Pulsed-field gel electrophoresis used to investigate genetic diversity of Haemophilus influenzae type b isolates in Australia shows differences between Aboriginal and non-Aboriginal isolates

Abstract

We used pulsed-field gel electrophoresis to study the epidemiology and population structure of Haemophilus influenzae type b. DNAs from 187 isolates recovered between 1985 and 1993 from Aboriginal children (n = 76), non-Aboriginal children (n = 106), and non-Aboriginal adults (n = 5) in urban and rural regions across Australia were digested with the SmaI restriction endonuclease. Patterns of 13 to 17 well-resolved fragments (size range, approximately 8 to 500 kb) defining 67 restriction fragment length polymorphism (RFLP) types were found. Two types predominated. One type (n = 37) accounted for 35 (46%) of the isolates from Aboriginals and 2 (2%) of the isolates from non-Aboriginals, and the other type (n = 41) accounted for 2 (3%) of the isolates from Aboriginals and 39 (35%) of the isolates from non-Aboriginals. Clustering revealed seven groups at a genetic distance of approximately 50% similarity in a tree-like dendrogram. They included two highly divergent groups representing 50 (66%) isolates from Aboriginals and 6 (5%) isolates from non-Aboriginals and another genetically distinct group representing 7 (9%) isolates from Aboriginals and 81 (73%) isolates from non-Aboriginals. The results showed a heterogeneous clonal population structure, with the isolates of two types accounting for 42% of the sample. There was no association between RFLP type and the diagnosis of meningitis or epiglottitis, age, sex, date of collection, or geographic location, but there was a strong association between the origin of isolates from Aboriginal children and RFLP type F2a and the origin of isolates from non-Aboriginal children and RFLP type A8b. The methodology discriminated well among the isolates (D = 0.91) and will be useful for the monitoring of postvaccine isolates of H. influenzae type b.

FIG. 1

Geographical map of Australia showing the locations from which isolates were obtained. The distance both between Perth and Sydney and between Bathurst Island and Melbourne is approximately 2,500 miles (4,000 km).

FIG. 2

SmaI-generated fragments obtained by CHEF PFGE of genomic DNAs of Hib isolates obtained from non-Aboriginals. A fragment size scale is shown on the right. Lanes 1 and 13, fragment standards from Hib HS0008; lane 2, RFLP type A6a; lane 3, RFLP type A1a; lane 4, RFLP type A8b; lane 5, RFLP type A3a; lane 6, RFLP type E1; lane 7, RFLP type A9a; lane 8, RFLP type E3; lane 9, RFLP type A1b; lane 10, RFLP type B6; lane 11, RFLP type C1a; lane 12, RFLP type A6b. The photograph seen here was prepared from a Polaroid picture. The original Polaroid picture does not have the very white background seen in the lanes here. The background is relatively faint in the original Polaroid, and a more faithful copy can be prepared.

FIG. 3

SmaI-generated fragments obtained by CHEF PFGE of genomic DNA of Hib isolates obtained from Aboriginals. A fragment size scale is shown on the right. Lanes 1 and 14, fragment standards from Hib HS0008; lanes 2, 3, 4, 8, 9, 11, and 12, RFLP type F2a; lane 5, RFLP type D1a; lane 6, RFLP type F2d; lane 7, RFLP type F2b; lanes 10 and 13, RFLP type G1. The photograph seen here was prepared from a Polaroid picture. The original Polaroid picture does not have the very white background seen in the lanes here. The background is relatively faint in the original Polaroid, and a more faithful copy can be prepared.

FIG. 4

Dendrogram showing the clustering of 67 SmaI RFLP types of 187 Hib isolates obtained from Aboriginals and non-Aboriginals from rural and urban regions around Australia. The dendrogram was generated with the mathematical model of Nei and Li (24), and distances were calculated by the neighbor-joining method. The distance between any two taxa is the sum of the horizontal lines between them. The bar indicates an F value of 0.1, or 10% similarity. Seven major groups cluster at an F value of ≤0.5, and 39 branches representing 39 clonal groups cluster at an F value of ≥0.9. Only types G1, F2a, A4b, A8b, and C3 share members from both Aboriginal and non-Aboriginal people.