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JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/99/$04.0010

May 1999, p. 1524–1531 Vol. 37, No. 5

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Pulsed-Field Gel Electrophoresis Used To Investigate GeneticDiversity of Haemophilus influenzae Type b Isolates in

Australia Shows Differences between Aboriginaland Non-Aboriginal Isolates

PATRICIA EZEKIEL MOOR,1* PETER C. COLLIGNON,2 AND GWENDOLYN L. GILBERT3

Division of Biochemistry and Molecular Biology, Australian National University, Australian Capital Territory 0200,1

Infectious Disease Unit, Canberra Hospital, Woden 2606,2 and Centre for Infectious Diseases and Microbiology,Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead 2145,3 Australia

Received 13 October 1998/Returned for modification 8 December 1998/Accepted 28 January 1999

We used pulsed-field gel electrophoresis to study the epidemiology and population structure of Haemophilusinfluenzae type b. DNAs from 187 isolates recovered between 1985 and 1993 from Aboriginal children (n 5 76),non-Aboriginal children (n 5 106), and non-Aboriginal adults (n 5 5) in urban and rural regions acrossAustralia were digested with the SmaI restriction endonuclease. Patterns of 13 to 17 well-resolved fragments(size range, ;8 to 500 kb) defining 67 restriction fragment length polymorphism (RFLP) types were found. Twotypes predominated. One type (n 5 37) accounted for 35 (46%) of the isolates from Aboriginals and 2 (2%) ofthe isolates from non-Aboriginals, and the other type (n 5 41) accounted for 2 (3%) of the isolates fromAboriginals and 39 (35%) of the isolates from non-Aboriginals. Clustering revealed seven groups at a geneticdistance of ;50% similarity in a tree-like dendrogram. They included two highly divergent groups representing50 (66%) isolates from Aboriginals and 6 (5%) isolates from non-Aboriginals and another genetically distinctgroup representing 7 (9%) isolates from Aboriginals and 81 (73%) isolates from non-Aboriginals. The resultsshowed a heterogeneous clonal population structure, with the isolates of two types accounting for 42% of thesample. 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 fromAboriginal children and RFLP type F2a and the origin of isolates from non-Aboriginal children and RFLP typeA8b. The methodology discriminated well among the isolates (D 5 0.91) and will be useful for the monitoringof postvaccine isolates of H. influenzae type b.

Before the introduction of conjugate vaccines in 1992, Hae-mophilus influenzae type b (Hib) disease was a major cause ofmorbidity and mortality in Australian children, particularlyamong Aboriginal populations, in whom the incidence of Hibdisease was among the highest in the world. Overall, 600 to 700cases of invasive Hib disease occurred each year in Australia.While the incidence of Hib disease has dropped dramaticallysince vaccination policies have been in place, invasive diseasedue to Hib still occurs. Between July 1993 and June 1996, 412cases of invasive disease due to Hib, including 18 deaths, werereported to the Hib Case Surveillance Scheme. Thirty-fourcases met the Australian case definition of a vaccine failure.However, a further 24 cases would meet the United Kingdomvaccine failure definition that includes cases occurring after theadministration of just two rather than three doses of vaccinewhen the first dose is given before the age of 7 months (14).

Studies on the epidemiology of Hib disease in Australia havedemonstrated significant differences in the incidence of epi-glottitis and meningitis between urban areas (7, 12, 20, 21), anextremely high incidence of invasive Hib disease among Ab-original children compared to that among non-Aboriginal chil-dren (9, 10, 11), and a lack of epiglottitis among Aboriginals(9). The reasons for the unique epidemiology of Hib diseaseremain largely unknown, and there is little information about

its population structure and genetic diversity in Australia. Thepurpose of this study was to characterize isolates of Hib, de-termine whether restriction fragment length polymorphism(RFLP) types correlated with the epidemiology of disease, andassess the usefulness of pulsed-field gel electrophoresis(PFGE) as the basis of a database of genetic types to monitorHib isolates in the postvaccine era.

We used contour-clamped homogeneous electric fieldPFGE (CHEF PFGE) to investigate the genetic diversity andpopulation structure of Hib and to develop a database ofgenetic types to use in the surveillance of Hib. CHEF PFGE isnow a well-established methodology in many pathology labo-ratories and is most often used to analyze relatively small setsof isolates related to outbreaks of disease. It has been used toconstruct a physical map of Hib strain Eagan and to charac-terize 10 genetically heterogeneous Hib isolates previouslytyped by multilocus enzyme electrophoresis (4). It has alsobeen used to study insertion mutations in the transferrin bind-ing system of Hib (5), and other types of PFGE have been usedto estimate its genome size (17). Results of PFGE for 29epidemiologically unrelated Hib isolates demonstrated evolu-tionary divergence consistent with that of multilocus enzymeelectrophoresis (1). PFGE, however, has not been applied pre-viously to the study of a large number of clinical isolates of Hibin population genetic studies. In conjunction with mathemati-cal analysis we used the RFLPs generated by PFGE to providenumerical estimates of genetic diversity among 187 isolates toconstruct a dendrogram and to assess the correlation of genetictypes with the epidemiology of Hib.

* Corresponding author. Mailing address: Gadi Research Centre,Faculty of Applied Science, University of Canberra, Canberra, ACT2601, Australia. Phone: 61-6-6201-5451. Fax: 61-6-6201-5727. E-mail:[email protected].

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MATERIALS AND METHODS

Bacterial isolates. One hundred eighty-eight previously characterized Hibisolates from collections representing urban and rural regions across Australiawere examined. All were recovered between 1985 and 1993. Thirty-four isolatesfrom Canberra and 37 isolates from Sydney were obtained from the CanberraHospital, Australian Capital Territory; 20 isolates from Melbourne and 10 iso-lates from the Alice Springs region were obtained from the Royal Children’sHospital in Melbourne, Victoria; 20 isolates from metropolitan Perth and ruralareas of Western Australia were obtained from the Princess Margaret Hospitalin Perth, Western Australia; 46 isolates from the Alice Springs Region wereobtained from the Queensland Institute of Medical Research, Brisbane, Queens-land; 19 isolates from Bathurst Island were obtained from the Menzies School ofHealth Research in Darwin, Northern Territory; and 2 isolates were obtainedfrom the Townsville General Hospital, Queensland. The geographic origins ofthe isolates are depicted in Fig. 1. The sample comprised 83 isolates recoveredfrom the cerebrospinal fluid of patients with meningitis, 29 isolates from theblood of patients with epiglottitis, and 39 isolates from patients with otherdiagnoses including 13 isolates from the blood of patients for whom no clinicalinformation was available. Twenty-four isolates were from healthy carriers. Allthe isolates were genotyped to confirm that they were type b. One was found tobe nonencapsulated and was excluded from the study. A breakdown of the Hib

sample (n 5 187) distribution by geographic location, origin from an Aboriginalor non-Aboriginal, and disease association is shown in Table 1. An isolate of Hib,HS0008, obtained from the Canberra Hospital collection was used as a sizestandard. All isolates except 20 isolates which had been lyophilized were storedat 270°C.

DNA preparation. The preparation of chromosomal DNA was performed bya modification of the method described by Bautsch (3). DNA was prepared in a1:1 ratio in an intact 1% agarose (SeaPlaque low-gelling-temperature agarose;FMC Bioproducts) matrix with cells harvested from an overnight growth onchocolate agar suspended in solution 21 (19) to an optical density of 1.8 at 650nm. The DNA-agarose plugs (1 by 8 by 19 mm) were lysed in a solution ofproteinase K (1 mg/ml) in 0.5 M EDTA–1% sodium lauryl sarcosine and wereincubated overnight at 50°C. The plugs were rinsed for 2 h in TE (10 mM Tris,1 mM EDTA [pH 8.0]) and were then stored in fresh TE at 4°C.

Restriction endonuclease digestion. H. influenzae has a base composition of 37mol% G1C. Therefore, enzymes recognizing 6-bp sequences comprising onlyG2C bases should produce a small number of distinct fragments. Initially, SmaI(CCC21GGG) and ApaI (G1GGCC2C) were used. Washing of plugs anddigestion of the DNA with endonucleases were performed as described by Ingliset al. (16). SmaI (Boehringer Mannheim) and ApaI (Boehringer Mannheim)were used according to the manufacturer’s instructions. For each analysis, one-half of a DNA-agarose plug was digested overnight with 30 U of enzyme at 30°C(SmaI) or 37°C (ApaI).

PFGE analysis. Separation of DNA fragments with a clamped homogeneouselectric field device (CHEF DRII; Bio-Rad) at 200 V was carried out in 1%agarose (type II-A; Medium EEO; Sigma) gels made with 0.53 TBE buffer(0.045 M Tris-borate, 0.001 M EDTA) in 0.53 TBE buffer at 12 to 14°C. Pulseparameters for both SmaI and ApaI digests included a ramp of pulses for 6 to 8 sfor 7 h followed by a ramp of 1 to 38 s for 17 h. The gels were stained for 30 minwith ethidium bromide (0.5 mg/ml) in distilled water and were then destained indistilled water for 1 h or more and photographed (Polaroid 667) with a UV lightsource. DNA size standards were lambda phage concatemers, a lambda phageHindIII digest, and the genomic fragments from Hib HS0008. Most strains wereindependently digested up to three times.

Estimation of genetic diversity between isolates and construction of dendro-grams. The numbers and mobilities of the fragments were determined by visualexamination of the Polaroid photographs of the stained gels. When comparisonof the sizes of the fragments from isolates from different gels was questionable,another gel was run so that the fragments could be compared while they were inclose proximity. Numerical analysis of RFLP patterns was performed by identi-fying the proportion of fragments shared by pairs of isolates by the method of Neiand Li (24) (also known as the Dice coefficient or coefficient of similarity) andcalculated as F 5 2nxy/(nx 1 ny), where nx is the total number of DNA fragmentsfrom isolate x, ny is the total number of fragments from isolate y, and nxy is thenumber of fragments identical in the two isolates.

A matrix of F values for all pairs of isolates was constructed, and a dendrogramwas constructed by the neighbor-joining method of Saitou and Nei (26) with theTDRAW program. The programs used for mathematical calculations and con-struction of the dendrogram are accessible in the package RAPDistance Pro-grams, distributed by J. Armstrong et al., Research School of Biological Sciences,Australian National University (2).

FIG. 1. Geographical map of Australia showing the locations from whichisolates were obtained. The distance both between Perth and Sydney and be-tween Bathurst Island and Melbourne is approximately 2,500 miles (4,000 km).

TABLE 1. Geographic location and disease association of 187 Hib isolates recovered from Aboriginals and non-Aboriginals

Group and geographic locationNo. of isolates

Meningitis Epiglottitis Other Carrier Total

Aboriginal 23 0 29 24 76Alice Springs region, Northern Territory 11 0 28a 6 45Bathurst Island, Northern Territory 0 0 1b 18 19Perth, Western Australia 5 0 0 0 5Rural Western Australia 7 0 0 0 7

Non-Aboriginal 60 29 22 0 111Canberra, Australian Capital Territory 20 14 0 0 34Melbourne, Victoria 10 10 0 0 20Sydney, New South Wales 21 1 15c 0 37Perth, Western Australia 4 4 0 0 8Townsville, Queensland 2 0 0 0 2Alice Springs region, Northern Territory 3 0 7d 0 10

Total 83 29 51 24 187

a The isolates were associated with pneumonia (n 5 9), gastroenteritis (n 5 9), acute lower respiratory tract infection (n 5 4), cellulitis (n 5 1), febrile disease (n 51), FTT (n 5 1), conjunctivitis (n 5 1), bronchiolitis (n 5 1), and otitis media (n 5 1).

b The isolate may be associated with otitis media.c Thirteen invasive isolates recovered from blood (diagnosis unknown) and 2 isolates associated with cellulitis.d Invasive isolates recovered from blood (diagnosis unknown).

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Southern blotting and hybridization. Capsular type was confirmed by hybrid-ization of Southern blots with 32P-labelled pU082, a cloned fragment which spanstype b-specific parts of the H. influenzae cap locus (18). Hybridization productswere detected by autoradiography. If no signal was detected, isolates were re-probed with pU038, which spans serotype-specific and non-serotype-specificparts of the cap locus (18). Isolates which did not hybridize with pU082 were notincluded in the study. The probes were kindly provided by E. Richard Moxon.

The DNA fragments separated by PFGE were acid depurinated in 0.25 M HCltwo times for 8 min each time followed by two 8-min rinses in 0.4 M NaOH andwere then transferred onto Hybond-N nylon membranes (Amersham) in 10 mMNaOH overnight. The membranes were prehybridized overnight at 70°C in abuffer containing 23 PE (13 PE is 0.133 M sodium phosphate plus 1 mMEDTA), 7% sodium dodecyl sulfate (SDS), and 1% bovine serum albumin. Theprobe consisted of 25 ng of pU082 or pU038 DNA labelled with [a-32P]dCTPwith the Megaprime labelling system (Amersham). The probe was incubatedovernight with the membranes at 70°C in the same hybridization buffer. Themembranes were then washed three times for 15 min each time in 23 SSC (13SSC is 0.15 M NaCl plus 0.015 M sodium citrate)–0.1% SDS at room tempera-ture and once in 53 SSC–1% SDS at 70°C and were analyzed by autoradiography.

RESULTS

Selection of isolates for examination. All of the isolates (n 5188) obtained for this study had previously been identified byconventional methods including serotyping. One failed to hy-bridize with either pU038 or pU082 and was excluded from thestudy, leaving 187 isolates for analysis.

Analysis of RFLPs. SmaI was used for the main analysis ofRFLPs because the number and size of fragments producedwith SmaI were fewer and more distinct than those producedwith ApaI. Comparison of the similarity matrices producedwith each enzyme with a subset of the sample by the programDIPLOMO (29) yielded a correlation coefficient of 0.90, andthe grouping of the isolates into major divisions establishedwith SmaI was confirmed with ApaI.

Well-resolved patterns of 13 to 17 SmaI fragments of ap-proximately 8 to 500 kb representing 67 RFLP types werefound among the 187 isolates. Figures 2 and 3 show represen-tative results of the SmaI digests. A total of 78 distinct frag-ments were observed on the gels. Only one of these fragmentswas present in all the RFLP patterns, and it did not carry the

cap locus detected in the hybridization studies. Forty-eight(72%) of the RFLP types had single isolates, and 19 (28%)types had multiple isolates. The isolates of the two types withthe most numbers of isolates, types F2a (n 5 37) and A8b (n 541), represented 42% of the sample. The remaining 17 typeswith multiple members had two to eight isolates each.

Nineteen types were found among the isolates from Aborig-inal children (n 5 76), and 53 types were found among thosefrom non-Aboriginal children and adults (n 5 111). The ratioof isolates to type was 4:1 and 2.1:1 among the Aboriginal andthe non-Aboriginal populations, respectively, with only fivetypes shared by both population groups. For the most part,within each of the five shared types, the isolates from Aborigi-nals and non-Aboriginals were from the same geographic re-gion.

Two types predominated. Type F2a accounted for 35 (46%)of the isolates from Aboriginals but only 2 (2%) of the isolatesfrom non-Aboriginals (Table 2). Type A8b accounted for 39(35%) of the isolates from non-Aboriginals and 2 (3%) of theisolates from Aboriginals (Table 3).

The number of fragments shared between pairs ranged fromall fragments (these pairs were considered to be identical) to 2of 29 fragments for the most diverse pairs. The RFLP patternswere stable. When the isolates were repeatedly subcultured,their DNAs yielded unchanged SmaI fragment patterns.

Genetic relationships determined by numerical analysis offragment length polymorphisms. F values ranged from 0.07 to1, and these correspond to a similarity range of from 7 to100%. An F value of 1, determined when all fragments be-tween a pair were shared, corresponded to identical fragmentpatterns on the gels. Thus, when F was equal to 1, the membersof the pair are considered to be the same RFLP type. Thesmallest F value found, 0.07, corresponded to pairs for whichonly 2 of the 14 and 15 fragments of the isolate pair wereshared. A similarity matrix based on pairwise comparison ofthe F values for all RFLP types was used to calculate a den-drogram (Fig. 4), as described in Materials and Methods.

The isolates fell into seven major clusters, designated groups

FIG. 2. SmaI-generated fragments obtained by CHEF PFGE of genomicDNAs of Hib isolates obtained from non-Aboriginals. A fragment size scale isshown on the right. Lanes 1 and 13, fragment standards from Hib HS0008; lane2, RFLP type A6a; lane 3, RFLP type A1a; lane 4, RFLP type A8b; lane 5, RFLPtype 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; lane12, RFLP type A6b. The photograph seen here was prepared from a Polaroidpicture. The original Polaroid picture does not have the very white backgroundseen 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 genomicDNA of Hib isolates obtained from Aboriginals. A fragment size scale is shownon 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 typeF2d; lane 7, RFLP type F2b; lanes 10 and 13, RFLP type G1. The photographseen here was prepared from a Polaroid picture. The original Polaroid picturedoes not have the very white background seen in the lanes here. The backgroundis relatively faint in the original Polaroid, and a more faithful copy can beprepared.

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A to G, at an F value of #0.5, i.e., at a genetic distancerepresenting less than 50% similarity. There were 39 branchesat an F value of $0.9, corresponding to a genetic distance ofgreater than 90% similarity, and these branches are referred toas clonal groups. Each of the clonal groups was represented bya single type or a cluster of closely related types whose mem-bers differed by three or fewer fragments. For comparativepurposes, each RFLP type was given a three-character desig-nation according to the major cluster (A, B, C, D, E, F, or G)in which it fell, the clonal group of which it was a member inthat cluster (numbered from 1), and its unique type in theclonal group (lettered from a).

Two highly divergent groups (designated F and G) compris-ing seven types represented 50 (66%) of the isolates fromAboriginals and 6 (5%) of the isolates from non-Aboriginals. Asingle genetically distinct group (designated A) comprising 37types represented 7 (9%) of the isolates from Aboriginals and81 (72%) of the isolates from non-Aboriginals.

The largest major cluster, group A, included 88 isolates andcomprised 37 types in 15 clonal groups, 13 of which comprisedmultiple types. Type A8b, which comprised 22% of the sample,and 35% of the isolates from non-Aboriginals, fell in group A.The distribution of RFLP types among groups B to E is shownin Fig. 4. Group F had 48 members among six types in fourclonal groups including type F2a, which comprised 20% of thesample and 46% of the isolates from Aboriginals. Group Gincluded a single type, type G1, which comprised six isolatesfrom Aboriginals isolates and two isolates from non-Aborigi-

nals from the Alice Springs region. Type G1 was the mosthighly divergent type in the sample, with less than 15% simi-larity to either of the two predominant types, types F2a andA8b (F 5 0.13 and 0.14, respectively).

The largest genetic distance estimate was between two mem-bers of group A (types A2b and A10) and the members ofgroup G. They were separated by genetic distances with Fvalues of 0.07, with only 2 of 29 fragments shared betweenpairs.

Genetic diversity and correlation to epidemiology. Cluster-ing of RFLP types was not associated with disease type. Iso-lates from patients with meningitis and epiglottitis, as ex-pected, were each represented by a diverse range of types, andno single type was associated with a particular diagnosis orcarriage. In addition, no association was found between age,sex, date of collection, or geographic location. A strong asso-ciation, however, between both the origin of isolates fromAboriginal children and RFLP type F2a and the origin ofisolates from non-Aboriginal children and RFLP type A8b(P , 0.001 by chi-square analysis) was found and three otherRFLP types (types F2d, G1, and C7a) with multiple memberswere composed of predominantly isolates from Aboriginals.

Type F2d comprised seven isolates and fell in the sameclonal group as type F2a. It accounted for six isolates fromAboriginal children living in the Alice Springs region and twoisolates from Aboriginal children living in rural Western Aus-tralia. Type G1 included six isolates from Aboriginals and twoisolates from non-Aboriginals from the Alice Springs region.

TABLE 2. Distribution of Hib RFLP type F2a (n 5 37), which accounted for 46% of the Aboriginal isolates

Geographic location

No. of isolates


Non-Aboriginal Aboriginal Non-

Aboriginal Aboriginal Non-Aboriginal Aboriginal Non-

Aboriginal Aboriginal Non-Aboriginal Aboriginal

CanberraMelbourneSydney 1 1Perth 3 3TownsvilleAlice Springs region 1 4 14a 4 1 22Bathurst Island 6 6Rural Western Australia 4 4

Total 1 11 1 0 0 14 0 10 2 35

a The isolates were associated with pneumonia (n 5 5), acute lower respiratory tract infection (n 5 3), gastrointestinal disorders (n 5 3), cellulitis (n 5 1),bronchiolitis (n 5 1), and FTT (n 5 1).

TABLE 3. Distribution of Hib RFLP type A8b (n 5 41), which accounted for 35% of the non-Aboriginal isolates

Geographic location

No. of isolates


Non-Aboriginal Aboriginal Non-

Aboriginal Aboriginal Non-Aboriginal Aboriginal Non-

Aboriginal Aboriginal Non-Aboriginal Aboriginal

Canberra 5 1 6Melbourne 4 7 11Sydney 11 6a 17Perth 1 1 1 2 1Townsville 1 1Alice Springs region 1 2a 2 1Bathurst IslandRural Western Australia

Total 22 2 9 0 8 0 0 0 39 2

a Invasive isolates (diagnosis unknown).



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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 regionsaround Australia. The dendrogram was generated with the mathematical model of Nei and Li (24), and distances were calculated by the neighbor-joining method. Thedistance 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 atan 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 bothAboriginal and non-Aboriginal people.



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Eight isolates from Aboriginal children shared RFLP type C7a,which fell in a group that comprised 13 isolates from Aborigi-nals and 11 isolates from non-Aboriginals. The association oftype C7a with origin in an Aboriginal or non-Aboriginal orgeographic location is not statistically significant; however,seven of the eight isolates of type C7a were from BathurstIsland in the Northern Territory, as was the single member ina clonally related type, type C7b.

DISCUSSION

Our aim was to ascertain whether CHEF PFGE could beuseful in distinguishing between isolates of Hib recovered inAustralia before the introduction of vaccines and to investigatetheir genetic diversity. It has been shown that the Hib popu-lation structure is clonal (23), which implies that the number ofdifferent types will be limited. One practical implication of thisis that general, nonspecific procedures such as RFLP analysiscan be used to identify bacteria in such populations (25).

In the analysis of the Hib isolates described here, RFLPswere determined for fragments from the entire genome exceptfor fragments that were present in such small amounts thatthey did not bind sufficient ethidium bromide to be visible orthat were so small (,6 kb) that they ran off the end of the gelduring electrophoresis; such fragments would account for asmall proportion of the genome.

The sample of 187 Hib isolates that we analyzed comprised67 different types of SmaI RFLP patterns with 17 or fewerfragments that were well resolved by PFGE. Closely relatedand more distantly related genomic types were easily discrim-inated. When Simpson’s index of diversity (15) was applied tothe system an index of D equal to 0.91 was calculated. TheSmaI RFLPs fell into seven major clusters in a tree-like den-drogram that is consistent with a clonal population structure,as is the presence of two predominant types among the 67types that were found. The two predominant types, referred toas types F2a and A8b, accounted for 42% of the sample andincluded almost one-half of the isolates from Aboriginals andover one-third of the isolates from non-Aboriginals, respec-tively. Not surprisingly, the results described a heterogeneousclonal population structure and extend the geographic rangeover which the clonality of Hib has been demonstrated.

Differences and similarities between Hib isolates werereadily visualized from the CHEF patterns, and a generalgrouping of the isolates could be established in this way. How-ever, a more quantitative approach can be derived by subject-ing the data to the analytical procedures described by Nei andLi (24). In an attempt to describe what are otherwise qualita-tive differences in fragment patterns among a large body ofcomparative data, we estimated DNA similarities on the basisof the fraction of common fragments generated by the endo-nuclease. The value generated is referred to as the F value.

It has been suggested by Tenover et al. (27) that, in thecontext of analyzing outbreak strains with one endonuclease, aone- to three-fragment difference can occur between closelyrelated strains and possibly related strains can be found amongthose that differ by four to six fragments. It has also beenreported that closely related strains of Enterococcus faecaliscan differ by up to six bands and that patterns have been shownto differ by up to seven bands and still be clonally related (28).Furthermore, using hybridization analysis and PFGE in theinvestigation of insertion mutations in the transferrin bindingsystems of Hib, Curran et al. (5) found five different changes ofzero to three bands between the SmaI fragments of five inser-tion mutants. These results remind us that several differentchromosomal mutations could generate the same phenotype.

Thus, the values of F obtained should not be seen as preciseestimates of genetic distance but should be seen as summaryvalues indicative of overall similarities and differences betweenisolates and a means of illustrating relationships within a largebody of comparative data. F values of $0.9 representing pairsof isolates that differ by up to three fragments were used todetermine closely related isolates in this study, and we describesuch closely related isolates as a clonal group. However, pairsof isolates that are closely related can be found as members ofdifferent clonal groups as well as within clonal groups, so thenumber of branches is not an absolute representation of allclosely related pairs. For example, the predominant RFLPtype, type A8b, is found within a clonal group that comprisestwo other closely related types. It is also closely related (F $0.9) to 16 types found in 10 other clonal groups within group A.

No association between RFLP type and age, sex, date ofcollection, or disease manifestation was found. Because of thelack of isolates from Aboriginals from urban areas and isolatesfrom non-Aboriginals from rural areas, the association be-tween geographic location and type was not found to be sig-nificant in this sample. The genetic distance separating theimportant major lineages ranged from a similarity of 20% toone of 50%, yet there is apparently an equivalent ability tocause disease among the lineages. One of the patterns of Hibdisease in Australia was a significant difference in the incidenceof epiglottitis among urban populations. The incidence of epi-glottitis in Victoria (7) and the Australian Capital Territory(20) was twice that in Sydney (21) and Western Australia (12).Another pattern of Hib disease in Australia was the lack ofepiglottitis among Aboriginal children, similar to the epidemi-ology of Hib disease among other high-risk indigenous popu-lations (9). The possibility that differences in the incidence ofepiglottitis among different population groups are related todifferences in the strains of Hib has not been demonstrated inthis study, and the patterns of the incidence of epiglottitis inAustralia remain unexplained. Other restriction enzymeswhich sample different areas of the genome may detect mo-lecular differences that demonstrate clonal disease specificity,but we did not detect such a relationship among the SmaI-generated RFLP types in this sample.

Analysis of isolates from Aboriginal children living in geo-graphically isolated regions of Australia revealed a relativelysmall number of genetically distinct RFLP types. A strongassociation was found between the origin of isolates from Ab-original children and one of these types. Isolates from non-Aboriginal populations were more diverse and genetically dis-tinct from most isolates from Aboriginal populations, and oneof these types, found in a cluster of closely related isolates, wasstrongly associated with the origin of isolates from non-Ab-original children. The data support the hypothesis described byMusser et al. (22) that a causal relationship exists between thedegree of ethnic mixing of human populations and the degreeof diversity in the clonal composition of Hib populations. Ac-cording to this hypothesis, a large component of the currentgeographic variation in the clonal composition of Hib reflectsan older pattern of differentiation that evolved in relative geo-graphic isolation before the Age of Exploration (beginningabout 450 years ago) and has not been completely obscured byrecent demographic changes (22). It explicitly predicts thatisolates from Aboriginal populations largely belong to a dis-tinctive set of clones, as described in this study.

If Hib had been present in Aboriginal populations beforethe settlement of Australia by Europeans, the striking differ-ence between the predominant Aboriginal type and the pre-dominant non-Aboriginal type suggests a separate evolution ofstrains. The time span since permanent settlement by Europe-



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ans that began on the east coast of Australia in 1788 does notsupport a long history of differentiation that may be needed forsuch a deep divergence between types. Therefore, it is morelikely that urban non-Aboriginal types have recently been in-troduced from other areas of the world rather than derivedfrom the Aboriginal types. Nonetheless, more extensive anal-ysis is needed to provide data to support the explanation of thephylogenetic relationship between the Hib types describedhere.

Prior to the introduction of vaccination, the incidence of Hibdisease in Aboriginal populations was reported to be extremelyhigh and varied among communities. The estimated annualincidence among Aboriginal children under age 5 in centralAustralia (an area corresponding to the Alice Springs region inthis study) was 991 cases per 100,000, with a high proportion ofcases of meningitis and a fatality rate of 8.3% (8). The corre-sponding rate among non-Aboriginal children in central Aus-tralia was 215 per 100,000, which was significantly higher thanthat in other areas of the Northern Territory and four timeshigher than that in Melbourne (9). It was concluded that en-vironmental factors and cultural factors were responsible forthe very high incidence of Hib disease in Aboriginal commu-nities (8). However, this does not explain the increased inci-dence in non-Aboriginal children. An alternative explanation,that the strains circulating among Aboriginal children and af-fecting some non-Aboriginal children in the same geographicarea are more virulent than urban strains, has not been inves-tigated.

Our data show that a few distinct types are responsible forHib disease among Aboriginal populations, and the high inci-dence of disease in very young children indicates that the veryhigh transmission rates are associated with environmental fac-tors. According to Ewald’s (6) cultural vector hypothesis, in-creased transmission rates and repeated passage through hu-man hosts may lead to the selection of more rapidlymultiplying and virulent strains. If so, such strains of Hib inAboriginal populations are also likely to spread to non-Aborig-inal children in the same geographic area and result in anincreased diversity of strains in this group as well as an in-creased incidence of disease.

The differences found between Hib types from Aboriginaland non-Aboriginal populations by PFGE demonstrate thepractical discriminatory power of PFGE for the analysis oflarge numbers of Hib isolates. Analysis of the huge similaritymatrices required for large samples has been facilitated by theavailability of computer programs and, with the standardiza-tion of PFGE methods, would allow inter- and intralaboratorycomparisons of results in a common database. This wouldimprove surveillance by increasing the capacity to detect newstrains of Hib and monitor populations for the presence of oldstrains. The usefulness of the system was shown when twoisolates of Hib recently recovered in a nursing home outbreakin the Sydney region (13) were analyzed, and both were shownto have patterns identical to that of SmaI type A8b. Thissuggested that these isolates were not a new genotype sincethey were identical to the predominant type found in Sydney inthe prevaccine era. We believe that this system will be usefulfor the monitoring of future Hib isolates from children inwhom the vaccine has failed, unimmunized children, andadults.

ACKNOWLEDGMENTS

We gratefully acknowledge Peter R. Stewart and Jan Bell for criticalassistance in the early stages of this work and Adrian Gibbs for in-valuable help in creating dendrograms. We also thank Michael Grattenof the Queensland Institute of Medical Research, Amanda Leach of

the Menzies School of Health Research in Darwin, Leonie Walpingtonof the Princess Margaret Hospital in Perth, and Chris Ashhurst-Smithof Townsville General Hospital for sending us isolates and FrancesOppedisano of the Royal Children’s Hospital in Melbourne for send-ing us isolates and the pU038 and pU082 probes originally provided toG. L. Gilbert by E. Richard Moxon of the John Radcliff Hospital,Oxford, United Kingdom.

The work presented here was undertaken in the Division of Bio-chemistry and Molecular Biology in the Faculty of Science at theAustralian National University with funds and resources provided bythat Division and the Graduate School. An Australian PostgraduateResearch Award provided financial support for P. E. Moor.

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