Korean Journal of Clinical Laboratory Science (대한임상검사과학회지)

Korean Society for Clinical Laboratory Science (대한임상검사과학회)
권호 표지
DOI QR코드

DOI QR Code

Detection of the Carbapenem Resistance Gene in Gram-negative Rod Bacteria Isolated from Clinical Specimens

임상검체에서 분리된 그람음성막대균으로부터 카바페넴 내성 유전자 검출

  • Received : 2022.07.13
  • Accepted : 2022.08.30
  • Published : 2022.09.30
Download PDF
⟨ Previous Next ⟩

Abstract

Carbapenem-resistant Enterobacteriaceae (CRE) poses an increasing public health threat and has limited treatment options with high associated mortality. Genotypes of carbapenemase that threaten public health (blaKPC, blaNDM, blaIMP, and blaVIM) and blaOXA-48-like genes were detected by phenotypic and molecular diagnosis, and related gene distribution patterns were investigated. Phenotypic testing using the modified Hodge test confirmed positivity in all 41 strains examined, and carbapenemase inhibitory testing using meropenem+phenyl boronic acid or meropenem+EDTA confirmed positivity in 18 and 8 strains, respectively. Polymerase chain reaction revealed the presence of amplification products in 28 strains of blaKPC, 25 strains of blaNDM, 5 strains of blaIMP, 1 strain of blaVIM, and 13 blaOXA-48-like strains. In addition, 7 strains of blaKPC+blaNDM, 1 strain of blaKPC+blaIMP, 1 strain of blaNDM+blaOXA-48-like, 1 strain of blaNDM+blaVIM, 4 strains of blaKPC+blaNDM+blaIMP, and 4 strains of blaKPC+blaNDM+blaOXA-48-like were identified. Melting curve analysis using real-time PCR was wholly consistent with PCR results. The study shows genetic identification of highly specific CRE by real-time PCR could be used to provide early diagnoses and infection control, improve surveillance, and prevent the transmission of CRE.

본 연구는 공중보건에 위협이 되고 있는 carbapenemase 유전자형 중 blaKPC, blaNDM, blaIMP, blaVIM, blaOXA-48-like 유전자의 표현형적 검사 및 분자진단으로 검출하고 관련 유전자 분포 양상에 대해 알아보았다. 표현형적 검사결과 MHT는 41균주 모두에서 양성을 확인하고, CIT는 meropenem+PBA는 18균주 및 meropenem+EDTA에서 8균주의 양성을 확인하였다. PCR 결과 blaKPC 28균주, blaNDM 25균주, blaIMP 5균주, blaVIM 1균주, blaOXA-48-like 13균주에서 증폭 산물을 확인하였다. 또한 blaKPC+blaNDM 7균주, blaKPC+blaIMP 1균주, blaNDM+blaOXA-48-like 1균주, blaNDM+blaVIM 1균주, blaKPC+blaNDM+blaIMP 4균주, blaKPC+blaNDM+blaOXA-48-like 4균주를 확인하였다. 실시간 중합효소 연쇄반응을 이용한 융해 곡선 분석결과는 PCR 결과와 100% 일치함을 확인하였다. 결론적으로 real-time PCR을 이용한 신속하고 특이성이 높은 CRE 조기진단을 통한 유전자 확인은 제한된 치료 옵션과 높은 사망률로 공중 보건 위협을 고조하는 CRE의 감시, 진단 및 치료를 개선할 수 있으며 전염을 방지하기 위한 효과적인 항균 치료 및 시기적절한 감염 통제가 가능할 것으로 사료된다.

Keywords

Acknowledgement

This paper was supported by the Jinju Health College in 2021. The pathogen resources for this study were provided by Gyeongsang National University Hospital Branch of the National Culture Collection for Pathogens (GNUH-NCCP).

References

  1. Feil EJ. Enterobacteriaceae: joining the dots with pan-European epidemiology. Lancet Infect Dis. 2016;17:118-119. https://doi.org/10.1016/S1473-3099(16)30333-4
  2. Weiner LM, Webb AK, Limbago B, Dudeck MA, Patel J, Kallen AJ, et al. Antimicrobial-resistant pathogens associated with healthcare-associated infections: summary of data reported to the national healthcare safety network at the centers for disease control and prevention, 2011-2014. Infect Control Hosp Epidemiol. 2016;37:1288-1301. https://doi.org/10.1017/ice.2016.174
  3. van Duin D, Doi Y. The global epidemiology of carbapenemase-producing Enterobacteriaceae. Virulence. 2016;8:460-469. https://doi.org/10.1080/21505594.2016.1222343
  4. Han R, Shi Q, Wu S, Yin D, Peng M, Dong D, et. al. Dissemination of carbapenemases (KPC, NDM, OXA-48, IMP, and VIM) among carbapenem-resistant Enterobacteriaceae isolated from adult and children patients in China. Front Cell Infect Microbiol. 2020;10:314. https://doi.org/10.3389/fcimb.2020.00314
  5. World Health Organization. New report calls for urgent action to avert antimicrobial resistance crisis. [Internet]. New York: World Health Organization; 2019[cited 2022 March 22]. Available from: https://www.who.int/news/item/29-04-2019-new-report-calls-for-urgent-action-to-avert-antimicrobial-resistance-crisis.
  6. Li J, Bi W, Dong G, Zhang Y, Wu Q, Dong T, et al. The new perspective of old antibiotic: in vitro antibacterial activity of TMP-SMZ against Klebsiella pneumoniae. J Microbiol Immunol Infect. 2020;53:757-765. https://doi.org/10.1016/j.jmii.2018.12.013
  7. Chen HY, Jean SS, Lee YL, Lu MC, Ko WC, Liu PY, et al. Carbapenem-resistant Enterobacterales in long-term care facilities: a global and narrative review. Front Cell Infect Microbiol. 2021;11:601968. https://doi.org/10.3389/fcimb.2021.601968
  8. Wang CH, Ma L, Huang LY, Yeh KM, Lin JC, Siu LK, et al. Molecular epidemiology and resistance patterns of bla OXA-48 Klebsiella pneumoniae and Escherichia coli: a nationwide multicenter study in Taiwan. J Microbiol Immunol Infect. 2021;54: 665-672. https://doi.org/10.1016/j.jmii.2020.04.006
  9. Zhang H, Jia P, Zhu Y, Zhang G, Zhang J, Kang W, et al. Susceptibility to imipenem/relebactam of Pseudomonas aeruginosa and Acinetobacter baumannii isolates from Chinese intra-abdominal, respiratory and urinary tract infections: SMART 2015 to 2018. Infect. Drug Resist. 2021;14:3509-3518. https://doi.org/10.2147/IDR.S325520
  10. Jean SS, Harnod D, Hsueh PR. Global threat of carbapenem resistant gram-negative bacteria. Front Cell Infect Microbiol. 2022;12:823684. https://doi.org/10.3389/fcimb.2022.823684
  11. Aurilio C, Sansone P, Barbarisi M, Pota V, Giaccari LG, Coppolino F, et al. Mechanisms of action of carbapenem resistance. Antibiotics. 2022;11:421. https://doi.org/10.3390/antibiotics11030421
  12. Diene SM, Rolain JM. Carbapenemase genes and genetic platforms in Gram-negative bacilli: Enterobacteriaceae, Pseudomonas and Acinetobacter species. Clin Microbiol Infect. 2014;20:831-838. https://doi.org/10.1111/1469-0691.12655
  13. Potron A, Poirel L, Nordmann P. Emerging broad-spectrum resistance to Pseudomonas aeruginosa and Acinetobacter baumannii: mechanisms and epidemiology. Int J Antimicrob Agents. 2015;45:568-585. https://doi.org/10.1016/j.ijantimicag.2015.03.001
  14. Yigit H, Queenan AM, Anderson GJ, Domenech-Sanchez A, Biddle JW, Steward CD, et al. Novel carbapenem-hydrolyzing β-Lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae. Antimicrob Agents Chemother. 2001;45: 1151-1161. https://doi.org/10.1128/AAC.45.4.1151-1161.2001
  15. Deshpande LM, Jones RN, Fritsche TR, Sader HS. Occurrence and characterization of carbapenemase-producing Enterobacteriaceae: Report from the SENTRY antimicrobial surveillance program (2000-2004). Microb Drug Resist. 2006;12:223-230. https://doi.org/10.1089/mdr.2006.12.223
  16. Qi Y, Wei Z, Ji S, Du X, Shen P, Yu Y. ST11, the dominant clone of KPC-producing Klebsiella pneumoniae in China. J Antimicrob Chemother. 2011;66:307-312. https://doi.org/10.1093/jac/dkq431
  17. Leavitt A, Chmelnitsky I, Carmeli Y, Navon-Venezia S. Complete nucleotide sequence of KPC-3-encoding plasmid pKpQIL in the epidemic Klebsiella pneumoniae sequence type 258. Antimicrob Agents Chemother. 2010;54:4493-4496. https://doi.org/10.1128/AAC.00175-10
  18. Walther-Rasmussen, J, Hoiby N. Class A carbapenemases. J Antimicrob Chemother. 2007;60:470-482. https://doi.org/10.1093/jac/dkm226
  19. Frere JM, Galleni M, Bush K, Dideberg O. Is it necessary to change the classification of beta-lactamases? J Antimicrob Chemother. 2005;55:1051-1053. https://doi.org/10.1093/jac/dki155
  20. Yoon EJ, Jeong SH. Mobile carbapenemase genes in Pseudomonas aeruginosa. Front Microbiol. 2021;12:614058. https://doi.org/10.3389/fmicb.2021.614058
  21. Thyrum PT, Yeh C, Birmingham B, Lasseter K. Pharmacokinetics of meropenem in patients with liver disease. Clin. Infect. Dis. 1997; 24:184-190. https://doi.org/10.1093/clinids/24.supplement_2.s184
  22. Queenan AM, Bush K. Carbapenemases: the versatile be- ta-lactamases. Clin Microbiol Rev. 2007;20:440-458. https://doi.org/10.1128/CMR.00001-07
  23. Moquet O, Bouchiat C, Kinana A, Seck A, Arouna O, Bercion R, et al. Class D OXA-48 carbapenemasein multidrug-resistant enterobacteria, Senegal. Emerg Infect Dis. 2011;17:143-144. https://doi.org/10.3201/eid1701.100244
  24. Poirel L, Potron A, Nordmann P. OXA-48-like carbapenemases: the phantom menace. J Antimicrob Chemother. 2012;67:1597-606. https://doi.org/10.1093/jac/dks121
  25. Banerjee R, Humphries R. Clinical and laboratory considerations for the rapid detection of carbapenem-resistant Enterobacteriaceae. Virulence. 2017;8:427-439. https://doi.org/10.1080/21505594.2016.1185577
  26. Sheu CC, Chang YT, Lin SY, Chen YH, Hsueh PR. Infections caused by carbapenem-resistant Enterobacteriaceae: an update on therapeutic options. Front Microbiol. 2019;10:80. https://doi.org/10.3389/fmicb.2019.00080
  27. Lin MY, Lyles-Banks RD, Lolans K, Hines DW, Spear JB, Petrak R, et al. The importance of long-term acute care hospitals in the regional epidemiology of Klebsiella pneumoniae carbapenemase-producing Enterobacteriaceae. Clin Infect Dis. 2013;57: 1246-52. https://doi.org/10.1093/cid/cit500
  28. Clinical Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; twenty-first informational supplement M100-S27. Wayne PA: Clinical Laboratory Standards Institute; 2017.
  29. Lee KW, Kim CK, Yong DE, Jeong SH, Yum JH, Seo YH, et al. Improved performance of the modified Hodge test with MacConkey agar for screening carbapenemase-producing Gram-negative bacilli. J Microbiol Methods. 2010;83:149-152. https://doi.org/10.1016/j.mimet.2010.08.010
  30. The Korean Society of Clinical Microbiology. Diagnostic instruction carbapenemase producing Enterobacteriaceae (CPE) [Internet]. Seoul: The Korean Society of Clinical Microbiology; 2015 [cited 2022 May 19]. Available from: http://kscm.or.kr/xe/kscmnotice/71241.
  31. Monteiro J, Widen RH, Pignatari ACC, Kubasek C, Silbert S. Rapid detection of carbapenemase genes by multiplex real-time PCR. J Antimicrob Chemother. 2012;67:906-909. https://doi.org/10.1093/jac/dkr563
  32. Poirel L, Revathi G, Bernabeu S, Nordmann P. Detection of NDM-1-Producing Klebsiella pneumoniae in Kenya. Antimicrob Agents Chemother. 2011;55:934-936. https://doi.org/10.1128/AAC.01247-10
  33. Doyle D, Peirano G, Lascols C, Lloyd T, Church DL, Pitout JD. Laboratory detection of Enterobacteriaceae that produce carbapenemases. J Clin Microbiol. 2012;50:3877-3880. https://doi.org/10.1128/JCM.02117-12
  34. Wang L, Gu H, Lu X. A rapid low-cost real-time PCR for the de-tection of Klebsiella pneumonia carbapenemase genes. Ann Clin Microbiol Antimicrob. 2012;11:9. https://doi.org/10.1186/1476-0711-11-9
  35. Kosykowska E, Dzieciatkowski T, Mlynarczyk G. Rapid detection of NDM, VIM, KPC and IMP carbapenemases by real-time PCR. J Bacteriol Parasitol. 2016;7:6. https://doi.org/10.4172/2155-9597.1000299
  36. Goudarzi H, Mirsamadi ES, Ghalavand Z, Vala MH, Mirjalali H, Hashemi A. Rapid detection and molecular survey of blaVIM, blaIMP and blaNDM genes among clinical isolates of Acinetobacter baumannii using new multiplex real-time PCR and melting curve analysis. BMC Microbiol. 2019;19:122. https://doi.org/10.1186/s12866-019-1510-y
  37. Bordin A, Trembizki E, Windsor M, Wee R, Tan LY, Buckley C, et al. Evaluation of the SpeeDx Carba (beta) multiplex real-time PCR assay for detection of NDM, KPC, OXA-48-like, IMP-4-like and VIM carbapenemase genes. BMC Infect Dis. 2019;19:571. https://doi.org/10.1186/s12879-019-4176-z
  38. Mutters NT, Tacconelli E. Infection prevention and control in Europe-the picture in the mosaic. Clin Microbiol Infect. 2015;21:1045-1046. https://doi.org/10.1016/j.cmi.2015.06.012
  39. Talapan D, Rafila A. Five-year survey of asymptomatic colonization with multidrug-resistant organisms in a Romanian tertiary care hospital. Infect Drug Resist. 2022;15:2959-2967. https://doi.org/10.2147/IDR.S360048
  40. Choi IH, Lee YS. Active surveillance for carbapenem-resistant Enterobacteriaceae at a single center for four years. Ann Lab Med. 2022;42:367-369. https://doi.org/10.3343/alm.2022.42.3.367
  41. Tamma PD, Simner PJ. Phenotypic detection of carbapenemase producing organisms from clinical isolates. J Clin Microbiol. 2018;56:e01140-18. https://doi.org/10.1128/JCM.01140-18
  42. Yang BS, Park JA. Detection of bla KPC and bla NDM genes from gram-negative rod bacteria isolated from a general hospital in Gyeongnam. Korean J Clin Lab Sci. 2021;53:49-59. https://doi.org/10.15324/kjcls.2021.53.1.49
  43. Tawfick MM, Alshareef WA, Bendary HA, Elmahalawy H, Abdulall AK. The emergence of carbapenemase blaNDM genotype among carbapenem-resistant Enterobacteriaceae isolates from Egyptian cancer patients. Eur J Clin Microbiol Infect Dis. 2020;39:1251-1259. https://doi.org/10.1007/s10096-020-03839-2
  44. El Solh AA, Alhajhusain A. Update on the treatment of Pseudomonas aeruginosa pneumonia. J Antimicrob Chemother. 2009;64:229-238. https://doi.org/10.1093/jac/dkp201
  45. Goodman KE, Simner PJ, Tamma PD, Milstone AM. Infection control implications of heterogeneous resistance mechanisms in carbapenem resistant Enterobacteriaceae (CRE). Expert Rev Anti Infect Ther. 2016;14:95-108. https://doi.org/10.1007/10.1586/14787210.2016.1106940
  46. Mangold KA, Santiano K, Broekman R, Krafft CA, Voss B, Wang V, et al. Real-time detection of blaKPC in clinical samples and surveillance specimens. J Clin Microbiol. 2011;49:3338-3339. https://doi.org/10.1128/JCM.00268-11