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http://dx.doi.org/10.4014/jmb.2103.03044

Duplex dPCR System for Rapid Identification of Gram-Negative Pathogens in the Blood of Patients with Bloodstream Infection: A Culture-Independent Approach  

Shin, Juyoun (Department of Microbiology, The Catholic University of Korea, College of Medicine)
Shin, Sun (Department of Microbiology, The Catholic University of Korea, College of Medicine)
Jung, Seung-Hyun (Department of Biochemistry, The Catholic University of Korea, College of Medicine)
Park, Chulmin (Vaccine Bio Research Institute, The Catholic University of Korea, College of Medicine, Seoul St. Mary's Hospital)
Cho, Sung-Yeon (Vaccine Bio Research Institute, The Catholic University of Korea, College of Medicine, Seoul St. Mary's Hospital)
Lee, Dong-Gun (Vaccine Bio Research Institute, The Catholic University of Korea, College of Medicine, Seoul St. Mary's Hospital)
Chung, Yeun-Jun (Department of Microbiology, The Catholic University of Korea, College of Medicine)
Publication Information
Journal of Microbiology and Biotechnology / v.31, no.11, 2021 , pp. 1481-1489 More about this Journal
Abstract
Early and accurate detection of pathogens is important to improve clinical outcomes of bloodstream infections (BSI), especially in the case of drug-resistant pathogens. In this study, we aimed to develop a culture-independent digital PCR (dPCR) system for multiplex detection of major sepsis-causing gram-negative pathogens and antimicrobial resistance genes using plasma DNA from BSI patients. Our duplex dPCR system successfully detected nine targets (five bacteria-specific targets and four antimicrobial resistance genes) through five reactions within 3 hours. The minimum detection limit was 50 ag of bacterial DNA, suggesting that 1 CFU/ml of bacteria in the blood can be detected. To validate the clinical applicability, cell-free DNA samples from febrile patients were tested with our system and confirmed high consistency with conventional blood culture. This system can support early identification of some drug-resistant gram-negative pathogens, which can help improving treatment outcomes of BSI.
Keywords
Digital PCR; bloodstream infection; Gram-negative; blood-stream infection; antimicrobial resistance;
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1 Horakova K, Mlejnkova H, Mlejnek P. 2008. Specific detection of Escherichia coli isolated from water samples using polymerase chain reaction targeting four genes: cytochrome bd complex, lactose permease, beta-D-glucuronidase, and beta-D-galactosidase. J. Appl. Microbiol. 105: 970-976.   DOI
2 Ling TK, Xiong J, Yu Y, Lee CC, Ye H, Hawkey PM. 2006. Multicenter antimicrobial susceptibility survey of gram-negative bacteria isolated from patients with community-acquired infections in the People's Republic of China. Antimicrob. Agents Chemother. 50: 374-378.   DOI
3 Purcell RV, Pearson J, Frizelle FA, Keenan JI. 2016. Comparison of standard, quantitative and digital PCR in the detection of enterotoxigenic bacteroides fragilis. Sci. Rep. 6: 34554.   DOI
4 Devonshire AS, O'Sullivan DM, Honeyborne I, Jones G, Karczmarczyk M, Pavsic J, et al. 2016. The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis. BMC Infect. Dis. 16: 366.   DOI
5 Wellinghausen N, Kochem AJ, Disque C, Muhl H, Gebert S, Winter J, et al. 2009. Diagnosis of bacteremia in whole-blood samples by use of a commercial universal 16S rRNA gene-based PCR and sequence analysis. J. Clin. Microbiol. 47: 2759-2765.   DOI
6 Lachmayr KL, Kerkhof LJ, Dirienzo AG, Cavanaugh CM, Ford TE. 2009. Quantifying nonspecific TEM beta-lactamase (blaTEM) genes in a wastewater stream. Appl. Environ. Microbiol. 75: 203-211.   DOI
7 Skvarc M, Stubljar D, Rogina P, Kaasch AJ. 2013. Non-culture-based methods to diagnose bloodstream infection: Does it work? Eur. J. Microbiol. Immunol. (Bp) 3: 97-104.   DOI
8 Dutour C, Bonnet R, Marchandin H, Boyer M, Chanal C, Sirot D, et al. 2002. CTX-M-1, CTX-M-3, and CTX-M-14 beta-lactamases from Enterobacteriaceae isolated in France. Antimicrob. Agents Chemother. 46: 534-537.   DOI
9 Chung B, Park C, Cho SY, Shin S, Yim SH, Jung GY, et al. 2016. Multiplex identification of drug-resistant Gram-positive pathogens using stuffer-free MLPA system. Electrophoresis 37: 3079-3083.   DOI
10 Chung B, Park C, Cho SY, Shin J, Shin S, Yim SH, et al. 2018. Multiplex identification of sepsis-causing Gram-negative pathogens from the plasma of infected blood. Electrophoresis 39: 645-652.   DOI
11 Leitner E, Kessler HH, Spindelboeck W, Hoenigl M, Putz-Bankuti C, Stadlbauer-Kollner V, et al. 2013. Comparison of two molecular assays with conventional blood culture for diagnosis of sepsis. J. Microbiol. Methods 92: 253-255.   DOI
12 Dong L, Wang S, Fu B, Wang J. 2018. Evaluation of droplet digital PCR and next generation sequencing for characterizing DNA reference material for KRAS mutation detection. Sci. Rep. 8: 9650.   DOI
13 Roberts CH, Last A, Molina-Gonzalez S, Cassama E, Butcher R, Nabicassa M, et al. 2013. Development and evaluation of a next-generation digital PCR diagnostic assay for ocular Chlamydia trachomatis infections. J. Clin. Microbiol. 51: 2195-2203.   DOI
14 Shin GW, Jung SH, Yim SH, Chung B, Yeol Jung G, Chung YJ. 2012. Stuffer-free multiplex ligation-dependent probe amplification based on conformation-sensitive capillary electrophoresis: a novel technology for robust multiplex determination of copy number variation. Electrophoresis 33: 3052-3061.   DOI
15 Lee A, Mirrett S, Reller LB, Weinstein MP. 2007. Detection of bloodstream infections in adults: how many blood cultures are needed? J. Clin. Microbiol. 45: 3546-3548.   DOI
16 Li Y, Yang X, Zhao W. 2017. Emerging microtechnologies and automated systems for rapid bacterial identification and antibiotic susceptibility testing. SLAS Technol. 22: 585-608.   DOI
17 System N. 1999. National Nosocomial Infections Surveillance (NNIS) system report, data summary from January 1990-May 1999, issued June 1999. A report from the NNIS system. Am. J. Infect. Control. 27: 520-532.   DOI
18 Talbot GH, Bradley J, Edwards JE, Jr., Gilbert D, Scheld M, Bartlett JG, et al. 2006. Bad bugs need drugs: an update on the development pipeline from the antimicrobial availability task force of the infectious diseases society of America. Clin. Infect. Dis. 42: 657-668.   DOI
19 Kollef MH. 2000. Inadequate antimicrobial treatment: an important determinant of outcome for hospitalized patients. Clin. Infect. Dis. 31 Suppl 4: S131-138.   DOI
20 Monteiro J, Widen RH, Pignatari AC, Kubasek C, Silbert S. 2012. Rapid detection of carbapenemase genes by multiplex real-time PCR. J. Antimicrob. Chemother. 67: 906-909.   DOI
21 Kwon JC, Kim SH, Choi JK, Cho SY, Park YJ, Park SH, et al. 2013. Epidemiology and clinical features of bloodstream infections in hematology wards: one year experience at the catholic blood and marrow transplantation center. Infect. Chemother. 45: 51-61.   DOI
22 Martin-Pena R, Dominguez-Herrera J, Pachon J, McConnell MJ. 2013. Rapid detection of antibiotic resistance in Acinetobacter baumannii using quantitative real-time PCR. J. Antimicrob. Chemother. 68: 1572-1575.   DOI
23 Mitchell G, Chen C, Portnoy DA. 2016. Strategies used by bacteria to grow in macrophages. Microbiol. Spectr. 4: 10.1128/microbiolspec.MCHD-0012-2015.   DOI
24 Kaufmann SHE, Dorhoi A. 2016. Molecular determinants in phagocyte-bacteria interactions. Immunity 44: 476-491.   DOI
25 Qin X, Emerson J, Stapp J, Stapp L, Abe P, Burns JL. 2003. Use of real-time PCR with multiple targets to identify Pseudomonas aeruginosa and other nonfermenting gram-negative bacilli from patients with cystic fibrosis. J. Clin. Microbiol. 41: 4312-4317.   DOI
26 Ricchi M, Bertasio C, Boniotti MB, Vicari N, Russo S, Tilola M, et al. 2017. Comparison among the quantification of bacterial pathogens by qPCR, dPCR, and cultural methods. Front. Microbiol. 8: 1174.   DOI
27 Cave L, Brothier E, Abrouk D, Bouda PS, Hien E, Nazaret S. 2016. Efficiency and sensitivity of the digital droplet PCR for the quantification of antibiotic resistance genes in soils and organic residues. Appl. Microbiol. Biotechnol. 100: 10597-10608.   DOI
28 Vincent JL, Brealey D, Libert N, Abidi NE, O'Dwyer M, Zacharowski K, et al. 2015. Rapid diagnosis of infection in the critically Ill, a multicenter study of molecular detection in bloodstream infections, pneumonia, and sterile site infections. Crit. Care Med. 43: 2283- 2291.   DOI
29 Pavlovic M, Luze A, Konrad R, Berger A, Sing A, Busch U, et al. 2011. Development of a duplex real-time PCR for differentiation between E. coli and Shigella spp. J. Appl. Microbiol. 110: 1245-1251.   DOI
30 McConnell MJ, Perez-Ordonez A, Perez-Romero P, Valencia R, Lepe JA, Vazquez-Barba I, et al. 2012. Quantitative real-time PCR for detection of Acinetobacter baumannii colonization in the hospital environment. J. Clin. Microbiol. 50: 1412-1414.   DOI
31 Kumar A. 2010. Early antimicrobial therapy in severe sepsis and septic shock. Curr. Infect. Dis. Rep. 12: 336-344.   DOI
32 Shorr AF, Micek ST, Welch EC, Doherty JA, Reichley RM, Kollef MH. 2011. Inappropriate antibiotic therapy in Gram-negative sepsis increases hosrufpital length of stay. Crit. Care Med. 39: 46-51.   DOI
33 Bassetti M, Righi E, Carnelutti A. 2016. Bloodstream infections in the Intensive Care Unit. Virulence 7: 267-279.   DOI
34 Kollef MH. 2001. Optimizing antibiotic therapy in the intensive care unit setting. Crit. Care. 5: 189-195.   DOI
35 Procop GW. 2007. Molecular diagnostics for the detection and characterization of microbial pathogens. Clin. Infect. Dis. 45 Suppl 2: S99-S111.   DOI
36 Opota O, Jaton K, Greub G. 2015. Microbial diagnosis of bloodstream infection: towards molecular diagnosis directly from blood. Clin. Microbiol. Infect. 21: 323-331.   DOI
37 El-Badawy MF, Tawakol WM, El-Far SW, Maghrabi IA, Al-Ghamdi SA, Mansy MS, et al. 2017. Molecular identification of aminoglycoside-modifying enzymes and plasmid-mediated quinolone resistance genes among Klebsiella pneumoniae clinical isolates recovered from Egyptian patients. Int. J. Microbiol. 2017: 8050432.
38 Lavenir R, Jocktane D, Laurent F, Nazaret S, Cournoyer B. 2007. Improved reliability of Pseudomonas aeruginosa PCR detection by the use of the species-specific ecfX gene target. J. Microbiol. Methods 70: 20-29.   DOI
39 Naas T, Ergani A, Carrer A, Nordmann P. 2011. Real-time PCR for detection of NDM-1 carbapenemase genes from spiked stool samples. Antimicrob. Agents Chemother. 55: 4038-4043.   DOI
40 Hansen WL, Beuving J, Bruggeman CA, Wolffs PF. 2010. Molecular probes for diagnosis of clinically relevant bacterial infections in blood cultures. J. Clin. Microbiol. 48: 4432-4438.   DOI
41 Itokazu GS, Quinn JP, Bell-Dixon C, Kahan FM, Weinstein RA. 1996. Antimicrobial resistance rates among aerobic gram-negative bacilli recovered from patients in intensive care units: evaluation of a national postmarketing surveillance program. Clin. Infect. Dis. 23: 779-784.   DOI