Evaluation of carbapenemase screening and confirmation tests in. Enterobacteriaceae and development of a practical diagnostic algorithm

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1 JCM Accepts, published online ahead of print on 29 October 2014 J. Clin. Microbiol. doi: /jcm Copyright 2014, American Society for Microbiology. All Rights Reserved. 1 2 Evaluation of carbapenemase screening and confirmation tests in Enterobacteriaceae and development of a practical diagnostic algorithm Florian P. Maurer 1, Claudio Castelberg 1, Chantal Quiblier 1, Guido V. Bloemberg 1, Michael Hombach 1, 6 7 1) Institut für Medizinische Mikrobiologie, Universität Zürich, 8006 Zürich, Schweiz 8 9 Running title: Diagnostic algorithm for carbapenemase detection Keywords: meropenem, imipenem, ertapenem, ESBL, AmpC, Carba NP, antibiotic resistance Corresponding author: Michael Hombach, M.D. Institut für Medizinische Mikrobiologie Universität Zürich Gloriastr. 30/ Zürich Switzerland Phone: Fax: [email protected]

2 30 Abstract Reliable identification of carbapenemase producing Enterobacteriaceae is necessary to limit their spread. This study aimed at developing a diagnostic flowchart suitable for implementation in different types of clinical laboratories using phenotypic screening and confirmation tests. In total, 334 clinical Enterobacteriaceae isolates genetically characterized with respect to carbapenemase, extended-spectrumbeta-lactamase (ESBL), and AmpC genes were analyzed. 142/334 isolates (42.2%) were suspicious for carbapenemase production, i.e. intermediate or resistant to ertapenem AND/OR meropenem AND/OR imipenem according to EUCAST clinical breakpoints (CBPs). A group of 193/334 isolates (57.8%) showing susceptibility to ertapenem AND meropenem AND imipenem was considered as negative control group for this study. CLSI and EUCAST carbapenem CBPs and the new EUCAST MEM screening cut-off were evaluated as screening parameters. ETP, MEM and IPM +/- aminophenylboronic acid (APBA) or EDTA combined-disc tests (CDTs), and the Carba NP-II test were evaluated as confirmation assays. EUCAST temocillin cut-offs were evaluated for OXA-48 detection. The EUCAST MEM screening cut-off (< 25 mm) showed a sensitivity of 100%. The ETP APBA-CDT on Muller-Hinton agar containing cloxacillin (MH-CLX) displayed 100% sensitivity and specificity for class A carbapenemase confirmation. ETP and MEM EDTA-CDTs showed 100% sensitivity and specificity for class B carbapenemases. Temocillin diameters/mic testing on MH-CLX was highly specific for OXA-48 producers. The overall sensitivity, specificity, PPV, and NPV of the Carba NP-II test were 78.9%, 100%, 100%, and 98.7%, respectively. Combining the EUCAST MEM carbapenemasescreening cut-off (< 25 mm), ETP (or MEM) APBA- and EDTA-CDTs, and 2

3 54 55 temocillin disk diffusion on MH-CLX agar promises excellent performance for carbapenemase detection. 3

4 56 Introduction In recent years, the emergence of diverse carbapenemases in Enterobacteriaceae has become a major challenge for healthcare systems (1). Carbapenemase producing bacterial isolates pose a severe clinical problem as non-susceptibility to beta-lactams is frequently accompanied by co-resistance to additional drug classes, e.g. aminoglycosides or quinolones (2, 3). As a consequence, treatment options for carbapenemase producers are alarmingly limited and often drugs displaying significant side effects need to be administered as a last resort (4) β-lactamases are classified according to their functional properties and molecular structure by Ambler and Bush (5, 6). Some of these enzymes also display hydrolytic activity towards carbapenems, e.g. Klebsiella pneumoniae carbapenemase (KPC, Ambler/Bush class A), the New Delhi metallo-β-lactamase (NDM-1), VIM, and GIM type enzymes (all Ambler/Bush class B), or OXA-48 (Ambler/Bush class D). A key characteristic used for discriminating enzymes belonging to different Ambler/Bush classes is the responsiveness to specific inhibitors: Class A enzymes are inhibited by clavulanic and aminophenylboronic acid (APBA), class B enzymes are inhibited by EDTA, and class D enzymes do not respond to any inhibitors used in β-lactamase diagnostics (5, 6) KPC enzymes were first detected in the USA in 1996 and have subsequently spread worldwide (7). In Europe, KPC is endemic in Italy, Greece, Poland, and northwestern England (7). In Central Europe, France, and Spain other carbapenemases are reported more frequently. NDM-1 is endemic in India, Bangladesh, and Pakistan. In Europe, most NDM-1 are being isolated in Great Britain (8). OXA-48 is endemic in Turkey and Morocco, but is increasingly reported from other European countries mostly in repatriated patients (8, 9). Scandinavian countries, the Netherlands, and other 4

5 countries such as Switzerland generally report low prevalence rates for all carbapenemases. Thus, rapid and reliable detection of carbapenemases is desirable in order to limit the spread of these enzymes Detection of carbapenemase producing bacteria comprises carrier screening and detection of carbapenemase production in routine antimicrobial susceptibility testing (AST). While chromogenic media are often used for carrier screening, laboratory strategies for β-lactamase detection in routine AST consist of a screening and a confirmation step (10-14) A variety of phenotypic and molecular, commercially available and in-house laboratory tests have been described for carbapenemase detection. Molecular techniques comprise end point and real-time PCRs as well as microarray techniques (15-17). Critical diameters/mics of ertapenem (ETP), meropenem (MEM), and imipenem (IPM), and automated microdilution expert systems have been evaluated as screening methods (14, 18-20). For carbapenemase confirmation, the modified Hodge test is recommended by CLSI and various commercial and in-house combined disk tests (CDTs) using boronic acid derivatives and EDTA/dipicolinic acid as specific inhibitors have been described (13, 19-25). In 2014, EUCAST published new guidelines for the detection of resistance mechanisms including carbapenemases, in which a CDT is recommended for carbapenemase confirmation (14, 22, 25). Recently, Nordmann et al. described a new inhibitor-based biochemical assay for carbapenemase detection, the Carba NP test, which has been published in two versions: The Carba NP-I assay provides a positive or negative result ( carbapenemase detected/not detected ) whereas the Carba NP-II test has been designed to also discriminate between carbapenemase classes A, B, and D (26-29). Apart from the original publications, few studies have systematically evaluated the Carba NP-I test for 5

6 Enterobacteriaceae, and both the originally published protocol and modified versions were used. Reported sensitivities varied between 72.5% and 100%, whereas specificity generally was reported to be 100% (30-33). Except for its original description the Carba NP-II assay has been systematically evaluated for Pseudomonas aeruginosa only (30, 31, 34-36) Several issues of carbapenemase detection remain challenging: i) Enterobacteriaceae overexpressing AmpC β-lactamases in combination with reduced cell-wall permeability compromise the specificity of APBA-CDTs as the inhibitor (APBA) affects both AmpC and carbapenemases (37-44); ii) Detection of OXA-48 and related enzymes remains problematic as no specific inhibitor is available. Temocillinresistance was suggested as an indicator for OXA-48 production, but not for OXA-48 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 various phenotypic screening and 120 confirmation tests for highly sensitive and specific carbapenemase detection

7 131 Materials and Methods Bacterial isolates. In total, 334 non-duplicate clinical isolates recovered in our laboratory from 2009 until 2014 were included in the study (Table 1). All isolates were genetically characterized for the presence of ESBL (TEM-ESBL, SHV-ESBL, and CTX-M types), plasmid-encoded AmpCs, chromosomal ampc promoter/attenuator mutations leading to overexpression (Escherichia coli only), and for the presence of carbapenemases (16, 47, 48). 142/334 isolates (42.2%) were considered suspicious for carbapenemase production due to non-susceptibility to ertapenem AND/OR meropenem AND/OR imipenem (intermediate or resistant zone diameters according to EUCAST CBPs), whereas 193/334 isolates (57.8%) considered non-suspicious for carbapenemase production (susceptible to ertapenem AND meropenem AND imipenem) served as a negative control group Genetic detection of carbapenemase, ESBL and ampc genes. Total DNA was extracted from bacterial colonies after growth on sheep blood agar medium using the InstaGene Matrix (Bio-Rad, Reinach, Switzerland). Genetic detection of carbapenemase genes was done by performing a carbapenemase multiplex PCR (16). For variant analysis OXA-48 genes were amplified with primers described(49). PCR amplicons were sequenced using PCR primers and sequences analyzed using GenBank and DNASTAR Lasergene software (DNASTAR Inc., Madison, Wisconsin USA). The AID ESBL line probe assay (AID Autoimmun Diagnostika GmbH, Germany) was used for the detection of ESBL genes (50). Bacterial isolates were genetically characterized for the presence of plasmid-mediated AmpC type β-lactamase genes by multiplex PCR (51). Chromosomal ampc promoter mutations of E. coli isolates were analyzed as described previously (52). 7

8 Susceptibility testing. Disk diffusion susceptibility testing was done according to EUCAST recommendations (53). Antibiotic disks and Mueller-Hinton (MH) agar were obtained from Becton Dickinson, Franklin Lakes, NJ. Cloxacillin supplemented Mueller-Hinton (MH-CLX) agar was obtained from Axonlab AG, Baden, Switzerland. Zone diameters were recorded using the Sirweb/Sirscan system (i2a, Montpellier, France). Minimal inhibitory concentrations (MICs) were determined by gradient diffusion (Etest, biomérieux, Marcy L Etoile, France) according to the manufacturer s 162 instructions Combined-disk tests (CDTs) for carbapenemase detection. CDTs were performed as described elsewhere (19, 24). Sets of two disks each containing IPM (10 μg), MEM (10 μg), or ETP (10 μg, all Becton Dickinson) were placed onto MH (EDTA-CDT) or both MH and MH-CLX (APBA-CDT) plates inoculated with a sample of the tested isolate (0.5 McFarland turbidity standard). Immediately after placing the disks onto the agar, 10 μl of a 29.2-mg/mL (0.1 M) EDTA solution (EDTA-CDT), or 10 μl of a 30- mg/ml APBA solution (APBA-CDT) were added to one of the two carbapenem disks in each set. Plates were incubated at 35 C for 16 to 20 hours, and zone diameters were recorded using the Sirweb/Sirscan system (i2a). Disc diameter differences of 5 mm between the APBA-free and APBA-containing discs or between the EDTA-free and EDTA-containing discs were considered indicative for production of a class A carbapenemases and class B carbapenemase, respectively Carba NP-II test. The Carba NP-II test was performed and interpreted as described (26). Reactions were read after 0, 30, 60 and 120 minutes of incubation. Color changes from red to yellow-orange were interpreted as follows: wells 2 and 4, positive (Ambler class A carbapenemase); wells 2 and 3, positive (Ambler class B carbapenemase); wells 2, 3 and 4: positive (probably Ambler class D carbapenemase); no well, 8

9 carbapenemase negative; all wells, test not interpretable. The Carba NP-II test was performed by experienced personal, and all discrepant results were additionally repeated at least 3 times. Software. All calculations were done using the IBM SPSS statistics software version 20 (IBM Corporation, Armonk, NY) and the Microsoft Excel 2010 software (Microsoft Corporation, Redmond, WA). 9

10 186 Results Evaluation of screening parameters for carbapenemase production The EUCAST non-susceptible ETP CBP (< 25 mm), and the EUCAST recommended carbapenemase MEM screening cut-off (< 25 mm) for carbapenemase production displayed highest sensitivity of all evaluated cut-offs (100%, Table 2). ETP, however, had a lower specificity (62.5%) than MEM (90.7%, Table 2). The ETP nonsusceptible CLSI CBP (< 22 mm) and the non-susceptible CLSI CBP for MEM (< 23 mm) displayed lower sensitivity (95.5% for both compounds, Table 2). The IPM nonsusceptible EUCAST CBP (< 22 mm) had the lowest sensitivity (81.8%), whereas the non-susceptible CLSI IPM CBP (23 mm) had a sensitivity of 90.9%. 196 Performance of carbapenemase confirmation tests Combined-disc tests (CDTs) The ETP APBA-CDT on MH-CLX agar displayed highest sensitivity and NPV for class A carbapenemase detection (100%, Table 2). Specificity of 100% was found for the ETP APBA-CDT, the IPM APBA-CDT, and the MEM APBA-CDT on MH-CLX, whereas the same CDTs on conventional MH agar showed a specificity of 96.9%, 99.4%, and 96.6%, respectively (Table 2). 9/10 false-positive ETP APBA-CDTs on conventional MH agar occurred in species with chromosomal AmpC (6 Enterobacter cloacae, 1 Enterobacter aerogenes, and 2 Hafnia alvei). 9/11 false-positive MEM APBA-CDTs on conventional MH agar were also found in AmpC positive species, i.e. 6 Enterobacter cloacae, 1 Enterobacter aerogenes, 1 Hafnia alvei, and 1 E. coli harboring a CIT type plasmid-encoded AmpC. One K. pneumoniae isolate lacking AmpC or ESBL was borderline positive in both ETP and MEM APBA-CDT on conventional MH (5 mm and 7 mm zone difference, respectively). Another 10

11 K. pneumoniae isolate producing an ESBL was borderline positive only in the MEM APBA-CDT on conventional MH (5 mm difference). Both the ETP and the MEM EDTA-CDTs displayed 100% sensitivity and specificity for class B carbapenemase detection, whereas the sensitivity of the IPM EDTA-CDT was significantly lower (70%, Table 2) Carba NP-II test The overall sensitivity, specificity, PPV, and NPV of the Carba NP-II test were 78.9%, 100%, 100%, and 98.7%, respectively (Table 2). The test created some reading problems resulting in ambiguous results that were treated as follows: One Enterobacter aerogenes isolate possessing a blavim gene gave ambiguous results in terms of class assignment (see isolate 8, Figure 1). After 30 min of incubation the pattern was consistent with a class B carbapenemase, while after 120 min of incubation the pattern was consistent with a class D carbapenemase (e.g. OXA-48).. For calculation of performance parameters this isolate was rated carbapenemase positive (Table 3). Three Klebsiella pneumoniae isolates co-producing OXA-48 and CTX-M ESBL gave inconclusive results (Table 3): the NP-II patterns were negative for carbapenemase production until 60 min of incubation. After 120 min of incubation, the patterns could either still be rated negative or weakly positive for class A carbapenemases (see Figure 1, isolates 20, 99, 51, results were reproduced three times with independent preparations); these isolates were excluded from the calculation of performance parameters. In addition, one OXA-48 producing Klebsiella pneumoniae (see isolate 19, Figure 1) and three NDM producing isolates of Providencia rettgeri, Providencia stuartti, and Proteus mirabilis, respectively, gave false-negative results with the NP-II test (see Table 3, isolates 136, 138, and 139, Figure 1). One Enterobacter cloacae isolate producing a GIM (class B) gave an OXA-48-like pattern (class D, see isolate 95, 11

12 Figure 1). For the calculation of sensitivity and specificity this isolate was rated carbapenemase positive (Table 3) Temocillin testing on MH-CLX agar Nineteen representative carbapenem non-susceptible isolates were tested for temocillin zone diameters and MICs on MH and MH-CLX agar as indicators for the presence of OXA-48. Five isolates harbored blaoxa-48 genes, nine isolates were blaoxa-48 gene negative but showed overexpression of a chromosomally encoded AmpC, and five isolates harbored ESBL genes (but not blaoxa-48, Table 4). All OXA-48 producers showed high-level temocillin resistance on both MH and MH-CLX agar (median diameter 6 mm, median MIC >1024 mg/l, Table 4). Five out of nine AmpC hyperproducers displayed temocillin zone diameters lower than 11 mm on MH (EUCAST screening cut-off for OXA-48 like enzymes) (14). On MH-CLX, the temocillin median diameter of the AmpC hyperproducers increased by 7 mm (corresponding to a median Etest-determined MIC decrease of 2 dilution steps, Table 4), and the five EUCAST OXA-48 screen false-positive isolates became true-negatives. Temocillin median diameters and gradient diffusion MICs of the five ESBL producers were not altered by the use of MH-CLX as compared to conventional MH agar. Median temocillin diameters/mics were 11 mm and 32 mg/l, respectively, on both media (Table 4). The only false-positive temocillin-based OXA-48 screening result originated from an CTX-M type ESBL-producing Klebsiella pneumoniae isolate displaying temocillin diameters/mics of 10 mm and 64 mg/l on both MH and MH-CLX agar Genetic characterization of isolates In total, 23 carbapenemase genes were detected in 22 Enterobacteriaceae isolates: 7 blakpc, 1 blaimi, 4 blavim, 4 blandm, 1 blagim, and 4 blaoxa-48; 1 isolate coproduced VIM and OXA-48 enzymes (Tables 1 and 2). Seventy-eight (23.4%) of the 12

13 studied isolates were genetically negative for ESBL, AmpC, and carbapenemases; 178 (53.3%) of the isolates produced an AmpC β-lactamase (including those species with chromosomally encoded AmpC, i.e. Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Hafnia alvei, Morganella morganii, Serratia marcescens, and Providencia stuartii, Table 1) (54); 105 (31.4%) of the isolates harbored an ESBL (Table 1). 13

14 266 Discussion Screening parameters for carbapenemases Disk diffusion critical diameters have been reported to display high sensitivity for the detection of carbapenemases (13, 20). This study found 100% sensitivity for the EUCAST critical MEM diameter (< 25 mm) with a comparably high specificity of 90.7% (Table 2). ETP screening using the EUCAST non-susceptible CBP (< 25 mm) also showed high sensitivity (100%), but low specificity (62.5%, Table 2). Thus, our results confirm the current EUCAST recommendation (15). CLSI non-susceptible ETP (< 22 mm) and MEM (< 23 mm) CBPs displayed lower sensitivity as compared to the current EUCAST recommendation (95.5%, Table 2). Based on the findings of this study, carbapenemase screening using MEM is recommended, whereas the use of IMP as screening drug is discouraged (IMP sensitivity EUCAST < 22 mm / CLSI < 23 mm 81.8% and 90.9%, respectively). Since automated microdilution AST reportedly lacks sensitivity and specificity due to antibiotic panel composition and drug concentrations tested (18, 55), disk diffusion critical MEM diameters promise the best performance for carbapenemase detection among all evaluated techniques. In addition, disk diffusion is cheap, simple, and widely implemented by many laboratories for routine AST. 283 Carbapenemase confirmation tests The modified Hodge test, which is recommended by CLSI for carbapenemase confirmation, is cheap and, in principle, simple to perform (23). However, it displays significant investigator dependence, practical interpretation is technically demanding, the test cannot distinguish between the different carbapenemase classes, and reportedly shows low specificity due to AmpC β-lactamase overproduction and decreased permeability, e.g. caused by porin loss (13, 20, 55). The problem of discriminating 14

15 carbapenemase activity from AmpC and impermeability is well known both for species possessing a chromosomal AmpC (e.g. Enterobacter spp., Citrobacter spp., or Hafnia alvei), and for producers of plasmid-encoded AmpC, in particular Klebsiella pneumoniae (39, 44, 56). Even E. coli overproducing AmpC due to mutations in the promoter/attenuator region and/or showing mutations in the active center of the enzyme resulting in an extended spectrum AmpC (ESAC) phenotype display carbapenem nonsusceptibility (41, 43). The same pattern accounts for ESBL producers in combination with porin loss (37, 38). AmpC and ESBL production interferes not only with carbapenemase screening, but also with APBA-CDT confirmation for class A carbapenemases (14, 19, 57). False positive results occur as APBA is not only an inhibitor of class A carbapenemases, but also of AmpC β-lactamases. To improve specificity of APBA-CDTs, MEM/CLX disks are used to check for AmpC interference (indirect approach) (13, 14, 20, 22, 25). However, based on the current EUCAST algorithm, class A carbapenemases in isolates co-producing AmpC may be missed as synergy of MEM with both CLX and APBA is interpreted as AmpC and porin loss (14). A recent study found two Enterobacter cloacae isolates overproducing AmpC, but also harboring KPC and NMC-A enzymes that would have been misclassified using this approach (13). Other authors pointed out that MEM-MEM/CLX zone diameter differences are relatively lower in AmpC hyperproducers co-expressing a class A carbapenemase (i.e. mean difference 1 mm) than in AmpC hyperproducers without a class A carbapenemase (mean difference 5 mm) (22). Another study, however, described MEM-MEM/CLX zone diameter differences of 6 to 7 mm and 0-7 mm for AmpC hyperproducing E. cloacae harboring class A carbapenemases and AmpC hyperproducers devoid of carbapenemases, respectively (13). Thus the discriminative power of relative MEM-MEM/CLX diameter differences may be insufficient. In addition, classification based on the relative degree of MEM-MEM/CLX diameter 15

16 differences is difficult to standardize and requires significant expertise. The present study on 178 (53.3%) AmpC producing isolates shows that APBA-CDTs performed on MH-CLX agar reliably detect class A carbapenemases with increased specificity (100%) due to suppression of AmpC activity (Table 2). The approach is simple to interpret as it uses a single critical zone diameter difference (5 millimeters), and it can be integrated in one step with ESBL confirmation testing on the same MH-CLX agar plate (48) In the present study, the Carba NP-II showed an overall sensitivity of 78.9% and a NPV of 98.7% (Table 2). Our results closely parallel those of a recent study, which found a sensitivity of 72.5% for the Carba NP-I and a NPV of 69.2%. The difference in NPV is well explained by the different prevalence of carbapenemase producers in the study populations, i.e. 6.6% (n = 22) in this study and >45% (n =145) in the study of Tijet et al. (31). Other authors found higher sensitivities for the Carba NP-I test using different types of protocols (32, 33). Our data confirm ambiguities in the reading of the Carba NP-I/II test in particular for OXA-48 producing isolates that tend to produce inconclusive, or false-negative results (see Figure 1, isolates 19, 20, 51, and 99) (31). If the inconclusive OXA-48 results from Figure 1 would have been rated negative (only a slight color-change was visible after 120 min of incubation), sensitivity would have been 68.2% (Table 2). If rated positive, the three ambiguous OXA-48 results would have been consistent with a class A carbapenemase pattern, most likely due to the simultaneous presence of a CTX-M type ESBL (class A enzyme), which may be responsible for the weak color-change in wells II and IV after 120 min of incubation, and which is inhibited by tazobactam in well III (see Figure 1). False-negative Carba NP results have also been described for mucoid colonies, e.g. of Providencia rettgeri, Providencia stuartii, or Proteus mirabilis isolates (29, 31). Negative results were 16

17 attributed to difficulties in protein extraction, species-specific traits, or the influence of the agar type and ion content on the Carba NP test (30, 31, 36). Besides OXA-48 producers, false-negative results in the present study also occurred in non-mucoid isolates of Providencia rettgeri, Providencia stuartii, and Proteus mirabilis producing NDM enzymes. All tests for these isolates were repeated three times with the standard protocol and additionally performed using colonies grown on various agar media of different manufacturers, i.e. MH (Becton Dickinson), MH-CLX (Axonlab), Columbia sheep blood, MacConkey (biomérieux), and Uriselect4 agar (BioRad). Despite reports that the Carba NP I test performed better from Columbia sheep blood and Uriselect4 agar results for these isolates remained false-negative for all media types pointing to species-specific issues related to Providencia and Proteus isolates, and a low sensitivity for OXA-48 enzymes (34). Other authors recently found a higher sensitivity and specificity for the detection of OXA-48 (28). In summary, due to the higher NPV, the Carba NP-II test may perform better in a low prevalence environment (i.e. our study) as compared to high prevalence settings such as those investigated by Tijet et al. (31). However, the issues of false-negative OXA-48 producers and species specific falsenegative results due to the unknown impact of different genetic backgrounds need to be further analyzed The phenotypic detection of OXA-48-like carbapenemases remains challenging. EUCAST recommends indirect OXA-48 confirmation by decreased zone diameters or increased MICs for temocillin (< 11 mm, and > 32 mg/l, respectively) to exclude ESBLs in combination with porin loss in cases where both APBA-CDT and EDTA- CDT are negative (14). Temocillin MICs, however, are not recommended to discriminate AmpC overproduction combined with porin loss from OXA-48 as temocillin MICs are variable in this setting resulting in poor specificity. By suppressing 17

18 potential AmpC activity, temocillin disk diffusion testing or MIC determination by a gradient diffusion method on MH-CLX can help to clearly increase specificity of temocillin-based OXA-48 screening without compromising sensitivity (Table 4). In summary, a combination of the EUCAST MEM carbapenemase-screening cut-off (< 25 mm) and ETP (or MEM) APBA- and EDTA-CDTs plus temocillin disk diffusion (or gradient diffusion-based MIC determination) on MH-CLX agar promises excellent performance for carbapenemase detection. The proposed diagnostic flow-chart (Figure 2) would have resulted in a sensitivity, specificity, PPV, and NPV of 100% in the study population. This algorithm is simple, easy to use, cost-efficient and applicable in the majority of clinical microbiology laboratories

19 Acknowledgments We are grateful to the laboratory technicians of the Institute of Medical Microbiology, University of Zurich for their dedicated help, and to Erik C. Böttger and Reinhard Zbinden for valuable discussions Funding This work was supported by the University of Zurich Transparency declaration All authors: No conflicts of interest to declare

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29 Tables and Figures Table 1: Species identification and beta-lactamase genotypes of studied isolates. Species N % ESBL, AmpC, Carbapenemase negative AmpC ESBL Carbapenemases KPC IMI VIM NDM GIM OXA-48 Escherichia coli total Enterobacter cloacae NA NA NA NA NA + + total Klebsiella pneumoniae total Enterobacter aerogenes NA NA NA + + total Klebsiella oxytoca total

30 Species N % ESBL, AmpC, Carbapenemase negative Table 1 continued AmpC ESBL Citrobacter freundii NA + + total NA NA + Hafnia alvei NA + total NA + + NA, not applicable, for species naturally harboring chromosomally-encoded AmpC beta-lactamases 30 Carbapenemases KPC IMI VIM NDM GIM OXA-48 Proteus mirabilis total Morganella morganii NA + + total NA + Serratia marcescens NA + Citrobacter koseri Salmonella spp Providencia rettgeri Providencia stuartii NA + + Enterobacter sp NA + Pantoea spp Citrobacter spp NA + Serratia spp NA + Total Genotypes (%)

31 Table 2: Performance parameters of screening and confirmation assays and the proposed diagnostic flow chart (see Figure 2). Parameter TP (N) FP (N) TN (N) FN (N) Total (N) Sensitivity (%) Specificity (%) PPV (%) NPV (%) Screening cut-offs / CBPs MEM Screen EUCAST (< 25 mm) ETP EUCAST I/R (< 25 mm) IPM EUCAST I/R (< 22 mm) MEM EUCAST I/R (< 22 mm) ETP CLSI I/R (< 22 mm) IPM CLSI I/R (< 23 mm) MEM CLSI I/R (< 23 mm) CDTs ETP-BA MH IPM-BA MH MEM-BA MH ETP-BA MH-CLX IPM-BA MH-CLX MEM-BA MH-CLX ETP-EDTA MH IPM-EDTA MH MEM-EDTA MH Carba NP-II Carba NP-II Proposed algorithm TP, true-positive; FP, false-positive; TN, true-negative; FN, false-negative; MEM, meropenem; ETP, ertapenem; CDT, combined-disk test; APBA, aminophenylboronic acid; MH-CLX agar, Muller Hinton agar supplemented with cloxacillin; MH agar, Muller Hinton agar without cloxacillin. 1 inconclusives were excluded from the calculation; 2 inconclusives rated negative. 31

32 Table 3: Carbapenemase-positive isolates with characteristics and confirmation test results. Isolate number Species AmpC ESBL Carbapenemase type Carbapenemase class NP-II CDTs (Δ mm) BA on MH BA on MH-CLX EDTA on MH ETP IMI MEM ETP IMI MEM ETP IMI MEM 7 Klebsiella pneumoniae - - KPC A Klebsiella pneumoniae - SHV-ESBL KPC A Klebsiella pneumoniae - - KPC A Klebsiella pneumoniae - - KPC A Klebsiella pneumoniae - - KPC A Enterobacter cloacae campc - IMI A Escherichia coli - - KPC A Klebsiella pneumoniae - CTX-M KPC A Enterobacter aerogenes campc - VIM B Klebsiella pneumoniae - - NDM B Enterobacter cloacae campc - VIM B Citrobacter freundii campc - VIM B Klebsiella pneumoniae - - VIM B Enterobacter cloacae campc - GIM-1 B Providencia rettgeri campc - NDM B Providencia stuartii campc - NDM B Proteus mirabilis CIT - NDM B Enterobacter cloacae campc SHV-ESBL VIM B Klebsiella pneumoniae - CTX-M OXA-48 D inconclusive Klebsiella pneumoniae - CTX-M OXA-48 D inconclusive Klebsiella pneumoniae - CTX-M OXA-48 D inconclusive Klebsiella pneumoniae - - OXA-48 D MEM, meropenem; ETP, ertapenem; CDT, combined-disk test; APBA, aminophenylboronic acid; MH-CLX, Muller Hinton agar supplemented with cloxacillin; MH, Muller Hinton agar without cloxacillin; ESBL, extended-spectrum beta-lactamase; campc, chromosomally encoded ampc gene 32

33 Table 4: Temocillin critical zone diameters and MICs for confirmation of OXA-48-like carbapenemases. 672 ID Species ESBL AmpC Carbapenemase Temocillin zone (mm) Temocillin MIC (mg/l) MH MH-CLX MH MH-CLX 19 Klebsiella pneumoniae - - OXA Klebsiella pneumoniae + - OXA Klebsiella pneumoniae + - OXA Klebsiella pneumoniae + - OXA Enterobacter cloacae + campc VIM Hafnia alvei - campc Enterobacter cloacae - campc Enterobacter cloacae - campc Enterobacter cloacae - campc Hafnia alvei - campc Enterobacter cloacae - campc Enterobacter cloacae - campc Enterobacter cloacae - campc Enterobacter aerogenes - campc Klebsiella pneumoniae Escherichia coli Proteus mirabilis Klebsiella pneumoniae Klebsiella pneumoniae median values OXA-48 positive isolates AmpC overexpression ESBL ID, isolate identification number, ESBL, extended-spectrum beta-lactamase, MH-CLX: Muller Hinton agar supplemented with cloxacillin, MH: Muller Hinton agar without cloxacillin, campc, chromosomally-encoded AmpC beta-lactamase

34 678 Figure 1: Discrepant test results of the Carba NP-II test and the carbapenemase genotype isolate number species genotype / Ambler class Carba NP-II result (examples of replicate testing) t = 0 minutes t = 30 minutes t = 60 minutes t = 120 minutes 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 interpretation (expected test result) negative result class A carbapenemase class B carbapenemase class D carbapenemase

35 681 Figure 2: Proposed diagnostic flow chart for carbapenemase detection. No No carbapenemase suspicion Enterobacteriaceae isolates Inhibition zone diameter MEM < 25mm Yes CDT ETP versus ETP/APBA on MH-CLX agar 1 Initial screening step Time to result 24 h (regular antibiogram) Carbapenemases excluded for 57.8% of study population Δ(ETP/APBA ETP) < 5 mm Δ(ETP/APBA ETP) 5 mm CDT ETP versus ETP/EDTA on MH agar 1 CDT ETP versus ETP/EDTA on MH agar 1 Δ(ETP/EDTA ETP) < 5 mm Carbapenemases class A class B No No class D? Δ(ETP/EDTA ETP) 5 mm Carbapenemases class A class B No Yes class D? Δ(ETP/EDTA ETP) < 5 mm Carbapenemases class A class B Yes No class D? Δ(ETP/EDTA ETP) 5 mm Carbapenemases class A class B Yes Yes class D? Phenotypic confirmation step 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 mm or 32 mg/l Oxa-48- like enzyme unlikely 2 < 11 mm or >32 mg/l Suspicion for Oxa-48- like enzyme Perform molecular assay for the detection of class D carbapenemases MEM, meropenem; ETP, ertapenem; CDT, combined-disk test; APBA, aminophenylboronic acid; MH-CLX agar, Muller Hinton agar supplemented with cloxacillin; MH agar, Muller Hinton agar without cloxacillin. 1 MEM can be used alternatively with slightly lower sensitivity. 2 Carbapenem resistance phenotype is most likely due to a combination of AmpC and/or ESBL overexpression and decreased permeability, e,g, due to porin deficiency. 35 Genotypic confirmation step 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 population