Multiplex identification of sepsis‐causing Gram‐negative pathogens from the plasma of infected blood

Abstract

Early and accurate detection of bacterial pathogens in the blood is the most crucial step for sepsis management. Gram-negative bacteria are the most common organisms causing severe sepsis and responsible for high morbidity and mortality. We aimed to develop a method for rapid multiplex identification of clinically important Gram-negative pathogens and also validated whether our system can identify Gram-negative pathogens with the cell-free plasm DNA from infected blood. We designed five MLPA probe sets targeting the genes specific to major Gram-negative pathogens (uidA and lacY for E. coli, ompA for A. baumannii, phoE for K. pneumoniae, and ecfX for P. aeruginosa) and one set targeting the CTX-M group 1 to identify the ESBL producing Gram-negative pathogens. All six target-specific peaks were clearly separated without any non-specific peaks in a multiplex reaction condition. The minimum detection limit was 100 fg of pathogen DNA. When we tested 28 Gram-negative clinical isolates, all of them were successfully identified without any non-specific peaks. To evaluate the clinical applicability, we tested seven blood samples from febrile patients. Three blood culture positive cases showed E. coli specific peaks, while no peak was detected in the other four culture negative samples. This technology can be useful for detection of major sepsis-causing, drug-resistant Gram-negative pathogens and also the major ESBL producing Gram-negatives from the blood of sepsis patients in a clinical setting. This system can help early initiation of effective antimicrobial treatment against Gram-negative pathogens for sepsis patients, which is very crucial for better treatment outcomes.

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

“…2C and 2D ). E. coli isolate cm66, known to produce CTX-M group 9 ESBL and bla TEM enzymes [ 16 ], showed signal for bla TEM , but bla CTX-M group 1 signal was not detected ( Figs. 2 E and 2F ).…”

“…Five reference strains were obtained from the American Type Culture Collection (USA) and Korean Collection of Type Cultures (Korea). Thirteen clinical isolates were obtained from Seoul St Mary’s Hospital in South Korea [ 16 ].…”

“…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 electrophoresis (CE) based single-strand conformation polymorphism (SSCP) [14][15][16]. Although this MLPA-CE-SSCP based system could achieve fast, multiplex bacterial identification, like other PCR-based tools [17][18][19], there is still room for improvement in sensitivity of the system for early diagnosis of BSI.…”

“…Multiplex identification and gram discrimination of clinically important drug-resistant bacterial pathogens are also important methods to control BSI. We have previously developed a novel tool for multiplex detection of antimicrobial resistant bacterial targets in the blood of BSI patients within 8-9 h, by integrating the principles of multiplex ligation-dependent probe amplification (MLPA) and capillary electrophoresis (CE) based single-strand conformation polymorphism (SSCP) [ 14 - 16 ]. Although this MLPA-CE-SSCP based system could achieve fast, multiplex bacterial identification, like other PCR-based tools [ 17 - 19 ], there is still room for improvement in sensitivity of the system for early diagnosis of BSI.…”