Rapid ResaImipenem/Acinetobacter NP Test for Detection of Carbapenem Susceptibility/Resistance in Acinetobacter baumannii

ABSTRACT

The rapid ResaImipenem/Acinetobacter NP test was developed for the identification of carbapenem resistance among Acinetobacter baumannii isolates. The principle of this test is based on the reduction of resazurin (a viability colorant) by metabolically active bacterial cells, hence detecting bacterial growth, in the presence of a defined concentration of imipenem chosen to be slightly above that defining imipenem resistance (6 μg/ml). Bacterial growth is visually detected by a color change from blue (resazurin) to purple or pink (resorufin product). A total of 110 A. baumannii isolates, among which 61 were imipenem resistant, were used to evaluate test performance. The sensitivity and specificity of the test were found to be 100%, in comparison with broth microdilution taken as the reference standard method. The rapid ResaImipenem/Acinetobacter NP test is highly specific and sensitive and is easy to implement in routine microbiology laboratories, and results are obtained within 2 h 30 min. It does not require any specific equipment.

INTRODUCTION

Acinetobacter baumannii belongs to the ESKAPE group of pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. baumannii, Pseudomonas aeruginosa, and Enterobacter species). Multidrug-resistant (MDR) A. baumannii is a significant nosocomial pathogen, especially in intensive care units, and is associated with prolonged hospitalization and increased mortality rates (1). Carbapenems are often considered to be drugs of choice for treating Acinetobacter-associated nosocomial infections (2), However, during the last few years, increasing reports of and outbreaks due to carbapenem-resistant Acinetobacter spp. represent a worldwide challenge. Carbapenem resistance in A. baumannii may be related to different mechanisms such as decreased permeability of the outer membrane due to porin loss/modification and/or the overexpression of efflux pumps, the production of carbapenemases (most cases), and, rarely, modifications of penicillin-binding proteins (1, 3). Noteworthy, and to the best of our knowledge, most of the A. baumannii isolates that are resistant to imipenem are concomitantly resistant to meropenem.

The carbapenemases identified in Acinetobacter spp. belong to Ambler class A (KPC or GES types), class B (VIM, IMP, SIM, and NDM types), or, mostly, class D enzymes. The latter enzymes, which are quite specific to Acinetobacter spp., are the main sources of acquired carbapenem resistance in Acinetobacter spp. The most common and acquired carbapenem-hydrolyzing class D β-lactamases (CHDLs) are OXA-23, OXA-24/40/72, and OXA-58 (1, 4, 5), whereas naturally occurring CHDLs in this species are of the OXA-51 type (OXA-51 and OXA-66), which may also be overexpressed (1, 4, 5).

The rapid, early, and accurate detection of carbapenem-resistant A. baumannii strains is critical in clinical microbiology to optimally adapt empirical treatment and also limit their further spread by the implementation of infection control measures. The determination of MICs by the broth microdilution (BMD) method (https://www.iso.org/standard/70464.html) is the currently used technique for their detection. However, this technique is time-consuming, requiring 18 h for obtaining results. Other phenotypic techniques, such as Etest strips, can be used, but they also require the same amount of time. Various selective culture media for the detection of carbapenem resistance among Acinetobacter isolates are available; however, these media suffer from some specificity and sensitivity issues, and they require an additional period of incubation of 24 h (6).

In this study, and in order to overcome these limitations of the currently available phenotypic tests, we developed a test, namely, the rapid ResaImipenem/Acinetobacter NP test, that may detect imipenem resistance among A. baumannii isolates, with corresponding results being obtained in only 2 h 30 min. The principle of this test is based on the reduction of resazurin (a viability colorant) by metabolically active bacterial cells, hence detecting bacterial growth, in the presence of a defined concentration of imipenem (6 μg/ml). Bacterial growth is visually detected by a color change from blue (resazurin) to purple or pink (resorufin product).

MATERIALS AND METHODS

Bacterial strains.

To evaluate the performance of the rapid ResaImipenem/Acinetobacter NP test, we used 110 well-characterized A. baumannii isolates from the Medical and Molecular Microbiology Unit, University of Fribourg, Switzerland (61 imipenem-resistant and 49 imipenem-susceptible A. baumannii isolates). They were obtained from various clinical sources and were of worldwide origins. These strains had previously been characterized at the molecular level for their β-lactamase content (Table 1). The imipenem-susceptible A. baumannii strain R1325 (wild type) and an imipenem-resistant A. baumannii OXA-23 strain (R639) were used as negative and positive controls, respectively, for the determination of MICs for imipenem and meropenem.

TABLE 1.

MICs of imipenem and meropenem and results of the rapid ResaImipenem/Acinetobacter NP test for the tested A. baumannii isolates^a

Isolate	Resistance determinant(s)	Geographical origin	Imipenem phenotype	BMD MIC range (μg/ml)		Rapid ResaImipenem/Acinetobacter NP test
				Imipenem	Meropenem	Result	Discrepancy with MIC imipenem result
R109	WTb	France	S	0.125	0.5	Susceptible	No
R112	WT	France	S	0.125	0.25	Susceptible	No
R1325	WT	Poland	S	0.25	0.5	Susceptible	No
R1386	WT	France	S	0.125	0.25	Susceptible	No
R1387	WT	France	S	0.125	0.5	Susceptible	No
R1388	WT	France	S	0.25	0.5	Susceptible	No
R1389	WT	France	S	0.125	0.5	Susceptible	No
R1390	WT	France	S	0.125	0.25	Susceptible	No
R1391	WT	France	S	0.125	0.25	Susceptible	No
R1392	WT	France	S	0.5	0.5	Susceptible	No
R1393	WT	France	S	0.125	0.5	Susceptible	No
R1394	WT	France	S	0.125	0.125	Susceptible	No
R1395	WT	France	S	0.125	0.125	Susceptible	No
R1396	WT	France	S	0.125	0.5	Susceptible	No
R1397	WT	Bahrain	S	0.125	0.25	Susceptible	No
R1402	WT	France	S	0.5	0.5	Susceptible	No
R2201	WT	France	S	0.125	0.125	Susceptible	No
R2513	WT	Hong Kong	S	0.125	0.125	Susceptible	No
R2464	WT	France	S	0.25	0.25	Susceptible	No
R2270	WT	France	S	0.125	0.125	Susceptible	No
N710	WT	Switzerland	S	0.5	0.5	Susceptible	No
N862	WT	Switzerland	S	0.25	0.5	Susceptible	No
N891	WT	Switzerland	S	1	0.5	Susceptible	No
N982	WT	Switzerland	S	1	1	Susceptible	No
R1322	OXA-51 overproduced (ISAba1-blaOXA-51)	France	S	2	2	Susceptible	No
R1323	OXA-51 overproduced (ISAba1-blaOXA-51)	France	S	2	2	Susceptible	No
N25	OXA-51 overproduced (ISAba1-blaOXA-51)	Switzerland	S	2	8	Susceptible	No
R5	OXA-51 overproduced (ISAba1-blaOXA-51)	France	S	2	2	Susceptible	No
R820	OXA-51 overproduced (ISAba1-blaOXA-51)	Bahrein	S	2	2	Susceptible	No
N58	OXA-51 overproduced (ISAba1-blaOXA-51)	Switzerland	S	2	8	Susceptible	No
R854	OXA-51-like overproduced (ISAba9-blaOXA-51)	Greece	S	1	2	Susceptible	No
R1398	AmpC overproduced (ISAba1-blaampC)	France	S	2	2	Susceptible	No
R1399	AmpC overproduced (ISAba1-blaampC)	France	S	2	2	Susceptible	No
R1400	AmpC overproduced (ISAba1-blaampC)	France	S	1	2	Susceptible	No
R1401	AmpC overproduced (ISAba1-blaampC)	France	S	2	2	Susceptible	No
R150	CTX-M-15	France	S	0.125	0.5	Susceptible	No
R1139	PER-1	Turkey	S	0.25	1	Susceptible	No
R1149	VEB-1a	Argentina	S	0.25	0.5	Susceptible	No
R1150	VEB-1a	Argentina	S	0.25	0.25	Susceptible	No
R1151	VEB-1a	Argentina	S	0.25	0.5	Susceptible	No
R1152	VEB-1a	Argentina	S	0.25	0.5	Susceptible	No
R152	VEB-1	France	S	0.5	0.5	Susceptible	No
R1144	VEB-1	France	S	0.25	0.5	Susceptible	No
R1145	VEB-1 + OXA-51 overproduced (ISAba1-blaOXA-51)	France	S	0.5	0.5	Susceptible	No
R1146	VEB-1 + OXA-51 overproduced (ISAba1-blaOXA-51)	France	S	0.5	1	Susceptible	No
R1147	VEB-1 + OXA-51 overproduced (ISAba1-blaOXA-51)	France	S	2	1	Susceptible	No
R1148	VEB-1 + OXA-51 overproduced (ISAba1-blaOXA-51)	France	S	1	2	Susceptible	No
R1259	ESAC	France	S	0.5	0.5	Susceptible	No
R1141	RTG-4 (CARB10)	France	S	0.25	0.5	Susceptible	No
R1143	GES-12	France	R	32	>32	Resistant	No
R187	GES-14	France	R	32	32	Resistant	No
R71	IMP-4	France	R	16	>32	Resistant	No
R176	NDM-1	France	R	32	>32	Resistant	No
R35	NDM-1	France	R	32	32	Resistant	No
R41	NDM-1	France	R	16	>32	Resistant	No
R43	NDM-1	France	R	16	32	Resistant	No
R3392	NDM-1	France	R	>32	>32	Resistant	No
R27	NDM-2	France	R	16	16	Resistant	No
N739	NDM-5	Switzerland	R	>32	>32	Resistant	No
R16	OXA-143	France	R	32	>32	Resistant	No
R1324	OXA-23	Poland	R	16	16	Resistant	No
R2512	OXA-23	France	R	16	>32	Resistant	No
R3382	OXA-23	Turkey	R	32	>32	Resistant	No
R3383	OXA-23	Turkey	R	>32	>32	Resistant	No
R3384	OXA-23	Turkey	R	>32	>32	Resistant	No
R3385	OXA-23	Turkey	R	>32	>32	Resistant	No
R3386	OXA-23	Turkey	R	>32	>32	Resistant	No
R3387	OXA-23	Turkey	R	>32	>32	Resistant	No
R3388	OXA-23	France	R	>32	>32	Resistant	No
R3389	OXA-23	Switzerland	R	>32	>32	Resistant	No
R3390	OXA-23	Switzerland	R	>32	>32	Resistant	No
R637	OXA-23	France	R	>32	>32	Resistant	No
R639	OXA-23	France	R	16	16	Resistant	No
R669	OXA-23	France	R	>32	>32	Resistant	No
R670	OXA-23	France	R	>32	>32	Resistant	No
R676	OXA-23	France	R	32	>32	Resistant	No
R622	OXA-23	France	R	>32	>32	Resistant	No
R623	OXA-23	Saudi Arabia	R	>32	>32	Resistant	No
R624	OXA-23	Colombia	R	>32	>32	Resistant	No
R625	OXA-23	France	R	>32	>32	Resistant	No
R626	OXA-23	Bahrein	R	>32	>32	Resistant	No
R695	OXA-24	France	R	>32	>32	Resistant	No
R696	OXA-24	France	R	>32	>32	Resistant	No
R697	OXA-25	France	R	16	16	Resistant	No
R698	OXA-25	Spain	R	>32	>32	Resistant	No
R699	OXA-26	France	R	32	32	Resistant	No
R700	OXA-26	Belgium	R	>32	>32	Resistant	No
R701	OXA-27	France	R	8	16	Resistant	No
R3391	OXA-40	Switzerland	R	32	>32	Resistant	No
R3393	OXA-40	Italy	R	>32	>32	Resistant	No
R3397	OXA-40	Switzerland	R	>32	>32	Resistant	No
R703	OXA-40	France	R	>32	>32	Resistant	No
R704	OXA-40	France	R	>32	>32	Resistant	No
N14	OXA-40	Switzerland	R	>32	>32	Resistant	No
N774	OXA-40	Switzerland	R	>32	>32	Resistant	No
R215	OXA-40	France	R	>32	>32	Resistant	No
R14	OXA-40	France	R	>32	>32	Resistant	No
R3398	OXA-51 overproduced (ISAba1-blaOXA-51)	France	R	6	4	Resistant	No
R817	OXA-51 overproduced (ISAba1- + ISAba9-blaOXA-51)	Greece	R	8	3	Resistant	No
R1142	SHV-5 + OXA-51 overproduced (ISAba1-blaOXA-51)	France	R	16	16	Resistant	No
N211	OXA-51 overproduced (ISAba1-blaOXA-51) + impermeability + efflux pump	Switzerland	R	8	6	Resistant	No
N671	OXA-51 overproduced (ISAba1-blaOXA-51) + impermeability + efflux pump	Switzerland	R	6	16	Resistant	No
R842	OXA-58	France	R	32	>32	Resistant	No
R843	OXA-58	France	R	>32	>32	Resistant	No
R845	OXA-58	France	R	16	32	Resistant	No
R857	OXA-72	France	R	>32	>32	Resistant	No
R858	OXA-72	Bahrain	R	>32	>32	Resistant	No
R859	OXA-72	Bahrain	R	>32	>32	Resistant	No
R860	OXA-72	Bahrain	R	>32	>32	Resistant	No
R3	OXA-97 (OXA-58-like)	France	R	32	>32	Resistant	No

^aS, susceptible; R, resistant; BMD, broth microdilution; WT, wild type; ESAC, extended-spectrum AmpC. The results for strains with MIC values of imipenem of 6 mg/liter were obtained after 3 h of incubation.

^bNonoverexpressed OXA-51-like β-lactamases are naturally occurring and chromosome-borne carbapenemases in A. baumannii.

Susceptibility testing.

Antimicrobial susceptibility testing was performed by determining MIC values with the reference broth microdilution method using cation-adjusted Mueller-Hinton (MH) broth (Bio-Rad, Marnes-la-Coquette, France). This method was considered the standard for comparison with the results obtained with the rapid ResaImipenem/Acinetobacter NP test. All experiments were repeated in triplicate in separate experiments. The latest breakpoints from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) were used as a reference (www.eucast.org/clinical_breakpoints). Isolates were categorized as susceptible when MICs of imipenem were ≤2 μg/ml and resistant when MICs were >4 μg/ml.

Rapid ResaImipenem/Acinetobacter NP test.

We first tested different parameters using two imipenem-susceptible isolates (A. baumannii R109 and A. baumannii R1325) and two imipenem-resistant isolates (A. baumannii R639 and A. baumannii R35) for this preliminary testing. These parameters included four different bacterial inocula (0.5, 1, 1.5, and 3 McFarland standards), two different resazurin dyes (alamarBlue and PrestoBlue; Thermo Fisher Scientific, Waltham, MA, USA), two different growth media (Luria-Bertani [LB] medium [Sigma, St. Louis, MO] and MH agar plates [Bio-Rad, Marnes-la-Coquette, France]), and, finally, four incubation durations (1 h, 2 h, 2 h 30 min, and 3 h). After comparison of the results with these different parameters, all experiments were performed in triplicate by two different persons with the optimal conditions obtained, as described below.

Preparation of solutions.

All the testing solutions were made in MH medium supplemented or not with imipenem monohydrate (Sigma-Aldrich) at a fixed final concentration of 6 μg/ml.

Bacterial inoculum preparation.

For each isolate to be tested, including the positive and negative controls, a standardized bacterial inoculum (1 McFarland standard) was prepared by using fresh bacterial colonies grown overnight on LB or MH plates. The bacterial colonies were resuspended into 5 ml of sterile NaCl (0.85%) to obtain a 1 McFarland standard optical density. A. baumannii R639 was used as a positive control (resistant isolate; MICs of imipenem and meropenem at 16 and 16 μg/ml, respectively), and A. baumannii R1325 was used as a negative control (susceptible isolate; MICs of imipenem and meropenem at 0.25 and 0.5 μg/ml, respectively). The bacterial suspensions were used within 15 min to 1 h after preparation, as recommended by EUCAST guidelines for susceptibility testing (www.eucast.org).

Tray inoculation.

Testing was performed in a 96-well polystyrene microplate (round base with lid, sterile; Sarstedt, Nuembrecht, Germany). For each isolate, the bacterial suspension was inoculated in parallel into two wells, with and without imipenem, in separate wells. The rapid ResaImipenem/Acinetobacter NP test was performed as follows: 180 μl of an imipenem-free MH solution was transferred to wells A1, B1, C1, and D1, and 180 μl of an imipenem-containing MH solution (6.67 μg/ml to obtain a final concentration of 6 μg/ml) was transferred to wells A2, B2, C2, and D2. Next, 20 μl of 0.85% NaCl was added to wells A1 and A2, 20 μl of the imipenem-susceptible isolate suspension used as a negative control was added to wells B1 and B2, 20 μl of the imipenem-resistant isolate suspension used as a positive control was added to wells C1 and C2, and 20 μl of the bacterial suspension to be tested was added to wells D1 and D2. For the previous steps, the bacterial suspension was mixed with the medium by pipetting up and down. Finally, the resazurin reagent PrestoBlue was added to each well at a concentration of 10% (vol/vol) (i.e., 20 μl per well), and each well was mixed by pipetting up and down.

Tray incubation.

The inoculated tray was incubated at 35°C ± 2°C for 2 h 30 min in ambient air without sealing or shaking.

Tray reading.

The tray was visually inspected every 30 min and then once per hour for 2 h 30 min. The test was considered positive (i.e., purple or pink color of the culture medium, indicating imipenem resistance) if the tested isolate grew in the presence of imipenem and negative (i.e., blue color of the culture medium, indicating susceptibility to imipenem) if the tested isolate did not grow in the presence of imipenem.

Results were considered interpretable if the following conditions were met: (i) both wells (A1 and A2) with 0.85% NaCl, without a bacterial suspension, remained unchanged (blue, indicating the absence of medium contamination); (ii) imipenem-free wells B1, C1, and D1 with a bacterial suspension turned from blue to purple or pink, confirming the growth and, thus, the viability of the isolate cells; (iii) well B2 with the imipenem-susceptible bacterial suspension (negative control) gave negative results (remaining blue), confirming the absence of growth of this isolate; (iv) well C3 with the imipenem-resistant bacterial suspension (positive control) gave a positive result (purple/pink), confirming the viability of the isolate in the presence of imipenem; and (v) the tested isolate that grew in the absence and the presence of imipenem (wells D1 and D2) was therefore reported to be imipenem resistant, or the tested isolates that grew in the absence but did not grow in the presence of imipenem were therefore reported to be imipenem susceptible. Results were also interpreted by a technician in a blind way.

Result analysis.

Results obtained with the rapid ResaImipenem/Acinetobacter NP test were compared to those obtained with the reference BMD method. Briefly, discrepancies were determined to assess the performances of the test to detect imipenem resistance. Errors (very major errors [VMEs] and major errors [MEs]) were calculated as described previously (7). A VME was considered when isolates were categorized as susceptible using the rapid ResaImipenem/Acinetobacter NP test but were resistant by the BMD method (false susceptibility result). An ME was considered when isolates were found to be resistant using the rapid ResaImipenem/Acinetobacter NP test but were susceptible by using the BMD method (false resistance result).

RESULTS

After comparison of the results with different parameters (different bacterial inocula, different resazurin dyes, different growth media, and different incubation durations) using two reference imipenem-susceptible isolates and two reference imipenem-resistant isolates, all experiments were performed in triplicate with the optimal conditions obtained to get the best endpoint for the test, as described above. Preliminary testing showed excellent results of the rapid ResaImipenem/Acinetobacter NP test, for both reference susceptible and resistant isolates, in comparison to broth microdilution taken as the reference standard method, and results are obtained within 2 h 30 min. A total of 110 A. baumannii isolates were used to evaluate the performance of the rapid ResaImipenem/Acinetobacter NP test (Table 1). All 49 imipenem-susceptible A. baumannii isolates (MICs of imipenem ranging from less than 0.125 to 2 μg/ml), according to the results of the BMD method, were identified as susceptible by the rapid ResaImipenem/Acinetobacter NP test (Table 1). The 61 imipenem-resistant A. baumannii isolates (MICs of imipenem ranging from 8 to >64 μg/ml) gave positive results (imipenem resistant) with the rapid ResaImipenem/Acinetobacter NP test (Table 1). All 61 imipenem-resistant and 49 imipenem-susceptible A. baumannii isolates were correctly identified as imipenem resistant and imipenem susceptible, respectively, by using the rapid ResaImipenem/Acinetobacter NP test (Table 1). Overall, no VMEs (false susceptibility) or MEs (false resistance) were observed. Therefore, there was an excellent agreement between the results of the reference BMD susceptibility test and those of the rapid ResaImipenem/Acinetobacter NP test, for both susceptible and resistant isolates. Under our conditions, the sensitivity and specificity were found to be 100%. All positive results were observed in a maximum of 2 h 30 min.

DISCUSSION

The present study showed that the rapid ResaImipenem/Acinetobacter NP test is a rapid and reliable test, combining excellent sensitivity and specificity. This test was able to detect imipenem resistance and subsequently carbapenem resistance as well (meaning cross-resistance to imipenem and meropenem) among A. baumannii isolates, regardless of their resistance mechanisms, within 2 h 30 min. Such a time frame is at least 16 h earlier (meaning 1 day earlier from a practical point of view) than with the reference BMD method or other phenotypic techniques such as Etest strips. This test is highly specific and sensitive, reliable, affordable, and easy to implement in routine microbiology laboratories. It allows a very rapid susceptibility/resistance categorization, which is the information needed for the implementation of an adequate carbapenem-containing treatment. The use of such a rapid accurate test may also contribute to improving the rapid implementation of infection control measures taking into account that virtually all imipenem-resistant A. baumannii strains actually produce a carbapenemase. Further studies with a broader set of resistant or susceptible Acinetobacter species isolates will be needed to further validate the accuracy of this test.