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http://dx.doi.org/10.15324/kjcls.2021.53.1.49

Detection of blaKPC and blaNDM Genes from Gram-Negative Rod Bacteria Isolated from a General Hospital in Gyeongnam  

Yang, Byoung Seon (Department of Medical Laboratory Science, Jinju Health College)
Park, Ji Ae (Department of Medical Laboratory Science, Jinju Health College)
Publication Information
Korean Journal of Clinical Laboratory Science / v.53, no.1, 2021 , pp. 49-59 More about this Journal
Abstract
This study investigated the use of real-time PCR melting curves for the diagnosis of blaKPC and blaNDM genes among the most frequently detected carbapenemase-producing Enterobacteriaceae in Korea. As a means of addressing the shortcomings of phenotype tests and conventional PCR. The modified Hodge test confirmed positivity in 25 of 35 strains, and carbapenemase inhibition testing confirmed positivity in 14 strains by meropenem+PBA or meropenem+EDTA. PCR analysis showed amplification products in 25 strains of Klebsiella pneumoniae carbapenemases (KPC), 10 of K. pneumoniae, 5 of E. coli, 5 of A. baumannii, 4 of P. aeruginosa, and 1 of P. putida. New Delhi metallo β-lactamase (NDM) identified amplification products in 8 strains, that is, 2 K. pneumoniae, 3 E. coli, 1 P. aeruginosa, 1 E. cloacae, and 1 P. retgeri strains. Real-time PCR melting curve analysis confirmed amplification in 25 strains of KPC and 8 strains of NDM, and these results were 100% consistent with PCR results. In conclusion, our findings suggest early diagnosis of carbapenem resistant Enterobacteriaceae by real-time PCR offers a potential means of antibacterial management that can prevent and control nosocomial infection spread.
Keywords
$bla_{KPC}$; $bla_{NDM}$; MHT; CIT; Real-time PCR;
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1 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   DOI
2 Papp-Wallace KM, Endimiani A, Taracila MA, Bonomo RA. Carbapenems: past, present, and future. Antimicrobial Agents and Chemotherapy. 2011;55:4943-4960. https://doi.org//AAC.00296-11   DOI
3 Lodise TP, Bonine NG, Ye JM, Folse HJ, Gillard P. Development of a bedside tool to predict the probability of drug-resistant pathogens among hospitalized adult patients with gram-negative infections. BMC Infectious Diseases. 2019;19:718. https://doi.org/10.1186/s12879-019-4363-y   DOI
4 Jeong SH, Kim HS, Kim JS, Shin DH, Kim HS, Park MJ, et al. Prevalence and molecular characteristics of carbapenemase producing Enterobacteriaceae from five hospitals in Korea. Ann. Lab. Med. 2016;36:529-535. https://doi.org/10.3343/alm.2016.36.6.529   DOI
5 Diene SM, Rolain JM. Carbapenemase genes and genetic platforms in gram-negative bacilli: Enterobacteriaceae, Pseudomonas, and Acinetobacter species. Clinical Microbiology and Infection. 2014;20:831-838. https://doi.org/10.1111/1469-0691.12655   DOI
6 Rhee JY, Park YK, Shin JY, Choi JY, Lee MY, Peck KR, et al. KPC producing extreme drug-resistant Klebsiella pneumoniae isolate from a patient with diabetes mellitus and chronic renal failure on hemodialysis in South Korea. Antimicrob Agents Chemother. 2010;54:2278-2279. https://doi.org/10.1128/AAC.00011-10   DOI
7 Kim JO, Song SA, Yoon EJ, Shin JH, Lee H, Jeong SH, et al. Outbreak of KPC-2-producing Enterobacteriaceae caused by clonal dissemination of Klebsiella pneumoniae ST307 carrying an IncX3-type plasmid harboring a truncated Tn4401a. Diagn. ZMicrobiol. Infect. Dis. 2017;87:343-348. https://doi.org/j.diagmicrobio.2016.12.012   DOI
8 Park SH, Kim JS, Kim HS, Yu YK, Han SH, Kang MJ, et. al. Prevalence of carbapenem-resistant Enterobacteriaceae in Seoul. J Bacteriol Virol. 2020;50:107-116. https://doi.org/10.4167/jbv.2020.50.2.107   DOI
9 Tacconelli E, Carrara E, Savoldi A, Harbarth S, Mendelson M, Monnet DL, et al. Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect Dis. 2018;18:318-327. https://doi.org/10.1016/S1473-3099(17)30753-3   DOI
10 Ahn SY, Sung JY, Kim HS, Kim MS, Hwang YJ, Jong SR, et al. Molecular epidemiology and characterization of carbapenemase-producing Enterobacteriaceae isolated at a university hospital in Korea during 4-year period. Ann Clin Microbiol. 2016;19:39-47. https://doi.org/10.5145/ACM.2016.19.2.39   DOI
11 Lodise TP, Zhao Q, Fahrbach K, Gillard PJ, Martin A. A systematic review of the association between delayed appropriate therapy and mortality among patients hospitalized with infections due to Klebsiella pneumoniae or Escherichia coli: how long is too long? BMC Infect Dis. 2018;18:625. https://doi.org/10.1186/s12879-018-3524-8   DOI
12 Seah C, Low DE, Patel SN, Melano RG. Comparative evaluation of a chromogenic agar medium, the modified Hodge test, and a battery of meropenem-inhibitor discs for detection of carbapenemase activity in Enterobacteriaceae. J Clin Microbiol. 2011;49:1965-1969. https://doi.org/10.1128/JCM.00203-11   DOI
13 Park JW, Lee EJ, et al. Status of carbapenemase producing Enterobacteriaceae incidences in Korea, 2015-2016. Research report. Cheongju: Korea Centers for Disease Control and Prevention; 2017;10:1243-1247.
14 Wang P, Chen S, Guo Y, Xiong Z, Hu F, Zhu D, et al. Occurrence of false positive results for the detection of carbapenemases in carbapenemasenegative Escherichia coli and Klebsiella pneumoniae isolates. PLoS One. 2011;6:e26356. https://doi.org/10.1371/journal.pone.0026356   DOI
15 Carvalhaes CG, Picao RC, Nicoletti AG, Xavier DE, Gales AC. Cloverleaf test (modified Hodge test) for detecting carbapenemase production in Klebsiella pneumoniae: be aware of false positive results. J Antimicrob Chemother. 2010;65:249-251. https://doi.org/10.1093/jac/dkp431   DOI
16 Nordmann P, Gniadkowski M, Giske CG, Poirel L, Woodford N, Miriagou V. Identification and screening of carbapenemase-producing Enterobacteriaceae. Clin Microbiol Infect. 2012;18:432-438. https://doi.org/10.1111/j.1469-0691.2012.03815.x   DOI
17 Pasteran F, Mendez T, Guerriero L, Rapoport M, Corso A. Sensitive screening tests for suspected class A carbapenemase production in species of Enterobacteriaceae. J Clin Microbiol. 2009;47:1631-1639. https://doi.org/10.1128/JCM.00130-09   DOI
18 Clinical Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; twenty-first informational supplement M100-S27. Wayne PA: Clinical Laboratory Standards Institute; 2017.
19 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   DOI
20 Bedenic B, Plecko V, Sardelic S, Uzunovic S, Godic Torkar K. Carbapenemases in gram-negative bacteria: laboratory detection and clinical significance. BioMed Research International. 2014;841951. https://doi.org/10.1155/2014/841951   DOI
21 Bora A, Sanjana R, Jha BK, Mahaseth SN, Pokharel K. Incidence of metallo-beta-lactamase producing clinical isolates of Escherichia coli and Klebsiella pneumoniae in central Nepal. BMC Res Notes 2014;7:557. https://doi.org/10.1186/1756-0500-7-557   DOI
22 The Korean Society of Clinical Microbiology. Diagnostic instruction carbapenemase producing Enterobacteriaceae (CPE) [Internet]. Seoul: The Korean Society of Clinical Microbiology; 2015 [cited 2021 February 9]. Available from: http://kscm.or.kr/xe/kscmnotice/71241.
23 Cha CH, Hae Kyong An HK, Kim JU. Detection of vancomycin-resistant Enterococci using multiplex real-time PCR assay and melting curve analysis. Korean J Lab Med. 2010;30:138-146. https://doi.org/10.3343/kjlm.2010.30.2.138   DOI
24 Ririe KM, Rasmussen RP, Wittwer CT. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem. 1997;245:154-60. https://doi.org/10.1006/abio.1996.9916   DOI
25 Zheng F, Sun J, Cheng C, Rui Y. The establishment of a duplex real-time PCR assay for rapid and simultaneous detection of blaNDM and blaKPC genes in bacteria. Ann Clin Microbiol Antimicrob. 2013;12:30. https://doi.org/10.1186/1476-0711-12-30   DOI
26 Lodise TP, Bonine NG, Ye JM, Folse HJ, Gillard P. Development of a bedside tool to predict the probability of drug-resistant pathogens among hospitalized adult patients with gram-negative infections. BMC Infectious Diseases. 2019;19:718. https://doi.org/10.1186/s12879-019-4363-y   DOI
27 Bonine NG, Berger A, Altincatal A, Wang R, Bhagnani T, Gillard P, et al. Impact of delayed appropriate antibiotic therapy on patient outcomes by antibiotic resistance status from serious gram-negative bacterial infections. Am J Med Sci. 2019;357:103-110. https://doi.org/10.1016/j.amjms.2018.11.009   DOI
28 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   DOI
29 Chu YW, Cheung TK, Ngan JY, Kam KM. EDTA susceptibility leading to false detection of metallo-beta-lactamase in Pseudomonas aeruginosa by Etest and an imipenem-EDTA disk method. Int J Antimicrob Agents 2005;26:340-341. https://doi.org/10.1016/j.ijantimicag.2005.07.004   DOI
30 Samuelsen O, Buaro L, Giske CG, Simonsen GS, Aasnaes B, Sundsfjord A. Evaluation of phenotypic tests for the detection of metallo-beta-lactamase-producing Pseudomonas aeruginosa in a low prevalence country. J Antimicrob Chemother. 2008;61:827-830. https://doi.org/10.1093/jac/dkn016   DOI
31 Ratkai C, Quinteira S, Grosso F, Monteiro N, Nagy E, Peixe L. Controlling for false positives: interpreting MBL Etest and MBL combined disc test for the detection of metallo-beta-lactamases. J Antimicrob Chemother 2009;64:657-658. https://doi.org/10.1093/jac/dkp229   DOI
32 Bonnin RA, Naas T, Poirel L, Nordmann P. Phenotypic, biochemical, and molecular techniques for detection of metallo-β-lactamase NDM in Acinetobacter baumannii. J Clin Microbiol. 2012;50:1419-1421. https://doi.org/10.1128/JCM.06276-11   DOI
33 Hansen F, Hammerum AM, Skov R, Haldorsen B, Sundsfjord A, Samuelsen O. Evaluation of the total MBL confirm kit (ROSCO) for detection of metallo-β-lactamases in Pseudomonas aeruginosa and Acinetobacter baumannii. Diagn Microbiol Infect Dis. 2014;79:486-488. https://doi.org/10.1016/j.diagmicrobio.2013.12.001   DOI
34 Goudarzi H, Mirsamadi ES, Ghalavand Z, Hakemi Vala M, 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 Microbiology. 2019;19:122. https://doi.org/10.1186/s12866-019-1510-y   DOI
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   DOI
36 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   DOI
37 Wang L, Gu H, Lu X. A rapid low-cost real-time PCR for the detection of Klebsiella pneumonia carbapenemase genes. Ann Clin Microbiol Antimicrob. 2012;11:9. https://doi.org/10.1186/1476-0711-11-9   DOI