JCM Accepts, published online ahead of print on 29 October 2014 J. Clin. Microbiol. doi:10.1128/JCM.01692-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Evaluation of carbapenemase screening and confirmation tests in
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Enterobacteriaceae and development of a practical diagnostic algorithm
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Florian P. Maurer1, Claudio Castelberg1, Chantal Quiblier1, Guido V.
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Bloemberg1, Michael Hombach1,‡
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1) Institut für Medizinische Mikrobiologie, Universität Zürich, 8006 Zürich, Schweiz
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Running title: Diagnostic algorithm for carbapenemase detection
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Keywords: meropenem, imipenem, ertapenem, ESBL, AmpC, Carba NP, antibiotic
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resistance
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‡
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Michael Hombach, M.D.
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Institut für Medizinische Mikrobiologie
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Universität Zürich
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Gloriastr. 30/32
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8006 Zürich
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Switzerland
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Phone: 0041 44 634 27 00
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Fax: 0041 634 49 06
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Email: [email protected]
Corresponding author:
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Abstract
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Reliable identification of carbapenemase producing Enterobacteriaceae is
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necessary to limit their spread. This study aimed at developing a diagnostic flow-
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chart suitable for implementation in different types of clinical laboratories using
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phenotypic screening and confirmation tests. In total, 334 clinical Enterobacteriaceae
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isolates genetically characterized with respect to carbapenemase, extended-spectrum-
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beta-lactamase (ESBL), and AmpC genes were analyzed. 142/334 isolates (42.2%)
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were suspicious for carbapenemase production, i.e. intermediate or resistant to
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ertapenem AND/OR meropenem AND/OR imipenem according to EUCAST clinical
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breakpoints (CBPs). A group of 193/334 isolates (57.8%) showing susceptibility to
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ertapenem AND meropenem AND imipenem was considered as negative control
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group for this study. CLSI and EUCAST carbapenem CBPs and the new EUCAST
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MEM screening cut-off were evaluated as screening parameters. ETP, MEM and IPM
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+/- aminophenylboronic acid (APBA) or EDTA combined-disc tests (CDTs), and the
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Carba NP-II test were evaluated as confirmation assays. EUCAST temocillin cut-offs
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were evaluated for OXA-48 detection. The EUCAST MEM screening cut-off (< 25
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mm) showed a sensitivity of 100%. The ETP APBA-CDT on Muller-Hinton agar
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containing cloxacillin (MH-CLX) displayed 100% sensitivity and specificity for class
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A carbapenemase confirmation. ETP and MEM EDTA-CDTs showed 100%
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sensitivity and specificity for class B carbapenemases. Temocillin diameters/MIC
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testing on MH-CLX was highly specific for OXA-48 producers. The overall
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sensitivity, specificity, PPV, and NPV of the Carba NP-II test were 78.9%, 100%,
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100%, and 98.7%, respectively. Combining the EUCAST MEM carbapenemase-
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screening cut-off (< 25 mm), ETP (or MEM) APBA- and EDTA-CDTs, and
2
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temocillin disk diffusion on MH-CLX agar promises excellent performance for
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carbapenemase detection.
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Introduction
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In recent years, the emergence of diverse carbapenemases in Enterobacteriaceae has
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become a major challenge for healthcare systems (1). Carbapenemase producing
59
bacterial isolates pose a severe clinical problem as non-susceptibility to beta-lactams is
60
frequently
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aminoglycosides or quinolones (2, 3). As a consequence, treatment options for
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carbapenemase producers are alarmingly limited and often drugs displaying significant
63
side effects need to be administered as a last resort (4).
accompanied
by
co-resistance
to
additional
drug
classes,
e.g.
64
β-lactamases are classified according to their functional properties and molecular
65
structure by Ambler and Bush (5, 6). Some of these enzymes also display hydrolytic
66
activity towards carbapenems, e.g. Klebsiella pneumoniae carbapenemase (KPC,
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Ambler/Bush class A), the New Delhi metallo-β-lactamase (NDM-1), VIM, and GIM
68
type enzymes (all Ambler/Bush class B), or OXA-48 (Ambler/Bush class D). A key
69
characteristic used for discriminating enzymes belonging to different Ambler/Bush
70
classes is the responsiveness to specific inhibitors: Class A enzymes are inhibited by
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clavulanic and aminophenylboronic acid (APBA), class B enzymes are inhibited by
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EDTA, and class D enzymes do not respond to any inhibitors used in β-lactamase
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diagnostics (5, 6).
74
KPC enzymes were first detected in the USA in 1996 and have subsequently spread
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worldwide (7). In Europe, KPC is endemic in Italy, Greece, Poland, and northwestern
76
England (7). In Central Europe, France, and Spain other carbapenemases are reported
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more frequently. NDM-1 is endemic in India, Bangladesh, and Pakistan. In Europe,
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most NDM-1 are being isolated in Great Britain (8). OXA-48 is endemic in Turkey
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and Morocco, but is increasingly reported from other European countries mostly in
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repatriated patients (8, 9). Scandinavian countries, the Netherlands, and other 4
81
countries such as Switzerland generally report low prevalence rates for all
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carbapenemases. Thus, rapid and reliable detection of carbapenemases is desirable in
83
order to limit the spread of these enzymes.
84
Detection of carbapenemase producing bacteria comprises carrier screening and
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detection of carbapenemase production in routine antimicrobial susceptibility testing
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(AST). While chromogenic media are often used for carrier screening, laboratory
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strategies for β-lactamase detection in routine AST consist of a screening and a
88
confirmation step (10-14).
89
A variety of phenotypic and molecular, commercially available and in-house
90
laboratory tests have been described for carbapenemase detection. Molecular
91
techniques comprise end point and real-time PCRs as well as microarray techniques
92
(15-17). Critical diameters/MICs of ertapenem (ETP), meropenem (MEM), and
93
imipenem (IPM), and automated microdilution expert systems have been evaluated as
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screening methods (14, 18-20). For carbapenemase confirmation, the modified Hodge
95
test is recommended by CLSI and various commercial and in-house combined disk
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tests (CDTs) using boronic acid derivatives and EDTA/dipicolinic acid as specific
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inhibitors have been described (13, 19-25). In 2014, EUCAST published new
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guidelines for the detection of resistance mechanisms including carbapenemases, in
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which a CDT is recommended for carbapenemase confirmation (14, 22, 25). Recently,
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Nordmann et al. described a new inhibitor-based biochemical assay for carbapenemase
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detection, the Carba NP test, which has been published in two versions: The Carba
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NP-I assay provides a positive or negative result (“carbapenemase detected/not
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detected”) whereas the Carba NP-II test has been designed to also discriminate
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between carbapenemase classes A, B, and D (26-29). Apart from the original
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publications, few studies have systematically evaluated the Carba NP-I test for
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Enterobacteriaceae, and both the originally published protocol and modified versions
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were used. Reported sensitivities varied between 72.5% and 100%, whereas specificity
108
generally was reported to be 100% (30-33). Except for its original description the
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Carba NP-II assay has been systematically evaluated for Pseudomonas aeruginosa
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only (30, 31, 34-36).
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Several issues of carbapenemase detection remain challenging: i) Enterobacteriaceae
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overexpressing AmpC β-lactamases in combination with reduced cell-wall
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permeability compromise the specificity of APBA-CDTs as the inhibitor (APBA)
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affects both AmpC and carbapenemases (37-44); ii) Detection of OXA-48 and related
115
enzymes remains problematic as no specific inhibitor is available. Temocillin-
116
resistance was suggested as an indicator for OXA-48 production, but not for OXA-48
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confirmation (14, 25, 31, 45, 46).
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This study aimed at developing a modular diagnostic flow-chart suitable for all types
119
of clinical laboratories, which integrates
120
confirmation tests for highly sensitive and specific carbapenemase detection.
121 122 123 124 125 126 127 128 129 130 6
various phenotypic screening and
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Materials and Methods
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Bacterial isolates. In total, 334 non-duplicate clinical isolates recovered in our
133
laboratory from 2009 until 2014 were included in the study (Table 1). All isolates were
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genetically characterized for the presence of ESBL (TEM-ESBL, SHV-ESBL, and
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CTX-M types), plasmid-encoded AmpCs, chromosomal ampC promoter/attenuator
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mutations leading to overexpression (Escherichia coli only), and for the presence of
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carbapenemases (16, 47, 48). 142/334 isolates (42.2%) were considered suspicious
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for carbapenemase production due to non-susceptibility to ertapenem AND/OR
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meropenem AND/OR imipenem (intermediate or resistant zone diameters according to
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EUCAST CBPs), whereas 193/334 isolates (57.8%) considered non-suspicious for
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carbapenemase production (susceptible to ertapenem AND meropenem AND
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imipenem) served as a negative control group.
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Genetic detection of carbapenemase, ESBL and ampC genes. Total DNA was
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extracted from bacterial colonies after growth on sheep blood agar medium using the
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InstaGene
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carbapenemase genes was done by performing a carbapenemase multiplex PCR (16).
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For variant analysis OXA-48 genes were amplified with primers described(49). PCR
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amplicons were sequenced using PCR primers and sequences analyzed using GenBank
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and DNASTAR Lasergene software (DNASTAR Inc., Madison, Wisconsin USA). The
150
AID ESBL line probe assay (AID Autoimmun Diagnostika GmbH, Germany) was
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used for the detection of ESBL genes (50). Bacterial isolates were genetically
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characterized for the presence of plasmid-mediated AmpC type β-lactamase genes by
153
multiplex PCR (51). Chromosomal ampC promoter mutations of E. coli isolates were
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analyzed as described previously (52).
Matrix
(Bio-Rad,
Reinach,
7
Switzerland).
Genetic
detection
of
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Susceptibility testing. Disk diffusion susceptibility testing was done according to
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EUCAST recommendations (53). Antibiotic disks and Mueller-Hinton (MH) agar were
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obtained from Becton Dickinson, Franklin Lakes, NJ. Cloxacillin supplemented
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Mueller-Hinton (MH-CLX) agar was obtained from Axonlab AG, Baden, Switzerland.
159
Zone diameters were recorded using the Sirweb/Sirscan system (i2a, Montpellier,
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France). Minimal inhibitory concentrations (MICs) were determined by gradient
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diffusion (Etest, bioMérieux, Marcy L’Etoile, France) according to the manufacturer´s
162
instructions.
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Combined-disk tests (CDTs) for carbapenemase detection. CDTs were performed
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as described elsewhere (19, 24). Sets of two disks each containing IPM (10 μg), MEM
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(10 μg), or ETP (10 μg, all Becton Dickinson) were placed onto MH (EDTA-CDT) or
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both MH and MH-CLX (APBA-CDT) plates inoculated with a sample of the tested
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isolate (0.5 McFarland turbidity standard). Immediately after placing the disks onto the
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agar, 10 μL of a 29.2-mg/mL (0.1 M) EDTA solution (EDTA-CDT), or 10 μL of a 30-
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mg/mL APBA solution (APBA-CDT) were added to one of the two carbapenem disks
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in each set. Plates were incubated at 35°C for 16 to 20 hours, and zone diameters were
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recorded using the Sirweb/Sirscan system (i2a). Disc diameter differences of ≥ 5 mm
172
between the APBA-free and APBA-containing discs or between the EDTA-free and
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EDTA-containing discs were considered indicative for production of a class A
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carbapenemases and class B carbapenemase, respectively.
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Carba NP-II test. The Carba NP-II test was performed and interpreted as described
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(26). Reactions were read after 0, 30, 60 and 120 minutes of incubation. Color changes
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from red to yellow-orange were interpreted as follows: wells 2 and 4, positive (Ambler
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class A carbapenemase); wells 2 and 3, positive (Ambler class B carbapenemase);
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wells 2, 3 and 4: positive (probably Ambler class D carbapenemase); no well, 8
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carbapenemase negative; all wells, test not interpretable. The Carba NP-II test was
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performed by experienced personal, and all discrepant results were additionally
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repeated at least 3 times.
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Software. All calculations were done using the IBM SPSS statistics software version
184
20 (IBM Corporation, Armonk, NY) and the Microsoft Excel 2010 software (Microsoft
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Corporation, Redmond, WA).
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Results
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Evaluation of screening parameters for carbapenemase production
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The EUCAST non-susceptible ETP CBP (< 25 mm), and the EUCAST
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recommended carbapenemase MEM screening cut-off (< 25 mm) for carbapenemase
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production displayed highest sensitivity of all evaluated cut-offs (100%, Table 2). ETP,
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however, had a lower specificity (62.5%) than MEM (90.7%, Table 2). The ETP non-
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susceptible CLSI CBP (< 22 mm) and the non-susceptible CLSI CBP for MEM (< 23
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mm) displayed lower sensitivity (95.5% for both compounds, Table 2). The IPM non-
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susceptible EUCAST CBP (< 22 mm) had the lowest sensitivity (81.8%), whereas the
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non-susceptible CLSI IPM CBP (23 mm) had a sensitivity of 90.9%.
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Performance of carbapenemase confirmation tests
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Combined-disc tests (CDTs)
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The ETP APBA-CDT on MH-CLX agar displayed highest sensitivity and NPV for
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class A carbapenemase detection (100%, Table 2). Specificity of 100% was found for
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the ETP APBA-CDT, the IPM APBA-CDT, and the MEM APBA-CDT on MH-CLX,
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whereas the same CDTs on conventional MH agar showed a specificity of 96.9%,
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99.4%, and 96.6%, respectively (Table 2). 9/10 false-positive ETP APBA-CDTs on
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conventional MH agar occurred in species with chromosomal AmpC (6 Enterobacter
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cloacae, 1 Enterobacter aerogenes, and 2 Hafnia alvei). 9/11 false-positive MEM
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APBA-CDTs on conventional MH agar were also found in AmpC positive species, i.e.
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6 Enterobacter cloacae, 1 Enterobacter aerogenes, 1 Hafnia alvei, and 1 E. coli
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harboring a CIT type plasmid-encoded AmpC. One K. pneumoniae isolate lacking
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AmpC or ESBL was borderline positive in both ETP and MEM APBA-CDT on
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conventional MH (5 mm and 7 mm zone difference, respectively). Another
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K. pneumoniae isolate producing an ESBL was borderline positive only in the MEM
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APBA-CDT on conventional MH (5 mm difference).
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Both the ETP and the MEM EDTA-CDTs displayed 100% sensitivity and specificity
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for class B carbapenemase detection, whereas the sensitivity of the IPM EDTA-CDT
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was significantly lower (70%, Table 2).
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Carba NP-II test
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The overall sensitivity, specificity, PPV, and NPV of the Carba NP-II test were
217
78.9%, 100%, 100%, and 98.7%, respectively (Table 2). The test created some reading
218
problems resulting in ambiguous results that were treated as follows: One Enterobacter
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aerogenes isolate possessing a blaVIM gene gave ambiguous results in terms of class
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assignment (see isolate 8, Figure 1). After 30 min of incubation the pattern was
221
consistent with a class B carbapenemase, while after 120 min of incubation the pattern
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was consistent with a class D carbapenemase (e.g. OXA-48).. For calculation of
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performance parameters this isolate was rated carbapenemase positive (Table 3). Three
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Klebsiella pneumoniae isolates co-producing OXA-48 and CTX-M ESBL gave
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inconclusive results (Table 3): the NP-II patterns were negative for carbapenemase
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production until 60 min of incubation. After 120 min of incubation, the patterns could
227
either still be rated negative or weakly positive for class A carbapenemases (see Figure
228
1, isolates 20, 99, 51, results were reproduced three times with independent
229
preparations); these isolates were excluded from the calculation of performance
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parameters. In addition, one OXA-48 producing Klebsiella pneumoniae (see isolate 19,
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Figure 1) and three NDM producing isolates of Providencia rettgeri, Providencia
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stuartti, and Proteus mirabilis, respectively, gave false-negative results with the NP-II
233
test (see Table 3, isolates 136, 138, and 139, Figure 1). One Enterobacter cloacae
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isolate producing a GIM (class B) gave an OXA-48-like pattern (class D, see isolate 95,
11
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Figure 1). For the calculation of sensitivity and specificity this isolate was rated
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carbapenemase positive (Table 3).
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Temocillin testing on MH-CLX agar
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Nineteen representative carbapenem non-susceptible isolates were tested for
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temocillin zone diameters and MICs on MH and MH-CLX agar as indicators for the
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presence of OXA-48. Five isolates harbored blaOXA-48 genes, nine isolates were
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blaOXA-48 gene negative but showed overexpression of a chromosomally encoded
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AmpC, and five isolates harbored ESBL genes (but not blaOXA-48, Table 4). All
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OXA-48 producers showed high-level temocillin resistance on both MH and MH-CLX
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agar (median diameter 6 mm, median MIC >1024 mg/L, Table 4). Five out of nine
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AmpC hyperproducers displayed temocillin zone diameters lower than 11 mm on MH
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(EUCAST screening cut-off for OXA-48 like enzymes) (14). On MH-CLX, the
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temocillin median diameter of the AmpC hyperproducers increased by 7 mm
248
(corresponding to a median Etest-determined MIC decrease of 2 dilution steps, Table
249
4), and the five EUCAST OXA-48 screen false-positive isolates became true-negatives.
250
Temocillin median diameters and gradient diffusion MICs of the five ESBL producers
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were not altered by the use of MH-CLX as compared to conventional MH agar. Median
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temocillin diameters/MICs were 11 mm and 32 mg/L, respectively, on both media
253
(Table 4). The only false-positive temocillin-based OXA-48 screening result originated
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from an CTX-M type ESBL-producing Klebsiella pneumoniae isolate displaying
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temocillin diameters/MICs of 10 mm and 64 mg/L on both MH and MH-CLX agar.
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Genetic characterization of isolates
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In total, 23 carbapenemase genes were detected in 22 Enterobacteriaceae isolates: 7
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blaKPC, 1 blaIMI, 4 blaVIM, 4 blaNDM, 1 blaGIM, and 4 blaOXA-48; 1 isolate co-
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produced VIM and OXA-48 enzymes (Tables 1 and 2). Seventy-eight (23.4%) of the 12
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studied isolates were genetically negative for ESBL, AmpC, and carbapenemases; 178
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(53.3%) of the isolates produced an AmpC β-lactamase (including those species with
262
chromosomally encoded AmpC, i.e. Enterobacter cloacae, Enterobacter aerogenes,
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Citrobacter freundii, Hafnia alvei, Morganella morganii, Serratia marcescens, and
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Providencia stuartii, Table 1) (54); 105 (31.4%) of the isolates harbored an ESBL
265
(Table 1).
13
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Discussion
267
Screening parameters for carbapenemases
268
Disk diffusion critical diameters have been reported to display high sensitivity for the
269
detection of carbapenemases (13, 20). This study found 100% sensitivity for the
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EUCAST critical MEM diameter (< 25 mm) with a comparably high specificity of
271
90.7% (Table 2). ETP screening using the EUCAST non-susceptible CBP (< 25 mm)
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also showed high sensitivity (100%), but low specificity (62.5%, Table 2). Thus, our
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results confirm the current EUCAST recommendation (15). CLSI non-susceptible ETP
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(< 22 mm) and MEM (< 23 mm) CBPs displayed lower sensitivity as compared to the
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current EUCAST recommendation (95.5%, Table 2). Based on the findings of this
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study, carbapenemase screening using MEM is recommended, whereas the use of IMP
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as screening drug is discouraged (IMP sensitivity EUCAST < 22 mm / CLSI < 23 mm
278
81.8% and 90.9%, respectively). Since automated microdilution AST reportedly lacks
279
sensitivity and specificity due to antibiotic panel composition and drug concentrations
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tested (18, 55), disk diffusion critical MEM diameters promise the best performance for
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carbapenemase detection among all evaluated techniques. In addition, disk diffusion is
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cheap, simple, and widely implemented by many laboratories for routine AST.
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Carbapenemase confirmation tests
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The modified Hodge test, which is recommended by CLSI for carbapenemase
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confirmation, is cheap and, in principle, simple to perform (23). However, it displays
286
significant investigator dependence, practical interpretation is technically demanding,
287
the test cannot distinguish between the different carbapenemase classes, and reportedly
288
shows low specificity due to AmpC β-lactamase overproduction and decreased
289
permeability, e.g. caused by porin loss (13, 20, 55). The problem of discriminating 14
290
carbapenemase activity from AmpC and impermeability is well known both for species
291
possessing a chromosomal AmpC (e.g. Enterobacter spp., Citrobacter spp., or Hafnia
292
alvei), and for producers of plasmid-encoded AmpC, in particular Klebsiella
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pneumoniae (39, 44, 56). Even E. coli overproducing AmpC due to mutations in the
294
promoter/attenuator region and/or showing mutations in the active center of the enzyme
295
resulting in an extended–spectrum AmpC (ESAC) phenotype display carbapenem non-
296
susceptibility (41, 43). The same pattern accounts for ESBL producers in combination
297
with porin loss (37, 38). AmpC and ESBL production interferes not only with
298
carbapenemase screening, but also with APBA-CDT confirmation for class A
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carbapenemases (14, 19, 57). False positive results occur as APBA is not only an
300
inhibitor of class A carbapenemases, but also of AmpC β-lactamases. To improve
301
specificity of APBA-CDTs, MEM/CLX disks are used to check for AmpC interference
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(indirect approach) (13, 14, 20, 22, 25). However, based on the current EUCAST
303
algorithm, class A carbapenemases in isolates co-producing AmpC may be missed as
304
synergy of MEM with both CLX and APBA is interpreted as AmpC and porin loss (14).
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A recent study found two Enterobacter cloacae isolates overproducing AmpC, but also
306
harboring KPC and NMC-A enzymes that would have been misclassified using this
307
approach (13). Other authors pointed out that MEM-MEM/CLX zone diameter
308
differences are relatively lower in AmpC hyperproducers co-expressing a class A
309
carbapenemase (i.e. mean difference 1 mm) than in AmpC hyperproducers without a
310
class A carbapenemase (mean difference 5 mm) (22). Another study, however,
311
described MEM-MEM/CLX zone diameter differences of 6 to 7 mm and 0-7 mm for
312
AmpC hyperproducing E. cloacae harboring class A carbapenemases and AmpC
313
hyperproducers devoid of carbapenemases, respectively (13). Thus the discriminative
314
power of relative MEM-MEM/CLX diameter differences may be insufficient. In
315
addition, classification based on the relative degree of MEM-MEM/CLX diameter 15
316
differences is difficult to standardize and requires significant expertise. The present
317
study on 178 (53.3%) AmpC producing isolates shows that APBA-CDTs performed on
318
MH-CLX agar reliably detect class A carbapenemases with increased specificity
319
(100%) due to suppression of AmpC activity (Table 2). The approach is simple to
320
interpret as it uses a single critical zone diameter difference (5 millimeters), and it can
321
be integrated in one step with ESBL confirmation testing on the same MH-CLX agar
322
plate (48).
323
In the present study, the Carba NP-II showed an overall sensitivity of 78.9% and a
324
NPV of 98.7% (Table 2). Our results closely parallel those of a recent study, which
325
found a sensitivity of 72.5% for the Carba NP-I and a NPV of 69.2%. The difference in
326
NPV is well explained by the different prevalence of carbapenemase producers in the
327
study populations, i.e. 6.6% (n = 22) in this study and >45% (n =145) in the study of
328
Tijet et al. (31). Other authors found higher sensitivities for the Carba NP-I test using
329
different types of protocols (32, 33). Our data confirm ambiguities in the reading of the
330
Carba NP-I/II test in particular for OXA-48 producing isolates that tend to produce
331
inconclusive, or false-negative results (see Figure 1, isolates 19, 20, 51, and 99) (31). If
332
the inconclusive OXA-48 results from Figure 1 would have been rated negative (only a
333
slight color-change was visible after 120 min of incubation), sensitivity would have
334
been 68.2% (Table 2). If rated positive, the three ambiguous OXA-48 results would
335
have been consistent with a class A carbapenemase pattern, most likely due to the
336
simultaneous presence of a CTX-M type ESBL (class A enzyme), which may be
337
responsible for the weak color-change in wells II and IV after 120 min of incubation,
338
and which is inhibited by tazobactam in well III (see Figure 1). False-negative Carba
339
NP results have also been described for mucoid colonies, e.g. of Providencia rettgeri,
340
Providencia stuartii, or Proteus mirabilis isolates (29, 31). Negative results were
16
341
attributed to difficulties in protein extraction, species-specific traits, or the influence of
342
the agar type and ion content on the Carba NP test (30, 31, 36). Besides OXA-48
343
producers, false-negative results in the present study also occurred in non-mucoid
344
isolates of Providencia rettgeri, Providencia stuartii, and Proteus mirabilis producing
345
NDM enzymes. All tests for these isolates were repeated three times with the standard
346
protocol and additionally performed using colonies grown on various agar media of
347
different manufacturers, i.e. MH (Becton Dickinson), MH-CLX (Axonlab), Columbia
348
sheep blood, MacConkey (bioMérieux), and Uriselect4 agar (BioRad). Despite reports
349
that the Carba NP I test performed better from Columbia sheep blood and Uriselect4
350
agar results for these isolates remained false-negative for all media types pointing to
351
species-specific issues related to Providencia and Proteus isolates, and a low sensitivity
352
for OXA-48 enzymes (34). Other authors recently found a higher sensitivity and
353
specificity for the detection of OXA-48 (28). In summary, due to the higher NPV, the
354
Carba NP-II test may perform better in a low prevalence environment (i.e. our study) as
355
compared to high prevalence settings such as those investigated by Tijet et al. (31).
356
However, the issues of false-negative OXA-48 producers and species specific false-
357
negative results due to the unknown impact of different genetic backgrounds need to be
358
further analyzed.
359
The phenotypic detection of OXA-48-like carbapenemases remains challenging.
360
EUCAST recommends indirect OXA-48 confirmation by decreased zone diameters or
361
increased MICs for temocillin (< 11 mm, and > 32 mg/L, respectively) to exclude
362
ESBLs in combination with porin loss in cases where both APBA-CDT and EDTA-
363
CDT are negative (14). Temocillin MICs, however, are not recommended to
364
discriminate AmpC overproduction combined with porin loss from OXA-48 as
365
temocillin MICs are variable in this setting resulting in poor specificity. By suppressing
17
366
potential AmpC activity, temocillin disk diffusion testing or MIC determination by a
367
gradient diffusion method on MH-CLX can help to clearly increase specificity of
368
temocillin-based OXA-48 screening without compromising sensitivity (Table 4).
369
In summary, a combination of the EUCAST MEM carbapenemase-screening cut-off
370
(< 25 mm) and ETP (or MEM) APBA- and EDTA-CDTs plus temocillin disk diffusion
371
(or gradient diffusion-based MIC determination) on MH-CLX agar promises excellent
372
performance for carbapenemase detection. The proposed diagnostic flow-chart (Figure
373
2) would have resulted in a sensitivity, specificity, PPV, and NPV of 100% in the study
374
population. This algorithm is simple, easy to use, cost-efficient and applicable in the
375
majority of clinical microbiology laboratories.
376
18
377
Acknowledgments
378
We are grateful to the laboratory technicians of the Institute of Medical
379
Microbiology, University of Zurich for their dedicated help, and to Erik C. Böttger and
380
Reinhard Zbinden for valuable discussions.
381 382
Funding
383
This work was supported by the University of Zurich.
384 385
Transparency declaration
386
All authors: No conflicts of interest to declare.
387 388 389 390 391 392 393 394
19
395
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593
Tables and Figures
594 595
Table 1: Species identification and beta-lactamase genotypes of studied isolates.
Species
Escherichia coli
N
%
ESBL, AmpC, Carbapenemase negative
Carbapenemases AmpC
KPC
5
1.5
+
26
7.8
+
34
10.2
45
13.5
1
0.3
IMI
VIM
NDM
GIM
OXA-48
+
+ + +
total
111 33.3
Enterobacter cloacae
59
17.7
NA
+
15
4.5
NA
+
1
0.3
NA
+
2
0.6
NA
+
1
0.3
NA
+
total
78
23.4
Klebsiella pneumoniae
24
7.2
22
6.6
13
3.9
+
2
0.6
+
2
0.6
4
1.2
1
0.3
3
0.9
1
0.3
1
0.3
total
73
21.9
Enterobacter aerogenes
11
3.3
NA
+
4
1.2
NA
+
1
0.3
NA
+
total
16
4.8
Klebsiella oxytoca
6
1.8
4
1.2
6
1.8
16
4.8
total
ESBL
+ + + +
+ +
+ +
+ + +
+
+ + +
+ +
+ + +
29
596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654
Table 1 continued
Species
N
%
Carbapenemases
ESBL, AmpC, Carbapenemase negative
AmpC
ESBL KPC
IMI
VIM
NDM
GIM
OXA-48
1
0.3
NA
+
3
0.9
NA
+
10
3.0
NA
+
total
14
4.2
Hafnia alvei
5
1.5
NA
+
1
0.3
NA
+
total
6
1.8
Proteus mirabilis
1
0.3
+
1
0.3
+
2
0.6
total
4
1.2
Morganella morganii
1
0.3
NA
+
2
0.6
NA
+
total
3
0.9
Serratia marcescens
3
0.9
NA
+
Citrobacter koseri
2
0.6
+
Salmonella spp.
2
0.6
+
Providencia rettgeri
1
0.3
+
Providencia stuartii
1
0.3
NA
+
Enterobacter sp.
1
0.3
NA
+
Pantoea spp.
1
0.3
+
Citrobacter spp.
1
0.3
NA
+
Serratia spp.
1
0.3
NA
+
Total
334
100
78
178
105
7
1
5
4
1
5
Genotypes (%)
100
23.4
53.3
31.4
2.1
0.3
1.5
1.2
0.3
1.5
Citrobacter freundii
+ +
+
+ + +
+
+ +
NA, not applicable, for species naturally harboring chromosomally-encoded AmpC beta-lactamases
30
655 656
Table 2: Performance parameters of screening and confirmation assays and the proposed
657
diagnostic flow chart (see Figure 2).
TP (N)
FP (N)
TN (N)
FN (N)
Total (N)
Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
MEM Screen EUCAST (< 25 mm)
22
29
283
0
334
100.0
90.7
43.1
100.0
ETP EUCAST I/R (< 25 mm)
22
117
195
0
334
100.0
62.5
15.8
100.0
IPM EUCAST I/R (< 22 mm)
18
16
296
4
334
81.8
94.9
52.9
98.7
MEM EUCAST I/R (< 22 mm)
20
18
294
2
334
90.9
94.2
52.6
99.3
ETP CLSI I/R (< 22 mm)
21
68
244
1
334
95.5
78.2
23.6
99.6
IPM CLSI I/R (< 23 mm)
20
19
293
2
334
90.9
93.9
51.3
99.3
MEM CLSI I/R (< 23 mm)
21
20
292
1
334
95.5
93.6
51.2
99.7
ETP-BA MH
6
10
316
2
334
75.0
96.9
37.5
99.4
IPM-BA MH
6
2
324
2
334
75.0
99.4
75.0
99.4
MEM-BA MH
7
11
315
1
334
87.5
96.6
38.9
99.7
ETP-BA MH-CLX
8
0
326
0
334
100.0
100.0
100.0
100.0
IPM-BA MH-CLX
6
0
326
2
334
75.0
100.0
100.0
99.4
MEM-BA MH-CLX
7
0
326
1
334
87.5
100.0
100.0
99.7
ETP-EDTA MH
10
0
324
0
334
100.0
100.0
100.0
100.0
IPM-EDTA MH
7
0
324
3
334
70.0
100.0
100.0
99.1
MEM-EDTA MH
10
0
324
0
334
100.0
100.0
100.0
100.0
Carba NP-II1
15
0
312
4
331
78.9
100.0
100.0
98.7
Carba NP-II
15
0
312
7
334
68.2
100.0
100.0
97.8
Proposed algorithm
22
0
312
0
334
100.0
100.0
100.0
100.0
Parameter Screening cut-offs / CBPs
CDTs
2
658 659 660
TP, true-positive; FP, false-positive; TN, true-negative; FN, false-negative; MEM, meropenem; ETP,
661
ertapenem; CDT, combined-disk test; APBA, aminophenylboronic acid; MH-CLX agar, Muller Hinton
662
agar supplemented with cloxacillin; MH agar, Muller Hinton agar without cloxacillin.
663
1
inconclusives were excluded from the calculation;
664
2
inconclusives rated negative.
665 31
666 667
Table 3: Carbapenemase-positive isolates with characteristics and confirmation test results. CDTs (Δ mm)
Isolate number
Species
AmpC
ESBL
Carbapenemase type Carbapenemase class
NP-II
BA on MH
BA on MH-CLX
EDTA on MH IMI
MEM
7
Klebsiella pneumoniae
-
-
KPC
A
+
7
5
8
8
6
10
0
0
0
29
Klebsiella pneumoniae
-
SHV-ESBL
KPC
A
+
6
5
5
11
7
11
0
1
0
31
Klebsiella pneumoniae
-
-
KPC
A
+
7
11
7
9
8
9
0
0
0
35
Klebsiella pneumoniae
-
-
KPC
A
+
4
5
8
7
5
6
0
0
1
37
Klebsiella pneumoniae
-
-
KPC
A
+
7
7
9
8
4
9
0
0
0
40
Enterobacter cloacae
cAmpC
-
IMI
A
+
11
13
13
11
8
10
2
1
1
55
Escherichia coli
-
-
KPC
A
+
4
2
6
7
3
6
2
0
0
137
Klebsiella pneumoniae
-
CTX-M
KPC
A
+
6
4
4
5
5
2
0
3
0
8
Enterobacter aerogenes cAmpC
-
VIM
B
+
0
0
0
0
0
0
7
5
8
9
Klebsiella pneumoniae
-
-
NDM
B
+
0
0
0
2
0
0
16
7
13
17
Enterobacter cloacae
cAmpC
-
VIM
B
+
0
0
0
3
0
0
5
3
5
70
Citrobacter freundii
cAmpC
-
VIM
B
+
0
0
0
0
0
0
5
4
7
82
Klebsiella pneumoniae
-
-
VIM
B
+
0
0
0
0
0
1
15
17
21
95
Enterobacter cloacae
cAmpC
-
GIM-1
B
+
0
0
0
2
0
2
10
3
10
136
Providencia rettgeri
cAmpC
-
NDM
B
-
0
0
0
0
0
0
10
19
19
138
Providencia stuartii
cAmpC
-
NDM
B
-
0
0
0
0
0
0
9
13
12
139
Proteus mirabilis
CIT
-
NDM
B
-
0
4
0
0
0
0
6
16
6
36
Enterobacter cloacae
VIM
B
+
0
0
0
3
0
1
5
6
8
20
Klebsiella pneumoniae
-
CTX-M
OXA-48
D
inconclusive
0
0
0
3
0
0
0
0
0
51
Klebsiella pneumoniae
-
CTX-M
OXA-48
D
inconclusive
0
0
0
2
0
3
0
0
0
99
Klebsiella pneumoniae
-
CTX-M
OXA-48
D
inconclusive
0
0
0
2
0
3
0
0
0
19
Klebsiella pneumoniae
-
-
OXA-48
D
-
3
0
1
3
0
2
0
0
0
cAmpC SHV-ESBL
ETP
IMI
MEM
ETP
IMI
MEM
ETP
668
MEM, meropenem; ETP, ertapenem; CDT, combined-disk test; APBA, aminophenylboronic acid; MH-CLX, Muller Hinton agar supplemented with cloxacillin; MH, Muller Hinton
669
agar without cloxacillin; ESBL, extended-spectrum beta-lactamase; cAmpC, chromosomally encoded ampC gene
32
670 Table 4: Temocillin critical zone diameters and MICs for confirmation of OXA-48-like 671 carbapenemases. 672 Temocillin zone (mm) ID
Species
ESBL
AmpC
Temocillin MIC (mg/L)
Carbapenemase MH
MH-CLX
MH
MH-CLX
19
Klebsiella pneumoniae
-
-
OXA-48
6
6
1024
1024
20
Klebsiella pneumoniae
+
-
OXA-48
6
6
1024
1024
51
Klebsiella pneumoniae
+
-
OXA-48
6
6
1024
1024
99
Klebsiella pneumoniae
+
-
OXA-48
6
6
1024
1024
36
Enterobacter cloacae
+
cAmpC
VIM
6
8
1024
128
16
Hafnia alvei
-
cAmpC
6
11
128
32
18
Enterobacter cloacae
-
cAmpC
9
17
32
16
5
Enterobacter cloacae
-
cAmpC
10
21
32
8
27
Enterobacter cloacae
-
cAmpC
10
12
32
32
25
Hafnia alvei
-
cAmpC
10
21
32
4
26
Enterobacter cloacae
-
cAmpC
11
18
32
8
2
Enterobacter cloacae
-
cAmpC
12
16
32
16
125 Enterobacter cloacae
-
cAmpC
14
22
16
4
1
Enterobacter aerogenes
-
cAmpC
16
20
8
8
39
Klebsiella pneumoniae
+
-
10
10
64
64
60
Escherichia coli
+
-
11
11
32
32
38
Proteus mirabilis
+
-
11
11
32
32
130 Klebsiella pneumoniae
+
-
14
13
16
16
128 Klebsiella pneumoniae
+
-
18
17
8
8
median values OXA-48 positive isolates
6
6
1024
1024
AmpC overexpression
10
18
32
8
ESBL
11
11
32
32
673 674
ID, isolate identification number, ESBL, extended-spectrum beta-lactamase, MH-CLX:
675
Muller Hinton agar supplemented with cloxacillin, MH: Muller Hinton agar without
676
cloxacillin, cAmpC, chromosomally-encoded AmpC beta-lactamase
677 33
Figure 1: Discrepant test results of the Carba NP-II test and the carbapenemase genotype
678
isolate number
species
genotype / Ambler class
8
Enterobacter aerogenes
VIM
B
20
Klebsiella pneumoniae
OXA-48, CTX-M
D
99
Klebsiella pneumoniae
OXA-48, CTX-M
D
51
Klebsiella pneumoniae
OXA-48, CTX-M
D
19
Klebsiella pneumoniae
OXA-48
D
136
Providencia rettgeri
NDM
B
138
Providencia stuartii
NDM
B
139
Proteus mirabilis
NDM
B
95
Enterobacter cloacae
GIM
B
Carba NP-II result (examples of replicate testing) t = 0 minutes
t = 30 minutes
negative result
class A carbapenemase
t = 60 minutes
t = 120 minutes
interpretation (expected test result)
679 680 34
class B carbapenemase
class D carbapenemase
Figure 2: Proposed diagnostic flow chart for carbapenemase detection.
681
Enterobacteriaceae isolates Initial screening step Inhibition zone diameter MEM < 25mm Time to result 24 h (regular antibiogram) No
Yes Carbapenemases excluded for 57.8% of study population
CDT ETP versus ETP/APBA on MH-CLX agar1
No carbapenemase suspicion
Δ(ETP/APBA – ETP) < 5 mm
Δ(ETP/APBA – ETP) ≥ 5 mm
CDT ETP versus ETP/EDTA on MH agar1
CDT ETP versus ETP/EDTA on MH agar1 Phenotypic confirmation step
Δ(ETP/EDTA – ETP) < 5 mm
Δ(ETP/EDTA – ETP) ≥ 5 mm
Δ(ETP/EDTA – ETP) < 5 mm
Δ(ETP/EDTA – ETP) ≥ 5 mm
Carbapenemases
Carbapenemases
Carbapenemases
Carbapenemases
class A
No
class A
No
class A
Yes
class A
Yes
class B
No
class B
Yes
class B
No
class B
Yes
class D
?
class D
?
class D
?
class D
?
Additional time to result 24 h (total 48 h) Carbapenemases excluded/confirmed for 98.5% of study population
Temocillin disk diffusion or MIC on MH-CLX agar
682
≥ 11 mm or ≤ 32 mg/L
< 11 mm or >32 mg/L
Oxa-48like enzyme unlikely2
Suspicion for Oxa-48like enzyme
Genotypic confirmation step Perform molecular assay for the detection of class D carbapenemases
683
Additional time to result 24 h (total 72 h) (1.5% of study population, OXA-48 only) Carbapenemases excluded/confirmed for 98.5% of study aminophenylboronic population
684
MEM, meropenem; ETP, ertapenem; CDT, combined-disk test; APBA,
685
acid; MH-CLX agar, Muller Hinton agar supplemented with cloxacillin; MH agar, Muller
686
Hinton agar without cloxacillin. 1 MEM can be used alternatively with slightly lower sensitivity.
687
2
688
overexpression and decreased permeability, e,g, due to porin deficiency.
Carbapenem resistance phenotype is most likely due to a combination of AmpC and/or ESBL
35
1
Evaluation of carbapenemase screening and confirmation tests in
2
Enterobacteriaceae and development of a practical diagnostic algorithm
3 4
Florian P. Maurer1, Claudio Castelberg1, Chantal Quiblier1, Guido V.
5
Bloemberg1, Michael Hombach1,‡
6 7
1) Institut für Medizinische Mikrobiologie, Universität Zürich, 8006 Zürich, Schweiz
8 9
Running title: Diagnostic algorithm for carbapenemase detection
10 11
Keywords: meropenem, imipenem, ertapenem, ESBL, AmpC, Carba NP, antibiotic
12
resistance
13 14
‡
15
Michael Hombach, M.D.
16
Institut für Medizinische Mikrobiologie
17
Universität Zürich
18
Gloriastr. 30/32
19
8006 Zürich
20
Switzerland
21
Phone: 0041 44 634 27 00
22
Fax: 0041 634 49 06
23
Email: [email protected]
Corresponding author:
24 25 26 27 28 29
1
30
Abstract
31
Reliable identification of carbapenemase producing Enterobacteriaceae is
32
necessary to limit their spread. This study aimed at developing a diagnostic flow-
33
chart suitable for implementation in different types of clinical laboratories using
34
phenotypic screening and confirmation tests. In total, 334 clinical Enterobacteriaceae
35
isolates genetically characterized with respect to carbapenemase, extended-spectrum-
36
beta-lactamase (ESBL), and AmpC genes were analyzed. 142/334 isolates (42.2%)
37
were suspicious for carbapenemase production, i.e. intermediate or resistant to
38
ertapenem AND/OR meropenem AND/OR imipenem according to EUCAST clinical
39
breakpoints (CBPs). A group of 193/334 isolates (57.8%) showing susceptibility to
40
ertapenem AND meropenem AND imipenem was considered as negative control
41
group for this study. CLSI and EUCAST carbapenem CBPs and the new EUCAST
42
MEM screening cut-off were evaluated as screening parameters. ETP, MEM and IPM
43
+/- aminophenylboronic acid (APBA) or EDTA combined-disc tests (CDTs), and the
44
Carba NP-II test were evaluated as confirmation assays. EUCAST temocillin cut-offs
45
were evaluated for OXA-48 detection. The EUCAST MEM screening cut-off (< 25
46
mm) showed a sensitivity of 100%. The ETP APBA-CDT on Muller-Hinton agar
47
containing cloxacillin (MH-CLX) displayed 100% sensitivity and specificity for class
48
A carbapenemase confirmation. ETP and MEM EDTA-CDTs showed 100%
49
sensitivity and specificity for class B carbapenemases. Temocillin diameters/MIC
50
testing on MH-CLX was highly specific for OXA-48 producers. The overall
51
sensitivity, specificity, PPV, and NPV of the Carba NP-II test were 78.9%, 100%,
52
100%, and 98.7%, respectively. Combining the EUCAST MEM carbapenemase-
53
screening cut-off (< 25 mm), ETP (or MEM) APBA- and EDTA-CDTs, and
2
54
temocillin disk diffusion on MH-CLX agar promises excellent performance for
55
carbapenemase detection.
3
56
Introduction
57
In recent years, the emergence of diverse carbapenemases in Enterobacteriaceae has
58
become a major challenge for healthcare systems (1). Carbapenemase producing
59
bacterial isolates pose a severe clinical problem as non-susceptibility to beta-lactams is
60
frequently
61
aminoglycosides or quinolones (2, 3). As a consequence, treatment options for
62
carbapenemase producers are alarmingly limited and often drugs displaying significant
63
side effects need to be administered as a last resort (4).
accompanied
by
co-resistance
to
additional
drug
classes,
e.g.
64
β-lactamases are classified according to their functional properties and molecular
65
structure by Ambler and Bush (5, 6). Some of these enzymes also display hydrolytic
66
activity towards carbapenems, e.g. Klebsiella pneumoniae carbapenemase (KPC,
67
Ambler/Bush class A), the New Delhi metallo-β-lactamase (NDM-1), VIM, and GIM
68
type enzymes (all Ambler/Bush class B), or OXA-48 (Ambler/Bush class D). A key
69
characteristic used for discriminating enzymes belonging to different Ambler/Bush
70
classes is the responsiveness to specific inhibitors: Class A enzymes are inhibited by
71
clavulanic and aminophenylboronic acid (APBA), class B enzymes are inhibited by
72
EDTA, and class D enzymes do not respond to any inhibitors used in β-lactamase
73
diagnostics (5, 6).
74
KPC enzymes were first detected in the USA in 1996 and have subsequently spread
75
worldwide (7). In Europe, KPC is endemic in Italy, Greece, Poland, and northwestern
76
England (7). In Central Europe, France, and Spain other carbapenemases are reported
77
more frequently. NDM-1 is endemic in India, Bangladesh, and Pakistan. In Europe,
78
most NDM-1 are being isolated in Great Britain (8). OXA-48 is endemic in Turkey
79
and Morocco, but is increasingly reported from other European countries mostly in
80
repatriated patients (8, 9). Scandinavian countries, the Netherlands, and other 4
81
countries such as Switzerland generally report low prevalence rates for all
82
carbapenemases. Thus, rapid and reliable detection of carbapenemases is desirable in
83
order to limit the spread of these enzymes.
84
Detection of carbapenemase producing bacteria comprises carrier screening and
85
detection of carbapenemase production in routine antimicrobial susceptibility testing
86
(AST). While chromogenic media are often used for carrier screening, laboratory
87
strategies for β-lactamase detection in routine AST consist of a screening and a
88
confirmation step (10-14).
89
A variety of phenotypic and molecular, commercially available and in-house
90
laboratory tests have been described for carbapenemase detection. Molecular
91
techniques comprise end point and real-time PCRs as well as microarray techniques
92
(15-17). Critical diameters/MICs of ertapenem (ETP), meropenem (MEM), and
93
imipenem (IPM), and automated microdilution expert systems have been evaluated as
94
screening methods (14, 18-20). For carbapenemase confirmation, the modified Hodge
95
test is recommended by CLSI and various commercial and in-house combined disk
96
tests (CDTs) using boronic acid derivatives and EDTA/dipicolinic acid as specific
97
inhibitors have been described (13, 19-25). In 2014, EUCAST published new
98
guidelines for the detection of resistance mechanisms including carbapenemases, in
99
which a CDT is recommended for carbapenemase confirmation (14, 22, 25). Recently,
100
Nordmann et al. described a new inhibitor-based biochemical assay for carbapenemase
101
detection, the Carba NP test, which has been published in two versions: The Carba
102
NP-I assay provides a positive or negative result (“carbapenemase detected/not
103
detected”) whereas the Carba NP-II test has been designed to also discriminate
104
between carbapenemase classes A, B, and D (26-29). Apart from the original
105
publications, few studies have systematically evaluated the Carba NP-I test for
5
106
Enterobacteriaceae, and both the originally published protocol and modified versions
107
were used. Reported sensitivities varied between 72.5% and 100%, whereas specificity
108
generally was reported to be 100% (30-33). Except for its original description the
109
Carba NP-II assay has been systematically evaluated for Pseudomonas aeruginosa
110
only (30, 31, 34-36).
111
Several issues of carbapenemase detection remain challenging: i) Enterobacteriaceae
112
overexpressing AmpC β-lactamases in combination with reduced cell-wall
113
permeability compromise the specificity of APBA-CDTs as the inhibitor (APBA)
114
affects both AmpC and carbapenemases (37-44); ii) Detection of OXA-48 and related
115
enzymes remains problematic as no specific inhibitor is available. Temocillin-
116
resistance was suggested as an indicator for OXA-48 production, but not for OXA-48
117
confirmation (14, 25, 31, 45, 46).
118
This study aimed at developing a modular diagnostic flow-chart suitable for all types
119
of clinical laboratories, which integrates
120
confirmation tests for highly sensitive and specific carbapenemase detection.
121 122 123 124 125 126 127 128 129 130 6
various phenotypic screening and
131
Materials and Methods
132
Bacterial isolates. In total, 334 non-duplicate clinical isolates recovered in our
133
laboratory from 2009 until 2014 were included in the study (Table 1). All isolates were
134
genetically characterized for the presence of ESBL (TEM-ESBL, SHV-ESBL, and
135
CTX-M types), plasmid-encoded AmpCs, chromosomal ampC promoter/attenuator
136
mutations leading to overexpression (Escherichia coli only), and for the presence of
137
carbapenemases (16, 47, 48). 142/334 isolates (42.2%) were considered suspicious
138
for carbapenemase production due to non-susceptibility to ertapenem AND/OR
139
meropenem AND/OR imipenem (intermediate or resistant zone diameters according to
140
EUCAST CBPs), whereas 193/334 isolates (57.8%) considered non-suspicious for
141
carbapenemase production (susceptible to ertapenem AND meropenem AND
142
imipenem) served as a negative control group.
143
Genetic detection of carbapenemase, ESBL and ampC genes. Total DNA was
144
extracted from bacterial colonies after growth on sheep blood agar medium using the
145
InstaGene
146
carbapenemase genes was done by performing a carbapenemase multiplex PCR (16).
147
For variant analysis OXA-48 genes were amplified with primers described(49). PCR
148
amplicons were sequenced using PCR primers and sequences analyzed using GenBank
149
and DNASTAR Lasergene software (DNASTAR Inc., Madison, Wisconsin USA). The
150
AID ESBL line probe assay (AID Autoimmun Diagnostika GmbH, Germany) was
151
used for the detection of ESBL genes (50). Bacterial isolates were genetically
152
characterized for the presence of plasmid-mediated AmpC type β-lactamase genes by
153
multiplex PCR (51). Chromosomal ampC promoter mutations of E. coli isolates were
154
analyzed as described previously (52).
Matrix
(Bio-Rad,
Reinach,
7
Switzerland).
Genetic
detection
of
155
Susceptibility testing. Disk diffusion susceptibility testing was done according to
156
EUCAST recommendations (53). Antibiotic disks and Mueller-Hinton (MH) agar were
157
obtained from Becton Dickinson, Franklin Lakes, NJ. Cloxacillin supplemented
158
Mueller-Hinton (MH-CLX) agar was obtained from Axonlab AG, Baden, Switzerland.
159
Zone diameters were recorded using the Sirweb/Sirscan system (i2a, Montpellier,
160
France). Minimal inhibitory concentrations (MICs) were determined by gradient
161
diffusion (Etest, bioMérieux, Marcy L’Etoile, France) according to the manufacturer´s
162
instructions.
163
Combined-disk tests (CDTs) for carbapenemase detection. CDTs were performed
164
as described elsewhere (19, 24). Sets of two disks each containing IPM (10 μg), MEM
165
(10 μg), or ETP (10 μg, all Becton Dickinson) were placed onto MH (EDTA-CDT) or
166
both MH and MH-CLX (APBA-CDT) plates inoculated with a sample of the tested
167
isolate (0.5 McFarland turbidity standard). Immediately after placing the disks onto the
168
agar, 10 μL of a 29.2-mg/mL (0.1 M) EDTA solution (EDTA-CDT), or 10 μL of a 30-
169
mg/mL APBA solution (APBA-CDT) were added to one of the two carbapenem disks
170
in each set. Plates were incubated at 35°C for 16 to 20 hours, and zone diameters were
171
recorded using the Sirweb/Sirscan system (i2a). Disc diameter differences of ≥ 5 mm
172
between the APBA-free and APBA-containing discs or between the EDTA-free and
173
EDTA-containing discs were considered indicative for production of a class A
174
carbapenemases and class B carbapenemase, respectively.
175
Carba NP-II test. The Carba NP-II test was performed and interpreted as described
176
(26). Reactions were read after 0, 30, 60 and 120 minutes of incubation. Color changes
177
from red to yellow-orange were interpreted as follows: wells 2 and 4, positive (Ambler
178
class A carbapenemase); wells 2 and 3, positive (Ambler class B carbapenemase);
179
wells 2, 3 and 4: positive (probably Ambler class D carbapenemase); no well, 8
180
carbapenemase negative; all wells, test not interpretable. The Carba NP-II test was
181
performed by experienced personal, and all discrepant results were additionally
182
repeated at least 3 times.
183
Software. All calculations were done using the IBM SPSS statistics software version
184
20 (IBM Corporation, Armonk, NY) and the Microsoft Excel 2010 software (Microsoft
185
Corporation, Redmond, WA).
9
186
Results
187
Evaluation of screening parameters for carbapenemase production
188
The EUCAST non-susceptible ETP CBP (< 25 mm), and the EUCAST
189
recommended carbapenemase MEM screening cut-off (< 25 mm) for carbapenemase
190
production displayed highest sensitivity of all evaluated cut-offs (100%, Table 2). ETP,
191
however, had a lower specificity (62.5%) than MEM (90.7%, Table 2). The ETP non-
192
susceptible CLSI CBP (< 22 mm) and the non-susceptible CLSI CBP for MEM (< 23
193
mm) displayed lower sensitivity (95.5% for both compounds, Table 2). The IPM non-
194
susceptible EUCAST CBP (< 22 mm) had the lowest sensitivity (81.8%), whereas the
195
non-susceptible CLSI IPM CBP (23 mm) had a sensitivity of 90.9%.
196
Performance of carbapenemase confirmation tests
197
Combined-disc tests (CDTs)
198
The ETP APBA-CDT on MH-CLX agar displayed highest sensitivity and NPV for
199
class A carbapenemase detection (100%, Table 2). Specificity of 100% was found for
200
the ETP APBA-CDT, the IPM APBA-CDT, and the MEM APBA-CDT on MH-CLX,
201
whereas the same CDTs on conventional MH agar showed a specificity of 96.9%,
202
99.4%, and 96.6%, respectively (Table 2). 9/10 false-positive ETP APBA-CDTs on
203
conventional MH agar occurred in species with chromosomal AmpC (6 Enterobacter
204
cloacae, 1 Enterobacter aerogenes, and 2 Hafnia alvei). 9/11 false-positive MEM
205
APBA-CDTs on conventional MH agar were also found in AmpC positive species, i.e.
206
6 Enterobacter cloacae, 1 Enterobacter aerogenes, 1 Hafnia alvei, and 1 E. coli
207
harboring a CIT type plasmid-encoded AmpC. One K. pneumoniae isolate lacking
208
AmpC or ESBL was borderline positive in both ETP and MEM APBA-CDT on
209
conventional MH (5 mm and 7 mm zone difference, respectively). Another
10
210
K. pneumoniae isolate producing an ESBL was borderline positive only in the MEM
211
APBA-CDT on conventional MH (5 mm difference).
212
Both the ETP and the MEM EDTA-CDTs displayed 100% sensitivity and specificity
213
for class B carbapenemase detection, whereas the sensitivity of the IPM EDTA-CDT
214
was significantly lower (70%, Table 2).
215
Carba NP-II test
216
The overall sensitivity, specificity, PPV, and NPV of the Carba NP-II test were
217
78.9%, 100%, 100%, and 98.7%, respectively (Table 2). The test created some reading
218
problems resulting in ambiguous results that were treated as follows: One Enterobacter
219
aerogenes isolate possessing a blaVIM gene gave ambiguous results in terms of class
220
assignment (see isolate 8, Figure 1). After 30 min of incubation the pattern was
221
consistent with a class B carbapenemase, while after 120 min of incubation the pattern
222
was consistent with a class D carbapenemase (e.g. OXA-48).. For calculation of
223
performance parameters this isolate was rated carbapenemase positive (Table 3). Three
224
Klebsiella pneumoniae isolates co-producing OXA-48 and CTX-M ESBL gave
225
inconclusive results (Table 3): the NP-II patterns were negative for carbapenemase
226
production until 60 min of incubation. After 120 min of incubation, the patterns could
227
either still be rated negative or weakly positive for class A carbapenemases (see Figure
228
1, isolates 20, 99, 51, results were reproduced three times with independent
229
preparations); these isolates were excluded from the calculation of performance
230
parameters. In addition, one OXA-48 producing Klebsiella pneumoniae (see isolate 19,
231
Figure 1) and three NDM producing isolates of Providencia rettgeri, Providencia
232
stuartti, and Proteus mirabilis, respectively, gave false-negative results with the NP-II
233
test (see Table 3, isolates 136, 138, and 139, Figure 1). One Enterobacter cloacae
234
isolate producing a GIM (class B) gave an OXA-48-like pattern (class D, see isolate 95,
11
235
Figure 1). For the calculation of sensitivity and specificity this isolate was rated
236
carbapenemase positive (Table 3).
237
Temocillin testing on MH-CLX agar
238
Nineteen representative carbapenem non-susceptible isolates were tested for
239
temocillin zone diameters and MICs on MH and MH-CLX agar as indicators for the
240
presence of OXA-48. Five isolates harbored blaOXA-48 genes, nine isolates were
241
blaOXA-48 gene negative but showed overexpression of a chromosomally encoded
242
AmpC, and five isolates harbored ESBL genes (but not blaOXA-48, Table 4). All
243
OXA-48 producers showed high-level temocillin resistance on both MH and MH-CLX
244
agar (median diameter 6 mm, median MIC >1024 mg/L, Table 4). Five out of nine
245
AmpC hyperproducers displayed temocillin zone diameters lower than 11 mm on MH
246
(EUCAST screening cut-off for OXA-48 like enzymes) (14). On MH-CLX, the
247
temocillin median diameter of the AmpC hyperproducers increased by 7 mm
248
(corresponding to a median Etest-determined MIC decrease of 2 dilution steps, Table
249
4), and the five EUCAST OXA-48 screen false-positive isolates became true-negatives.
250
Temocillin median diameters and gradient diffusion MICs of the five ESBL producers
251
were not altered by the use of MH-CLX as compared to conventional MH agar. Median
252
temocillin diameters/MICs were 11 mm and 32 mg/L, respectively, on both media
253
(Table 4). The only false-positive temocillin-based OXA-48 screening result originated
254
from an CTX-M type ESBL-producing Klebsiella pneumoniae isolate displaying
255
temocillin diameters/MICs of 10 mm and 64 mg/L on both MH and MH-CLX agar.
256
Genetic characterization of isolates
257
In total, 23 carbapenemase genes were detected in 22 Enterobacteriaceae isolates: 7
258
blaKPC, 1 blaIMI, 4 blaVIM, 4 blaNDM, 1 blaGIM, and 4 blaOXA-48; 1 isolate co-
259
produced VIM and OXA-48 enzymes (Tables 1 and 2). Seventy-eight (23.4%) of the 12
260
studied isolates were genetically negative for ESBL, AmpC, and carbapenemases; 178
261
(53.3%) of the isolates produced an AmpC β-lactamase (including those species with
262
chromosomally encoded AmpC, i.e. Enterobacter cloacae, Enterobacter aerogenes,
263
Citrobacter freundii, Hafnia alvei, Morganella morganii, Serratia marcescens, and
264
Providencia stuartii, Table 1) (54); 105 (31.4%) of the isolates harbored an ESBL
265
(Table 1).
13
266
Discussion
267
Screening parameters for carbapenemases
268
Disk diffusion critical diameters have been reported to display high sensitivity for the
269
detection of carbapenemases (13, 20). This study found 100% sensitivity for the
270
EUCAST critical MEM diameter (< 25 mm) with a comparably high specificity of
271
90.7% (Table 2). ETP screening using the EUCAST non-susceptible CBP (< 25 mm)
272
also showed high sensitivity (100%), but low specificity (62.5%, Table 2). Thus, our
273
results confirm the current EUCAST recommendation (15). CLSI non-susceptible ETP
274
(< 22 mm) and MEM (< 23 mm) CBPs displayed lower sensitivity as compared to the
275
current EUCAST recommendation (95.5%, Table 2). Based on the findings of this
276
study, carbapenemase screening using MEM is recommended, whereas the use of IMP
277
as screening drug is discouraged (IMP sensitivity EUCAST < 22 mm / CLSI < 23 mm
278
81.8% and 90.9%, respectively). Since automated microdilution AST reportedly lacks
279
sensitivity and specificity due to antibiotic panel composition and drug concentrations
280
tested (18, 55), disk diffusion critical MEM diameters promise the best performance for
281
carbapenemase detection among all evaluated techniques. In addition, disk diffusion is
282
cheap, simple, and widely implemented by many laboratories for routine AST.
283
Carbapenemase confirmation tests
284
The modified Hodge test, which is recommended by CLSI for carbapenemase
285
confirmation, is cheap and, in principle, simple to perform (23). However, it displays
286
significant investigator dependence, practical interpretation is technically demanding,
287
the test cannot distinguish between the different carbapenemase classes, and reportedly
288
shows low specificity due to AmpC β-lactamase overproduction and decreased
289
permeability, e.g. caused by porin loss (13, 20, 55). The problem of discriminating 14
290
carbapenemase activity from AmpC and impermeability is well known both for species
291
possessing a chromosomal AmpC (e.g. Enterobacter spp., Citrobacter spp., or Hafnia
292
alvei), and for producers of plasmid-encoded AmpC, in particular Klebsiella
293
pneumoniae (39, 44, 56). Even E. coli overproducing AmpC due to mutations in the
294
promoter/attenuator region and/or showing mutations in the active center of the enzyme
295
resulting in an extended–spectrum AmpC (ESAC) phenotype display carbapenem non-
296
susceptibility (41, 43). The same pattern accounts for ESBL producers in combination
297
with porin loss (37, 38). AmpC and ESBL production interferes not only with
298
carbapenemase screening, but also with APBA-CDT confirmation for class A
299
carbapenemases (14, 19, 57). False positive results occur as APBA is not only an
300
inhibitor of class A carbapenemases, but also of AmpC β-lactamases. To improve
301
specificity of APBA-CDTs, MEM/CLX disks are used to check for AmpC interference
302
(indirect approach) (13, 14, 20, 22, 25). However, based on the current EUCAST
303
algorithm, class A carbapenemases in isolates co-producing AmpC may be missed as
304
synergy of MEM with both CLX and APBA is interpreted as AmpC and porin loss (14).
305
A recent study found two Enterobacter cloacae isolates overproducing AmpC, but also
306
harboring KPC and NMC-A enzymes that would have been misclassified using this
307
approach (13). Other authors pointed out that MEM-MEM/CLX zone diameter
308
differences are relatively lower in AmpC hyperproducers co-expressing a class A
309
carbapenemase (i.e. mean difference 1 mm) than in AmpC hyperproducers without a
310
class A carbapenemase (mean difference 5 mm) (22). Another study, however,
311
described MEM-MEM/CLX zone diameter differences of 6 to 7 mm and 0-7 mm for
312
AmpC hyperproducing E. cloacae harboring class A carbapenemases and AmpC
313
hyperproducers devoid of carbapenemases, respectively (13). Thus the discriminative
314
power of relative MEM-MEM/CLX diameter differences may be insufficient. In
315
addition, classification based on the relative degree of MEM-MEM/CLX diameter 15
316
differences is difficult to standardize and requires significant expertise. The present
317
study on 178 (53.3%) AmpC producing isolates shows that APBA-CDTs performed on
318
MH-CLX agar reliably detect class A carbapenemases with increased specificity
319
(100%) due to suppression of AmpC activity (Table 2). The approach is simple to
320
interpret as it uses a single critical zone diameter difference (5 millimeters), and it can
321
be integrated in one step with ESBL confirmation testing on the same MH-CLX agar
322
plate (48).
323
In the present study, the Carba NP-II showed an overall sensitivity of 78.9% and a
324
NPV of 98.7% (Table 2). Our results closely parallel those of a recent study, which
325
found a sensitivity of 72.5% for the Carba NP-I and a NPV of 69.2%. The difference in
326
NPV is well explained by the different prevalence of carbapenemase producers in the
327
study populations, i.e. 6.6% (n = 22) in this study and >45% (n =145) in the study of
328
Tijet et al. (31). Other authors found higher sensitivities for the Carba NP-I test using
329
different types of protocols (32, 33). Our data confirm ambiguities in the reading of the
330
Carba NP-I/II test in particular for OXA-48 producing isolates that tend to produce
331
inconclusive, or false-negative results (see Figure 1, isolates 19, 20, 51, and 99) (31). If
332
the inconclusive OXA-48 results from Figure 1 would have been rated negative (only a
333
slight color-change was visible after 120 min of incubation), sensitivity would have
334
been 68.2% (Table 2). If rated positive, the three ambiguous OXA-48 results would
335
have been consistent with a class A carbapenemase pattern, most likely due to the
336
simultaneous presence of a CTX-M type ESBL (class A enzyme), which may be
337
responsible for the weak color-change in wells II and IV after 120 min of incubation,
338
and which is inhibited by tazobactam in well III (see Figure 1). False-negative Carba
339
NP results have also been described for mucoid colonies, e.g. of Providencia rettgeri,
340
Providencia stuartii, or Proteus mirabilis isolates (29, 31). Negative results were
16
341
attributed to difficulties in protein extraction, species-specific traits, or the influence of
342
the agar type and ion content on the Carba NP test (30, 31, 36). Besides OXA-48
343
producers, false-negative results in the present study also occurred in non-mucoid
344
isolates of Providencia rettgeri, Providencia stuartii, and Proteus mirabilis producing
345
NDM enzymes. All tests for these isolates were repeated three times with the standard
346
protocol and additionally performed using colonies grown on various agar media of
347
different manufacturers, i.e. MH (Becton Dickinson), MH-CLX (Axonlab), Columbia
348
sheep blood, MacConkey (bioMérieux), and Uriselect4 agar (BioRad). Despite reports
349
that the Carba NP I test performed better from Columbia sheep blood and Uriselect4
350
agar results for these isolates remained false-negative for all media types pointing to
351
species-specific issues related to Providencia and Proteus isolates, and a low sensitivity
352
for OXA-48 enzymes (34). Other authors recently found a higher sensitivity and
353
specificity for the detection of OXA-48 (28). In summary, due to the higher NPV, the
354
Carba NP-II test may perform better in a low prevalence environment (i.e. our study) as
355
compared to high prevalence settings such as those investigated by Tijet et al. (31).
356
However, the issues of false-negative OXA-48 producers and species specific false-
357
negative results due to the unknown impact of different genetic backgrounds need to be
358
further analyzed.
359
The phenotypic detection of OXA-48-like carbapenemases remains challenging.
360
EUCAST recommends indirect OXA-48 confirmation by decreased zone diameters or
361
increased MICs for temocillin (< 11 mm, and > 32 mg/L, respectively) to exclude
362
ESBLs in combination with porin loss in cases where both APBA-CDT and EDTA-
363
CDT are negative (14). Temocillin MICs, however, are not recommended to
364
discriminate AmpC overproduction combined with porin loss from OXA-48 as
365
temocillin MICs are variable in this setting resulting in poor specificity. By suppressing
17
366
potential AmpC activity, temocillin disk diffusion testing or MIC determination by a
367
gradient diffusion method on MH-CLX can help to clearly increase specificity of
368
temocillin-based OXA-48 screening without compromising sensitivity (Table 4).
369
In summary, a combination of the EUCAST MEM carbapenemase-screening cut-off
370
(< 25 mm) and ETP (or MEM) APBA- and EDTA-CDTs plus temocillin disk diffusion
371
(or gradient diffusion-based MIC determination) on MH-CLX agar promises excellent
372
performance for carbapenemase detection. The proposed diagnostic flow-chart (Figure
373
2) would have resulted in a sensitivity, specificity, PPV, and NPV of 100% in the study
374
population. This algorithm is simple, easy to use, cost-efficient and applicable in the
375
majority of clinical microbiology laboratories.
376
18
377
Acknowledgments
378
We are grateful to the laboratory technicians of the Institute of Medical
379
Microbiology, University of Zurich for their dedicated help, and to Erik C. Böttger and
380
Reinhard Zbinden for valuable discussions.
381 382
Funding
383
This work was supported by the University of Zurich.
384 385
Transparency declaration
386
All authors: No conflicts of interest to declare.
387 388 389 390 391 392 393 394
19
395
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28
593
Tables and Figures
594 595
Table 1: Species identification and beta-lactamase genotypes of studied isolates.
Species
Escherichia coli
N
%
ESBL, AmpC, Carbapenemase negative
Carbapenemases AmpC
KPC
5
1.5
+
26
7.8
+
34
10.2
45
13.5
1
0.3
IMI
VIM
NDM
GIM
OXA-48
+
+ + +
total
111 33.3
Enterobacter cloacae
59
17.7
NA
+
15
4.5
NA
+
1
0.3
NA
+
2
0.6
NA
+
1
0.3
NA
+
total
78
23.4
Klebsiella pneumoniae
24
7.2
22
6.6
13
3.9
+
2
0.6
+
2
0.6
4
1.2
1
0.3
3
0.9
1
0.3
1
0.3
total
73
21.9
Enterobacter aerogenes
11
3.3
NA
+
4
1.2
NA
+
1
0.3
NA
+
total
16
4.8
Klebsiella oxytoca
6
1.8
4
1.2
6
1.8
16
4.8
total
ESBL
+ + + +
+ +
+ +
+ + +
+
+ + +
+ +
+ + +
29
596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654
Table 1 continued
Species
N
%
Carbapenemases
ESBL, AmpC, Carbapenemase negative
AmpC
ESBL KPC
IMI
VIM
NDM
GIM
OXA-48
1
0.3
NA
+
3
0.9
NA
+
10
3.0
NA
+
total
14
4.2
Hafnia alvei
5
1.5
NA
+
1
0.3
NA
+
total
6
1.8
Proteus mirabilis
1
0.3
+
1
0.3
+
2
0.6
total
4
1.2
Morganella morganii
1
0.3
NA
+
2
0.6
NA
+
total
3
0.9
Serratia marcescens
3
0.9
NA
+
Citrobacter koseri
2
0.6
+
Salmonella spp.
2
0.6
+
Providencia rettgeri
1
0.3
+
Providencia stuartii
1
0.3
NA
+
Enterobacter sp.
1
0.3
NA
+
Pantoea spp.
1
0.3
+
Citrobacter spp.
1
0.3
NA
+
Serratia spp.
1
0.3
NA
+
Total
334
100
78
178
105
7
1
5
4
1
5
Genotypes (%)
100
23.4
53.3
31.4
2.1
0.3
1.5
1.2
0.3
1.5
Citrobacter freundii
+ +
+
+ + +
+
+ +
NA, not applicable, for species naturally harboring chromosomally-encoded AmpC beta-lactamases
30
655 656
Table 2: Performance parameters of screening and confirmation assays and the proposed
657
diagnostic flow chart (see Figure 2).
TP (N)
FP (N)
TN (N)
FN (N)
Total (N)
Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
MEM Screen EUCAST (< 25 mm)
22
29
283
0
334
100.0
90.7
43.1
100.0
ETP EUCAST I/R (< 25 mm)
22
117
195
0
334
100.0
62.5
15.8
100.0
IPM EUCAST I/R (< 22 mm)
18
16
296
4
334
81.8
94.9
52.9
98.7
MEM EUCAST I/R (< 22 mm)
20
18
294
2
334
90.9
94.2
52.6
99.3
ETP CLSI I/R (< 22 mm)
21
68
244
1
334
95.5
78.2
23.6
99.6
IPM CLSI I/R (< 23 mm)
20
19
293
2
334
90.9
93.9
51.3
99.3
MEM CLSI I/R (< 23 mm)
21
20
292
1
334
95.5
93.6
51.2
99.7
ETP-BA MH
6
10
316
2
334
75.0
96.9
37.5
99.4
IPM-BA MH
6
2
324
2
334
75.0
99.4
75.0
99.4
MEM-BA MH
7
11
315
1
334
87.5
96.6
38.9
99.7
ETP-BA MH-CLX
8
0
326
0
334
100.0
100.0
100.0
100.0
IPM-BA MH-CLX
6
0
326
2
334
75.0
100.0
100.0
99.4
MEM-BA MH-CLX
7
0
326
1
334
87.5
100.0
100.0
99.7
ETP-EDTA MH
10
0
324
0
334
100.0
100.0
100.0
100.0
IPM-EDTA MH
7
0
324
3
334
70.0
100.0
100.0
99.1
MEM-EDTA MH
10
0
324
0
334
100.0
100.0
100.0
100.0
Carba NP-II1
15
0
312
4
331
78.9
100.0
100.0
98.7
Carba NP-II
15
0
312
7
334
68.2
100.0
100.0
97.8
Proposed algorithm
22
0
312
0
334
100.0
100.0
100.0
100.0
Parameter Screening cut-offs / CBPs
CDTs
2
658 659 660
TP, true-positive; FP, false-positive; TN, true-negative; FN, false-negative; MEM, meropenem; ETP,
661
ertapenem; CDT, combined-disk test; APBA, aminophenylboronic acid; MH-CLX agar, Muller Hinton
662
agar supplemented with cloxacillin; MH agar, Muller Hinton agar without cloxacillin.
663
1
inconclusives were excluded from the calculation;
664
2
inconclusives rated negative.
665 31
666 667
Table 3: Carbapenemase-positive isolates with characteristics and confirmation test results. CDTs (Δ mm)
Isolate number
Species
AmpC
ESBL
Carbapenemase type Carbapenemase class
NP-II
BA on MH
BA on MH-CLX
EDTA on MH IMI
MEM
7
Klebsiella pneumoniae
-
-
KPC
A
+
7
5
8
8
6
10
0
0
0
29
Klebsiella pneumoniae
-
SHV-ESBL
KPC
A
+
6
5
5
11
7
11
0
1
0
31
Klebsiella pneumoniae
-
-
KPC
A
+
7
11
7
9
8
9
0
0
0
35
Klebsiella pneumoniae
-
-
KPC
A
+
4
5
8
7
5
6
0
0
1
37
Klebsiella pneumoniae
-
-
KPC
A
+
7
7
9
8
4
9
0
0
0
40
Enterobacter cloacae
cAmpC
-
IMI
A
+
11
13
13
11
8
10
2
1
1
55
Escherichia coli
-
-
KPC
A
+
4
2
6
7
3
6
2
0
0
137
Klebsiella pneumoniae
-
CTX-M
KPC
A
+
6
4
4
5
5
2
0
3
0
8
Enterobacter aerogenes cAmpC
-
VIM
B
+
0
0
0
0
0
0
7
5
8
9
Klebsiella pneumoniae
-
-
NDM
B
+
0
0
0
2
0
0
16
7
13
17
Enterobacter cloacae
cAmpC
-
VIM
B
+
0
0
0
3
0
0
5
3
5
70
Citrobacter freundii
cAmpC
-
VIM
B
+
0
0
0
0
0
0
5
4
7
82
Klebsiella pneumoniae
-
-
VIM
B
+
0
0
0
0
0
1
15
17
21
95
Enterobacter cloacae
cAmpC
-
GIM-1
B
+
0
0
0
2
0
2
10
3
10
136
Providencia rettgeri
cAmpC
-
NDM
B
-
0
0
0
0
0
0
10
19
19
138
Providencia stuartii
cAmpC
-
NDM
B
-
0
0
0
0
0
0
9
13
12
139
Proteus mirabilis
CIT
-
NDM
B
-
0
4
0
0
0
0
6
16
6
36
Enterobacter cloacae
VIM
B
+
0
0
0
3
0
1
5
6
8
20
Klebsiella pneumoniae
-
CTX-M
OXA-48
D
inconclusive
0
0
0
3
0
0
0
0
0
51
Klebsiella pneumoniae
-
CTX-M
OXA-48
D
inconclusive
0
0
0
2
0
3
0
0
0
99
Klebsiella pneumoniae
-
CTX-M
OXA-48
D
inconclusive
0
0
0
2
0
3
0
0
0
19
Klebsiella pneumoniae
-
-
OXA-48
D
-
3
0
1
3
0
2
0
0
0
cAmpC SHV-ESBL
ETP
IMI
MEM
ETP
IMI
MEM
ETP
668
MEM, meropenem; ETP, ertapenem; CDT, combined-disk test; APBA, aminophenylboronic acid; MH-CLX, Muller Hinton agar supplemented with cloxacillin; MH, Muller Hinton
669
agar without cloxacillin; ESBL, extended-spectrum beta-lactamase; cAmpC, chromosomally encoded ampC gene
32
670 Table 4: Temocillin critical zone diameters and MICs for confirmation of OXA-48-like 671 carbapenemases. 672 Temocillin zone (mm) ID
Species
ESBL
AmpC
Temocillin MIC (mg/L)
Carbapenemase MH
MH-CLX
MH
MH-CLX
19
Klebsiella pneumoniae
-
-
OXA-48
6
6
1024
1024
20
Klebsiella pneumoniae
+
-
OXA-48
6
6
1024
1024
51
Klebsiella pneumoniae
+
-
OXA-48
6
6
1024
1024
99
Klebsiella pneumoniae
+
-
OXA-48
6
6
1024
1024
36
Enterobacter cloacae
+
cAmpC
VIM
6
8
1024
128
16
Hafnia alvei
-
cAmpC
6
11
128
32
18
Enterobacter cloacae
-
cAmpC
9
17
32
16
5
Enterobacter cloacae
-
cAmpC
10
21
32
8
27
Enterobacter cloacae
-
cAmpC
10
12
32
32
25
Hafnia alvei
-
cAmpC
10
21
32
4
26
Enterobacter cloacae
-
cAmpC
11
18
32
8
2
Enterobacter cloacae
-
cAmpC
12
16
32
16
125 Enterobacter cloacae
-
cAmpC
14
22
16
4
1
Enterobacter aerogenes
-
cAmpC
16
20
8
8
39
Klebsiella pneumoniae
+
-
10
10
64
64
60
Escherichia coli
+
-
11
11
32
32
38
Proteus mirabilis
+
-
11
11
32
32
130 Klebsiella pneumoniae
+
-
14
13
16
16
128 Klebsiella pneumoniae
+
-
18
17
8
8
median values OXA-48 positive isolates
6
6
1024
1024
AmpC overexpression
10
18
32
8
ESBL
11
11
32
32
673 674
ID, isolate identification number, ESBL, extended-spectrum beta-lactamase, MH-CLX:
675
Muller Hinton agar supplemented with cloxacillin, MH: Muller Hinton agar without
676
cloxacillin, cAmpC, chromosomally-encoded AmpC beta-lactamase
677 33
Figure 1: Discrepant test results of the Carba NP-II test and the carbapenemase genotype
678
isolate number
species
genotype / Ambler class
8
Enterobacter aerogenes
VIM
B
20
Klebsiella pneumoniae
OXA-48, CTX-M
D
99
Klebsiella pneumoniae
OXA-48, CTX-M
D
51
Klebsiella pneumoniae
OXA-48, CTX-M
D
19
Klebsiella pneumoniae
OXA-48
D
136
Providencia rettgeri
NDM
B
138
Providencia stuartii
NDM
B
139
Proteus mirabilis
NDM
B
95
Enterobacter cloacae
GIM
B
Carba NP-II result (examples of replicate testing) t = 0 minutes
t = 30 minutes
negative result
class A carbapenemase
t = 60 minutes
t = 120 minutes
interpretation (expected test result)
679 680 34
class B carbapenemase
class D carbapenemase
Figure 2: Proposed diagnostic flow chart for carbapenemase detection.
681
Enterobacteriaceae isolates Initial screening step Inhibition zone diameter MEM < 25mm Time to result 24 h (regular antibiogram) No
Yes Carbapenemases excluded for 57.8% of study population
CDT ETP versus ETP/APBA on MH-CLX agar1
No carbapenemase suspicion
Δ(ETP/APBA – ETP) < 5 mm
Δ(ETP/APBA – ETP) ≥ 5 mm
CDT ETP versus ETP/EDTA on MH agar1
CDT ETP versus ETP/EDTA on MH agar1 Phenotypic confirmation step
Δ(ETP/EDTA – ETP) < 5 mm
Δ(ETP/EDTA – ETP) ≥ 5 mm
Δ(ETP/EDTA – ETP) < 5 mm
Δ(ETP/EDTA – ETP) ≥ 5 mm
Carbapenemases
Carbapenemases
Carbapenemases
Carbapenemases
class A
No
class A
No
class A
Yes
class A
Yes
class B
No
class B
Yes
class B
No
class B
Yes
class D
?
class D
?
class D
?
class D
?
Additional time to result 24 h (total 48 h) Carbapenemases excluded/confirmed for 98.5% of study population
Temocillin disk diffusion or MIC on MH-CLX agar
682
≥ 11 mm or ≤ 32 mg/L
< 11 mm or >32 mg/L
Oxa-48like enzyme unlikely2
Suspicion for Oxa-48like enzyme
Genotypic confirmation step Perform molecular assay for the detection of class D carbapenemases
683
Additional time to result 24 h (total 72 h) (1.5% of study population, OXA-48 only) Carbapenemases excluded/confirmed for 98.5% of study aminophenylboronic population
684
MEM, meropenem; ETP, ertapenem; CDT, combined-disk test; APBA,
685
acid; MH-CLX agar, Muller Hinton agar supplemented with cloxacillin; MH agar, Muller
686
Hinton agar without cloxacillin. 1 MEM can be used alternatively with slightly lower sensitivity.
687
2
688
overexpression and decreased permeability, e,g, due to porin deficiency.
Carbapenem resistance phenotype is most likely due to a combination of AmpC and/or ESBL
35
