Binding of complement regulators to invasive nontypeable Haemophilus influenzae isolates is not increased compared to nasopharyngeal isolates, but serum resistance is linked to disease severity

Abstract

The aim of the present study was to analyze the importance of nontypeable Haemophilus influenzae (NTHi) isolated from patients with sepsis (invasive isolates) compared to nasopharyngeal isolates from patients with upper respiratory tract infection for resistance to complement-mediated attack in human serum and to correlate this result with disease severity. We studied and characterized cases of invasive NTHi disease in detail. All patients with invasive NTHi isolates were adults, and 35% had a clinical presentation of severe sepsis according to the ACCP/SCCM classification of sepsis grading. Moreover, 41% of the patients had evidence of immune deficiency. The different isolates were analyzed for survival in human serum and for binding of 125I-labeled, purified human complement inhibitors C4b-binding protein (C4BP), factor H, and vitronectin, in addition to binding of regulators directly from serum. No significant differences were found when blood-derived and nasopharyngeal isolates were compared, suggesting that interactions with the complement system are equally important for NTHi strains, irrespective of isolation site. Interestingly, a correlation between serum resistance and invasive disease severity was found. The ability to resist the attack of the complement system seems to be important for NTHi strains infecting the respiratory tract as well as the bloodstream.

FIG. 1.

Bactericidal activity against a series of randomly selected blood-derived isolates. NTHi bacteria were incubated in the presence of increasing concentrations of NHS (5 to 20%). Four typical strains are shown in panels A to D. After incubation for 20 min, bacteria were spread on chocolate agar plates to allow determination of the number of surviving bacteria. The number of bacteria (CFU) at the initiation of the experiment was defined as 100%. The mean values from three independent experiments are shown, with error bars indicating standard deviations (SD).

FIG. 2.

NTHi isolates from blood samples (invasive disease) or nasopharynxes of patients with upper respiratory tract infection show variation in serum resistance independent of the presence of IgG in NHS. (A and B) No significant difference in survival rate in NHS was observed between NTHi isolates from blood samples and those from nasopharynxes. (C) All strains were resistant to hiNHS. NTHi isolates from blood samples (n = 21) (A) and nasopharynx (n = 21) (B) were analyzed for survival in human serum. The strains were incubated in the presence of 5% NHS or 5% hiNHS for 20 min. Thereafter, bacteria were spread on chocolate agar plates to allow determination of the number of surviving bacteria. The numbers of bacteria (CFU) at the initiation of the experiment was defined as 100%. (D) The NHS contained IgG directed against NTHi strains independently of whether they were isolated from blood samples or nasopharynxes. Flow cytometry profiles show the deposition of IgG and the binding of a rabbit anti-NTHi antiserum to the surfaces of the NTHi strains from patients 11 (blood) and 4 (nasopharynx). Bacteria were incubated with mouse anti-human IgG or rabbit anti-NTHi antiserum, followed by FITC-conjugated anti-mouse pAb or anti-rabbit pAb. Results for one representative experiment out of three independent ones performed are shown.

FIG. 3.

Both blood-derived and nasopharyngeal NTHi isolates bind complement regulators. A series of clinical strains isolated from blood samples (n = 21; cases with invasive disease and an available patient history are listed in Table 1) or nasopharynxes (n = 21; patients with upper respiratory tract infection) was tested for C4BP (A), factor H (B), and vitronectin (C) binding. The different NTHi strains were grown overnight and incubated with ¹²⁵I-labeled C4BP, factor H, or vitronectin. The binding level was calculated as the percentage of bound radioactivity relative to the level of added radioactivity, measured after separation of free and bound ¹²⁵I-labeled protein over a sucrose column. The mean values of results from three independent experiments are shown. The mean value for all isolates in the same group is indicated by a line.