Characterization of genetic and phenotypic diversity of invasive nontypeable Haemophilus influenzae

Abstract

The ability of unencapsulated (nontypeable) Haemophilus influenzae (NTHi) to cause systemic disease in healthy children has been recognized only in the past decade. To determine the extent of similarity among invasive nontypeable isolates, we compared strain R2866 with 16 additional NTHi isolates from blood and spinal fluid, 17 nasopharyngeal or throat isolates from healthy children, and 19 isolates from middle ear aspirates. The strains were evaluated for the presence of several genetic loci that affect bacterial surface structures and for biochemical reactions that are known to differ among H. influenzae strains. Eight strains, including four blood isolates, shared several properties with R2866: they were biotype V (indole and ornithine decarboxylase positive, urease negative), contained sequence from the adhesin gene hia, and lacked a genetic island flanked by the infA and ksgA genes. Multilocus sequence typing showed that most biotype V isolates belonged to the same phylogenetic cluster as strain R2866. When present, the infA-ksgA island contains lipopolysaccharide biosynthetic genes, either lic2B and lic2C or homologs of the losA and losB genes described for Haemophilus ducreyi. The island was found in most nasopharyngeal and otitis isolates but was absent from 40% of invasive isolates. Overall, the 33 hmw-negative isolates were much more likely than hmw-containing isolates to have tryptophanase, ornithine decarboxylase, or lysine decarboxylase activity or to contain the hif genes. We conclude (i) that invasive isolates are genetically and phenotypically diverse and (ii) that certain genetic loci of NTHi are frequently found in association among NTHi strains.

FIG. 1.

Gene arrangement at two variable loci. (A) Variable content of the genetic island between infA and ksgA. Of the 99 NTHi isolates studied, 31 lack this island, as depicted for Rd KW20 and R2866. Forty-five isolates contain lic2B and lic2C, as shown for RM7004 (16, 20). Seventeen contain losA and losB, as shown for R2846. Six isolates contain lic2C without lic2B (not shown). (B) The urease locus of Rd KW20 and R2846 is replaced in R2866 by mtrF. We amplified the region between aspA and groES of 12 urease-negative isolates, and all yielded a 4-kb product consistent with mtrF. Homology with mtrF was confirmed by sequencing for seven of the isolates.

FIG. 2.

UPGMA (unweighted pair-group method with arithmetic averages) dendrogram based on the pairwise differences in the MLST allelic profiles of 24 NTHi isolates. Eleven strains that share the properties of belonging to biotype V, lacking hmw-related sequence, and lacking a genetic island flanked by infA and ksgA are clustered on the dendrogram. Of these, eight are most closely related: R3569, C1344, R2866, C470, R3157, R3176, R3567, and R3631. R3174 is less closely related, and R3265 and R3101 are closely related to each other but not to the other biotype V isolates. The remaining isolates for which MLST was determined belong to biotype I, II, or III. Those containing hmw-related sequence are separated on the dendrogram from the strains lacking hmw.

FIG. 3.

Bactericidal activity of pooled normal human serum for throat, middle ear, and invasive isolates of NTHi compared with that for laboratory isolates. Strain Eagan is an encapsulated type b strain, and S2 is an unencapsulated mutant derived from Eagan. Sd is the encapsulated type d strain (Garf) from which Rd KW20 was derived. A total of 17 throat, 19 otitis, and 17 invasive isolates were incubated with normal human serum, and IC₅₀s were calculated as described in Materials and Methods. The results of 22 replicate IC₅₀ determinations for strains Rd KW20 and R2866 are shown to illustrate the reproducibility of the assay. Horizontal lines indicate the median IC₅₀ for each group of data points. The geometric mean IC₅₀s for the clinical isolates were 2.97% for the throat isolates, 4.27% for the otitis media isolates, and 4.66% for the invasive isolates.