Service de Microbiologie, CHU Côte de Nacre, Avenue Côte de Nacre, 14033 Caen cedex, France
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 MuellerHinton 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 lAntibiogramme 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mutants of S. pyogenes could be selected on agar medium containing various concentrations of clindamycin. The frequency of mutation was 107 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 109 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 stemloop structures (Figure 1). 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 stemloop 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 stemloop 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 stemloop structure composed of IR5 and IR6 (Figure 1
).
|
|
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 |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Kaplan, E. L., Johnson, D. R., Beyer, J., Flamm, R. K. & Shortridge, D. (1999). Characterization of macrolide resistance in upper respiratory tract (URT) isolates of endemic Streptococcus pyogenes from the United States, 19961998. In Program and Abstracts of the Thirty-ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1999. Abstract 104, p. 154. American Society for Microbiology, Washington, DC.
3
.
Bingen, E., Fitoussi, F., Doit, C., Cohen, R., Tanna, A., George, R. et al. (2000). Resistance to macrolides in Streptococcus pyogenes in France in pediatric patients. Antimicrobial Agents and Chemotherapy 44, 14537.
4
.
De Azavedo, J. C., Yeung, R. H., Bast, D. J., Duncan, C. L., Borgia, S. B. & Low, D. E. (1999). Prevalence and mechanisms of macrolide resistance in clinical isolates of group A streptococci from Ontario, Canada. Antimicrobial Agents and Chemotherapy 43, 21447.
5
.
Seppälä, H., Skurnik, M., Soini, H., Roberts, M. C. & Huovinen, P. (1998). A novel erythromycin resistance methylase gene (ermTR) in Streptococcus pyogenes. Antimicrobial Agents and Chemotherapy 42, 25762.
6
.
Giovanetti, E., Montanari, M. P., Mingoia, M. & Varaldo, P. E. (1999). Phenotypes and genotypes of erythromycin-resistant Streptococcus pyogenes strains in Italy and heterogeneity of inducibly resistant strains. Antimicrobial Agents and Chemotherapy 43, 193540.
7
.
Kataja, J., Huovinen, P., Skurnik, M. & Seppälä, H. (1999). Erythromycin resistance genes in group A streptococci in Finland. The Finnish Study Group for Antimicrobial Resistance. Antimicrobial Agents and Chemotherapy 43, 4852.
8 . Murphy, E. (1985). Nucleotide sequence of ermA, a macrolide- lincosamide-streptogramin B determinant in Staphylococcus aureus. Journal of Bacteriology 162, 63340.[ISI][Medline]
9
.
Roberts, M. C., Sutcliffe, J., Courvalin, P., Jensen, L. B., Rood, J. & Seppälä, H. (1999). Nomenclature for macrolide and macrolidelincosamide-streptogramin B resistance determinants. Antimicrobial Agents and Chemotherapy 43, 282330.
10 . Horinouchi, S., Byeon, W. H. & Weisblum, B. (1983). A complex attenuator regulates inducible resistance to macrolides, lincosamides and streptogramin type B antibiotics in Streptococcus sanguis. Journal of Bacteriology 154, 125262.[ISI][Medline]
11 . Leclercq, R., Bauduret, F. & Soussy, C. J. (1989). Selection of constitutive mutants of gram-positive cocci inducibly resistant to macrolides, lincosamides and streptogramins (MLS): comparison of the selective effects of the MLS. Pathologie Biologie (Paris) 37, 56872.[ISI][Medline]
12 . Clewell, D. B., Tomich, P. K., Gawron-Burke, M. C., Franke, A. E., Yagi, Y. & An, F. Y. (1982). Mapping of Streptococcus faecalis plasmids pAD1 and pAD2 and studies relating to transposition of Tn917. Journal of Bacteriology 152, 122030.[ISI][Medline]
13 . Datta, N., Hedges, R. W., Becker, D. & Davies, J. (1974). Plasmid-determined fusidic acid resistance in Enterobacteriaceae. Journal of Genetic Microbiology 83, 1916.
14
.
Rosato, A., Vicarini, H. & Leclercq, R. (1999). Inducible or constitutive expression of resistance in clinical isolates of streptococci and enterococci cross-resistant to erythromycin and lincomycin. Journal of Antimicrobial Chemotherapy 43, 55962.
15 . Comité de lAntibiogramme de la Société Française de Microbiologie. (1996). 1996 report of the Comité de lAntibiogramme de la Société Française de Microbiologie. Technical recommendations for in vitro susceptibility testing. Clinical Microbiology and Infection 2, Suppl. 1, 1125.
16 . Zuker, M., Mathews, D. H. & Turner, D. H. (1999). Algorithms and thermodynamics for RNA secondary structure prediction: a practical guide. In RNA Biochemistry and Biotechnology, (Barciszewski, J. &. Clark, B. F. C., Eds), pp. 1143. NATO ASI Series, Kluwer Academic Publishers, Dordrecht, The Netherlands.
17
.
De Rijk, P. & De Wachter, R. (1997). RnaViz, a program for the visualisation of RNA secondary structure. Nucleic Acids Research 25, 467984.
18 . Watanakunakorn, C. (1976). Clindamycin therapy of Staphylococcus aureus endocarditis. Clinical relapse and development of resistance to clindamycin, lincomycin and erythromycin. American Journal of Medicine 60, 41925.[ISI][Medline]
19 . Sutcliffe, J., Tait-Kamradt, A. & Wondrack, L. (1996). Streptococcus pneumoniae and Streptococcus pyogenes resistant to macrolides but sensitive to clindamycin: a common resistance pattern mediated by an efflux system. Antimicrobial Agents and Chemotherapy 40, 181724.[Abstract]
20
.
Werckenthin, C., Schwarz, S. & Westh, H. (1999). Structural alterations in the translational attenuator of constitutively expressed ermC genes. Antimicrobial Agents and Chemotherapy 43, 16815.
Received 15 December 2000; returned 28 March 2001; revised 25 April 2001; accepted 21 May 2001