Mutual antagonism between Mannheimia haemolytica and Pasteurella multocida when forming a biofilm on bovine bronchial epithelial cells in vitro.

Mannheimia haemolytica and Pasteurella multocida are two bacterial species implicated in the bovine respiratory disease complex (BRDC) that is costly to the beef and dairy cattle industries. Both bacterial species are thought to occupy a similar niche as commensals in the upper respiratory tract. Many bacteria are thought to exist as biofilms in their hosts, perhaps in close proximity with other bacterial species. We previously showed that M. haemolytica forms biofilm on bovine respiratory epithelial cells in vitro. We are interested in the possibility that M. haemolytica and P. multocida co-exist as biofilms in the upper respiratory tract of cattle. In this study, we begin to explore this possibility by assessing the ability of M. haemolytica and P. multocida to form a biofilm on bovine respiratory epithelial cells in vitro. We found that M. haemolytica and P. multocida are separately able to form biofilms on bovine respiratory epithelial cells, but mutually inhibit one another when incubated together as a biofilm. Both the biofilm matrix (crystal violet stain) and bacterial numbers (CFU and PCR) were reduced when M. haemolytica and P. multocida were incubated together on fixed epithelial cells. This inhibition does not appear to result from a soluble factor, as neither conditioned medium nor separation of the two species by a transwell filter membrane reproduced the effect. We infer that when located in close proximity on the epithelial surface, M. haemolytica and P. multocida mutually regulate one another.

Mannheimia haemolytica biofilm formation on bovine respiratory epithelial cells.

Mannheimia haemolytica is the most important bacterial agent associated with the bovine respiratory disease complex (BRDC), which causes worldwide economic losses to the cattle industry. M. haemolytica cells initially colonize the tonsillar crypts in the upper respiratory tract of cattle, from where they can subsequently descend into the lungs to cause disease. Many bacteria exist as biofilms inside their hosts. We hypothesize that M. haemolytica colonization of cattle during its commensal state may include biofilm formation. To begin to assess this possibility, we developed an in vitro system to study biofilm formation directly on bovine respiratory epithelial cells. Using fixed primary bovine bronchial epithelial cells, we observed M. haemolytica biofilm formation after a 48h incubation period at 37°C. Addition of mucin, the main component of mucus present in the upper respiratory tract, decreased M. haemolytica biofilm formation on bovine epithelial cells. We investigated the effects of prior viral infection of the epithelial cells on subsequent biofilm formation by M. haemolytica and found negligible effects. Utilization of this model system will provide new insights into the potential role of biofilm formation by M. haemolytica in the pathogenesis of BRDC.

Characterization of Mannheimia haemolytica biofilm formation in vitro.

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/cm(2) of protein, 0.81 µg/cm(2) of total carbohydrate, and 0.47 µg/cm(2) 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.