Molecular basis of rifampicin resistance in Haemophilus influenzae

Susana Cruchaga, María Pérez-Vázquez, Federico Román and José Campos*

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Haemophilus influenzae causes a variety of diseases in humans, ranging from conjunctivitis and chronic respiratory infection to meningitis.1 Rifampicin is an effective antibiotic for the eradication of H. influenzae nasopharyngeal colonization and is recommended for those in close contact with patients with invasive disease to prevent the spread of this infection.1

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 507–533) and II (amino acids 563–572), 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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Twelve rifampicin-resistant (Rifr), one rifampicin-intermediate and four rifampicin-susceptible H. influenzae isolates (including the reference strain ATCC 51907) were analysed in this study, as defined by current NCCLS criteria.6 These isolates were selected from a large group of consecutive clinical isolates collected at the Reference Laboratory of Haemophilus, Majadahonda, Spain, during the years 1994–2001. Species were identified and their serotype and biotype determined according to standard techniques.1 Two of the rifampicin-resistant isolates were serotype f and 10 were non-capsulated. The sources of the rifampicin-resistant isolates were sputum (five isolates), conjunctiva (five), blood (one) and bronchial aspirate (one); the intermediate isolate was isolated from sputum. Four rifampicin-susceptible isolates were chosen for comparative purposes: ATCC 51907, and the clinical isolates 83498, 53700 and 33598; all were non-typeable isolates isolated from sputum except for 33598, which was a serotype f isolated from blood. The isolates belonged to different biotypes.

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 manufacturer’s 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 (Sigma–Aldrich, 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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
According to PFGE fingerprinting analysis, most of the isolates were genetically unrelated (data not shown).

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 492–632), 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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Table 1. Rifampicin susceptibility and amino acid changes in clusters I, II and III, and outside these clusters, in the sequenced fragment of RpoB in H. influenzae
 


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Figure 1. Comparison between the same fragment of RpoB in E. coli K12 (E.C. 2.7.7.6) (Ec) and H. influenzae Rd (HI0515) (Hi). Amino acids that differ are underlined. Clusters I and II are in italics. Positions and amino acid changes found in the rifampicin-resistant H. influenzae isolates are shown below the sequence.

 
Eleven of the 12 rifampicin-resistant isolates had amino acid changes in the RpoB cluster I (amino acids 507–533), of these one isolate had an additional mutation in cluster II (amino acids 563–572) and four had one mutation downstream from cluster II (Table 1), but this mutation is also found in some of the susceptible isolates, so cannot be associated with increased rifampicin MIC. One of the isolates (27400) had no amino acid modifications. None of the four rifampicin-susceptible isolates revealed amino acid changes within either of the clusters associated with rifampicin resistance. In the rifampicin-resistant isolates, 12 amino acid changes at positions 513, 516, 518, 526 and 533 of cluster I were identified. The position with most amino acid changes was Asp-516, and the most common, Asp->Val, was observed in four isolates (Table 1). Three more distinct substitutions were found at codon 516: Asp-516->Asn, Asp-516->Tyr and Asp-516->Ala (Table 1). Other substitutions found were Gln-513->Leu, His-526->Leu and Leu-534->Ser (Table 1). The rifampicin-resistant isolate 2495 presented a second mutation, Asn-518->Asp, within cluster I. All of these amino acid changes were detected in isolates with a high level of resistance to rifampicin (MIC >= 32 mg/L), except the Asp-516->Ala mutation, which was observed in the low-level resistant isolate 11200 (MIC 4 mg/L).

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-526->Asn) 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-516->Val and Asn-518->Asp) and had MICs > 32 mg/L.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
For a variety of bacteria25 the majority of mutations leading to rifampicin resistance have been detected in the conserved cluster I and II regions of the rpoB gene; however, no information about the molecular mechanism of rifampicin resistance in H. influenzae has been available until now.

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-516->Ala 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-572->Asn) 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-516->Ala 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.


    Acknowledgements
 
This work was supported by a fellowship from the Instituto Carlos III (reference 02/16), and the Autonomous Community of Madrid (reference 08.2/0007/2001 1), Spain.


    Footnotes
 
* Corresponding author. Tel: +34-91-8223650; Fax: +34-91-5097966; E-mail: jcampos{at}isciii.es Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Campos, J. & Sáez-Nieto J. A. (2001). Gram-negative infections: Haemophilus and other clinically relevant Gram negative coccobacilli. In Laboratory Diagnosis of Bacterial Infections (Cimolai, N., Ed.), pp. 557–80. Marcel Dekker, Inc., New York, NY, USA.

2 . Ding, J. J. & Gross, C. A. (1988). Mapping and sequencing of mutations in the Escherichia coli rpoB gene that lead to rifampin resistance. Journal of Molecular Biology 202, 45–58.[ISI][Medline]

3 . Padayachee, T. & Klugman, K. P. (1999). Molecular basis of rifampicin resistance in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 43, 2361–5.[Abstract/Free Full Text]

4 . Stefanelli, P., Fazio, C., La Rosa, G. et al. (2001). Rifampicin-resistant meningococci causing invasive disease: detection of point mutations in the rpoB gene and molecular characterization of strains. Journal of Antimicrobial Chemotherapy 47, 219–22.[Abstract/Free Full Text]

5 . Wichelhaus, T. A., Schäfer, V., Brade, V. et al. (2001). Differential effect of rpoB mutations on antibacterial activities of rifampicin and KRM-1648 against Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 47, 153–6.[Abstract/Free Full Text]

6 . National Committee for Clinical Laboratory Standards. (2001). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Sixth Edition: Approved Standard M7-A6. NCCLS, Wayne, PA, USA.

7 . National Committee for Clinical Laboratory Standards. (2002). Performance Standards for Antimicrobial Susceptibility Testing: Eleventh Informational Supplement M100-S12. NCCLS, Wayne, PA, USA.

8 . Fleischmann, R. D., Adams, M. D., White, O. et al. (1995). Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269, 496–512.[ISI][Medline]

9 . Herriott, R. M., Meyer, E. M. & Vogt, M. (1970). Defined nongrowth media for stage II development of competence in H.influenzae. Journal of Bacteriology 101, 517–24.[ISI][Medline]

10 . Wilson, K. (1994). Preparation of genomic DNA for bacteria. In Current Protocols in Molecular Biology, Vol. 1 (Ausubel, F. M., Brent, R., Kingston, R. E. et al., Eds), pp. 2.4.1–2.4.2. John Wiley & Sons, Inc., Harvard, MA, USA.





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