume of collagen. These structural changes and in umbilical vessels were associated with greater volumes of regions immunostained for isoprostane, ETAR and ETBR. Findings indicate that the adverse effects of PM on birth weight may be mediated in part by alterations in UC structure or imbalances in the endogenous regulators of vascular tone and oxidative stress. (C) 2012 Elsevier Inc. All rights reserved.

Analysis of Two Component Systems in Group B Streptococcus Shows that RgfAC and the Novel FspSR Modulate Virulence and Bacterial Fitness

Group B Streptococcus (GBS), in its transition from commensal to pathogen, will encounter diverse host environments and thus require coordinately controlling its transcriptional responses to these changes. This work was aimed at better understanding the role of two component signal transduction systems (TCS) in GBS pathophysiology through a systematic screening procedure. We first performed a complete inventory and sensory mechanism classification of all putative GBS TCS by genomic analysis. Five TCS were further investigated by the generation of knock-out strains, and in vitro transcriptome analysis identified genes regulated by these systems, ranging from 0.1-3% of the genome. Interestingly, two sugar phosphotransferase systems appeared differently regulated in the knock-out mutant of TCS-16, suggesting an involvement in monitoring carbon source availability. High throughput analysis of bacterial growth on different carbon sources showed that TCS-16 was necessary for growth of GBS on fructose-6-phosphate. Additional transcriptional analysis provided further evidence for a stimulus-response circuit where extracellular fructose-6-phosphate leads to autoinduction of TCS-16 with concomitant dramatic up-regulation of the adjacent operon encoding a phosphotransferase system. The TCS-16-deficient strain exhibited decreased persistence in a model of vaginal colonization and impaired growth/survival in the presence of vaginal mucoid components. All mutant strains were also characterized in a murine model of systemic infection, and inactivation of TCS-17 (also known as RgfAC) resulted in hypervirulence. Our data suggest a role for the previously unknown TCS-16, here named FspSR, in bacterial fitness and carbon metabolism during host colonization, and also provide experimental evidence for TCS-17/RgfAC involvement in virulence.

Vaccination of cystic fibrosis patients against Pseudomonas aeruginosa reduces the proportion of patients infected and delays time to infection

INTRODUCTION: Cystic fibrosis (CF) almost always leads to chronic airway infection with Pseudomonas aeruginosa. Despite advances in antibiotic therapy, after chronic infection rapid deterioration in lung function occurs, increasing morbidity and mortality. Prevention of infection by vaccination is desirable, but earlier trials produced disappointing results. The promising short term immunogenicity and safety of a new P. aeruginosa vaccine prompted us to evaluate its long term efficacy. We conducted a 10-year retrospective analysis of outcomes in a group of vaccinated patients. MATERIALS AND METHODS: In 1989-1990, 30 young children with CF, mean age 7 years, with no prior history of infection with P. aeruginosa, were vaccinated against P. aeruginosa with a polyvalent conjugate vaccine. We report the follow-up of 26 of these patients from 1989 to 2001. The patients were given yearly vaccine boosters. Comparisons were made with a CF patient control group matched for gender, age and, where possible, genetic mutation. Vaccinated patients and controls were attending a single CF clinic and received the same clinical management throughout the study period. Main outcomes were time to infection, proportion of patients infected, development of P. aeruginosa mucoid phenotype, lung function and body weight. RESULTS: The time to infection with P. aeruginosa was longer in the vaccination group than in the control group, and fewer vaccinated patients than controls became chronically infected (32% versus 72%; P < 0.001). The proportion of mucoid infections was higher in the control group (44%) than in the vaccinated group (25%). Patients >/=18 years of age at the end of the study had a lower mean forced expiratory volume at 1 s (FEV1) than did those 13-17 years of age, but this difference was small in the vaccinated group (73.6% versus 83.7%) compared with the controls (48.0% versus 78.7%). In the >/=18 year age category the mean FEV1% at 10 years was 73.6% (vaccinated) and 48.0% (controls) (P < 0.05). In the vaccinated group only 11 (44%) of 25 patients were underweight at the 10-year follow-up compared with 18 (72%) of 25 at the beginning of the study. In the control group 17 (68%) of 25 patients were underweight at 10-year follow-up compared with 16 (64%) of 25 at the beginning of the study. CONCLUSION: Regular vaccination of young CF patients for a period of 10 years with a polyvalent conjugate vaccine reduced the frequency of chronic infection with P. aeruginosa. This was associated with better preservation of lung function. Vaccinated patients gained more weight during the study period, a possible indication of an improved overall health status.