Study On The Mechanisms Of Drug Resistance In Mycoplasma Pneumoniae Isolates

【Objectives】 To further study the mechanisms of macrolide resistance and predict thepossible mechanisms of tetracyclines resistance in Mp isolates in China; to investigate therole (if any) of Mp in subacute cough to help analyze the etiology of subacute cough.【Methods】(1) Oropharyngeal swab specimens were obtained from the patients. Twomethods of the real-time PCR and cultivation with broth and agar alternate medium wereused to identify the purified Mp isolates.(2) The MICs of four classes of antibioticsincluding macrolides (erythromycin, azithromycin and medicamycin), tetracyclines(tetracycline and minocycline), fluoroquinolones (ofloxacin, levofloxaxin andmoxifloxacin) and aminoglycosides (amikacin and etimicin) were detected for72Mpstrains by employing96-well plates broth microdilution assay.(3) The nested PCR andone-step multiplex PCR were used to genotype drug-resistant Mp strains. The nucleotidesequencing of RepMP4and RepMP2/3elements in p1gene were applied to analyze thehomology of drug-resistant Mp strains.(4) Three common mechanisms of macrolideresistance (efflux pump, target modification and inactivated enzyme) were researched bynucleotide sequencing of23SrRNA and5SrRNA genes, observing the changes of MICsinfluenced by the efflux pump inhibitor (reserpine), and screening the genes of mef andmsr and mph(C).(5) Sequencing the16S rRNA genes and screening the tet (M) and effluxpump were analyzed to identify such the mechanisms in the clinical obtainedtetracyclines-resistant Mp strains and the Mp strains by selecting in vitro for tetracyclines-resistant mutants.(6) Oropharyngeal swab specimens were obtained to detect Mp from thepatients with subacute cough and from the healthy subjects in the Affiliated Hospital ofAcademy of Military and Medical Sciences in the period between July2010and June2011.We analyzed the role of Mp in subacute cough by detection, genotyping, MICsdetermination and observing the efficacy of azithromycin in Mp isolates.【Results】(1)72Mp isolates were obtained from this study.(2) The MICs testing results showed the macrolide-resistant rate for Mp was41.7%(30/72) and nofluoroquinolone-resistant Mp was found; one Mp strain with tetracycline-resistance for thefirst time was found; aminoglycosides had little effect on inhibiting the growth of Mp.(3)Genotyping of30Mp isolates showed type I in29Mp strains and type IIa in1strain.Homology was found among the most drug-resistant Mp strains.(4) All the30macrolide-resistant Mp strains had a point transition at position2063, of which29strainshad an A–G transition and1strain had a heterozygote mutation. One Mp strain waspositive for the presence of the msrA/B gene. Nucleotide sequencing of the gene showedhighly similarity with the acquired macrolide resistance-like protein gene of Enterococcusfaecium strain TX2465and the translation of the gene showed it had some similarity in theconserved sequence of P-loop＿NTPase superfamily. No inactivated enzyme of mph(C)was detected.(5) There was point mutation in the16SrRNA gene of Mp withtetracyclines-induced resistance. No tet (M) gene was found and the efflux pump inhibitor(reserpine) had no influence on the MIC of antibiotics for the tetracyclines-resistant Mpisolates.(6) The Mp detection rates in the group of patients with subacute cough and in thegroup of healthy subjects were55.4%and5%(P<0.001), respectively.46Mp strainsisolated from patients were genotyped into type I in38strains (82.6%), type II in6strains(13.0%), and type IIa2strains (4.3%).23of46(50%) Mp strains were resistant tomacrolides. Mp detection rate was not associated with sex and age but with seasons. Mpinfections tended to occur in winter and autumn. There was no significant difference oncough resolution time after azithromycin were taken (3days for1course,0.5g/day)between the12patients group who were infected with macrolide-resistant Mp and the18patients group who were infected with macrolide-susceptible Mp.【Conclusions】Two mechanisms of point mutation and efflux pump were presented in themacrolide-resistant Mp in China. Tetracyclines could induce drug-resistant Mp strains invitro. Point mutation in the16SrRNA gene may be the possible mechanism oftetracyclines-resistance for Mp. Mp infection may play an important role in subacutecough.