JAC Advance Access originally published online on November 12, 2003
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Journal of Antimicrobial Chemotherapy (2003) 52, 1011-1014
© 2003 The British Society for Antimicrobial Chemotherapy
Molecular basis of rifampicin resistance in Haemophilus influenzae
Centro Nacional de Microbiología, Instituto de Salud Carlos III, Carretera Majadahonda a Pozuelo, Km 2, 28220 Majadahonda, Madrid, Spain
Received 31 July 2003; returned 15 September 2003; revised 6 October 2003; accepted 6 October 2003
Objectives: To determine the molecular basis of rifampicin resistance in Haemophilus influenzae.
Methods: Mutations in the rifampicin-resistance determining region of the rpoB gene of H. influenzae were analysed by gene amplification and sequencing in 12 rifampicin resistant, one intermediate and four susceptible isolates.
Results: All clinical resistant isolates except one had at least one amino acid substitution in the ß-subunit of RNA polymerase. Eleven resistant isolates had amino acid changes at codons 513, 516, 518, 526 and 533 of cluster I, with the most common amino acid substitution being Asp-516
Val. Only one resistant isolate also had a second mutation Asn-518
Asp in cluster I; transformants obtained with DNA of this isolate also had both mutations. All the amino acid changes in cluster I were detected in isolates with a high level of rifampicin resistance (MIC
32 mg/L), except the Asp-516
Ala mutation in a low-level resistant isolate (MIC 4 mg/L). Only one serotype f isolate with an MIC of 2 mg/L had a mutation in cluster II. Cluster III presented no amino acid changes. In in vitro-generated high-level rifampicin-resistant mutants, only amino acid changes at codons 516 and 526 were seen, with new amino acid changes appearing at codon 526 of cluster I, while His-526
Asn was associated with low-level resistance.
Conclusions: Rifampicin resistance in H. influenzae is due to point mutations in the rpoB gene, and the resistance levels are dependent on both the location and the nature of amino acid substitution.
Keywords: H. influenzae, RNA polymerase, rpoB gene
* Corresponding author. Tel: +34-91-8223650; Fax: +34-91-5097966; E-mail: jcampos{at}isciii.es
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