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Different antimicrobial susceptibility testing methods to detect ertapenem resistance in enterobacteriaceae: VITEK2, MicroScan, Etest, disk diffusion, and broth microdilution
Journal of Microbiological Methods 112 (2015) 87–91
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Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Different antimicrobial susceptibility testing methods to detect ertapenem resistance in enterobacteriaceae: VITEK2, MicroScan, Etest, disk diffusion, and broth microdilution Miae Lee, Hae-Sun Chung ⁎ Department of Laboratory Medicine, Ewha Womans University School of Medicine, Republic of Korea
a r t i c l e
i n f o
Article history: Received 7 January 2015 Received in revised form 16 March 2015 Accepted 16 March 2015 Available online 17 March 2015 Keywords: Ertapenem Enterobacteriaceae Antimicrobial susceptibility test Antimicrobial resistance
a b s t r a c t We investigated different antimicrobial susceptibility testing methods to detect ertapenem resistance in Enterobacteriaceae. A total of 72 Enterobacteriaceae isolates were collected from a clinical microbiology laboratory of a tertiary university hospital, all of which were detected ertapenem resistance by the VITEK2 system. Bacterial identification and antimicrobial susceptibility were determined using the VITEK2. Ertapenem susceptibility test was performed using the MicroScan, Etest and a disk diffusion test. Ertapenem MICs were confirmed using the broth microdilution (BMD). Sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) of each method for the detection of ertapenem resistance were calculated. Carbapenemases and AmpC β-lactamase were screened using phenotypic methods. Among the 72 isolates, 20 isolates (27.8%) were resistant to ertapenem. Etest showed high sensitivity and specificity (85.0% and 88.5%, respectively) and excellent concordance with BMD. The disk diffusion test had the lowest sensitivity of 50.0%. The VITEK2 showed the lowest essential and categorical agreement (30.5% and 27.8%, respectively). The MicroScan showed relatively good agreement with BMD compared to the VITEK2. Most category disagreements were minor errors. There were 3 very major errors in both the MicroScan and disk diffusion test. Only 1 isolate was positive for carbapenemase screening test and all of the isolates were positive for AmpC screening test. In conclusion, the detection of ertapenem resistance in Enterobacteriaceae has limitations using routine testing such as an automated system or disk diffusion. Confirmation of results by an additional MIC test is recommended for accurate resistance results of ertapenem. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Carbapenem-resistant Enterobacteriaceae (CRE) has emerged and been threatening the successful treatment of major infection (CDC, 2012; Schwaber and Carmeli, 2008; van Duin et al., 2013). Invasive CRE infections are associated with poorer outcomes compared to carbapenem-susceptible Enterobacteriaceae infections, which is likely due in part to limited treatment options (Schwaber and Carmeli, 2008; van Duin et al., 2013). Very few options remain for the treatment of these carbapenem-resistant organisms. Ertapenem is a carbapenem developed to overcome the pharmacokinetic shortcomings (short half-life) of imipenem and meropenem. The extensive protein binding of ertapenem extends the half-life and allows once-daily dosing (Nix et al., 2004; Zhanel et al., 2005, 2007). Ertapenem has been widely used since the early 2000s, but it was only recently introduced to the routine antimicrobial susceptibility ⁎ Corresponding author at: Department of Laboratory Medicine, Ewha Womans University School of Medicine, 1071, Anyangcheon-ro, Yangcheon-gu, Seoul 158-710, Republic of Korea. E-mail address: [email protected] (H.-S. Chung).
http://dx.doi.org/10.1016/j.mimet.2015.03.014 0167-7012/© 2015 Elsevier B.V. All rights reserved.
tests performed by automated systems. Since then, ertapenemresistant Enterobacteriaceae isolates have been detected (Behera et al., 2009; Wu et al., 2011). Antimicrobial susceptibilities can be tested using various methods. In clinical microbiology laboratories, especially those in large tertiary hospitals, antimicrobial susceptibility tests are performed using commercially available automated systems, including the VITEK2 (bioMérieux) and MicroScan (Siemens) systems. The Etest and disk diffusion test are also relatively easy to use. Broth microdilution (BMD) is a well-established, standard method, but it is too laborintensive for use in clinical laboratories (Versalovic et al., 2011; Garcia and Isenberg, 2010). Antimicrobial susceptibility testing for ertapenem can be performed using all of the above methods; however, many laboratories lack the essential experience to do so, and there is little data comparing the efficacies of the different methods (Pailhories et al., 2014; Samuel et al., 2005; Vading et al., 2011). The breakpoints for ertapenem have changed in recent years. In June 2010, the Clinical and Laboratory Standards Institute (CLSI) published new interpretive criteria for carbapenems of Enterobacteriaceae (CLSI, 2010). Two years later, in January 2012, the CLSI revised the breakpoints for ertapenem (CLSI M100-S22, 2012a). However, there have been little
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M. Lee, H.-S. Chung / Journal of Microbiological Methods 112 (2015) 87–91
data of evaluation of antimicrobial susceptibility tests for isolates close to the new breakpoints. In this study, we investigated different antimicrobial susceptibility testing methods including the VITEK2, MicroScan, BMD, Etest, and disk diffusion tests to detect ertapenem resistance in Enterobacteriaceae isolates from a clinical microbiology laboratory at a tertiary university hospital. 2. Methods 2.1. Isolates Clinical Enterobacteriaceae isolates were collected from a clinical microbiology laboratory of a tertiary university hospital, all of which were detected ertapenem resistance by the VITEK2 system during August 2012 and July 2013. A total of 72 Enterobacteriaceae isolates were analyzed; 43 (59.7%) Enterobacter spp., 13 (18.1%) Klebsiella pneumoniae, 8 (11.1%) Escherichia coli, 3 (4.2%) Citrobacter freundii, 2 (2.8%) Serratia marcescens, and 3 others (4.2%). Bacterial identification was performed using the VITEK2 with the GN card (bioMérieux). 2.2. Antimicrobial susceptibility test 2.2.1. Interpretive criteria and quality control Susceptibility results were interpreted using the CLSI guideline recommended in 2013: ≤0.5 μg/mL for susceptible, 1 μg/mL for intermediate, and ≥2 μg/mL for resistant (CLSI, 2013). Enterococcus faecalis ATCC 29212, E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as controls for BMD, Etest and disk diffusion test. Quality controls for the two automated systems were performed according to the manufacturers instructions. 2.2.2. VITEK2 Antimicrobial susceptibility was tested by the VITEK2 using ASTN224 card (bioMérieux), according to the manufacturer's instructions. The MIC range for ertapenem on the AST-N224 card was ≤0.25 μg/mL to ≥ 8 μg/mL in doubling dilutions. Colonies from an overnight agar plate culture of each isolate were suspended in 3 mL of 0.45% saline and adjusted to a turbidity of 0.5 McFarland standard with VITEK Densicheck (bioMérieux). 2.2.3. MicroScan Antimicrobial susceptibility was tested by MicroScan WalkAway 96 SI instrument using the Neg MIC 37 panel (Siemens) containing, according to the manufacturer's instructions. The MIC range for ertapenem on the Neg MIC 37 panel was ≤ 0.5 μg/mL to N4 μg/mL in doubling dilutions. Colonies from an overnight agar plate culture of each isolate were suspended in 3 mL of distilled water and adjusted to a turbidity of 0.5 McFarland standard with MicroScan Turbidity Meter (Siemens). 2.2.4. Etest The MICs for ertapenem were determined with Etest (bioMérieux) carried out on Mueller-Hinton agar (ASAN Pharm.), according to the manufacturer's instructions. Etest strips were applied when the agar surface was completely dry. The strips were incubated at 35 °C for 18 h. The MIC was determined as the point where inhibition of the growth intersected with the Etest strip. The Etest MIC test range was 0.002 μg/mL to 32 μg/mL. For categorical interpretation and comparison, Etest MICs between standard dilutions were rounded up to the nearest 2-fold BMD dilution. 2.2.5. Disk diffusion test Disk diffusion susceptibility testing was performed using commercial ertapenem disks (Becton Dickinson) containing 10 μg and Muller-Hinton agar. Isolates with a zone diameter ≤ 18 mm were considered to be
resistant, those having a zone diameter of 19–21 mm were intermediate, and those with a zone diameter of ≥22 mm were susceptible (CLSI, 2013). 2.2.6. Broth microdilution (BMD) BMD was performed by using 96-well broth microdilution panels according to the CLSI guidelines (CLSI M07-A9, 2012b). Solvent and diluent for preparation of stock solutions of ertapenem were prepared as described in the CLSI document, in cation-adjusted Mueller-Hinton broth (Becton Dickinson). The BMD MIC test range was 0.25 μg/mL to 128 μg/mL. The results of BMD were considered the reference standard against which all other results were compared. 2.3. Carbapenemase and AmpC screening test For carbapenemase detection, a modified Hodge test (MHT) and a carbapenemase inhibition test (Rosco Diagnostica) were performed. AmpC detection was done by cefoxitin-boronic acid disk synergy test (Coudron, 2005). 2.4. Data analysis The results of different antimicrobial susceptibility testing methods were compared to those of BMD. Sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) of each method for the detection of ertapenem resistance were calculated to evaluate performance. Agreement was assessed in two manners: category (qualitative) and MIC (quantitative) comparison. Category disagreements were classified as follows. A minor error was listed when an isolate was categorized as intermediate by one test method but either susceptible or resistant by another test method. A major error occurred when the isolate was categorized as being resistant by the test method but susceptible by the reference method (false-resistant result). A very major error happened when the isolate was categorized as susceptible by the test method but resistant by the reference method (false-susceptible result). If the MIC of the test method was within a single 2-fold dilution (±1 doubling dilution) of the reference result, then the MIC for that isolate was defined as being in agreement (essential agreement) (Garcia and Isenberg, 2010). To compare MICs, the isolates that had MICs out of the determinable range of the test were excluded due to uncertainty; this included isolates with MIC ≥ 8 μg/mL by the VITEK2 (13 isolates) and isolates with MIC ≤ 0.5 μg/mL and N4 μg/mL by MicroScan (21 and 6 isolates, respectively). One isolate with an MIC N 32 μg/mL by Etest was also excluded. Isolates with MICs ≤ 0.25 μg/mL by both BMD and Etest were not excluded because the MICs of these isolates were considered to be very low. 3. Results The resistant rates (%) to ertapenem as determined by the BMD, MicroScan, Etest and disk diffusion test were 27.8%, 29.2%, 31.9% and 15.3%, respectively. The performance for the detection of ertapenem resistance in Enterobacteriaceae of different antimicrobial susceptibility testing methods is shown in Table 1. Etest showed high sensitivity and specificity; 85.0% and 88.5%, respectively. The PPVs of two automated systems were low; 27.8% and 61.9% for the VITEK2 and MicroScan, respectively. Disk diffusion had the lowest sensitivity of 50.0% but the highest specificity and PPV among the testing methods (98.1% and 90.9%, respectively). The testing methods were compared to BMD as the reference method (Table 2). Comparing MICs of BMD with other tests, essential agreement was the lowest for the VITEK2 (30.5%), while the Etest and the MicroScan showed essential agreement greater than 90%. VITEK2 showed the lowest categorical agreement (27.8%), followed by MicroScan (55.6%). Most category disagreements were minor errors. There were 3 very major errors in both the MicroScan and disk diffusion test.
M. Lee, H.-S. Chung / Journal of Microbiological Methods 112 (2015) 87–91
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Table 1 The performance for the detection of ertapenem resistance in Enterobacteriaceae of different antimicrobial susceptibility testing methods. Testing method
BMD (no. of isolates)
VITEK2 MicroScan
R R NR R NR R NR
Etest Disk diffusion
R
NR
20 13 7 17 3 10 10
52 8 44 6 46 1 51
Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
NAa 65.0
NAa 84.6
27.8 61.9
NAa 86.3
85.0
88.5
73.9
93.9
50.0
98.1
90.9
83.6
Abbreviations: BMD, broth microdilution; NPV, negative predictive value; NR, not resistant (susceptible and intermediate); PPV, positive predictive value; R, resistant. a Not applicable; all of the isolates were detected ertapenem resistance by the VITEK2 system in this study, therefore sensitivity, specificity and NPV were not calculated.
4. Discussion Table 2 Agreements of ertapenem susceptibility results between different antimicrobial susceptibility testing methods and broth microdilution. Testing method
VITEK2 MicroScan Etest Disk diffusion
% Agreement (no.)
No. of error
Essential agreementa
Category agreementb
Very major
Major
Minor
30.5 93.3 93.1
27.8 55.6 75.0 59.7
NAc 3 0 3
32 1 0 0
20 28 18 26
(18/59) (42/45) (67/72)
(20/72) (40/72) (54/72) (43/72)
a Essential agreement was defined when the MIC of the test method was within a single 2-fold dilution (±1 doubling dilution) of the reference result. b Category disagreements were classified as follows. A minor error was listed when an isolate was categorized as intermediate by one test method but either susceptible or resistant by another test method. A major error occurred when the isolate was categorized as being resistant by the test method but susceptible by the reference method (false-resistant result). A very major error happened when the isolate was categorized as susceptible by the test method but resistant by the reference method. c Not applicable; there were no susceptible isolates according to the VITEK2 in this study.
Comparing BMD with Etest, the interpretive category results were matched in 54 isolates, and minor errors were observed in 18 isolates. Neither major errors nor very major errors were noted. The distributions of MICs determined by BMD and Etest are shown in Table 3. For isolates with MICs of 2 or 4 μg/mL by the VITEK2, the resistance rates determined by BMD (MIC ≥ 2 μg/mL) were 14.3% (3/21) and 23.7% (9/38), respectively. Among the 21 isolates that were resistant to ertapenem by the MicroScan, only 1 isolate was susceptible to ertapenem (Table 4). Among the 8 intermediate isolates identified by the disk diffusion test, all but one were resistant according to BMD. Among the 20 isolates with an MIC of 1 μg/mL according to BMD (i.e., intermediate), 18 were susceptible based on the disk diffusion test (Fig. 1). Only 1 isolate was positive for both the MHT and carbapenemase inhibition test, and all isolates were positive for AmpC.
Table 3 Comparison between ertapenem MICs of broth microdilution and Etest. MIC (μg/mL) determined by Etest
No. of isolates with ertapenem MIC (μg/mL) determined by broth microdilution ≤0.25
≤0.25 0.25 b ≤0.5 0.5 b ≤1 1 b ≤2 2 b ≤4 4 b ≤8 8 b ≤16 16 b ≤32 N32 Sum
9 1 1
0.5 11 8 2
1
2 1 5 8 4 2
3 2 6
4
8
21
20
11
32
64
4
1
1 1 2
Table 4 Distribution of ertapenem MICs determined by the VITEK2 and MicroScan system compared to broth microdilution. Test
MIC (μg/mL)
VITEK2
2 4 ≥8 ≤0.5 1 2 4 N4 Sum
128
3 1 1
11
16
Sum
In this study, ertapenem susceptibility results were different depending on the methodology used. Overall, the VITEK2 results showed higher ertapenem MICs than those determined by BMD, thereby producing a high number of discrepancies: major errors in 33 isolates and minor errors in 20 isolates. A significant portion of ertapenemresistant Enterobacteriaceae isolates initially detected by the VITEK2, especially isolates with an MIC of 2 or 4 μg/mL by the VITEK2, were susceptible when examined using other testing methods. Caution should be taken when interpreting ertapenem-resistant Enterobacteriaceae isolates using VITEK2, especially for isolates with an MIC of 2 or 4 μg/mL, because they are likely to be susceptible when tested with other antimicrobial susceptibility tests. These findings were also observed in a recent study (Pailhories et al., 2014); the MICs determined by VITEK2 were higher than those determined by the Etest and agar dilution method. Therefore, confirmation of testing results by other methods is encouraged for resistance results determined by the VITEK2. An accurate MIC may be required for antimicrobial dosing optimization. Given the busy environment of today's clinical microbiology laboratory, automated systems such as the VITEK2 and the MicroScan have become popular for antimicrobial susceptibility testing. However, these systems may produce non-reliable results with ertapenem due to low PPV. In this study, the MicroScan showed MICs and interpretive category results in relatively good agreement with BMD compared to the VITEK2. This may be due to differences in measurement methods of MICs; the MicroScan is more similar to BMD than to the VITEK2. Etest is relatively simple and very accurate compared to BMD (Garcia and Isenberg, 2010). Therefore, it is a good alternative method for susceptibility tests of ertapenem. In our study, Etest showed the best performance among the testing methods. An excellent concordance between BMD and Etest was observed. There were no falseresistant or false-susceptible results by Etest. In previous studies, Etest has been validated as a method with good concordance with BMD and the agar dilution method (Livermore et al., 2001; Pailhories et al., 2014).
1 1 2
21 14 14 6 11 1 3 1 1 72
MicroScan
No. of isolates with ertapenem MIC (μg/mL) determined by broth microdilution ≤0.25
0.5
1
2 5 4 10 1
10 11
6 13 1 2 11 6 1
3 3 4 1
20
11
11
6 14 1
21
2
4 3 8
1 3
8
16
32
64
Sum 128
1
2
2
1 1
2 2
2 2
1 2 1 4
21 38 13 21 30 11 4 6 72
90
M. Lee, H.-S. Chung / Journal of Microbiological Methods 112 (2015) 87–91
Fig. 1. Scatterplot of ertapenem MICs (μg/mL) determined by broth microdilution compared with ertapenem disk zone diameters (mm). The vertical solid line represents the Clinical and Laboratory Standards Institute (CLSI) breakpoint for the 10 μg ertapenem disk diffusion (susceptible (S) ≥ 22 mm, resistant (R) ≤ 18 mm). The horizontal solid line represents the CLSI breakpoints for ertapenem MIC (S ≤ 0.5 μg/mL, R ≥ 2 μg/mL).
The disk diffusion test tended to show more susceptible results. Considering high specificity and PPV, it would be helpful to confirm the resistant results of ertapenem. However, there is a risk of missing ertapenem-resistant Enterobacteriaceae due to low sensitivity. Further studies are needed to determine the most appropriate method for ertapenem susceptibility testing for Enterobacteriaceae in clinical microbiology laboratories. Careful consideration regarding not only accuracy, but also cost and labor intensiveness is required. It is important to develop an easy-to perform methodology that can be routinely used in the laboratory. Ertapenem is not included in the interim surveillance definition of CRE developed by the Centers for Disease Control and Prevention (CDC), but the CDC recommends that, if the older CLSI breakpoints (pre-dating M100-S20-U) are being used to determine carbapenem susceptibility, then consideration should be given to including ertapenem in the CRE definition in order to increase sensitivity (CDC, 2012). The CDC guideline is more focused on infection control of carbapenemase-producing Enterobacteriaceae. Ertapenem resistance in Enterobacteriaceae is mainly known to be caused by mechanisms other than carbapenemases, the most common of which is expression of β-lactamases, such as an AmpC-type enzyme or an ESBL, combined with porin loss (Doumith et al., 2009; Garcia-Fernandez et al., 2010; Wu et al., 2011). In this study, only 1 isolate was suggested to produce carbapenemase. A possible reason for the discrepancy in susceptibility results among the test methods might involve the inoculum size. AmpC β-lactamases were prevalent among the tested isolates in this study, which has been well known to influence antimicrobial susceptibility test results according to the inoculum size (Kang et al., 2004; Queenan et al., 2004). In addition, previous studies showed false susceptibilities for carbapenem-resistant KPC-harboring K. pneumoniae isolates by the MicroScan and the VITEK systems due to small inoculum size, demonstrating that this factor had a major influence on the outcomes of these automated systems (Bratu et al., 2005a,b). Many discrepancies observed in this study are due in part to the selection of organisms, which favors resistant strains and includes many isolates for which ertapenem MICs are close to the intermediate breakpoint. In this study, a large number of resistant and intermediate isolates were included to investigate the performance for the detection of ertapenem resistance in Enterobacteriaceae. In this sense, the majority of discrepancies between the methods in this study occurred in intermediate isolates (minor errors). Unlike this study, the data of the manufacturer showed that the agreement between ertapenem susceptibility results of the VITEK2 and reference method was high; 98.8% of category agreement. And there was no discordance between ertapenem susceptibility results of the VITEK2 and other methods in previous study (Behera et al., 2009).
In summary, the performance of the antimicrobial susceptibility testing methods to detect ertapenem resistance in Enterobacteriaceae was variable among the methods. The detection of ertapenem-resistant Enterobacteriaceae has limitations when using routine testing such as an automated system or disk diffusion. Confirmation of results by additional MIC tests is recommended for resistant results. It would provide valuable information about susceptibility tests for ertapenem. Funding No funding sources. Conflict of interest None declared. Ethical approval Not required. References Behera, B., Mathur, P., Das, A., Kapil, A., 2009. Ertapenem susceptibility of extended spectrum beta-lactamase-producing Enterobacteriaceae at a tertiary care centre in India. Singapore Med. J. 50, 628–632. Bratu, S., Landman, D., Haag, R., Recco, R., Eramo, A., Alam, M., Quale, J., 2005a. Rapid spread of carbapenem-resistant Klebsiella pneumoniae in New York City: a new threat to our antibiotic armamentarium. Arch. Intern. Med. 165, 1430–1435. Bratu, S., Mooty, M., Nichani, S., Landman, D., Gullans, C., Pettinato, B., Karumudi, U., Tolaney, P., Quale, J., 2005b. Emergence of KPC-possessing Klebsiella pneumoniae in Brooklyn, New York: epidemiology and recommendations for detection. Antimicrob. Agents Chemother. 49, 3018–3020. CDC (Centers for Disease Control and Prevention), 2012. Guidance for Control of Carbapenem-resistant Enterobacteriaceae (CRE). Centers for Disease Control and Prevention, Atlanta, GA (http://www.cdc.gov/hai/pdfs/cre/CRE-guidance-508.pdf). CLSI (Clinical and Laboratory Standards Institute), 2010. Performance Standards for Antimicrobial Susceptibility Testing; Twentieth Informational Supplement, M100-S20-U. Clinical and Laboratory Standards Institute, Wanye, PA. CLSI (Clinical and Laboratory Standards Institute), 2012a. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard-ninth Edition, M07-A9. Clinical and Laboratory Standards Institute, Wanye, PA. CLSI (Clinical and Laboratory Standards Institute), 2012b. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-second Informational Supplement, M100-S22. Clinical and Laboratory Standards Institute, Wanye, PA. CLSI (Clinical and Laboratory Standards Institute), 2013. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-third Informational Supplement, M100-S23. Clinical and Laboratory Standards Institute, Wanye, PA. Coudron, P.E., 2005. Inhibitor-based methods for detection of plasmid-mediated AmpC beta-lactamases in Klebsiella spp., Escherichia coli, and Proteus mirabilis. J. Clin. Microbiol. 43, 4163–4167. Doumith, M., Ellington, M.J., Livermore, D.M., Woodford, N., 2009. Molecular mechanisms disrupting porin expression in ertapenem-resistant Klebsiella and Enterobacter spp. clinical isolates from the UK. J. Antimicrob. Chemother. 63, 659–667. Garcia, L.S., Isenberg, H.D., 2010. Clinical Microbiology Procedures Handbook. ASM Press, Washington, DC.
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Different antimicrobial susceptibility testing methods to detect ertapenem resistance in enterobacteriaceae: VITEK2, MicroScan, Etest, disk diffusion, and broth microdilution Miae Lee, Hae-Sun Chung ⁎ Department of Laboratory Medicine, Ewha Womans University School of Medicine, Republic of Korea
a r t i c l e
i n f o
Article history: Received 7 January 2015 Received in revised form 16 March 2015 Accepted 16 March 2015 Available online 17 March 2015 Keywords: Ertapenem Enterobacteriaceae Antimicrobial susceptibility test Antimicrobial resistance
a b s t r a c t We investigated different antimicrobial susceptibility testing methods to detect ertapenem resistance in Enterobacteriaceae. A total of 72 Enterobacteriaceae isolates were collected from a clinical microbiology laboratory of a tertiary university hospital, all of which were detected ertapenem resistance by the VITEK2 system. Bacterial identification and antimicrobial susceptibility were determined using the VITEK2. Ertapenem susceptibility test was performed using the MicroScan, Etest and a disk diffusion test. Ertapenem MICs were confirmed using the broth microdilution (BMD). Sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) of each method for the detection of ertapenem resistance were calculated. Carbapenemases and AmpC β-lactamase were screened using phenotypic methods. Among the 72 isolates, 20 isolates (27.8%) were resistant to ertapenem. Etest showed high sensitivity and specificity (85.0% and 88.5%, respectively) and excellent concordance with BMD. The disk diffusion test had the lowest sensitivity of 50.0%. The VITEK2 showed the lowest essential and categorical agreement (30.5% and 27.8%, respectively). The MicroScan showed relatively good agreement with BMD compared to the VITEK2. Most category disagreements were minor errors. There were 3 very major errors in both the MicroScan and disk diffusion test. Only 1 isolate was positive for carbapenemase screening test and all of the isolates were positive for AmpC screening test. In conclusion, the detection of ertapenem resistance in Enterobacteriaceae has limitations using routine testing such as an automated system or disk diffusion. Confirmation of results by an additional MIC test is recommended for accurate resistance results of ertapenem. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Carbapenem-resistant Enterobacteriaceae (CRE) has emerged and been threatening the successful treatment of major infection (CDC, 2012; Schwaber and Carmeli, 2008; van Duin et al., 2013). Invasive CRE infections are associated with poorer outcomes compared to carbapenem-susceptible Enterobacteriaceae infections, which is likely due in part to limited treatment options (Schwaber and Carmeli, 2008; van Duin et al., 2013). Very few options remain for the treatment of these carbapenem-resistant organisms. Ertapenem is a carbapenem developed to overcome the pharmacokinetic shortcomings (short half-life) of imipenem and meropenem. The extensive protein binding of ertapenem extends the half-life and allows once-daily dosing (Nix et al., 2004; Zhanel et al., 2005, 2007). Ertapenem has been widely used since the early 2000s, but it was only recently introduced to the routine antimicrobial susceptibility ⁎ Corresponding author at: Department of Laboratory Medicine, Ewha Womans University School of Medicine, 1071, Anyangcheon-ro, Yangcheon-gu, Seoul 158-710, Republic of Korea. E-mail address: [email protected] (H.-S. Chung).
http://dx.doi.org/10.1016/j.mimet.2015.03.014 0167-7012/© 2015 Elsevier B.V. All rights reserved.
tests performed by automated systems. Since then, ertapenemresistant Enterobacteriaceae isolates have been detected (Behera et al., 2009; Wu et al., 2011). Antimicrobial susceptibilities can be tested using various methods. In clinical microbiology laboratories, especially those in large tertiary hospitals, antimicrobial susceptibility tests are performed using commercially available automated systems, including the VITEK2 (bioMérieux) and MicroScan (Siemens) systems. The Etest and disk diffusion test are also relatively easy to use. Broth microdilution (BMD) is a well-established, standard method, but it is too laborintensive for use in clinical laboratories (Versalovic et al., 2011; Garcia and Isenberg, 2010). Antimicrobial susceptibility testing for ertapenem can be performed using all of the above methods; however, many laboratories lack the essential experience to do so, and there is little data comparing the efficacies of the different methods (Pailhories et al., 2014; Samuel et al., 2005; Vading et al., 2011). The breakpoints for ertapenem have changed in recent years. In June 2010, the Clinical and Laboratory Standards Institute (CLSI) published new interpretive criteria for carbapenems of Enterobacteriaceae (CLSI, 2010). Two years later, in January 2012, the CLSI revised the breakpoints for ertapenem (CLSI M100-S22, 2012a). However, there have been little
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data of evaluation of antimicrobial susceptibility tests for isolates close to the new breakpoints. In this study, we investigated different antimicrobial susceptibility testing methods including the VITEK2, MicroScan, BMD, Etest, and disk diffusion tests to detect ertapenem resistance in Enterobacteriaceae isolates from a clinical microbiology laboratory at a tertiary university hospital. 2. Methods 2.1. Isolates Clinical Enterobacteriaceae isolates were collected from a clinical microbiology laboratory of a tertiary university hospital, all of which were detected ertapenem resistance by the VITEK2 system during August 2012 and July 2013. A total of 72 Enterobacteriaceae isolates were analyzed; 43 (59.7%) Enterobacter spp., 13 (18.1%) Klebsiella pneumoniae, 8 (11.1%) Escherichia coli, 3 (4.2%) Citrobacter freundii, 2 (2.8%) Serratia marcescens, and 3 others (4.2%). Bacterial identification was performed using the VITEK2 with the GN card (bioMérieux). 2.2. Antimicrobial susceptibility test 2.2.1. Interpretive criteria and quality control Susceptibility results were interpreted using the CLSI guideline recommended in 2013: ≤0.5 μg/mL for susceptible, 1 μg/mL for intermediate, and ≥2 μg/mL for resistant (CLSI, 2013). Enterococcus faecalis ATCC 29212, E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as controls for BMD, Etest and disk diffusion test. Quality controls for the two automated systems were performed according to the manufacturers instructions. 2.2.2. VITEK2 Antimicrobial susceptibility was tested by the VITEK2 using ASTN224 card (bioMérieux), according to the manufacturer's instructions. The MIC range for ertapenem on the AST-N224 card was ≤0.25 μg/mL to ≥ 8 μg/mL in doubling dilutions. Colonies from an overnight agar plate culture of each isolate were suspended in 3 mL of 0.45% saline and adjusted to a turbidity of 0.5 McFarland standard with VITEK Densicheck (bioMérieux). 2.2.3. MicroScan Antimicrobial susceptibility was tested by MicroScan WalkAway 96 SI instrument using the Neg MIC 37 panel (Siemens) containing, according to the manufacturer's instructions. The MIC range for ertapenem on the Neg MIC 37 panel was ≤ 0.5 μg/mL to N4 μg/mL in doubling dilutions. Colonies from an overnight agar plate culture of each isolate were suspended in 3 mL of distilled water and adjusted to a turbidity of 0.5 McFarland standard with MicroScan Turbidity Meter (Siemens). 2.2.4. Etest The MICs for ertapenem were determined with Etest (bioMérieux) carried out on Mueller-Hinton agar (ASAN Pharm.), according to the manufacturer's instructions. Etest strips were applied when the agar surface was completely dry. The strips were incubated at 35 °C for 18 h. The MIC was determined as the point where inhibition of the growth intersected with the Etest strip. The Etest MIC test range was 0.002 μg/mL to 32 μg/mL. For categorical interpretation and comparison, Etest MICs between standard dilutions were rounded up to the nearest 2-fold BMD dilution. 2.2.5. Disk diffusion test Disk diffusion susceptibility testing was performed using commercial ertapenem disks (Becton Dickinson) containing 10 μg and Muller-Hinton agar. Isolates with a zone diameter ≤ 18 mm were considered to be
resistant, those having a zone diameter of 19–21 mm were intermediate, and those with a zone diameter of ≥22 mm were susceptible (CLSI, 2013). 2.2.6. Broth microdilution (BMD) BMD was performed by using 96-well broth microdilution panels according to the CLSI guidelines (CLSI M07-A9, 2012b). Solvent and diluent for preparation of stock solutions of ertapenem were prepared as described in the CLSI document, in cation-adjusted Mueller-Hinton broth (Becton Dickinson). The BMD MIC test range was 0.25 μg/mL to 128 μg/mL. The results of BMD were considered the reference standard against which all other results were compared. 2.3. Carbapenemase and AmpC screening test For carbapenemase detection, a modified Hodge test (MHT) and a carbapenemase inhibition test (Rosco Diagnostica) were performed. AmpC detection was done by cefoxitin-boronic acid disk synergy test (Coudron, 2005). 2.4. Data analysis The results of different antimicrobial susceptibility testing methods were compared to those of BMD. Sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) of each method for the detection of ertapenem resistance were calculated to evaluate performance. Agreement was assessed in two manners: category (qualitative) and MIC (quantitative) comparison. Category disagreements were classified as follows. A minor error was listed when an isolate was categorized as intermediate by one test method but either susceptible or resistant by another test method. A major error occurred when the isolate was categorized as being resistant by the test method but susceptible by the reference method (false-resistant result). A very major error happened when the isolate was categorized as susceptible by the test method but resistant by the reference method (false-susceptible result). If the MIC of the test method was within a single 2-fold dilution (±1 doubling dilution) of the reference result, then the MIC for that isolate was defined as being in agreement (essential agreement) (Garcia and Isenberg, 2010). To compare MICs, the isolates that had MICs out of the determinable range of the test were excluded due to uncertainty; this included isolates with MIC ≥ 8 μg/mL by the VITEK2 (13 isolates) and isolates with MIC ≤ 0.5 μg/mL and N4 μg/mL by MicroScan (21 and 6 isolates, respectively). One isolate with an MIC N 32 μg/mL by Etest was also excluded. Isolates with MICs ≤ 0.25 μg/mL by both BMD and Etest were not excluded because the MICs of these isolates were considered to be very low. 3. Results The resistant rates (%) to ertapenem as determined by the BMD, MicroScan, Etest and disk diffusion test were 27.8%, 29.2%, 31.9% and 15.3%, respectively. The performance for the detection of ertapenem resistance in Enterobacteriaceae of different antimicrobial susceptibility testing methods is shown in Table 1. Etest showed high sensitivity and specificity; 85.0% and 88.5%, respectively. The PPVs of two automated systems were low; 27.8% and 61.9% for the VITEK2 and MicroScan, respectively. Disk diffusion had the lowest sensitivity of 50.0% but the highest specificity and PPV among the testing methods (98.1% and 90.9%, respectively). The testing methods were compared to BMD as the reference method (Table 2). Comparing MICs of BMD with other tests, essential agreement was the lowest for the VITEK2 (30.5%), while the Etest and the MicroScan showed essential agreement greater than 90%. VITEK2 showed the lowest categorical agreement (27.8%), followed by MicroScan (55.6%). Most category disagreements were minor errors. There were 3 very major errors in both the MicroScan and disk diffusion test.
M. Lee, H.-S. Chung / Journal of Microbiological Methods 112 (2015) 87–91
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Table 1 The performance for the detection of ertapenem resistance in Enterobacteriaceae of different antimicrobial susceptibility testing methods. Testing method
BMD (no. of isolates)
VITEK2 MicroScan
R R NR R NR R NR
Etest Disk diffusion
R
NR
20 13 7 17 3 10 10
52 8 44 6 46 1 51
Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
NAa 65.0
NAa 84.6
27.8 61.9
NAa 86.3
85.0
88.5
73.9
93.9
50.0
98.1
90.9
83.6
Abbreviations: BMD, broth microdilution; NPV, negative predictive value; NR, not resistant (susceptible and intermediate); PPV, positive predictive value; R, resistant. a Not applicable; all of the isolates were detected ertapenem resistance by the VITEK2 system in this study, therefore sensitivity, specificity and NPV were not calculated.
4. Discussion Table 2 Agreements of ertapenem susceptibility results between different antimicrobial susceptibility testing methods and broth microdilution. Testing method
VITEK2 MicroScan Etest Disk diffusion
% Agreement (no.)
No. of error
Essential agreementa
Category agreementb
Very major
Major
Minor
30.5 93.3 93.1
27.8 55.6 75.0 59.7
NAc 3 0 3
32 1 0 0
20 28 18 26
(18/59) (42/45) (67/72)
(20/72) (40/72) (54/72) (43/72)
a Essential agreement was defined when the MIC of the test method was within a single 2-fold dilution (±1 doubling dilution) of the reference result. b Category disagreements were classified as follows. A minor error was listed when an isolate was categorized as intermediate by one test method but either susceptible or resistant by another test method. A major error occurred when the isolate was categorized as being resistant by the test method but susceptible by the reference method (false-resistant result). A very major error happened when the isolate was categorized as susceptible by the test method but resistant by the reference method. c Not applicable; there were no susceptible isolates according to the VITEK2 in this study.
Comparing BMD with Etest, the interpretive category results were matched in 54 isolates, and minor errors were observed in 18 isolates. Neither major errors nor very major errors were noted. The distributions of MICs determined by BMD and Etest are shown in Table 3. For isolates with MICs of 2 or 4 μg/mL by the VITEK2, the resistance rates determined by BMD (MIC ≥ 2 μg/mL) were 14.3% (3/21) and 23.7% (9/38), respectively. Among the 21 isolates that were resistant to ertapenem by the MicroScan, only 1 isolate was susceptible to ertapenem (Table 4). Among the 8 intermediate isolates identified by the disk diffusion test, all but one were resistant according to BMD. Among the 20 isolates with an MIC of 1 μg/mL according to BMD (i.e., intermediate), 18 were susceptible based on the disk diffusion test (Fig. 1). Only 1 isolate was positive for both the MHT and carbapenemase inhibition test, and all isolates were positive for AmpC.
Table 3 Comparison between ertapenem MICs of broth microdilution and Etest. MIC (μg/mL) determined by Etest
No. of isolates with ertapenem MIC (μg/mL) determined by broth microdilution ≤0.25
≤0.25 0.25 b ≤0.5 0.5 b ≤1 1 b ≤2 2 b ≤4 4 b ≤8 8 b ≤16 16 b ≤32 N32 Sum
9 1 1
0.5 11 8 2
1
2 1 5 8 4 2
3 2 6
4
8
21
20
11
32
64
4
1
1 1 2
Table 4 Distribution of ertapenem MICs determined by the VITEK2 and MicroScan system compared to broth microdilution. Test
MIC (μg/mL)
VITEK2
2 4 ≥8 ≤0.5 1 2 4 N4 Sum
128
3 1 1
11
16
Sum
In this study, ertapenem susceptibility results were different depending on the methodology used. Overall, the VITEK2 results showed higher ertapenem MICs than those determined by BMD, thereby producing a high number of discrepancies: major errors in 33 isolates and minor errors in 20 isolates. A significant portion of ertapenemresistant Enterobacteriaceae isolates initially detected by the VITEK2, especially isolates with an MIC of 2 or 4 μg/mL by the VITEK2, were susceptible when examined using other testing methods. Caution should be taken when interpreting ertapenem-resistant Enterobacteriaceae isolates using VITEK2, especially for isolates with an MIC of 2 or 4 μg/mL, because they are likely to be susceptible when tested with other antimicrobial susceptibility tests. These findings were also observed in a recent study (Pailhories et al., 2014); the MICs determined by VITEK2 were higher than those determined by the Etest and agar dilution method. Therefore, confirmation of testing results by other methods is encouraged for resistance results determined by the VITEK2. An accurate MIC may be required for antimicrobial dosing optimization. Given the busy environment of today's clinical microbiology laboratory, automated systems such as the VITEK2 and the MicroScan have become popular for antimicrobial susceptibility testing. However, these systems may produce non-reliable results with ertapenem due to low PPV. In this study, the MicroScan showed MICs and interpretive category results in relatively good agreement with BMD compared to the VITEK2. This may be due to differences in measurement methods of MICs; the MicroScan is more similar to BMD than to the VITEK2. Etest is relatively simple and very accurate compared to BMD (Garcia and Isenberg, 2010). Therefore, it is a good alternative method for susceptibility tests of ertapenem. In our study, Etest showed the best performance among the testing methods. An excellent concordance between BMD and Etest was observed. There were no falseresistant or false-susceptible results by Etest. In previous studies, Etest has been validated as a method with good concordance with BMD and the agar dilution method (Livermore et al., 2001; Pailhories et al., 2014).
1 1 2
21 14 14 6 11 1 3 1 1 72
MicroScan
No. of isolates with ertapenem MIC (μg/mL) determined by broth microdilution ≤0.25
0.5
1
2 5 4 10 1
10 11
6 13 1 2 11 6 1
3 3 4 1
20
11
11
6 14 1
21
2
4 3 8
1 3
8
16
32
64
Sum 128
1
2
2
1 1
2 2
2 2
1 2 1 4
21 38 13 21 30 11 4 6 72
90
M. Lee, H.-S. Chung / Journal of Microbiological Methods 112 (2015) 87–91
Fig. 1. Scatterplot of ertapenem MICs (μg/mL) determined by broth microdilution compared with ertapenem disk zone diameters (mm). The vertical solid line represents the Clinical and Laboratory Standards Institute (CLSI) breakpoint for the 10 μg ertapenem disk diffusion (susceptible (S) ≥ 22 mm, resistant (R) ≤ 18 mm). The horizontal solid line represents the CLSI breakpoints for ertapenem MIC (S ≤ 0.5 μg/mL, R ≥ 2 μg/mL).
The disk diffusion test tended to show more susceptible results. Considering high specificity and PPV, it would be helpful to confirm the resistant results of ertapenem. However, there is a risk of missing ertapenem-resistant Enterobacteriaceae due to low sensitivity. Further studies are needed to determine the most appropriate method for ertapenem susceptibility testing for Enterobacteriaceae in clinical microbiology laboratories. Careful consideration regarding not only accuracy, but also cost and labor intensiveness is required. It is important to develop an easy-to perform methodology that can be routinely used in the laboratory. Ertapenem is not included in the interim surveillance definition of CRE developed by the Centers for Disease Control and Prevention (CDC), but the CDC recommends that, if the older CLSI breakpoints (pre-dating M100-S20-U) are being used to determine carbapenem susceptibility, then consideration should be given to including ertapenem in the CRE definition in order to increase sensitivity (CDC, 2012). The CDC guideline is more focused on infection control of carbapenemase-producing Enterobacteriaceae. Ertapenem resistance in Enterobacteriaceae is mainly known to be caused by mechanisms other than carbapenemases, the most common of which is expression of β-lactamases, such as an AmpC-type enzyme or an ESBL, combined with porin loss (Doumith et al., 2009; Garcia-Fernandez et al., 2010; Wu et al., 2011). In this study, only 1 isolate was suggested to produce carbapenemase. A possible reason for the discrepancy in susceptibility results among the test methods might involve the inoculum size. AmpC β-lactamases were prevalent among the tested isolates in this study, which has been well known to influence antimicrobial susceptibility test results according to the inoculum size (Kang et al., 2004; Queenan et al., 2004). In addition, previous studies showed false susceptibilities for carbapenem-resistant KPC-harboring K. pneumoniae isolates by the MicroScan and the VITEK systems due to small inoculum size, demonstrating that this factor had a major influence on the outcomes of these automated systems (Bratu et al., 2005a,b). Many discrepancies observed in this study are due in part to the selection of organisms, which favors resistant strains and includes many isolates for which ertapenem MICs are close to the intermediate breakpoint. In this study, a large number of resistant and intermediate isolates were included to investigate the performance for the detection of ertapenem resistance in Enterobacteriaceae. In this sense, the majority of discrepancies between the methods in this study occurred in intermediate isolates (minor errors). Unlike this study, the data of the manufacturer showed that the agreement between ertapenem susceptibility results of the VITEK2 and reference method was high; 98.8% of category agreement. And there was no discordance between ertapenem susceptibility results of the VITEK2 and other methods in previous study (Behera et al., 2009).
In summary, the performance of the antimicrobial susceptibility testing methods to detect ertapenem resistance in Enterobacteriaceae was variable among the methods. The detection of ertapenem-resistant Enterobacteriaceae has limitations when using routine testing such as an automated system or disk diffusion. Confirmation of results by additional MIC tests is recommended for resistant results. It would provide valuable information about susceptibility tests for ertapenem. Funding No funding sources. Conflict of interest None declared. Ethical approval Not required. References Behera, B., Mathur, P., Das, A., Kapil, A., 2009. Ertapenem susceptibility of extended spectrum beta-lactamase-producing Enterobacteriaceae at a tertiary care centre in India. Singapore Med. J. 50, 628–632. Bratu, S., Landman, D., Haag, R., Recco, R., Eramo, A., Alam, M., Quale, J., 2005a. Rapid spread of carbapenem-resistant Klebsiella pneumoniae in New York City: a new threat to our antibiotic armamentarium. Arch. Intern. Med. 165, 1430–1435. Bratu, S., Mooty, M., Nichani, S., Landman, D., Gullans, C., Pettinato, B., Karumudi, U., Tolaney, P., Quale, J., 2005b. Emergence of KPC-possessing Klebsiella pneumoniae in Brooklyn, New York: epidemiology and recommendations for detection. Antimicrob. Agents Chemother. 49, 3018–3020. CDC (Centers for Disease Control and Prevention), 2012. Guidance for Control of Carbapenem-resistant Enterobacteriaceae (CRE). Centers for Disease Control and Prevention, Atlanta, GA (http://www.cdc.gov/hai/pdfs/cre/CRE-guidance-508.pdf). CLSI (Clinical and Laboratory Standards Institute), 2010. Performance Standards for Antimicrobial Susceptibility Testing; Twentieth Informational Supplement, M100-S20-U. Clinical and Laboratory Standards Institute, Wanye, PA. CLSI (Clinical and Laboratory Standards Institute), 2012a. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard-ninth Edition, M07-A9. Clinical and Laboratory Standards Institute, Wanye, PA. CLSI (Clinical and Laboratory Standards Institute), 2012b. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-second Informational Supplement, M100-S22. Clinical and Laboratory Standards Institute, Wanye, PA. CLSI (Clinical and Laboratory Standards Institute), 2013. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-third Informational Supplement, M100-S23. Clinical and Laboratory Standards Institute, Wanye, PA. Coudron, P.E., 2005. Inhibitor-based methods for detection of plasmid-mediated AmpC beta-lactamases in Klebsiella spp., Escherichia coli, and Proteus mirabilis. J. Clin. Microbiol. 43, 4163–4167. Doumith, M., Ellington, M.J., Livermore, D.M., Woodford, N., 2009. Molecular mechanisms disrupting porin expression in ertapenem-resistant Klebsiella and Enterobacter spp. clinical isolates from the UK. J. Antimicrob. Chemother. 63, 659–667. Garcia, L.S., Isenberg, H.D., 2010. Clinical Microbiology Procedures Handbook. ASM Press, Washington, DC.
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