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
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Abstract |
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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
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Introduction |
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Materials and methods |
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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 manufacturers 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
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Results |
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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.
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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%).
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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).
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Discussion |
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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.
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Acknowledgements |
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Footnotes |
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References |
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