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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-g temocillin alone and
supplemented with 5-g 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 conrmatory 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 conrm Ambler class D
OXA-48 production (Giske et al., 2011). Avibactam (formerly NXL104) is a recently developed non -lactam diazabicyclooctane
inhibitor of class A, C, and selected D -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 veried 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 conrmed 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.
Conict of interest declaration: None to declare.
Parts of this study were published as an abstract in the 33rd Runion
Interdisciplinaire de Chimiothrapie Anti-Infectieuse, Paris, November 2122, 2013.
Corresponding author. Tel.: +32-81-423212; fax: +32-81-423204.
E-mail address: te-din.huang@uclouvain.be (T.-D. Huang).
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) xxxxxx
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
VIM (n = 20)
NDM (n = 4)
KPC (n = 17)
OXA-48 (n = 72)
Negative (n = 100)
13 (620)
15 (625)
6 (615)
0 (06)
1 (015)
2 (012)
15 (920)
18 (1720)
17 (625)
0 (03)
0 (02)
0 (06)
10 (615)
12 (619)
15 (620)
15 (017)
14 (016)
6 (07))
18 (726)
23 (929)
6 (616)
5 (216)
3 (116)
11 (615)
17
23
21
5
2
2
(638)
(1037)
(630)
(015)
(2 to 12)
(2 to 11)
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 conrming highlevel resistance to temocillin stated previously (Huang et al., 2014;
Woodford et al., 2014).
Phenotypical conrmatory test using Rosco CDT with inhibitors
correctly detected all 17 KPC producers and 21/24 metallo-lactamase (MBL) producers (2 E. cloacae and 1 Providencia rettgeri
VIM-positive isolates gave false-negative results), but it misidentied
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-48producing 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 noncarbapenemase-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 nonOXA-48
carbapenemase, and 22/100 carbapenemase-negative isolates. When
applying a modied cut-off of 8 mm of diameter increase, 97% (70/72)
of OXA-48 producers, 5% (2/41) of CPE expressing nonOXA-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 specicity
for the detection of OXA-48positive CPE of 97% and 93%, respectively.
Among OXA-48 producers, no signicant 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
Conrmation Kit and that were temocillin-resistant is shown in
Table 2. No change in sensitivity (97%) was observed using the modied
cut-off of 8 mm with temocillin avibactam, and the specicity
remained high at 84% (4 carbapenemase-negative and 1 VIM-31
producing isolates would have been misidentied 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 conrm the carbapenemase gene.
Table 2
Distribution (number of isolates) of temocillin 30 g + avibactam 5 g inhibition zone increase and of resistance mechanisms to -lactams for carbapenem-non-susceptible a and
temocillin-resistantb Enterobacteriaceae isolates tested negative by the Rosco KPC/MBL Conrmation 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)
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
1
1
1
1
2
1
ESBL = extended-spectrum -lactamase; AmpC = hyperproduced chromosomal or plasmidic AmpC cephalosporinase; TMC + AVB/TMC = temocillin + avibactam/temocillin;
in grey, the modied cut-off (8 mm) of diameter increase for this CDT.
a
According to the EUCAST interpretative criteria (ertapenem 10 g inhibition zone b25 mm).
b
According to the breakpoint (temocillin 30 g inhibition zone b20 mm) published by Vanstone et al. (2013).
c
One K-OXY -lactamase hyperproducing Klebsiella oxytoca and 1 OXA-1 -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) xxxxxx
Acknowledgments
We are thankful to Wright Nichols, Aztra-Zeneca, for providing the
avibactam for the testing.
References
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Validation of carbapenemase and extended-spectrum beta-lactamase multiplex
endpoint PCR assays according to ISO 15189. J Antimicrob Chemother. 2013;68:157682.
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):41331.
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