Conformational features of cepacian: the exopolysaccharide produced by clinical strains of Burkholderia cepacia

Conformational energy calculations and molecular dynamics investigations, both in water and in dimethyl sulfoxide, were carried out on the exopolysaccharide cepacian produced by the majority of the clinical strains of Burkholderia cepacia, an opportunistic pathogen causing serious lung infection in patients affected by cystic fibrosis, the investigation was aimed at defining the structural and conformational features, which might be relevant for clarification of the structure-function relationships of the polymer. The molecular dynamics calculations were carried out by Ramachandran-type energy plots of the disaccharides that constitute the polymer repeating unit. The dynamics of an oligomer composed of three repeating units were investigated in water and in Me2SO, a non-aggregating solvent. Analysis of the time persistence of hydrogen bonds showed the presence of a large number of favourable interactions in water, which were less evident in Me2SO. The calculations on the cepacian chain indicated that polymer conformational features in water were affected by the lateral chains, but were also largely dictated by the presence of solvent. Moreover, the large number of intra-chain hydrogen bonds in water disappeared in Me2SO solution, increasing the average dimension of the polymer chains. (c) 2005 Elsevier Ltd. All rights reserved.

Chloroquine inhibits Paracoccidioides brasiliensis survival within human monocytes by limiting the availability of intracellular iron

The mechanisms used by Paracoccidioides brasiliensis (Pb 18) to survive into monocytes are not clear. Cellular iron metabolism is of critical importance to the growth of several intracellular pathogens, including P. brasiliensis, whose capacity to multiply in mononuclear phagocytes is dependent on the availability of intracellular iron. Chloroquine, by virtue of its basic properties, has been shown to prevent release of iron from holotransferrin by raising endocytic and lysosomal pH, and thereby interfering with normal iron metabolism. Then, in view of this, we have studied the effects of CHLOR on P. brasiliensis multiplication in human monocytes and its effect on the murine paracoccidioidomycosis. CHLOR induced human monocytes to kill P. brasiliensis. The effect of CHLOR was reversed by FeNTA, an iron compound that is soluble at neutral to alkaline pH, but not by holotransferrin, which releases iron only in an acidic environment. CHLOR treatment of Pb 18-infected BALB/c mice significantly reduced the viable fungi recovery from lungs, during three different periods of evaluation, in a dose-dependent manner. This study demonstrates that iron is of critical importance to the survival of P. brasiliensis yeasts within human monocytes and the CHLOR treatment in vitro induces Pb 18 yeast-killing by monocytes by restricting the availability of intracellular iron. Besides, the CHLOR treatment in vivo significantly reduces the number of organisms in the lungs of Pb-infected mice protecting them from several infections. Thus, CHLOR was effective in the treatment of murine paracoccidioidomycosis, suggesting the potential use of this drug in patients' treatment.

Implicações do parasitismo por nematódeos do gênero Rhabdias(Nematoda, Rhabdiasidae) em Crotalus durissus terrificus (Serpentes, Viperidae): alterações pulmonares, microbiológicas e hematológicas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

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.

Função pulmonar dos pacientes com fibrose cística colonizados por Pseudomonas aeruginosa no Pará

A Pseudomonas aeruginosa aparece como um patógeno oportunista na fibrose cística, sendo a maior fonte de infecção pulmonar e é responsável por altas taxas de mortalidade nestes pacientes. O objetivo deste trabalho é de mostrar as características espirométricas e clínicas dos pacientes com fibrose cística atendidos no Hospital Universitário João de Barros Barreto (HUJBB), no Pará. Foi realizado um estudo retrospectivo nos prontuários de 44 pacientes acompanhados no HUJBB, no período de 1997-2007, que atenderam aos critérios de inclusão, sendo 14 colonizados por P. aeruginosa, com a ocorrência de 9 óbitos. Com nível de significância de 5%. No grupo colonizado 10 eram do sexo feminino, a idade média dos sintomas iniciais foi de 0.3±0.6 anos, com diferença significativa quando comparado com pacientes não colonizados (p<0.05). A idade média ao diagnóstico foi de 13.1±10.8 nos colonizados todos apresentavam sintomas respiratórios persistentes ao diagnóstico. A média dos valores percentuais preditos das espirometrias, referentes à avaliação inicial e final, do grupo colonizado foi VEF1(60.0± 25.0%) e (47,82±16.1%) e FEF25-75%(42.5± 22.9%) e (26,5±17.9%) e no grupo não colonizado foi VEF1(79.2± 21.0%) e (79,6±18.0%) e FEF25-75%(69.2± 26.7%) e (68,9±26.8%), respectivamente (p<0.005). A média do escore de Shwachman inicial nos colonizados foi de 42.9±13.5 e nos não colonizados foi de 68.4±15.1(p<0.0001) e na avaliação final foi de 36.6±18.7 e 73.6±12.3 (p<0.0001), respectivamente, com diferença significativa entre os grupos. Os fatores relacionados aos óbitos encontrados foram a colonização por P. aeruginosa, estado nutricional deficiente e VEF1 reduzido. Na casuística estudada foi encontrado maior comprometimento da função pulmonar e idade média mais elevada ao diagnóstico do que em outros estados brasileiros. Estes dados reforçam a necessidade de ações para um diagnóstico precoce no estado do Pará, propiciando uma abordagem terapêutica eficaz e direcionada, buscando aumento de sobrevida e melhoria da qualidade de vida destes indivíduos, objetivo principal das ações dos profissionais envolvidos nesta assistência.

Diversidade genética das espécies de micobactérias não tuberculosas identificadas em laboratório de referência para o diagnóstico da tuberculose na região Norte

Nos últimos anos tem sido observado um aumento de relatos de infecções associadas às micobactérias não tuberculosas (MNT). No entanto, o conhecimento da frequência e as espécies envolvidas nas infecções pulmonares no Brasil são limitados. Neste trabalho foi avaliada a ocorrência de espécies de MNT isoladas no Laboratório de Micobactérias do Instituto Evandro Chagas, Laboratório de Regional de Saúde Pública da Região Norte. Foram analisadas todas as MNT isoladas de espécimes clínicos pulmonares e não pulmonares de indivíduos com infecção, de acordo com os critérios da American Thoracic Society e Ministério da Saúde entre os anos de 2004 a 2007. As MNT foram caracterizadas por PCR-restriction analysis (PRA-hsp65) e sequenciamento dos genes do RNAr 16S, hsp65, rpoB. Foram identificados 51 indivíduos com infecção pulmonar, sendo as seguintes MNT envolvidas: M. abscessus (n=2), M. bolletii (n=4), M. massiliense (n=9), M. avium (n=5), M. colombiense (n=5), M. fortuitum (n=4), M. simiae (n=2), M. interjectum (n=4), M. intracellulare (n=5), M. kansasii (n=1), M. scrofulaceum (n=1) e M. terrae (n=1). Em oito indivíduos foram isoladas MNT não identificadas ao nível de espécie, podendo representar nova entidade taxonômica pertencente ao complexo M.simiae. O presente estudo descreveu a diversidade de MNT isoladas de espécimes clínicos pulmonares no estado do Pará, Região Amazônica Brasileira. O achado de casos infecções pulmonares diagnosticados e tratados sem sucesso por vários meses como tuberculose apontam para a necessidade de isolamento e identificação da micobactéria envolvida antes estabelecimento de falência terapêutica.

Preclinical in vitro and in vivo characterization of the fully human monoclonal IgM antibody KBPA101 specific for Pseudomonas aeruginosa serotype IATS-O11

Pseudomonas aeruginosa infection in ventilator-associated pneumonia is a serious and often life-threatening complication in intensive care unit patients, and new treatment options are needed. We used B-cell-enriched peripheral blood lymphocytes from a volunteer immunized with a P. aeruginosa O-polysaccharide-toxin A conjugate vaccine to generate human hybridoma cell lines producing monoclonal antibodies specific for individual P. aeruginosa lipopolysaccharide serotypes. The fully human monoclonal antibody secreted by one of these lines, KBPA101, is an IgM/kappa antibody that binds P. aeruginosa of International Antigenic Typing System (IATS) serotype O11 with high avidity (5.81 x 10(7) M(-1) +/- 2.8 x 10(7) M(-1)) without cross-reacting with other serotypes. KBPA101 specifically opsonized the P. aeruginosa of IATS O11 serotype and mediated complement-dependent phagocytosis in vitro by the human monocyte-like cell line HL-60 at a very low concentration (half-maximal phagocytosis at 0.16 ng/ml). In vivo evaluation of KBPA101 demonstrated a dose-response relationship for protection against systemic infections in a murine burn wound sepsis model, where 70 to 100% of animals were protected against lethal challenges with P. aeruginosa at doses as low as 5 microg/animal. Furthermore, a high efficacy of KBPA101 in protection from local respiratory infections in an acute lung infection model in mice was demonstrated. Preclinical toxicology evaluation on human tissue, in rabbits, and in mice did not indicate any toxicity of KBPA101. Based on these preclinical findings, the first human clinical trials have been initiated.

Increased surfactant protein D fails to improve bacterial clearance and inflammation in serpinB1-/- mice

Previously, we described the protective role of the neutrophil serine protease inhibitor serpinB1 in preventing early mortality of Pseudomonas aeruginosa lung infection by fostering bacterial clearance and limiting inflammatory cytokines and proteolytic damage. Surfactant protein D (SP-D), which maintains the antiinflammatory pulmonary environment and mediates bacterial removal, was degraded in infected serpinB1-deficient mice. Based on the hypothesis that increased SP-D would rescue or mitigate the pathological effects of serpinB1 deletion, we generated two serpinB1(-/-) lines overexpressing lung-specific rat SP-D and inoculated the mice with P. aeruginosa. Contrary to predictions, bacterial counts in the lungs of SP-D(low)serpinB1(-/-) and SP-D(high) serpinB1(-/-) mice were 4 logs higher than wild-type and not different from serpinB1(-/-) mice. SP-D overexpression also failed to mitigate inflammation (TNF-α), lung injury (free protein, albumin), or excess neutrophil death (free myeloperoxidase, elastase). These pathological markers were higher for infected SP-D(high)serpinB1(-/-) mice than for serpinB1(-/-) mice, although the differences were not significant after controlling for multiple comparisons. The failure of transgenic SP-D to rescue antibacterial defense of serpinB1-deficient mice occurred despite 5-fold or 20-fold increased expression levels, largely normal structure, and dose-dependent bacteria-aggregating activity. SP-D of infected wild-type mice was intact in 43-kD monomers by reducing SDS-PAGE. By contrast, proteolytic fragments of 35, 17, and 8 kD were found in infected SP-D(low)serpinB1(-/-), SP-D(high) serpinB1(-/-) mice, and serpinB1(-/-) mice. Thus, although therapies to increase lung concentration of SP-D may have beneficial applications, the findings suggest that therapy with SP-D may not be beneficial for lung inflammation or infection if the underlying clinical condition includes excess proteolysis.

Ciprofloxacin-induced hematuria

We used ciprofloxacin, a quinolone-derivative, to treat a lung infection due to Pseudomonas aeruginosa in an adult cystic fibrosis patient. On three different occasions the use of ciprofloxacin was associated with the development of an asymptomatic hematuria with red blood cell casts. The mechanism responsible for this hematuria is presently unknown, but clinicians should be aware of this potential adverse effect of ciprofloxacin.

ALLERGEN SENSITIZATION AND IMMUNOMODULATION BY SYNTHETIC LIGANDS, PUL-042, IN AN ALLERGEN-INDUCED ASTHMA MURINE MODEL

Allergen-induced asthma is the leading form of asthma and a chronic condition worldwide. Common allergens are known to contribute to the pathogenesis of this disease. Murine models of allergic asthma have mostly used an intraperitoneal route of sensitization (not airway) to study this disease. Allergic asthma pathophysiology involves the activation of TH2-specific cells, which triggers production of IgE antibodies, the up-regulation of TH2-specific cytokines (i.e. IL-4, IL-5, IL-9 and IL-13), increased airway eosinophilia, and mucin hypersecretion. Although there are several therapeutics currently treating asthmatic patients, some of these treatments can result in drug tolerance and may be linked to increased mortality. CpG oligodeoxynucleotides (ODNs) is a synthetic ligand that targets Toll-like Receptor (TLR) 9. It has been evaluated as a therapeutic agent for the treatment of cancer, infectious diseases, and for treating allergy and asthma. PUL-042 is also a synthetic TLR ligand and is composed of two agonists against TLR2/6 heterodimer and TLR9. Previous studies have evaluated PUL-042 for its ability to confer resistance against bacterial and viral lung infection. These findings, combined with studies performed using CpG ODNs, led to speculation that PUL-042 dampens the immune response in allergen-induced asthma. My thesis research investigated airway route sensitization and airway delivery of PUL-042 to evaluate its effects in reducing an allergen-induced asthma phenotype in a murine model. The results of this study contribute to the foundation for future investigations to evaluate the efficacy of PUL-042 as a novel therapy in allergic-asthma disease.

Protease IV production in Pseudomonas aeruginosa from the lungs of adults with cystic fibrosis

Protease IV is important in the pathogenesis of Pseudomonas aeruginosa-induced microbial keratitis, but little is known of its role in cystic fibrosis (CF) lung infection. In this study protease IV production was examined in 43 P. aeruginosa isolates (24 non-clonal and 19 clonal) from the lungs of chronically infected adult patients attending the Royal Prince Alfred Hospital CF Clinic, Sydney, Australia. Overall, 32/43 (74 %) isolates were positive for protease IV protein by Western blotting and 22/43 (51 %) had evidence of active protease IV on gelatin zymography. Clonal strains were 1.6 times more likely than non-clonal strains to produce protease IV [18/19 (95 %) versus 14/24 (58 %), RR=1.6, CI 1.1–2.3, P=0.007] and 3 times more likely to secrete the protein [16/19 (84 %) versus 6/24 (25 %), RR=3.4, CI 1.6–6.9, P

Eradication and phenotypic tolerance of Burkholderia cenocepacia biofilms exposed to atmospheric pressure non-thermal plasma.

Chronic lung infection with bacteria from the Burkholderia cepacia complex (BCC), and in particular B. cenocepacia, is associated with significant morbidity and mortality in patients with cystic fibrosis (CF). B. cenocepacia can spread from person to person and exhibits intrinsic broad-spectrum antibiotic resistance. Recently, atmospheric pressure non-thermal plasmas (APNTPs) have gained increasing attention as a novel approach to the prevention and treatment of a variety of hospital-acquired infections. In this study, we evaluated an in-house-designed kHz-driven plasma source for the treatment of biofilms of a number of clinical CF B. cenocepacia isolates. The results demonstrated that APNTP is an effective and efficient tool for the eradication of B. cenocepacia biofilms but that efficacy is highly variable across different isolates. Determination of phenotypic differences between isolates in an attempt to understand variability in plasma tolerance revealed that isolates which are highly tolerant to APNTP typically produce biofilms of greater biomass than their more sensitive counterparts. This indicates a potential role for biofilm matrix components in biofilm tolerance to APNTP exposure. Furthermore, significant isolate-dependent differences in catalase activity in planktonic bacteria positively correlated with phenotypic resistance to APNTP by isolates grown in biofilms.

Oral re-vaccination of Eurasian wild boar with Mycobacterium bovis BCG yields a strong protective response against challenge with a field strain

BACKGROUND Field vaccination trials with Mycobacterium bovis BCG, an attenuated mutant of M. bovis, are ongoing in Spain, where the Eurasian wild boar (Sus scrofa) is regarded as the main driver of animal tuberculosis (TB). The oral baiting strategy consists in deploying vaccine baits twice each summer, in order to gain access to a high proportion of wild boar piglets. The aim of this study was to assess the response of wild boar to re-vaccination with BCG and to subsequent challenge with an M. bovis field strain. RESULTS BCG re-vaccinated wild boar showed reductions of 75.8% in lesion score and 66.9% in culture score, as compared to unvaccinated controls. Only one of nine vaccinated wild boar had a culture-confirmed lung infection, as compared to seven of eight controls. Serum antibody levels were highly variable and did not differ significantly between BCG re-vaccinated wild boar and controls. Gamma IFN levels differed significantly between BCG re-vaccinated wild boar and controls. The mRNA levels for IL-1b, C3 and MUT were significantly higher in vaccinated wild boar when compared to controls after vaccination and decreased after mycobacterial challenge. CONCLUSIONS Oral re-vaccination of wild boar with BCG yields a strong protective response against challenge with a field strain. Moreover, re-vaccination of wild boar with BCG is not counterproductive. These findings are relevant given that re-vaccination is likely to happen under real (field) conditions.

Characterising novel anti-biofilm targets for the treatment of chronic Pseudomonas aeruginosa infection in the cystic fibrosis lung

The global rise in antibiotic resistance is a significant problem facing healthcare professionals. In particular within the cystic fibrosis (CF) lung, bacteria can establish chronic infection and resistance to a wide array of antibiotic therapies. One of the principle pathogens associated with chronic infection in the CF lung is Pseudomonas aeruginosa. P. aeruginosa can establish chronic infection in the CF lung partly through the use of the biofilm mode of growth. This biofilm mode of growth offers a considerable degree of protection from a wide variety of challenges such as the host immune system or antibiotic therapy. The threat posed by the emergence of chronic pathogens is prompting the development of next generation antimicrobials. The biofilm mode of growth is often central to the establishment of chronic infection and the development of antibiotic resistance. Thus, targeting biofilm formation has emerged as one of the principle strategies for the development of next generation antimicrobials. In this thesis two separate approaches were used to identify potential anti - biofilm targets. The first strategy focused on the identification of novel genes with a role in a biofilm formation. High throughput screening identified almost 300 genes which had a role in biofilm formation. A number of these genes were characterised at a phenotypic and a molecular level. The second strategy focused on the identification of compounds capable of inhibiting biofilm formation. A collection of marine sponge isolated bacteria were screened for the ability to inhibit the central pathway regulating biofilm formation, quorum sensing. A number of distinct isolates were identified that had quorum sensing inhibition activity from which, a Pseudomonas isolate was selected for further characterisation. A specific compound capable of inhibiting quorum sensing was identified using chemical analytical technologies in the supernatant of this marine isolate.