An improved method for rapid detection and characterization of carbapenemase-producing Enterobacterales directly from...

In this study, a new phenotypic method "dBLIMplus" performed by using combination disks for both detection of carbapenemase activity and determination of the carbapenemase enzyme of carbapenemase-producing Enterobacterales strains from positive blood culture bottles was introduced and evaluated relative to the carbapenemase enzyme determined by the BD MAX Check-Points CPO test. Sixty strains (bla_KPC [n = 15], bla_NDM [n = 13], bla_VIM/IMP [n = 2], bla_OXA-48 [n = 15], and non-carbapenemase-producing Enterobacterales [n = 15]) were used for validation of dBLIMplus, and zone diameter interpretation criteria were determined. dBLIMplus was applied to 100 Enterobacterales clinical isolates to evaluate its applicability in routine laboratory practices. The bacterial suspension to be tested was obtained by stepwise centrifugation applied to the blood culture fluid. Two hundred microliters of the suspension was added to D73C-MASTDISCS Combi Carba Plus disks. Simultaneously, 200 microliters of Escherichia coli ATCC 25922 reference strain adjusted to 1.0 McFarland was transferred to Mueller-Hinton agar, and all were incubated at 35°C ± 2°C for 2 h. After incubation, disks were placed on preincubated Mueller-Hinton agar and then incubated at 35°C ± 2°C. Inhibition zone diameters were measured at 6, 12, and 18 h and interpreted according to the evaluation criteria determined by validation. The sensitivity and specificity of dBLIMplus for detecting carbapenemase-producing strains and for identifying the individual carbapenemase enzymes were as follows: 100% and 100% for carbapenemase producers; 77.8% and 100% for bla_KPC; 22.6% and 100% for metallo-beta-lactamases; and 79.2% and 98.1% for bla_OXA-48. In conclusion, although the sensitivity of dBLIMplus is relatively low for isolates harboring more than one carbapenemase enzyme, it may still serve as a useful phenotypic tool in laboratories where PCR is not available thanks to its 100% sensitivity in detecting carbapenemase activity, nearly 80% sensitivity, and close to 100% specificity in identifying bla_KPC and bla_OXA-48.IMPORTANCEThe significance of this study lies in the development and validation of a novel phenotypic method, dBLIMplus, for the rapid detection of carbapenemase-producing Enterobacterales directly from positive blood culture bottles. Carbapenemase-producing Enterobacterales are associated with high morbidity and mortality rates. Rapid and accurate identification is critical for timely infection control and effective treatment decisions. Given its ability to detect carbapenemase producers directly from positive blood culture bottles using only combination disks, dBLIMplus is presented as a viable alternative to molecular techniques, which are generally more costly. With 100% sensitivity and specificity in detecting carbapenemase-producing Enterobacterales, dBLIMplus demonstrates strong potential for routine clinical use. Its ability to deliver accurate results as early as 6 h makes it a valuable tool for microbiology laboratories, especially in resource-limited settings.