Characterization of Mannheimia haemolytica biofilm formation in vitro

• Mannheimia haemolytica forms a biofilm in vitro.
• Biofilm formation is greater in tissue culture medium than in bacteriological broth.
• The biofilm is largely protein with lesser amounts of carbohydrate and nucleic acid.
• Biofilm cells are more resistant than planktonic cells to some antibiotics.

Mannheimia haemolytica is the primary bacterial agent in the bovine respiratory disease complex. It is thought that M. haemolytica colonizes the tonsillar crypts of cattle as a commensal and subsequently descends into the lungs to cause disease. Many bacterial species persist in the host as biofilms. There is limited information about the ability of M. haemolytica to form biofilms. The aim of this study was to develop an in vitro model for M. haemolytica biofilm formation. We found that M. haemolytica required at least 36 h to form robust biofilms on plastic in vitro when incubated in RPMI-1640 tissue culture medium at 37 °C, with maximal biofilm formation being evident at 48 h. Biofilm formation was inhibited by adding the monosaccharides d(+) galactose and d(+) mannose to the growth medium. Addition of antibodies to the M. haemolytica surface protein OmpA also reduced biofilm formation. Upon evaluating the macromolecules within the biofilm extracellular polymeric substance we found it contained 9.7 μg/cm2 of protein, 0.81 μg/cm2 of total carbohydrate, and 0.47 μg/cm2 of extracellular DNA. Furthermore, proteinase K treatment significantly decreased biofilms (P < 0.05) while α-amylase and micrococcal nuclease decreased biofilms to a lesser extent. M. haemolytica biofilm cells were more resistant than planktonic cells to the antibiotics florfenicol, gentamicin, and tulathromycin. These results provide evidence that M. haemolytica can form biofilms, which could contribute to its ability to persist as a commensal in the bovine upper respiratory tract.