Carbapenem Disks on MacConkey Agar in Screening Methods for Detection of Carbapenem-Resistant Gram-Negative Rods in Stools

Abstract

Direct plating of simulated stool specimens on MacConkey agar (MCA) with 10-μg ertapenem, meropenem, and imipenem disks allowed the establishment of optimal zone diameters for the screening of carbapenem-resistant Gram-negative rods (CRGNR) of ≤24 mm (ertapenem), ≤34 mm (meropenem), and ≤32 mm (imipenem).

TEXT

Screening of stool specimens is recommended by the Centers for Disease Control and Prevention as well as the Institut national de santé publique du Québec to identify carriers of carbapenem-resistant Gram-negative rods (CRGNR) and initiate appropriate infection control measures (1, 2). Lolans and colleagues reported that an ertapenem zone diameter of ≤27 mm on MacConkey agar (MCA) was highly sensitive for the detection of Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae in rectal swab specimens (3). However, the zone diameter interpretive criteria for imipenem and meropenem placed directly on MCA have not yet been established. This study compares the performances of the screening method using MCA and 10-μg carbapenem disks (ertapenem, meropenem, and imipenem) and defines the optimal inhibition zone diameters for detecting CRGNR using simulated stool specimens.

Thirty-nine clinical isolates have been well characterized, phenotypically and genotypically, as described in Table 1. Twenty carbapenemase-producing isolates (17 Enterobacteriaceae and 3 nonfermenters) were selected based on the presence of genes coding for different carbapenemases. Nineteen non-carbapenemase-producing Enterobacteriaceae (18 extended-spectrum β-lactamase [ESBL]- or plasmid-mediated AmpC [pAmpC] lactamase-producing isolates) and a susceptible wild-type Escherichia coli strain were also selected as negative controls. The MICs of ceftazidime, cefotaxime, ertapenem, meropenem, and imipenem were determined by the microdilution method according to the Clinical and Laboratory Standards Institute (4).

Table 1.

Characteristics of the bacterial isolates tested at the Laboratoire de santé publique du Québec and National Microbiology Laboratory, Public Health Agency of Canada^a

Isolate	Type of β-lactamase	Gene	MIC (μg/ml) of:					Inhibition zone diam (mm) at 102 dilution with:
			CAZ	CTX	ERTA	MERO	IMI	ERTA	MERO	IMI
Citrobacter freundii	Carbase	blaKPC	>64	>32	16	16	16	16	23	26
Enterobacter cloacae	Carbase	blaNMC	0.5	1	16	16	32	20	30	25
Enterobacter cloacae	Carbase	blaKPC	>64	>32	8	4	4	17	28	28
Escherichia coli	Carbase	blaKPC	>64	>32	8	4	16	21	34	30
Escherichia coli	Carbase	blaKPC	>64	>32	4	4	4	20	30	31
Escherichia coli	Carbase	blaKPC	>64	>32	4	4	8	20	32	27
Escherichia coli	Carbase	blaKPC	>64	>32	4	4	16	26	33	32
Klebsiella pneumoniae	Carbase	blaKPC	>64	>32	>32	>32	32	13	22	29
Klebsiella pneumoniae	Carbase	blaKPC	>64	>32	>32	>32	>32	15	14	25
Klebsiella pneumoniae	Carbase	blaKPC	>64	>32	>32	32	32	18	29	26
Klebsiella pneumoniae	Carbase	blaKPC	>64	>32	32	32	32	31	15	24
Klebsiella pneumoniae	Carbase	blaKPC	>64	>32	>32	>32	>32	6	16	21
Klebsiella pneumoniae	Carbase	blaOXA-48	1	1	4	2	8	20	27	30
Klebsiella pneumoniae	Carbase	blaNDM-1	>64	>32	>32	>32	>32	6	15	20
Klebsiella pneumoniae	Carbase	blaNDM-1	64	32	32	32	32	13.5	25	30
Klebsiella oxytoca	Carbase	blaKPC	>64	>32	>32	16	16	20	27	32
Serratia marcescens	Carbase	blaKPC	16	8	8	16	16	20	32	32
Acinetobacter baumanii	Carbase	blaIMP-4	>64	>32	>32	>32	>32	9	20	24
Acinetobacter baumanii	Carbase	blaOXA-23, blaOXA-51	>64	>32	>32	>32	>32	16	11	16
Pseudomonas aeruginosa	Carbase	blaVIM-2	>64	>32	>32	>32	>32	12	20	11
Escherichia coli	ESBL	blaTEM-26	>64	4	0.06	<0.03	0.25	33	44	43
Escherichia coli	ESBL	blaTEM-1, blaDHA	32	32	<0.03	<0.03	0.12	38	48	40
Escherichia coli	ESBL	blaSHV-2a	8	4	<0.03	<0.03	0.12	40	40	44
Escherichia coli	ESBL	blaTEM-1, blaCTX-M	>64	>32	0.12	0.12	0.25	33	41	40
Escherichia coli	ESBL	blaCTX-M	>64	>32	<0.03	<0.03	0.25	36	44	43
Escherichia coli	ESBL	blaTEM-19	4	4	<0.03	<0.03	0.25	39	40	41
Klebsiella pneumoniae	ESBL	blaSHV-11, blaCTX-M	>64	>32	4	0.06	0.25	26	38	40
Klebsiella pneumoniae	ESBL	blaSHV-18	>64	8	0.06	0,06	0,12	34	44	43
Klebsiella pneumoniae	ESBL	blaSHV-5	0.5	0.06	<0.03	0.06	0.5	36.5	44	47
Citrobacter freundii	pAmpC	blaCMY-2	0.25	0.12	<0.03	<0.03	0.5	39	46	43
Escherichia coli	pAmpC	blaTEM-1, blaCMY-2	64	8	0.06	<0.03	0.5	32	42	37
Escherichia coli	pAmpC	blaCMY-2	>64	16	0.12	0.06	0.5	34	44	38
Klebsiella pneumoniae	pAmpC	blaSHV-1, blaCMY-2	>64	16	0.25	0.06	0.5	28	40	41
Klebsiella pneumoniae	pAmpC	blaSHV-1, blaFOX	>64	16	0.06	0.06	0.12	38	45	44
Morganella morganii	pAmpC	blaDHA	<0.06	0.06	0.06	0.25	4	41	49	37
Proteus mirabilis	pAmpC	blaCMY-2	8	8	1	1	4	36	44	38
Proteus mirabilis	pAmpC	blaCMY-2	64	32	0.5	4	32	35	38	35
Escherichia coli	None	None	1	0.06	<0.03	<0.03	0.25	36	46	46

^aCarb_ase, carbapenemase; ESBL, extended-spectrum β-lactamase; pAmpC, plasmid-mediated AmpC β-lactamase; CAZ, ceftazidime; CTX, cefotaxime; ERTA, ertapenem; MERO, meropenem; IMI, imipenem.

A stool specimen obtained from a normal volunteer was used to prepare all the simulated clinical specimens. To ensure that the specimen did not harbor any β-lactam-resistant bacteria, screening tests were performed using ChromID ESBL, CHROMagar KPC, and MCA with ertapenem, meropenem, and imipenem disks. Each plate was inoculated with 100 μl of liquefied stool and incubated 24 h aerobically at 35°C. There was no growth on the two selective chromogenic agar plates. For the MCA, inhibition diameters around the antibiotic disks were 29 mm, 39 mm, and 35 mm for ertapenem, meropenem, and imipenem, respectively.

Dilutions of the 39 isolates were mixed with aliquots of stool. The simulated fecal material was inoculated onto the screening MCA medium to obtain final challenge concentrations of 10⁴ to 10¹ CFU/ml for each strain. The fecal inoculum was spread on MCA by rotation using a rake spreader, and disks of the carbapenems were individually placed onto MCA. After incubation, the diameters of the inhibition zones around each carbapenem disk were measured.

The results of all inhibition diameters obtained with the 4 dilutions tested for each strain were used to construct a receiver operating characteristic (ROC) curve. A zone diameter breakpoint was identified in order to maximize the sensitivity and specificity for the detection of CRGNR for each carbapenem disk. All ROC curve analyses followed the methodology of DeLong et al., and the binomial exact test for the determination of confidence interval for the area under the ROC curve was used (5). To evaluate the relative performances of the 3 different carbapenem disks, the ROC curves were compared using a pairwise comparison. Statistical analysis was conducted using MedCalc software version 12.1.0 (Mariakerke, Belgium). For all statistical tests, significance was set at an alpha value of 0.05 and a 95% confidence interval (95% CI).

The optimal breakpoints for the CRGNR screening on MCA were zone diameters of ≤24 mm for ertapenem, ≤34 mm for meropenem, and ≤32 mm for imipenem. The areas under the ROC curve were 0.94 with a 95% CI (0.89 to 0.97) for ertapenem (P value, <0.0001), 0.92 with a 95% CI (0.87 to 0.96) for meropenem (P value, <0.0001), and 0.90 with a 95% CI (0.84 to 0.94) for imipenem (P value, <0.0001) (Fig. 1). There was no statistical difference when comparing the ROC curves of the three carbapenems: ertapenem versus imipenem (P value, 0.14), ertapenem versus meropenem (P value, 0.40), and meropenem versus imipenem (P value, 0.42). Using the optimal breakpoint identified for each carbapenem disk, the respective sensitivity and specificity for each dilution tested were also calculated as shown in Table 2.

Fig 1.

ROC curves for zones of inhibition around a 10-μg ertapenem disk (A), 10-μg meropenem disk (B), and 10-μg imipenem disk (C) for 39 challenge strains at different dilutions (10¹, 10², 10³, and 10⁴ CFU/ml).

Table 2.

Sensitivity and specificity of zone diameters around a 10-μg ertapenem (≤24 mm), meropenem (≤34 mm), or imipenem (≤32 mm) disk for the detection of CRGNR^a

Dilution (CFU/ml)	Ertapenem		Meropenem		Imipenem
	Sen (%)	Spe (%)	Sen (%)	Spe (%)	Sen (%)	Spe (%)
101	55.0	89.5	52.5	94.7	40	100
102	92.5	92.1	95.0	94.7	100	94.7
103	100.0	89.5	100.0	94.7	100	89.5
104	100.0	84.2	100,0	94.7	100	89.5
All dilutions	86.3	90.8	88.8	94.7	85.0	93.4

^aSen, sensitivity; Spe, specificity.

The use of ROC curve analysis has allowed the establishment of an optimal zone diameter for each carbapenem disk that discriminates fairly well between CRGNR and non-CRGNR. However, comparison of each curve did not demonstrate a significant statistical superiority of any of the carbapenem disks for the detection of CRGNR in stools. We might have been able to show a difference in performance among the three disks if we had tested a larger number of strains.

A major strength of our study is the diversity of the carbapenemase enzymes produced by the strains tested. Furthermore, the CRGNR displayed a wide range of carbapenem MICs. Using standardized inocula, we were also able to identify breakpoint diameters for screening CRGNR in stools with the ertapenem, meropenem, or imipenem disk (respectively, ≤24 mm, ≤34 mm, and ≤32 mm). Our study has some limitations. First, the species selected for this study may not correspond to those that would be isolated from clinical specimens. Second, we used standardized dilutions of collection strains (10¹, 10², 10³, and 10⁴ CFU/ml) and the correlation with real specimen inoculum concentrations in patient stools or rectal swabs remains unknown. Third, screening for CRGNR by direct carbapenem disk testing using the breakpoints established was not performed on rectal swab specimens, owing to the very low prevalence of CRGNR in our hospital setting. Therefore, this might limit the external validity of our results. We should also underline the fact that our population of strains contained a very high proportion of CRGNR (51.3%). The overrepresentation of CRGNR may nevertheless underestimate the negative predictive value, given that this parameter varies according to prevalence, which is usually the most important value for a screening test.

In conclusion, screening stool specimens for CRGNR using a direct carbapenem disk method on MCA is reliable and easy to perform. Actually, the main limitation of this method is its poor sensitivity when a CRGNR is present at a low concentration (10¹ CFU/ml). To maximize the likelihood of finding CRGNR, two carbapenem disks per plate could be used. Our results suggest that the carbapenem disk method can be a useful tool for screening CRGNR in stool specimens.