Development of a Rapid Reverse Blot Hybridization Assay for Detection of Clinically Relevant Antibiotic Resistance Genes in Blood Cultures Testing Positive for Gram-Negative Bacteria

Abstract

Rapid and accurate identification of the causative pathogens of bloodstream infections is crucial for the prompt initiation of appropriate antimicrobial therapy to decrease the related morbidity and mortality rates. The aim of this study was to evaluate the performance of a newly developed PCR-reverse blot hybridization assay (REBA) for the rapid detection of Gram-negative bacteria (GNB) and their extended-spectrum β-lactamase (ESBL), AmpC β-lactamase, and carbapenemase resistance genes directly from the blood culture bottles. The REBA-EAC (ESBL, AmpC β-lactamase, carbapenemase) assay was performed on 327 isolates that were confirmed to have an ESBL producer phenotype, 200 positive blood culture (PBCs) specimens, and 200 negative blood culture specimens. The concordance rate between the results of REBA-EAC assay and ESBL phenotypic test was 94.2%. The sensitivity, specificity, positive predictive value, and negative predictive value of the REBA-EAC assay for GNB identification in blood culture specimens were 100% (95% CI 0.938-1.000, P < 0.001), 100% (95% CI 0.986-1.000, P < 0.001), 100% (95% CI 0.938-1.000, P < 0.001), and 100% (95% CI 0.986-1.000, P < 0.001), respectively. All 17 EAC-producing GNB isolates from the 73 PBCs were detected by the REBA-EAC assay. The REBA-EAC assay allowed easy differentiation between EAC and non-EAC genes in all isolates. Moreover, the REBA-EAC assay was a rapid and reliable method for identifying GNB and their β-lactamase resistance genes in PBCs. Thus, this assay may provide essential information for accelerating therapeutic decisions to achieve earlier appropriate antibiotic treatment during the acute phase of bloodstream infection.

Keywords: AmpC β-lactamase; REBA; blood culture; carbapenemase; extended-spectrum β-lactamase; qPCR.

Figure 1

Results of the REBA-EAC assay. (A) Results of the REBA-EAC assay for the detection of TEM/SHV-ESBLs: Lane 1, E. coli; 2, TEM-1; 3, TEM-73; 4, TEM-ESBL (Glu104Lys); 5, TEM-ESBL (Gly238Ser); 6-7, TEM-52 and TEM-66 (Glu104Lys and Gly238Ser mixed); 8, K. pneumoniae; 9, SHV-1; 10, SHV-ESBL (Asp179Ala); 11-12, SHV-12 and SHV-2a (Gly238Ser). (B) Specificity of the REBA-EAC assay as tested with DNA from 16 EAC-producing and 6 reference bacterial species: Lane 1, CTX-M-1-2; 2, CTX-M-9; 3, TEM; 4, SHV; 5, ACT; 6, CMY-2-like; 7, DHA; 8, ACC-1; 9, CMY-1-like/MOX; 10, FOX; 11, IMP; 12, VIM; 13, NDM; 14, KPC; 15, OXA-48-like; 16, SPM; 17, E. coli; 18, Enterobacter aerogenes; 19, K. pneumoniae; 20, Serratia marcescens; 21, P. aeruginosa; 22, A. baumannii; N, negative control.

Figure 2

Detection limits of the REBA-EAC assay for the 16 EAC gene probes. Limits were determined using 10-fold serial dilutions of DNA samples from EAC-producing strains. A, CTX-M-1; B, CTX-M-9; C, TEM; D, SHV; E, ACT; F, CMY-2-like; G, DHA; H, ACC-1; I, CMY-1-like/MOX; J, FOX; K, IMP; L, VIM; M, NDM; N, KPC; O, OXA-48-like; and P, SPM. Lane 1, 10 ng; Lane 2, 1 ng; Lane 3, 100 pg; Lane 4, 10 pg; Lane 5, 1 pg; Lane 6, 100 fg; Lane 7, 10 fg, Lane 8, negative control.

Figure 3

Representative results for the REBA-EAC assay performed with clinical isolates and positive blood culture samples. Lanes 1–7, 11–17, and 20–23, EAC-producing strains; lanes 8–10, 18, and 19, non-EAC-producing strains; N, negative control.