Capsule gene analysis of invasive Haemophilus influenzae: accuracy of serotyping and prevalence of IS1016 among nontypeable isolates

Abstract

We evaluated the accuracy of serologic capsule typing by analyzing capsule genes and related markers among invasive Haemophilus influenzae isolates before and after the introduction of H. influenzae serotype b (Hib) conjugate vaccines. Three hundred and sixty invasive H. influenzae isolates were collected as part of Active Bacterial Core surveillance within the Georgia Emerging Infections Program between 1 January 1989 and 31 July 1998. All isolates were biotyped, serotyped by slide agglutination serotyping (SAST), and evaluated using PCR capsule typing. Nontypeable H. influenzae (NTHi) isolates were probed with Hib cap-gene-containing plasmid pUO38 and with IS1016; a subset was examined with phosphoglucose isomerase (pgi) genotyping and pulsed-field gel electrophoresis (PFGE). Discrepancies between SAST and PCR capsule typing were found for 64/360 (17.5%) of the isolates; 48 encapsulated by SAST were NTHi by PCR, 8 NTHi by SAST were encapsulated by PCR, 6 encapsulated by SAST were a different capsule type by PCR, and 2 encapsulated by SAST were capsule-deficient Hib variants (Hib-minus). None of the PCR-confirmed NTHi isolates demonstrated homology with residual capsule gene sequences; 19/201 (9.5%) had evidence of IS1016, an insertion element associated with division I H. influenzae capsule serotypes. The majority of IS1016-positive NTHi were biotypes I and V and showed some genetic relatedness by PFGE. In conclusion, PCR capsule typing was more accurate than SAST and Hib-minus variants were rare. IS1016 was present in 9.5% of NTHi isolates, suggesting that this subset may be more closely related to encapsulated organisms. A better understanding of NTHi may contribute to vaccine development.

FIG. 1.

Summary of capsule gene analysis on NTHi strains by Southern hybridization with probes pUO38, pBR322, and IS1016.

FIG. 2.

Southern hybridization of EcoRI-digested chromosomal DNA from Hib strain 1007 and Hib-minus strain GA346 probed with DIG-labeled pUO38. GA346 contains the major hybridizing bands corresponding to the Hib cap locus (20, 10.2, 4.4, 2.7, and 2.1 kb) and is missing the 9-kb central bridge fragment.

FIG. 3.

Southern hybridization analysis of representative isolates demonstrating hybridization with IS1016. Chromosomal DNA was digested with EcoRI from Hib 1007 (lane 2), Rd (lane 3), GA858 (lane 4), GA1354 (lane 5), GA4891 (lane 6), GA2078 (lane 7), GA3204 (lane 8), GA2014 (lane 10), GA2761 (lane 11), GA2873 (lane 12)ç GA2924 (lane 13), GA3108 (lane 14), GA3925 (lane 15), GA4837 (lane 16), GA5500 (lane 17), GA3701 (lane 18), and GA10071 (lane 19). Lanes 1 and 9, DIG-labeled DNA molecular weight marker II (Roche Diagnostics GmbH). DNA was probed with IS1016 that was PCR DIG labeled using a kit from Roche Diagnostics GmbH. Molecular size markers are noted at the left. Lanes 6 and 18 are NTHi strains without IS1016.

FIG. 4.

Evolutionary analysis of invasive NTHi strains. Dendrogram generated by BioNumerics software showing the genetic relationships between seventeen IS1016-positive and nineteen IS1016-negative NTHi strains. Dendrogram was constructed by cluster analysis of PFGE patterns (data not shown) obtained after digestion of chromosomal DNA with SmaI enzyme, using UPGMA. Strain number, capsule type by PCR, biotype, presence or absence of IS1016, and pgi alleles are shown. Four different clusters (I to IV) of patterned branches were identified by the cluster cutoff method of BioNumerics software. A group of solid lines branching from a dashed line root represent a cluster. Solid lines in the dendrogram link entries in a cluster while dashed lines link different clusters. NT, nontypeable.