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Antimicrobial Agents and Chemotherapy, October 1999, p. 2361-2365, Vol. 43, No. 10
Pneumococcal Diseases Research Unit of the
MRC, SAIMR, and the University of the Witwatersrand, Johannesburg,
South Africa
Received 26 February 1999/Returned for modification 2 June
1999/Accepted 27 July 1999
Rifampin resistance among South African clinical isolates of
Streptococcus pneumoniae was shown to be due to missense
mutations within the rpoB gene. Sequence analysis of 24 rifampin-resistant isolates revealed the presence of mutations within
cluster I as well as novel mutations in an area designated pneumococcus
cluster III. Of the 24 isolates characterized, only 1 resistant isolate did not contain any mutations in the regions sequenced. Either the
cluster I or the cluster III mutations separately conferred MICs of 32 to 128 µg/ml. Clinical isolate 55, for which the MIC was 256 µg/ml,
was noted to contain 9 of the 10 mutations identified, which included
the cluster I and cluster III mutations. As in Escherichia
coli, it is possible that cluster I (amino acids 406 to 434) and
cluster III (amino acids 523 to 600) of S. pneumoniae interact to form part of the antibiotic binding site, thus accounting for the very high MIC observed for isolate 55. PCR products containing cluster I or cluster III mutations were able to transform
rifampin-susceptible S. pneumoniae to resistance. Although
many of the isolates studied displayed identical sequences,
pulsed-field gel electrophoresis analysis revealed that the isolates
were not of clonal origin.
Rifampin, a semisynthetic derivative
of the rifamycins, is active against both gram-positive and
gram-negative bacteria. It has principally been advocated for use in
the treatment of tuberculosis but has nevertheless found use in therapy
for infections caused by staphylococci (28), legionellae
(29), and brucellae (1) and as a prophylactic
treatment for meningococcus (25) and Haemophilus influenzae (34) carriers. Rifampin is widely used in
South Africa for the treatment of tuberculosis in children. The
majority of young children have colonization with pneumococci in their
nasopharynx, a fact which results in the bacteria being indirectly
exposed to the selective pressures of the antimicrobial agent. Rifampin is, however, not routinely used in the treatment of pneumococcal infections, although it may be used in combination with ceftriaxone or
cefotaxime for cephalosporin-resistant pneumococcal meningitis (16, 18). Its mechanism of action is based on its ability to
bind to and inactivate the bacterial DNA-dependent RNA polymerase, a
mode of action not shared by any other antibiotic in use (10, 35).
The RNA polymerase molecule consists of five subunits, namely,
Bacterial resistance to rifampin has been investigated extensively with
Escherichia coli (15), Mycobacterium
tuberculosis (4, 17, 31, 32), M. smegmatis
(12, 22), M. leprae (13), and
Neisseria meningitidis (5). The mutations within the rpoB gene appear to cluster into three distinct areas
and are generally numbered according to the E. coli protein
coordinates. The principal clusters, I (amino acids 505 to 532) and II
(amino acids 560 to 572), harbor most of the mutations, while a single mutation at position 687 defines cluster III (11, 14, 15, 38). The discovery of numerous other mutations (23)
has made the delineation of the central rifampin resistance region
difficult. In E. coli, the majority of the mutations map to
clusters I and II, while in other species, rifampin resistance appears
to be due to cluster I mutations.
Until recently, little was known about the mechanism of rifampin
resistance in pneumococci. We thus investigated rifampin resistance
among South African clinical strains of Streptococcus pneumoniae.
Bacterial strains.
Twenty-four rifampin-resistant and 8 rifampin-susceptible isolates were randomly selected from a group of
clinical isolates collected from 1987 to 1996 and kept at the South
African Institute for Medical Research, a reference center for
laboratories in South Africa. S. pneumoniae R6, an
unencapsulated laboratory strain, was also included in this study. All
isolates were serotyped by use of the Quellung reaction (8)
with specific antisera from the Staten Seruminstitut (Copenhagen,
Denmark). Serogroups, serotypes, and antibiotic data for the isolates
are presented in Table 1.
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Molecular Basis of Rifampin Resistance in
Streptococcus pneumoniae
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
2
'
. Rifampin binds directly to the subunit
of the polymerase (38), and bacterial resistance to rifampin
has been shown to be due to mutations occurring in the rpoB
gene. Mutations to rifampin resistance result in alterations of
rifampin binding and render the organism highly resistant. These
mutations occur at a frequency of approximately 10
6 to
108 in naturally occurring cells (19, 28).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Properties of the clinical isolates used in this study as
well as the mutations identified in rifampin-resistant isolates
Susceptibility testing. The MICs were determined by the agar dilution method according to the guidelines of the National Committee for Clinical Laboratory Standards (26). Antibiotic plates were made by use of Mueller-Hinton agar supplemented with 5% horse blood.
DNA isolation.
Chromosomal DNA was extracted by the salt
lysis method of Ausubel et al. (3). DNA was precipitated at
37°C for 10 min with 0.6 volume of a solution containing 20%
polyethylene glycol (PEG) and 2.5 M NaCl. DNA was resuspended in water
containing 20 µg of RNase A per ml and stored at 
70°C for further use.
PCR. The PCR primers were designed from the S. pneumoniae rpoB gene sequence obtained from the TIGR database (contig no BSP131). The primers rpoBF1 (5'-GACAATGAAGTCTTGACACC-3') (nucleotides 1144 to 1163; S. pneumoniae coordinates) and rpoBR1 (5'-CGTGACAACACCTGTTTC-3') (nucleotides 1486 to 1503) were designed to flank clusters I and II. For isolates which carried no mutations within this central region, the primers rpoBF2 (5'-CCTGAAACTGGAGAAATCTTG-3') (nucleotides 721 to 741), rpoBR2 (5'-TGTTACAGGACGGATATTGAT-3') (nucleotides 1174 to 1194), rpoBF3 (5'-GTTCAAACACCATACCGTAAG-3') (nucleotides 1456 to 1176), and rpoBR3 (5'-CAATGAACCATCTTCACGACG-3') (nucleotides 1906 to 1926) were designed to amplify the regions upstream and downstream of clusters I and II.
PCR amplification was carried out with 50-µl volumes containing 1 µM (each) primer, 1.5 mM MgCl2, 150 µM (each) deoxynucleoside triphosphate (dNTP), 1 U of Taq polymerase, and 1 µl of DNA in the buffer provided. PCR products for use in cloning were amplified with mixtures containing 2.5 U of Taq polymerase with proofreading activity (Pwo Taq; Boehringer GmbH, Mannheim, Germany), 1 µM (each) primer, 1.5 mM MgSO4, 150 µM (each) dNTP, and 1 µl of DNA in the buffer provided by the manufacturer. Reactions were performed with a Cetus thermocycler and the following program: 1 cycle at 95°C for 5 min; 30 cycles at 95°C for 1 min, of primer-specific annealing for 2 min, and at 72°C for 2 min; and a final extension at 72°C for 2 min. Primer pairs rpoBF1-rpoBR1 and rpoBF3-rpoBR3 were annealed at 58°C, while primer pair rpoBF2-rpoBR2 was annealed at 57°C.PFGE. Pulsed-field gel electrophoresis (PFGE) for S. pneumoniae was carried out by the method of Lefevre and coworkers (21). Separation of the SmaI restriction fragments was achieved by electrophoresis for 21 h at 200 V, and visualization was done with UV illumination after ethidium bromide staining.
Nucleotide sequence analysis. Single-stranded PCR products were prepared for sequencing with streptavidin-coated magnetic particles (Boehringer). Briefly, a standard amplification reaction (100 µl) was performed with a 5'-biotinylated forward primer (rpoBF1, rpoBF2, or rpoBF3) and an unlabelled reverse primer. Nonincorporated dNTPs and primers were removed by precipitation with PEG as described previously (3), and the pellet was resuspended in 40 µl of Tris-EDTA buffer. Single-stranded DNA bound to the magnetic beads was prepared according to the manufacturer's recommendations. The unlabelled DNA was salt precipitated from the supernatant and resuspended in 20 µl of water.
Sequence analysis was carried out by use of the Sequenase version 2.0 sequencing kit (United States Biochemicals, Cleveland, Ohio) with 6.5 µl of single-stranded DNA and 2 µl of primer (10 µM stock) per dideoxy chain termination sequencing reaction according to the manufacturer's recommendations.Cloning of PCR products.
The amplified PCR products of the
rpoB gene were PEG precipitated (3). Blunt-end
ligations into SmaI (Boehringer)-digested expression vector
pGEM-7Zf(+) were performed. The ligations were carried out with 12-µl
reaction volumes containing 0.1 µg of SmaI-restricted vector, 0.3 µg of PCR product, 8% PEG 6000; 4.34 mM ATP, 5 U of SmaI, and 2 U of T4 DNA ligase (United States Biochemicals)
in ligation buffer provided by the manufacturer. The mixtures were subjected to cycling overnight at between 10 and 30°C for 30 s by the method described by Lund et al. (24). Competent
E. coli JM109 cells were electrotransformed with purified
ligated DNA as described previously (6). Transformants were
selected for -complementation on Luria-Bertani agar containing 50 µg of ampicillin per ml, 20 mg of -galactosidase per ml, and 100 mM isopropyl--D-thiogalactopyranoside (IPTG).
Transformations. Transformations were performed with competent S. pneumoniae R6 as detailed by Tomasz and Hotchkiss (33). Thawed competent cells were incubated with 1 µg of cloned PCR product per ml at 30°C for 30 min. The cells were allowed to express resistance at 37°C for 60 min before plating of 100 µl of cells onto selection media. The plates were incubated for 48 h at 37°C. DNA-free controls were also tested in order to confirm that any growth observed was the result of transformation and not mutation. Cells were also plated on antibiotic-free medium for the determination of viable counts. The MICs for transformants were determined as described above.
Nucleotide sequence accession numbers. The accession numbers AJ236789, AJ236790, AJ236791, and AJ236792 have been assigned to the sequences submitted to the EMBL database.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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PCR amplification and sequence analysis. The amino acid numbering used in this study is that of the S. pneumoniae sequence obtained from the TIGR database. The corresponding E. coli numbering has been inserted in some instances to allow for alignment with previously identified mutations.
Amplification of genomic DNA with primer pair rpoBF1-rpoBR1 yielded a 360-bp fragment that included the area designated clusters I and II; these clusters map to S. pneumoniae amino acid positions 406 to 434 and 462 to 472, respectively. Sequence analysis of the 24 rifampin-resistant and 8 rifampin-susceptible isolates, including S. pneumoniae R6, revealed numerous polymorphisms (data not shown). Some of these changes caused amino acid alterations, but most were single base substitutions that had no effect at the protein level. Eighteen of the 24 resistant isolates contained mutations within rpoB cluster I, and no mutations were identified within cluster II. Of these 18 resistant isolates, 11 displayed Asp415-Glu (equivalent to E. coli Asp516) as well as His425-Asn (equivalent to E. coli His526) substitutions (Table 1 and Fig. 1). For strains that contained the His425-Asn mutation, the MICs were found to be in the range of 32 to 128 µg/ml. The presence of the Asp415-Glu mutation did not increase the MICs for strains containing both mutations. Of the remaining seven resistant isolates, six contained the single His425-Asn mutation (isolates 2, 26940, 37277, 85991, 8085, and 17), while the other (isolate 55) contained His425-Asn as well as novel downstream mutations (described below). Six of the 24 rifampin-resistant isolates (isolates 17236, 13619, 45, 81519, 16997, and 13897) lacked these mutations and did not contain any other mutations within this PCR-amplified region. All of the rifampin-susceptible isolates lacked the mutations.
|
) and cluster III
(
) are indicated above the amino
acid residues. The shaded codons correspond to the E. coli
Asp516 and His526 amino acids. Mutations that
have been reported previously are indicated by asterisks (Transformations.
The significance of the mutations observed
was confirmed by transformation. The rpoBF1-rpoBR1 PCR product
containing cluster I mutations and the fragment obtained with
rpoBF3-rpoBR3 were able to transform rifampin-susceptible R6 to
resistance. The mean transformation efficiencies were 6.3 × 104 and 1.4 × 104, respectively. The
MICs for the transformants obtained with the cluster I product were
equivalent to the MICs for the donor, while the downstream fragment was
only able to transform S. pneumoniae R6 so that the MIC was
16 µg/ml.
Genetic relatedness of isolates. The observation that many of the rpoB gene sequences obtained were identical led us to investigate the clonal relationship of the isolates. PFGE patterns (Fig. 2) were obtained for isolates representative of the different mutation groups. Contrary to expectations, the PFGE patterns of the isolates were very different. Isolates 85991, 8085, and 17 were related on the basis of their PFGE pattern, but none of the other isolates were of clonal origin.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Rifampin resistance among South African clinical isolates of
S. pneumoniae appears to involve mutations within the subunit of the rpoB gene, as is the case in numerous other bacteria.
It was observed that 75% of the rifampin-resistant isolates studied
contained cluster I mutations
both Asp415-Glu (equivalent to E. coli Asp516) and His425-Asn
(equivalent to E. coli His526) or
His425-Asn alone. Numerous mutations at both of these
locations have been reported previously, both for E. coli
(15) and, more recently, while this study was being
conducted, for S. pneumoniae (7). The
substitution Asp415-Asn has been reported to confer low-level resistance (MIC, 8 µg/ml) in S. pneumoniae
(7) on its own. We report for the first time the presence in
S. pneumoniae of an Asp415-Glu substitution, a
mutation reported previously to occur in M. tuberculosis and
shown to be involved in the resistance phenotype (17, 32).
The second cluster I mutation, His425-Asn, has been
reported for S. pneumoniae (7), N. meningitidis (5), and M. tuberculosis
(31, 32). It is interesting to note that a large proportion
of the isolates studied contained both mutations, an observation made
previously for M. tuberculosis (36). The presence
of both Asp415-Glu and His425-Asn mutations
does not, however, appear to contribute to higher MICs, and the results obtained suggest that the presence of His425-Asn alone is
sufficient to confer high-level resistance to rifampin. These results
are consistent with other findings in that mutations at the
Asp516 and His526 codons lead to high-level
rifampin resistance in both E. coli and M. tuberculosis (15, 27, 36). The predominance of the
His425-Asn mutation in the rifampin-resistant isolates used
in this study suggests that this mutation plays an important role in
the selection of antibiotic resistance, as has been alluded to
previously by Carter and colleagues (5).
Resistant isolates that did not contain mutations in cluster I or cluster II have been reported previously for S. pneumoniae (7) and M. tuberculosis (17, 36), but further investigation of their nucleotide sequences was not attempted. Sequence analysis of the six isolates that lacked cluster I or cluster II mutations in this study revealed the presence of eight mutations, Arg523-Lys, Glu526-Ala, Iso534-Val, Asn549-Ser, Iso550-Ser, Asn595-Glu, Gln597-Lys, and Tyr600-Phe, downstream of cluster II, in a region which we now designate S. pneumoniae cluster III. This area is distinct from the E. coli cluster III region (15). The presence of all eight mutations in all six isolates lends support to the assumption that this cluster of mutations involved in rifampin resistance was acquired by homologous recombination of transformed DNA. The MIC for the transformants of these isolates was 16 µg/ml, lower than that for the parent strain, suggesting that these changes may operate in conjunction with other, as-yet-unidentified mutations within rpoB or elsewhere to confer resistance to rifampin. This idea is consistent with the findings for E. coli (30), where the cluster III mutation Arg687-His confers only very low levels of rifampin resistance, suggesting that it might be located at the periphery of the region of RNA polymerase that binds to rifampin. Genetic evidence also suggests that cluster I (the region around E. coli Arg529) and cluster III (the region around E. coli Arg687) are in close proximity in the native protein, suggesting that these two regions of the polypeptide are both involved in forming the binding site for rifampin. A similar scenario may be envisaged to occur in S. pneumoniae, with cluster I (positions 406 to 434) and cluster III (positions 523 to 600) interacting to form the antibiotic binding site. Such a scenario may account for the very high MIC observed for isolate 55 (256 µg/ml), which was found to contain mutations at His425 and in the region from Arg523 to Tyr600.
One isolate, 13897 (MIC, 64 µg/ml), was sequenced from Lys247 to Tyr627 (sequence not shown) and was found to contain no mutations within this region. It is also conceivable that mutations that could play a role in conferring resistance exist elsewhere in rpoB. Substitutions of amino acids 146 and 687, located outside clusters I and III, have been described for E. coli (15, 23). Further characterization of the rpoB gene from isolate 13897 is currently in progress.
Several alternatives have been proposed to account for the lack of
mutations observed in rpoB in rifampin-resistant strains. These include the modification of rifampin (2, 37),
alterations in drug uptake or efflux mechanisms (9, 20), and
changes in antibiotic permeability or metabolism (17). It
has also been suggested that mutations in other subunits of the
polymerase could contribute to rifampin resistance, perhaps by altering
the conformation of the rifampin binding region of the subunit
(12). Although these mechanisms have only been hinted at for
organisms other than S. pneumoniae, we cannot rule out the
possibility that they may play a role in rifampin resistance in pneumococci.
Sequence analysis revealed the possibility that some isolates may be of clonal origin, but the PFGE fingerprints show that these isolates are in fact unrelated to each other and reference clones. It appears, therefore, that rifampin resistance among South African clinical isolates is not due to the clonal spread of resistant strains.
These molecular studies support the idea that rifampin-resistant strains could be selected by a mutational event occurring among susceptible strains. The shared sequence of mutations identified for the first time in cluster III may indicate a single transformational event that has occurred among a variety of strains.
Our study has documented a second area of mutations in the rpoB gene that confers rifampin resistance in S. pneumoniae and suggests that other mechanisms have yet to be clarified.
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ACKNOWLEDGMENTS |
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We thank Lesley McGee for assistance with the PFGE and Avril Wasas for performing the serotyping.
This work was supported by the Medical Research Council, the South African Institute for Medical Research, and the University of the Witwatersrand.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Clinical Microbiology and Infectious Diseases, SAIMR, P.O. Box 1038, Johannesburg 2000, South Africa. Phone: 27-11-4899335. Fax: 27-11-4899332. E-mail: thanup{at}hotmail.com.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Antimicrobial Agents and Chemotherapy, October 1999, p. 2361-2365, Vol. 43, No. 10
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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57: 849-854
[Abstract] [Full Text] -
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50: 827-834
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49: 2237-2245
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Achour, W., Guenni, O., Fines, M., Leclercq, R., Ben Hassen, A.
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Dreses-Werringloer, U., Padubrin, I., Kohler, L., Hudson, A. P.
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Martin-Galiano, A. J., de la Campa, A. G.
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