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
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Abstract |
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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-516Val. 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
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Introduction |
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Rifampicin inhibits chain initiation of bacterial DNA-dependent RNA polymerase by binding to the ß-subunit of RNA polymerase. Previous studies25 have shown that point mutations located in a short conserved region in the rpoB gene render the enzyme less susceptible to rifampicin. The majority of mutation sites are clustered in three distinct areas, numbered according to the Escherichia coli protein coordinates: the principal clusters are I (amino acids 507533) and II (amino acids 563572), which harbour most mutations, while a single mutation at position 687 defines cluster III.2
The aims of this study were to describe the molecular basis of rifampicin resistance in clinical isolates of H. influenzae and to determine the genetic relationship between rifampicin-resistant isolates.
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Materials and methods |
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Rifampicin susceptibility data were initially obtained by the Etest method (AB Biodisk, Solna, Sweden) and further confirmed by the reference broth microdilution method according to NCCLS guidelines.6,7
H. influenzae DNA was digested with SmaI (MBI Fermentans, Madrid, Spain) and pulsed-field gel electrophoresis (PFGE) performed with the CHEF-DRII system (Bio-Rad, Hemel Hempstead, UK). The genetic relationships were calculated by the Dice correlation coefficient (Molecular Analyst program; Bio-Rad, Madrid, Spain).
Two DNA fragments including the clusters I, II and III of rpoB were amplified by PCR: one fragment of 410 bp included cluster I and another fragment of 531 bp included clusters II and III. Two sets of primers were designed from the sequenced H. influenzae rpoB gene (TIGR database; locus HI0515), flanking the equivalent positions involved in resistance to rifampicin in Escherichia coli. These primers were: RpoF-1 (5'-gtaaccgtcgtatccgtagcg-3'), RpoR-1 (5'-gcacgtactaatgattatggt-3'), RpoF-2 (5'-gcaacacctgagtcaagtgc-3') and RpoR-2 (5'-gcacttgactcaggtgttgc-3'). PCR products were purified with a purification Kit (Qiagen, Hilden, Germany). Fragments were sequenced on both DNA strands with the Big Dye Terminator Cycle Sequencing Kit (Perkin-Elmer, Warrington, UK) according to the manufacturers instructions. The products were resolved and analysed with an ABI PRISMR 377 DNA sequencer. Nucleotide sequences were analysed with the DNAstar program (DNASTAR, Inc., Madison, WI, USA).
H. influenzae ATCC 519078 was used as a reference isolate for the molecular procedures.
Rifampicin-resistant mutants were obtained in vitro by plating 106 cfu/mL of exponentially growing H. influenzae ATCC 51907 onto supplemented Haemophilus test medium agar base, containing 1 and 10 mg/L of rifampicin (SigmaAldrich, Madrid, Spain). After 48 h of incubation at 37°C, single colonies were re-plated and the MICs of the isolates determined by the Etest method. In all mutant isolates with rifampicin MICs > 1 mg/L, the two rpoB fragments including clusters I, II and III were amplified by PCR and sequenced.
H. influenzae ATCC 51907 was transformed by the MIV media procedure9 using whole-cell DNA from isolate 2495, extracted and purified as described.10 This isolate was the only one with two mutations in cluster I. Transformants were selected in 10 mg/L of rifampicin.
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Results |
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The MICs of rifampicin and amino acid changes in the H. influenzae isolates studied are presented in Table 1. The percentage of similarity between RpoB of E. coli K12 and H. influenzae Rd was 80% (Figure 1). We found 84.3% similarity in the fragment of RpoB (amino acids 492632), in which the amino acid changes were found. In the region responsible for rifampicin resistance (clusters I, II and III) the homology was 97.5% (Figure 1).
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Within cluster II, replacement of Ile-572 by Asn was identified in the serotype f isolate 17300 with intermediate susceptibility to rifampicin (MIC 2 mg/L). No amino acid changes were observed in cluster III. Substitution of Val by Ala was found outside these clusters at position 608 (Table 1), but since this mutation was found in some of the susceptible isolates, it has no role in rifampicin resistance.
We also determined the rifampicin MICs and the presence of amino acid modifications in 10 in vitro-selected rifampicin-resistant or -intermediate mutants. All five mutant isolates with a rifampicin MIC of 4 mg/L had one unique amino acid substitution (His-526Asn) in cluster I. The amino acid modification Asp-516
Ala was found in two intermediately resistant mutants with an MIC of 2 mg/L. In three mutants with a rifampicin MIC > 32 mg/L, the amino acid mutations obtained were at codon 516 (Asp
Tyr or Asn) and at codon 526 (His
Tyr).
Transformation of ATCC 51907 with donor DNA of isolate 2495 yielded six transformants; all had the same two amino acid changes found in the donor isolate (Asp-516Val and Asn-518
Asp) and had MICs > 32 mg/L.
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Discussion |
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In clinical rifampicin-resistant H. influenzae isolates we found 12 mutational changes at five positions (513, 516, 518, 526, 533) in cluster I and one in cluster II (572). Asp-516Ala represents a previously unreported amino acid substitution; this change was apparently related to low-level rifampicin resistance in H. influenzae both in clinical isolates and in vitro mutants. The other amino acid changes found have been reported in other rifampicin-resistant microorganisms.25
Only one amino acid substitution was observed in this study in cluster II (Ile-572Asn) of one H. influenzae serotype f isolate with an intermediate level of rifampicin resistance. The predominance of the Asp-516
Val mutation and the occurrence of different non-synonymous substitutions at this same position in resistant isolates demonstrated that amino acid 516 may be the most important rifampicin-binding site in H. influenzae, followed by amino acid 526.
With the exception of three mutations (Asp-516Ala and Ile-572
Asn in clinical isolates, and His-526
Asn in the in vitro-selected mutants), only one other mutation confers a high level of rifampicin resistance on H. influenzae. In vitro-selected rifampicin-resistant mutants of H. influenzae did not exactly reproduce the amino acid changes observed in clinical isolates as the most frequent position at which amino acid changes were detected was codon 526, while in clinical isolates it was 516. The most frequent amino acid change in clinical isolates (Asp-516
Val) was not seen in any of the 10 mutants analysed, while in vitro mutants revealed new mutations at codon 526. In five low-level resistant mutants (MIC 4 mg/L), the His-526
Asn substitution was obtained; this mutation has been observed in Streptococcus pneumoniae,3 and in other species such as Neisseria meningitidis4 and Staphylococcus aureus.5 A new amino acid change (His-526
Tyr) appeared in one mutant isolate but not in the clinical isolates; this mutation has been reported in several microorganisms, such as E. coli,2 N. meningitidis4 and S. aureus.5
In one isolate, 27400 (MIC 16 mg/L), no mutations were found within rpoB. In this case, mutations outside the DNA regions sequenced, alterations in drug uptake and efflux mechanisms and changes in rifampicin permeability of the outer membrane could play a role in resistance to rifampicin.
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Acknowledgements |
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Footnotes |
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References |
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