Investigations into the growth and formation of biofilm by Leptospira biflexa at temperatures encountered during infection

The genus <i>Leptospira</i> comprises unique atypical spirochete bacteria that includes the etiological agent of leptospirosis, a globally important zoonosis. Biofilms are microecosystems composed of microorganisms embedded in a self-produced matrix that offers protection against hostile factors. Leptospires form biofilms <i>in vitro, in situ</i> in rice fields and unsanitary urban areas, and <i>in vivo</i> while colonizing rodent kidneys. The complex three-dimensional biofilm matrix includes secreted polymeric substances such as proteins, extracellular DNA (eDNA), and saccharides. The genus <i>Leptospira</i> comprises pathogenic and saprophytic species with the saprophytic <i>L. biflexa</i> being commonly used as a model organism for the genus. In this study, the growth and formation of biofilms by <i>L. biflexa</i> was investigated not just at 29 °C, but at 37 °C/5 % CO₂, a temperature similar to that encountered during host infection. Planktonic free-living <i>L. biflexa</i> grow in HAN media at both 29 °C and 37 °C/5 % CO_2, but cells grown at 37 °C/5 % CO₂ are longer (18.52 μm ± 3.39) compared to those at 29 °C (13.93 μm ± 2.84). Biofilms formed at 37 °C/5 % CO₂ had more biomass compared to 29 °C, as determined by crystal violet staining. Confocal microscopy determined that the protein content within the biofilm matrix was more prominent than double-stranded DNA, and featured a distinct layer attached to the solid substrate. Additionally, the model enabled effective protein extraction for proteomic comparison across different biofilm phenotypes. Results highlight an important role for proteins in biofilm matrix structure by leptospires and the identification of their specific protein components holds promise for strategies to mitigate biofilm formation.