Research letters
combining antibiotic resistance and putative virulence functions is highly
related to virulence plasmids identified in pathogenic E. coli isolates.
Plasmid 2013; 69: 127– 37.
9 Antonelli A, D’Andrea MM, Vaggelli G et al. OXA-372, a novel
carbapenem-hydrolysing class D b-lactamase from a Citrobacter freundii
isolated from a hospital wastewater plant. J Antimicrob Chemother
2015; 70: 2749– 56.
10 Carattoli A, Bertini A, Villa L et al. Identification of plasmids by
PCR-based replicon typing. J Microbiol Methods 2005; 63: 219– 28.
J Antimicrob Chemother 2016
doi:10.1093/jac/dkv283
Advance Access publication 15 September 2015
Detection of carbapenemase activity in
Enterobacteriaceae: comparison of the
carbapenem inactivation method versus
the Carba NP test
Nathalie Tijet1, Samir N. Patel1,2 and
Roberto G. Melano1–3*
1
Public Health Ontario Laboratory, Toronto, Ontario, Canada;
2
Department of Laboratory Medicine and Pathobiology, University
of Toronto, Toronto, Ontario, Canada; 3Department of Microbiology,
Mt Sinai Hospital, Toronto, Ontario, Canada
*Corresponding author. Public Health Ontario Laboratory, 661 University
Ave., Rm 20 –53, Toronto, Ontario M5G 1M1, Canada.
Tel: +1-647-792-3430; Fax: +1-416-235-5800;
E-mail: roberto.melano@oahpp.ca
Sir,
Carbapenem resistance mediated by plasmid-encoded carbapene-
mases has emerged worldwide and become a major concern.1 The
detection of the activity of carbapenemases has a strong impact on
hospital infection control, because the detection of their presence
can initiate measures to avoid potential outbreaks and lateral
spread of the resistance. Although molecular detection by PCR is
considered the gold standard for carbapenemase gene identifica-
tion, some limitations are clearly recognized. Between them, false-
negative results (the presence of a carbapenemase gene not tested
in the PCR reaction, or mutations affecting annealing of primers) or
the detection of inactive genes (i.e. no carbapenemase expression)
can delay infection-control measures or, oppositely, initiate them
when they are not required. A fast and accurate phenotypic
method, the Carba NP test (CNPt), was developed; it detects carba-
penemase activity with very high sensitivity and specificity and
lower costs compared with those of PCR.2 This method is now
being recommended in the CLSI guidelines for carbapenemase
activity detection.3 However, recent studies have shown that this
test has lower sensitivity particularly against isolates expressing
b-lactamases with low carbapenemase activity, such as OXA-48-
like, or expressing mucoid colonies.4,5 Another new test, the carba-
penem inactivation method (CIM), has shown very promising
results based on its sensitivity, specificity, low cost and easy
274
interpretation.6 Briefly, the CIM consists of two steps: (i) incubation
of a meropenem disc with the isolate to be tested; and (ii) incuba-
tion of this meropenem disc with the Escherichia coli ATCC strain.
After this second incubation step, the presence of carbapenemase
activity can be easily detected: the absence of an inhibition zone
indicates enzymatic hydrolysis of meropenem during the first incu-
bation step, whereas a ‘clear inhibition zone’ appears when the
tested isolate does not express carbapenemase activity.6 The
goal of this study was to compare the performance of the CIM
and the CNPt against a panel of well-characterized enterobacteria.
A total of 182 Enterobacteriaceae were tested (Table 1). Of these,
82 were negative controls with variable carbapenem susceptibilities,
which were determined by Etest and their results interpreted using
the CLSI guidelines.3 These 82 isolates had PCR results that were
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negative for carbapenemase genes, and none displayed carbapene-
mase activity by phenotypic tests (modified Hodge test, CNPt and
KPC/MBL Confirm Kit, Rosco Diagnostica).4 We also included 100
carbapenemase-producing isolates (including KPC, NDM, VIM, IMP,
OXA-48-like, NMC/IMI and SME producers), all confirmed by PCR
results and sequence analysis (Table 1). Both the CNPt and the CIM
were performed in triplicate for each isolate as described previously.3,6
All non-carbapenemase producers were negative by both pheno-
typic methods (Table 1). The fact that some of these negative con-
trols had high carbapenem MICs (≥32 mg/L) suggests the presence
of other mechanisms of resistance, such as AmpC and/or ESBL pro-
duction plus impermeability. These results confirm the specificity
and a positive predictive value of 100% for both methods. For carba-
penemase producers, the CNPt had a few false-negative results,
most of them related to OXA-48-like producers (two OXA-48, two
OXA-181, two OXA-232 and one OXA-244), but also with two
NDM-1 producers (one of which showed mucoid colonies) and one
KPC producer (mucoid). Both methods failed to detect a swarming-
producing Proteus mirabilis positive for IMP-27. However, the carba-
penemase activities of this enzyme as well as the NDM-1 from the
mucoid Providencia rettgeri were identified by both methods in trans-
formant or transconjugant E. coli strains, as described previously.4
These results gave a sensitivity (90.1%) and negative predictive
value (88.2%) for the CNPt that were similar to those previously
reported.4,5 However, considerably higher sensitivity (98.8%)
and negative predictive value (99%) were obtained by the CIM.
The CIM proves to be an accurate method for detection of carba-
penemase activity. In our hands, its main disadvantage was that, in
practice, it required an overnight incubation of the plates to obtain
results (in contrast to the 8 h incubation period claimed in the ori-
ginal report6) versus a maximum incubation of 2 h using the CNPt.
On the other hand, the CIM had multiple advantages: (i) it is an
easier-to-perform test; (ii) only water and a 10 mg meropenem
susceptibility-testing disc per isolate were necessary to perform
the test; (iii) four isolates plus positive and negative controls can
be tested on the same Mueller–Hinton agar plate (standard size)
inoculated with E. coli ATCC 25922 (these three first points highlight
the low costs of the test); and (iv) the interpretation of results is easy
(no inhibition zones versus ≥20 mm for negative carbapenemase
activity; in our tests, with the exception of one false-negative result,
we did not detect a carbapenemase producer exhibiting merope-
nem inhibition zones .6 mm).
In summary, both methods proved to be very efficient in the
detection of carbapenemase activity, with pros and cons for their
implementation in microbiological laboratories. Although the
CNPt showed an excellent specificity and a short turnaround time
Research letters JAC
Table 1. Results of the CIM and CNPt

MIC, mg/L Test results (n)a

Carbapenemase
Species (n)a detected by PCR (n)a imipenem meropenem ertapenem CNPtb CIMc

Non-carbapenemase producers (82)d

Citrobacter freundii (1) none 2 0.5 1.5 neg neg
Enterobacter aerogenes (4) none 0.5– 24 0.06 –32 0.12 to ≥32 neg neg
Enterobacter asburiae (1) none 0.5 0.064 0.19 neg neg
Enterobacter cloacae (22) none 0.19 –24 0.06 –16 0.25 to ≥32 neg neg
Enterobacter spp. (1) none ≥32 ≥32 ≥32 neg neg
Escherichia coli (12) none 0.023 –6 0.012 to ≥32 0.008 to ≥32 neg neg
Klebsiella oxytoca (1) none 0.25 0.25 1 neg neg

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Klebsiella pneumoniae (34) none 0.125 to ≥32 0.016– 24 0.016 to ≥32 neg neg
Morganella morganii (1) none 2 0.12 0.25 neg neg
Providencia stuartii (1) none 1.5 0.12 0.5 neg neg
Pantoea spp. (1) none 0.38 0.047 0.19 neg neg
Serratia marcescens (3) none 0.75 –2 0.12 –0.75 0.25 –1.5 neg neg

Carbapenemase producers (100)d

C. freundii (2) KPC 6 – 12 2 –6 4–6 pos pos
E. cloacae (13) KPC (3) 12 to ≥32 24 to ≥32 8 to ≥32 pos pos
NDM (2) ≥32 ≥32 ≥32 pos pos
NMC/IMI (4) ≥32 16 to ≥32 24 –32 pos pos
VIM (4) 6 – 24 6 –32 2 – 24 pos pos
E. coli (26) IMP-27 (1) 0.5 4 3 pos pos
KPC (2) 1–4 0.25 –1.5 0.5– 1.5 pos pos
NDM (4) 6 to ≥32 3 to ≥32 12 to ≥32 pos pos
OXA-48 (11) 0.5– 6 0.25 –6 0.5 to ≥32 pos (10); neg (1) pos
OXA-181 (4) 1–4 0.75 –1 1.5– 8 pos (2); neg (2) pos
OXA-244 (1) 3 3 16 neg pos
VIM (3) 2 – 12 0.5–24 0.125 –16 pos pos
K. oxytoca (1) OXA-48 3 0.75 3 pos pos
K. pneumoniae (38) KPC (4)e 0.5 to ≥32 0.125– 32 0.19 –32 pos (3); neg (1) pos
NDM (2) ≥32 ≥32 ≥32 pos pos
OXA-48 (15) 0.38 –32 0.125– 32 0.38 to ≥32 pos (13); neg (1) pos
OXA-181 (10) 6 to ≥32 2 to ≥32 3 to ≥32 pos pos
OXA-232 (7) 3 – 32 3 to ≥32 16 to ≥32 pos (5); neg (2) pos
M. morganii (1) NDM 32 2 2 pos pos
Proteus mirabilis (1) IMP-27 4 4 4 neg neg
Providencia rettgeri (1)e NDM-1 ≥32 ≥32 16 neg pos
P. stuartii (1) NDM-1 ≥32 ≥32 ≥32 neg pos
Raoultella ornithinolytica (1) KPC 2 0.75 0.75 pos pos
S. marcescens (15) KPC (1) ≥32 24 ≥32 pos pos
SME (14) ≥32 12 to ≥32 3 to ≥32 pos pos

a
n, number of isolates.
b
CNPt results: neg, negative (red); and pos, positive (yellow/orange). All the results shown in this table were obtained following the CLSI guidelines.3
False-negative results are shown in bold.
c
CIM results: neg, negative (inhibition zone ≥20 mm); and pos, positive (no inhibition zone).3 False-negative results are shown in bold.
d
PCR analysis was performed for the following carbapenemase genes: blaKPC, blaNDM, blaOXA-48-like, blaVIM, blaIMP, blaGES,7,8 blaSME and blaNMC/IMI.9
e
Includes one mucoid isolate.

for carbapenemase detection (from 10 min to 2 h), it was unable to or skills are necessary), but it requires at least 8 h (overnight in
detect around 10% of the carbapenemase (mainly OXA-48-like) our hands) of incubation before the results may be read. In both
producers. The CIM, on the other hand, showed excellent specificity cases, molecular tests are needed for identification of the
and sensitivity and low costs (no specialized reagents, equipment carbapenemase-producing genes in the clinical isolate.

275
Research letters
Funding
This work was supported by Public Health Ontario Laboratory internal
funding.
Transparency declarations
None to declare.
References
1 Patel G, Bonomo RA. “Stormy waters ahead”: global emergence of car-
bapenemases. Front Microbiol 2013; 4: 48.
2 Nordmann P, Poirel L, Dortet L. Rapid detection of carbapenemase-
producing Enterobacteriaceae. Emerg Infect Dis 2012; 18: 1503 –7.
3 Clinical and Laboratory Standards Institute. Performance Standards for
Antimicrobial Susceptibility Testing: Twenty-fifth Informational Supplement
M100-S25. CLSI, Wayne, PA, USA, 2015.
4 Tijet N, Boyd D, Patel SN et al. Evaluation of the Carba NP test for rapid detec-
tion of carbapenemase-producing Enterobacteriaceae and Pseudomonas
aeruginosa. Antimicrob Agents Chemother 2013; 57: 4578–80.
5 Osterblad M, Hakanen AJ, Jalava J. Evaluation of the Carba NP test for
carbapenemase detection. Antimicrob Agents Chemother 2014; 58:
7553– 6.
6 van der Zwaluw K, de Haan A, Pluister GN et al. The carbapenem inacti-
vation method (CIM), a simple and low-cost alternative for the Carba NP
test to assess phenotypic carbapenemase activity in Gram-negative
rods. PLoS One 2015; 10: e0123690.
7 Tijet N, Alexander DC, Richardson D et al. New Delhi metallo-b-lactamase,
Ontario, Canada. Emerg Infect Dis 2011; 17: 306–7.
8 Dallenne C, Da Costa A, Decre D et al. Development of a set of multiplex
PCR assays for the detection of genes encoding important b-lactamases in
Enterobacteriaceae. J Antimicrob Chemother 2010; 65: 490–5.
9 Voets GM, Fluit AC, Scharringa J et al. A set of multiplex PCRs for geno-
typic detection of extended-spectrum b-lactamases, carbapenemases,
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Antimicrob Agents 2011; 37: 356–9.
J Antimicrob Chemother 2016
doi:10.1093/jac/dkv303
Advance Access publication 7 October 2015
Update on dalbavancin activity tested
against Gram-positive clinical isolates
responsible for documented skin and
skin-structure infections in US and
European hospitals (2011 –13)
Rodrigo E. Mendes*, Mariana Castanheira, David J. Farrell,
Robert K. Flamm, Helio S. Sader and Ronald N. Jones
JMI Laboratories, North Liberty, IA, USA
*Corresponding author. Tel: +1-319-665-3370, ext. 218;
Fax: +1-319-665-3371; E-mail: rodrigo-mendes@jmilabs.com
276
Sir,
Dalbavancin was approved in the USA (2014) and Europe (2015)
for the treatment of adults with acute bacterial skin and skin-
structure infections (ABSSSIs) caused by susceptible isolates of
Staphylococcus aureus, including MSSA, MRSA, Streptococcus pyo-
genes, Streptococcus agalactiae and the Streptococcus anginosus
group.1 The ABSSSI indication was based on two identically
designed non-inferiority trials comparing dalbavancin safety and
efficacy to vancomycin/linezolid.2 Results showed that dalbavan-
cin was non-inferior to comparators, and 79.7% (525 of 659) and
79.8% (521 of 653) of patients in the dalbavancin and vancomy-
cin/linezolid arms had an early clinical response indicating treat-
ment success in the pooled analysis, respectively. Moreover,
Downloaded from http://jac.oxfordjournals.org/ at University of Toronto Library on January 5, 2016
dalbavancin is under consideration for other indications, including
paediatric osteomyelitis and community-acquired pneumonia.
A total of 8527 isolates from documented skin and skin-
structure infections (SSSIs) were collected from 29 sites in the
USA and 39 sites in European (Belgium, Czech Republic, France,
Germany, Greece, Hungary, Ireland, Italy, Poland, Portugal,
Romania, Slovenia, Spain, Sweden, UK and Ukraine), Israeli,
Russian and Turkish regions (herein described as Europe). Isolates
were determined clinically significant based on local guidelines and
submitted to a central monitoring laboratory (JMI Laboratories,
North Liberty, IA, USA), as part of the SENTRY Antimicrobial
Surveillance Program (2011–13). Isolates were initially identified by
the participating laboratory and identifications confirmed by the
monitoring laboratory using standard algorithms and supported by
MALDI–TOF–MS (Bruker Daltonics, Bremen, Germany). Broth microdi-
lution susceptibility testing followed the CLSI M07-A10 guidelines.3
Quality assurance was performed by concurrent testing of S. aureus
ATCC 29213, Enterococcus faecalis ATCC 29212 and Streptococcus
pneumoniae ATCC 49619.4 The dalbavancin approved breakpoints
for susceptibility were applied: S. aureus, ≤0.12 mg/L; S. anginosus
group [also applied for viridans group streptococci (VGS)],
≤0.12 mg/L; S. pyogenes and S. agalactiae, ≤0.12 mg/L [also used
for b-haemolytic streptococci and Streptococcus dysgalactiae].1,5
Breakpoint criteria for comparator agents were those from EUCAST.6
Dalbavancin had MIC50 and MIC90 values of 0.06 and 0.06 mg/L
for S. aureus from the USA or Europe, irrespective of the methicillin
(oxacillin) susceptibility (Table 1). When compared with other
agents with similar clinical indications, dalbavancin (MIC50/90,
0.06/0.06 mg/L) was 4- to 8-fold more potent than daptomycin
(MIC50/90, 0.25/0.5 mg/L) and 16-fold more potent than vancomy-
cin (MIC50/90, 1/1 mg/L) or linezolid (MIC50/90, 1/1 mg/L) against
MRSA (all 99.7% – 100.0% susceptible; Table S1, available as
Supplementary data at JAC Online). Clindamycin, gentamicin and
tetracycline were more active against MRSA isolates from the
USA (80.4%–97.8% susceptible) than Europe (68.4%–83.4% sus-
ceptible; Table S1).
MRSA subsets from the USA and Europe displaying vancomycin
MICs of 2 mg/L had dalbavancin modal MIC and MIC50 values
(0.06 mg/L for both) similar to the populations of S. aureus, MSSA
or those MRSA with vancomycin MIC values of ≤1 mg/L; however,
the dalbavancin MIC90 increased to 0.12 mg/L (Table 1). Linezolid
also demonstrated similar MIC50 values (i.e. 1 mg/L) for MRSA sub-
sets showing a vancomycin MIC of ≤1 or 2 mg/L, while daptomycin
tested against the MRSA subset with a vancomycin MIC of 2 mg/L
had MIC50 results 2-fold higher than those with an MIC of ≤1 mg/L
(Table S1). Of note, gentamicin, tetracycline and trimethoprim/