RESUMO
The available pneumococcal conjugate vaccines provide protection against only those serotypes that are included in the vaccine, which leads to a selective pressure and serotype replacement in the population. An alternative low-cost, safe and serotype-independent vaccine was developed based on a nonencapsulated pneumococcus strain. This study evaluates process intensification to improve biomass production and shows for the first time the use of perfusion-batch with cell recycling for bacterial vaccine production. Batch, fed-batch, and perfusion-batch were performed at 10 L scale using a complex animal component-free culture medium. Cells were harvested at the highest optical density, concentrated and washed using microfiltration or centrifugation to compare cell separation methods. Higher biomass was achieved using perfusion-batch, which removes lactate while retaining cells. The biomass produced in perfusion-batch would represent at least a fourfold greater number of doses per cultivation than in the previously described batch process. Each strategy yielded similar vaccines in terms of quality as evaluated by western blot and animal immunization assays, indicating that so far, perfusion-batch is the best strategy for the intensification of pneumococcal whole-cell vaccine production, as it can be integrated to the cell separation process keeping the same vaccine quality.
Assuntos
Técnicas de Cultura Celular por Lotes/instrumentação , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Animais , Técnicas de Cultura Celular por Lotes/métodos , Biomassa , Reatores Biológicos , Desenho de Equipamento , Feminino , Humanos , Imunização , Camundongos Endogâmicos C57BL , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/uso terapêutico , Pneumonia Pneumocócica/imunologia , Pneumonia Pneumocócica/prevenção & controle , Streptococcus pneumoniae/citologiaRESUMO
Streptococcus pneumoniae is a human pathogen whose principal virulence factor is its capsule. This structure allows the bacterium to evade the human immune system. Treatment of infections caused by this bacterium is based on antibiotics; however, the emergence of antibiotic-resistant strains makes this task increasingly difficult. Therefore, it is necessary to investigate new therapies, such as those based on gold nanoparticles, for which unfortunately the mechanisms involved have not yet been investigated. As far as we know, this study is the first that attempts to explain how gold nanoparticles destroy the bacterium Streptococcus pneumoniae. We found that the mean particle size was an important issue, and that the effect on the bacterium was dose-dependent. Cellular growth was inhibited by the presence of the nanoparticles, as was cell viability. The pH of the bacterial growth media was acidified, but interestingly the reactive species were not affected. A transmission electron microscopy analysis revealed the presence of inclusion bodies of gold nanoparticles within the bacterium. We present the first findings that attempt to explain how gold nanoparticles lyse Gram-positive bacteria.
Assuntos
Ouro/química , Ouro/farmacologia , Nanopartículas Metálicas/química , Streptococcus pneumoniae/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Nanopartículas Metálicas/ultraestrutura , Infecções Pneumocócicas/tratamento farmacológico , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/citologia , Streptococcus pneumoniae/crescimento & desenvolvimentoRESUMO
Streptococcus pneumoniae, a human pathogen bacterium, can support its growth using haemoglobin (Hb) and haem as sole iron sources, but not when holo-transferrin or holo-lactoferrin is supplied. For this reason, it is easy to think that the principal iron sources for this pathogen inside humans are Hb and haem. Unfortunately, the mechanism has been poorly studied. The findings presented in this study are the first efforts that attempted to explain the mechanism involved in iron acquisition of this pathogen. This pathogen was capable of supporting its viability when iron sources such as Hb or haem were supplied. Membranes of S. pneumoniae were separated and their respective proteins were solubilized in order to be purified by haem-affinity chromatography. This strategy allowed us to purify seven membrane proteins. An experiment of competence with haem and iron showed two potential haem and Hb-binding proteins. Their Hb-binding function was confirmed by overlay assay using Hb and their respective identities were obtained by mass spectrometry. Then by amino acid alignment analysis, the motif involved in binding of Hb or haem was revealed. These results are the first findings that attempt to explain the mechanisms developed by S. pneumoniae to acquire iron from Hb or haem in the host, which could allow a better understanding of the biology of this bacterium.
Assuntos
Proteínas de Bactérias/metabolismo , Heme/metabolismo , Hemoglobinas/metabolismo , Ferro/farmacologia , Proteínas de Membrana/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Streptococcus pneumoniae/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Humanos , Espectrometria de Massas , Proteínas de Membrana/química , Dados de Sequência Molecular , Peso Molecular , Ligação Proteica/efeitos dos fármacos , Streptococcus pneumoniae/citologia , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/crescimento & desenvolvimentoRESUMO
BACKGROUND: Streptococcus pneumoniae, particularly penicillin-resistant strains (PRSP), constitute one of the most important causes of serious infections worldwide. It is a fastidious microorganism with exquisite nutritional and environmental requirements to grow, a characteristic that prevents the development of useful animal models to study the biology of the microorganism. This study was designed to determine optimal conditions for culture and growth of PRSP. RESULTS: We developed a simple and reproducible method for culture of diverse strains of PRSP representing several invasive serotypes of clinical and epidemiological importance in Colombia. Application of this 3-step culture protocol consistently produced more than 9 log10 CFU/ml of viable cells in the middle part of the logarithmic phase of their growth curve. CONCLUSION: A controlled inoculum size grown in 3 successive steps in supplemented agar and broth under 5% CO2 atmosphere, with pH adjustment and specific incubation times, allowed production of great numbers of PRSP without untimely activation of autolysis mechanisms.