Considerable evidence has implicated Streptococcus pneumoniae neuraminidase in the pathogenesis of otitis media (OM); however, its exact role has not been conclusively established. Recently, an S. pneumoniae neuraminidase-deficient mutant, ⌬NA1, has been constructed by insertion-duplication mutagenesis of the nanA gene of S. pneumoniae strain D39. The relative ability of ⌬NA1 and the D39 parent strain to colonize the nasopharynx and to induce OM subsequent to intranasal inoculation and to survive in the middle ear cleft after direct challenge of the middle ear were evaluated in the chinchilla model. Nasopharyngeal colonization data indicate a significant difference in the ability of the ⌬NA1 mutant to colonize as well as to persist in the nasopharynx. The neuraminidase-deficient mutant was eliminated from the nasopharynx 2 weeks earlier than the D39 parent strain. Both the parent and the mutant exhibited similar virulence levels and kinetics during the first week after direct inoculation of the middle ear. The ⌬NA1 neuraminidase-deficient mutant, however, was then completely eliminated from the middle ear by day 10 postchallenge, 11 days before the D39 parent strain. Data from this study indicate that products of the nanA gene have an impact on the ability of S. pneumoniae to colonize and persist in the nasopharynx as well as the middle ear.Streptococcus pneumoniae is one of the primary bacterial pathogens associated with otitis media (OM) and accounts for approximately 30% of all cases of this disease (11).Neuraminidase is an enzyme which cleaves N-acetylneuraminic acid from mucin, glycolipids, glycoproteins, and oligosaccharides on host cell surfaces. Although a precise role for S. pneumoniae neuraminidase in the pathogenesis of S. pneumoniae-caused diseases has not been established, it has been proposed that neuraminidase could enhance colonization by decreasing the viscosity of mucus (13) or by exposing cell surface receptors for S. pneumoniae (1,7,8). All clinical S. pneumoniae isolates examined to date have been shown to produce neuraminidase (6). Production of neuraminidase is believed to contribute to the poor prognosis for pneumococcal meningitis (12). Moreover, S. pneumoniae neuraminidase has been detected in 78% of culture-positive middle ear effusions from patients with acute OM and in 96% of S. pneumoniaepositive middle ear effusions from patients with chronic OM (4). Two recent reports from our laboratory indicate that during S. pneumoniae-induced OM in the chinchilla model, terminal sialic acid residues are removed from the epithelial surface lining the lumen of the eustachian tube, presumably as a result of S. pneumoniae neuraminidase production (7, 8). Similar data have been derived clinically from adenoidal tissue obtained from children with chronic OM with effusion. Adenoids colonized with S. pneumoniae demonstrated removal of sialic acid and exposure of N-acetylglycosamine (9). The result is exposure of GlcNAc␤1-4Gal, which is part of one of the eukaryotic receptors of S. pneumoniae (1). More...

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The trachea whole organ perfusion technique was used to study the effect of the disruption of the Streptococcus pneumoniae neuraminidase nanA gene on bacterial adherence and alteration of the carbohydrate surface structures of respiratory epithelial cells. Six different lectin probes were used to examine alterations of the cell surface carbohydrates in chinchilla tracheal epithelium incubated in vitro with S. pneumoniae deltaNA1, a neuraminidase-deficient mutant, or its D39 parent strain. The labeling pattern revealed that the binding of wheat germ agglutinin (WGA), Erythrina cristagalli lectin (ECL), peanut agglutinin (PNA), Bandeiraea simplicifolia lectin II (BSL II) and succinylated WGA was significantly increased in the luminal surface of the trachea in the D39-incubated cohort compared with the uninfected control, which indicated that GlcNAc and D-galactose residues were exposed. Concurrently, decreased labeling with Sambucus nigra agglutinin (SNA) indicated that there were few sialic acid residues remaining in the tracheal epithelium subsequent to incubation with D39. The deltaNA1 neuraminidase-deficient mutant, however, did not induce any significant changes in the lectin labeling patterns, which were comparable to those of the control cohort. Moreover, adherence data expressed as colony-forming units (CFU) of S. pneumoniae per millimeter of trachea indicated a significant decline in the ability of deltaNA1 to adhere in vitro. We propose that products of the nanA gene have a significant impact on changes in the carbohydrate moieties in the tracheal epithelium, and may be responsible for the previously reported increased ability of the D39 parent to colonize the nasopharynx and invade the middle ear.

Nontypeable Haemophilus influenzae (NTHI) has four loci, lic-1 to lic-3 and lgtC, that generate phase-variable lipooligosaccharide (LOS) structures. lic-1, which is required for the expression of phosphorylcholine (ChoP), is the best characterized and is associated with an enhanced ability of H. influenzae to persist within the nasopharynges of infant rats. Recent data indicate that LOS impacts various aspects of NTHI virulence in the chinchilla model of nasopharyngeal colonization and otitis media (OM). In this study the effects of ChoP expression and the sequences of lic-1 to lic-3 and lgtC of NTHI strain 2019 were evaluated in the chinchilla OM model. Nasopharyngeal colonization data showed that a switch from the ChoP ؊ to the ChoP ؉ phenotype was observed as early as day 3 after intranasal inoculation. Chinchillas colonized by strains with the ChoP Otitis media (OM) or inflammation of the middle ear (ME), in one of its various clinical forms, is one of the most common childhood diseases. Nontypeable Haemophilus influenzae (NTHI) is a major OM pathogen and accounts for 25 to 30% of all cases of this disease. The initial event in the pathogenesis of NTHI OM is the colonization of the host mucosal surface; however, the bacterial factors that contribute to the colonization of the nasopharynx, retrograde ascension of the eustachian tube, and invasion of the ME during the natural progression of the disease are not well characterized. NTHI cells are frequently isolated from the upper respiratory tract, especially the nasopharynges of healthy children, with a reported rate of colonization of approximately 80% (8). Recent data suggested that there is a strong relationship between the frequency of colonization and the incidence of OM in children (5).NTHI lipooligosaccharide (LOS) is a major virulence determinant and may play a role in colonization and invasion of mucosal surfaces in the respiratory tract (3, 16, 23). NTHI LOS is analogous to the lipopolysaccharide (LPS) of enteric gramnegative bacteria in that it contains lipid A linked by 3-deoxy-D-manno-octulosonic acid to a heterogeneous sugar polymer (7). NTHI LOS, however, differs from classic enterobacterial LPS in that it does not contain repeating O-antigen units and is therefore more similar to that derived from Neisseria and Bordetella species (14). Moreover, H. influenzae cells demonstrate a propensity to alter or modulate their surface-expressed antigens including LOS (10, 12). NTHI cells express, on their outer surfaces, a number of LOS core oligosaccharide epitopes, and the expression of these epitopes is subject to frequent, reversible phase variation. Four chromosomal loci, lic-1 to lic-3 and lgtC, which contain long stretches of 4-bp tandem repeats within their 5Ј coding regions, have been reported to generate phase-variable LOS structures (9,11,19). lic-1 functions to add phosphorylcholine (ChoP) to the LOS molecule (22), lic-2 and lgtC are necessary for the expression of , and the effect of variation in lic-3 is unknown. Phase variation may represen...

Phase variation in the colonial opacity of Streptococcus pneumoniae has been implicated as a factor in bacterial adherence, colonization, and invasion in the pathogenesis of pneumococcal disease. Additionally, the synergistic effects of influenza A virus and S. pneumoniae in the development of otitis media (OM) have been reported. This study examined the ability of opaque or transparent S. pneumoniae from the same strain in combination with an antecedent influenza A virus infection to colonize the nasopharynx and invade the middle ear in the chinchilla model. Our data indicated that there was no significant difference in the level of nasopharyngeal colonization and induction of OM between the opaque and transparent variants unless there was a prior challenge with influenza A virus. Subsequent to influenza A virus infection, there was a significant difference between the variants in the ability to colonize and persist in the nasopharynx and middle ear. The concentrations of the opaque variant in nasopharyngeal-lavage samples and middle-ear fluid remained consistently higher than those of the transparent variant for 10 days postinoculation. Data from this study indicate that the effects of influenza A virus on the pathogenesis of experimental S. pneumoniae-induced OM differ depending on the opacity phenotype involved.Otitis media (OM) is one of the most common childhood diseases. The prevalence, medical care costs, and hearing-related morbidity of OM are significant. Streptococcus pneumoniae, the primary etiological agent, has been isolated from approximately 20 to 50% of middle-ear effusions from children with OM (4). The process whereby S. pneumoniae becomes established in the human nasopharynx and then affects the transition from a colonized to a diseased state in the middle ear is not yet known.