Exopolysaccharide Production and Biofilm Formation by Histophilus somni.

The biofilm matrix of Histophilus somni is a complex architecture that differs substantially in structure between a pathogenic and commensal isolate examined. Overall, most pathogenic isolates produce more biofilm than commensal isolates. A major component of the biofilm is exopolysaccharide (EPS), which is also produced in greater quantity in the pathogenic isolate than in the commensal isolate studied. The EPS is composed of a D-mannan polymer, with occasional galactose residues present on side chains, similar in composition to that of yeast mannan. When grown in the presence of sialic acid, the biofilm EPS becomes sialylated and the amino sugars N-acetylglucosamine and N-acetylgalactosamine can be detected. In vitro biofilm formation follows a typical 4-stage growth curve, characterized by attachment, growth, maturation, and detachment. Following experimental challenge, formation of an H. somni biofilm has been demonstrated in cardiopulmonary tissue, often with Pasteurella multocida cohabitating the biofilm. A recently developed diagnostic test can detect antibodies to the EPS only in animals with systemic disease due to H. somni and is therefore capable of distinguishing between healthy animals colonized with H. somni and animals with systemic disease.

Polymicrobial Biofilm Interaction Between Histophilus somni and Pasteurella multocida.

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.

Biofilm formation and avian immune response following experimental acute and chronic avian cholera due to Pasteurella multocida.

Pasteurella multocida is the causative agent of avian cholera, an important economic and ecological disease that can present as a peracute, acute, chronic, or asymptomatic infection. Acute avian cholera is associated with encapsulated P. multocida, while chronic and asymptomatic cases of avian cholera may be associated with capsule-deficient P. multocida isolates. We hypothesize that biofilm formation is also associated with chronic and asymptomatic avian cholera. Experimental infections of chickens with encapsulated, biofilm-deficient P. multocida strain X73, proficient biofilm forming P. multocida strain X73ΔhyaD, and proficient biofilm forming clinical strains 775 and 756 showed that virulence was inversely correlated with biofilm formation. Biofilm-proficient isolates induced chronic avian cholera in the chicken host. Histopathological analysis was used to show that biofilm-proficient isolates induced little inflammation in the lungs, heart, and liver, while biofilm-deficient isolates induced greater inflammation and induced the recruitment of heterophil granulocytes. Putative biofilm matrix material and exopolysaccharide was detected in pulmonary tissue of chickens diagnosed with chronic avian cholera using scanning electron microscopy and a fluorescently-tagged lectin, respectively, supporting a role for biofilm in chronic avian cholera. P. multocida induced Th1 and Th17 immune responses during acute and chronic avian cholera, as determined by quantitative real-time PCR of splenic cytokine genes. Chickens that succumbed to acute avian cholera after experimental challenge with strain X73 had high levels of INF-γ, IL-1ß, IL-6, IL-12A, IL-22, IL-17A, and IL-17RA expressed in the spleen compared to all other experimental groups. Birds infected with capsule-deficient strains had chronic infections lasting 7 days or longer, and had increased levels of IL-17RA, CCR6, and IL-16 compared to non-infected control chickens. However, specific antibody titers increased only transiently to capsule-deficient strains and were low, indicating that antibodies are less important in managing and clearing P. multocida infections.

Capsular Polysaccharide Interferes with Biofilm Formation by Pasteurella multocida Serogroup A.

Pasteurella multocida is an important multihost animal and zoonotic pathogen that is capable of causing respiratory and multisystemic diseases, bacteremia, and bite wound infections. The glycosaminoglycan capsule of P. multocida is an essential virulence factor that protects the bacterium from host defenses. However, chronic infections (such as swine atrophic rhinitis and the carrier state in birds and other animals) may be associated with biofilm formation, which has not been characterized in P. multocida Biofilm formation by clinical isolates was inversely related to capsule production and was confirmed with capsule-deficient mutants of highly encapsulated strains. Capsule-deficient mutants formed biofilms with a larger biomass that was thicker and smoother than the biofilm of encapsulated strains. Passage of a highly encapsulated, poor-biofilm-forming strain under conditions that favored biofilm formation resulted in the production of less capsular polysaccharide and a more robust biofilm, as did addition of hyaluronidase to the growth medium of all of the strains tested. The matrix material of the biofilm was composed predominately of a glycogen exopolysaccharide (EPS), as determined by gas chromatography-mass spectrometry, nuclear magnetic resonance, and enzymatic digestion. However, a putative glycogen synthesis locus was not differentially regulated when the bacteria were grown as a biofilm or planktonically, as determined by quantitative reverse transcriptase PCR. Therefore, the negatively charged capsule may interfere with biofilm formation by blocking adherence to a surface or by preventing the EPS matrix from encasing large numbers of bacterial cells. This is the first detailed description of biofilm formation and a glycogen EPS by P. multocidaIMPORTANCEPasteurella multocida is an important pathogen responsible for severe infections in food animals, domestic and wild birds, pet animals, and humans. P. multocida was first isolated by Louis Pasteur in 1880 and has been studied for over 130 years. However, aspects of its lifecycle have remained unknown. Although formation of a biofilm by P. multocida has been proposed, this report is the first to characterize biofilm formation by P. multocida Of particular interest is that the biofilm matrix material contained a newly reported amylose-like glycogen as the exopolysaccharide component and that production of capsular polysaccharide (CPS) was inversely related to biofilm formation. However, even highly mucoid, poor-biofilm-forming strains could form abundant biofilms by loss of CPS or following in vitro passage under biofilm growth conditions. Therefore, the carrier state or subclinical chronic infections with P. multocida may result from CPS downregulation with concomitant enhanced biofilm formation.