Pasteurella multocida is an important multi-host animal and zoonotic pathogen that is capable of causing respiratory and multi-systemic diseases, bacteremia, and infections resulting from bite wounds. The glycosaminoglycan capsule (CPS) of P. multocida is an essential virulence factor, protecting the bacterium from host defenses. However, chronic infections such as bovine respiratory disease (BRD) and avian cholera may be associated with biofilm formation.
Biofilm formation was inversely related to capsule production (determined by uronic acid and N-acetylglucosamine assays), and was confirmed with capsule-deficient mutants of mucoid strains. Capsule-deficient mutants formed biofilms with a larger biomass that was much thicker and smoother than encapsulated strains.
Gas chromatography-mass spectrometry, nuclear magnetic resonance, and enzymatic digestion demonstrated that the matrix material of the biofilm was composed predominately of a glycogen exopolysaccharide (EPS). Therefore, CPS may interfere with biofilm formation by blocking adherence to a surface or by preventing the EPS matrix to encase large numbers of bacterial cells.
Chemical mutagenesis was performed on P. multocida strain P1059, resulting in isolation of an acapsular mutant designated as P1059-R8. A uridyltransferase encoded by gene P1059_01979 was mutated in such a way that a polar amino acid was changed to a non-polar amino acid near the active site. The protein product of P1059_01979 is important for the biosynthesis of the CPS subunit N-acetylglucosamine. CPS quantification revealed that the subunit glucuronic acid was produced in equal concentrations to the parent, but the CPS subunit N-acetylglucosamine was not detected in the chemical mutant. Biofilm formation in the chemical mutant was significantly higher than in WT P1059 and the capsule-deficient mutant. We hypothesize that P1059_01979 is essential for CPS production in P. multocida serogroup A.
Histophilus somni and Pasteurella multocida cause bovine respiratory disease (BRD) and systemic infections in cattle. Following respiratory infection of calves with H. somni, P. multocida is also often isolated from the lower respiratory tract. Because H. somni normally forms a biofilm during BRD, we suspected that P. multocida may co-exist with H. somni in a polymicrobial biofilm. Interactions between the two species in the biofilm were characterized and quantified by fluorescence in situ hybridization (FISH), and the biofilm matrix of each species examined by fluorescently-tagged lectins (FTL), confocal scanning laser microscopy of in vitro biofilms and bovine pulmonary tissue following dual H. somni and P. multocida infection. FISH and FTL were used to show that P. multocida and H. somni were evenly distributed in the in vitro biofilm, and both species contributed to the polymicrobial biofilm matrix. COMSTAT z-stack image analysis revealed that the average biomass and biofilm thickness of the individual and polymicrobial biofilms were greatest when both species were present. Encapsulated P. multocida isolates not capable of forming a biofilm still formed a polymicrobial biofilm with H. somni, but only the EPS of H. somni could be detected by FTL staining of bovine tissues from which both species were isolated. Bacteria within a biofilm are more quiescent than during planktonic growth and induce less of an inflammatory response, indicating encapsulated P. multocida may take advantage of the H. somni biofilm to persist in the host during less severe, but more chronic, BRD. These results may have important implications for the management of BRD.
Acute avian cholera is associated with encapsulated P. multocida, while chronic and asymptomatic cases of avian cholera are associated with acapsular P. multocida isolates. We hypothesize that biofilm formation is present and an important factor for chronic and asymptomatic avian cholera. Experimental infections of chickens with biofilm deficient P. multocida strain WT X73, proficient biofilm forming P. multocida strain X73ΔhyaD, and proficient biofilm forming clinical isolates 775 and 756 showed that virulence inversely correlated with biofilm formation. Histopathological analysis showed that biofilm forming isolates induced little inflammation in the lungs, heart, and liver, while biofilm deficient isolates induced greater inflammation. Biofilm material was located in pulmonary tissues of chickens diagnosed with chronic avian cholera using FTL staining.. Quantitative real-time PCR for expression of cytokine genes in the spleens of infected chickens indicated that P. multocida induced Th1 and Th17 immune responses during acute and chronic avian cholera. Chickens that succumbed to acute avian cholera after experimental challenge with WT X73 had high levels of INF-ƴ, IL-1β, IL-6, IL-12A, IL-22, IL-17A, and IL-17RA expression in the spleen compared to all other experimental groups. Antibody titers were low, indicating that antibodies may be less important in managing and clearing P. multocida infections. / Ph. D. / Pasteurella multocida is a zoonotic pathogen, which means it can be transferred from animals to humans as part of the normal flora of many animals including household pets such as cats and dogs, and agriculture species such as cattle. P. multocida is responsible for infected animal bites, especially those resulting from household and large cats. Additionally, P. multocida is responsible for several diseases of veterinary importance, including avian cholera and bovine respiratory disease (BRD).
Capsule, composed of capsular polysaccharide (CPS), is an essential virulence factor for P. multocida. Virulence factors are genetically encoded attributes that aid the bacteria in causing an infection. Capsule covers the surface of bacterial cells, which allows P. multocida to survive within the host and avoid detection by the immune system. The P. multocida capsular serogroup A is composed of hyaluronic acid.
Biofilms are communities of bacteria that survive within a hydrated matrix composed of polysaccharides, proteins, enzymes, antimicrobial compounds, extracellular DNA, and other bacterial and host components. Biofilms can be compared to multicellular organs of eukaryotes. While less complex, biofilms similarly regulate nutrients, water, composition, remove waste, and perform other processes such as DNA transfer. Biofilms protect bacterial communities by shielding them from the host immune response. Bacteria living in biofilms also grow slowly, and as a result are protected from many antibiotic treatments. While biofilm formation has been suggested for P. multocida, the biofilm has not yet been characterized. The work reported here characterizes biofilm formation by P. multocida isolates of capsular serogroup A. Biofilms formed by P. multocida were stained with fluorescently-tagged lectins, DNA stain, and other fluorescent dyes, as well as crystal violet stain. Biofilms were imaged using several microscopy techniques. Biofilm formation was prominent for serogroup A strains of P. multocida that were acapsular. However, in the presence of CPS, biofilm formation was inhibited.
H. somni forms a biofilm during BRD that allows the bacterium to survive within the heart and lungs of the bovine host. BRD is often caused by several different bacterial, viral, and even parasitic microbes – resulting in a polymicrobial disease. Polymicrobial diseases are more difficult to diagnose and treat, which is a challenge when trying to control this economically important disease. Experimental infections of bovines with H. somni have resulted in polymicrobial infections with P. multocida. We hypothesize that these two bacterial species may form a mutualistic or commensalistic interaction together during BRD to improve the survival of one or both species within the host. The polymicrobial biofilm was observed using fluorescent microscopy techniques. We confirmed that H. somni and P. multocida form a polymicrobial biofilm.
Avian cholera can be an acute, chronic, or asymptomatic disease that affects poultry farms and migratory flocks around the world. The spread of P. multocida and avian cholera is thought to occur through infected water, infected insects, and through other infected animals surrounding water supplies such as deer, raccoon, and even fish. We hypothesize that P. multocida can produce a biofilm and survive within the respiratory tract of birds for extended periods of time, that biofilm formation is important for the establishment of chronic and asymptomatic avian cholera, and that a biofilm assists in the spread of disease between flocks of birds. Chickens were challenged in the respiratory tract with a highly encapsulated, poor biofilm forming strain, or a prominent biofilm forming strain. After 7, 14, and 28 days chicken lungs were examined to identify bacteria, biofilm material, and inflammation. Biofilm-forming P. multocida strains were less virulent and caused less inflammation than non-biofilm forming P. multocida strains. Biofilms were visible in the airways of pulmonary tissue by scanning electron microscopy. Biofilm formation by P. multocida was observed within the pulmonary tissue of chickens with chronic and acute avian cholera.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/92695 |
| Date | 08 February 2018 |
| Creators | Petruzzi, Briana Lynn |
| Contributors | Biomedical and Veterinary Sciences, Inzana, Thomas J., Pierson, Frank W., Edgar, Kevin J., Caswell, Clayton C. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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