Reduced susceptibility to tetracyclines is associated in vitro with the presence of 16S rRNA mutations in Mycoplasma hominis and Mycoplasma pneumoniae

Sir, Mycoplasma pneumoniae and Mycoplasma hominis are aetiological agents of respiratory and genitourinary tract infections, respectively, for which tetracyclines present potential for empirical treatment.1 As mycoplasmas possess a small number of rrn operons, one for M. pneumoniae and two for M. hominis, the target-related mechanism of resistance to tetracyclines caused by 16S rRNA mutations could be expected as previously described for Helicobacter pylori.2 The purpose of this study was to identify such a mechanism in the reference strains M. hominis PG21 and M. pneumoniae FH by selecting in vitro for tetracycline-resistant mutants and sequencing the 16S rRNA genes of the obtained mutants. Growth conditions and antibiotic susceptibility testing of the mycoplasma strains have been described previously.3 Two selection methods, with either broth or agar medium, were used for M. hominis PG21 (ATCC 23114), although only the broth-based selection was done for M. pneumoniae FH (ATCC 15531).4 Broth-selected mutants were obtained by serial transfers of M. hominis PG21 and M. pneumoniae FH in appropriate Hayflick-modified broth medium containing increasing subinhibitory concentrations of doxycycline. Stepwise selection of doxycycline-resistant mutants was performed onto Hayflick-modified agar medium containing increasing inhibitory concentrations of doxycycline, as described previously.4 Two steps were performed with doxycycline concentrations at 2× and 8× MIC for the respective parent strain. Amplifications of M. hominis and M. pneumoniae 16S rRNA genes were performed with primers described previously.5 For operon rrnA, inverse PCR (IPCR) was carried out on self-ligated genomic DNA of M. hominis with two divergent primers H2pu1 (5′-GGTGCATGGTTGTCGTCAGC-3′) and Mh16S-6 (5′-GCCAGCGTTCATCCTGAGCC-3′), located at the 5′ and 3′ ends of the 16S rRNA gene, respectively. PCR products were cloned into the pGEM-T cloning vector (Promega). For operon rrnB, libraries of HindIII-digested M. hominis total DNA were constructed with the pGEM7ZF(+) cloning vector (Promega). Recombinant clones were selected by colony hybridization with a labelled PCR product, obtained with primers Mh16S-1 and Mh16S-3.5 Plasmids and the purified PCR products were directly sequenced by using an ABI PRISM dRhodamine terminator cycle sequencing ready reaction kit (Applied Biosystems). The nucleotide sequences of the fragments encompassing the 16S rRNA of operons rrnA and rrnB of M. hominis have been deposited in GenBank under accession nos AF443616 and AF443617, respectively. The molecular organization in the vicinity of both 16S rRNA copies was determined to differentiate them in M. hominis. IPCR allowed us to amplify a fragment of 1447 bp named X4 encompassing the region directly upstream of the 16S rRNA gene of operon rrnA. The screening of a representative library of HindIII-digested total DNA, with the 16S-specific probe amplified with primers Mh16S-1 and Mh16S-3, led to the identification of a 4472 bp genomic fragment encompassing the region directly upstream of the 16S rRNA of operon rrnB. Subsequently, two specific primers, Mh16S-A (5′-CCAAGCATGTGAAAACTGCGG-3′) and Mh16S-B (5′-GCTAGCTAAATTTAAAGCAGG-3′), were designed to amplify, in association with Mh16S-7,5 the 16S rRNA genes of operons rrnA and rrnB, respectively. For both species, after serial passages in subinhibitory concentrations of doxycycline, we were able to select mutants having reduced susceptibility to tetracyclines and harbouring a variety of mutations in 16S rRNA. No resistant strains could be obtained in vitro (tetracycline MICs >8 mg/L), as previously described for H. pylori.2 Most of the mutations were located in the primary tetracycline binding site6 formed by the 16S rRNA residues 1054–1056 and 1196–1200 of helix 34 and residues 964–967 of helix 31. Table 1 shows the MICs of tetracyclines for each mutant studied and the mutations observed in the 16S rRNA genes. According to the ‘Comité de l'Antibiogramme de la Société Française de Microbiologie’, all M. hominis and M. pneumoniae selected mutants remained categorized as susceptible to the three tetracyclines (MICs ≤ 2 mg/L), except the M. hominis mutant DH12A which was categorized as intermediate to tetracycline (MIC 8 mg/L, Table 1). Characteristics of doxycycline-selected mutants of M. hominis and M. pneumoniae aBroth-selected mutants are designated by the initial of the selector doxycycline (D) followed by the initial of the mycoplasmal species (H for M. hominis or P for M. pneumoniae) and the passage number. Fifteen and 18 passages were performed for M. hominis and M. pneumoniae, respectively. Only passages with a significant MIC increase (at least 4-fold) and a 16S rRNA mutation are presented. For these passages, two of the five clones, named A and B, subcultured were studied. When both clones from one passage were identical, only one clone is represented in this table. M. hominis agar-selected mutants are designated by a prefix corresponding to the selection step (I or II), followed by the initial of the selector doxycycline (D). Only clones with significant increased MICs or 16S rRNA mutations are shown. bTET, tetracycline; DOX, doxycycline; MIN, minocycline. cEscherichia coli numbering. dLetters in parentheses indicate the nucleotides found in both rrs genes of M. hominis. M. pneumoniae has only one copy of the rrs gene. Characteristics of doxycycline-selected mutants of M. hominis and M. pneumoniae aBroth-selected mutants are designated by the initial of the selector doxycycline (D) followed by the initial of the mycoplasmal species (H for M. hominis or P for M. pneumoniae) and the passage number. Fifteen and 18 passages were performed for M. hominis and M. pneumoniae, respectively. Only passages with a significant MIC increase (at least 4-fold) and a 16S rRNA mutation are presented. For these passages, two of the five clones, named A and B, subcultured were studied. When both clones from one passage were identical, only one clone is represented in this table. M. hominis agar-selected mutants are designated by a prefix corresponding to the selection step (I or II), followed by the initial of the selector doxycycline (D). Only clones with significant increased MICs or 16S rRNA mutations are shown. bTET, tetracycline; DOX, doxycycline; MIN, minocycline. cEscherichia coli numbering. dLetters in parentheses indicate the nucleotides found in both rrs genes of M. hominis. M. pneumoniae has only one copy of the rrs gene. For M. hominis, single (at positions 966, 967 or 1054), double (positions 346 and 965) and triple mutants (positions 346, 965 and 966) were obtained and showed various levels of tetracycline MIC increases. Positions 965–967 correspond to the triple-base-pair mutation involved in the high-level tetracycline resistance in H. pylori,2 whereas nucleotide 1054 contacts the A-site tRNA.6 In contrast, the G346A mutation is located in a 16S rRNA region not closely associated with tetracycline binding. For M. pneumoniae, two mutations were found in the broth-selected mutants and were associated with 2- to 16-fold increases in MICs of the three tetracyclines studied, in comparison with those of the parental strain FH. The two mutations described for this species, G1193A and T968C, are located very close to or contact the primary tetracycline binding site, respectively. In summary, this is the first description of mutations in 16S rRNA associated with decreased susceptibility to tetracyclines in human mycoplasmas. However, what real effect these new mutations have on the tetracycline susceptibility of both mycoplasmas has yet to be determined. No specific funding was received for the study. None to declare.