Mechanism Of Drug Resistance Of Mycoplasma Pneumoniae Against Tetreacyclines

Objective: Mycoplasma pneumoniae is the most common causative pathogen in respiratory tract infections,prone to infect all people.Mycoplasma pneumoniae is also the most common pathogen to cause community acquired pneumonia(CAP)in China,which tends to have an outbreak and worldwide epidemic every 2-6 years.There is also an increasing number of severe cases reported that are caused by or related to Mycoplasma pneumoniae infections.Generally,three categories of antibiotics are applied in the treatment of Mp infections: macrolides,quinolones and tetracyclines.Recent years,Mycoplasma pneumoniae is becoming increasingly resistant against macrolides,which are the first choice in the treatment of Mp infection.In China,the rate of macrolide-resistance is up to 92%,highest in the world.It is of utter importance that we take measures to prevent Mp from being resistant to the other two categories of antibiotics.It takes less than a decade since 2001,the first time Okazaki N openly reported the isolation of macrolide-resistant Mp,until 2009,92% resistance rate in China was reported.Although no quinolone-resistant Myplasma pneumnoiae has been isolated clinically,other human mycoplasmas have already been identified and isolated as clinically quinolone-resistant.In addition,in vitro induced quinolone-resistant Mp has already been successfully acquired both domestic and abroad.Quinolones are also considered unfit for people under age 18,due to its side effect to influence bone growth.So,tetracyclines become the only choice for the treatment of macrolide-resistant Mycoplasma pneumoniae infections.Tetracycline-resistant Mp has not yet been identified both in vitro and in vivo.Our research is to collect clinical samples,screen for tetracycline-resisant Mp strains,while developing tetracycline-resistance in vitro at the same time.By investigating and comparing the possible mechanism of their resistance,we will be able to offer suggestion for clinical usage of tetracyclines,thus prevent or delay Mycoplasma pneumoniae from developing resistance against these antibiotics.Methods: 1.Our research group has formerly isolated several dozens of Mp strains from clinical samples from the respiratory department of a hospital during the year 2010-2014,and established a library of Mp strains.This research revives Mycoplasma pneumoniae strains formerly stored at-70? in this strain library;Broth-agar-broth culture were carried out to acquire purified Mp strains.Meanwhile,clinical samples(throat swab and BALF)were collected,using real-time PCR and broth culture to identify,isolate and acquire new Mp strains.2.Minimum inhibitory concentration(MIC)of Mp strains were determined by broth-microdilution.Mp strains were considered resistant to tetracyclines if MIC ? 8 ?g/m L.3.Develop resistance from tetracycline-sensitive Mp strains by culturing them in the existence of tetracyclines with subinhibitory concentration.4.Determine the MIC of resistant Mp strains against antibiotics in the presence of reserpine and CCCP,categorized as efflux pump inhibitors(EPI),as a screening process to indicate the presence of efflux pumps in resistant strains.5.Resistant strains and their parent strains were performed whole-genome sequencing to identify resistance-related genes,including ribosomal protection genes,efflux pump genes and tetracycline degrading gene(tet X),also to identify mutations in 16 S r RNA,where the binding site of tetracyclines locates.6.2-dimentional electrophoresis was performed between a tetracycline-sensitive strain and a tetracycline-resistant strain,to identify differential proteins between them,thus to determine if any tetracycline-resistance related functional proteins are involved.7.Ribosome binding assay: Incubate [7-3H]tetracycline with ribosomes isolated from tetracycline-induced strain(point mutation in binding area in 16 S r RNA already affirmed)and its parent strain,then filter the incubated ribosome through a GF/C filter(Whatman).After three washes with buffer TMMKA-100,the filters were dried under 80?,then immersed in 1m L of scintillation fluid.CPM were measured with a Tricarb 9100 scintillation counter(Perkin Elmer,USA).Results: 1.Totally 33 strains of Mp were acquired.132 clinical samples were collected,12 of which are detected positive(9.1%)with real-time PCR.No new Mp strains were isolated from these samples.2.Antibiotic susceptibility testing showed that all Mp strains were sensitive to tetracyclines.Tigecycline of all,presents relatively lower MIC,showing higher antibacterial activity than first and second generation tetracycline and quinolene.All Mp strains were sensitive to levofloxacin,while 54.8% of them are resistant to erythromycin.3.Eight strains of induced tetracycline-resistant Mp were acquired(MIC ? 8 ?g/m L)by cultivating sensitive strains in the presence of sub-inhibitory concentration of tetracyclines.All tetracycline-induced strains(including the ones not identified as “resistant”)have shown a significant increase in MIC against tetracyclines.And induced-reisistant strains show a cross-resistance against other tetracyclines.4.Reserpine can significantly lower the MIC of some of the resistant strainsagainst tetracycline,minocycline and tigecycline(4-fold or more).Carbonylcyanide-p-chlorophenyl hydrazone(CCCP)shows no similar effect.5.Whole genome sequencing indicates: of 8 strains of induced tetracycline-resistant Mp,6 of them present point mutation A?T in site 1173 of 16 S r RNA.The other 2 strains present point mutations in two sites: 964T?C,1169G?A.These point mutations do not occur in any of their parent strains.Point mutations in these sites exist only in resistant strains,and these sites are reportedly within the binding area of tetracylines with 16 S r RNA.By blasting the whole genome of resistant strains with resistance gene bank using Res Finder2.1,we found that none of the 8 resistant strains' genome consist of ribosomal protection genes,efflux pump genes,tetracycline degrading gene,or other resistance genes related to tetracycline resisitance.6.The binding assay of ribosome with tetracycline shows that the ribosome of resistant strain exhibits lower affinity to tetracycline than that of the sensitive strain.7.We established the method to study the proteomics of Mycoplasma pneumoniae using 2-D electrophoresis.Nineteen differential protein spots were chosen and identified between sensitive strain and resistant strain.No tetracycline-resistance related proteins were identified.However,we observed that,overexpressed proteins(e.g.Chaperone protein,Transcription elongation factor Gre A)found in the resistant strain are mostly related to protein translation and synthesis,while overexpressed proteins(e.g.Pyruvate dehydrogenase,enolase)found in the sensitive strain are mostly related to energy metabolism.Conclusion: 1.No clinical tetracycline-resistant Mycoplasma pneumoniae strains were identified in our research.2.Tetracyclines can induce resistance in Mycoplasma pneumoniae,and can result in cross resistance in other tetracyclines.3.Complete genome sequences of 8 strains of tetracycline-resistant Mycoplasma pneumoniae were obtained for the first time worldwide and were submitted to Genbank.4.No ribosomal protection genes,efflux pump genes and tetracycline degrading gene were yet identified in the resistant strains.5.The most probable mechanism of resistance in tetracycline-induced Mp is point mutation in the binding area of 16 S r RNA.6.Ribosome binding assay indicates that point mutations within binding area of 16 S r RNA can lower the affinity of tetracyclines to ribosome.7.No proteins were identified in the resistant strain that are directly related to tetracycline-resistance.