a Service de Bactériologie-Virologie, Hôpital Henri Mondor-Université Paris XII, 94010 Créteil b Service de Microbiologie, Hôpital Côte de Nacre, Université de Caen, 14033 Caen, France
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Abstract |
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Introduction |
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In streptococci, MLS B resistance is encoded by genes belonging to the ermB group of genes. The ermB group includes closely related genes such as ermAM, ermB and ermBC genes which have been reported in a variety of Gram-positive and Gram-negative organisms. 1 ,2 ,3 ,4 ,5 The inducible expression of MLS B resistance is putatively controlled at a post-transcriptional level by a regulatory region located upstream from the structural erm gene. 2 Constitutive expression of the MLS B resistance mediated by plasmid pAMß1 from Enterococcus faecalis has been related to a deletion of most of the regulatory region of the erm gene. 6 In a recent study, we have shown that a similar deletion was responsible for constitutive expression of the erm gene from Streptococcus agalactiae HM1081. 7 In this report, we have determined the inducibility of 40 streptococci and enterococci that carry MLS B resistance and determined the molecular structure of the regulatory regions of three erm genes expressed constitutively.
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Materials and methods |
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Forty clinical isolates of streptococci and enterococci which had no zone of inhibition around discs containing erythromycin (30 µg), spiramycin (100 µg) and lincomycin (15 µg) (all discs from Sanofi Diagnostics Pasteur, Marnes-La Coquette, France) were studied. The strains were epidemiologically unrelated. They included 11 E. faecalis, two Enterococcus faecium, 14 Streptococcus pneumoniae six oral streptococci and seven S. agalactiae (including S. agalactiae HM1081, as a control) from various hospitals located in France, Spain, the UK and the USA.
Antibiotic susceptibility testing
MICs of erythromycin, rokitamycin and lincomycin were determined by a standard agar-dilution technique using Mueller- Hinton medium (Sanofi Diagnostic Pasteur) supplemented with 5% horse blood. 8 Erythromycin (Hoechst-Marion-Roussel, Romainville, France), lincomycin (Upjohn Laboratories, Val de Reuil, France) and rokitamycin (Pierre Fabre Laboratories, Boulogne, France) were provided by their respective manufacturers.
Induction assays
To test inducibility, MICs of erythromycin, lincomycin and rokitamycin against the strains cultured in the absence or in the presence of erythromycin (0.06 mg/L) were determined.
Analysis of the regulatory regions of ermAM-related genes
The sequences of the erm attenuators were amplified from bacterial DNA by PCR using primers SR3 (5'-CTTAGAAGCAAACTTAAGAGTGTGT-3') and SR5 (5'-GGTTGAGTACCTTTTCATTCGTTAA-3') designed to amplify a fragment including the regulatory region and the 24 first nucleotides of the ermB group genes. PCR experiments were carried out as follows: 35 cycles of amplification including 30 s of denaturation at 94°C, 1 min of annealing at 50°C and 1 min of elongation at 72°C. Nucleotide sequencing was performed with an ABI PRISM 310 sequencer.
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Results and discussion |
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All 40 streptococci and enterococci were cross-resistant to erythromycin (MICs 16-8000 mg/L) and lincomycin (MICs 128-1000 mg/L). MICs of these antibiotics against streptococci and enterococci were already high in the absence of induction with erythromycin and did not increase or increased only by one dilution after induction, probably because of rapid self-induction by the tested antibiotic. By contrast, MICs of rokitamycin were lower and had a greater range (1-2000 mg/L). After induction, the rokitamycin MICs increased for 32 out of the 40 strains studied by more than two dilutions, probably because of a weaker inducing capacity of this antibiotic. These 32 strains were thus considered inducibly resistant to erythromycin. This study confirms that for streptococci and enterococci, inducible MLS B resistance cannot be distinguished from constitutive resistance on the sole basis of elevated MICs of erythromycin and lincomycin. High-level cross-resistance to erythromycin and lincomycin in our clinical isolates of streptococci and enterococci was mostly due to inducible expression of MLS B resistance. This is in contrast to staphylococci where the MLS B phenotype is related to constitutive synthesis of the ribosomal methylase. 1 ,2 The high prevalence of the MLS B-inducible phenotype in streptococci explains why macrolides that are very weak inducers, such as the ketolides, are active against most of these Gram-positive organisms despite MLS B cross-resistance. 7,9
Analysis of the regulatory regions of erm genes
The ermB group genes are preceded by an attenuator composed of an ORF encoding a putative control peptide (36 amino acids) and of a set of numerous inverted repeats which could fold into several successive stem- loops. 3 Using oligonucleotides SR3 and SR5, we obtained an amplification product from the 40 strains studied. DNA fragments of approximately 380 bp were amplified for 38 strains whereas 170 bp fragments were amplified from the constitutive strains of S. pneumoniae HM36 and S. agalactiae HM1081. The sequence of the PCR product from three of the constitutive strains, E. faecalis UMH1 and S. pneumoniae HM36 and HM35, and from four of the inducible strains, S. agalactiae HM1 and HM12, and oral streptococci HM6 and HM18, was determined and compared with that from gene banks. Alignment of the sequence of the constitutive erm gene of S. pneumoniae HM36 with those of the inducible genes from plasmid pAM77 and transposons Tn917 and Tn1545 revealed a large deletion within the regulatory region similar to that observed upstream from the erm genes of constitutively resistant strains of E. faecalis harbouring plasmid pAMß1 and of S. agalactiae HM1081 (Figures 1 and 2). 6 ,7 This deletion could result in constitutive expression of MLS B resistance since it removed the inverted repeats and the sequence of the leader peptide, thus making the ribosome binding site and the start codon for the methylase accessible to the ribosomes. However, occurrence of a deletion was not the only explanation for constitutive expression of MLS B resistance, as shown by the analysis of nucleotide sequences of the two remaining constitutive strains, E. faecalis UMH1 and S. pneumoniae HM35. E. faecalis UMH1 displayed a duplication of a CGTAATGT sequence which generated two successive stop codons, resulting in a truncated leader peptide of 12 or 20 amino acids (Figures 1 and 2). The contribution of this marked alteration in constitutive expression of MLS B resistance remains unknown. The sequence of the regulatory region of the last constitutive strain, S. pneumoniae HM35, was similar to that of inducible strains (Figure 1). Therefore, modifications of other still unidentified structures could be responsible for constitutive expression of MLS resistance.
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Acknowledgments |
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Notes |
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References |
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Received 9 July 1998; returned 17 September 1998; revised 26 October 1998; accepted 9 December 1998