Characterization of Mycoplasma hominis mutations involved in resistance to fluoroquinolones.
ABSTRACT: Fluoroquinolone-resistant mutants of Mycoplasma hominis were selected in vitro from the PG21 susceptible reference strain either by multistep selection on increasing concentrations of various fluoroquinolones or by one-step selection on agar medium with ofloxacin. The quinolone resistance-determining regions (QRDR) of the structural genes encoding the A and b subunits of DNA gyrase were amplified by PCR, and the nucleotide sequences of eight multistep-selected resistant strains were compared to those of susceptible strain PG21. Four high-level resistant mutants that were selected on norfloxacin or ofloxacin contained a C-to-T transition in the gyrA QRDR, leading to substitution of Ser-83 by Leu in the GyrA protein. Analysis of the sequence of the gyrB QRDR of the eight multistep-selected mutants did not reveal any difference compared to that of the gyrB QRDR of the reference strain M. hominis PG21. Similar analyses of eight one-step-selected mutants did not reveal any base change in the gyrA and gyrB QRDRs. These results suggest that in M. hominis, like in other bacterial species, a gyrA mutation at Ser-83 is associated with fluoroquinolone resistance.
Project description:Mycoplasma hominis mutants were selected stepwise for resistance to ofloxacin and sparfloxacin, and their gyrA, gyrB, parC, and parE quinolone resistance-determining regions were characterized. For ofloxacin, four rounds of selection yielded six first-, six second-, five third-, and two fourth-step mutants. The first-step mutants harbored a single Asp426-->Asn substitution in ParE. GyrA changes (Ser83-->Leu or Trp) were found only from the third round of selection. With sparfloxacin, three rounds of selection generated 4 first-, 7 second-, and 10 third-step mutants. In contrast to ofloxacin resistance, GyrA mutations (Ser83-->Leu or Ser84-->Trp) were detected in the first-step mutants prior to ParC changes (Glu84-->Lys), which appeared only after the second round of selection. Further analysis of eight multistep-selected mutants of M. hominis that were previously described (2) revealed that they carried mutations in ParE (Asp426-->Asn), GyrA (Ser83-->Leu) and ParE (Asp426-->Asn), GyrA (Ser83-->Leu) and ParC (Ser80-->Ile), or ParC (Ser80-->Ile) alone, depending on the fluoroquinolone used for selection, i.e., ciprofloxacin, norfloxacin, ofloxacin, or pefloxacin, respectively. These data indicate that in M. hominis DNA gyrase is the primary target of sparfloxacin whereas topoisomerase IV is the primary target of pefloxacin, ofloxacin, and ciprofloxacin.
Project description:Fluoroquinolone resistance in Mycobacterium tuberculosis can be conferred by mutations in gyrA or gyrB. The prevalence of resistance mutations outside the quinolone resistance-determining region (QRDR) of gyrA or gyrB is unclear, since such regions are rarely sequenced. M. tuberculosis isolates from 1,111 patients with newly diagnosed culture-confirmed tuberculosis diagnosed in Tennessee from 2002 to 2009 were screened for phenotypic ofloxacin resistance (>2 ?g/ml). For each resistant isolate, two ofloxacin-susceptible isolates were selected: one with antecedent fluoroquinolone exposure and one without. The complete gyrA and gyrB genes were sequenced and compared with M. tuberculosis H37Rv. Of 25 ofloxacin-resistant isolates, 11 (44%) did not have previously reported resistance mutations. Of these, 10 had novel polymorphisms: 3 in the QRDR of gyrA, 1 in the QRDR of gyrB, and 6 outside the QRDR of gyrA or gyrB; 1 did not have any gyrase polymorphisms. Polymorphisms in gyrA codons 1 to 73 were more common in fluoroquinolone-susceptible than in fluoroquinolone-resistant strains (20% versus 0%; P = 0.016). In summary, almost half of fluoroquinolone-resistant M. tuberculosis isolates did not have previously described resistance mutations, which has implications for genotypic diagnostic tests.
Project description:Fluoroquinolone antibiotics are among the most potent second-line drugs used for treatment of multidrug-resistant tuberculosis (MDR TB), and resistance to this class of antibiotics is one criterion for defining extensively drug resistant tuberculosis (XDR TB). Fluoroquinolone resistance in Mycobacterium tuberculosis has been associated with modification of the quinolone resistance determining region (QRDR) of gyrA. Recent studies suggest that amino acid substitutions in gyrB may also play a crucial role in resistance, but functional genetic studies of these mutations in M. tuberculosis are lacking. In this study, we examined twenty six mutations in gyrase genes gyrA (seven) and gyrB (nineteen) to determine the clinical relevance and role of these mutations in fluoroquinolone resistance. Transductants or clinical isolates harboring T80A, T80A+A90G, A90G, G247S and A384V gyrA mutations were susceptible to all fluoroquinolones tested. The A74S mutation conferred low-level resistance to moxifloxacin but susceptibility to ciprofloxacin, levofloxacin and ofloxacin, and the A74S+D94G double mutation conferred cross resistance to all the fluoroquinolones tested. Functional genetic analysis and structural modeling of gyrB suggest that M330I, V340L, R485C, D500A, D533A, A543T, A543V and T546M mutations are not sufficient to confer resistance as determined by agar proportion. Only three mutations, N538D, E540V and R485C+T539N, conferred resistance to all four fluoroquinolones in at least one genetic background. The D500H and D500N mutations conferred resistance only to levofloxacin and ofloxacin while N538K and E540D consistently conferred resistance to moxifloxacin only. Transductants and clinical isolates harboring T539N, T539P or N538T+T546M mutations exhibited low-level resistance to moxifloxacin only but not consistently. These findings indicate that certain mutations in gyrB confer fluoroquinolone resistance, but the level and pattern of resistance varies among the different mutations. The results from this study provide support for the inclusion of the QRDR of gyrB in molecular assays used to detect fluoroquinolone resistance in M. tuberculosis.
Project description:The topoisomerase IV parC and parE genes from the wall-less organism Mycoplasma hominis PG21 were cloned and sequenced. The coupled genes are located far from the DNA gyrase genes gyrA and gyrB. They encode proteins of 639 and 866 amino acids, respectively. As expected, the encoded ParE and ParC proteins exhibit higher homologies with the topoisomerase IV subunits of the gram-positive bacteria Staphylococcus aureus and Streptococcus pneumoniae than with their Escherichia coli counterparts. The conserved regions include the Tyr residue of the active site and the region involved in quinolone resistance (quinolone resistance-determining region [QRDR]) in ParC and the ATP-binding site and the QRDR in ParE.
Project description:<h4>Background</h4>Fluoroquinolone resistance in Mycobacterium tuberculosis (Mtb) is conferred by DNA gyrase mutations, but not all fluoroquinolone-resistant Mtb isolates have mutations detected. The optimal allele frequency threshold to identify resistance-conferring mutations by whole-genome sequencing is unknown.<h4>Methods</h4>Phenotypically ofloxacin-resistant and lineage-matched ofloxacin-susceptible Mtb isolates underwent whole-genome sequencing at an average coverage depth of 868 reads. Polymorphisms within the quinolone-resistance-determining region (QRDR) of gyrA and gyrB were identified. The allele frequency threshold using the Genome Analysis Toolkit pipeline was ~8%; allele-level data identified the predominant variant allele frequency and mutational burden (ie, sum of all variant allele frequencies in the QRDR) in gyrA, gyrB, and gyrA + gyrB for each isolate. Receiver operating characteristic (ROC) curves assessed the optimal measure of allele frequency and potential thresholds for identifying phenotypically resistant isolates.<h4>Results</h4>Of 42 ofloxacin-resistant Mtb isolates, area under the ROC curve (AUC) was highest for predominant variant allele frequency, so that measure was used to evaluate optimal mutation detection thresholds. AUCs for 8%, 2.5%, and 0.8% thresholds were 0.8452, 0.9286, and 0.9069, respectively. Sensitivity and specificity were 69% and 100% for 8%, 86% and 100% for 2.5%, 91% and 91% for 0.8%. The sensitivity of the 2.5% and 0.8% thresholds were significantly higher than the 8% threshold (P = .016 and .004, respectively) but not significantly different between one another (P = .5).<h4>Conclusions</h4>A predominant mutation allele frequency threshold of 2.5% had the highest AUC for detecting DNA gyrase mutations that confer ofloxacin resistance, and was therefore the optimal threshold.
Project description:To characterize mechanisms of resistance to fluoroquinolones by Mycobacterium tuberculosis, mutants of strain H37Ra were selected in vitro with ofloxacin. Their quinolone resistance-determining regions for gyrA and gyrB were amplified and sequenced to identify mutations in gyrase A or B. Three types of mutants were obtained: (i) one mutant (TKp1) had no mutations in gyrA or gyrB; (ii) mutants that had single missense mutations in gyrA, and (iii) mutants that had two missense mutations resulting in either two altered gyrase A residues or an altered residue in both gyrases A and B. The TKp1 mutant had slightly reduced levels of uptake of [14C]norfloxacin, which was associated with two- to fourfold increases in the MICs of ofloxacin, ciprofloxacin, and sparfloxacin. Gyrase mutations caused a much greater increase in the MICs of fluoroquinolones. For mutants with single gyrA mutations, the increases in the MICs were 4- to 16-fold, and for mutants with double gyrase mutations, the MICs were increased 32-fold or more compared with those for the parent. A gyrA mutation in TKp1 secondary mutants was associated with 32- to 128-fold increases in the MICs of ofloxacin and ciprofloxacin compared with the MICs for H37Ra and an eight-fold increase in the MIC of sparfloxacin. Sparfloxacin was the most active fluoroquinolone tested. No sparfloxacin-resistant single-step mutants were selected at concentrations of > 2.5 micrograms/ml, and high-level resistance (i.e., MIC, > and = 5 micrograms/ml) was associated with two gyrase mutations. Mutations in gyrB and possibly altered levels of intracellular accumulation of drug are two additional mechanisms that may be used by M. tuberculosis in the development of fluoroquinolone resistance. Because sparfloxacin is more active in vitro and selection of resistance appears to be less likely to occur, it may have important advantage over ofloxacin or ciprofloxacin for the treatment of tuberculosis.
Project description:Fluoroquinolone (FQ) resistance is emerging in Mycobacterium tuberculosis. The main mechanism of FQ resistance is amino acid substitution within the quinolone resistance-determining region (QRDR) of the GyrA subunit of DNA gyrase, the sole FQ target in M. tuberculosis. However, substitutions in GyrB whose implication in FQ resistance is unknown are increasingly being reported. The present study clarified the role of four GyrB substitutions identified in M. tuberculosis clinical strains, two located in the QRDR (D500A and N538T) and two outside the QRDR (T539P and E540V), in FQ resistance. We measured FQ MICs and also DNA gyrase inhibition by FQs in order to unequivocally clarify the role of these mutations in FQ resistance. Wild-type GyrA, wild-type GyrB, and mutant GyrB subunits produced from engineered gyrB alleles by mutagenesis were overexpressed in Escherichia coli, purified to homogeneity, and used to reconstitute highly active gyrase complexes. MICs and DNA gyrase inhibition were determined for moxifloxacin, gatifloxacin, ofloxacin, levofloxacin, and enoxacin. All these substitutions are clearly implicated in FQ resistance, underlining the presence of a hot spot region housing most of the GyrB substitutions implicated in FQ resistance (residues NTE, 538 to 540). These findings help us to refine the definition of GyrB QRDR, which is extended to positions 500 to 540.
Project description:The L2 reference strain of Chlamydia trachomatis was exposed to subinhibitory concentrations of ofloxacin (0.5 microg/ml) and sparfloxacin (0.015 microg/ml) to select fluoroquinolone-resistant mutants. In this study, two resistant strains were isolated after four rounds of selection. The C. trachomatis mutants presented with high-level resistance to various fluoroquinolones, particularly to sparfloxacin, for which a 1,000-fold increase in the MICs for the mutant strains compared to the MIC for the susceptible strain was found. The MICs of unrelated antibiotics (doxycycline and erythromycin) for the mutant strains were identical to those for the reference strain. The gyrase (gyrA, gyrB) and topoisomerase IV (parC, parE) genes of the susceptible and resistant strains of C. trachomatis were partially sequenced. A point mutation was found in the gyrA quinolone-resistance-determining region (QRDR) of both resistant strains, leading to a Ser83-->Ile substitution (Escherichia coli numbering) in the corresponding protein. The gyrB, parC, and parE QRDRs of the resistant strains were identical to those of the reference strain. These results suggest that in C. trachomatis, DNA gyrase is the primary target of ofloxacin and sparfloxacin.
Project description:We report the cloning and characterization of the gyrA gene of the Mycoplasma hominis DNA gyrase, which was previously shown to be associated with quinolone resistance in this organism. The 2,733-bp gyrA gene encodes a protein of 911 amino acids with a calculated molecular mass of 102.5 kDa. As expected, M. hominis GyrA exhibits higher homology with the GyrA subunits of the gram-positive bacteria Clostridium acetobutylicum, Bacillus subtilis, Streptococcus pneumoniae, and Staphylococcus aureus than with its Escherichia coli counterpart. Knowing the entire sequence of the gyrA gene of M. hominis could be very useful for confirming the role of the GyrA subunit in fluoroquinolone resistance. Twenty-nine mutants of M. hominis were selected stepwise for resistance to trovafloxacin, a new potent fluoroquinolone, and their gyrA, gyrB, parC, and parE quinolone resistance-determining regions were characterized. Three rounds of selection yielded 3 first-step, 12 second-step, and 14 third-step mutants. The first-step mutants harbored a single substitution, Glu460-->Lys (E. coli coordinates), in ParE. GyrA changes, Ser83-->Leu, Glu87-->Lys, and Ala119-->Glu or Val, were found only in the second round of selection. At the third step, additional substitutions, at ParC Ser80, Ser81, and Glu84 and ParE Leu440, associated with high-level resistance to fluoroquinolones, appeared. Thus, high-level resistance to trovafloxacin required three steps and was associated with alterations in both fluoroquinolone targets. According to these genetic data, in M. hominis, as in Staphylococcus aureus and Streptococcus pneumoniae, topoisomerase IV seems to be the primary target of trovafloxacin.
Project description:Fluoroquinolone-resistant mutants of Mycobacterium smegmatis have been obtained in vitro by using ofloxacin as a selecting agent. Two types of mutants were identified according to their quinolone resistance patterns. Type 1 showed a low level of resistance to ofloxacin (MIC of 8 micrograms/ml), whereas a high level of resistance to this drug (MICs of 32 to 64 micrograms/ml) characterized type 2. By using two oligonucleotide primers homologous to DNA sequences flanking the quinolone resistance-determining region (QRDR) in the gyrA gene of Escherichia coli and Staphylococcus aureus, a 150-bp DNA fragment was obtained by PCR amplification from total DNA of two wild-type and five mutant strains of M. smegmatis. The nucleotide sequences of the amplified fragments were determined. The deduced amino acid sequence from the wild-type strains showed ca. 79% similarity with the QRDR in the gyrase A subunit from other gram-positive and gram-negative bacteria. The DNA sequences obtained from the fluoroquinolone-resistant mutants of M. smegmatis exhibited nucleotide modifications compared with the wild-type QRDR. The QRDR from type 1 mutants had a C-T or an A-G transition leading to a change from Ala-83 to Val or Asp-87 to Gly, respectively. The QRDR from type 2 mutants had a Val-83 mutation or both Val-83 and Gly-87 mutations detected in the type 1 mutants. These results suggest that point mutations in the QRDR of the mycobacterial gyrA gene are responsible for acquired quinolone resistance in M. smegmatis.