Diagnostic Microbiology and Infectious Disease xxx (2014) xxxxxx

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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-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).

carbapenemase producers (43 with extended-spectrum -lactamase

[ESBL], 26 with hyperproduced plasmidic or derepressed chromosomal AmpC -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 Conrmation 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 g,
meropenem 10 g, and temocillin 30 g, each alone (BioRad) or
supplemented with avibactam 5 g (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 noncarbapenemaseproducing 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

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

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 (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)

ETP = ertapenem 10 g; MEM = meropenem 10 g; TMC = temocillin 30 g; AVB = avibactam 5 g.

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)

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

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

High-level resistance to temocillin has been proposed as an

excellent surrogate marker for the detection of OXA-48producing
CPE (Woodford et al., 2014). The CDT using temocillin 30 g alone and
supplemented with avibactam 5 g 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 conrmatory 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.

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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