High level of sequence diversity in the 16S rRNA genes of Haemophilus influenzae isolates is useful for molecular subtyping.

A molecular typing method based on the 16S rRNA sequence diversity was developed for Haemophilus influenzae isolates. A total of 330 H. influenzae isolates were analyzed, representing a diverse collection of U.S. isolates. We found a high level of 16S rRNA sequence heterogeneity (up to 2.73%) and observed an exclusive correlation between 16S types and serotypes (a to f); no 16S type was found in more than one serotype. Similarly, no multilocus sequence typing (MLST) sequence type (ST) was found in more than one serotype. Our 16S typing and MLST results are in agreement with those of previous studies showing that serotypable H. influenzae isolates behave as highly clonal populations and emphasize the lack of clonality of nontypable (NT) H. influenzae isolates. There was not a 1:1 correlation between 16S types and STs, but all H. influenzae serotypable isolates clustered similarly. This correlation was not observed for NT H. influenzae; the two methods clustered NT H. influenzae isolates differently. 16S rRNA gene sequencing alone provides a level of discrimination similar to that obtained with the analysis of seven genes for MLST. We demonstrated that 16S typing is an additional and complementary approach to MLST, particularly for NT H. influenzae isolates, and is potentially useful for outbreak investigation.