Phylogenetic relatedness and diversity of non-typable Haemophilus influenzae in the nasopharynx and middle ear fluid of children with acute otitis media

Abstract

The phylogenetic relationships of non-typable Haemophilus influenzae (NTHi) strains prospectively isolated from healthy children and children with acute otitis media (AOM) were analysed using multilocus sequence typing (MLST). A total of 165 NTHi isolates were collected over a 3.5 year time frame during 2006 through 2009. The strains were tested for β-lactamase production; 28.5% were positive. Seventy different NTHi sequence types (STs) were identified of which 29 (41.4%) were novel. NTHi strains did not show any phylogenetic grouping or clustering among asymptomatic colonizing strains or strains that caused AOM, or based on β-lactamase enzyme production. Evaluation of triplets and other siblings over time demonstrated relatively frequent genetic exchanges in NTHi isolates in vivo in a short time frame and subsequent transfer among children in a family. Comparison of the MLST STs isolated at different time points showed that in ~85% of the nasopharynx (NP) colonizations, NTHi strains cleared from the host within 3 months, that sequential colonization in the same child involved different strains in all cases except one, and that NP and middle ear isolates were identical STs in 84% of cases. In this first study of its type to our knowledge, we could not identify predominant MLST types among strains colonizing the NP versus those causing AOM or expressing a β-lactamase enzyme conferring penicillin resistance in children.

Fig. 1.

Phylogenetic tree from concatenated sequences of MLST of 70 NTHi strains isolated from Rochester NY children. The linkage distance is shown at the bottom of the dendrogram and is in the units of the number of base substitutions per site. The number of isolates cultured for each ST is shown in parentheses. The strains that were found in MEF of children with AOM are indicated by an asterisk. β-Lactamase results are also shown for each strain with a + and – symbol after the strain number.

Fig. 2.

Comparison of MLST STs obtained from NTHi isolates at different time points from the same subjects. The x-axis represents the child’s age in months and the y-axis indicates the child’s identification number. Circles represent NTHi strains colonizing the NP; squares represent NTHi strains found in MEF. Two isolates of NTHi connected by a line are the same ST, and those connected by a dashed line are different STs, but the same clone (single-locus variant). Inc means it was not possible to obtain a sequence for all seven genes for that isolate and therefore the ST was not obtained, but partial analysis clearly allowed us to determine that the strain was different from the other strains in that particular child.