Rapid identification of carbapenemase-producing Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii using a modified Carba NP test.

Biochemical tests have been previously developed to identify carbapenemase-producing Enterobacteriaceae, Pseudomonas spp. (Carba NP test) and Acinetobacter spp. (CarbAcineto NP test). We evaluated a modified Carba NP test to detect carbapenemase production in Enterobacteriaceae, Pseudomonas and Acinetobacter species using a single protocol with rapid results and found good reliability and speed.

Multidrug-resistant Gram-negative bacteria (GNB) are increasingly being reported worldwide. The spread of carbapenemase-producing Enterobacteriaceae, Pseudomonas and Acinetobacter species have become a global threat. The emergence of resistance to carbapenems makes the treatment for infections caused by these carbapenem-resistant strains very limited [1-3]. Different types of carbapenemases have been reported, such as Ambler class A Klebsiella pneumoniae carbapenemase (KPC) and Guiana extended spectrum (GES) β-lactamase, Ambler class B metallo-β-lactamases (MBL) and Ambler class D oxacillinase type [1].

Rapid methods for detecting carbapenemase producers have been described, such as the MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) carbapenemase assay [4]. Previous studies have described a rapid biochemical carbapenemase detection method based on imipenem hydrolysis, the Carba NP test, for Enterobacteriaceae[5] and Pseudomonas species [6], as well as the CarbAcineto NP test for Acinetobacter species [7]. Recently, however, several authors have published evaluations of the Carba NP and the CarbAcineto NP tests; their criticisms focussed essentially on the absence of detection of oxacillinase (OXA) type carbapenemases [8-10].

Here we describe a modified Carba NP (MCNP) test which enables the rapid detection of different carbapenemases (KPC, MBL and OXA types) from Enterobacteriaceae, Pseudomonas and Acinetobacter species using a single protocol.

One hundred ten previously characterized GNB, including 69 carbapenemase-producing GNB (Enterobacteriaceae n = 14, Pseudomonas aeruginosa n = 11 and Acinetobacter baumannii n = 44), and 41 non-carbapenemase-producing GNB, including Enterobacteriaceae (n = 24), P. aeruginosa (n = 5) and A. baumannii (n = 12), were tested in two laboratories including Unité de recherche sur les maladies infectieuses et tropicales émergentes (URMITE), Aix-Marseille University, Marseille, France, and Microbial Ecology laboratory, Béjaia University, Béjaia, Algeria (Table 1). Carbapenemase activity was assessed using phenotypic and genotypic tests, including the modified Hodge test, MALDI-TOF MS assay, PCR amplification and sequencing [4,11].

Table 1

Test results for Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii strains

Group	Species	Carbapenemase or other β-lactamase gene	Test result by:
			MHT	MALDI-TOF MS	MCNP
Enterobacteriaceae	Klebsiella pneumoniae H5500M	NDM-1	+	+	+
	K. pneumoniae KpnaseyM	NDM-1	+	+	+
	K. pneumoniae U2A 2246M	KPC-3	+	+	+
	K. pneumoniaeA	KPC-3	+	+	+
	K. pneumoniae 360M	KPC-2	+	+	+
	K. pneumoniaeM	OXA-48	+	+	+
	K. pneumoniae 413A	TEM-1/CTX-M-3/SHV-12/DHA-1	−	−	−
	K. pneumoniae 473A	TEM-1/CTX-M-15/OXA-1	−	−	−
	K. pneumoniae 463A	TEM-1/CTX-M-15	−	−	−
	K. pneumoniae 550A	TEM-1/CTX-M-3	−	−	−
	K. pneumoniae 47A	TEM-1/CMY-4	−	−	−
	K. pneumoniae 123A	TEM-1/CMY-4	−	−	−
	K. pneumoniae 318A	CTX-M-15	−	−	−
	K. pneumoniae 476A	CTX-M-15	−	−	−
	K. pneumoniae 613A	CMY-4	−	−	−
	K. pneumoniae 615A	CMY-4	−	−	−
	Escherichia coli H5636M	NDM-1	+	+	+
	E. coli 181A	NDM-5	+	+	+
	E. coliM	NDM-5	+	+	+
	E. coli 99A	OXA-48	+	+	+
	E. coli 100A	OXA-48	+	+	+
	E. coli 132A	OXA-48	+	+	+
	E. coli 192A	OXA-48	+	+	+
	E. coliM	OXA-48	+	+	+
	E. coli 544A	TEM-1/CTX-M-15/OXA-1	−	−	−
	E. coli 469A	TEM-1/CTX-M-3/OXA-1	−	−	−
	E. coli 472A	TEM-1/CTX-M-3/OXA-1	−	−	−
	E. coli 234A	TEM-1/CTX-M-15/CMY-4	−	−	−
	E. coli 611A	TEM-1/CTX-M-15/CMY-4	−	−	−
	E. coli 536A	TEM-1/CTX-M-15	−	−	−
	E. coli 534A	CTX-M-15/OXA-1	−	−	−
	E. coli 542A	CTX-M-15/OXA-1	−	−	−
	E. coli 560A	TEM-1/CMY-4	−	−	−
	E. coli 606A	TEM-1/CMY-4	−	−	−
	E. coli ATCC 25922M	—	−	−	−
	E. coli 7624M	—	−	−	−
	E. cloacae 308A	TEM-1/CTX-M-15/OXA-1	−	−	−
	E. cloacae 570A	TEM-1/CTX-M-15	−	−	−
Pseudomonas	P. aeruginosaM	VIM-2	+	+	+
	P. aeruginosaM	VIM-2	+	+	+
	P. aeruginosaM	VIM-2	+	+	+
	P. aeruginosaM	VIM-2	+	+	+
	P. aeruginosaM	VIM-2	+	+	+
	P. aeruginosaM	VIM-2	+	+	+
	P. aeruginosaM	VIM-2	+	+	+
	P. aeruginosaM	VIM-2	+	+	+
	P. aeruginosaM	VIM-2	+	+	+
	P. aeruginosaM	VIM-2	+	+	+
	P. aeruginosaM	—	−	−	−
	P. aeruginosaM	—	−	−	−
	P. aeruginosaM	—	−	−	−
	P. aeruginosaM	—	−	−	−
	P. aeruginosaM	—	−	−	−
	P. aeruginosa UAA 2257M	IMP-1	+	+	+
Acinetobacter	A. baumanniiA	OXA-23	+	+	+
	A. baumanniiA	OXA-23	+	+	+
	A. baumanniiA	OXA-23	+	+	+
	A. baumanniiA	OXA-23	+	+	+
	A. baumanniiA	OXA-23	+	+	+
	A. baumanniiA	OXA-23	+	+	+
	A. baumanniiM	OXA-23	+	+	+
	A. baumanniiM	OXA-23	+	+	+
	A. baumanniiM	OXA-23	+	+	+
	A. baumanniiM	OXA-23	+	+	+
	A. baumanniiM	OXA-24	+	+	+
	A. baumanniiM	OXA-24	+	+	+
	A. baumanniiM	OXA-24	+	+	+
	A. baumanniiM	OXA-24	+	+	+
	A. baumanniiM	OXA-24	+	+	+
	A. baumanniiM	OXA-24	+	+	+
	A. baumanniiA	OXA-24	+	+	+
	A. baumanniiA	OXA-24	+	+	+
	A. baumanniiA	OXA-24	+	+	+
	A. baumanniiA	OXA-24	+	+	+
	A. baumanniiM	OXA-58	+	+	+
	A. baumanniiM	OXA-23/OXA-24	+	+	+
	A. baumanniiA	OXA-23/OXA-24	+	+	+
	A. baumanniiA	OXA-23/OXA-24	+	+	+
	A. baumanniiA	NDM-1	+	+	+
	A. baumanniiA	NDM-1	+	+	+
	A. baumanniiA	NDM-1	+	+	+
	A. baumanniiA	NDM-1	+	+	+
	A. baumanniiA	NDM-1	+	+	+
	A. baumanniiM	NDM-1	+	+	+
	A. baumanniiM	NDM-1	+	+	+
	A. baumanniiM	NDM-1	+	+	+
	A. baumanniiM	NDM-1	+	+	+
	A. baumanniiM	NDM-1	+	+	+
	A. baumanniiM	OXA-23/NDM-1	+	+	+
	A. baumanniiM	OXA-23/NDM-1	+	+	+
	A. baumanniiM	OXA-23/NDM-1	+	+	+
	A. baumanniiM	OXA-23/NDM-1	+	+	+
	A. baumanniiM	OXA-23/NDM-1	+	+	+
	A. baumanniiM	OXA-23/NDM-1	+	+	+
	A. baumanniiA	OXA-23/NDM-1	+	+	+
	A. baumanniiA	OXA-23/NDM-1	+	+	+
	A. baumanniiA	OXA-23/NDM-1	+	+	+
	A. baumanniiA	OXA-23/NDM-1	+	+	+
	A. baumanniiA	TEM-128	−	−	−
	A. baumanniiA	TEM-128	−	−	−
	A. baumanniiA	TEM-128	−	−	−
	A. baumanniiA	TEM-128	−	−	−
	A. baumanniiA	TEM-128	−	−	−
	A. baumanniiA	TEM-128	−	−	−
	A. baumanniiA	TEM-128	−	−	−
	A. baumanniiM	TEM-128	−	−	−
	A. baumanniiM	TEM-128	−	−	−
	A. baumanniiM	TEM-128	−	−	−
	A. baumannii AYEM	VEB-1	−	−	−
	A. baumannii SDFM	—	−	−	−

MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MCNP, modified Carba NP test; MHT, modified Hodge test.

AStrains tested in Microbial Ecology Laboratory, Béjaia University, Béjaia, Algeria.

MStrains tested in Unité de recherche sur les maladies infectieuses et tropicales émergentes (URMITE), Aix-Marseille University, Marseille, France.

The Carba NP and the CarbAcineto NP tests are straightforward biochemical tests which identify carbapenemase production in GNB by detecting imipenem hydrolysis using phenol red solution as a colour indicator and a bacterial lysis buffer (B-PER II, Bacterial Protein Extraction Reagent) for Enterobacteriaceae and Pseudomonas species (Carba NP test) [5,6] and 5 M NaCl for Acinetobacter species (CarbAcineto NP test) [7].

In order to use a single protocol to detect the production of carbapenemases in the three types of bacteria (Enterobacteriaceae, Pseudomonas and Acinetobacter) and to accelerate the speed with which results are produced, the lysis buffer and pH of the colour indicator solution used in the Carba NP and CarbAcineto NP tests were changed.

In the MCNP test, the lysis buffers used for the Carba NP test and CarbAcineto NP test, B-PER II, Bacterial Protein Extraction Reagent and NaCl 5 M, respectively, were replaced by cetyl trimethyl ammonium bromide (CTAB) 0.02%, and the pH value of the phenol red solution was adjusted to 7.5 (instead of 7.8). In addition, two steps used in the previous protocols [5,6], centrifugation and incubation at room temperature for 30 minutes, were eliminated in our method. These modifications simplify the lysis step and produce results more quickly.

The MCNP test was performed as follows. One inoculation loop (10 μL) of the tested strain, directly recovered from a Mueller Hinton agar plate (bioMérieux, Marcy l'Étoile, France), was resuspended in 200 μL of 0.02% CTAB (Sigma-Aldrich Chimie, Saint-Quentin-Fallavier, France) and vortexed for 1 to 2 minutes. Subsequently, 100 μL of the bacterial suspension was mixed with 100 μL of diluted phenol red solution (2 mL of phenol red (Sigma-Aldrich) solution 0.5% (wt/vol) with 16.6 mL of distilled water) containing 0.1 mM ZnSO4 (pH 7.5) in the first tube, tube 1, used as negative control, and a diluted phenol red solution containing 0.1 mM ZnSO4 (pH 7.5) supplemented with 6 mg/mL of commercially available imipenem (Tienam 500; Merck Sharp & Dohme, Paris, France) in the second tube, tube 2. Tubes 1 and 2 were vortexed, then incubated at 37°C for a maximum of 2 hours.

Carbapenemase activity was revealed when the test and negative control solutions, respectively, were yellow vs. red or orange vs. red. In contrast, both solutions remained red in the case of noncarbapenemase producers (Fig. 1).

Fig. 1

Modified Carba NP test results for Enterobacteriaceae, Pseudomonas and Acinetobacter species. (A) Escherichia coli ATCC 25922. (B) NDM-5-positive E. coli. (C) KPC-2-positive Klebsiella pneumoniae 360. (D) IMP-1-positive Pseudomonas aeruginosa UAA 2257. (E) OXA-23-positive Acinetobacter baumannii. (F) OXA-24-positive A. baumannii. (G) NDM-1-positive A. baumannii. (a) Tube containing phenol red solution 0.1 mM ZnSO4 (pH 7.5) and cetyl trimethyl ammonium bromide (CTAB) 0.02%. (b) Tube containing phenol red solution 0.1 mM ZnSO4 (pH 7.5) supplemented with 6 mg/mL of imipenem and CTAB 0.02%.

The results showed that the MCNP method detected all carbapenemases produced by carbapenem-resistant strains with 100% sensitivity and 100% specificity. Positive results were observed at different times for the different carbapenemases types (MBL, KPC and OXA-48 at 10 to 30 minutes vs. 1 to 2 hours for OXA type). The most interesting aspect of this method is that the colour changed from red to orange or yellow (positive result) even before incubation in some cases (NDM-5-producing Escherichia coli, NDM-1-producing Klebsiella pneumoniae (Kpnasey) and imipenem-producing P. aeruginosa UAA2257). Moreover, a higher inoculums (two inoculation loops (10 μL)) is recommended for Acinetobacter species tests.

Currently, the MCNP test is routinely used in Timone Hospital, Marseille, France. It was performed when antibiotic susceptibility testing revealed a resistance to ertapenem and susceptibility or resistance to imipenem. The suspicion of carbapenemase producers, in particular OXA-48, was based on this phenotype. Between November 2014 and May 2015, a total of 233 strains were tested. Among them, 35 positives strains with carbapenemase producers were detected (Table 2). These positives strains were isolated from 25 different patients. These results confirm the efficiency of the MCNP test with high sensitivity, given the detection of all strains producing OXA-48-type carbapenemases. Also, two carbapenemase-producing A. baumannii were detected, thus confirming the advantages of the MCNP test.

Table 2

Results of MCNP test applied for carbapenem-resistant strains isolated in La Timone Hospital, Marseille, France

Date	Sample source	Strain	Antibiotic susceptibility testing results		MCNP result	Carbapenemases gene detected
			ETP	IMP
10/11/2014	Urine	Klebsiella pneumoniae	R	R	+	OXA-48
20/11/2014	Rectal swab	Escherichia coli	R	S	+	OXA-48
25/11/2014	Bronchoalveolar lavage fluid	K. pneumoniae	R	S	+	OXA-48
05/12/2014	Bronchoalveolar lavage fluid	K. pneumoniae	R	R	+	OXA-48
08/12/2014	Rectal swab	K. pneumoniae	R	S	+	OXA-48
09/12/2014	Blood culture	K. pneumoniae	R	R	+	OXA-48
11/12/2014	Urine	Enterobacter cloacae	R	S	+	OXA-48
21/12/2014	Stools	K. pneumoniae	R	S	+	OXA-48
31/12/2014	Spittle	K. pneumoniae	R	R	+	OXA-48
12/01/2015	Urine	E. coli	R	S	+	OXA-48
24/01/2015	Armpit swab	K. pneumoniae	R	S	+	OXA-48
02/02/2015	Rectal swab	K. pneumoniae	R	R	+	NDM
06/02/2015	Urine	E. coli	R	S	+	OXA-48
17/02/2015	Urine	E. coli	R	S	+	OXA-48
19/02/2015	Spittle	K. pneumoniae	R	S	+	OXA-48
20/02/2015	Rectal swab	K. pneumoniae	R	S	+	OXA-48
23/02/2015	Urine	K. pneumoniae	R	S	+	OXA-48
04/03/2015	Rectal swab	E. cloacae	R	R	+	OXA-48
04/03/2015	Rectal swab	K. pneumoniae	R	R	+	OXA-48
16/03/2015	Rectal swab	K. pneumoniae	R	S	+	OXA-48
23/03/2015	Bronchial aspirate	K. pneumoniae	R	S	+	OXA-48
30/03/2015	Armpit swab	K. pneumoniae	R	S	+	OXA-48
31/03/2015	Urine	K. pneumoniae	R	S	+	OXA-48
13/04/2015	Sinus	Serratia marcescens	R	R	+	OXA-48
13/04/2015	Blood culture	E. cloacae	R	S	+	OXA-48
18/04/2015	Blood culture	E. coli	R	I	+	OXA-48
18/04/2015	Rectal swab	K. pneumoniae	R	S	+	OXA-48
18/04/2015	Blood culture	E. coli	R	I	+	OXA-48
04/05/2015	Urine	K. pneumoniae	R	R	+	OXA-48
30/04/2015	Urine	K. pneumoniae	R	I	+	NDM-1
07/05/2015	Bronchial aspirate	K. pneumoniae	R	I	+	OXA-48
08/05/2015	Blood culture	Acinetobacter baumannii	NT	R	+	OXA-23
11/05/2015	Urine	A. baumannii	NT	R	+	OXA-23
18/05/2015	Rectal swab	K. pneumoniae	R	I	+	OXA-48

ETP, ertapenem; IMP, imipenem; MCNP, modified Carba NP test; NT, not tested; R, resistant; S, susceptible; I, Intermediate.

In conclusion, the advantages of the MCNP test are the detection of different carbapenemase types from Enterobacteriaceae, Pseudomonas and Acinetobacter species using a single protocol, as well as the short time to results, particularly in the case of MBL-producing Enterobacteriaceae and Pseudomonas species. In addition, the effectiveness of this test on a large series of bacteria may allow us to identify the production of carbapenemase enzymes even before identification of the bacterial strain.

Interestingly, as well as using this test in developed countries such as France (URMITE laboratories, La Timone Hospital), given the simplicity and the low cost of the MCNP test, it could be used by any laboratory, including laboratories in developing countries. In Algeria, this test has been used in the Microbial Ecology Laboratory of Béjaia University since May 2014, and it will soon be introduced to laboratories in Algerian hospitals.

Conflict of interest

None declared.