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Polymicrobial Biofilm Interaction Between Histophilus somni and Pasteurella multocida
Briana Petruzzi1, Allan Dickerman2, Kevin Lahmers1
1Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, United States.
Abstract:
Histophilus somni and Pasteurella multocida are two of multiple agents responsible for bovine respiratory disease (BRD) in cattle. Following respiratory infection of calves with H. somni, P. multocida may also be isolated from the lower respiratory tract. Because H. somni may form a biofilm during BRD, we sought to determine if P. multocida can co-exist with H. somni in a polymicrobial biofilm in vitro and in vivo. Interactions between the two species in the biofilm were characterized and quantified by fluorescence in situ hybridization (FISH). The biofilm matrix of each species was examined using fluorescently tagged lectins (FTL) specific for the exopolysaccharide (EPS) using confocal laser scanning microscopy. Bacterial interactions were determined by auto-aggregation and biofilm morphology. Pasteurella multocida and H. somni were evenly distributed in the in vitro biofilm, and both species contributed to the polymicrobial biofilm matrix. The average biomass and biofilm thickness, and the total carbohydrate and protein content of the biofilm, were greatest when both species were present. Polymicrobial bacterial suspensions auto-aggregated faster than single species suspensions, suggesting physical interactions between the two species. Almost 300 P. multocida genes were significantly differentially regulated when the bacteria were in a polymicrobial biofilm compared to a mono-species biofilm, as determined by RNA-sequencing. As expected, host genes associated with inflammation and immune response were significantly upregulated at the infection site following H. somni challenge. Encapsulated P. multocida isolates not capable of forming a substantial biofilm enhanced an in vitro polymicrobial biofilm with H. somni, indicating they contributed to the polymicrobial biofilm matrix. Indirect evidence indicated that encapsulated P. multocida also contributed to a polymicrobial biofilm in vivo. Only the EPS of H. somni could be detected by FTL staining of bovine tissues following challenge with H. somni. However, both species were isolated and an immune response to the biofilm matrix of both species was greater than the response to planktonic cells, suggesting encapsulated P. multocida may take advantage of the H. somni biofilm to persist in the host during chronic BRD. These results may have important implications for the management and prevention of BRD.
Insights
Histophilus somni and Pasteurella multocida form enhanced biofilms together, increasing biomass and gene regulation. This polymicrobial biofilm aids Pasteurella multocida persistence in cattle with bovine respiratory disease.
Area of Science:
- Veterinary Microbiology
- Bacterial Pathogenesis
- Host-Pathogen Interactions
Background:
- Bovine respiratory disease (BRD) is a significant economic concern in cattle, often involving multiple bacterial agents.
- Histophilus somni is known to form biofilms, potentially influencing disease chronicity.
- Pasteurella multocida frequently co-infects cattle with H. somni, but its role in polymicrobial biofilms is less understood.
Purpose of the Study:
- To investigate the in vitro and in vivo coexistence of Histophilus somni and Pasteurella multocida in polymicrobial biofilms.
- To characterize the interactions and matrix contributions of these two species within a biofilm.
- To determine the impact of polymicrobial biofilm formation on bacterial gene expression and host immune response.
Main Methods:
- In vitro and in vivo biofilm formation assays.
- Fluorescence in situ hybridization (FISH) for bacterial localization and quantification.
- Confocal laser scanning microscopy with fluorescently tagged lectins (FTL) to analyze biofilm matrix composition (exopolysaccharide - EPS).
- RNA-sequencing to assess differential gene regulation in polymicrobial vs. monospecies biofilms.
- Host gene expression analysis for inflammation and immune response markers.
Main Results:
- Histophilus somni and Pasteurella multocida were evenly distributed in in vitro biofilms, with both contributing to the matrix.
- Polymicrobial biofilms exhibited increased biomass, thickness, and total carbohydrate/protein content compared to monospecies biofilms.
- Polymicrobial suspensions showed faster auto-aggregation, indicating physical interactions.
- RNA-seq revealed significant differential regulation of nearly 300 P. multocida genes in polymicrobial biofilms.
- Host immune response genes were upregulated at the infection site following H. somni challenge.
- Encapsulated P. multocida enhanced H. somni biofilms in vitro and showed indirect evidence of contribution in vivo.
- While only H. somni EPS was detected in bovine tissues, both species persisted, and immune response to the polymicrobial biofilm was heightened.
Conclusions:
- Histophilus somni and Pasteurella multocida can form robust polymicrobial biofilms, enhancing structural integrity and bacterial gene expression.
- Pasteurella multocida, particularly encapsulated strains, may leverage H. somni biofilms for persistence during chronic BRD.
- Understanding these interactions is crucial for developing effective BRD management and prevention strategies.
