Diagnostic Microbiology and Infectious Disease xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Evaluation of avibactam-supplemented combination disk tests for the detection of OXA-48 carbapenemase-producing Enterobacteriaceae☆,☆☆,★ Te-Din Huang ⁎, Catherine Berhin, Pierre Bogaerts, Youri Glupczynski National Reference Laboratory for Monitoring of Antimicrobial Resistance in Gram-negative bacteria, CHU Dinant-Godinne|UCL Namur, 1 Avenue Dr. G. Therasse, 5530 Yvoir, Belgium
a r t i c l e
i n f o
Article history: Received 13 January 2014 Received in revised form 17 February 2014 Accepted 9 March 2014 Available online xxxx Keywords: Temocillin Inhibitor OXA-type beta-lactamase
a b s t r a c t The ability of various combination disk tests (CDTs) incorporating avibactam to detect OXA-48 carbapenemase-producing Enterobacteriaceae was evaluated. The CDT using 30-μg temocillin alone and supplemented with 5-μg avibactam showed good performance and could be an adjunctive test to the classic CDT containing class A and class B carbapenemase inhibitors for the positive discrimination of OXA-48 carbapenemase producers from carbapenemase-negative strains. © 2014 Elsevier Inc. All rights reserved.
Accurate phenotypical detection of carbapenemases in Enterobacteriaceae is of great importance (Canton et al., 2012). Current phenotypic confirmatory methods of carbapenemase-producing Enterobacteriaceae (CPE) are based on the use of inhibitor-supplemented antimicrobial disks and allow differentiation between Ambler class A and B carbapenemase producers but fail to confirm Ambler class D OXA-48 production (Giske et al., 2011). Avibactam (formerly NXL104) is a recently developed non β-lactam diazabicyclooctane inhibitor of class A, C, and selected D β-lactamases including some carbapenemases (Livermore et al., 2011). We evaluated various combination disk tests (CDTs) incorporating avibactam to detect OXA-48 CPE. We selected 213 well-characterized Enterobacteriaceae collected from clinical laboratories across Belgium in 2012 to represent the diversity of the beta-lactams resistance mechanisms. The selection included 116 Klebsiella spp., 51 Enterobacter spp., 27 Escherichia coli, 7 Citrobacter spp., 5 Serratia marcescens, 3 Proteus spp., 3 Morganella morganii, and 1 Providencia spp. All isolates had been verified for the presence of carbapenemase by multiplex PCR targeting blaVIM, blaIMP, blaNDM, blaKPC, and blaOXA-48 and/or by imipenem hydrolysis using the Carba NP assay previously described (Bogaerts et al., 2013; Nordmann et al., 2012). A total of 113 were confirmed as CPE (OXA-48 [n = 72], VIM [n = 20], KPC [n = 17], and NDM [n = 4]), and 100 were non-
☆ Funding: The study was supported in part by the research grant of Fondation Mont-Godinne. The national reference centre is partially supported by the Belgian Ministry of Social Affairs through a fund within the Health Insurance System. ☆☆ Conflict of interest declaration: None to declare. ★ Parts of this study were published as an abstract in the 33rd Réunion Interdisciplinaire de Chimiothérapie Anti-Infectieuse, Paris, November 21–22, 2013. ⁎ Corresponding author. Tel.: +32-81-423212; fax: +32-81-423204. E-mail address: [email protected] (T.-D. Huang).
carbapenemase producers (43 with extended-spectrum β-lactamase [ESBL], 26 with hyperproduced plasmidic or derepressed chromosomal AmpC β-lactamase [AmpC], 7 with ESBL associated to AmpC, 14 with other narrow-spectrum penicillinases, and 10 with wild-type pattern). All isolates were tested using EUCAST disk diffusion methodology against the KPC/MBL Confirmation Kit® (containing meropenem, meropenem + dipicolinic acid, meropenem + phenylboronic acid, and meropenem + cloxacillin tablets; Rosco Diagnostics, Taastrup, Denmark) and against 6 paper disks including ertapenem 10 μg, meropenem 10 μg, and temocillin 30 μg, each alone (BioRad) or supplemented with avibactam 5 μg (home-made solution). CDT results using Rosco tablets were interpreted according to the manufacturers’ instructions. A cut-off of ≥5-mm diameter increase in paper disk containing avibactam was arbitrarily selected to assign positive result for the CDT. The distribution of ertapenem, meropenem, and temocillin paper disks zone diameter and the different CDT diameter increase on all isolates tested are shown in Table 1. 111/113 CPE and 57/100 noncarbapenemase producers isolates would have been categorized as carbapenem non-susceptible Enterobacteriaceae (CNSE) according to the EUCAST interpretative criteria (European Society of Clinical Microbiology and Infectious Diseases, 2013a). Among the 113 CPE isolates, 111 and 83, respectively, would have been detected by the ertapenem or meropenem screening breakpoint (b25 mm) proposed by EUCAST (European Society of Clinical Microbiology and Infectious Diseases, 2013b). All but 1 (VIM-producing Enterobacter cloacae) of the CPE isolates missed by the EUCAST screening breakpoint were OXA-48 producers. Concerning temocillin, 36 of 100 non–carbapenemaseproducing isolates were resistant according to the breakpoint (inhibition zone b20 mm) recently published (Vanstone et al., 2013), while all but 2 (1 NDM-positive M. morganii and 1 KPC-
0732-8893/© 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.diagmicrobio.2014.03.010
Please cite this article as: Huang T-D, et al, Evaluation of avibactam-supplemented combination disk tests for the detection of OXA-48 carbapenemase-producing Enterobacteriaceae, Diagn Microbiol Infect Dis (2014), http://dx.doi.org/10.1016/j.diagmicrobio.2014.03.010
2
T.-D. Huang et al. / Diagnostic Microbiology and Infectious Disease xxx (2014) xxx–xxx
Table 1 Distribution of ertapenem, meropenem, and temocillin paper disks zone diameter and the CDTs diameter increase on Enterobacteriaceae isolates tested (n = 213). Median (range) value in mm per carbapenemase enzyme
Paper disks
Combination disk tests
ETP zone diameter MEM zone diameter TMC zone diameter ETP + AVB/ETP diameter increase MEM + AVB/MEM diameter increase TMC + AVB/TMC diameter increase
VIM (n = 20)
NDM (n = 4)
KPC (n = 17)
OXA-48 (n = 72)
Negative (n = 100)
13 (6–20) 15 (6–25) 6 (6–15) 0 (0–6) 1 (0–15) 2 (0–12)
15 (9–20) 18 (17–20) 17 (6–25) 0 (0–3) 0 (0–2) 0 (0–6)
10 (6–15) 12 (6–19) 15 (6–20) 15 (0–17) 14 (0–16) 6 (0–7))
18 (7–26) 23 (9–29) 6 (6–16) 5 (2–16) 3 (1–16) 11 (6–15)
17 23 21 5 2 2
(6–38) (10–37) (6–30) (0–15) (−2 to 12) (−2 to 11)
ETP = ertapenem 10 μg; MEM = meropenem 10 μg; TMC = temocillin 30 μg; AVB = avibactam 5 μg.
producing Klebsiella pneumoniae) of the 113 CPE strains were temocillinresistant including 95% of the VIM (19/20) and of OXA-48 (68/72) producers having temocillin zone diameter b12 mm and confirming highlevel resistance to temocillin stated previously (Huang et al., 2014; Woodford et al., 2014). Phenotypical confirmatory test using Rosco CDT with inhibitors correctly detected all 17 KPC producers and 21/24 metallo-βlactamase (MBL) producers (2 E. cloacae and 1 Providencia rettgeri VIM-positive isolates gave false-negative results), but it misidentified 4 carbapenemase-negative (3 AmpC- and 1 ESBL-producing) as MBLproducing isolates. 55/100 non-carbapenemase producers, 15/17 KPC-positive, and 54/72 OXA-48–producing isolates showed increased diameter of ≥5 mm when avibactam was added to ertapenem, while 18/ 100 carbapenemase-negative, 15/17 KPC-positive, and 23/72 OXA-48– producing isolates had increased diameter of ≥5 mm when avibactam was combined to meropenem. No differences in diameter increase were observed between OXA-48 producers and non–carbapenemase-producing isolates with ertapenem ± avibactam nor with meropenem ± avibactam CDT since the distribution of diameter increase is similar for the 2 groups (see Table 1). The use of an arbitrary cut-off of ≥5 mm diameter increase yielded positive result with temocillin ± avibactam CDT for all 72 OXA-48 producers, 21/41 CPE expressing non–OXA-48 carbapenemase, and 22/100 carbapenemase-negative isolates. When applying a modified cut-off of ≥8 mm of diameter increase, 97% (70/72) of OXA-48 producers, 5% (2/41) of CPE expressing non–OXA-48
carbapenemase (1 VIM-1 and 1 VIM-31), and 8% (8/100) carbapenemase-negative isolates showed a positive CDT result with temocillin ± avibactam resulting in the overall calculated sensitivity and specificity for the detection of OXA-48–positive CPE of 97% and 93%, respectively. Among OXA-48 producers, no significant variation in diameter increase with temocillin ± avibactam CDT was observed between those isolates harbouring a blaOXA-48 gene alone (n = 15) and those co-producing an ESBL and/or AmpC (n = 57) in addition to OXA-48 (identical median value of 11-mm diameter increase for both groups). The distribution of diameter increase with temocillin ± avibactam focusing on the subset of 102 CNSE (72 OXA-48– producing, 30 carbapenemase-negative, and 2 VIM-producing) strains that yielded a negative result with the Rosco KPC/MBL Confirmation Kit® and that were temocillin-resistant is shown in Table 2. No change in sensitivity (97%) was observed using the modified cut-off of ≥8 mm with temocillin ± avibactam, and the specificity remained high at 84% (4 carbapenemase-negative and 1 VIM-31– producing isolates would have been misidentified as class D carbapenemase producers) within this group. Our study had some limitations. The current unavailability of avibactam for diagnostic purpose and the current home-made format of the disk tests requiring preparation skill could hold up its use as routine screening assay. We also believe that CDT used to detect betalactamases is a phenotypic test that should be backed by molecular methods to confirm the carbapenemase gene.
Table 2 Distribution (number of isolates) of temocillin 30 μg + avibactam 5 μg inhibition zone increase and of resistance mechanisms to β-lactams for carbapenem-non-susceptible a and temocillin-resistantb Enterobacteriaceae isolates tested negative by the Rosco KPC/MBL Confirmation Kit® (n = 102).
TMC + AVB/TMC diameter increase (mm) −2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
OXA-48 carbapenemase (n = 70)
VIM carbapenemase (n = 2)
Carbapenemase-negative (n = 30) ESBL (n = 14)
AmpC (n = 9)
ESBL and AmpC (n = 5)
Non-ESBL and non-AmpCc (n = 2)
1 1
1 1 6 11 9 14 15 10 2 1
1 1 3 2 2 2 1
1 2 2
1 1 1
2
1 1
1 1
1
2 1
ESBL = extended-spectrum β-lactamase; AmpC = hyperproduced chromosomal or plasmidic AmpC cephalosporinase; TMC + AVB/TMC = temocillin + avibactam/temocillin; in grey, the modified cut-off (≥8 mm) of diameter increase for this CDT. a According to the EUCAST interpretative criteria (ertapenem 10 μg inhibition zone b25 mm). b According to the breakpoint (temocillin 30 μg inhibition zone b20 mm) published by Vanstone et al. (2013). c One K-OXY β-lactamase hyperproducing Klebsiella oxytoca and 1 OXA-1 β-lactamase producing K. pneumoniae.
Please cite this article as: Huang T-D, et al, Evaluation of avibactam-supplemented combination disk tests for the detection of OXA-48 carbapenemase-producing Enterobacteriaceae, Diagn Microbiol Infect Dis (2014), http://dx.doi.org/10.1016/j.diagmicrobio.2014.03.010
T.-D. Huang et al. / Diagnostic Microbiology and Infectious Disease xxx (2014) xxx–xxx
High-level resistance to temocillin has been proposed as an excellent surrogate marker for the detection of OXA-48–producing CPE (Woodford et al., 2014). The CDT using temocillin 30 μg alone and supplemented with avibactam 5 μg showed good performance for the detection of OXA-48 CPE isolates when considering a diameter increase cut-off of ≥8 mm. This combination could constitute an adjunctive test to the confirmatory CDTs containing class A and class B carbapenemase inhibitors for the positive discrimination of class D carbapenemase producers from carbapenemase-negative strains.
Acknowledgments We are thankful to Wright Nichols, Aztra-Zeneca, for providing the avibactam for the testing.
References Bogaerts P, Rezende de Castro R, de Mendonca R, Huang TD, Denis O, Glupczynski Y. Validation of carbapenemase and extended-spectrum beta-lactamase multiplex endpoint PCR assays according to ISO 15189. J Antimicrob Chemother. 2013;68:1576–82. Canton R, Akova M, Carmeli Y, Giske CG, Glupczynski Y, Gniadkowski M, et al. Rapid evolution and spread of carbapenemases among Enterobacteriaceae in Europe. Clin Microbiol Infect 2012;18(5):413–31.
3
European Society of Clinical Microbiology and Infectious Diseases. Clinical breakpoints version 3.0. In European Committee on Antimicrobial Susceptibility Testing. 2013a [1 February 2013, date last accessed).]. Available from: http://www.eucast.org. European Society of Clinical Microbiology and Infectious Diseases. EUCAST guidelines for detection of resistance mechanisms and specific resistances of clinical and/or epidemiological importance. Version 1.0. 2013b [11 December 2013, date last accessed).]. Available from: http://www.eucast.org. Giske CG, Gezelius L, Samuelsen O, Warner M, Sundsfjord A, Woodford N. A sensitive and specific phenotypic assay for detection of metallo-beta-lactamases and KPC in Klebsiella pneumoniae with the use of meropenem disks supplemented with aminophenylboronic acid, dipicolinic acid and cloxacillin. Clin Microbiol Infect 2011;17(4):552–6. Huang TD, Poirel L, Bogaerts P, Berhin C, Nordmann P, Glupczynski Y. Temocillin and piperacillin/tazobactam resistance by disc diffusion as antimicrobial surrogate markers for the detection of carbapenemase-producing Enterobacteriaceae in geographical areas with a high prevalence of OXA-48 producers. J Antimicrob Chemother 2014;69(2):445–50. Livermore DM, Mushtaq S, Warner M, Zhang J, Maharjan S, Doumith M, et al. Activities of NXL104 combinations with ceftazidime and aztreonam against carbapenemaseproducing Enterobacteriaceae. Antimicrob Agents Chemother 2011;55(1):390–4. Nordmann P, Poirel L, Dortet L. Rapid detection of carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis 2012;18(9):1503–7. Vanstone GL, Dilley R, Schwenk S, Williams A, Balakrishnan I. Temocillin disc diffusion susceptibility testing by EUCAST methodology. J Antimicrob Chemother 2013;68 (11):2688–9. Woodford N, Pike R, Meunier D, Loy R, Hill R, Hopkins KL. In vitro activity of temocillin against multidrug-resistant clinical isolates of Escherichia coli, Klebsiella spp. and Enterobacter spp., and evaluation of high-level temocillin resistance as a diagnostic marker for OXA-48 carbapenemase. J Antimicrob Chemother 2014;69(2): 564–7.
Please cite this article as: Huang T-D, et al, Evaluation of avibactam-supplemented combination disk tests for the detection of OXA-48 carbapenemase-producing Enterobacteriaceae, Diagn Microbiol Infect Dis (2014), http://dx.doi.org/10.1016/j.diagmicrobio.2014.03.010
Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Evaluation of avibactam-supplemented combination disk tests for the detection of OXA-48 carbapenemase-producing Enterobacteriaceae☆,☆☆,★ Te-Din Huang ⁎, Catherine Berhin, Pierre Bogaerts, Youri Glupczynski National Reference Laboratory for Monitoring of Antimicrobial Resistance in Gram-negative bacteria, CHU Dinant-Godinne|UCL Namur, 1 Avenue Dr. G. Therasse, 5530 Yvoir, Belgium
a r t i c l e
i n f o
Article history: Received 13 January 2014 Received in revised form 17 February 2014 Accepted 9 March 2014 Available online xxxx Keywords: Temocillin Inhibitor OXA-type beta-lactamase
a b s t r a c t The ability of various combination disk tests (CDTs) incorporating avibactam to detect OXA-48 carbapenemase-producing Enterobacteriaceae was evaluated. The CDT using 30-μg temocillin alone and supplemented with 5-μg avibactam showed good performance and could be an adjunctive test to the classic CDT containing class A and class B carbapenemase inhibitors for the positive discrimination of OXA-48 carbapenemase producers from carbapenemase-negative strains. © 2014 Elsevier Inc. All rights reserved.
Accurate phenotypical detection of carbapenemases in Enterobacteriaceae is of great importance (Canton et al., 2012). Current phenotypic confirmatory methods of carbapenemase-producing Enterobacteriaceae (CPE) are based on the use of inhibitor-supplemented antimicrobial disks and allow differentiation between Ambler class A and B carbapenemase producers but fail to confirm Ambler class D OXA-48 production (Giske et al., 2011). Avibactam (formerly NXL104) is a recently developed non β-lactam diazabicyclooctane inhibitor of class A, C, and selected D β-lactamases including some carbapenemases (Livermore et al., 2011). We evaluated various combination disk tests (CDTs) incorporating avibactam to detect OXA-48 CPE. We selected 213 well-characterized Enterobacteriaceae collected from clinical laboratories across Belgium in 2012 to represent the diversity of the beta-lactams resistance mechanisms. The selection included 116 Klebsiella spp., 51 Enterobacter spp., 27 Escherichia coli, 7 Citrobacter spp., 5 Serratia marcescens, 3 Proteus spp., 3 Morganella morganii, and 1 Providencia spp. All isolates had been verified for the presence of carbapenemase by multiplex PCR targeting blaVIM, blaIMP, blaNDM, blaKPC, and blaOXA-48 and/or by imipenem hydrolysis using the Carba NP assay previously described (Bogaerts et al., 2013; Nordmann et al., 2012). A total of 113 were confirmed as CPE (OXA-48 [n = 72], VIM [n = 20], KPC [n = 17], and NDM [n = 4]), and 100 were non-
☆ Funding: The study was supported in part by the research grant of Fondation Mont-Godinne. The national reference centre is partially supported by the Belgian Ministry of Social Affairs through a fund within the Health Insurance System. ☆☆ Conflict of interest declaration: None to declare. ★ Parts of this study were published as an abstract in the 33rd Réunion Interdisciplinaire de Chimiothérapie Anti-Infectieuse, Paris, November 21–22, 2013. ⁎ Corresponding author. Tel.: +32-81-423212; fax: +32-81-423204. E-mail address: [email protected] (T.-D. Huang).
carbapenemase producers (43 with extended-spectrum β-lactamase [ESBL], 26 with hyperproduced plasmidic or derepressed chromosomal AmpC β-lactamase [AmpC], 7 with ESBL associated to AmpC, 14 with other narrow-spectrum penicillinases, and 10 with wild-type pattern). All isolates were tested using EUCAST disk diffusion methodology against the KPC/MBL Confirmation Kit® (containing meropenem, meropenem + dipicolinic acid, meropenem + phenylboronic acid, and meropenem + cloxacillin tablets; Rosco Diagnostics, Taastrup, Denmark) and against 6 paper disks including ertapenem 10 μg, meropenem 10 μg, and temocillin 30 μg, each alone (BioRad) or supplemented with avibactam 5 μg (home-made solution). CDT results using Rosco tablets were interpreted according to the manufacturers’ instructions. A cut-off of ≥5-mm diameter increase in paper disk containing avibactam was arbitrarily selected to assign positive result for the CDT. The distribution of ertapenem, meropenem, and temocillin paper disks zone diameter and the different CDT diameter increase on all isolates tested are shown in Table 1. 111/113 CPE and 57/100 noncarbapenemase producers isolates would have been categorized as carbapenem non-susceptible Enterobacteriaceae (CNSE) according to the EUCAST interpretative criteria (European Society of Clinical Microbiology and Infectious Diseases, 2013a). Among the 113 CPE isolates, 111 and 83, respectively, would have been detected by the ertapenem or meropenem screening breakpoint (b25 mm) proposed by EUCAST (European Society of Clinical Microbiology and Infectious Diseases, 2013b). All but 1 (VIM-producing Enterobacter cloacae) of the CPE isolates missed by the EUCAST screening breakpoint were OXA-48 producers. Concerning temocillin, 36 of 100 non–carbapenemaseproducing isolates were resistant according to the breakpoint (inhibition zone b20 mm) recently published (Vanstone et al., 2013), while all but 2 (1 NDM-positive M. morganii and 1 KPC-
0732-8893/© 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.diagmicrobio.2014.03.010
Please cite this article as: Huang T-D, et al, Evaluation of avibactam-supplemented combination disk tests for the detection of OXA-48 carbapenemase-producing Enterobacteriaceae, Diagn Microbiol Infect Dis (2014), http://dx.doi.org/10.1016/j.diagmicrobio.2014.03.010
2
T.-D. Huang et al. / Diagnostic Microbiology and Infectious Disease xxx (2014) xxx–xxx
Table 1 Distribution of ertapenem, meropenem, and temocillin paper disks zone diameter and the CDTs diameter increase on Enterobacteriaceae isolates tested (n = 213). Median (range) value in mm per carbapenemase enzyme
Paper disks
Combination disk tests
ETP zone diameter MEM zone diameter TMC zone diameter ETP + AVB/ETP diameter increase MEM + AVB/MEM diameter increase TMC + AVB/TMC diameter increase
VIM (n = 20)
NDM (n = 4)
KPC (n = 17)
OXA-48 (n = 72)
Negative (n = 100)
13 (6–20) 15 (6–25) 6 (6–15) 0 (0–6) 1 (0–15) 2 (0–12)
15 (9–20) 18 (17–20) 17 (6–25) 0 (0–3) 0 (0–2) 0 (0–6)
10 (6–15) 12 (6–19) 15 (6–20) 15 (0–17) 14 (0–16) 6 (0–7))
18 (7–26) 23 (9–29) 6 (6–16) 5 (2–16) 3 (1–16) 11 (6–15)
17 23 21 5 2 2
(6–38) (10–37) (6–30) (0–15) (−2 to 12) (−2 to 11)
ETP = ertapenem 10 μg; MEM = meropenem 10 μg; TMC = temocillin 30 μg; AVB = avibactam 5 μg.
producing Klebsiella pneumoniae) of the 113 CPE strains were temocillinresistant including 95% of the VIM (19/20) and of OXA-48 (68/72) producers having temocillin zone diameter b12 mm and confirming highlevel resistance to temocillin stated previously (Huang et al., 2014; Woodford et al., 2014). Phenotypical confirmatory test using Rosco CDT with inhibitors correctly detected all 17 KPC producers and 21/24 metallo-βlactamase (MBL) producers (2 E. cloacae and 1 Providencia rettgeri VIM-positive isolates gave false-negative results), but it misidentified 4 carbapenemase-negative (3 AmpC- and 1 ESBL-producing) as MBLproducing isolates. 55/100 non-carbapenemase producers, 15/17 KPC-positive, and 54/72 OXA-48–producing isolates showed increased diameter of ≥5 mm when avibactam was added to ertapenem, while 18/ 100 carbapenemase-negative, 15/17 KPC-positive, and 23/72 OXA-48– producing isolates had increased diameter of ≥5 mm when avibactam was combined to meropenem. No differences in diameter increase were observed between OXA-48 producers and non–carbapenemase-producing isolates with ertapenem ± avibactam nor with meropenem ± avibactam CDT since the distribution of diameter increase is similar for the 2 groups (see Table 1). The use of an arbitrary cut-off of ≥5 mm diameter increase yielded positive result with temocillin ± avibactam CDT for all 72 OXA-48 producers, 21/41 CPE expressing non–OXA-48 carbapenemase, and 22/100 carbapenemase-negative isolates. When applying a modified cut-off of ≥8 mm of diameter increase, 97% (70/72) of OXA-48 producers, 5% (2/41) of CPE expressing non–OXA-48
carbapenemase (1 VIM-1 and 1 VIM-31), and 8% (8/100) carbapenemase-negative isolates showed a positive CDT result with temocillin ± avibactam resulting in the overall calculated sensitivity and specificity for the detection of OXA-48–positive CPE of 97% and 93%, respectively. Among OXA-48 producers, no significant variation in diameter increase with temocillin ± avibactam CDT was observed between those isolates harbouring a blaOXA-48 gene alone (n = 15) and those co-producing an ESBL and/or AmpC (n = 57) in addition to OXA-48 (identical median value of 11-mm diameter increase for both groups). The distribution of diameter increase with temocillin ± avibactam focusing on the subset of 102 CNSE (72 OXA-48– producing, 30 carbapenemase-negative, and 2 VIM-producing) strains that yielded a negative result with the Rosco KPC/MBL Confirmation Kit® and that were temocillin-resistant is shown in Table 2. No change in sensitivity (97%) was observed using the modified cut-off of ≥8 mm with temocillin ± avibactam, and the specificity remained high at 84% (4 carbapenemase-negative and 1 VIM-31– producing isolates would have been misidentified as class D carbapenemase producers) within this group. Our study had some limitations. The current unavailability of avibactam for diagnostic purpose and the current home-made format of the disk tests requiring preparation skill could hold up its use as routine screening assay. We also believe that CDT used to detect betalactamases is a phenotypic test that should be backed by molecular methods to confirm the carbapenemase gene.
Table 2 Distribution (number of isolates) of temocillin 30 μg + avibactam 5 μg inhibition zone increase and of resistance mechanisms to β-lactams for carbapenem-non-susceptible a and temocillin-resistantb Enterobacteriaceae isolates tested negative by the Rosco KPC/MBL Confirmation Kit® (n = 102).
TMC + AVB/TMC diameter increase (mm) −2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
OXA-48 carbapenemase (n = 70)
VIM carbapenemase (n = 2)
Carbapenemase-negative (n = 30) ESBL (n = 14)
AmpC (n = 9)
ESBL and AmpC (n = 5)
Non-ESBL and non-AmpCc (n = 2)
1 1
1 1 6 11 9 14 15 10 2 1
1 1 3 2 2 2 1
1 2 2
1 1 1
2
1 1
1 1
1
2 1
ESBL = extended-spectrum β-lactamase; AmpC = hyperproduced chromosomal or plasmidic AmpC cephalosporinase; TMC + AVB/TMC = temocillin + avibactam/temocillin; in grey, the modified cut-off (≥8 mm) of diameter increase for this CDT. a According to the EUCAST interpretative criteria (ertapenem 10 μg inhibition zone b25 mm). b According to the breakpoint (temocillin 30 μg inhibition zone b20 mm) published by Vanstone et al. (2013). c One K-OXY β-lactamase hyperproducing Klebsiella oxytoca and 1 OXA-1 β-lactamase producing K. pneumoniae.
Please cite this article as: Huang T-D, et al, Evaluation of avibactam-supplemented combination disk tests for the detection of OXA-48 carbapenemase-producing Enterobacteriaceae, Diagn Microbiol Infect Dis (2014), http://dx.doi.org/10.1016/j.diagmicrobio.2014.03.010
T.-D. Huang et al. / Diagnostic Microbiology and Infectious Disease xxx (2014) xxx–xxx
High-level resistance to temocillin has been proposed as an excellent surrogate marker for the detection of OXA-48–producing CPE (Woodford et al., 2014). The CDT using temocillin 30 μg alone and supplemented with avibactam 5 μg showed good performance for the detection of OXA-48 CPE isolates when considering a diameter increase cut-off of ≥8 mm. This combination could constitute an adjunctive test to the confirmatory CDTs containing class A and class B carbapenemase inhibitors for the positive discrimination of class D carbapenemase producers from carbapenemase-negative strains.
Acknowledgments We are thankful to Wright Nichols, Aztra-Zeneca, for providing the avibactam for the testing.
References Bogaerts P, Rezende de Castro R, de Mendonca R, Huang TD, Denis O, Glupczynski Y. Validation of carbapenemase and extended-spectrum beta-lactamase multiplex endpoint PCR assays according to ISO 15189. J Antimicrob Chemother. 2013;68:1576–82. Canton R, Akova M, Carmeli Y, Giske CG, Glupczynski Y, Gniadkowski M, et al. Rapid evolution and spread of carbapenemases among Enterobacteriaceae in Europe. Clin Microbiol Infect 2012;18(5):413–31.
3
European Society of Clinical Microbiology and Infectious Diseases. Clinical breakpoints version 3.0. In European Committee on Antimicrobial Susceptibility Testing. 2013a [1 February 2013, date last accessed).]. Available from: http://www.eucast.org. European Society of Clinical Microbiology and Infectious Diseases. EUCAST guidelines for detection of resistance mechanisms and specific resistances of clinical and/or epidemiological importance. Version 1.0. 2013b [11 December 2013, date last accessed).]. Available from: http://www.eucast.org. Giske CG, Gezelius L, Samuelsen O, Warner M, Sundsfjord A, Woodford N. A sensitive and specific phenotypic assay for detection of metallo-beta-lactamases and KPC in Klebsiella pneumoniae with the use of meropenem disks supplemented with aminophenylboronic acid, dipicolinic acid and cloxacillin. Clin Microbiol Infect 2011;17(4):552–6. Huang TD, Poirel L, Bogaerts P, Berhin C, Nordmann P, Glupczynski Y. Temocillin and piperacillin/tazobactam resistance by disc diffusion as antimicrobial surrogate markers for the detection of carbapenemase-producing Enterobacteriaceae in geographical areas with a high prevalence of OXA-48 producers. J Antimicrob Chemother 2014;69(2):445–50. Livermore DM, Mushtaq S, Warner M, Zhang J, Maharjan S, Doumith M, et al. Activities of NXL104 combinations with ceftazidime and aztreonam against carbapenemaseproducing Enterobacteriaceae. Antimicrob Agents Chemother 2011;55(1):390–4. Nordmann P, Poirel L, Dortet L. Rapid detection of carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis 2012;18(9):1503–7. Vanstone GL, Dilley R, Schwenk S, Williams A, Balakrishnan I. Temocillin disc diffusion susceptibility testing by EUCAST methodology. J Antimicrob Chemother 2013;68 (11):2688–9. Woodford N, Pike R, Meunier D, Loy R, Hill R, Hopkins KL. In vitro activity of temocillin against multidrug-resistant clinical isolates of Escherichia coli, Klebsiella spp. and Enterobacter spp., and evaluation of high-level temocillin resistance as a diagnostic marker for OXA-48 carbapenemase. J Antimicrob Chemother 2014;69(2): 564–7.
Please cite this article as: Huang T-D, et al, Evaluation of avibactam-supplemented combination disk tests for the detection of OXA-48 carbapenemase-producing Enterobacteriaceae, Diagn Microbiol Infect Dis (2014), http://dx.doi.org/10.1016/j.diagmicrobio.2014.03.010
