In vitro selection of resistance to clindamycin related to alterations in the attenuator of the erm(TR) gene of Streptococcus pyogenes UCN1 inducibly resistant to erythromycin

Marguerite Fines*,, Marie Gueudin, Aude Ramon and Roland Leclercq

Service de Microbiologie, CHU Côte de Nacre, Avenue Côte de Nacre, 14033 Caen cedex, France


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
A clinical isolate of Streptococcus pyogenes UCN1 intermediate to erythromycin (MIC 1 mg/L) and susceptible to clindamycin (MIC 0.03 mg/L) harboured an inducible erm(TR) gene encoding a ribosomal methylase. We have selected in vitro, in the presence of concentrations of clindamycin ranging from 0.12 to 1 mg/L, one-step mutants that are highly resistant to this antibiotic (MIC 64 mg/L) at a frequency of 10–7. By contrast, in an erythromycin-susceptible strain of S. pyogenes UCN5, mutants could be selected only by a low concentration of clindamycin (0.12 mg/L) at a frequency of 10–9. Clindamycin resistance in four of six S. pyogenes UCN1 mutants was associated with deletions of 163 and 6 bp, as well as a tandem duplication of 101 bp in the regulatory sequence of the erm(TR) gene. The role of these structural alterations in clindamycin resistance was demonstrated by cloning the erm(TR) gene from the wild-type and mutant strains in Escherichia coli DB10, a mutant susceptible to macrolides. Clindamycin resistance was expressed only when the erm(TR) gene was preceded by an altered attenuator. Mutations could lead to the formation of mRNA secondary structures accounting for the accessibility of the ribosome-binding site and the initiation codon of the ErmTR methylase to the ribosomes, and subsequently for the translation of the erm(TR) transcripts. The easy selection in one step of mutants resistant to high levels of clindamycin by concentrations of this antibiotic ranging from four to 40 times the MIC leads us to recommend caution in the use of clindamycin therapy in group A Streptococcus infections.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Erythromycin and related molecules such as clindamycin are the second-line choice of drug used in penicillin-hypersensitive patients for treatment of pharyngitis and other infections caused by Streptococcus pyogenes or group A Streptococcus (GAS).1 Recent emergence of macrolide resistance among GAS is a cause of concern. Having remained at a low incidence for a long time, resistance to erythromycin in GAS has emerged during the last decade in several countries, particularly in Europe. Erythromycin resistance ranges from as low as between 1 and <10% in countries such as Argentina, Canada, USA and France, up to >45% in parts of Italy.24

In GAS, the mechanism of acquired resistance to erythromycin involves either an efflux pump encoded by the mef(A) gene, leading to the M-resistance phenotype, or a methylase encoded by erm genes, which modifies the ribosomal target site of macrolides and confers the MLSB phenotype. The recently described erm(TR) gene has only been found in ß-haemolytic streptococci and accounts for nearly 30% and 38% of erythromycin-resistant GAS in Italy and Finland, respectively.57 The erm(TR) gene shares 82.5% nucleotide sequence identity with the staphylococcal erm(A) gene and possesses a similar leader sequence, probably involved in post-transcriptional regulation of the methylase expression.5,8 Therefore, the two methylases form two subclasses recently assigned to the same class, class A, of rRNA methylase.9 The expression of erm(A) is thought to be regulated in a similar way to that of erm(C), which is the prototype of inducible erm genes.8 However, the attenuator structures of erm(A) and erm(TR) are more complex than that of erm(C) and are composed of two peptides (15 and 19 amino acids) as opposed to one for erm(C). Expression of the ErmTR methylase is usually inducible in GAS, therefore conferring resistance to the 14-membered ring (clarithromycin, dirithromycin, erythromycin and roxithromycin) and 15-membered ring (azithromycin) macrolides, but not to lincosamides (clindamycin and lincomycin), which are non-inducers. This phenotype contrasts with the usual cross-resistance between these drugs conferred by the streptococcal erm(B) gene.10

In vitro studies have shown that clindamycin resistance due to constitutive synthesis of methylase can be obtained from inducible strains harbouring staphylococcal erm(A) or erm(C) genes by selection on agar plates containing this antibiotic.11

In this report, we have studied the in vitro conditions of emergence of clindamycin resistance in GAS harbouring an inducible erm(TR) gene and showed that clindamycin resistance was mostly related to alterations in the structure of the attenuator, which controls the expression of the methylase gene. The easy selection of clindamycin-resistant mutants might lead one to question the use of clindamycin as an alternative treatment of infection due to GAS harbouring the inducible erm(TR) gene.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial strains

S. pyogenes UCN5 is a clinical isolate susceptible to erythromycin (MIC 0.06 mg/L) and clindamycin (MIC 0.03 mg/L), used as a control strain. S. pyogenes UCN1 is a clinical isolate inducibly intermediate to erythromycin (MIC 1 mg/L) but fully susceptible to clindamycin (MIC 0.03 mg/L) and containing an erm(TR)-like gene, the sequence of which is identical to the prototype erm(TR) gene from S. pyogenes A200.5 Amplification from S. pyogenes UCN1 DNA of erm(A), erm(B) or erm(C) genes with specific primers was unsuccessful. No plasmid could be found in the strain using a technique described previously by Clewell et al.12 Escherichia coli DB10 is a mutant susceptible to erythromycin (MIC 4 mg/L) and clindamycin (MIC 1 mg/L) that was used as a recipient for cloned erm(TR) genes.13

One-step selection of mutants resistant to clindamycin

S. pyogenes strains were grown overnight on blood agar plates and colonies were suspended in saline. After concentration by centrifugation, c. 109 to 5 x 109 cfu were spread onto trypticase soy agar plates supplemented with 5% horse blood and containing increasing concentrations of clindamycin (0.125, 0.25, 0.5 and 1 mg/L). The bacterial inoculum was measured using a spiral system (Interscience, Saint-Nom-la-Bretèche, France). After 48 h of incubation at 37°C in CO2, growing colonies were counted and studied for susceptibility to erythromycin and clindamycin. Frequency of mutation was expressed as the ratio of the number of mutants to the inoculum. The constitutive or inducible expression of macrolide resistance in S. pyogenes UCN1 and its derivatives was tested as described previously.14

Susceptibility tests

MICs of erythromycin and clindamycin were determined by the agar dilution method with Mueller–Hinton agar (Bio-Rad, Marnes-la-Coquette, France) supplemented with 5% horse blood for S. pyogenes strains and not supplemented for E. coli DB10 strains, according to the recommendations of the Comité de l’Antibiogramme de la Société Française de Microbiologie.15

Nucleotide sequence of the erm(TR) attenuator

Oligonucleotides TRRG1 5'-GCATAAGGAGGAGTTAAATATGTG-3' and TRRG2 5'-TTTTATCTTGTTTATTGATATTCG-3' (Eurobio, Les Ullis, France) complementary to sequences flanking the regulatory region of the erm(TR) gene were used to amplify this region. DNA amplification was performed in a GeneAmp PCR System 2400 (Perkin-Elmer Cetus, Norwalk, CT, USA). The PCR mixture of 50 µL contained 2 mM MgCl2, 20 pmol of each primer, 200 µL of desoxyribonucleotides, 5 µL of Taq polymerase buffer, 2.5 U of Taq polymerase and 5 µL of DNA template obtained by Chelex100 extraction (Bio-Rad, Hercules, CA, USA). A total of 35 cycles was performed, with denaturation at 94°C for 30 s, annealing at 48°C for 30 s and extension at 72°C for 30 s. PCR products were resolved by electrophoresis on 1% agarose gels; the expected size was 340 bp. PCR products were then purified on Microcon 100 columns (Millipore Corp., Bedford, MA, USA) and sequenced in an automated ABI PRISM 310 system (Perkin-Elmer). The secondary structure of the sequence of attenuator mRNA was analysed using the Mu-fold software.16 Drawing was done using RnaViz software.17

Cloning experiments

Oligonucleotides TR1 5'-AGTCGACTAAGGAGGAGTTAAATATGTG-3' and TR2 5'-TTATTGAAATAATTTGTAACTATT-3' (Eurobio) were used to amplify the entire erm(TR) gene and its attenuator from S. pyogenes UCN1 (amplicon size equal to c. 1 kb) and from four clindamycin-resistant derivatives. DNA amplification was performed as described above. The MgCl2 concentration was 1.5 mM and annealing temperature was 57°C. The amplified fragments were cloned into plasmid pCR2.1 following the manufacturer's recommendations (Invitrogen, Groningen, The Netherlands). The recombinant plasmid was introduced into competent E. coli DB10 cells by electrotransformation.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
In vitro selection of mutants resistant to clindamycin

Mutants of S. pyogenes could be selected on agar medium containing various concentrations of clindamycin. The frequency of mutation was 10–7 in the presence of 0.12, 0.25, 0.5 and 1 mg/L of clindamycin for S. pyogenes UCN1, which harboured the erm(TR) gene, while mutants could be selected only by the lowest concentration of clindamycin (0.12 mg/L) and at a frequency of 10–9 for S. pyogenes UCN5 susceptible to erythromycin. This hundred times difference in the mutation frequencies could be related to the presence of the erm(TR) gene as shown by further analysis of the erm(TR) attenuator in several mutants (see below). S. pyogenes UCN1 mutants were highly resistant to clindamycin (MIC 64 mg/L), whereas MICs of erythromycin remained unchanged (MIC 1 mg/L). This increase in the MIC of clindamycin was probably due to constitutive expression of resistance. Growth rates were similar in mutants, whether cells grown in the presence of subinhibitory concentrations of clindamycin or erythromycin were induced by these antimicrobials or not. By contrast, for the wild-type strain the lag phase of the growth curves was reduced after induction with erythromycin.

We have noted a similar frequency of mutation to clindamycin resistance in a clinical isolate of group G streptococcus highly resistant to erythromycin (MIC > 128 mg/L), susceptible to clindamycin (MIC 0.06 mg/L) and containing a plasmid-borne erm(TR) gene (data not shown). The observation that resistant mutants could be readily selected in one step by clindamycin concentrations ranging from four to 40 times the MIC might have clinical relevance for clindamycin therapy of infections with heavy inoculum of GAS strains with an inducible erm(TR) gene. Selection of constitutive mutants during clindamycin therapy of an infection caused by an inducibly erythromycin-resistant S. aureus strain has been reported previously.18 Of note, the frequency of in vitro selection of clindamycin resistance in staphylococci containing inducible erm(A) or erm(C) genes is similar to that obtained in this study with S. pyogenes UCN1.11 The report of rare clinical isolates of GAS containing an erm(TR) gene and cross-resistant to erythromycin and clindamycin showed that this type of resistance has already spread.7 It should be stressed that the use of clindamycin in infections due to GAS strains resistant to erythromycin by an efflux mechanism does not present the same risk for selection of clindamycin resistance, since clindamycin is not a substrate for the efflux pump.19 Thus, it is clinically relevant to distinguish the inducible MLSB phenotype from the efflux phenotype. The existence of a D-shaped zone between clindamycin and erythromycin is indicative of the inducible MLSB phenotype. However, in the case of S. pyogenes UCN1, this antagonism was barely visible, probably because of the intermediate level of resistance to erythromycin.

Analysis of structural alterations in the regulatory region of the erm(TR) gene

Attenuators of the erm(TR) gene of S. pyogenes UCN1 and of six clindamycin-resistant mutants were amplified by PCR and sequenced three times in both directions in independent replicons. The erm(TR) attenuator sequence of the wild-type strain showed a 100% identity with the corresponding sequence determined by Seppälä et al.5 The analysis of this region located upstream of the methylase gene confirmed that it contained two ORFs, each preceded by a ribosome-binding site (RBS), which could encode two leader peptides, LP1 and LP2, of 15 and 19 amino acids, respectively. In addition, the mRNA sequence contained a series of inverted repeats that could form stem–loop structures (Figure 1Go). The similarity of the structure with that of the erm(A) gene attenuator indicated that the two genes might share the same mode of regulation of methylase expression. In the erm(A) gene model, it has been suggested that two stem–loop structures could be formed in the regulatory region, which could sequester the RBS and the initiation codons of the second leader peptide and of the methylase gene.8 The presence of inducing concentrations of erythromycin would result in a stalling of a ribosome while translating the first leader peptide, disrupting the stem–loop structure comprised of IR1 and IR2 and then allowing translation of the second leader peptide. In turn, stalling of ribosome translating this leader peptide would lead to the translation of erm(A) gene by destabilization of the stem–loop structure composed of IR5 and IR6 (Figure 1Go).



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Figure 1. Proposed inactive conformation for the mRNA secondary structure of the attenuator of erm(TR). Pairs of associated inverted repeats (IR) are numbered from 1 to 6; RBS1 and RBS2, ribosome-binding sites for leader peptides 1 and 2 (LP1 and LP2), respectively; RBS3, ribosome-binding site for ErmTR methylase. Nucleotides composing the RBS are underlined. ORFs of LP1, LP2 and erm(TR) are in bold. The initiation sequence for synthesis of methylase is sequestered in a stem–loop structure.

 
One of the amplification products obtained from the six UCN1 mutants was greater and another one smaller in size than expected (340 bp). The last four amplification products and the fragment amplified from the wild-type attenuator were similar in size. Nucleotide sequence analysis revealed that one of the six mutants, S. pyogenes UCN1-1, had a large 163 bp deletion that encompassed the terminal portion of LP1, LP2 entirely and the RBS of erm(TR) (Figure 2Go). This deletion eliminated most of the stem–loop structures and produced a single new open reading frame (ORF) composed of the fusion of the 5' portion (54 nucleotides) of LP1, nine nucleotides preceding erm(TR) and erm(TR) in its entirety. The wild-type RBS of the erm(TR) gene was replaced by the RBS of LP1, which is accessible to the ribosomes; this most likely explains the constitutive expression of resistance. Two other clindamycin-resistant mutants, S. pyogenes UCN1-2 and UCN1-3, had a 6 bp deletion from nucleotides 189–194, four nucleotides upstream from the erm(TR) RBS (Figure 2Go). This deletion might destabilize the stem–loop structure by modifying free energy from –13.6 to –3.4 kcal/mol. This could result in an energetically stable conformation where the sequestered initiation sequence for methylase became accessible to ribosomes. In S. pyogenes UCN1-4, a 101 bp tandem duplication of the region between the 3' end of LP2 and the 5' end of the erm(TR) gene was identified. This alteration led to the generation of a new putative ORF designated LP3 (14 amino acids) preceded by a RBS. Most probably, the displacement of the first two leader peptides and the first inverted repeats towards the 5' end of the messenger had a role in the changes in expression of resistance. Similar deletions and duplications in the regulatory region of constitutive erm(C) genes have been found in erythromycin-resistant isolates of staphylococci of human and animal origin and explained by homologous recombination.20



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Figure 2. Schematic representation of the erm(TR) attenuator of the inducibly expressed erm(TR) gene of S. pyogenes UCN1 and of the four clindamycin resistant mutants S. pyogenes UCN1-1 to UCN1-4 described in this study. The arrows indicate the inverted repeats (IR) 1–6 responsible for the stem–loop structure of mRNA. Deletions of 163 bp in S. pyogenes UCN1-1 and of 6 bp in S. pyogenes UCN1-2 and UCN1-3 were observed. S. pyogenes UCN1-4 displayed a 101 bp duplication.

 
The nucleotide sequences of the structural erm(TR) gene of the wild-type and mutant strains were identical, indicating that only alterations of the attenuators were responsible for clindamycin resistance. In order to confirm that these alterations were sufficient to confer clindamycin resistance, erm(TR) genes with the corresponding attenuator were amplified from the wild-type strain and the three different types of mutants, cloned on plasmid pCR2.1 and introduced by electrotransformation into E. coli DB10, an erythromycin- and clindamycin-susceptible E. coli strain. Acquisition by E. coli DB10 of erm(TR) genes with altered attenuators resulted in an MIC of clindamycin >128 mg/L as compared with an MIC of 1 mg/L for E. coli DB10 harbouring the wild-type erm(TR) gene. By contrast, the MIC of erythromycin was the same (MIC 4 mg/L) for all constructs, consistent with the results obtained for the S. pyogenes mutants. The last two clindamycin-resistant mutants that had no mutation in the erm(TR) attenuator did not have any alteration in the structural gene sequence either and the mechanism for acquisition of clindamycin resistance in these mutants remained unexplained. Hypothetically, mutations in the gene promoter leading to a dramatic increase in the basal level of methylase production or in the ribosomal target of macrolides or mutations increasing the number of erm(TR) gene copy might be involved.

In conclusion, high frequency of selection of clindamycin resistance in S. pyogenes UCN1 was related to the presence of an inducible erm(TR) gene. The selective pressure of clindamycin leads to selection of constitutively resistant derivatives with alterations in the attenuator of the erm(TR) gene. Therefore, the use of clindamycin in therapy of infections caused by S. pyogenes strains harbouring an inducible erm(TR) gene should be discouraged.


    Notes
 
* Corresponding author. Tel: +33-2-31-06-45-72; Fax: +33-2-31-06-45-73; E-mail: fines-m{at}chu-caen.fr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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Received 15 December 2000; returned 28 March 2001; revised 25 April 2001; accepted 21 May 2001