In Vitro Selection and Characterization of Resistance to Macrolides and Related Antibiotics in Mycoplasma pneumoniae

Abstract

Macrolide-resistant mutants of Mycoplasma pneumoniae were selected in vitro from the susceptible reference strain M129, by 23 to 50 serial passages in subinhibitory concentrations of macrolides and related antibiotics, erythromycin A, azithromycin, josamycin, clindamycin, quinupristin, quinupristin-dalfopristin, pristinamycin, and telithromycin. Mutants for which the MICs are increased could be selected with all antibiotics except the streptogramin B quinupristin. Portions of genes encoding 23S rRNA (domains II and V) and ribosomal proteins L4 and L22 of mutants were amplified by PCR, and their nucleotide sequences were compared to those of the susceptible strain M129. No mutation could be detected in domain II of 23S rRNA. Two point mutations in domain V of 23S rRNA, C2611A and A2062G, were selected in the presence of erythromycin A, azithromycin, josamycin, quinupristin-dalfopristin, and telithromycin. Mutants selected in the presence of clindamycin and telithromycin harbored a single amino acid change (H70R or H70L, respectively) in ribosomal protein L4, whereas insertions of one, two, or three adjacent glycines at position 60 (M. pneumoniae numbering) were selected in the presence of both streptogramin combinations. Telithromycin was the sole antibiotic that selected for substitutions (P112R and A114T) and deletions ( 111 IPRA 114 ) in ribosomal protein L22. Three sequential mutational events in 23S rRNA and in both ribosomal proteins were required to categorize the strain as resistant to the ketolide. Azithromycin and erythromycin A were the only selector antibiotics that remained active (MICs, 0.06 and 1 g/ml, respectively) on their mutants selected after 50 passages.Mycoplasma pneumoniae is a common etiological agent of community-acquired respiratory tract infections in children and young adults. Macrolide and related antibiotics are the drugs of choice for the treatment of these infections, and strains with acquired resistance to macrolides have been rarely described. However, resistant strains of M. pneumoniae have been obtained in vitro, by selection in the presence of erythromycin A (20,22,27), and few macrolide-resistant M. pneumoniae isolates have been reported from patients treated with these antibiotics (8,22,27).Macrolide, lincosamide, streptogramin, and ketolide antibiotics (MLSKs) inhibit protein synthesis by binding to domain V of 23S rRNA, and domain II is in the vicinity of this binding site (1,12,17,26). Three main mechanisms of resistance have been reported: drug inactivation, active efflux, and modification of the target sites by methylation or mutation (29, 34). Mutations in domains II and V of 23S rRNA and in ribosomal proteins L4 and L22 were involved in resistance to MLSKs, first in bacteria with a small number of rrn operons and more recently in bacteria like Streptococcus pneumoniae and Staphylococcus aureus with four and six rrn operons, respectively (25,29,32). In mycoplasmas, gram-positive related bacteria which possess one or two copies of 23S rRNA, only resistance by point muta...

Emergence of Macrolide-Resistant Mycoplasma pneumoniae with a 23S rRNA Gene Mutation

“…MICs of 10 agents for M. pneumoniae strains were determined by microdilution methods with PPLO broth (17,21). The following agents were provided by the indicated manufacturer: erythromycin (Shionogi Pharmaceutical Co., Ltd., Osaka, Japan), clarithromycin (Taisho Pharmaceutical Co., Ltd., Tokyo, Japan), josamycin (Yamanouchi Pharmaceutical Co., Ltd., Tokyo, Japan), midecamycin (Meiji Seika Kaisha, Ltd., Tokyo, Japan), rokitamycin (Asahi Kasei Co., Tokyo, Japan), azithromycin (Pfizer Japan, Inc., Tokyo, Japan), telithromycin (Aventis Pharma, Ltd., Bridgewater, N.J.), minocycline (Wyeth Japan, Tokyo, Japan), and levofloxacin and sitafloxacin (Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan).…”

Macrolide resistance mechanisms inEnterobacteriaceae: Focus on azithromycin

“…Similarly, it has been shown that the C2611U alteration results in erythromycin resistance but does not affect 16C macrolides such as spiramycin or tylosin (Vannuffel et al, 1992). In species such as M. pneumoniae or S. pneumoniae it has been found that C2611A and C2611G mutations result in a similar pattern of resistance (Pereyre et al, 2004;Tait-Kamradt et al, 2000), and may thus predict a similar scenario in Enterobacteriaceae. Meanwhile, the U2609C mutation has a differential effect, resulting in slightly higher erythromycin and azithromycin susceptibility levels but an increase in the telithromycin and cethromycin resistance (Garza-Ramos et al, 2001).…”

First Report of Macrolide-Resistant Strains and Description of a Novel Nucleotide Sequence Variation in the P1 Adhesin Gene in Mycoplasma pneumoniae Clinical Strains Isolated in France over 12 Years

“…Macrolide-resistant isolates were characterized by PCR amplification and DNA sequencing of three DNA fragments of interest in the 23S rRNA gene, one fragment in domain II (primers MP23S-17b and MP23S-24) and two fragments in domain V (primers MH23S-11 and MP23S-22 and primers MH23S-9 and MP23S-23) (15). Fragments of ribosomal proteins L4 and L22 were also amplified and sequenced with primers MPL4-1 and MPL4-2 and primers MPL22-1 and MPL22-2, respectively (15).…”