title: Characterization of Mannheimia haemolytica biofilm formation in vitro.
abstract: 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.
authors.affiliation: Department of Pathobiological Sciences, University of Wisconsin-Madison, School of Veterinary Medicine, 2015 Linden Drive, Madison, WI 53706, USA. Electronic address: iboukahil@wisc.edu.
authors.forename: Ismail
authors.initials: I
authors.lastname: Boukahil
authors.affiliation: Department of Pathobiological Sciences, University of Wisconsin-Madison, School of Veterinary Medicine, 2015 Linden Drive, Madison, WI 53706, USA. Electronic address: czuprync@svm.vetmed.wisc.edu.
authors.forename: Charles J
authors.initials: CJ
authors.lastname: Czuprynski
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000907:Antibodies, Bacterial / Q000276:immunology*; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology*; D018441:Biofilms / Q000187:drug effects*; D048090:Bovine Respiratory Disease Complex / Q000382:microbiology*; D002417:Cattle; D002418:Cattle Diseases / Q000382:microbiology*; D024881:Drug Resistance, Bacterial; D016978:Mannheimia haemolytica / Q000187:drug effects / Q000276:immunology / Q000502:physiology*; D008954:Models, Biological; D009005:Monosaccharides / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins; D009005:Monosaccharides; C078167:OMPA outer membrane proteins
keywords: Adhesin; Biofilm; Mannheimia haemolytica; OmpA