RESUMO
El objetivo de este estudio fue evaluar la formación de biopelículas por micoplasmas de interés médico.Métodos. La formación de las biopelículas se realizó en microplacas, fueron enjuagadas con solución PBS para remover las células que no se adhirieron y se tiñeron con soluciónde cristal violeta al 0,5% durante 30 minutos. La formación de biopelículas por parte de Mycoplasma pneumoniae, Mycoplasma fermentans y Mycoplasma penetrans se analizó por medio de microscopía óptica y microscopía electrónica de barrido. Resultados. Los micoplasmas evaluados mostraron la capacidad para formar biopelículas,lo cual se evidenció por medio de la tinción de cristal violeta. Las biopelículas formadas en las microplacas y analizadas por microscopía mostraron agregados de microcolonias. La formación de biopelículas por parte de M. fermentans, M. pneumoniae y M. penetrans se presentó a las 72 horas de incubación. Se comparó la formación de biopelícula y la cuantificación de plancton, y se encontró correlación. Conclusión. Se debe considerar que este estudio se hizo bajo condiciones de laboratorioy que, para trabajos futuros, se recomienda utilizar modelos animales para definir cómo contribuyen estos agregados en la persistencia en los huéspedes. Estos resultados sugierenque la capacidad de M. fermentans, M. pneumoniae y M. penetrans para formar biopelículas puede considerarse un factor de virulencia, y un evento importante en la patogénesis yevolución en infecciones asociadas con el uso de dispositivos médicos.
The purpose of this study was to evaluate the development of biofilms by mycoplasmas of medical importance.Methods: Biofilms grown in microtiter plates were rinsed briefly in PBS to remove non-adherent cells and stained with 0,5% crystal violet solution for 30 minutes. The biofilm formation bymycoplasma species were analyzed by optical and scanning electron microscopy. Results: Three mycoplasma species were assessed for their ability to form biofilms. Crystal violet staining of biofilms in microtiter plates revealed the ability of mycoplasma to form a biofilm. Microscopic analysis of crystalviolet stained biofilms on microtiters indicated aggregation to form microcolonies. Biofilm growth by Mycoplasma fermentans, Mycoplasmapneumoniae and Mycoplasmapenetrans was followed over a time course of 72 hours. Mycoplasma were also analyzed quantitatively for biofilm formation and cell counts compared for both biofilm and plankton cells. Cell counts for biofilms showed a goodcorrelation with results obtained using crystal violet staining. Conclusion: This study has examined biofilm formation under in vitro laboratory conditions.Further studies on animal models will be crucial to determine if biofilms form in vivo and whether they contribute to mycoplasma persistence in thehost. These results suggest that the ability of M. fermentans, M. pneumoniae and M. penetrans to form a biofilm may be a virulence factor, and an important event in the pathogenesis and evolutionin infections associated with the use of medical devices.
Assuntos
Mycoplasma pneumoniae , Biofilmes , MycoplasmaRESUMO
Mycoplasmas are a heterogeneous group of the smallest organisms capable of self replication and are known to cause many detrimental diseases in both animals and humans. These wall-less prokaryotes are enveloped by a lipoprotein membrane and their small genomes are sufficient to synthesize molecules required for growth and self-replication. Among sixteen species isolated from humans, Mycoplasma pneumoniae, an agent of primary atypical pneumonia, and the urogenital tract species Mycoplasma hominis,Ureaplasma urealyticum and Ureaplasma parvum have been confirmed to be pathogenic. Mycoplasma penetrans and Mycoplasma fermentans, which are species associated with HIV, have been investigated mainly in research laboratories. In this study we have characterized lipid-associated membrane proteins (LAMP) of Mycoplasma penetrans and Mycoplasma fermentans, in view of the importance of mycoplasmas in human diseases and the peculiar antigenic variation observed in these species. To characterize proteins with possible diagnostic value, we used ELISA and Western blot in sera of pregnant women whose cervical samples were positive for these species of mycoplasmas when tested by PCR. ELISA showed IgG anti-LAMP-M. fermentans antibodies to be present in 57.5% of cases and IgM antibodies to be present in 74.5% of cases. The three samples that were PCR positive for M. penetrans showed IgG anti-LAMP-M. penetrans antibodies, and one sample was positive for IgM. No IgA antibodies against either species were detected in any of the samples. LAMP analysis by Western blot revealed the 35, 38, 42, 61 and 103 kDa proteins of M. penetrans and the 29, 38, 41, 61, 78 and 95 kDa proteins of M. fermentans. Among these, will be considered p35 to M. penetrans and 29 kDa protein to M. fermentans, the main immunoreactive proteins and therefore useful markers for further laboratory diagnosis.
Micoplasmas são procariotos diminutos, desprovidos de parede celular e envoltos por uma membrana lipoproteica cujo pequeno genoma sintetiza a maioria das moléculas necessárias para crescimento e replicação. Dentre as dezesseis espécies isoladas do homem, Mycoplasma pneumoniae, agente causador da pneumonia atípica primária, e as espécies do trato urogenital como Mycoplasma hominis,Ureaplasma urealyticum e Ureaplasma parvum têm definido seu papel patogênico. M. penetrans e M. fermentans, espécies associadas ao HIV, têm sido investigadas principalmente em laboratórios de pesquisa. Considerando a importância dos micoplasmas nas doenças humanas e a peculiar variação antigênica observada em tais espécies, foram caracterizadas, neste estudo, as lipoproteínas associadas a membranas (LAMP) de Mycoplasma penetrans e Mycoplasma fermentans. Para definir peptídeos com possível valor diagnóstico, empregamos as técnicas de ELISA e de Western blot usando soros de gestantes cujas amostras cervicais foram positivas por PCR. Por meio do ELISA foram observados anticorpos IgG anti-LAMP-M. fermentans em 57,5% e IgM em 74,5% das amostras. As três amostras PCR positivas para M. penetrans apresentaram anticorpos IgG anti-LAMP-M. penetrans e uma amostra positiva para IgM. IgA não foi detectada em nenhuma das espécies. A análise da LAMP, por Western blot, revelou como principais proteinas imunoreativas: 35, 38, 42, 61 and 103 kDa para M. penetrans e 29, 38, 41, 61, 78 and 95 kDa de M. fermentans. Dentre estas podemos considerar p35 específica para M. penetrans e p29, M. fermentans.Tais proteinas são promissoras como marcadores em diagnóstico.