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Journal of Clinical Microbiology, April 2003, p. 1623-1636, Vol. 41, No. 4
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.4.1623-1636.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Characterization of Encapsulated and Noncapsulated Haemophilus influenzae and Determination of Phylogenetic Relationships by Multilocus Sequence Typing

Emma Meats,1 Edward J. Feil,2 Suzanna Stringer,3 Alison J. Cody,4 Richard Goldstein,5 J. Simon Kroll,6 Tanja Popovic,7 and Brian G. Spratt1*

Department of Infectious Disease Epidemiology,1 Molecular Infectious Diseases Group, Department of Paediatrics, Imperial College London, St. Mary's Hospital, London W2 1PG,6 Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY,2 PHLS Haemophilus Reference Unit, Academic Department of Microbiology and Infectious Disease, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU,3 Molecular Infectious Diseases Group, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom,4 Section of Molecular Genetics, Division of Pediatric Infectious Diseases, Maxwell Finland Laboratory for Infectious Diseases, Boston University School of Medicine and Boston Medical Center, Boston Massachusetts 02118,5 Meningitis and Special Pathogens Branch, Centers for Disease Control and Prevention, Atlanta, Georgia 303337

Received 10 October 2002/ Returned for modification 9 December 2002/ Accepted 7 January 2003

A multilocus sequence typing (MLST) scheme has been developed for the unambiguous characterization of encapsulated and noncapsulated Haemophilus influenzae isolates. The sequences of internal fragments of seven housekeeping genes were determined for 131 isolates, comprising a diverse set of 104 serotype a, b, c, d, e, and f isolates and 27 noncapsulated isolates. Many of the encapsulated isolates had previously been characterized by multilocus enzyme electrophoresis (MLEE), and the validity of the MLST scheme was established by the very similar clustering of isolates obtained by these methods. Isolates of serotypes c, d, e, and f formed monophyletic groups on a dendrogram constructed from the differences in the allelic profiles of the isolates, whereas there were highly divergent lineages of both serotype a and b isolates. Noncapsulated isolates were distinct from encapsulated isolates and, with one exception, were within two highly divergent clusters. The relationships between the major lineages of encapsulated H. influenzae inferred from MLEE data could not be discerned on a dendrogram constructed from differences in the allelic profiles, but were apparent on a tree reconstructed from the concatenated nucleotide sequences. Recombination has not therefore completely eliminated phylogenetic signal, and in support of this, for encapsulated isolates, there was significant congruence between many of the trees reconstructed from the sequences of the seven individual loci. Congruence was less apparent for noncapsulated isolates, suggesting that the impact of recombination is greater among noncapsulated than encapsulated isolates. The H. influenzae MLST scheme is available at www.mlst.net, it allows any isolate to be compared with those in the MLST database, and (for encapsulated isolates) it assigns isolates to their phylogenetic lineage, via the Internet.

* Corresponding author. Mailing address: Department of Infectious Disease Epidemiology, Faculty of Medicine, Imperial College London, St. Mary's Hospital, Norfolk Pl., London W2 1PG, United Kingdom. Phone: 44 207 594 3629. Fax: 44 207 594 3693. E-mail: b.spratt{at}imperial.ac.uk.

Journal of Clinical Microbiology, April 2003, p. 1623-1636, Vol. 41, No. 4
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.4.1623-1636.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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