Adhesin expression in matched nasopharyngeal and middle ear isolates of nontypeable Haemophilus influenzae from children with acute otitis media

Abstract

The HMW1 and HMW2 proteins, Hia, and hemagglutinating pili are important adherence factors in nontypeable Haemophilus influenzae. To gain insight into the relative importance of these adhesins in nasopharyngeal colonization and localized respiratory tract disease, we assessed their expression in matched nasopharyngeal and middle ear isolates of nontypeable H. influenzae from 17 children with acute otitis media. In all patients, including 11 with bilateral disease, the matched isolates were isogenic based on total protein profiles and genomic fingerprints. Of the nasopharyngeal isolates, 14 expressed only HMW1/HMW2-like proteins, 1 expressed only Hia, 1 expressed only pili, and 1 expressed both Hia and pili. Further analysis revealed concordance between nasopharyngeal isolates and the matched middle ear isolates for expression of the HMW1/HMW2-like proteins and Hia. In contrast, in the two children whose nasopharynges were colonized by piliated organisms, the corresponding middle ear isolates were nonpiliated and could not be enriched for piliation. Nevertheless, Southern analysis revealed that these two middle ear isolates contained all five hif genes required for pilus biogenesis and had no evidence of major genetic rearrangement. In summary, the vast majority of isolates of nontypeable H. influenzae associated with acute otitis media express HMW1/HMW2-like proteins, with expression present in both the nasopharynx and the middle ear. A smaller fraction of nasopharyngeal isolates express pili, while isogenic strains recovered from the middle ear are often refractory to enrichment for piliation. We speculate that the HMW adhesins and Hia are important at multiple steps in the pathogenesis of otitis media while pili contribute to early colonization and then become dispensable.

FIG. 1

Analysis of paired nasopharyngeal (N/P) and middle ear (ME) isolates of nontypeable H. influenzae recovered from two neighbors with concurrent otitis media and comparison with isolates from two other patients. (A) Whole-cell lysates of the matched N/P and ME isolates were resolved on sodium dodecyl sulfate–0.7% polyacrylamide gels and stained with Coomassie brilliant blue. The positions of molecular mass standards are shown to the left of the gel. (B) Chromosomal DNA from the matched N/P and ME isolates was digested with EcoRI and resolved on a 0.7% agarose gel. A 1-kb ladder (Gibco BRL, Gaithersburg, Md.) is in the leftmost lane (St). (C) Chromosomal DNA from the matched N/P and ME isolates was digested with XbaI and then resolved on a 0.7% agarose gel. For all three gels (A, B, and C), samples were loaded in the same order. A 1-kb ladder is in the leftmost lane. Lanes: 1 to 3, samples of the N/P, LME, and RME isolates, respectively, from patient 6; 4 to 6, samples of the N/P, LME, and RME isolates, respectively, from patient 7; 7 to 9, samples of the N/P, LME, and RME isolates, respectively, from patient 8; 10 and 11, samples of the N/P and LME isolates, respectively, from patient 10. Patients 6 and 7 were neighbors (as well as cousins) and had concurrent infections.

FIG. 2

Southern analysis of matched nontypeable H. influenzae isolates from patients 1 and 2, probing with hifA to hifE genes from H. influenzae type b strain Eagan. Chromosomal DNA was digested with either EcoRI (hifA, -B, -D, and -E blots) or BglII (hifC blot) prior to being probed. All gels were loaded as follows: lane 1, strain Eagan (positive control); lane 2, strain 11 (negative control); lane 3, patient 1, nasopharyngeal specimen; lane 4, patient 1, left middle ear specimen; lane 5, patient 1, right middle ear specimen; lane 6, patient 2, nasopharyngeal specimen; and lane 7, patient 2, right middle ear specimen. The positions of molecular size standards are indicated on the left.