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




PPPP < 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: Microbiology
Keywords: extended-spectrum β-lactamase, AmpC β-lactamase, carbapenemase, REBA, qPCR, blood culture
Introduction

Antibiotic resistance among pathogenic organisms has become a major healthcare problem worldwide, with serious consequences for the treatment of infectious diseases (Gottlieb and Nimmo, 2011). Early targeted antibiotic treatment is an important prognostic factor, especially for seriously ill patients (Cosgrove, 2006). Among the GNB, the Enterobacteriaceae family includes the most widespread pathogens causing infectious diseases; its members are among the most common causes of nosocomial infection. Beta-lactamase production is the major resistance mechanism of the Enterobacteriaceae. Specifically, extended-spectrum β-lactamases (ESBLs), plasmid-mediated cephalosporinases (pAmpCs), and carbapenemases pose major therapeutic challenges in the treatment of hospitalized and community-based patients. Rapid and accurate identification of β-lactamase-producing Enterobacteriaceae is crucial for reducing clinical failure due to the use of inappropriate antimicrobial therapy and for preventing nosocomial outbreaks.

The CLSI has released recommendations for the detection of ESBLs in clinical isolates of Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, and Proteus mirabilis using clavulanic acid (CLSI, 2015). However, no guidelines have yet been issued for the detection of AmpCs. Moreover, the occurrence of bacteria that co-produce ESBLs and AmpCs has become a serious issue in the context of Enterobacteriaceae β-lactam resistance. Furthermore, overproduction of AmpCs in association with a porin defect can lead to carbapenem resistance (Jacoby, 2009). Although the modified Hodge test for carbapenemases has been found to be a useful tool for the detection of carbapenemase-producing Enterobacteriaceae, this test lacks sensitivity for NDM carbapenemases. Moreover, false positive results can occur in isolates that produce ESBL or AmpC enzymes coupled with porin loss.

Currently, molecular diagnostic methods such as real-time PCR (qPCR) (Beuving et al., 2011), mass spectrometry (Chong et al., 2015), microarray (Card et al., 2013), and loop-mediated isothermal amplification-based systems (García-Fernández et al., 2015) are available for bacterial identification and discrimination of antimicrobial susceptibility. However, DNA-based ESBL identification is technically challenging because new ESBLs are derived from pre-existing class A β-lactamases by several single amino acid substitutions (Rubtsova et al., 2010).

In this study, we evaluated a PCR-based reverse blot hybridization assay (REBA) that was recently developed for the rapid detection of GNB and their ESBL, AmpC, and carbapenemase (EAC) resistance genes. Specifically, the REBA-EAC assay was designed to detect ESBLs (CTX-M-, TEM-, and SHV-type), AmpCs (ACT, CMY-2-like, DHA, ACC-1, CMY-1-like/MOX, and FOX), and carbapenemases (IMP, VIM, NDM, KPC, OXA-48-like, and SPM).


Materials and methods

Archived isolates

To determine the specificity of the REBA-EAC assay, we tested archived frozen stocks of 29 EAC-producing Enterobacteriaceae strains (Song et al., 2015) and 128 non-EAC-producing clinical isolates belonging to 35 different genera [Citrobacter (1), Enterobacter (2), Escherichia (9), Klebsiella (3), Leclercia (1), Proteus (3), Salmonella (5), Serratia (1), Shigella (4), Bordetella (1), Haemophilus (1), Acinetobacter (1), Pseudomonas (2), Enterococcus (17), Micrococcus (1), Staphylococcus (12), Streptococcus (5), Corynebacterium (1), Mycobacterium (23), Absidia (1), Aspergillus (7), Aureobasidium (1), Beauveria (1), Bipolaris (1), Candida (8), Cryptococcus (1), Curvularia (1), Epidermophyton (1), Fusarium (1), Kodamaea (1), Malassezia (1), Microsporum (3), Penicillium (2), Saccharomyces (1), and Trichophyton (4)] from various specimen types. The 29 EAC-producing Enterobacteriaceae isolates included 11 ESBL-producing, 9 AmpC-producing, and 9 carbapenemase-producing strains (Table 1).


Table: Table 1

Characteristics of the 29 EAC-producing Enterobacteriaceae isolates with ESBL, AmpC β-lactamase, and carbapenemase resistance genes
Table: .

Organism type (no. of isolates)	Stock no	.Resistance gene	
ESBL STRAINS (11)	
Escherichia coli		DML1101	CTX-M-1
E. coli		DML1786	CTX-M-1
E. coli		DML1100	CTX-M-9
E. coli		DML1780	SHV-2a
E. coli		DML1783	SHV-2a
E. coli		DML1784	SHV-151
E. coli		DML1785	SHV-28
Klebsiella pneumoniae		DML1098	CTX-M-1
K. pneumoniae		DML1095	SHV-12
K. pneumoniae		DML1098	SHV-28
Shigella	 spp.	DML1096	TEM-52
AmpC β-LACTAMASES (9)	
E. coli		DLMH 71	CMY-2-like
E. coli		DML1784	CMY-11
E. coli		DLMH 173	CMY-1-like/MOX
E. coli		DLM 256	ACC-1
K. pneumoniae		DLMH 70	DHA-1
K. pneumoniae		DLMH 585	DHA-1
K. pneumoniae		DLMH 492	CMY-1-like/MOX
Enterobacter cloacae		DML1099	ACT
E. cloacae		DML1781	ACT
CARBAPENEMASES (9)	
K. pneumoniae		DLMH 411	OXA-232
K. pneumoniae		DLMH 107	OXA-48-like
K. pneumoniae		DLMH 108	OXA-181
K. pneumoniae		DLMH 492	NDM
E. coli		DLMH 410	OXA-232
E. coli		DLMH 744	KPC
E. cloacae		DML 1781	VIM
E. cloacae		DLMH 568	VIM
Pseudomonas aeruginosa		DLMH 155	IMP
DML, Diagnostic Microbiology Laboratory of Yonsei University (Wonju, Republic of Korea); DLMH, Department of Laboratory Medicine of Hallym University College of Medicine (Seoul, Republic of Korea).



Clinical isolates

To assess the diagnostic performance of the REBA-EAC assay, 327 ESBL phenotype clinical isolates obtained from 2010 to 2014 at Wonju Severance Christian Hospital (WSCH) were used. Isolates were identified by conventional identification method including phenotypic characteristics such as colony morphology, Gram staining result, and commercial identification kit results. MicroScan (Beckman Coulter, Brea, CA, USA) overnight Pos BP Combo 28, MICroSTREP Plus, overnight Neg Combo 53 and Neg Combo 54 panels were used for the identification of GPB, streptococci and GNB. For the identification of Candida spp., VITEK 2 (bioMérieux, Durham, NC, USA) yeast identification card was used. The ESBL phenotype of each isolate was confirmed by the microbroth dilution method, which is the test recommended by the CLSI. AmpC β-lactamase and carbapenemase production was confirmed using qPCR and by sequencing analyses with EAC primers (Table 2). This study was approved by the Institutional Ethics Committee at Yonsei University Wonju College of Medicine (approval no. CR312055-002) and samples were de-identified.


Table: Table 2

Primers and probes for REBA-EAC and qPCR
Table: .

Target and primer/probe name	Nucleotide sequence (5′-3′)	Product size	
ESBLs	
	CTX-M-1-210-F	GATGTGCAGCACCAGTAAAGTGATG	203 bp
	CTX-M-1-413-R	ATCGCCACGTTATCGCTGTACTG	
	CTX-M-1/2-258-P	AAGTGAAAGCGAACCGAATCTGTTA	
	CTX-M-9-210-F	TGTGCAGTACCAGTAAAGTTATGGCG	170 bp
	CTX-M-9-380-R	GCGCTAAGCTCAGCCAGTGACA	
	CTX-M-9-261-P	GAAACGCAAAAGCAGCTGCTT	
	TEM-520-F	TGCTCACCCAGAAACGCTGGT	705 bp
	TEM-1225-R	ACGATACGGGAGGGCTTACCAT	
	Gln39Lys-P	AAAGATGCTGAAGATAAGTTGGGTG	
	Glu104Lys-P	CCAACTATCAAGGCGGGTTACA	
	Gly164His-P	TTCCCAATGATCAAGGCGAGTTA	
	Gly238Ser-P	GAGATCCATGCTCACCGGCTC	
	Glu240Lys-P	GAGATCCATGCTCACCGGCTC	
TEM-127-Fa		GGTTACATCGAGCTGGATCTCAACA	360bp
TEM-488-Ra		CAACGATCAAGGCGGGTTACAT	
	SHV-243-F	TATTATCTCCCTGTTAGCCACCCT	717 bp
	SHV-960-R	CCAAGCAGGGCGACAATCC	
	Leu35Gln-P	GGCTTTCGCTTTGTTTAATTTGCTC	
	Asp179Ala-P	GCCCGCACCACCACTA	
SHV-147-Fb		GCAAATTAAACAAAGCGAAAGCCA	133bp
SHV-280-Rb		GCACTACTTTAAAGGTGCTCATCATGG	
AmpC β-LACTAMASES	
	ACT-MIR-416-F	TACAGGTRCCGGATGAGGTCACG	205 bp
	ACT-MIR-620-R	CTATGGTCCAGCTTGAGCGGCTTAA	
	ACT-MIR-464-P	ATCAAAACTGGCAGCCGCAG	
	CMY-2-like-261-F	AAGACGTTTAACGGCGTGTTGG	175 bp
	CMY-2-like-436-R	TAACGTCATCGGGGATCTGCA	
	CMY2-like-328-P	ACGAAATACTGGCCAGAACTGACA	
	DHA-261-F	TTTCACAGGTGTGCTGGGTGC	200 bp
	DHA-460-R	AGAAATTCAGCAGATCCGCACGG	
	DHA-299-P	AAGAGATGGCGCTGAATGATCC	
	ACC-1-410-F	ACGT AGCGTAACAAGTCGCTGGAG	210 bp
	ACC-1-620-R	TGGGTGTGAGCTATGAAGATGCGATT	
	ACC-1-439-P	GCTTCGTTACCCAAAATCTCCATATTC	
	CMY-1-like/MOX-128-F	CTGCTCAAGGAGCACMGGATC	243 bp
	CMY-1-like/MOX-371-R	CGGATCCCTTGAGCCAGGG	
	CMY-1-like/MOX-172-P	AAAGGATGGCAAGGCCCACTA	
	FOX-182-F	ATTTCAACTATGGGGTTGCCAACC	208 bp
	FOX-390-R	AAGCTCGGCCATRGTCACACCATC	
	FOX-239-P	TGTTCGAGATTGGCTCGGTCAG	
CARBAPENEMASES	
	IMP-263-F	TAAAAGGCAGTATYTCCTCWCATTT	232 bp
	IMP-495-R	CCAAACCACTACGTTATCTKGAG	
	IMP-307-P	GGAATAGAGTGGCTTAATTCTCRATCTAT	
	VIM-136-F	CTTTACCAGATTGCCGATGGTGTT	214 bp
	VIM-350-R	TCATGAAAGTGCGTGGAGACTGCA	
	VIM-198-P	CTACCCGTCCAATGGTCTCATTGT	
	KPC-269-F	TGGACACACCCATCCGTTACG	211 bp
	KPC-480-R	ACGGAACGTGGTATCGCCGATAG	
	KPC-329-P	AATATCTGACAACAGGCATGACGGT	
	NDM-327-F	AGATCCTCAACTGGATCAAGCAGG	223 bp
	NDM-550-R	TGCTGGTTCGACCCAGCCATTGGC	
	NDM-385-P	TCACGCGCATCAGGACAAGA	
	OXA-48-like-131-F	TTGTGCTCTGGAATGAGAATAAGCAG	210 bp
	OXA-48-like-340-R	CGGTGATTAGATTATGATCGCA	
	OXA-48-186-P	GAACCAAGCATTTTTACCCGCA	
	SPM-50-F	CGGATCATGTCGACTTGCCCT	200 bp
	SPM-250-R	ACTACTTTCTTCGGCTTCATAGTC	
	SPM-107-P	TCGTCACAGACCGCGATTTCT	
a,bPrimers for qPCR.



DNA preparation

To prepare DNA templates for the REBA-EAC assay, one colony from each frozen stock and one colony from each clinical isolate was suspended in 100 μL of DNA extraction solution (Optipharm, Osong, Republic of Korea). The bacterial suspension was boiled for 10 min. After centrifugation at 13,000 × g for 10 min, the supernatant was used as a source of DNA template.


REBA-EAC primers and probes

The 26 specific primers and probes were used for the detection of GNB (10 probes) and their antibiotic resistance genes (16 EAC gene probes) and an example of the result is shown in Figure 1. The sequences of the primers and probes were compared by nucleotide-nucleotide NCBI BLAST (BLASTn) searches to determine their sequence homologies. Primers and probes were selected by multiple alignments using the MultAlign program (http://multalin.toulouse.inra.fr/multalin/). All publicly available nucleotide sequences of the TEM and SHV alleles related to the ESBL-production phenotype (http://www.lahey.org/studies/) were retrieved from GenBank. Previous reports have concluded that 95% of all TEM-variants with an established ESBL phenotype have one or more amino acid substitutions at positions 39, 104, 164, 238, and/or 240; moreover, 77% of all ESBL SHV-variants have substitutions at positions 179, 238, and/or 240 (Cohen Stuart et al., 2010; Rubtsova et al., 2010). Based on these findings, 7 oligonucleotide probes (Figure 1) were designed to identify TEM-variants (one TEM-all probe and 6 probes for ESBL variants). Specifically, the probes were designed to detect the following ESBL-associated amino acid substitutions: Gln39Lys, Glu104Lys, Arg164Ser/His, Gly238Ser, and/or Glu240Lys. To identify SHV-variants, 6 probes (Figure 1) were designed (one SHV-all probe and 5 probes for ESBL variants). Specifically, the probes were designed to detect the following ESBL-associated substitutions: Leu35Gln, Asp179, Gly238Ser, and/or Glu240Lys (Cohen Stuart et al., 2010; Rubtsova et al., 2010).

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.


PCR

PCR was performed in a final reaction volume of 20 μL. Each reaction contained 2 × master mix (10 μL) (GeNet Bio, Daejeon, Republic of Korea), 5 μL primer mixture, and 5 μL sample DNA. The first 10 PCR cycles were comprised of denaturation at 95°C for 30 s, followed by annealing and extension at 60°C for 30 s. These 10 cycles were followed by 40 cycles of denaturation at 95°C for 30 s and annealing/extension at 54°C for 30 s. After the final cycle, samples were maintained at 72°C for 10 min to complete the synthesis of all strands. The amplicon was visualized as a single 230 bp band using a ChemiDoc system (Bio-Rad, Hercules, CA, USA).


REBA-EAC assay

To validate the efficiency of the selected probes, target DNA samples amplified from EAC-producing strains were applied to REBA membrane strips spotted with the selected probes. Two genes (FOX and SPM) were synthesized (Bioneer, Daejeon, Republic of Korea) and amplified with custom PCR primers (FOX, F-5′-ATGCAACAACGACGTGCGTTCGC-3′ and R-5′-TGGCAAGCTCGGCCATGGTCAC-3′; SPM, F-5′-CGTTTTGTTTGTTGCTCGTTGCG-3′ and R-5′-TCAGCTGCTTTTATCCGGTCTTTC-3′), resulting in amplicons of 395 and 400 bp, respectively. The resultant products were mutagenized after subcloning into the pBHA vector. Two plasmids were extracted from the transformants, and the mutated sequences were confirmed by sequence analysis (CosmoGenetech, Seoul, South Korea).

For the REBA-EAC assay, hybridization and washing were performed according to the manufacturer's instructions. Each sample was tested in duplicate and all REBA experiments were performed twice. In brief, biotinylated PCR products were denatured at 25°C for 5 min in denaturation solution. The resulting single-stranded PCR products were transferred to hybridization solution and then incubated with REBA-EAC membrane strips at 55°C with shaking at 90 rpm for 30 min in the provided blotting tray. The strips were then washed twice with gentle shaking in 1 mL of washing solution for 10 min at 55°C, incubated at 25°C with a 1:2,000 dilution of streptavidin-alkaline phosphatase (AP) conjugate (Roche Diagnostics, Mannheim, Germany) in conjugate diluent solution (CDS) for 30 min, and finally, washed twice with 1 mL CDS at room temperature for 1 min. The colorimetric hybridization signals were visualized by the addition of a 1:50 dilution of the NBT/BCIP AP-mediated staining solution (Roche Diagnostics), and incubated until a color change was detected. Finally, the banding pattern was read and interpreted.


Real time PCR (qPCR) assay

All phenotypic ESBL producers were screened by qPCR (CFX-96 qPCR system; Bio-Rad) to identify their non-ESBL and ESBL-carrying genes using primers specific for detecting predominant subtypes of CTX-M, TEM, and SHV. Next, the AmpC- and carbapenemase-carrying genes were analyzed using qPCR (CFX-96 qPCR system; Bio-Rad). Specifically, the presence of genes encoding ACT, CMY-2-like, DHA, ACC-1, CMY-1-like/MOX, FOX, IMP, VIM, NDM, KPC, OXA-48-like, and SPM was analyzed (Table 2). qPCR amplification was performed in a total volume of 20 μL, including 10 μL of 2 × Thunderbird SYBR master mix (Toyobo, Osaka, Japan), 2 μL primer mixture, 5 μL template DNA, and 3 μL sterile distilled water. Each qPCR assay included internal control DNA that was used to simulate pure nucleic acid extraction and high sample quality, thus monitoring the effect of PCR inhibitors in the reaction. Positive and negative controls were also included throughout the procedure. Template controls containing sterile distilled water were also incorporated in each assay. The thermocycling conditions were as follows: 95°C for 3 min, followed by 40 cycles of 95°C for 3 s and 60°C for 30 s. Each sample was tested in duplicate and all PCR assays were performed twice. The relative bacterial load was quantified by determining the cycle threshold (CT), i.e., the number of PCR cycles required for the fluorescence to exceed a value significantly higher than the background fluorescence. A positive result was interpreted as when the CT value was less than 30 after fluorescence signal above background was observed.


Sequence analysis

The PCR amplicons of all clinical isolates in this study were sequenced using an ABI 3730 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA) and an ABI Prism BigDye Terminator (Applied Biosystems) system. The obtained sequences were compared with sequences in the NCBI GenBank database for species assignment.


Blood culture samples

To evaluate the diagnostic performance of the REBA-EAC assay on blood culture samples, 200 positive blood culture (PBC), and 200 negative blood culture (NBC) samples were collected at WSCH from April 2015 to June 2015. Blood samples were drawn from the patient at the bedside. Three or two pairs of culture bottles were inoculated with the blood samples and then incubated in a BacT/Alert 3D (bioMérieux, Durham, NC, USA) or BACTEC FX (Becton Dickinson Diagnostic Systems, Spark, MD, USA) blood culture system for 5 days. If no bacterial growth was detected within 5 days, the blood culture was considered to be negative. When bacterial growth was noted, the culture sample was inoculated onto blood and MacConkey agar plates (Becton Dickinson) and cultured overnight at 37°C in a 5% CO2 incubator.

For the preparation of DNA templates from the PBC and NBC samples, 0.5 mL of blood suspension was withdrawn directly from the culture bottle. Next, 1 mL of phosphate-buffered saline (pH 8.0) was added and the sample was spun by centrifugation at 13,000 × g for 1 min. The supernatant was then removed, after which the pellet was resuspended in 1 mL of erythrocyte lysis buffer (Sigma, St. Louis, MO, USA) and spun by centrifugation at 13,000 × g for 1 min. This wash step was repeated twice. The pellet was then resuspended in DNA extraction solution, as described above for the clinical isolates. To confirm the positive results from the REBA-EAC assay, qPCR and sequence analyses were performed using the same DNA samples.


Statistical analysis

All statistical analyses were performed using Prism 5 software (GraphPad, La Jolla, CA, USA) and SPSS statistics software version 21.0 (IBM, Armonk, NY, USA). The sensitivity, specificity, negative predictive value, and positive predictive value of the qPCR and REBA-EAC assays were analyzed by calculating the kappa coefficient of the predictive ability, its corresponding P value, and the 95% confidence interval (CI).



Results

Analytical sensitivity and specificity of the REBA-EAC assay

Twenty-nine EAC-producing strains were identified using the designated target probes in the REBA-EAC assay, whereas 128 non-EAC-producing clinical isolates yielded negative results by the REBA-EAC assay (data not shown). No cross-genus or cross-species reactivity was observed.

The REBA-EAC assay was able to detect single nucleotide polymorphisms found in the most prevalent TEM- and SHV-type ESBLs, including Gln39Lys, Glu104Lys, Arg164Ser, Gly238Ser, and/or Glu240Lys in the TEMs, and Asp179Ala/Asn/Gly, Gly238Ser/Ala, and/or Glu240Lys in the SHVs. TEM-52, TEM-66 (Glu104Lys and Gly238Ser mixed), and SHV-12 (Gly238Ser), which were identified as TEM/SHV-ESBLs by the REBA-EAC assay, were also confirmed ESBL-associated amino acid substitutions by sequence analyses. The target DNA samples hybridized strongly with the probes derived from their targets and showed no cross-reactivity (Figure 1). The analytical sensitivities of the 16 target probes (4 ESBL, 6 AmpC, and 6 carbapenemase probes) were determined using a standard curve of 10-fold dilutions (10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, and 10 fg) of DNA extracted from EAC-producing strains. The detection limit of the REBA-EAC assay for each probe ranged from 100 fg to 10 fg DNA per reaction (Figure 2).

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.


Diagnostic performance of the REBA-EAC assay for clinical isolates

Based on the genus-specific or species-specific REBA-EAC assay probes, the overall concordance rate of the assay for species identification of the 327 Enterobacteriaceae isolates between the conventional identification test and the REBA-EAC assay was 100%. Of the 327 ESBL-phenotypically confirmed isolates, 308 isolates (94.2%) were positive by the REBA-EAC assay for ESBL genes, 16 K. pneumoniae isolates had only AmpC plasmid-mediated DHA genes, and three E. coli isolates did not have any EAC genes by the REBA-EAC assay (Table 3). Among the 324 isolates with at least one EAC gene, exclusive ESBL-producing isolates accounted for up to 70.0% of all EAC-producers (one-type ESBL producer, 154 isolates; two-type ESBL co-producer, 73 isolates), followed by 23.1% for ESBL and AmpC β-lactamase; 4.9% for AmpC β-lactamase; 1.5% for ESBL and carbapenemase; and 0.3% for ESBL, AmpC, and carbapenemase (Table 3). Overall, 80.2% (247/308) of ESBL-producing isolates were the CTX-M type, with CTX-M-9 group (57.5%) being the most prevalent ESBL gene. For the TEM and SHV genes, the REBA-EAC assay was able to differentiate between non-ESBL TEM/SHV, and their ESBL derivatives for all isolates. Among the 144 pan-TEM-positive isolates tested by qPCR, 104 (72.2%) were positive for G104L (TEM-3, -22, -52, -66), G238S, and/or G240L (TEM-22, -46, -52). Moreover, remaining 40 (27.8%) isolates, which were negative for G104L, G238S, and/or G240L, were considered to be non-ESBL producers by both the REBA-EAC assay and sequence analysis. The SHV-ESBL specific amino acid substitution, G238S, was detected in 31 (26.1%) of the 119 SHV-producing Klebsiella spp., and was confirmed in the SHV-2a, -5, and -12 genes by sequence analysis (Table 4).


Table: Table 3

REBA-EAC assay results for the detection of ESBLs, AmpC β-lactamases, and carbapenemases in the 327 ESBL-phenotypically confirmed isolates
Table: .

ESBL-phenotypically confirmed isolate (no. of isolates)	REBA-EAC assay	
GN-ID (no. of isolates)	β-lactamase content (no. of isolates)	
Escherichia coli	 (196)E. coli	 (196)	CTX-M only (109)
			CTX-M + TEM-type ESBL (68)
			TEM-type ESBL (5)
			CTX-M + CMY2 (4)
			CTX-M + DHA (2)
			CTX-M + NDM (2)
			CTX-M + ACT (1)
			CTX-M + KPC (1)
			CTX-M + VIM (1)
			ND (3)
Klebsiella pneumoniae	 (120)K. pneumoniae	 (120)	CTX-M + DHA (58)
			TEM-type ESBL (11)
			CTX-M only (10)
			SHV-type ESBL (9)
			CTX-M + CMY2 (7)
			CTX-M + SHV-type ESBL (5)
			CTX-M + DHA + CMY2 (2)
			CTX-M + IMP (1)
			CTX-M + DHA + NDM (1)
			DHA only (16)
Proteus mirabilis	 (4)	Gram negative (4)	CTX-M (4)
Klebsiella oxytoca	 (3)K. oxytoca	 (2)	SHV-type ESBL (2)
	K. oxytoca	 (1)	CTX-M (1)
Enterobacter	 spp. (3)Enterobacter	 spp. (2)	CTX-M (2)
	Enterobacter	 spp. (1)	CTX-M + ACT (1)
Providencia stuartii	 (1)	Gram negative (1)	CTX-M (1)
ND, not detected.



Table: Table 4

Comparison of the performances of the REBA-EAC assay versus sequence analysis for detecting β-lactamase genes in the 327 ESBL-phenotypically confirmed isolates
Table: .

Class of β-lactamase (no. of isolates)	Type of β-lactamase gene	Microorganism	No. of isolates	
	REBA-EAC assay	Sequence analysis			
	ESBL (382)	CTX-M-1/2	CTX-M-1E. coli		97
		CTX-M-1/2	CTX-M-1K. pneumoniae		3
		CTX-M-1/2	CTX-M-1K. oxytoca		1
		CTX-M-1/2	CTX-M-1E. aerogenes		1
		CTX-M-9	CTX-M-9E. coli		86
		CTX-M-9	CTX-M-9K. pneumoniae		49
		CTX-M-9	CTX-M-9P. mirabilis		4
		CTX-M-9	CTX-M-9E. cloacae		2
		CTX-M-9	CTX-M-9P. mirabilis		4
		CTX-M-1/2 + CTX-M-9	CTX-M-1 + CTX-M-9E. coli		2
		CTX-M-1/2 + CTX-M-9	CTX-M-1 + CTX-M-9K. pneumoniae		1
		TEM	TEM-3K. pneumoniae		3
		TEM	TEM-22E. coli		21
		TEM	TEM-22K. pneumoniae		5
		TEM	TEM-46E. coli		9
		TEM	TEM-46K. pneumoniae		5
		TEM	TEM-52E. coli		25
		TEM	TEM-52K. pneumoniae		10
		TEM	TEM-66E. coli		21
		TEM	TEM-66K. pneumoniae		5
		SHV	SHV-2aK. pneumoniae		14
		SHV	SHV-5K. pneumoniae		5
		SHV	SHV-12K. pneumoniae		10
		SHV	SHV-12K. oxytoca		2
	AmpC β-lactamase (92)	DHA	DHA-1K. pneumoniae		61
		DHA	DHA-23K. pneumoniae		8
		DHA	DHA-15K. pneumoniae		5
		DHA	DHA-3K. pneumoniae		1
		DHA	DHA-1E. coli		2
		CMY-2-like	CMY-2K. pneumoniae		7
		CMY-2-like	CMY-2E. coli		4
		DHA+CMY-2-like	DHA+CMY-2K. pneumoniae		2
		ACT	ACT-1E. coli		1
		ACT	ACT-1E. aerogenes		1
	Carbapenemase (6)	NDM	NDM-1E. coli		2
		NDM	NDM-1K. pneumoniae		1
		KPC	KPC-18E. coli		1
		VIM	VIM-2E. coli		1
		IMP	IMP-1K. pneumoniae		1


Diagnostic performance of the REBA-EAC assay for blood culture samples

Typical results of the REBA-EAC assay using blood culture samples are shown in Figure 3. Among the 200 PBC samples, 120 (60%), 73 (36.5%), and 7 (3.5%) samples contained GPB, GNB, and Candida species by the conventional identification test, respectively. None of the GPB or Candida species were detected by the REBA-EAC assay (Table 5). Based on the genus-specific or species-specific REBA-EAC assay probes, 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. Among the 57 Enterobacteriaceae isolates, only 12 (12/57; 21.1%) isolates were phenotypically confirmed as ESBL producers, whereas 16 (16/57; 28.1%) isolates were confirmed as carrying EAC genes by both the REBA-EAC assay and sequence analysis (Table 6). Eleven of the twelve (91.7%) phenotypically ESBL-positive isolates also showed positive results by the REBA-EAC assay, with CTX-M type genes (8 CTX-M-1 and 3 CTX-M-9) being the most prevalent ESBL genes. One Pseudomonas aeruginosa and five Enterobacteriaceae isolates (3 Enterobacter cloacae, 1 Citrobacter freundii, and 1 Providencia stuartii) were positive for an IMP and AmpC genes (ACT, CMY, or DHA), respectively, by the REBA-EAC assay. This finding was confirmed by qPCR and Sanger sequencing.

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.


Table: Table 5

Comparison of species identification results obtained using the conventional method versus the REBA-EAC assay on 400 blood culture samples
Table: .

Conventional method (no.)	REBA-EAC assay no. (%)	
	Positive	Negative	
	Positive blood culture (200)	73 (36.5)	127 (63.5)
	 Gram positive (120)	0 (0)	120 (100)
	 Gram negative (73)	73 (100)	0 (0)
 Candida	 spp. (7)	0 (0)	7 (100)
	Negative blood culture (200)	0 (0)	200 (100)
	Total (400)	73 (18.3)	327 (81.7)


Table: Table 6

Comparison of the results of phenotypic versus molecular assays for the detection of β-lactamases in 73 Gram-negative bacteria isolates from blood culture samples
Table: .

Microorganism	No. (%) of isolates	Detection of β-lactamases, no. (%) of isolates	
		Phenotypic assay	REBA-EAC assay	qPCR	Sequence analysis	
		Positive	Negative	Positive	Negative	Positive	Negative	Positive	Negative	
E. coli		33 (45.2)	11 (33.3)	22 (66.7)10a	 (30.3)	23 (69.7)20b	 (60.6)	13 (39.4)10a	 (30.3)	23 (69.7)
Klebsiella	 spp.	14 (19.1)1c	 (7.1)	13 (92.9)1c	 (7.1)	13 (92.9)12b	 (85.7)	2 (14.3)1c	 (7.1)	13 (92.9)
Enterobacter	 spp.	5 (6.8)	0 (0)	5 (100)3d	 (60)	2 (40)3d	 (60)	2 (40)3d	 (60)	2 (40)
Providencia	 spp.	2 (2.7)	0 (0)	2 (100)1e	 (50)	1 (50)1e	 (50)	1 (50)1e	 (50)	1 (50)
Citrobacter freundii		1 (1.4)	0 (0)	1 (100)1f	 (100)	0 (0)1f	 (100)	0 (0)1f	 (100)	0 (0)
Serratia marcescens		1 (1.4)	0 (0)	1 (100)	0 (0)	1 (100)	0 (0)	1 (100)	0 (0)	1 (100)
Proteus mirabilis		1 (1.4)	0 (0)	1 (100)	0 (0)	1 (100)	0 (0)	1 (100)	0 (0)	1 (100)
Campylobacter	 spp.	1 (1.4)	NA	NA	0 (0)	1 (100)	0 (0)	1 (100)	0 (0)	1 (100)
Acinetobacter	 spp.	7 (9.6)	NA	NA	0 (0)	7 (100)	0 (0)	7 (100)	0 (0)	7 (100)
Pseudomonas aeruginosa		6 (8.2)	NA	NA1g	 (16.7)	5 (83.3)1g	 (16.7)	5 (83.3)1g	 (16.7)	5 (83.3)
Sphingomonas paucimobilis		1 (1.4)	NA	NA	0 (0)	1 (100)	0 (0)	1 (100)	0 (0)	1 (100)
Burkholderia cepacia		1 (1.4)	NA	NA	0 (0)	1 (100)	0 (0)	1 (100)	0 (0)	1 (100)
aIsolates with CTX-M gene (8 CTX-M-1 and 2 CTX-M-9).

bSome of isolates with non-ESBL type TEM or SHV genes were included in the positive results.

cIsolate with CTX-M gene (CTX-M-9).

dIsolates with ACT gene.

eIsolate with DHA gene.

fIsolate with CMY gene

gIsolate with IMP gene.

NA, not available.




Discussion

Presently, antimicrobials are usually selected empirically for the initial treatment of bacteremia when a bloodstream infection is strongly suspected (Park et al., 2014). Furthermore, since the antimicrobial susceptibility results usually cannot be provided in < 48–72 h, clinicians continue to rely on Gram stain results for selection of an appropriate antibiotic treatment for patients with PBCs (Uehara et al., 2009). Rapid and accurate EAC detection is crucial for effective infection control measures and for the selection of appropriate antimicrobial therapy, because the emergence of EAC-producing strains is a vital factor in the treatment of infections associated with sepsis. Like other mechanisms of antibiotic resistance, the genes for EACs are most often encoded on plasmids that can be readily transferred between bacteria (Haenni et al., 2014). The use of semiautomated systems for the identification and antimicrobial susceptibility testing of GNB is now routine practice in many laboratories. Compared to more recently developed molecular tests, traditional phenotypic susceptibility results usually take an additional 1–2 days after a PBC is reported. Since phenotypic detection of ESBLs, AmpCs, and carbapenemases is time-consuming and the results may be difficult to interpret, a faster and more accurate detection method is highly desirable for routine use in clinical laboratories (Cohen Stuart et al., 2010). In general, commercially available molecular based tests have been developed for one of ESBL (CTX-M types, TEM, and SHV), AmpC β-lactamase (CMY-2-like, DHA, FOX, ACC-1, ACT/MIR, and CMY-1-like/MOX), and carbapenemase (VIM, IMP, NDM, KPC, and OXA-48-type) genes (Diekema and Pfaller, 2013). In comparison, REBA-EAC assay can provide information about GNB identification and broad range of clinically relevant antibiotic resistance genes in GNB.

Although the prevalence of carbapenemase-producing Enterobacteriaceae strains is still very low (< 1%) in Korea, various types of carbapenemases have been identified (Bae et al., 2015). In this study, ESBL and AmpC β-lactamase co-production was seen in 23.1%; ESBL and carbapenemase co-production in 1.5%; and ESBL, AmpC β-lactamase, and carbapenemase co-production in 0.3% of the phenotypically confirmed ESBL-producing Enterobacteriaceae isolates. Several studies have demonstrated that AmpC β-lactamase with ESBLs causes treatment failure of cefepime and that inadequate antimicrobial therapy is an independent risk factor for mortality or microbiological failure in severely ill patients with life-threatening infections (Fraser et al., 2006; Park et al., 2010). Therefore, rapid and accurate identification of drug resistance genes directly from PBCs is critical for the selection of appropriate antibiotics in the treatment of bacterial infections.

In this study, we confirmed that the REBA-EAC assay is a highly sensitive and specific probe-based method that provides rapid results and can detect multiple species and drug-resistant genes simultaneously (Ajbani et al., 2011). The REBA-EAC assay also showed complete agreement with the conventional identification test and sequence analysis for the detection of ESBL (CTX-M groups, TEM-ESBL derivatives, SHV-ESBL derivatives) and non-ESBL (TEM and SHV) genes. For qPCR, TEM/SHV show positive results for both ESBL and non-ESBL, which makes it difficult to differentiate between them. However, as REBA-EAC assay detects one or more amino acid substitutions in targeted region, it can discriminate between ESBL TEM/SHV derivatives and non-ESBL TEM/SHV types. In general, our results are similar to previous assessments of REBA assay (sensitivity of 95–99% and specificity of 100%) (Kim et al., 2016; Wang et al., 2016) and other methods such as microarray-based check-points assay (sensitivity of 94–95% and specificity of 83–100%) (Cohen Stuart et al., 2010; Card et al., 2013) and MALDI-TOF MS-based assay (sensitivity of 97–99% and specificity of 94–99%) (Bork et al., 2015; Chong et al., 2015). Moreover, the performance of the REBA-EAC assay was better than the E-test (sensitivity of 71% and specificity of 73%) (Garrec et al., 2011). We also found that CTX-M type genes (CTX-M-1 and CTX-M-9) were the most prevalent ESBL-encoding genes. These genes were detected in almost all of the ESBL-producing Enterobacteriaceae (in particular, 48.4% of all E. coli isolates), whereas ESBL-type TEM and SHV genes were present in 20.4 and 6.1% of the isolates, respectively. These findings are in agreement with other recent studies showing that CTX-M type genes are the ESBL genes most frequently expressed by Enterobactericeae (Cantón et al., 2012). In many Enterobacteriaceae, AmpC expression is low but inducible in response to β-lactam exposure (Haenni et al., 2014). Using the REBA-EAC assay, we observed various types of AmpC β-lactamases (DHA, CMY-2, DHA, and ACT) in different Enterobacteriaceae species: CMY-2 in E. coli, DHA-1 in K. pneumoniae or E. coli, and ACT in Enterobacter aerogenes. In particular, DHA-producing K. pneumoniae isolates were predominant in Enterobacteriaceae, a finding that is similar to the results reported in other studies (Park et al., 2010).

Our study confirmed that REBA-EAC assay can be applied directly in blood culture samples. As shown in Table 5, the results of conventional identification test and REBA-EAC assay were completely concordant in species identification. Furthermore, REBA-EAC assay detected isolates harboring AmpC or carbapenema genes (1 Pseudomonas aeruginosa, 3 Enterobacter cloacae, 1 Citrobacter freundii, and 1 Providencia stuartii).

One potential limitation of our study compared to previous studies is the limited number of samples, since the study was carried out at a single institution. Neither the AmpC β-lactamase ACC-1, CMY-1-like/MOX, or FOX genes, nor the carbapenemase OXA-48-like gene, were detected in clinical isolates of this study. Moreover, since AmpC and carbapenemase genes are less prevalent than ESBL genes in Korea, we confirmed and included only ESBL-phenotype isolates in screening. Thus, there is a possibility that some of the non-ESBL isolates producing AmpC and/or carbapenemase could not be identified. Therefore, larger studies are required to confirm our results and limitations.

In conclusion, the recently developed REBA-EAC assay is an accurate, rapid, and convenient tool for identifying GNB. This assay can also discriminate ESBL, AmpC β-lactamase, and carbapenemase genes and directly detect pathogen species from PBC samples in only 2 h. Therefore, the REBA-EAC assay can provide essential information that can accelerate therapeutic decisions for earlier appropriate antibiotic treatment in the acute phase of pathogen infection.


Author contributions

YU, WS, JBK, and HL designed the experimental strategy. HW and GY performed the experiments. HW, GY, and JK analyzed and interpreted the data. HW and GY wrote the paper with input from all co-authors. All authors have read and approved the final version of the manuscript.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.



This work was supported by a grant from the Korea Health Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (Grant A121030 to HL). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.


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