Clinical and Experimental Pediatrics

The Korean Pediatric Society (대한소아청소년과학회)
권호 표지
DOI QR코드

DOI QR Code

Detection of genetic mutations associated with macrolide resistance of Mycoplasma pneumoniae

Mycoplasma pneumoniae의 macrolide 내성과 연관된 유전자 변이의 검출

  • Oh, Chi Eun (Department of Pediatrics, Seoul National University College of Medicine) ;
  • Choi, Eun Hwa (Department of Pediatrics, Seoul National University College of Medicine) ;
  • Lee, Hoan Jong (Department of Pediatrics, Seoul National University College of Medicine)
  • 오지은 (서울대학교 의과대학 소아과학교실) ;
  • 최은화 (서울대학교 의과대학 소아과학교실) ;
  • 이환종 (서울대학교 의과대학 소아과학교실)
  • Received : 2009.11.30
  • Accepted : 2010.01.14
  • Published : 2010.02.15
Download PDF
⟨ Previous Next ⟩

Abstract

Purpose : The aim of this study was to identify mutations associated with macrolide resistance in Mycoplasma pneumoniae (MP) and to establish a cultural method to determine antimicrobial susceptibility. Methods : Nasopharyngeal aspirates (NPAs) were collected from 62 children diagnosed with MP pneumonia by a serologic method or polymerase chain reaction. The 23S rRNA and L4 ribosomal protein genes of MP were amplified and sequenced. To identify mutations in these 2 genes, their nucleotide sequences were compared to those of the reference strain M129. MP cultivation was carried out for 32 (28 frozen and 5 refrigerated) NPAs and M129 strain using Chanock's glucose broth and agar plate in a 5% $CO_2$ incubator at $37^{\circ}C$ and examined at 2-3 day intervals for 6 weeks. Results : Among the 62 specimens, 17 had M144V mutations in ribosomal protein L4. The A2064G mutation was observed in 1 specimen; its 23S rRNA gene was successfully sequenced. Culture for MP was successful from the M129 strain and 2 of the 5 NPAs that were refrigerated for no longer than 3 days. However, MP did not grow from the 28 NPAs that were kept frozen at $-80^{\circ}C$ since 2003. Conclusion : We found the M144V mutation of L4 protein to be common and that of domain V of 23S rRNA gene was relatively rare among MP. Studies on the prevalence of macrolide-resistant MP and the relationship between the mutations of 23S rRNA gene and ribosomal protein L4 will aid in understanding the mechanism of macrolide resistance in MP.

목 적 : 최근에 macrolide계 항균제에 내성인 M. pneumoniae 균주가 증가한다는 외국의 보고가 있었으며, 국내에서 수행된 한 연구에서도 M. pneumoniae의 macrolide 내성률을 49% 정도로 보고한 바 있다. 이에, 본 연구는 M. pneumoniae 폐렴으로 진단된 소아의 비인두 흡인물에서 M. pneumoniae의 macrolide 계항균제 내성에 연관된 것으로 알려진 유전자 변이 유무를 확인하고, M. pneumoniae의 macrolide계 항균제에 대한 최소억제농도를 측정하기 위한 기초 연구로 M. pneumoniae 배양법을 구축하고자 시행되었다. 방 법 : 2000년과 2003년 M. pneumoniae 감염의 유행기에 급성 호흡기 증상을 주소로 서울대학교 어린이병원과 분당서울대학교병원에서 치료받은 소아 중 혈청학적 검사와 M. pneumoniae PCR을 통해 M. pneumoniae 폐렴으로 진단받은 환아 62명으로 부터 채취하여 $-80^{\circ}C$에 보관되었던 비인두 흡인물을 대상으로 하였다. M. pneumoniae의 23S rRNA domain V의 peptidyl transferase 부위와 ribosomal protein L4를 M. pneumoniae 특이 PCR로 증폭한 후 염기서열분석을 시행하였다. 염기서열의 분석은 M. pneumoniae 표준 균주와 비교하여, 23S rRNA domain V의 A2063G, A2064G 변이와 ribosomal protein L4의 M144V변이 유무를 확인하였다. 또한, M. pneumoniae 표준 균주와 33개의 비인두흡인물($-80^{\circ}C$에 보관되었던 28검체와 1-2일간 냉장보관되었던 비인두흡인물 5 검체)을 Chanock's glucose 액체배지와 한천배지에 접종하고 $37^{\circ}C$의 5% $CO_2$ 항온기에서 6주간 관찰하여 배양을 확인하였다. 결 과 : 총 62 검체 중 23S rRNA gene에 대한 염기서열분석이 가능했던 61 검체 중 1검체(1.6%)에서 A2064G변이가 관찰되었고, 62 검체의 ribosomal protein L4에 대한 염기서열분석 결과 17검체(27.4%)에서 M144V 아미노산 변이가 확인되었다. M. pneumoniae 배양 결과, 표준 균주는 Chanock's glucose 액체배지와 한천배지 모두에서 배양되었고 2009년에 채취된 5검체 중 2검체에서 배양이 확인되었으나, $-80^{\circ}C$에 보관되었던 28검체는 모두 배양되지 않았다. 결 론 : 본 연구에서 23S rRNA gene의 유전자 변이 빈도는 매우 낮았고, ribosomal protein L4의 M144V 변이는 좀 더 많은 검체에서 확인되었다. Macrolide계 항균제에 내성인 M. pneumoniae의 분포와 M. pneumoniae의 23S rRNA gene과 ribosomal protein L4의 변이에 대한 추가적인 연구들을 통해 M. pneumoniae의 macrolide 항균제에 대한 내성기전을 이해하는데 도움을 줄 수 있을 것으로 생각된다.

Keywords

Acknowledgement

Supported by : Seoul National University Hospital

References

  1. Baum SG. Mycoplasma pneumoniae and atypical pneumonia. In: Mandell GL, Bennett JE, Dolin R, ed. Principles and Practice of Infectious Diseases. 6th ed. Philadelphia: Elsevier Churchill Livingstone; 2005:2271-8
  2. Waites KB, Crabb DM, Duffy LB. In vitro activities of ABT- 773 and other antimicrobials against human mycoplasmas. Antimicrob Agents Chemother 2003;47:39-42 https://doi.org/10.1128/AAC.47.1.39-42.2003
  3. Niitu Y, Hasegawa S, Suetake T, Kubota H, Komatsu S, Horikawa M. Resistance of Mycoplasma pneumoniae to erythromycin and other antibiotics. J Pediatr 1970;76:438-43 https://doi.org/10.1016/S0022-3476(70)80485-1
  4. Stopler T, Gerichter CB, Branski D. Antibiotic-resistant mutants of Mycoplasma pneumoniae. Isr J Med Sci 1980;16:169-73
  5. Morozumi M, Iwata S, Hasegawa K, Chiba N, Takayanagi R, Matsubara K, et al. Increased macrolide resistance of Mycoplasma pneumoniae in pediatric patients with communityacquired pneumonia. Antimicrob Agents Chemother 2008;52:348-50 https://doi.org/10.1128/AAC.00779-07
  6. Peuchant O, Menard A, Renaudin H, Morozumi M, Ubukata K, Bebear CM, et al. Increased macrolide resistance of Mycoplasma pneumoniae in France directly detected in clinical specimens by real-time PCR and melting curve analysis. J Antimicrob Chemother 2009;64:52-8 https://doi.org/10.1093/jac/dkp160
  7. Chang MW, Kim KH, Park ID, Song GY, Kim SW, Lee EY, et al. Isolation of Mycoplasma pneumoniae and antimicrobial susceptibilities of the isolates. J Life Science 2005;15:863-70
  8. Kim NH, Lee JA, Eun BW, Shin SH, Chung EH, Park KW, et al. Comparison of polymerase chain reaction and the indirect particle agglutination antibody test for the diagnosis of Mycoplasma pneumoniae pneumonia in children during twooutbreaks. Pediatr Infect Dis J 2007;26:897-903 https://doi.org/10.1097/INF.0b013e31812e4b81
  9. Morozumi M, Hasegawa K, Kobayashi R, Inoue N, Iwata S, Kuroki H, et al. Emergence of macrolide-resistant Mycoplasma pneumoniae with a 23S rRNA gene mutation. Antimicrob Agents Chemother 2005;49:2302-6 https://doi.org/10.1128/AAC.49.6.2302-2306.2005
  10. Leclercq R, Courvalin P. Resistance to Macrolides and Related Antibiotics in Streptococcus pneumoniae. Antimicrob Agents Chemother 2002;46:2727-34 https://doi.org/10.1128/AAC.46.9.2727-2734.2002
  11. Hansen LH, Mauvais P, Douthwaite S. The macrolide-ketolide antibiotic binding site is formed by structures in domains II and V of 23S ribosomal RNA. Molecular Microbiology 1999;31:623-31 https://doi.org/10.1046/j.1365-2958.1999.01202.x
  12. Gobel U, Butler GH, Stanbridge EJ. Comparative analysis of mycoplasma ribosomal RNA operons. Isr J Med Sci 1984;20:762-4
  13. Lucier TS, Heitzman K, Liu SK, Hu PC. Transition mutations in the 23S rRNA of erythromycin-resistant isolates of Mycoplasma pneumoniae. Antimicrob Agents Chemother 1995;39:2770-3
  14. Okazaki N, Narita M, Yamada S, Izumikawa K, Umetsu M, Kenri T, et al. Characteristics of macrolide-resistant Mycoplasma pneumoniae strains isolated from patients and induced with erythromycin in vitro. Microbiol Immunol 2001;45:617-20
  15. Li X, Atkinson TP, Hagood J, Makris C, Duffy LB, Waites KB. Emerging macrolide resistance in Mycoplasma pneumoniae in children: detection and characterization of resistant isolates. Pediatr Infect Dis J 2009;28:693-6 https://doi.org/10.1097/INF.0b013e31819e3f7a
  16. Liu Y, Ye X, Zhang H, Xu X, Li W, Zhu D, et al. Antimicrobial susceptibility of Mycoplasma pneumoniae isolates and molecular analysis of macrolide-resistant strains from Shanghai, China. Antimicrob Agents Chemother 2009;53:2160-2 https://doi.org/10.1128/AAC.01684-08
  17. Pereyre S, Guyot C, Renaudin H, Charron A, Bebear C, Bebear CM. In vitro selection and characterization of resistance to macrolides and related antibiotics in Mycoplasma pneumoniae. Antimicrob Agents Chemother 2004;48:460-5 https://doi.org/10.1128/AAC.48.2.460-465.2004
  18. Chang MW, Kim KH, Park ID, Joh MH. Detection of Mycoplasma pneumoniae in clinical specimens of patients by PCR and culture method. J Korean Soc Microbiol 1995;30:517-25
  19. Chang MW, Kim KH, Park ID, Kang KH, Kong EH, Jeong MH, et al. Rapid detection of Mycoplasma pneumoniae and antimicobial susceptibilities of the M. pneumoniae isolates. J Bacteriol Virol 2003;33:183-91
  20. Dular R, Kajioka R, Kasatiya S. Comparison of Gen-Probe commercial kit and culture technique for the diagnosis of Mycoplasma pneumoniae infection. J Clin Microbiol 1988;26:1068-9
  21. Eun BW, Kim NH, Choi EH, Lee HJ. Mycoplasma pneumoniae in Korean children: the epidemiology of pneumonia over an 18-year period. J Infect 2008;56:326-31. https://doi.org/10.1016/j.jinf.2008.02.018

Cited by

  1. Clinical and Epidemiologic Characteristics ofMycoplasma pneumoniaePneumonia in Adults During 2011 Epidemic vol.44, pp.5, 2010, https://doi.org/10.3947/ic.2012.44.5.367
  2. Mycoplasma pneumoniaePneumonia Unresponsive to Macrolide Treatment vol.2, pp.4, 2010, https://doi.org/10.3343/lmo.2012.2.4.226
  3. Literature review and future strategies of childhood respiratory diseases in Korea vol.6, pp.suppl1, 2018, https://doi.org/10.4168/aard.2018.6.s1.s66
  4. Clinical analysis of risk factors in refractory mycoplasma pneumonia in children vol.6, pp.1, 2018, https://doi.org/10.4168/aard.2018.6.1.62