Mutations of the rpoB gene in rifampicin-resistant Streptococcus pneumoniae in Taiwan

J.-Y. Chen1, Chang-Phone Fung2, Feng-Yee Chang3, Li-Yueh Huang1, Jen-Chang Chang1 and L. K. Siu1,*

1 Division of Clinical Research, National Health Research Institutes; 2 Section of Infectious Diseases, Department of Medicine, Taipei Veterans General Hospital and National Yang-Ming University; 3 Division of Infectious Diseases and Tropical Medicine, Department of Internal Medicine, Tri-Services General Hospital, Taipei, Taiwan

Received 30 October 2003; returned 3 November 2003; revised 17 November 2003; accepted 17 November 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective: To determine the mechanism of rifampicin resistance in Streptococcus pneumoniae in Taiwan.

Methods: Rifampicin resistance was investigated with respect to the rpoB gene in 23 invasive S. pneumoniae isolates collected from 1996 to 2001. PCR and molecular typing were used for genetic and epidemiological analyses. Transformation was used to determine the functional gene for resistance.

Results: Twenty-two of 23 isolates carried at least one mutation at either cluster I or III of rpoB; the most frequent mutation found in 21 isolates (91%) was histidine (H499) to asparagine or tyrosine at position 499, followed by isoleucine to valine (I624V) at position 624 in 16 isolates (70%), tyrosine to phenylalanine (Y589F) at position 589 in 14 isolates (60.9%) and isoleucine to valine (I608V) at position 608 in 13 isolates (56.5%). Less-frequent mutations were also identified: D489V, R597F, N623E, N623S, N669D, Q671K, Y674F and A683V. High-level rifampicin resistance was observed in isolates with a mutation at position 499 or 489. Mutations other than at position 499 or 489 played little role in or had no relation to rifampicin resistance. No dominant epidemic strain was observed with ribotyping, multilocus sequence typing, or serotyping.

Conclusions: Rifampicin resistance among multidrug-resistant S. pneumoniae in Taiwan was mostly caused by rpoB mutations.

Keywords: S. pneumoniae, rifampicin resistance, rpoB gene


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Streptococcus pneumoniae is an important causative pathogen for infections such as otitis, meningitis, bacteraemia and pneumonia; many antibiotics have been suggested for pneumococcal infections. However, S. pneumoniae isolates resistant to many kinds of antibiotics are increasing, and failure of treatment has been reported worldwide.1 Rifampicin has been suggested for multidrug-resistant streptococcal infections2,3 and has shown encouraging results in a rabbit model and in clinical treatments when combined with either ß-lactam antibiotics or vancomycin.2,3 However, few studies have been conducted worldwide on the molecular epidemiology and mechanism of rifampicin resistance in S. pneumoniae, whereas none has been reported from Taiwan. In this study, we attempted to understand the molecular epidemiology and mechanism of rifampicin resistance of S. pneumoniae in Taiwan.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains and antimicrobial susceptibility testing

Rifampicin-resistant, invasive S. pneumoniae isolates were obtained from a previous study of S. pneumoniae in Taiwan from 1996 to 2001.4,5 Antimicrobial susceptibility was determined by Sensititre Susceptibility Plates (TREK Diagnostic Systems, UK) according to the National Committee for Clinical Laboratory Standards (NCCLS).6 The following antibiotics were used: cefotaxime, vancomycin and rifampicin. The ATCC 49619 control strain of S. pneumoniae was included in each test run. Susceptibility against high-level rifampicin was tested using Etest strips (PDM Epsilometer; AB Biodisk, Solna, Sweden).

PCR amplification of rpoB gene clusters and DNA sequencing

PCR was carried out under conditions specified by the manufacturer (Amersham Pharmacia Biotech, UK). The reactions were denatured for 1 min at 94°C, annealed for 1 min at 58°C, and incubated for 1 min at 72°C for 35 cycles in a Programmable Thermal Controller PTC-100 (MJ Research, USA). A 789 bp DNA fragment of rpoB that included clusters I and II and pneumococcus III was amplified with the forward primer, rpoBF1 (5'-GACAATGAAGTCTTGACACC-3'), and the reverse primer, rpoBR3 (5'-CAATGAACCATCTTCACGACG-3').7 Primers specific for clusters I and II were rpoBF1 and rpoBR1 (5'-CGTGACAACACCTGTTTC-3'), and those for pneumococcus cluster III were rpoBF3 (5'-GTTCAAACACCATACCGTAAG-3') and rpoBR3.7 For direct DNA sequencing, sequencing reactions were carried out with an automated sequencer (ABI Prism 377; Perkin-Elmer, CT, USA).

Restriction fragment length polymorphism (RFLP) of the rrn gene

Ribotyping was carried out using the automated Riboprinter Microbial Characterization System (Qualicon, Wilmington, DE, USA) according to the manufacturer’s instructions. Total DNA was digested with the HindIII enzyme; DNA was separated by electrophoresis and transferred directly to nylon membranes. Assignment to a particular ribotype was based upon differences in band numbers, band position and signal intensity at a given banding position. To reveal ribotyping polymorphism, each sample was analysed by Molecular Analyst Fingerprinting, Fingerprinting Plus, and Fingerprinting DST Software (Bio-Rad Laboratories, Richmond, CA, USA). The grouping method was carried out to deduce a dendrogram from the matrix via the Unweighted Pair Group Method using Arithmetic Averages (UPGMA) clustering technique after calculation of similarities using Pearson correlation coefficients between every pair of organisms.5

Multilocus sequence typing (MLST)

MLST was carried out as described by Enright & Spratt.8 In the interpretation of results, strains with identical allelic profiles, or those differing at a single locus, were considered likely to be genetically related, whereas strains differing in two of the seven loci were considered likely to be genetically unrelated.

Transformation

To investigate whether point mutations found in cluster I mutants mediated high-level rifampicin resistance, PCR fragments using primers rpoBF1 and rpoBR1 were purified and cloned into the TOPO vector (Invitrogen, San Diego, CA, USA). Mini-prep DNA (Wizard Miniprep, Qiagen, CA, USA) was then used to transform S. pneumoniae strain R6.9


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Epidemiological data of the collected rifampicin-resistant S. pneumoniae isolates

Twenty-three rifampicin-resistant isolates were obtained from 1- to 76-year-old patients and were evenly distributed among northern, southern, and central parts of Taiwan. MICs of rifampicin ranged from 4 to >256 mg/L. The coexistence of vancomycin and cefotaxime resistance was not detected. Among these invasive isolates, 11, 7 and 5 were collected in 1996, 1998 and 2000, respectively. Serotype 23F was the most frequent type encountered (11/23), followed by serotypes 19F (4/23), 14 and non-typeable (3/23 of each type). Only one isolate of each belonged to serotypes 3 and 15. Seventeen of the 23 isolates were obtained from blood, whereas two isolates were from CSF. The rest were from pus or urine.

Ribotype pattern polymorphism

Ribotyping of the 23 rifampicin-resistant S. pneumoniae isolates was analysed. In total, 13 different ribotypes were observed. Four different clusters with identical ribotypes were found for 12 isolates overall (52.2%): one major cluster which included six isolates (26.1%) and three minor clusters which each included two isolates (8.7%).

MLST

Among 23 rifampicin-resistant isolates, nine different sequence types were identified, among which four had not previously been described (Table 1). Sequence types 718 and 719 are probably variants of sequence type 242, which has been found in Taiwan, Italy and Brazil. A novel spi allelic sequence (77) was found in one non-typeable isolate and was designated sequence type 884. No predominant sequence type was associated with rifampicin resistance.


View this table:
[in this window]
[in a new window]
 
Table 1. MLST of the 23 rifampicin-resistant Streptococcus pneumoniae isolates
 
Molecular mechanism of rifampicin resistance

The distribution of mutations and results of rifampicin susceptibility tests for each isolate are summarized in Table 2. Twenty-two of the 23 isolates carried at least one mutation at either cluster I or III of rpoB. These caused amino acid substitutions; histidine (H499) to asparagine or tyrosine at position 499 was the most frequent mutation found in 21 isolates (91%), followed by isoleucine to valine (I624V) at position 624 in 16 isolates (70%), tyrosine to phenylalanine (Y589F) at position 589 in 14 isolates (60.9%), and isoleucine to valine (I608V) at position 608 in 13 isolates (56.5%). Less-frequent mutations were seen at position 669 from asparagine to aspartate (N669D) in five isolates (21.7%) and four isolates (17.4%) each at position 623 from asparagine to glutamate (N623E) or serine (N623S), at position 671 from glutamine to lysine (Q671K), and at position 683 from alanine to valine (A683V). Mutations at position 489 from aspartate to valine (D489V), position 597 from arginine to phenylalanine (R597F), and position 674 from tyrosine to phenylalanine (Y674F) were relatively rare events which occurred only once each (4.3%).


View this table:
[in this window]
[in a new window]
 
Table 2. RpoB mutations of rifampicin-resistant Streptococcus pneumoniae isolates
 
Transformation

The transformation study demonstrated that the mutations of H499N, H499Y and D489V were involved in rifampicin resistance. MICs of transformants showed that the H499Y and D489V mutations conferred high-level rifampicin resistance (>=256 mg/L), whereas H499N was associated with a relatively low level of resistance with MICs of 8 and 16 mg/L (Table 3).


View this table:
[in this window]
[in a new window]
 
Table 3. rpoB gene mutations and amino acid changes leading to rifampicin resistance in Streptococcus pneumoniae in vitro
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study of rifampicin-resistant S. pneumoniae, cluster I mutation at position 499 of RpoB from histidine to asparagine alone increased resistance significantly to 8 mg/L (Table 3), as described previously.10 An isolate with the histidine substituted by tyrosine (H499Y) at the same position also showed an extremely high level of resistance to rifampicin (>=256 mg/L).11 A mutation from aspartate to glutamate (D489E) at position 489 of RpoB has been described to increase resistance to 8 mg/L. Here we confirmed the importance of D489V mutation in high-level resistance.12 Mutation R597F in cluster III is a novel mutation that has not been reported, although a few rifampicin-resistant isolates were previously described that had a mutation to lysine at the same position.7 This mutation was accompanied by another seven serial mutations of I608, N623, I624, N669, Q671, Y674 and A683 (Table 2). However, positions Y589, I608, N623, I624, N669, Q671, Y674 and A683 seemed to have little effect on increasing rifampicin resistance. This may have been because the amino acid substitutions at these positions either shared identical physicochemical properties to those at positions I608 and I624, or had a similar polarity (to a greater extent) and/or a similar size (to a less extent) as those of positions N623, N669, Q671 and A683, according to detailed inspection through examples from the literature and this study.7 One isolate was determined to have no RpoB mutation. Its low-level resistance to rifampicin was probably the result of other mechanisms such as multidrug efflux.13

According to the ribotyping and MLST results, there was no evidence of involvement of any specific clonal prevalence in rifampicin-resistant isolates (Table 1). The small population of one major cluster suggested that the rifampicin resistance of S. pneumoniae in Taiwan is a factor in individual cases rather than clonal spreading. Combined with different patterns of amino acid mutations, rifampicin resistance is probably the result of spontaneous mutations or drug-selective pressures.

In conclusion, rifampicin resistance among the multidrug-resistant isolates was mostly caused by RpoB mutations. Although resistance to cefotaxime, ceftriaxone and vancomycin has yet to arise in any of these rifampicin-resistant isolates, physicians should carefully evaluate the use of rifampicin combination therapy for meningitis and severe illnesses when multidrug resistance is identified. Attention should be paid to increasing incidences of resistance as a result of the selective pressures possibly leading to the spread of rifampicin resistance in clinical settings.


    Acknowledgements
 
This work was supported by a grant from the National Health Research Institutes, Taiwan.


    Footnotes
 
* Corresponding author. Tel: +886-2-26524094; Fax: +886-2-27890254; E-mail: lksiu{at}mail.nhri.org.tw Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Mathai, D., Lewis, M. T., Kugler, K. C. et al. (2001). Antibacterial activity of 41 antimicrobials tested against over 2773 bacterial isolates from hospitalized patients with pneumonia: I—results from the SENTRY Antimicrobial Surveillance Program (North America, 1998). Diagnostic Microbiology and Infectious Disease 39, 105–16.[CrossRef][ISI][Medline]

2 . Bradley, J. S. & Scheld, W. M. (1997). The challenge of penicillin-resistant Streptococcus pneumoniae meningitis: current antibiotic therapy in the 1990s. Clinical Infectious Diseases 24, Suppl. 2, S213–21.[ISI][Medline]

3 . Paris, M. M., Hickey, S. M., Uscher, M. I. et al. (1994). Effect of dexamethasone on therapy of experimental penicillin- and cephalosporin-resistant pneumococcal meningitis. Antimicrobial Agents and Chemotherapy 38, 1320–4.[Abstract]

4 . Fung, C. P., Hu, B. S., Lee, S. C. et al. (2000). Antimicrobial resistance of Streptococcus pneumoniae isolated in Taiwan: an island-wide surveillance study between 1996 and 1997. Journal of Antimicrobial Chemotherapy 45, 49–55.[Abstract/Free Full Text]

5 . Siu, L. K., Chu, M. L., Ho, M. et al. (2002). Epidemiology of invasive pneumococcal infection in Taiwan: antibiotic resistance, serogroup distribution, and ribotypes analyses. Microbial Drug Resistance 8, 201–8.[CrossRef][ISI][Medline]

6 . National Committee for Clinical Laboratory Standards. (2002). Performance Standards for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard M7-A4. NCCLS, Villanova, PA, USA.

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

8 . Enright, M. C. & Spratt, B. G. (1998). A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease. Microbiology 144, 3049–60.[Abstract]

9 . Barcus, V. A., Ghanekar, K., Yeo, M. et al. (1995). Genetics of high level penicillin resistance in clinical isolates of Streptococcus pneumoniae. FEMS Microbiology Letters 126, 299–303.[CrossRef][ISI][Medline]

10 . Enright, M., Zawadski, P., Pickerill, P. et al. (1998). Molecular evolution of rifampicin resistance in Streptococcus pneumoniae. Microbial Drug Resistance 4, 65–70.[ISI][Medline]

11 . Meier, P. S., Utz, S., Aebi, S. et al. (2003). Low-level resistance to rifampin in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 47, 863–8.[Abstract/Free Full Text]

12 . Martin-Galiano, A. J. & De La Campa, A. G. (2003). High-efficiency generation of antibiotic-resistant strains of Streptococcus pneumoniae by PCR and transformation. Antimicrobial Agents and Chemotherapy 47, 1257–61.[Abstract/Free Full Text]

13 . Putman, M., van Veen, H. W. & Konings, W. N. (2000). Molecular properties of bacterial multidrug transporters. Microbiology and Molecular Biology Reviews 64, 672–93.[Abstract/Free Full Text]