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Journal of Clinical Microbiology, August 2003, p. 3473-3480, Vol. 41, No. 8
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.8.3473-3480.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Typing of Nonencapsulated Haemophilus Strains by Repetitive-Element Sequence-Based PCR Using Intergenic Dyad Sequences

Guillaume Bruant, Stephane Watt, Roland Quentin, and Agnes Rosenau*

Département de Microbiologie Médicale et Moléculaire, Unité de Bactériologie, Centre Hospitalo-Universitaire Bretonneau, 37044 Tours Cedex, France

Received 17 January 2003/ Returned for modification 26 February 2003/ Accepted 2 May 2003

Intergenic dyad sequences (IDS) are short repeated elements that have been described for several Haemophilus genomes and for only two other bacterial genera. We developed a repetitive-element sequence-based PCR using an IDS-specific primer as a typing method (IDS-PCR) for nonencapsulated Haemophilus strains and compared this technique with pulsed-field gel electrophoresis (PFGE) of DNA restricted with SmaI. IDS-PCR was rapid, easy to perform, and reproducible, with a high discriminatory capacity for nontypeable Haemophilus influenzae (NTHI) strains. The 69 NTHI strains tested generated 65 different banding patterns. Epidemiologically related strains gave similar or identical fingerprints, and all of the unrelated strains except two showed different patterns. These results were in agreement with those obtained by PFGE. For 20 genital strains usually identified as being biotype IV NTHI and belonging to a cryptic genospecies of Haemophilus with remarkable genetic homogeneity, four bands were significantly present and six bands were significantly absent from the fingerprints. The 20 strains were gathered in 11 closely related profiles, whereas PFGE provided no band when DNA was treated with SmaI. IDS-PCR improved the differentiation previously obtained within this species by ribotyping and multilocus enzyme electrophoresis. Our findings suggest that IDS-PCR is a rapid, reliable, and discriminatory method for typing NTHI strains and is currently the most efficient method for distinguishing strains within the cryptic genospecies of Haemophilus.

* Corresponding author. Mailing address: Département de Microbiologie Médicale et Moléculaire, EA3250, Unité de Bactériologie, Centre Hospitalo-Universitaire Bretonneau, 37044 Tours Cedex, France. Phone: (33) 2 47 47 80 59. Fax: (33) 2 47 47 38 12. E-mail: rosenau{at}med.univ-tours.fr.

Journal of Clinical Microbiology, August 2003, p. 3473-3480, Vol. 41, No. 8
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.8.3473-3480.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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