Accepted Manuscript

Rapid detection of carbapenemase-producing Acinetobacter

baumannii and carbapenem-resistant Enterobacteriaceae using a
bioluminescence-based phenotypic method

Vincent van Almsick, Beniam Ghebremedhin, Niels

Pfennigwerth, Parviz Ahmad-Nejad

PII: S0167-7012(18)30092-7
DOI: https://doi.org/10.1016/j.mimet.2018.02.004
Reference: MIMET 5329
To appear in: Journal of Microbiological Methods
Received date: 17 November 2017
Revised date: 5 February 2018
Accepted date: 6 February 2018

Please cite this article as: Vincent van Almsick, Beniam Ghebremedhin, Niels
Pfennigwerth, Parviz Ahmad-Nejad , Rapid detection of carbapenemase-producing
Acinetobacter baumannii and carbapenem-resistant Enterobacteriaceae using a
bioluminescence-based phenotypic method. The address for the corresponding author
was captured as affiliation for all authors. Please check if appropriate. Mimet(2017),
https://doi.org/10.1016/j.mimet.2018.02.004

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 ACCEPTED MANUSCRIPT
Rapid detection of carbapenemase-producing Acinetobacter baumannii and carbapenem-

resistant Enterobacteriaceae using a bioluminescence-based phenotypic method

Vincent van Almsicka, Beniam Ghebremedhina, Niels Pfennigwerthb, Parviz Ahmad-Nejada

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a
Institute for Medical Laboratory Diagnostics, Centre for Clinical and Translational Research

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(CCTR), HELIOS University Hospital Wuppertal, Witten/Herdecke University, Heusnerstraße 40,

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42283 Wuppertal, Germany.
b
Department of Medical Microbiology, National Reference Centre for Multidrug-resistant Gram-
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negative Bacteria, Ruhr-University Bochum, Universitätsstraße 150, 44801 Bochum, Germany.
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Running title:
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Rapid phenotypic detection of carbapenem resistance

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Corresponding author:

Ahmad-Nejad, Parviz, Prof. Dr. med.

Heusnerstraße 40

D-42283 Wuppertal, Germany

Phone: 0049-202-2525

Fax: 0049-202-896-2726

Email: parviz.ahmad-nejad@helios-kliniken.de

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Abstract

Accurate detection of carbapenem-resistant Enterobacteriaceae (CRE) and carbapenemase-

producing carbapenem-resistant Acinetobacter spp. (CP-CRA) constitutes a major challenge in

laboratory diagnostics. We developed a bioluminescence-based carbapenem susceptibility detection

assay (BCDA) which allows identification of CRE (carbapenemase-producing-CRE (CP-CRE) as

well as non-carbapenemase-producing-CRE (non-CP-CRE) and CP-CRA in 2.5 h from culture

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media. This laboratory method was evaluated with CP-CRE and CP-CRA isolates producing

different β-lactamases of different Ambler classes (A, n=16; B, n=25; D, n=67) and 22 non-CP-

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CRE. The results were correlated with those obtained by BD Phoenix™ and genotypic analysis

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results. The performance of BCDA on 123 validated CRE (except C. freundii isolates) and CP-CPA

isolates revealed that 122 of 123 isolates were identified correctly. Only one OXA-48-producing
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Klebsiella pneumoniae was falsely classified. Among 45 meropenem susceptible
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Enterobacteriaceae (except C. freundii isolates) and meropenem susceptible Acinetobacter spp.

strains tested, 44 were confirmed as susceptible by our BCDA. Overall, our BCDA had a sensitivity
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of 99% and a specificity of 98% and is a rapid and accurate assay which distinguished CRE/CP-
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CRA from meropenem susceptible Enterobacteriaceae and Acinetobacter spp. .

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Keywords: Gram-negative bacteria, multidrug resistance, carbapenemase, rapid detection,

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phenotypic screening
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Abbreviations

CRE = carbapenem-resistant Enterobacteriaceae

CP-CRE = carbapenemase-producing carbapenem-resistant Enterobacteriaceae

non-CP-CRE = non-carbapenemase-producing carbapenem-resistant Enterobacteriaceae

CP-CRA = carbapenemase-producing Acinetobacter spp.

BCDA = bioluminescence-based carbapenem susceptibility detection assay

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MRSA = methicillin-resistant Staphylococcus aureus

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VRE = vancomycin-resistant enterococci

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MIC = minimal inhibitory concentration

NRC = National Reference Centre

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AST = antibiotic susceptibility testing
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KPC = Klebsiella-pneumoniae-carbapenemase

NDM = New Delhi metallo-β-lactamase

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VIM = Verona integron-encoded metallo-β-lactamase

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IMP = imipenemase
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OXA = oxacillinase
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Introduction

Infections caused by carbapenemase-producing carbapenem-resistant Enterobacteriaceae (CP-

CRE), non-carbapenemase-producing carbapenem-resistant Enterobacteriaceae (non-CP-CRE) and

carbapenemase-producing carbapenem-resistant Acinetobacter spp. (CP-CRA) are considerably

life-threatening. The number of bacteria that form carbapenemases is increasing worldwide and

poses a challenge for today´s healthcare facilities (van Duin and Doi, 2016). An increase in

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resistance to carbapenems significantly impairs the therapeutic possibilities which are particularly

used for patients with severe diseases. Early identification and treatment leads to significantly lower

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mortality as well as lower cost in healthcare systems (Falagas et al., 2014, Lapointe-Shaw et al.,

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2017, Tamma et al., 2017).

Therefore, the detection and identification of CP-CRA, CP-CRE and non-CP-CRE has become a
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major and important task, as well as controlling the spread of these pathogens (Nordmann et al.,
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2011). Effective screening of patients based on rapid detection methods is indispensable and the

assays used to achieve these goals should be affordable and rapidly provide accurate results.
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At the same time, mechanisms for carbapenem resistance are genetically heterogeneous (Miller and
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Humphries, 2016, Woodford et al., 2006).

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Carbapenem resistance can be caused by carbapenemase production but also because of loss of

porin in combination with overexpression of AmpC (Nordmann et al., 2012). Carbapenemases are
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classified biochemically according to the Ambler classification. Many carbapenemases belong to

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Ambler class A (e.g. Klebsiella-pneumoniae-carbapenemase (KPC)), which hydrolyses all β-

lactams. Ambler class B comprises the metallo-β-lactamases [e.g. New Delhi metallo-β-lactamase

(NDM), Verona integron-encoded metallo-β-lactamase (VIM) and imipenemase (IMP)]. All these

enzymes hydrolyze β-lactams in dependency of zinc (with the exception of aztreonam). Some

oxacillinases have the potential to hydrolyse carbapenemas. These are included in Ambler class D,

which contain serine β-lactamases (oxacillinase (OXA)) that also hydrolyze carbapenems (Bush and

Jacoby, 2010, Nordmann, et al., 2012).

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Phenotypic susceptibility testing of Enterobacteriaceae to carbapenems is sophisticated, and that is

shown, for example, by the different breakpoints in meropenem minimal inhibition concentration

(MIC) for Enterobacteriaceae (European Committee on Antimicrobial Susceptibility Testing vs.

Clinical and Laboratory Standards Institute) to classify them as resistant or susceptible.

PCR-based detection methods do exist (Avlami et al., 2010, Queenan and Bush, 2007, van der Zee

et al., 2014) but are cost intensive and will not detect unknown resistance genes. Screening of

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patient samples based on culture is time consuming and the results are sometimes difficult to

interpret. An alternative is the CarbaNP test (Nordmann et al., 2012, Yamada et al., 2016), which is

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based on the hydrolysis of imipenem. However, this test will not detect other types of resistance

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such as loss of porin or overexpression of AmpC.

Considering the fact of global spread of multidrug resistant bacilli with genotypic heterogeneity in
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their resistance patterns, there is currently no laboratory test available which combines the attributes
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of celerity, equity and high diagnostic accuracy. Therefore, we developed a new and rapid

phenotypic detection method based on the measurement of bacterial growth.

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We developed the bioluminescence based carbapenem susceptibility detection assay (BCDA).

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Bioluminescence based methods can detect bacterial ATP-level very accurately (Lehtinen et al.,
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2006). Because of this high degree of accuracy, bacterial viability in presence and absence of

meropenem can be measured indirectly by ATP-level differences in a short period of time. This
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allows identification of CRE (CP-CRE as well as non-CP-CRE) and CP-CPA in 2.5 hours from
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culture media.

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Material and Method:

A total of 106 carbapenemase producing microbes and 22 non-CP-CRE were included in this study.

Strains with susceptibility to meropenem were used as controls (n= 47). Confirmed CRE and CP-

CRA were provided in part from the National Reference Centre for Multidrug-resistant Gram-

negative Bacteria at Ruhr-University Bochum (NRC) as well as CP-CRE and CP-CRA from our

own laboratory, also confirmed by NRC. The control group was collected from our laboratory and

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were confirmed by the BD-PhoenixTM System (MIC <=0,125 mg/L for meropenem and no

indication for carbapenemase forming by the system).

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All carbapenemase producing strains were genetically characterized with respect to the type of

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carbapenemase by NRC (KPC (Woodford et al., 2004, Yigit et al., 2001); IMP (Pitout et al., 2005),

NDM (unpublished primer design by NRC), VIM (Juan et al., 2008, Pitout, et al., 2005); OXA for
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Enterobacteriaceae (Poirel et al., 2004, Queenan, et al., 2007) and Acinetobacter spp. (Woodford,
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et al., 2006)). Non-CP-CRE were provided by NRC and were confirmed as resistant and the

presence of carbapenemases was disproved by performing PCRs for carbapenemases (as written
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above) with negative results. In the cases of E. coli and K. pneumoniae isolates, AmpC associated
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genetic elements were confirmed by PCR (primer reference (Perez-Perez and Hanson, 2002)). The
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study population comprising resistant and susceptible bacilli (Enterobacteriaceae n=110;

A.baumannii n=65) is shown in Table 1.

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Supplementary tables 1 and 2 display the investigated microbes including the MIC for meropenem,
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the relative induction calculated from the BCDA and the resistance associated genes detected for

each isolate.

In our experimental setup, we prepared an inoculum of the bacterium to be investigated by

suspending a colony of one isolate directly from the nutrient medium (Columbia (BD, Columbia

Agar with 5% Sheep Blood, Becton Dickinson GmbH, Heidelberg, Germany) or McConkey (BD,

MacConkey III Agar, Becton Dickinson GmbH, Heidelberg, Germany) both were used) using a 10

μl loop transferring it from nutrition medium into one milliliter Antibiotic-Susceptibility-Testing

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(AST) Broth (BD AST Broth, Becton Dickinson GmbH, Heidelberg, Germany). The main content

of BD AST Broth is Müller-Hinton Broth. The inoculum was made from cultured bacilli at room-

temperature. Figure 1 shows a scheme of the procedure: 10 μl of the inoculum were added to an

Eppendorf tube (1.5 mL) as well as 990 μl pure AST Broth, leading to 1 mL filled testing tube. We

called them “sample growth control” [G]. “Sample antibiotic” [A] was formed as sample G only

adding antibiotic.

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We used two different concentrations of antibiotics for the different microbes. For Acinetobacter,

we used a solution of 10 mg/L meropenem using a 10 μg meropenem disk (BD BBLTM Sensi-

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DiscTM Meropenem MEM-10, Becton Dickinson GmbH, Heidelberg, Germany) which was added

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in the tube. For Enterobacteriaceae we formed a solution of 1 mg/L meropenem by using liquid-

antibiotic (Meronem Kabi, FRESENIUS KABI Deutschland GmbH, Bad Homburg, Germany). The
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Acinetobacter- and Enterobacteriaceae-protocols were established because of different reaction by
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Enterobacteriaceae and Acinetobacter spp. to BCDA. This is explained below. Sample G served as

a growth control and in combination with sample A for the determination of relative induction [ri]
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(see Fig. 1).

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The samples were incubated for 2.5 h at 37 ° C and swirled at 350 rpm. Measurements were
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performed at time 0 h and 2.5 h. The bacterial growth was indirectly determined via the adenosine

tri-phosphate content of the solution.

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The direct correlation of the ATP content of the solution and the number of bacteria by means of
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bioluminescence (cleavage of luciferin by means of ATP-dependent luciferase) has been

demonstrated in previous studies (Cai et al., 2015, Lafond et al., 2010, Lehtinen, et al., 2006).

Bioluminescence was generated by BacTiter-Glo® microbial viability assay (Promega, Madison,

Wisconsin, USA). This is a lytic assay which lysis cell walls completely so that intracellular ATP

can dissolve. The reagent was prepared according to the manufacturer’s instructions. For

measurement, 75 μl of the BacTiter-Glo® reagent were pipetted into a full white colored 96-well

plate (Sarstedt ELISA-plate white, Sarstedt AG & Co., Nümbrecht, Germany) together with 75 μl

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of testing material (Sample G and A, see Fig. 1). After blending sample and reagent, the 96-well

plate was swirled at room temperature for five minutes to obtain an optimal light signal (complete

lysis of all cell walls and full activation of ATP-dependent luciferase). Light emission (measured in

relative light units) was detected by the GloMax® Integrated Luminescence System (Promega)

using an integration time of 0.5 second as recommended by the manufacturer. Each measurement

was repeated after one minute and the mean value from both measurements was used for evaluation.

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Relative and absolute inductions were calculated as written in Figure 1. If absolute induction was

less than 20, measurements were excluded from the evaluation due to lack of growth (8 samples in

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total, data not shown).

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By applying calculation of relative growth, the results were nearly the same as obtained by

calculation of ri. We used ri for scientific correctness, but the application of relative growth will
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reduce hands-on time and consumption of reagents. Only one measurement using G is needed at
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time-point 0h to collect information about absolute induction.

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Results:
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In 172 out of 177 tested Gram-negative bacteria the susceptibility to meropenem was successfully
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shown.

In the first protocol (=Acinetobacter-Protocol) of our experimental setup we used meropenem disk
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to simplify setup and used CP-CRE/CRA to check feasibility.

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For A. baumannii isolates a sensitivity and specificity of 100% can be demonstrated for a

measurement with meropenem disk (10 mg/L) and a breakpoint of 36% ri (see Fig. 2 and Table 2).

Using the first protocol some CP-CRE showed low relative inductions using meropenem disks (10

mg/L) leading to potentially false results. Therefore we adapted the protocol for CRE by reducing

the antibiotic concentration from 10 mg/L to 1 mg/L (CLSI breakpoint for meropenem (Institute,

2014) and literature recommendation (Nordmann et al., 2012)) to get better accuracy in detecting

CP-CRE.

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By applying the reduced meropenem concentration in the BCDA on the CP-CRE (which shown low

induction under high meropenem concentration), we could characterize 86 out of 88 samples

correctly. All class A and B carbapenemase-producers were classified correctly. For CP-CRE, we

achieved a sensitivity of 98% and a specificity of 96% (Fig. 3, Table 2) with a cut off value of 23%

ri. One K. pneumoniae with a low MIC (0,25 mg/L) and a OXA-48 resistance could not be assigned

precisely. Also, one E. coli with no antibiotic resistance (MIC <=0,125 mg/L; no indication by BD

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PhoenixTM for carbapenemase forming; 32% ri) was not classified correctly by BCDA. We do not

have a satisfying explanation for this but suspect a specific reaction of this distinct pathogen with

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our assay leading to a false positive classification. In a second step, we included non-CP-CRE using

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the adapted protocol (=Enterobacteriaceae-protocol). We could identify all isolates correctly with

the exception of non-CP- CR C. freundii isolates. They did not show induction.
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Two microbes (E. coli, OXA-48, ri 17%, E. coli, OXA-181, ri 18%) were excluded because they
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were classified as susceptible and not flagged as carbapenemase producing in the BD PhoenixTM

system.
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Discussion:

The presented BCDA is a robust phenotypic screening method that can reliably detect a reduced
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susceptibility to meropenem. Detection of CP-CPA displayed high sensitivity and specificity also in
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the case of OXA-associated CP-CRA isolates, where other phenotypic methods often fail (Boelens

et al., 2016, Campana et al., 2016, Knox et al., 2014, Lafeuille et al., 2015).

Results generated by testing Enterobacteriaceae showed a slight overlap of the boxplots (Figure 3)

from 19% to 32% ri. Here, both, carbapenem-resistant and susceptible Enterobacteriaceae isolates

could be identified. In our assay, all of them carried class D carbapenemase genes (5% of

measurements, supplementary Table 1). Also, non-CP-CR C. freundii isolates were not classified

correctly. They did not show induction in BCDA. Only CP-CR C. freundii showed growth over

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32% ri. If the results are in this transition zone between resistant and susceptible (19-32% ri), a

diagnostic uncertainty must be assumed and further diagnostic procedures should be included in the

detection of OXA-associated carbapenemase resistance testing Enterobacteriaceae. C. freundii

isolates should not classified as susceptible to meropenem only using BCDA because non-CP-CR

C.freundii and susceptible C. freundii isolates could not be distinguished.

The two excluded isolates (E. coli, OXA-48, ri 17%, no indication by BD PhoenixTM for

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carbapenemase forming; E. coli, OXA-181, ri 18%, no indication by BD PhoenixTM for

carbapenemase forming) could not be detected by BCDA and BD PhoenixTM. Enterobacteriaceae

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with OXA carbapenemases are known to be difficult to identify by phenotypic methods (Dortet et

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al., 2015, Girlich et al., 2013, Poirel et al., 2012, Tijet et al., 2013). OXA-genetic elements

sometimes not translated from DNA to active enzymes (Dortet et al., 2015). This observation is not
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consistent with the results of Woodford et al. (Woodford et al., 2010) who demonstrated sensitivity
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of 100% in the detection of carbapenemases by the BD PhoenixTM automated antimicrobial

susceptibility testing and should be examined in follow-up studies to clarify whether there was a
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lack of expression (and no carbapenemase was produced) or our reference method and BCDA were
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unable to identify it.

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In some cases of CRE and over 50% in CP-CRA ri was more than 100%. This phenomenon could

be interpreted as an antibiotic triggered growth boost, which was described already by Reding-
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Roman et al. using doxycycline investigating E. coli isolates (Reding-Roman et al., 2017). Since
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BCDA is a phenotypic assay which allows interpretation of the microbial growth in a quantitative

fashion it would be interesting to see if the aforementioned results by Reding-Roman et al. can be

reproduced using BCDA.

Since this method is based on the detection of growth of bacteria, the application of BCDA for other

samples is, in principle, conceivable e.g. for blood-culture testing.

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BCDA is faster than the usual agar based phenotypic methods and reduces the evaluation time by at

least one working day. Compared to genotypic methods, it is also possible to identify antimicrobial

resistances caused by unknown genes or mechanisms, e.g. loss of porin and increased efflux.

We did not include carbapenem-resistant Pseudomonas aeruginosa in this study since this species

cannot be classified as carbapenem-resistant by testing meropenem alone. Moreover, the method

has not yet been clinically evaluated. Different antibiotic concentrations were used to optimize

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identification of meropenem susceptibility in different bacilli groups. We tested CP-CRA/CRE

before non-CP-CRE to get high sensitivity in this group of bacilli because they are more relevant in

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clinical settings (Biehle et al., 2015, de Maio Carrilho et al., 2016, Goodman et al., 2016, Tamma, et

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al., 2017). Our method does not appear to offer reliable detection testing CP-CR K. pneumoniae

isolates with very low MIC (≤ 0,25 mg/L). It is questionable whether this is clinically relevant
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because the CSLI classified Enterobacteriaceae with MIC < 1 mg/L as susceptible (which does not
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exclude the possibility of carbapenemase formation).

BCDA can be implemented in diagnostic algorithms for carbapenem resistance in

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Enterobacteriaceae and A. baumannii e.g. as a confirmation assay if a chromogenic screening

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medium is used and displays a color change. This will reduce the time to appropriate therapy which
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can provide a benefit for patients’ treatment and outbreak control of CRE and CP-CRA, including

cost optimization.
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Summary:

The potential of this new method lies in the rapid and reliable detection of antimicrobial resistance,

especially in CP-CRA, which can be implemented within one working day in the laboratory

workflow. It does not have to rely on PCR-based methods, which are significantly more expensive

than our assay. Difficulties may occur in the detection of CRE with low MIC combined with OXA-

associated carbapenem resistance. C. freundii should not classified as susceptible only using

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BCDA.

Acknowledgments:

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This work was funded by Stiftung für Pathobiochemie und Molekulare Diagnostik (scholarship).

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The funders had no role in study design, data collection and interpretation, or the decision to submit

the work for publication. We thank Dr Martin Kaase, former member of the National Reference
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Centre for Multidrug-resistant Gram-negative Bacteria in Bochum, and Dr Niels Pfennigwerth,
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member of the National Reference Centre for Multidrug-resistant Gram-negative Bacteria in

Bochum, for providing us carbapenem resistant bacterial isolates.

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Figure legends

Figure 1

Shows the experimental setup and calculation of results generated by BCDA.

Figure 2:

Results generated by BCDA investigating Acinetobacter sp.. Green colored results were confirmed by BD

PhoenixTM as susceptible against meropenem and were not flagged as carbapenemase-producer. Red colored

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results were confirmed by BD PhoenixTM as resistant against meropenem. Value in parentheses describes

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used concentration of meropenem.

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Figure 3:
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Results given by BCDA investigating Enterobacteriaceae. Green colored results were confirmed by BD

PhoenixTM as susceptible against meropenem and were not flagged as carbapenemase-producer. Red colored
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results were confirmed by BD PhoenixTM as resistant against meropenem as well as by NRC.

Value in parentheses describes used concentration of meropenem. 23 CP-CRE were tested again with
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reduced concentration of meropenem.

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Tables
Table 1 Investigated bacilli

Study population
Ambler class no carbapenemase gene
A B D Resistance by
susceptible to
AmpC with
KPC IMP NDM VIM OXA meropenem n=
Porinloss
Enterobacteriaceae
C. freundii - - - 1 1 3 2 7
E. asburiae - - 1 - - - - 1
E. cloacae - 2 1 2 - 4 1 10

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E. coli 1 - 3 - 7 6 23 40
K. oxytoca 1 - - 1 - - - 2

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K. pneumoniae 14 - 4 4 15 6 2 45
S. marcescens - - - 1 1 3 - 5

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A. baumannii - - 5 - 43 - 19 65*
n= 16 2 14* 9 67* 22 47 175
* 2 isolates have coexpression of OXA-23 and NDM-1/-6
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Table 1 Investigated bacilli
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Table 2:

Results of susceptibility testing to meropenem by BCDA for Enterobacteriaceae

(excepted C. freundii) and Acinetobacter spp. compared with BD PhoenixTM results
BCDA
Enterobacteriaceae Acinetobacter spp.
(Breakpoint: 23% ri) (Breakpoint: 36% ri)
susceptible resistant susceptible resistant
BD susceptible 25 1 19 0

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PhoenixTM resistant 1 76 0 46
n=103; =0.95 (CI 95%: 0.89-1.02) n=65; =1(CI 95%: 1.00-1.00)
Cohens kappa with 95% confidence interval (CI) was calculated for statistical accordance of BCDA

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compared with BD PhoenixTM results using formulas given by Watson et al. (Watson and Petrie, 2010).

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Enterobacteriaceae were tested using the Enterbacteriaceae-protocol. Acinetobacter spp. were
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tested using the Acinetobacter-protocol.
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Figure 1
Figure 2
Figure 3