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Antimicrobial Agents and Chemotherapy, August 2000, p. 2118-2125, Vol. 44, No. 8
Department of Infectious
Diseases1 and Department of Genomic and
Proteomic Sciences,3 Pfizer, Inc., Groton,
Connecticut; Hershey Medical Center, Hershey,
Pennsylvania;2 and Case Western
Reserve University, Cleveland, Ohio4
Received 7 March 2000/Returned for modification 11 April
2000/Accepted 3 May 2000
The mechanisms responsible for macrolide resistance in
Streptococcus pneumoniae mutants, selected from susceptible
strains by serial passage in azithromycin, were investigated. These
mutants were resistant to 14- and 15-membered macrolides, but
resistance could not be explained by any clinically relevant
resistance determinant [mef(A),
erm(A), erm(B), erm(C),
erm(TR), msr(A), mph(A),
mph(B), mph(C), ere(A),
ere(B)]. An investigation into the sequences of 23S
rRNAs in the mutant and parental strains revealed individual changes of
C2611A, C2611G, A2058G, and A2059G (Escherichia coli numbering) in four mutants. Mutations at these residues in domain V of
23S rRNA have been noted to confer erythromycin resistance in other
species. Not all four 23S rRNA alleles have to contain the mutation to
confer resistance. Some of the mutations also confer
coresistance to streptogramin B (C2611A, C2611G, and A2058G), 16-membered macrolides (all changes), and clindamycin (A2058G and A2059G). Interestingly, none of these mutations confer high-level resistance to telithromycin (HMR-3647). Further, two of the
mutants which had no changes in their 23S rRNA sequences had
changes in a highly conserved stretch of amino acids
(63KPWRQKGTGRAR74) in ribosomal protein L4. One
mutant contained a single amino acid change (G69C), while the other
mutant had a 6-base insert, resulting in two amino acids
(S and Q) being inserted between amino acids Q67 and K68. To our
knowledge, this is the first description of mutations in 23S rRNA
genes or ribosomal proteins in macrolide-resistant S. pneumoniae strains.
The predominant forms of macrolide
resistance in Streptococcus pneumoniae are mediated by
mef(A), a gene encoding an efflux pump in the
major facilitator superfamily, or by erm(B), an rRNA methylase. [Note that the mef(A) and mef(E)
genes, originally named for the macrolide efflux determinants in
Streptococcus pyogenes (6) and S. pneumoniae (44), respectively, have been classified into one group, mef(A) (32).] The prevalence
of macrolide resistance varies geographically, being high in
Japan (73%), Hong Kong (81.5%), France (47%), Italy (42%), and the
United States (19 to 34%) (9; M. R. Jacobs, D. Felminghan, P. C. Appelbaum, and T. A. P. Group, Abstr.
39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1044, 1999).
In some countries, mef(A) predominates, while in others, erm(B) is the major resistance determinant
(7, 19). Although the levels of prevalence of resistance to
clarithromycin, erythromycin, and azithromycin are sometimes reported
to be slightly different in surveillance studies, isolates
containing either mef(A) or erm(B) should be
regarded as coresistant to erythromycin, clarithromycin, and
azithromycin regardless of the absolute MICs of these compounds
(42; L. Brennan, J. Duignan, J. Petitpas, M. Anderson, W. Fu, J. Retsema, J. Rainville, D. Smyth, W. Su, and J. Sutcliffe, Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother.,
abstr. F-124, 1998). However, isolates containing mef(A) are
susceptible to clindamycin and 16-membered macrolides while those
containing erm(B) are resistant to these
agents. Virtually all clinical isolates ofmacrolide-resistant S. pneumoniae that have been examined for macrolide resistance
mechanisms have contained either mef(A) or
erm(B), and occasional strains have contained both genes
(7).
In a previous study we described the selection of
azithromycin-resistant mutants from several macrolide-susceptible
clinical strains of S. pneumoniae containing neither
mef(A) nor erm(B) (29). Examination of
these passage-derived mutants by PCR for mef(A) and
erm(B) sequences showed that neither determinant
accounted for the resistance. In this study we investigated the
resistance mechanisms of these mutants by testing for the presence of
other known macrolide resistance determinants, including three genes that encode macrolide phosphorylases (22, 26,
27; J. Cheng, T. Grebe, L. Wondrack, P. Courvalin, and
J. Sutcliffe, Abstr. 39th Intersci. Conf. Antimicrob. Agents
Chemother., abstr. 837, 1999; K. O'Hara, T. Kawabe, K. Taniguchi, A. Nakamura, and T. Sawai, Abstr. 37th Intersci. Conf.
Antimicrob. Agents Chemother., abstr. C-67, 1997), two genes that
encode macrolide esterases, erm(TR) [now
reclassified as erm(A) (32)], a newly described methylase found in Streptococcus pyogenes (38),
and msr(A), which encodes an ABC-type transporter
(36). Failing to find genes previously characterized to
confer macrolide resistance in these mutants, we reasoned that
mutations in the 23S rRNA alleles and/or in the ribosomal proteins L4
and L22 might account for resistance. A mutation in the ribosome seemed
likely based on the precedents that rRNA mutations do exist in other
species of bacteria (17, 18, 23, 24, 34, 35, 41, 45, 48, 49)
and that mutations in L4 or L22 exist in erythromycin-resistant laboratory-derived mutants of Escherichia coli and
Bacillus spp. (5, 30, 37, 39, 46, 50, 51).
Bacterial strains.
The clinical isolates of S. pneumoniae used in the passage study and the mutants derived from
them have been described previously (29). Briefly, the
mutants were identified after they were subcultured 50 times in
Mueller-Hinton broth (Difco) plus 5% lysed horse blood containing
doubling dilutions of azithromycin. For each subsequent passage, an
inoculum was taken from the tube with the MIC nearest in opacity to
that of the antibiotic-free control. When the MIC increased fourfold,
strains were subcultured in antibiotic-free medium for 10 serial
passages. Strains for which MICs showed MIC determinations.
MICs were determined with microtiter
trays using Mueller-Hinton broth supplemented with 2.5% lysed horse
blood according to the recommendations of the National Committee for
Clinical Laboratory Standards (NCCLS) (25). All
compounds were purchased from Sigma or made by published methods at
Pfizer, Inc.
PCR primers and DNA sequencing.
Primers were purchased from
Sigma/Genosys Biotechnologies (The Woodlands, Tex.). Primers for
erm(A), erm(B), erm(C),
msr(A), mef(A), mph(A),
mph(B), ere(A), and ere(B) have been
described previously (43). Primers for erm(TR)
have been designed for S. pyogenes and were used in this
study (38). Primers for mph(C) are based on the
sequence of a putative macrolide phosphorylase from
Staphylococcus aureus clinical strains
(22; Cheng et al., 39th ICAAC). The primers used to
detect erm(TR) and mph(C) are listed in Table
1. Preliminary sequence data for 23S rRNA
and for L4 and L22 ribosomal proteins in S. pneumoniae and
E. coli were obtained from The Institute for Genomic
Research (TIGR) website (http://www.tigr.org). Primers were designed
from these sequences and are listed in Table 1. All other L4 and L22
sequences were obtained courtesy of Incyte Pharmaceuticals (unpublished
data, 1999). To locate mutations in 23S rRNAs, the genes were initially amplified from total genomic DNA as three overlapping contigs. Unique
primers downstream of each 23S rRNA gene were designed from sequences
beyond the 3' end of 23S rRNA. The use of these primers enabled the
peptidyl transferase region from each allele of 23S rRNA to be
separately amplified and purified for DNA sequencing. For each primer
pair, the concentration of MgCl2 was optimized and is
listed in Table 1. All PCRs were subjected to 35 cycles of 94°C (1 min), either 54 or 44°C (1 min), 72°C (1 min), and then 72°C (10 min) for elongation. PCR products were purified using a QIAquick PCR
purification kit (Qiagen Inc., Valencia, Calif.). Sequencing of
purified DNA was performed on an ABI 373XL automated sequencing
apparatus with stretch upgrade (PE Biosystems, Foster City, Calif.).
Cycle sequencing using the BigDye Terminator TaqFS (BDT)
chemistry (PE Biosystems) was performed according to the
manufacturer's protocol with the following modifications: half-reaction BDT reaction mixtures (50% BDT, 50% ABI 5× buffer [PE
Biosystems]) contained 5% dimethyl sulfoxide (Fisher Scientific, Fair
Lawn, N.J.), and the cycle sequencing profile with a hot start was
95°C for 1 min (1 cycle); 98°C for 45 s, 50°C for 10 s,
and 60°C for 4 min (1 cycle); and 98°C for 15 s, 50°C for
10 s, and 60°C for 4 min (29 cycles) on a PTC-225 DNA Engine
Tetrad Thermal Cycler (MJ Research, Watertown, Mass.). Sequencing
reaction mixtures were purified by following the manufacturer's
protocol over 96-well gel filtration blocks (Edge BioSystems,
Gaithersburg, Md.) and electrophoresed for 18 h at 2,500 V, 40 mA,
on polyacrylamide gels made of 5.75% PAGE-Plus acrylamide (Amresco,
Solon, Ohio)-6 M urea (Boehringer Mannheim, Indianapolis, Ind.) in 1×
Tris-borate-EDTA (Roche Diagnostics Corp., Indianapolis, Ind.), with a
4% mobility file and ABI 100 base calling (PE Biosystems). Clones were
assembled and edited using the ABI Factura and AutoAssembler programs
(PE Biosystems). Sequence comparisons were carried out using the
Lasergene sequence analysis software (DNASTAR, Inc., Madison, Wis.).
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Mutations in 23S rRNA and Ribosomal Protein L4
Account for Resistance in Pneumococcal Strains Selected In Vitro
by Macrolide Passage
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
4-fold increases were
determined to be of the same clonal type as that of the parental
strains by pulsed-field gel electrophoresis. S. pneumoniae
R6 was used as a recipient in transformation experiments, and strain
A9, a spontaneous streptomycin-resistant derivative of R6, served as a
control (3).
TABLE 1.
Primer descriptions and MgCl2 concentrations
used in this study
Southern hybridization. S. pneumoniae genomic DNA was prepared as described previously (44). DNA was digested with restriction endonucleases from New England Biolabs (Beverly, Mass.) according to manufacturer's directions, and fragments were resolved by gel electrophoresis. DNA fragments were transferred to a Magnagraph nylon membrane (Micron Separations Inc., Westboro, Mass.). The 23S ribosomal DNA probe was a PCR product derived with the primer pair encompassing the 3' end of the 23S rRNA gene (1,003 bp) (Table 1). The probe was purified, labeled, and detected by chemiluminescence with a digoxigenin DNA random prime labeling and detection kit (Boehringer Mannheim) according to manufacturer's directions. Hybridization was carried out at 60°C.
Transformation. Synthetic competence-stimulating peptide I and the method of Havarstein et al. (15) were used to induce S. pneumoniae into a transformation-competent state. Briefly, cells were inoculated at a very low density (optical density at 550 nm [OD550], ~0.005) and grown to early log phase (OD550, ~0.02) in Todd-Hewitt medium plus 0.5% yeast extract. The cells were triggered to the competence state by the addition of NaOH (0.006 N final concentration), bovine serum albumin (0.2% final concentration), CaCl2 (1 mM final concentration), and synthetic competence-stimulating peptide I (0.2-µg/ml final concentration). After 15 min, DNA was added at a final concentration of 1 µg/ml to aliquots of cells and the mixture was incubated for an hour at 30°C. Cells with DNA were diluted fourfold and further incubated for 2 h at 37°C. They were then plated on Todd-Hewitt agar containing 5% sheep blood and appropriate antibiotics (0.3 to 0.5 µg of azithromycin per ml or 100 µg of streptomycin per ml) and incubated overnight at 37°C in 5% CO2. Cell suspensions were monitored for competence by determining the frequency of transformation of the streptomycin resistance gene from S. pneumoniae A9, a streptomycin-resistant mutant spontaneously derived from R6. Transformation frequencies for introduction of streptomycin resistance ranged from 0.1 to 0.5%.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Passaged mutants do not contain known erythromycin resistance determinants. Genomic DNAs from all of the resistant isolates and parental strains were isolated and subjected to PCR analysis using primers specific for macrolide esterases [ere(A) and ere(B)], phosphotransferases [mph(A), mph(B), and mph(C)], an ABC-binding transporter [msr(A)], and rRNA methylases [erm(TR), erm(A), and erm(C)] (38, 43; Cheng et al., 39th ICAAC). None of the isolates had a PCR product specific to any of the known resistance determinants described for enterics, staphylococci, or streptococci (data not shown). These results, in conjunction with our previous data [the absence of mef(A) and erm(B)], suggested that these isolates contained a novel mechanism(s) of resistance.
Copy number determination of 23S rRNA.
Since the resistant
isolates were negative for all known macrolide resistance determinants,
we reasoned that the isolates could have mutations at the level of the
ribosome. Alteration in 23S rRNA seemed a possibility, and we initially
sought to determine the number of 23S rRNA alleles present in S. pneumoniae. The copy number of 23S rRNA was uncertain, as there
had been reports that pneumococcal strains contained either six
(10; P. Matsushima and R. H. Baltz, Abstr.
96th Gen. Meet. Am. Soc. Microbiol. 1996, abstr. H-63, 1996) or four
(4) copies of 23S rRNA. Search of the TIGR database for
S. pneumoniae with the published 23S rRNA sequence from
staphylococci yielded six contigs: a single contig (spn_23) with
sequence beyond the expected 5' and 3' ends of a canonical rRNA operon
(16S-23S-5S rRNA), three copies with homology to a portion of the 23S
rRNA sequence but each possessing unique 3' ends, and two contigs with
sequence homology to internal regions of 23S rRNA
(http://www.tigr.org). The last two, being internal, were not
considered to be additional individual copies. To determine the
number of alleles of 23S rRNA, genomic DNA was restricted with 18 enzymes; digestion with ApaI or ClaI revealed
four bands, while the majority of enzymes gave less than four bands
following digestion and Southern blot analysis with a probe specific
for 23S rRNA (data not shown). Since some of the fragment sizes were large, we employed a double-restriction enzyme strategy to help distinguish if multiple alleles were present on one fragment. I-CeuI from New England Biolabs is an intron-encoded protein
from the chloroplast large rRNA gene of Chlamydomonas
eugametos (20). This enzyme has a 26-bp recognition
sequence that is unique and conserved only within 23S ribosomal genes.
Restriction with I-CeuI was coupled with EcoRI,
which cuts outside of 23S rRNA. After enzyme digestion with
I-CeuI and EcoRI, Southern blot analysis with a
probe specific for 23S rRNA revealed four bands in each of three
different isolates of S. pneumoniae (Fig.
1). Four 23S-rRNA-specific bands were
observed at 8.8, 7.3, 5.6, and 3.3 kb in isolate 6T0, and bands of 9.9, 3.9, 2.9, and 2.0 kb were observed in isolate 8T0; a doublet band at
11.4 kb, with singlet fragments of 7.4 and 3.4 kb, were seen in R6.
These results are consistent with the presence of four copies of 23S
rRNA genes in S. pneumoniae.
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Analysis of 23S rRNA and L4 and L22 sequences in passaged
mutants.
DNAs from parental strains and macrolide-resistant
passaged isolates were amplified using three pairs of primers specific to overlapping regions of 23S rRNA in pneumococci (Table 1). Although
there was some strain-to-strain variation in 23S rRNA sequences among
susceptible isolates, the only change between susceptible and resistant
pairs was seen in the peptidyl transferase region within domain V
(E. coli nucleotides 2042 to 2628) of the 23S rRNA. Four of
the six mutants showed changes in this region (Fig.
2). Two resistant strains, 3Az and 8Az,
showed changes in A2059G and A2058G, respectively (Table
2). Resistance to 14-membered (erythromycin, clarithromycin), 15-membered (azithromycin), and 16-membered (spiramycin) macrolides was noted for both of these mutants, and resistance to lincosamides (lincomycin and clindamycin) was also noted for strain 8Az, according to NCCLS guidelines
(where available). For strain 3Az, the MIC of streptogramin B was not increased and, although there was decreased susceptibility to the
ketolide telithromycin, the MIC of the ketolide remained low. Conversion of A2058 to guanine in 8Az provided a significant increase in the MICs of 14- and 15-membered macrolides and more modest increases in the MICs of spiramycin, lincosamides, streptogramin B, and
telithromycin. C2611 in two other strains, 1Az and 6Az, was
changed to adenine and guanine, respectively. Changes to adenine in 1Az
conferred low-level resistance to the 14-, 15-, and 16-membered macrolides, with slight and large increases in the MICs of
telithromycin and streptogramin B, respectively. The C2611G change in
6Az resulted in notable resistance to 14- and 15-membered macrolides
and streptogramin B but less resistance to lincosamides and spiramycin.
Further, there was a 130-fold increase in the MIC of telithromycin.
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4-fold, but
the MICs of the ketolide telithromycin and the lincosamides were
increased not more than 2-fold (Table 4). In some cases, the increase
in resistance to 14- and 15-membered macrolides would not be sufficient to reveal these isolates as macrolide resistant, according to NCCLS
guidelines (25).
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Effects of mutations on growth physiology. Mutations in ribosomal proteins and alterations to 23S rRNA have been shown to alter growth rate and to confer temperature sensitivity to growth (5, 30, 39, 46, 50). To determine if any of the mutants were temperature sensitive, each mutant was evaluated for the ability to form colonies on four media (brain heart infusion, Todd-Hewitt, Trypticase soy, and Mueller-Hinton) supplemented with 5% sheep red blood cells at three different temperatures (25, 35, and 42°C) (data not shown). The mutants grew well on each medium at all temperatures with the exception of mutant 5Az. This mutant did not form colonies on any of the media at 25°C. Further, neither 5Az nor its parent, 5T0, were able to sustain growth at 42°C on any medium. As a confirmation of the phenotype, 5Az and 5T0 were grown in Todd-Hewitt medium containing 0.5% yeast extract at 35°C for ~1.5 generations and then shifted to either 25 or 42°C. Growth was monitored every hour by measuring the number of CFU on Todd-Hewitt broth-blood agar plates at 35°C. Both 5T0 and 5Az stopped increasing in cell number upon the temperature shift to 25°C, and the cell number remained constant for 8 h. However, parent 5T0 was capable of forming colonies on plates incubated at 25°C whereas 5Az was not. Upon being shifted to 42°C, both strains lost viability. 5Az had a longer doubling time (110 min versus 60 min) at 35°C than that of its isogenic parent. Thus, it appears that the insertion mutation (SQ) in 5Az resulted in a protein that impacted the growth rate at 35°C and viability at 25°C.
Transformation of L4 mutations into R6. To determine if the mutations in L4 were sufficient to confer a macrolide resistance phenotype, the sequences for L4 from strains 4Az and 5Az were amplified. Purified PCR products were introduced into R6 by transformation, and azithromycin-resistant transformants were selected on Todd-Hewitt broth-blood agar containing 0.5 µg of azithromycin per ml. Transformants were selected, streaked onto selective medium, and phenotyped. The L4 sequences from MS transformants were amplified, and the PCR products were purified and sequenced. All transformants had the same MS phenotypes and mutations in L4 as their parental version, 4Az or 5Az (Table 4).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The S. pneumoniae parental strains and the mutants obtained by exposure to azithromycin did not contain any known mechanism of resistance that has been described for clinical strains, namely, rRNA methylases, efflux genes, esterases, or phosphorylases. Since these resistance determinants are generally acquired, this finding is not totally unexpected. Mutations in 23S rRNA conferring erythromycin resistance have been described for clinical strains of Mycobacterium avium (24), Mycobacterium intracellulare (23), Mycobacterium chelonae (49), Mycobacterium abscessus (49), Brachyspira (Serpulina) hyodysenteriae (18), Helicobacter pylori (17, 41, 45, 48), and Proprionibacterium spp. (34, 35). These mutations have been mapped to the peptidyl transferase region, specifically, G2057, A2058, or A2059 in the E. coli numbering scheme. Changes of A2058G have also been seen in chloroplasts from either Chlamydomonas reinhardtii or Nicotiana plumbaginafolia, mitochondria from Saccharomyces cerevisiae, and laboratory mutants of E. coli (50) and Mycoplasma pneumoniae (21). In addition, the A2058G mutation was selected in Streptomyces ambofaciens, a spiramycin producer, by another 16-membered macrolide, chalcomycin (31). Methylation at A2058 by one of many erythromycin methylases is a major mechanism of resistance that is widespread in many bacterial species, including macrolide producer strains (32, 50). The A2059G transition in N. plumbaginafolia chloroplast rRNA has been described previously (50). Four of the six passage-derived strains in our study contain mutations in A2058, A2059, and C2611, a residue that pairs with G2507 in the secondary structure of 23S rRNA (13). The mutations C2611U and -G have been described for laboratory-derived mutants of E. coli, as chloroplast mutations in Chlamydomonas moewusei or C. reinhardtii, and as mitochondrial mutations in S. cerevisiae (50). The conversion of C2611A is described for the first time for the S. pneumoniae mutant 1Az.
To determine if one or more alleles encoding 23S rRNA contained a mutation conferring macrolide resistance, we first had to ascertain how many 23S rRNA operons S. pneumoniae contained. The literature was confusing, with one reference reporting results consistent with four copies of 23S rRNA (4) but with others claiming six alleles (10; Matsushima and Baltz, Abstr. 96th Gen. Meet. Am. Soc. Microbiol., 1996). We reexamined the number of 23S rRNA alleles and found in three different isolates that four copies of 23S rRNA were present. Other clinical species with 23S rRNA mutations have either one, two, or three copies of the 23S rRNA operon. For those species containing either one copy (Mycobacterium, Brachyspira) or two copies (Helicobacter, Mycoplasma pneumoniae), a change in one of the copies has been shown to be sufficient to confer erythromycin resistance. Although Proprionibacterium avidum contains one copy and Proprionibacterium granulosum contains two copies, Proprionibacterium acnes contains three alleles of 23S rRNA (34). However, there was no evidence of heterozygosity in Proprionibacterium granulosum or Proprionibacterium acnes isolates that were macrolide resistant. A mutation in one of the seven alleles of rRNA in E. coli was recessive, with erythromycin resistance being conferred only when point mutations were provided in trans on a multicopy plasmid (40). It was Streptomyces ambofaciens, with one of four rRNA alleles carrying A2058G, that provided the precedent that changes in one of the four alleles could be sufficient to confer macrolide resistance (31).
Using a strategy that allowed us to amplify each allele independently, we found that all but one of the 23S rRNA mutants were heterozygous. Two mutants, 3Az and 8Az, had changes of guanine for A2059 and A2058, respectively, in two of their four alleles. The resistance phenotype was notably different for these two mutants; change at position 2058 resulted in a stronger phenotype to macrolide, lincosamide, and streptogramin B antibiotics, while the change at position 2059 did not confer resistance to streptogramin B. Interestingly, erythromycin-resistant Proprionibacterium acnes mutants with A2059G were homozygous at all three loci and highly resistant to both 14- and 16-membered macrolides (34) whereas the pneumococcal isolate carrying half of its alleles as 2059G was not as highly resistant to 14-membered macrolides as to the 15-membered macrolide azithromycin and the 16-membered macrolide spiramycin. Clarithromycin-resistant H. pylori clinical isolates with the mutations A2058G, A2058C, and A2059G have been described previously (17, 41, 45, 48). Heterozygosity was evident in some isolates, but the majority contained both alleles with their respective mutations. The MICs of clarithromycin were generally higher for isolates with A2058 mutations than for those with A2059 mutations. Cross-resistance to other 14-membered macrolides was not seen in every case. In B. hyodysenteriae, resistance to tylosin, erythromycin, and clindamycin was associated with an A-to-T transversion mutation in nucleotide position 2058 (18). Susceptible strains subcultured on agar containing tylosin yielded resistant mutants with an A-to-G transition at 2058.
Two passage-derived mutants had changes to either adenine or guanine at C2611, a residue important in maintaining the stem preceding the single-stranded portion of the peptidyl transferase region containing positions A2058 and A2059 (Fig. 2) (13). The C2611A and C2611G mutants contained either three alleles of adenine or four alleles of guanine. Strain 6Az, containing the homozygous transversion, appeared to be more resistant to 14- and 15-membered macrolides, telithromycin, and streptogramin B than strain 1Az (ratio of A to C at 2611, 3:1). However, whether the higher level of resistance was due to homozygosity of a purine or the presence of guanine rather than adenine at this residue is not known.
The 50S subunit of bacterial ribosomes is composed of 23S and 5S rRNAs and more than 30 proteins (11). An affinity-labeling study with two photoreactive erythromycin analogs identified a strong interaction of both compounds with the proteins L22 and L15, while only one of two derivatives labeled proteins L2 and L4 (2). L4 has been reported to interact largely with the 5' end (domains I and II) of 23S rRNA (28, 33). Erythromycin-resistant mutants of E. coli or Bacillus stearothermophilus have been described to have mutations in L4 (5, 30, 37, 50, 51) or, with E. coli or Bacillus subtilis, mutations in protein L22 (5, 39, 46, 50). The presence of erythromycin resistance mutations in either the L4 or L22 ribosomal protein is consistent with the interpretation that these proteins are also in contact with or near the peptidyl transferase region in domain V since erythromycin footprints at A2058 and A2059. The only mutations conferring macrolide resistance that have been identified in ribosomal DNA sequences are those in E. coli; the L4 mutation was a point mutation that changed Lys63 to Glu in L4, while the L22 mutation was a deletion of 3 amino acids, Met82, Lys83, and Arg84 (5). Both mutations appear to be in regions predicted to interact with RNA, suggesting that the mutations in the ribosomal proteins may act indirectly to alter 23S rRNA conformation (47). Interestingly, when the E. coli L4 and L22 proteins carrying the mutations were evaluated in ribosome footprinting experiments, they were found to profoundly alter the conformation of 23S rRNA as assessed by chemical modifications in domains II, III, and V (12), not previously described. Notably, there were no detectable effects at or near A2058 in domain V.
It is interesting that a number of passages (31 to 45 passages for 1Az,
3Az, 4Az, and 5Az; 13 and 17 passages for 8Az and 6Az, respectively)
appeared to be necessary before stable mutations in either 23S rRNA or
L4 occurred. However, since the 23S rRNA genes in S. pneumoniae are identical in sequence, it is likely that a mutation
had to occur in only one copy and that, by homologous recombination,
other alleles were converted. We have not yet isolated a laboratory
mutant or seen in a clinical strain a mutational change in one 23S rRNA
allele conferring macrolide resistance. However, only passaged isolates
for which MICs of azithromycin exhibited a 4-fold change were
examined in the previous study (29). It is possible that one
of four changes confers a weaker phenotype and was not examined. As for
L4 gene copy number, there appears to be only one L4 sequence per
species (Incyte Pharmaceuticals, unpublished data, 1999; TIGR website).
The mutations that we detected in L4 were either a change at amino acid
69 from glycine (GGA) to cysteine (TGC) in 4Az or a 6-nucleotide insert
(AGTCAA) in 5Az. It is not clear how the 2-nucleotide change
occurred in 4Az, but TGA would encode a stop triplet and GGC would be
silent, as it still encodes glycine. The 6-nucleotide insert may result
from slippage, as the preceding 6 nucleotides (CGTCAA) are
nearly identical to those in the insert (TIGR website).
Like 5Az, the E. coli strain with a mutation in L4 did not form colonies at low temperatures (5). The inability of the E. coli L4 mutant to form colonies at 20 to 25°C was associated with a 50S subunit assembly defect at the reduced temperature (5, 14). In addition, the E. coli ribosomes containing the altered L4 bound erythromycin poorly and purified ribosomes from the mutant had reduced peptidyltransferase activities (5, 51). No mutations were found in L22 in this study, but two previously unidentified mutations in L4 were defined in two independently passage-derived isolates. Interestingly, neither L4 mutation conferred cross-resistance to telithromycin or the lincosamides, clindamycin, or lincomycin by NCCLS guidelines (where available). We have not examined subunit assembly in the S. pneumoniae mutants or the ability of altered L4 proteins to bind erythromycin.
This study characterized mutations in azithromycin-derived isolates; however, similar results can be obtained with mutants derived from passage with erythromycin or clarithromycin (8; W. Fu, M. Anderson, S. Williams, A. Tait-Kamradt, J. Sutcliffe, and J. Retsema, Prog. Abstr. 5th Int. Conf. Macrolides Azalides Streptogramins Ketolides Oxazolidinones, abstr. 07-10, 2000). Although some may question the utility of laboratory-derived mutants to predict the nature and frequency of resistance in clinical strains, laboratory experiments with nonfermentation-derived antibiotics like quinolones have identified mutations in gyrase, topoisomerase, and efflux genes that have been subsequently observed in clinical strains (16). It seems likely that, when there are resistance determinants that have mobility via plasmids or transposons, experiments in the laboratory might not predict the frequency of resistance or even the nature of resistance in the clinic. However, laboratory-derived mutants can be predictive for mutations observed in clinical strains, as has been noted for isolates that are linezolid resistant (E. faecium) (S. M. Swaney, D. L. Shinabarger, R. D. Schaadt, J. H. Bock, J. L. Slightom, and G. E. Zurenko, Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. C-104, 1998; G. E. Zurenko, W. M. Todd, B. Hafkin, B. Meyers, C. Kauffman, J. Bock, J. Slightom, and D. Shinabarger, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 0848, 1999) or everninomicin resistant (S. pneumoniae) (1; P. Adrian, C. Mendrick, D. Loebenberg, K. J. Shaw, K. P. Klugman, and R. S. Hare, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 0845, 1999; P. M. McNicholas, P. A. Mann, D. J. Najarian, L. Miesel, T. A. Black, R. S. Hare, and K. J. Shaw, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 0846, 1999). Perhaps not surprisingly, we have identified some clinical strains of pneumococci that have either L4 or 23S rRNA mutations (A. Tait-Kamradt, T. Davies, L. Brennan, F. Depardieu, P. Courvalin, J. Duignan, J. Petitpas, L. Wondrack, M. Jacobs, P. Appelbaum, and J. Sutcliffe, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. LB-8, 1999). Although the frequency of these mutations is unknown, the presence of clinical strains with similar mutations does serve to verify that what we have seen in the laboratory is of some predictive value in the clinic.
In summary, we established that mutations in either 23S rRNA alleles or ribosomal protein L4 were responsible for the macrolide resistance in S. pneumoniae isolates passaged with azithromycin. Furthermore, we determined that there are four copies of 23S rRNA in S. pneumoniae and that macrolide resistance is conferred when at least two or more alleles carry a mutation in A2058, A2059, or C2611, important residues for binding of macrolides and maintaining the conformation of the peptidyl transferase region within 23S rRNA. The application of these findings in characterizing macrolide resistance in clinical isolates will enhance our understanding of the nature and transmission of clones of macrolide-resistant pneumococci.
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ACKNOWLEDGMENTS |
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Sequencing of the S. pneumoniae genome was accomplished with support from TIGR, The National Institute of Allergy and Infectious Diseases, and the Merck Genome Research Institute.
We are also indebted to Incyte Pharmaceuticals for allowing the publication of portions of the L4 sequences from many bacterial species. We thank Robert Kessler for reviewing the manuscript and Paul Miller for enthusiastic support of the study. We also thank all members of the DNA sequencing lab for their consummate patience and careful work.
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FOOTNOTES |
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*
Corresponding author. Mailing address: Pfizer Central
Research, Department of Infectious Diseases
RAIIID, Eastern Point Rd., Groton, CT 06340. Phone: (860) 441-4693. Fax: (860) 441-6159. E-mail:
joyce_a_sutcliffe{at}groton.pfizer.com.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Littauer, P., Sangvik, M., Caugant, D. A., Hoiby, E. A., Simonsen, G. S., Sundsfjord, A., the Norwegian Macrolide Study Group,
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43: 2125-2132
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Pfister, P., Corti, N., Hobbie, S., Bruell, C., Zarivach, R., Yonath, A., Bottger, E. C.
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Reinert, R. R., Ringelstein, A., van der Linden, M., Cil, M. Y., Al-Lahham, A., Schmitz, F.-J.
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43: 1294-1300
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Monaco, M., Camilli, R., D'Ambrosio, F., Del Grosso, M., Pantosti, A.
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55: 256-259
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Haanpera, M., Huovinen, P., Jalava, J.
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49: 457-460
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Okitsu, N., Kaieda, S., Yano, H., Nakano, R., Hosaka, Y., Okamoto, R., Kobayashi, T., Inoue, M.
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43: 168-173
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Riska, P. F., Kutlin, A., Ajiboye, P., Cua, A., Roblin, P. M., Hammerschlag, M. R.
(2004). Genetic and Culture-Based Approaches for Detecting Macrolide Resistance in Chlamydia pneumoniae. Antimicrob. Agents Chemother.
48: 3586-3590
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Farrell, D. J., Morrissey, I., Bakker, S., Buckridge, S., Felmingham, D.
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48: 3169-3171
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Drago, L., De Vecchi, E., Nicola, L., Colombo, A., Gismondo, M. R.
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54: 542-545
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Reinert, R. R.
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53: 918-927
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Misyurina, O. Y., Chipitsyna, E. V., Finashutina, Y. P., Lazarev, V. N., Akopian, T. A., Savicheva, A. M., Govorun, V. M.
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48: 1347-1349
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Reinert, R. R., Lutticken, R., Sutcliffe, J. A., Tait-Kamradt, A., Cil, M. Y., Schorn, H. M., Bryskier, A., Al-Lahham, A.
(2004). Clonal Relatedness of Erythromycin-Resistant Streptococcus pyogenes Isolates in Germany. Antimicrob. Agents Chemother.
48: 1369-1373
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Pereyre, S., Guyot, C., Renaudin, H., Charron, A., Bebear, C., Bebear, C. M.
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48: 460-465
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Canu, A., Abbas, A., Malbruny, B., Sichel, F., Leclercq, R.
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48: 297-304
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Waites, K. B., Jones, K. E., Kim, K. H., Moser, S. A., Johnson, C. N., Hollingshead, S. K., Kang, E.-S., Hong, K. S., Benjamin, W. H. Jr.
(2003). Dissemination of Macrolide-Resistant Streptococcus pneumoniae Isolates Containing Both erm(B) and mef(A) in South Korea. J. Clin. Microbiol.
41: 5787-5791
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Arrieta, A., Arguedas, A., Fernandez, P., Block, S. L., Emperanza, P., Vargas, S. L., Erhardt, W. A., de Caprariis, P. J., Rothermel, C. D.
(2003). High-Dose Azithromycin versus High-Dose Amoxicillin-Clavulanate for Treatment of Children with Recurrent or Persistent Acute Otitis Media. Antimicrob. Agents Chemother.
47: 3179-3186
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Walsh, F., Willcock, J., Amyes, S.
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Clark, J. P., Langston, E.
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Dunne, M. W., Khurana, C., Mohs, A. A., Rodriguez, A., Arrieta, A., McLinn, S., Krogstad, J. A., Blatter, M., Schwartz, R., Vargas, S. L., Emparanza, P., Fernandez, P., Gooch, W. M. III, Aspin, M., Podgore, J., Roine, I., Blumer, J. L., Ehrlich, G. D., Chow, J.
(2003). Efficacy of Single-Dose Azithromycin in Treatment of Acute Otitis Media in Children after a Baseline Tympanocentesis. Antimicrob. Agents Chemother.
47: 2663-2665
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Reinert, R. R., Wild, A., Appelbaum, P., Lutticken, R., Cil, M. Y., Al-Lahham, A.
(2003). Ribosomal Mutations Conferring Resistance to Macrolides in Streptococcus pneumoniae Clinical Strains Isolated in Germany. Antimicrob. Agents Chemother.
47: 2319-2322
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Farrell, D. J., Douthwaite, S., Morrissey, I., Bakker, S., Poehlsgaard, J., Jakobsen, L., Felmingham, D.
(2003). Macrolide Resistance by Ribosomal Mutation in Clinical Isolates of Streptococcus pneumoniae from the PROTEKT 1999-2000 Study. Antimicrob. Agents Chemother.
47: 1777-1783
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Pihlajamaki, M., Jalava, J., Huovinen, P., Kotilainen, P.
(2003). Antimicrobial Resistance of Invasive Pneumococci in Finland in 1999-2000. Antimicrob. Agents Chemother.
47: 1832-1835
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Peric, M., Bozdogan, B., Jacobs, M. R., Appelbaum, P. C.
(2003). Effects of an Efflux Mechanism and Ribosomal Mutations on Macrolide Susceptibility of Haemophilus influenzae Clinical Isolates. Antimicrob. Agents Chemother.
47: 1017-1022
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Bartkus, J. M., Juni, B. A., Ehresmann, K., Miller, C. A., Sanden, G. N., Cassiday, P. K., Saubolle, M., Lee, B., Long, J., Harrison, A. R. Jr., Besser, J. M.
(2003). Identification of a Mutation Associated with Erythromycin Resistance in Bordetella pertussis: Implications for Surveillance of Antimicrobial Resistance. J. Clin. Microbiol.
41: 1167-1172
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Ackermann, G., Rodloff, A. C.
(2003). Drugs of the 21st century: telithromycin (HMR 3647)--the first ketolide. J Antimicrob Chemother
51: 497-511
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Reinert, R. R., Lutticken, R., Bryskier, A., Al-Lahham, A.
(2003). Macrolide-Resistant Streptococcus pneumoniae and Streptococcus pyogenes in the Pediatric Population in Germany during 2000-2001. Antimicrob. Agents Chemother.
47: 489-493
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Montanari, M. P., Mingoia, M., Cochetti, I., Varaldo, P. E.
(2003). Phenotypes and Genotypes of Erythromycin-Resistant Pneumococci in Italy. J. Clin. Microbiol.
41: 428-431
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Giovanetti, E., Brenciani, A., Burioni, R., Varaldo, P. E.
(2002). A Novel Efflux System in Inducibly Erythromycin-Resistant Strains of Streptococcus pyogenes. Antimicrob. Agents Chemother.
46: 3750-3755
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Pereyre, S., Gonzalez, P., de Barbeyrac, B., Darnige, A., Renaudin, H., Charron, A., Raherison, S., Bebear, C., Bebear, C. M.
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46: 3142-3150
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Leclercq, R., Courvalin, P.
(2002). Resistance to Macrolides and Related Antibiotics in Streptococcus pneumoniae. Antimicrob. Agents Chemother.
46: 2727-2734
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Kozlov, R. S., Bogdanovitch, T. M., Appelbaum, P. C., Ednie, L., Stratchounski, L. S., Jacobs, M. R., Bozdogan, B.
(2002). Antistreptococcal Activity of Telithromycin Compared with Seven Other Drugs in Relation to Macrolide Resistance Mechanisms in Russia. Antimicrob. Agents Chemother.
46: 2963-2968
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Ng, L.-K., Martin, I., Liu, G., Bryden, L.
(2002). Mutation in 23S rRNA Associated with Macrolide Resistance in Neisseria gonorrhoeae. Antimicrob. Agents Chemother.
46: 3020-3025
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Prunier, A.-L., Malbruny, B., Tande, D., Picard, B., Leclercq, R.
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46: 3054-3056
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Clark, C. L., Nagai, K., Dewasse, B. E., Pankuch, G. A., Ednie, L. M., Jacobs, M. R., Appelbaum, P. C.
(2002). Activity of cefditoren against respiratory pathogens. J Antimicrob Chemother
50: 33-41
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Malbruny, B., Nagai, K., Coquemont, M., Bozdogan, B., Andrasevic, A. T., Hupkova, H., Leclercq, R., Appelbaum, P. C.
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Pihlajamaki, M., Kaijalainen, T., Huovinen, P., Jalava, J., the Finnish Study Group for Antimicrobial Resistan,
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49: 785-792
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Nilius, A. M., Hensey-Rudloff, D. M., Reimann, M. A., Flamm, R. K.
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Bingen, E., Leclercq, R., Fitoussi, F., Brahimi, N., Malbruny, B., Deforche, D., Cohen, R.
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46: 1199-1203
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Low, D. E., de Azavedo, J., Weiss, K., Mazzulli, T., Kuhn, M., Church, D., Forward, K., Zhanel, G., Simor, A., McGeer, A.
(2002). Antimicrobial Resistance among Clinical Isolates of Streptococcus pneumoniae in Canada during 2000. Antimicrob. Agents Chemother.
46: 1295-1301
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Pihlajamaki, M., Kataja, J., Seppala, H., Elliot, J., Leinonen, M., Huovinen, P., Jalava, J.
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46: 654-658
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Shortridge, V. D., Zhong, P., Cao, Z., Beyer, J. M., Almer, L. S., Ramer, N. C., Doktor, S. Z., Flamm, R. K.
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46: 783-786
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Reinert, R. R., Al-Lahham, A., Lemperle, M., Tenholte, C., Briefs, C., Haupts, S., Gerards, H. H., Lutticken, R.
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Canu, A., Malbruny, B., Coquemont, M., Davies, T. A., Appelbaum, P. C., Leclercq, R.
(2002). Diversity of Ribosomal Mutations Conferring Resistance to Macrolides, Clindamycin, Streptogramin, and Telithromycin in Streptococcus pneumoniae. Antimicrob. Agents Chemother.
46: 125-131
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Garza-Ramos, G., Xiong, L., Zhong, P., Mankin, A.
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Nagai, K., Davies, T. A., Ednie, L. M., Bryskier, A., Palavecino, E., Jacobs, M. R., Appelbaum, P. C.
(2001). Activities of a New Fluoroketolide, HMR 3787, and Its (Des)-Fluor Derivative RU 64399 Compared to Those of Telithromycin, Erythromycin A, Azithromycin, Clarithromycin, and Clindamycin against Macrolide-Susceptible or -Resistant Streptococcus pneumoniae and S. pyogenes. Antimicrob. Agents Chemother.
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Fluit, A. C., Visser, M. R., Schmitz, F.-J.
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Farrell, D. J., Morrissey, I., Bakker, S., Felmingham, D.
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48: 541-544
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Morosini, M.-I., Canton, R., Loza, E., Negri, M.-C., Galan, J.-C., Almaraz, F., Baquero, F.
(2001). In Vitro Activity of Telithromycin against Spanish Streptococcus pneumoniae Isolates with Characterized Macrolide Resistance Mechanisms. Antimicrob. Agents Chemother.
45: 2427-2431
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Gregory, S. T., Cate, J. H. D., Dahlberg, A. E.
(2001). Spontaneous Erythromycin Resistance Mutation in a 23S rRNA Gene, rrlA, of the Extreme Thermophile Thermus thermophilus IB-21. J. Bacteriol.
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Fitoussi, F., Loukil, C., Gros, I., Clermont, O., Mariani, P., Bonacorsi, S., Le Thomas, I., Deforche, D., Bingen, E.
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45: 1889-1891
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Dinos, G. P., Michelinaki, M., Kalpaxis, D. L.
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Ip, M., Lyon, D. J., Yung, R. W. H., Chan, C., Cheng, A. F. B.
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45: 1578-1580
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McGee, L., Klugman, K. P., Wasas, A., Capper, T., Brink, A., The Antibiotics Surveillance Forum Of South Africa,
(2001). Serotype 19F Multiresistant Pneumococcal Clone Harboring Two Erythromycin Resistance Determinants [erm(B) and mef(A)] in South Africa. Antimicrob. Agents Chemother.
45: 1595-1598
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Jalava, J., Kataja, J., Seppälä, H., Huovinen, P.
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45: 789-793
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Reinert, R. R., Simic, S., Al-Lahham, A., Reinert, S., Lemperle, M., Lütticken, R.
(2001). Antimicrobial Resistance of Streptococcus pneumoniae Recovered from Outpatients with Respiratory Tract Infections in Germany from 1998 to 1999: Results of a National Surveillance Study. J. Clin. Microbiol.
39: 1187-1189
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Fitoussi, F., Doit, C., Geslin, P., Brahimi, N., Bingen, E.
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45: 636-638
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Vester, B., Douthwaite, S.
(2001). Macrolide Resistance Conferred by Base Substitutions in 23S rRNA. Antimicrob. Agents Chemother.
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Depardieu, F., Courvalin, P.
(2001). Mutation in 23S rRNA Responsible for Resistance to 16-Membered Macrolides and Streptogramins in Streptococcus pneumoniae. Antimicrob. Agents Chemother.
45: 319-323
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Syrogiannopoulos, G. A., Grivea, I. N., Tait-Kamradt, A., Katopodis, G. D., Beratis, N. G., Sutcliffe, J., Appelbaum, P. C., Davies, T. A.
(2001). Identification of an erm(A) Erythromycin Resistance Methylase Gene in Streptococcus pneumoniae Isolated in Greece. Antimicrob. Agents Chemother.
45: 342-344
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Tait-Kamradt, A., Davies, T., Appelbaum, P. C., Depardieu, F., Courvalin, P., Petitpas, J., Wondrack, L., Walker, A., Jacobs, M. R., Sutcliffe, J.
(2000). Two New Mechanisms of Macrolide Resistance in Clinical Strains of Streptococcus pneumoniae from Eastern Europe and North America. Antimicrob. Agents Chemother.
44: 3395-3401
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Tsiodras, S., Gold, H. S., Coakley, E. P. G., Wennersten, C., Moellering, R. C. Jr., Eliopoulos, G. M.
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38: 3991-3993
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