Nontypeable Haemophilus influenzae Strain 2019 Produces a Biofilm Containing N -Acetylneuraminic Acid That May Mimic Sialylated O-Linked Glycans

Abstract

Previous studies suggested that nontypeable Haemophilus influenzae (NTHI) can form biofilms during human and chinchilla middle ear infections. Microscopic analysis of a 5-day biofilm of NTHI strain 2019 grown in a continuous-flow chamber revealed that the biofilm had a diffuse matrix interlaced with multiple water channels. Our studies showed that biofilm production was significantly decreased when a chemically defined medium lacking N-acetylneuraminic acid (sialic acid) was used. Based on these observations, we examined mutations in seven NTHI strain 2019 genes involved in carbohydrate and lipooligosaccharide biosynthesis. NTHI strain 2019 with mutations in the genes encoding CMP-N-acetylneuraminic acid synthetase (siaB), one of the three NTHI sialyltransferases (siaA), and the undecaprenyl-phosphate ␣-N-acetylglucosaminyltransferase homolog (wecA) produced significantly smaller amounts of biofilm. NTHI strain 2019 with mutations in genes encoding phosphoglucomutase (pgm), UDP-galactose-4-epimerase, and two other NTHI sialyltransferases (lic3A and lsgB) produced biofilms that were equivalent to or larger than the biofilms produced by the parent strain. The biofilm formed by the NTHI strain 2019pgm mutant was studied with Maackia amurensis fluorescein isothiocyanate (FITC)-conjugated and Sambucus nigra tetramethyl rhodamine isocyanate (TRITC)-conjugated lectins. S. nigra TRITC-conjugated lectin bound to this biofilm, while M. amurensis FITC-conjugated lectin did not. S. nigra TRITC-conjugated lectin binding was inhibited by incubation with ␣2,6-neuraminyllactose and by pretreatment of the biofilm with Vibrio cholerae neuraminidase. Matrix-assisted laser desorption ionization-time of flight mass spectometry analysis of lipooligosaccharides isolated from a biofilm, the planktonic phase, and plate-grown organisms showed that the levels of most sialylated glycoforms were twoto fourfold greater when the lipooligosaccharide was derived from planktonic or biofilm organisms. Our data indicate that NTHI strain 2019 produces a biofilm containing ␣2,6-linked sialic acid and that the sialic acid content of the lipooligosaccharides increases concomitant with the transition of organisms to a biofilm form.

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

“…More detailed analysis by scanning electron microscopy showed dense multilayered bacterial communities (Fig. 5C) that are reminiscent of those observed by our group and others in vitro (12,38,43,48).…”

“…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).…”

Antimicrobial Effect of Fluoroquinolones for the Eradication of Nontypeable Haemophilus Influenzae Isolates within Biofilms

“…However, the pathogenesis of NTHi infection of airway cells involves both an intracellular life cycle as well as biofi lm formation on the surface of the airway epithelia. Therefore, because NTHi invade human bronchial epithelial cells by macropinocytosis (Ketterer et al 1999) and form biofi lms both in vitro and in vivo (Greiner et al 2004;Jurcisek et al 2005;Hall-Stoodley et al 2006), it is not clear whether drug resistance alone causes treatment diffi culty and failure in otitis media among young children. To our knowledge, the antimicrobial effect of antibiotics against NTHi within biofi lms remains unclear.…”

“…Recent reports state that NTHi forms biofi lms both in vitro and in vivo, suggesting that biofi lm formation in vivo might play an important role in the pathogenesis and chronicity of otitis media (Greiner et al 2004;Jurcisek et al 2005;Hall-Stoodley et al 2006). Very little information is available concerning the effectiveness of various antimicrobials for the eradication of NTHi within biofi lms.…”

“…Biofi lm formation in the H99 and H01 strains were assessed using a 96-well microplate, as previously described (Murphy and Kirkham 2002;Greiner et al 2004). After being cultured for 48 hrs, the biofi lms were exposed for 1 hr to differing concentrations (0.1 x MIC, 1 x MIC and 10 x MIC) of each antibiotics (AMP, CTX, ERY, CLR, LVX and GAT) solution every 12 hrs for 2 days (totally 4 doses).…”