RESUMEN
Extracellular DNA (eDNA) is a major component of bacterial biofilms. In this study, we performed a three-dimensional analysis of Leptospira biofilm using advanced imaging by confocal laser scanning microscopy (CLSM) and multi-parameter analysis by COMSTAT 2 software, with quantification of Leptospira and eDNA fluorescence. To investigate the role of eDNA in Leptospira biofilm, we treated Leptospira biflexa biofilms with DNase I enzyme (DNase), which digested eDNA, and compared DNase treated biofilms and controls. There was a significant reduction of the biomass of biofilms treated with DNase, by spectrophotometry and COMSTAT analysis. The multiparameter analysis evidenced for DNase-treated biofilms a significant decrease in the surface area and the average thickness; opposing to a significant augmentation of the surface/biovolume ratio and the roughness coefficient (Ra*), when compared to controls. We analyzed the parameters of DNase-treated biofilms by Pearson's correlation coefficient and found significant positive correlations between biomass and average thickness; biomass and surface area; surface area and average thickness. On the other hand, there were significant negative correlations between Ra* and biomass; Ra* and average thickness; Ra* and surface area. These findings suggest that eDNA digestion results in biofilm instability and alteration of the three-dimensional architecture, justifying the negative correlation between Ra* and the above-mentioned parameters. In conclusion, our study showed that eDNA digestion produced a massive structural loss, instability, and dramatic changes in the three-dimensional architecture of Leptospira biflexa biofilm. These findings contribute to a better understanding of the role of eDNA and highlight the importance of eDNA as a key component in Leptospira biofilms.
RESUMEN
Leptospirosis is a zoonosis caused by bacteria of the genus Leptospira. The disease is globally distributed and a major public health concern. The Norway rat (Rattus norvegicus) is the main reservoir of the pathogen in urban slums of developing and developed countries. The potential routes of intra-specific leptospire transmission in rats are largely unknown. Herein, we identified pathogenic Leptospira spp. in breast tissue and milk of naturally infected rats. We examined kidney, breast tissue and milk from 24 lactating rats for the presence of leptospires using immunofluorescence, immunohistochemistry, polymerase chain reaction (PCR) and scanning electronic microscopy. All 24 rats had evidence for Leptospira in the kidneys, indicating chronic carriage. The majority of kidney-positive rats had detectable leptospires in milk (18, 75%) and breast tissue (16, 67%), as evidenced by immunofluorescence assay and immunohistochemistry. Four (17%) milk samples and two (8%) breast tissue samples were positive by quantitative real-time PCR. Scanning electron microscopy confirmed the presence of leptospires in breast tissue. No major pathological changes in breast tissue were found. This study, for the first time, identified leptospires in the milk and breast tissue of wild Norway rats, suggesting the possibility of milk-borne transmission of leptospirosis to neonates.
