Role of Sialic Acid and Complex Carbohydrate Biosynthesis in Biofilm Formation by Nontypeable Haemophilus influenzae in the Chinchilla Middle Ear

Abstract

Nontypeable Haemophilus influenzae (NTHI) is an important pathogen in respiratory tract infections, including otitis media (OM). NTHI forms biofilms in vitro as well as in the chinchilla middle ear, suggesting that biofilm formation in vivo might play an important role in the pathogenesis and chronicity of OM. We've previously shown that SiaA, SiaB, and WecA are involved in biofilm production by NTHI in vitro. To investigate whether these gene products were also involved in biofilm production in vivo, NTHI strain 2019 and five isogenic mutants with deletions in genes involved in carbohydrate biosynthesis were inoculated into the middle ears of chinchillas. The wild-type strain formed a large, well-organized, and viable biofilm; however, the wecA, lsgB, siaA, pgm, and siaB mutants were either unable to form biofilms or formed biofilms of markedly reduced mass, organization, and viability. Despite their compromised ability to form a biofilm in vivo, wecA, lsgB, and siaA mutants survived in the chinchilla, inducing culture-positive middle ear effusions, whereas pgm and siaB mutants were extremely sensitive to the bactericidal activity of chinchilla serum and thus did not survive. Lectin analysis indicated that sialic acid was an important component of the NTHI 2019 biofilm produced in vivo. Our data suggested that genes involved in carbohydrate biosynthesis and assembly play an important role in the ability of NTHI to form a biofilm in vivo. Collectively, we found that when modeled in a mammalian host, whereas biofilm formation was not essential for survivability of NTHI in vivo, lipooligosaccharide sialylation was indispensable.

Phosphorylcholine Decreases Early Inflammation and Promotes the Establishment of Stable Biofilm Communities of Nontypeable Haemophilus influenzae Strain 86-028NP in a Chinchilla Model of Otitis Media

“…However, it is noteworthy that we were consistently able to recover viable bacteria from middle ear effusions of those animals infected with NTHi 86-028NP licD that had recoverable middle ear effusion fluids. This finding is reminiscent of prior work showing that some variants can persist in the planktonic phase within the middle ear despite failing to form biofilms (18).…”

“…A large body of work shows that populations of NTHi in vivo contain variants that are sialylated (18,24) and PCho ϩ (45,52,53). Moreover, prior work has clearly shown that NeuAc promotes biofilm formation and bacterial persistence in animal models of NTHi disease (4,18,43).…”

“…Bacterial counts were obtained from tapped middle ear fluids by serial dilution and plate count, and all remaining middle ear fluids were stored at Ϫ80°C until further study. Examination of biofilms within the middle ear was performed essentially as described previously (18). Briefly, after euthanasia, the bullae were aseptically opened to expose the middle ear cavity.…”

“…OM infections include chronic OM that is difficult to resolve with antibiotic therapy, and it has long been postulated that chronic OM involves the formation of bacterial biofilm communities (5,35). In support of that hypothesis, biofilms have been visualized in tympanostomy drain tubes removed from patients with OM and on middle ear tissue from experimentally infected chinchillas (7,18,33). More recent evidence shows that NTHi and other bacterial agents are present within biofilms on tissue specimens obtained from patients with chronic and recurrent OM (13).…”

“…LOS confers resistance to host killing (8,9,37) and is also the primary target of the Toll-like receptor 4 pathway that mediates protection against H. influenzae in the airways (47). It has been established that NTHi strains that express NeuAc-LOS forms comprise a greater proportion of biofilm communities than of planktonic cultures, and that mutations eliminating these forms decrease biofilm formation and bacterial persistence in animal models of OM (4,12,18,43). More recently, we showed that LOS purified from biofilms has decreased potency as an inflammatory agonist, which correlated with an increase in PCho ϩ LOS forms that were present within biofilms (55).…”