Direct RNA-based detection of CTX-M β-lactamases in human blood samples

Accepted Manuscript Title: Direct RNA-based detection of CTX-M ␤-lactamases in human blood samples Author: Claudia Stein Oliwia Makarewicz Yvonne Pfeifer Christian Brandt Mathias W. Pletz PII: DOI: Reference:

S1438-4221(15)00016-8 http://dx.doi.org/doi:10.1016/j.ijmm.2015.02.005 IJMM 50931

To appear in: Received date: Revised date: Accepted date:

17-9-2014 8-12-2014 14-2-2015

Please cite this article as: Stein, C., Makarewicz, O., Pfeifer, Y., Brandt, C., Pletz, M.W.,Direct RNA-based detection of CTX-M rmbeta-lactamases in human blood samples, International Journal of Medical Microbiology (2015), http://dx.doi.org/10.1016/j.ijmm.2015.02.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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aDirect RNA-based detection of CTX-M -lactamases in human blood samples.

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Running title: Detection of CTX-M in blood samples

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4 Authors:

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Claudia Stein1, Oliwia Makarewicz1, Yvonne Pfeifer2, Christian Brandt1, Mathias W.

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Pletz1

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8 Affiliations: 1

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Erlanger Allee 101, D-07747 Jena, Germany

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37, D-38855 Wernigerode

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Center for Infectious Diseases and Infection´s Control, Jena University Hospital,

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Nosocomial Pathogens and Antibiotic Resistance, Robert Koch Institute, Burgstraße

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14 Corresponding author:

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Claudia Stein

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Jena University Hospital, Erlanger Allee 101, D-07747 Jena, Germany

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[email protected]

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Phone number 00 49 3641- 9 32 47 93

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Fax 0049 3641 9 32 46 52

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CS and OM contributed equally to the manuscript.

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Keywords:

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Extended-spectrum beta-lactamases (ESBL), degenerate primers, pyrosequencing,

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qRT-PCR, EDTA blood, blood culture

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27 Abstract:

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Bloodstream infections with ESBL-producers are associated with increased mortality,

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which is due to delayed appropriate treatment resulting in clinical failure. Current

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routine diagnostics for detection of bloodstream infections consists of blood culture

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followed by species identification and susceptibility testing. In attempts to improve

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and accelerate diagnostic procedures, PCR-based methods have been developed.

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These methods focus on species identification covering only a limited number of

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ESBL coding genes. Therefore they fail to cover the steadily further evolving genetic

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diversity of clinically relevant -lactamases. We have recently designed a fast and

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novel RNA targeting method to detect and specify CTX-M alleles from bacterial

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cultures, based on an amplification-pyrosequencing approach.

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We further developed this assay towards a diagnostic tool for clinical use and

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evaluated its sensitivity and specificity when applied directly to human blood

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samples. An optimized protocol for mRNA isolation allows detection of specific CTX-

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M groups from as little as 100 CFU/mL blood via reverse transcription, amplification,

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and pyrosequencing directly from human EDTA blood samples as well as from pre-

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incubated human blood cultures with a turnaround time for test results of <7 h.

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53 Introduction

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The spread of Gram-negative bacteria producing extended-spectrum -lactamases

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(ESBLs) is a rising problem worldwide (Valentin et al., 2014). In some regions up to

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20 % of the population is already colonized with ESBL producers (Overdevest et al.,

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2011; Schoevaerdts et al., 2011) which is associated with an increased risk for ESBL

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bloodstream infection: a study demonstrated that 8.5 % of colonized patients

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developed a bloodstream infection caused by ESBL-producing Enterobacteriaceae

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(Freeman et al., 2012; Reddy et al., 2007; Vehreschild et al., 2014). Bloodstream

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infections with ESBL-producers are associated with increased mortality (Marra et al.,

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2006; Tumbarello et al., 2007), which is due to often inappropriate initial treatment

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resulting in clinical failure (Hyle et al., 2005; Tumbarello et al., 2006). Empiric

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treatment

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antipseudomonal activity in optional combination with an aminoglycoside or a

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fluoroquinolone, does not cover ESBL producers except for carbapenems

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(ATS/IDSA, 2005; Dalhoff et al., 2012; Dellinger et al., 2008; Reinhart et al., 2010).

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Hence, early identification of the resistance profile of the causative pathogen is

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required to minimize the delay until onset of appropriate treatment and, subsequently,

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to reduce mortality.

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The current routine diagnostic procedure for detection of bloodstream infections

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consists of blood culture followed by species identification and susceptibility testing.

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Combined analysis is provided by some automated systems, e.g. Vitek®2

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(bioMérieux, Inc) or PhoenixTM (Becton Dickinson and Company) (Drieux et al., 2008;

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Gazin et al., 2012). These microbiological, phenotypic methods are not optimal for

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diagnosing and characterising -lactamases: The tests provide no information about

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the resistance genes, and often require labour-intensive and time-consuming

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sepsis

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beta-lactam

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additional tests, such as the modified Hodge-test, Etest or combined disc diffusion

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test to confirm carbapenemases (Garrec et al., 2011). Test interpretation needs

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trained personnel, but still certain constellations are easily misinterpreted (Nordmann

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et al., 2011; Oliver et al., 2002). Finally, the accuracy of results is prone to inoculum

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effects (Kellogg et al., 1984). In attempts to improve diagnostic procedures, PCR-

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based methods have been developed, most of which aim for species identification.

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From a clinical point of view, however, this is insufficient in the era of increasing

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antimicrobial resistance. The decisive information is, if the empirically started

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treatment is effective or not (Pletz et al., 2010).

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Moreover, most current molecular biological methods still exhibit unsatisfactory

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sensitivity compared to culture-dependent methods, in particular in the case of low

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bacterial counts, as have to be expected in bloodstream infections (<1-100 CFU/mL)

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(Yagupsky and Nolte, 1990). In addition, many compounds in blood samples or blood

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cultures, such as bile salts, haemoglobin, leucocyte DNA, immune globulins, and

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anticoagulants, interfere with molecular biological applications (Rolfs et al., 1991).

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Thus, most PCR-based methods require bacterial cultures, resulting in an increased

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time-to-result.

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In European countries, class A -lactamases represent the most prevalent ESBLs

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(Canton et al., 2008; Machado et al., 2013), with CTX-M variants (CTX-M-1, CTX-M-

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14, CTX-M-15) as most frequent types in ESBL colonization and infection (Babic et

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al., 2006; Canton and Coque, 2006; Canton et al., 2012b; Overdevest et al., 2011).

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CTX-M variants are mainly detected in Escherichia coli and Klebsiella pneumoniae

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and have been reported to be associated with hospital-acquired as well as with

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community-acquired bloodstream infections (Rodriguez-Bano et al., 2006; Son et al.,

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2010).

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We have recently established a novel method to detect and specify CTX-M alleles

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from bacterial cultures, based on an amplification-sequencing approach. This method

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differs from conventional multiplex approaches by i) detecting resistance genotypes

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instead of species, ii) combining PCR using degenerate primers with consecutive

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pyrosequencing, and iii) targeting mRNA instead of DNA (Stein et al., 2013).

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The work presented here further developed this assay towards a diagnostic tool for

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clinical use and evaluated its sensitivity and specificity when applied directly to

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human blood samples. The optimized protocol for mRNA isolation allows detection of

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specific CTX-M groups from as little as 100 CFU/mL blood via reverse transcription,

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amplification, and pyrosequencing directly from human EDTA blood samples as well

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as from pre-incubated human blood cultures.

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115 Material and methods

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Ethics statement

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The usage of human blood from six healthy volunteers was approved by the ethics

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committee of the Jena University Hospital with the file number 4016-02/14. Written

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informed consent was obtained from all study participants prior blood sampling.

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Bacterial strains, preparation of spiked human blood samples, and colony counting

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The study included 13 clinical isolates producing different CTX-M-type ESBLs (Table

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1). Bacterial strains were obtained from the Robert Koch Institute (Wernigerode,

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Germany). All β-lactamase genes were confirmed by sequencing. Escherichia coli

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K12J53 was used as a β-lactamase negative control; one SHV-4 bearing strain was

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used as CTX-M negative control. All strains were cultivated on Mueller-Hinton (MH)

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agar plates or MH broth (Roth). Media were supplemented with 16 µg/mL cefotaxime

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(Fresenius Kabi) for selection of strains harbouring CTX-M -lactamases, 100 g/mL 5

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ampicillin (Roth) for the SHV-4 producing strain, or 50 µg/mL sodium azide for strain

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K12J53.

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Cultures were grown over night at 37 °C under constant rotation (200 rpm), then

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diluted 1:20 in fresh MH broth, and grown for additional 3 hours. The cultures were

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adjusted to an optical density of 0.05 at 600 nm (Schultz et al., 1987).This bacterial

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suspension was serially diluted in 0.9 % sodium chloride. The viable bacterial cells

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(CFU/mL) were determined by plating 100 µl of each bacterial dilution step on

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selective MH agar plates. 1 mL of each dilution step was added to 3 mL of fresh

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EDTA blood (Monovette, Sarstedt), obtained from healthy volunteers. Resulting

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bacterial concentrations ranged from 104 to 100 CFU/mL blood. Non-spiked blood

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samples were used as negative controls.

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141 Plasmid standard

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A plasmid DNA standard was constructed to quantify the real-time PCR (qPCR)

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reaction. The blaCTX-M-15 gene of strain 25/08 was amplified using CTX-M-15 primers

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(for ATGGTTAAAAAATCACTGCGCCA; rev TTACAAACCGTCGGTGACGATTT) in a

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colony PCR. The amplicon was purified using the NucleoSpin PCR clean-up kit

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(Machery Nagel) and ligated into the pGEMT vector according to the manufacturer’s

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protocol (Promega). The generated pCTXM15 plasmid was transformed into electro-

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competent E. coli JM109 cells and transformants were selected on MH agar,

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supplemented with 8 µg/mL cefotaxime. After plasmid isolation with the NucleoSpin

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Plasmid Kit (Machery Nagel), the pCTXM15 plasmid was linearized by SacI (Life

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Technologies) and stored in 1 x TE buffer (10 mM TrisHCl, 1 mM EDTA, pH 7.5) at -

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20°C. The plasmid concentration was determined by NanoDrop2000 (PeqLab).

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RNA isolation from EDTA blood samples 6

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Total RNA was directly extracted from 3 mL EDTA blood using TempusTM Blood RNA

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Tube and TempusTM Spin RNA Isolation Kit according to manufacturer’s protocols

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(Life Technologies). DNase I digestion was carried out using AbsoluteRNA Wash

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Solution (Life Technologies) on column. RNA samples were stored at -80 °C after

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elution with 100 µl RNase-free water.

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To maximize the yield of RNA for reverse transcription, we performed an ammonium

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acetate precipitation. Therefore, 95 l of isolated RNA were precipitated by adding

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355 l ethanol (98%) and 47 l ammonium acetate (7.5 mM), mixing and storing at -

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20 °C for 30 min. The RNA was centrifuged for 15 min at 15000 rpm and 4 °C;

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supernatants were removed carefully. RNA pellets were dried at room temperature

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and resuspended in 7.7 µl RNase-free water.

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RNA isolation from spiked blood culture bottles

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Aerobe (BacT/Alert® SA, bioMérieux) and anaerobe (BacT/Alert® SN, bioMérieux)

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blood culture bottles were inoculated with 1 mL bacteria suspension (10 CFU/mL)

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and 4 mL blood (without anticoagulants). Blood cultures were routinely incubated at

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37 °C until positivity, as determined by Bact/AlertTM (bioMérieux). .0.5 mL of the

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culture were subsequently used for the total RNA isolation. Erythrocytes were lysed

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by adding 3 volumes of buffer EL (Qiagen) and incubation at room temperature for 5

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min. Bacteria were pelleted at 3000 rpm and washed with PBS (137 M NaCl, 3 mM

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KCl, 10 mM NaH2PO4, 1.6 mM K2HPO4, pH 7). Bacteria were lysed by adding 800 μL

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TRIzol (Life Technologies) and 160 μL chloroform (Roth). Cell debris was removed

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by centrifugation for 20 minutes at 15000 rpm and careful removal of the aqueous

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phase. The supernatant was mixed with 250 μL ethanol (absolute) and total RNA

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was further purified using RNeasy Mini Kit columns (Qiagen) according to

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manufacturer's protocol.

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182 RNA quality control

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Quality and quantity of the RNA preparations were evaluated using Agilent 2100

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Bioanalyzer (Agilent Technologies) and the Agilent Eukaryote Total RNA Nano Assay

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according to the manufacturer’s instruction and as described previously (Stein et al.,

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2013). Additionally, the quantity was determined using NanoDrop2000 (PeqLab).

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188 Reverse transcription (RT)

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Reverse transcription reactions were performed in 10 µL volumes, containing 6.7 µL

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of total RNA and 0.16 µM of the 5'-biotinylated, degenerate reverse primer revCTX-M314

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as previously described (Stein et al., 2013).

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193 Quantitative real-time PCR

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The real-time PCR was performed in a Rotor-GeneQ cycler (Qiagen) by directly

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applying 2 µL cDNA and the degenerate primer sets blaCTX-M67 and revCTX-M314 (Stein

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et al., 2013). The PCR reaction mixture was composed of 1.5 mM MgCl2, 1 x PCR

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buffer (Life Technologies), 0.2 mM dNTPs (Roth), 1.9 µM of each primer (Sigma

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Aldrich), 0.15x SybrGreen (Life Technologies), 0.08 U/µl Platinum Taq DNA

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Polymerase (Life Technologies), and 0.1 mg/mL BSA (Life Technologies). The PCR

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was run as follows: pre-denaturation at 99 °C for 10 s and 93 °C for 20 s; 50 cycles

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composed of 93 °C for 11 s, 55 °C for 11 s, and 72 °C for 14 s. The melting

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temperatures of the PCR products were determined by stepwise of the temperature

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increasing (0.5 °C / 4 s) from 75 °C to 99 °C.

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Pyrosequencing

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Pyrosequencing analysis was performed on the PyroMark Q24 (Qiagen). The whole

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PCR reaction volume was added to the pyrosequencing reaction mixture (PyroMark

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Gold Q24 reagent, Qiagen). The CTX-M sequences were analysed using the

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sequencing primer seqCTX-M211 (Stein et al., 2013). Measurements were performed

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according the manufacturer’s protocol, except for increased concentration of the

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primer (fourfold) and streptavidin sepharose (threefold).

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213 Results

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RNA isolation from EDTA blood samples

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The TempusTM Blood RNA Tube method is a single-step isolation method based on

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selective RNA precipitation (Prezeau et al., 2006). This method allows purification of

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total RNA from blood samples without pre-treatments, such as selective erythrocyte

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lysis. After additional chromatographic purification, the RNA samples contained both

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eukaryotic and bacterial total RNA that was further enriched by precipitation. This

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procedure yielded 2.5 g to 7 g total RNA from 3 mL EDTA blood, independently of

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the inoculated bacteria counts due to the high content of eukaryotic material. All

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obtained RNA samples exhibited high RNA integrity numbers (RIN >7, see Table 2),

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indicating high quantity for further analysis and confirming the good performance of

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this single-step method. Despite the loss of approximately 20 % of total RNA, the

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sensitivity of the downstream experiments could be increased 13-fold by the

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additional enrichment via precipitation (data not shown).

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RNA isolation from spiked blood cultures

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The TempusTM technology was insufficient for isolation of high-quality total RNA from

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the blood culture, probably due to the high content of organic and inorganic

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compounds in the culture medium (see Table 3). No PCR products could be obtained 9

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from these samples. We hence compared the performance of the TempusTM

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technology and TRIzol-chloroform extraction (Chomczynski and Sacchi, 1987), an

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alternative single-step protocol for RNA isolation from spiked blood cultures. Using

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different sample volumes (0.5 mL, 1 mL, and 2 mL) of a positive blood culture (spiked

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with CTX-M-14-positive strain RKI 443/08), we quantified the isolated RNA samples

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by NanoDrop and determined the CTX-M mRNA by RT and PCR.

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TRIzol-chloroform extraction, combined with chromatographic purification (RNeasy

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Mini columns, Qiagen) resulted in adequate quantities and qualities of the total RNA

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for reverse transcription and real-time PCR. The lower sample volumes (0.5 mL and

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1 mL) yielded a higher quality (ratio260/280) of the total RNA compared to 2 mL blood

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culture sample resulting in lower CT values (Table 3).

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Optimization of the RT-qPCR conditions

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For optimization of the RT-qPCR, we used a linearized plasmid DNA (pCTXM15, ~ 3

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kbp) containing the CTX-M-15 gene (~ 0.8 kbp) and blood samples spiked with

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various concentrations of pathogens carrying different variants of the CTX-M -

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lactamase. Standard RT and qPCR protocols yielded a high extent of unspecific

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transcription products due to the degenerate reverse primer in the RT reaction and

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the high eukaryotic RNA content of the total RNA (Fig. 1 B-D). Specificity of the RT

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reaction could be increased by reducing the degenerate primer concentration to 0.16

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M (see Fig. 1 B, lanes 7 to 10).

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The efficiency and specificity of the amplification reaction was analysed by real-time

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PCR sensograms and the integrated melting curve, respectively. To improve the

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sensitivity, we increased the primer concentration to 1.9 µM and the cycle number to

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50, whereas MgCl2 concentration was decreased to 1.5 mM. To maintain the

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polymerase activity over the 50 cycles, we reduced the denaturation temperature to

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93°C and periods of the cycle steps (see Material and Methods).

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This optimized RT-qPCR protocol was sufficient to amplify a CTX-M-15 fragment

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using 0.1 fg (~23 molecules) of the pCTXM15 plasmid DNA. The PCR efficiency was

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high (0.92), despite the use of the degenerate primer set (see Fig. 1 A).

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263 Specificity and sensitivity of the RT-qPCR

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Total RNA isolated from EDTA blood and from blood culture was used for the RT-

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qPCR. Specific PCR products of the CTX-M melted between 89 °C and 92 °C, as

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described previously (Stein et al., 2013). To ensure that the PCR products were

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amplified from transcribed cDNA and not from contaminant DNA, RT controls without

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reverse transcriptase were included. These controls yielded no products, indicating

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that DNA contamination was negligible in both RNA isolation procedures

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(supplementary data, Fig. A.1).

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EDTA blood samples were used to further test the sensitivity of the method and to

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determine the detection limits. Non-specific PCR products increased with decreasing

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bacterial concentrations (104 – 101 CFU/mL), but exhibited comparable CT values

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(see Fig. 2). Electrophoretic analysis on agarose gels confirmed the unspecific PCR

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products at lower template concentration (Fig. 2 B). To assess assay specificity,

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blood samples that were spiked with strains bearing non-CTX-M -lactamases were

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subjected to RT-qPCR analysis. These samples yielded no CTX-M-corresponding

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PCR products within the tested bacterial concentration range (101 – 104 CFU/mL

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blood; Fig. 3) and only very low intensities of unspecific products were detected.

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These results confirm that the degenerate primers are highly specific for the CTX-M

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group and unspecific products due to the co-isolated high content of human blood

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RNA or other -lactamases are negligible.

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The optimized protocols for RNA isolation, RT, and qPCR were sufficient to reversely

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transcribe CTX-M mRNA and to amplify the corresponding cDNA from EDTA blood

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samples spiked with 101 – 104 bacteria/mL blood. Given the obviously higher

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bacterial RNA contents, sensitivity limitation was no issue in positive blood culture

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samples. However, we also analysed blood culture samples incubated for 0 h, 2 h, 4

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h, 6 h, and 8 h. Specific PCR products were detectable after 6 and 8 h incubation,

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which corresponds to a bacterial concentration range from 105 to 107 CFU/mL (see

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supplementary data, Fig. A.2).

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292 Specificity and sensitivity of pyrosequencing

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PCR products generated by RT-qPCR were used for the pyrosequencing reaction.

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No signals were detectable in the sterile blood and all non-CTX-M-bearing samples

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during the pyrosequencing for RNA isolation systems. All samples containing specific

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CTX-M PCR products yielded evaluable pyrograms. Based on the pyrograms, we

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could distinguish the phylogenetic groups of CTX-M variants as described previously

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(Stein et al., 2013).

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Again, sensitivity limitations in the pyrogram signals were no issue for positive blood

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culture samples. For samples obtained from spiked EDTA blood, the lower limit of

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detection (LOD) was determined at 101 – 102 CFU/mL blood (see Table 2). This

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correlated with the limit of the qPCR in our setup. The observed differences (20 to

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447 CFU/mL for E. coli and S. enterica strains) in the LOD resulted mainly from the

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colony counting method that was performed by serial 10-fold dilution and thus

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exhibited a high variance. Although the optimized protocols displayed increased

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sensitivity, we could not reach the level of <10 CFU/mL that has been described for

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conventional blood samples from untreated adult patients with bloodstream infections

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(Kellogg et al., 1984).

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The LODs differed depending on the species. For K. pneumoniae strains (RKI

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346/12, 93/07) carrying more than one ESBL, we reached a detection limit of 103

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CFU/ml; the LODs for E. cloacae ranged from 573 to 630 CFU/mL and those for E.

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coli from 20 to 447 CFU/mL. This might be caused by an differing lysis efficiency in

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these species, due to the varying capsule construction (Gerasimenko et al., 2004).

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315 Analysis time required

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Analysis of EDTA blood samples, which included amplification to saturation in the

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RT-qPCR and pyrosequencing, took 6 – 7 hours (see supplementary data, Fig. A.3).

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The time-to-result for blood culture samples was below 19 h. The time-limiting step in

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this case was the time-to-positivity of the blood culture: this usually takes 10-15 h for

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Enterobacteriaceae (Defrance et al., 2013). The subsequent RT-qPCR and

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pyrosequencing analyses were performed within 5 hours (see supplementary data,

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Fig. A.4) without sensitivity and specificity limitations.

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Costs of the RT-qPCR-pyrosequencing method

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The material costs for one sequence of reverse transcription, qPCR, and

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pyrosequencing reaction can be estimated at around 15 €. The purification costs

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range between 13.50 € / sample for the TempusTM technology and 5 € / sample for

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the TRIzol/RNeasy method. All quotes were calculated based on catalogue prices.

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Discussion

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We present a rapid protocol for a novel RNA-based PCR-pyrosequencing method

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that directly detects all subtypes of CTX-M, the most frequent ESBL family, in blood

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samples. The achieved lower limit of detection lies between 2x101 and 5x102

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CFU/mL, which is above those for blood culture, but within the range of other

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multiplex PCRs.

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In the era of increasing antibiotic resistance, particularly in Gram-negative bacteria,

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rapid information about the susceptibility of the pathogen causing bloodstream

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infection is required. As E. coli as the most frequent causative agent of bloodstream

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infections (Fennell et al., 2012; Gastmeier et al., 2012), the rapidly increasing rate of

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ESBL-producing E. coli (currently ranging between 5 – 20 %), is complicating the

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selection of an appropriate antibiotic for empiric treatment in critically ill patients:

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General first line use of carbapenems increases the risk of selection of

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carbapenemase

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inappropriate treatment, particularly in patients with severe sepsis.

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Therefore, accurate and fast identification of ESBL-producing Enterobacteriaceae

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and characterisation of their resistance profile are of major importance. Since

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infections with bacteria expressing certain ESBL variants (e.g. CTX-M) can be

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successfully treated with piperacillin / tazobactam, identification of the individual CTX-

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M variant is of greater benefit than just diagnosing presence of ESBLs (Leclercq et

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al., 2013). Importantly, the presented approach will also detect potentially novel

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variants that may evolve by occurrence of point mutations. Furthermore, we can

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apply the knowledge about the individual CTX-M variant to manage and control

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outbreaks and to reconstruct the molecular epidemiology, thereby contributing to

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increased surveillance.

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Some currently available PCR-based methods, such as the DNA microarray platform

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Check-Points (Cuzon et al., 2012; Lascols et al., 2012; Naas et al., 2010) or the

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Verigene System by Nanoshere, Inc, provide information on the presence of

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individual resistance determinants. However, only few resistance determinants are

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included, e.g. methicillin resistance (mecA) of staphylococci and vancomycin (vanA

whereas

using

other antibiotics

may result in

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and vanB) resistance of entrococci (Afshari et al., 2012; Paolucci et al., 2010). Two

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systems identify single -lactamases SHV (Vyoo®) or carbapenemase KPC (Plex-

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IDTM). Only VAPchip includes a broad range of -lactamases: SHV (four codons),

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TEM (8 codons), and CTX-M (groups 1, 2, and 9) without variant discrimination as

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well as the most frequent carbapenemases (OXA-23, OXA-40, OXA-48, OXA-58,

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OXA-72, KPC-2, VIM-1, VIM-2, VIM-like, IMP-4, and IMP-13). However, these tests

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fail to cover the steadily further evolving genetic diversity of clinically relevant -

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lactamases, which also displays regional and temporal variation (Canton et al.,

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2012a).

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Compared to culture-based systems, the higher limit of detection is a disadvantage of

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PCR-based techniques. However, only few commercially available techniques and

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platforms using blood cultures do detect resistance determinants. Some include

373

mecA and van genes (Afshari et al., 2012), which is owed to the interest of MRSA

374

and VRE in the last decades; but ESBLs and carbapenemases are currently

375

becoming more relevant. Thus, some recent efforts aimed to improve the

376

performance of various platforms for -lactamase detection in blood cultures. Fujita et

377

al. determined CTX-M in positive blood culture bottles by amplification and microchip

378

gel electrophoresis within 1.5 h after blood cultures turned positive (Fujita et al.,

379

2011). Fishbain et al. used the Check-KPC/ESBL microarray systems (Check-Points,

380

Inc., The Netherlands) to determine the corresponding genes in blood culture

381

(Fishbain et al., 2012). When we investigated positive blood culture bottles by our

382

RT-qPCR-pyrosequencing method, the LODs were negligible and all CTX-M variants

383

could be accurately assigned to the correct CTX-M-group even after only 6-8 hours of

384

culturing.

385

To improve sensitivity we focus on mRNA as target molecule since it is usually

386

present in a higher copy number per cell compared to the encoding gene. The time-

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15

Page 15 of 30

to-result of the presented RT-qPCR-pyrosequencing method was comparable to

388

automated multiplex-PCR systems for direct use of EDTA blood. However, the LOD

389

needs further improvement. This especially holds true for K. pneumoniae, since this

390

species is a frequent cause of nosocomial infections and hospital outbreaks (Filippa

391

et al., 2013; Muro et al., 2012; Rettedal et al., 2012).

ip t

387

cr

392 Conclusions

394

Beside the direct use of EDTA blood, the presented RT-qPCR-pyrosequencing

395

method has some advantages compared to other PCR-based methods for -

396

lactamase detection (Table 4). Due to the degenerate primers, all CTX-M variants

397

can be determined and the pyrosequencing step allows further discrimination of the

398

phylogenetic groups.

399

Our results demonstrate that RNA is a promising template for a direct utilization of

400

blood samples. Using degenerate primers helps to overcome the challenging

401

emergence of new -lactamase variants and changing local epidemiology. In the

402

context of the highly mobile -lactamases, discrimination of pathogens in clinical

403

specimen alone is of minor use. Direct determination of the expressed resistance

404

genes seems to be more appropriate to immediately adjust the adequate antibiotic

405

therapy. However, this approach does currently not detect simultaneous expression

406

of AmpC. Therefore using piperacillin/tazobactam after detection a susceptible CTX-

407

M variant may not be appropriate in species known to harbour frequently AmpC, e.g.

408

Enterobacter spp. Therefore we presently work on expansion of the mRNA-based

409

approaches to other -lactamase variants including OXA that harbour many

410

carbapenemases, and AmpC that can be plasmidal or chromosomal encoded and

411

inducible or constitutively expressed.

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412 16

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Acknowledgement

414

The work was supported by a grant from the German Ministry of Education and

415

Research (BMBF); grant numbers 01KI1204. We thank Dr. Margit Leitner (Center for

416

Sepsis Control and Care, UKJ) for critical reading of the manuscript.

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418 References

420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456

Afshari, A., Schrenzel, J., Ieven, M., Harbarth, S., 2012. Bench-to-bedside review: Rapid molecular diagnostics for bloodstream infection - a new frontier? Crit Care 16, 222. ATS/IDSA, 2005. Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia. Am J Respir Crit Care Med 171, 388-416. Babic, M., Hujer, A.M., Bonomo, R.A., 2006. What's new in antibiotic resistance? Focus on beta-lactamases. Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy 9, 142-156. Canton, R., Akova, M., Carmeli, Y., Giske, C.G., Glupczynski, Y., Gniadkowski, M., Livermore, D.M., Miriagou, V., Naas, T., Rossolini, G.M., Samuelsen, O., Seifert, H., Woodford, N., Nordmann, P., 2012a. Rapid evolution and spread of carbapenemases among Enterobacteriaceae in Europe. Clin Microbiol Infect 18, 413-431. Canton, R., Coque, T.M., 2006. The CTX-M beta-lactamase pandemic. Current opinion in microbiology 9, 466-475. Canton, R., Gonzalez-Alba, J.M., Galan, J.C., 2012b. CTX-M Enzymes: Origin and Diffusion. Front Microbiol 3, 110. Canton, R., Novais, A., Valverde, A., Machado, E., Peixe, L., Baquero, F., Coque, T.M., 2008. Prevalence and spread of extended-spectrum beta-lactamase-producing Enterobacteriaceae in Europe. Clin Microbiol Infect 14 Suppl 1, 144-153. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162, 156-159. Cuzon, G., Naas, T., Bogaerts, P., Glupczynski, Y., Nordmann, P., 2012. Evaluation of a DNA microarray for the rapid detection of extended-spectrum -lactamases (TEM, SHV and CTX-M), plasmid-mediated cephalosporinases (CMY-2-like, DHA, FOX, ACC-1, ACT/MIR and CMY-1-like/MOX) and carbapenemases (KPC, OXA-48, VIM, IMP and NDM). J Antimicrob Chemoth 67, 1865-1869. Dalhoff, K., Abele-Horn, M., Andreas, S., Bauer, T., von Baum, H., Deja, M., Ewig, S., Gastmeier, P., Gatermann, S., Gerlach, H., Grabein, B., Hoffken, G., Kern, W.V., Kramme, E., Lange, C., Lorenz, J., Mayer, K., Nachtigall, I., Pletz, M., Rohde, G., Rosseau, S., Schaaf, B., Schaumann, R., Schreiter, D., Schutte, H., Seifert, H., Sitter, H., Spies, C., Welte, T., 2012. Epidemiologie, Diagnostik und Therapie erwachsener Patienten mit nosokomialer Pneumonie. Pneumologie 66, 707-765. Defrance, G., Birgand, G., Ruppe, E., Billard, M., Ruimy, R., Bonnal, C., Andremont, A., Armand-Lefevre, L., 2013. Time-to-positivity-based discrimination between Enterobacteriaceae, Pseudomonas aeruginosa and strictly anaerobic Gram-negative bacilli in aerobic and anaerobic blood culture vials. J Microbiol Methods 93, 77-79.

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Dellinger, R.P., Levy, M.M., Carlet, J.M., Bion, J., Parker, M.M., Jaeschke, R., Reinhart, K., Angus, D.C., Brun-Buisson, C., Beale, R., Calandra, T., Dhainaut, J.F., Gerlach, H., Harvey, M., Marini, J.J., Marshall, J., Ranieri, M., Ramsay, G., Sevransky, J., Thompson, B.T., Townsend, S., Vender, J.S., Zimmerman, J.L., Vincent, J.L., 2008. Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008. Intensive Care Med 34, 1760. Drieux, L., Brossier, F., Sougakoff, W., Jarlier, V., 2008. Phenotypic detection of extended-spectrum beta-lactamase production in Enterobacteriaceae: review and bench guide. Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases 14 Suppl 1, 90103. Fennell, J., Vellinga, A., Hanahoe, B., Morris, D., Boyle, F., Higgins, F., Lyons, M., O'Connell, K., Keady, D., Cormican, M., 2012. Increasing prevalence of ESBL production among Irish clinical Enterobacteriaceae from 2004 to 2008: an observational study. BMC Infect Dis 12, 116. Filippa, N., Carricajo, A., Grattard, F., Fascia, P., El Sayed, F., Defilippis, J.P., Berthelot, P., Aubert, G., 2013. Outbreak of multidrug-resistant Klebsiella pneumoniae carrying qnrB1 and blaCTX-M15 in a French intensive care unit. Annals of intensive care 3, 18. Fishbain, J.T., Sinyavskiy, O., Riederer, K., Hujer, A.M., Bonomo, R.A., 2012. Detection of extended-spectrum beta-lactamase and Klebsiella pneumoniae Carbapenemase genes directly from blood cultures by use of a nucleic acid microarray. J Clin Microbiol 50, 2901-2904. Freeman, J.T., McBride, S.J., Nisbet, M.S., Gamble, G.D., Williamson, D.A., Taylor, S.L., Holland, D.J., 2012. Bloodstream infection with extended-spectrum betalactamase-producing Enterobacteriaceae at a tertiary care hospital in New Zealand: risk factors and outcomes. Int J Infect Dis 16, e371-374. Fujita, S., Yosizaki, K., Ogushi, T., Uechi, K., Takemori, Y., Senda, Y., 2011. Rapid identification of gram-negative bacteria with and without CTX-M extended-spectrum beta-lactamase from positive blood culture bottles by PCR followed by microchip gel electrophoresis. J Clin Microbiol 49, 1483-1488. Garrec, H., Drieux-Rouzet, L., Golmard, J.L., Jarlier, V., Robert, J., 2011. Comparison of nine phenotypic methods for detection of extended-spectrum betalactamase production by Enterobacteriaceae. J Clin Microbiol 49, 1048-1057. Gastmeier, P., Schwab, F., Meyer, E., Geffers, C., 2012. [Excess mortality and prolongation of stay due to bloodstream infections caused by multiresistant pathogens in Germany]. Dtsch Med Wochenschr 137, 1689-1692. Gazin, M., Paasch, F., Goossens, H., Malhotra-Kumar, S., 2012. Current trends in culture-based and molecular detection of extended-spectrum-beta-lactamaseharboring and carbapenem-resistant Enterobacteriaceae. Journal of clinical microbiology 50, 1140-1146. Gerasimenko, D.V., Avdienko, I.D., Bannikova, G.E., Zueva, O., Varlamov, V.P., 2004. [Antibacterial effects of water-soluble low-molecular-weight chitosans on different microorganisms]. Prikl Biokhim Mikrobiol 40, 301-306. Hyle, E.P., Lipworth, A.D., Zaoutis, T.E., Nachamkin, I., Bilker, W.B., Lautenbach, E., 2005. Impact of inadequate initial antimicrobial therapy on mortality in infections due to extended-spectrum beta-lactamase-producing enterobacteriaceae: variability by site of infection. Arch Intern Med 165, 1375-1380.

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Kellogg, J.A., Manzella, J.P., McConville, J.H., 1984. Clinical laboratory comparison of the 10-ml isolator blood culture system with BACTEC radiometric blood culture media. Journal of clinical microbiology 20, 618-623. Klouche, M., Schroder, U., 2008. Rapid methods for diagnosis of bloodstream infections. Clin Chem Lab Med 46, 888-908. Lascols, C., Hackel, M., Hujer, A.M., Marshall, S.H., Bouchillon, S.K., Hoban, D.J., Hawser, S.P., Badal, R.E., Bonomo, R.A., 2012. Using nucleic acid microarrays to perform molecular epidemiology and detect novel beta-lactamases: a snapshot of extended-spectrum beta-lactamases throughout the world. J Clin Microbiol 50, 16321639. Leclercq, R., Canton, R., Brown, D.F., Giske, C.G., Heisig, P., MacGowan, A.P., Mouton, J.W., Nordmann, P., Rodloff, A.C., Rossolini, G.M., Soussy, C.J., Steinbakk, M., Winstanley, T.G., Kahlmeter, G., 2013. EUCAST expert rules in antimicrobial susceptibility testing. Clin Microbiol Infect 19, 141-160. Machado, E., Coque, T.M., Canton, R., Sousa, J.C., Peixe, L., 2013. Commensal Enterobacteriaceae as reservoirs of extended-spectrum beta-lactamases, integrons, and sul genes in Portugal. Front Microbiol 4, 80. Marra, A.R., Wey, S.B., Castelo, A., Gales, A.C., Cal, R.G., Filho, J.R., Edmond, M.B., Pereira, C.A., 2006. Nosocomial bloodstream infections caused by Klebsiella pneumoniae: impact of extended-spectrum beta-lactamase (ESBL) production on clinical outcome in a hospital with high ESBL prevalence. BMC Infect Dis 6, 24. Muro, S., Garza-Gonzalez, E., Camacho-Ortiz, A., Gonzalez, G.M., Llaca-Diaz, J.M., Bosques, F., Rositas, F., 2012. Risk factors associated with extended-spectrum betalactamase-producing Enterobacteriaceae nosocomial bloodstream infections in a tertiary care hospital: a clinical and molecular analysis. Chemotherapy 58, 217-224. Naas, T., Cuzon, G., Truong, H., Bernabeu, S., Nordmann, P., 2010. Evaluation of a DNA microarray, the check-points ESBL/KPC array, for rapid detection of TEM, SHV, and CTX-M extended-spectrum beta-lactamases and KPC carbapenemases. Antimicrob Agents Chemother 54, 3086-3092. Nordmann, P., Naas, T., Poirel, L., 2011. Global spread of Carbapenemaseproducing Enterobacteriaceae. Emerg Infect Dis 17, 1791-1798. Oliver, A., Weigel, L.M., Rasheed, J.K., McGowan Jr, J.E., Jr., Raney, P., Tenover, F.C., 2002. Mechanisms of decreased susceptibility to cefpodoxime in Escherichia coli. Antimicrob Agents Chemother 46, 3829-3836. Overdevest, I., Willemsen, I., Rijnsburger, M., Eustace, A., Xu, L., Hawkey, P., Heck, M., Savelkoul, P., Vandenbroucke-Grauls, C., van der Zwaluw, K., Huijsdens, X., Kluytmans, J., 2011. Extended-spectrum beta-lactamase genes of Escherichia coli in chicken meat and humans, The Netherlands. Emerg Infect Dis 17, 1216-1222. Paolucci, M., Landini, M.P., Sambri, V., 2010. Conventional and molecular techniques for the early diagnosis of bacteraemia. Int J Antimicrob Agents 36 Suppl 2, S6-16. Pletz, M.W., Bloos, F., Burkhardt, O., Brunkhorst, F.M., Bode-Boger, S.M., MartensLobenhoffer, J., Greer, M.W., Stass, H., Welte, T., 2010. Pharmacokinetics of moxifloxacin in patients with severe sepsis or septic shock. Intensive Care Med 36, 979-983. Prezeau, N., Silvy, M., Gabert, J., Picard, C., 2006. Assessment of a new RNA stabilizing reagent (Tempus Blood RNA) for minimal residual disease in oncohematology using the EAC protocol. Leuk Res 30, 569-574. Reddy, P., Malczynski, M., Obias, A., Reiner, S., Jin, N., Huang, J., Noskin, G.A., Zembower, T., 2007. Screening for extended-spectrum beta-lactamase-producing

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Enterobacteriaceae among high-risk patients and rates of subsequent bacteremia. Clin Infect Dis 45, 846-852. Reinhart, K., Brunkhorst, F.M., Bone, H.G., Bardutzky, J., Dempfle, C.E., Forst, H., Gastmeier, P., Gerlach, H., Grundling, M., John, S., Kern, W., Kreymann, G., Kruger, W., Kujath, P., Marggraf, G., Martin, J., Mayer, K., Meier-Hellmann, A., Oppert, M., Putensen, C., Quintel, M., Ragaller, M., Rossaint, R., Seifert, H., Spies, C., Stuber, F., Weiler, N., Weimann, A., Werdan, K., Welte, T., 2010. Prevention, diagnosis, therapy and follow-up care of sepsis: 1st revision of S-2k guidelines of the German Sepsis Society (Deutsche Sepsis-Gesellschaft e.V. (DSG)) and the German Interdisciplinary Association of Intensive Care and Emergency Medicine (Deutsche Interdisziplinare Vereinigung fur Intensiv- und Notfallmedizin (DIVI)). Ger Med Sci 8, Doc14. Rettedal, S., Lohr, I.H., Natas, O., Giske, C.G., Sundsfjord, A., Oymar, K., 2012. First outbreak of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in a Norwegian neonatal intensive care unit; associated with contaminated breast milk and resolved by strict cohorting. Apmis 120, 612-621. Rodriguez-Bano, J., Navarro, M.D., Romero, L., Muniain, M.A., de Cueto, M., Rios, M.J., Hernandez, J.R., Pascual, A., 2006. Bacteremia due to extended-spectrum beta -lactamase-producing Escherichia coli in the CTX-M era: a new clinical challenge. Clin Infect Dis 43, 1407-1414. Rolfs, A., Schuller, I., Finckh, U., Weber-Rolfs, I., 1991. PCR principles and reaction components., in: Rolfs, A., Schumacher, H.C., Marx, P. (Eds.), PCR topics. Usage of PCR in genetic and infectious diseases. Springer-Verlag, New York, N.Y., pp. 1-21. Schoevaerdts, D., Bogaerts, P., Grimmelprez, A., de Saint-Hubert, M., Delaere, B., Jamart, J., Swine, C., Glupczynski, Y., 2011. Clinical profiles of patients colonized or infected with extended-spectrum beta-lactamase producing Enterobacteriaceae isolates: a 20 month retrospective study at a Belgian University Hospital. BMC Infect Dis 11, 12. Schultz, S.C., Dalbadie-McFarland, G., Neitzel, J.J., Richards, J.H., 1987. Stability of wild-type and mutant RTEM-1 beta-lactamases: effect of the disulfide bond. Proteins 2, 290-297. Son, J.S., Song, J.H., Ko, K.S., Yeom, J.S., Ki, H.K., Kim, S.W., Chang, H.H., Ryu, S.Y., Kim, Y.S., Jung, S.I., Shin, S.Y., Oh, H.B., Lee, Y.S., Chung, D.R., Lee, N.Y., Peck, K.R., 2010. Bloodstream infections and clinical significance of healthcareassociated bacteremia: a multicenter surveillance study in Korean hospitals. J Korean Med Sci 25, 992-998. Stein, C., Makarewicz, O., Pfeifer, Y., Brandt, C., Ramos, J.C., Klinger, M., Pletz, M.W., 2013. Direct RNA-based detection and differentiation of CTX-M-type extendedspectrum beta-lactamases (ESBL). PLoS One 8, e80079. Tumbarello, M., Sanguinetti, M., Montuori, E., Trecarichi, E.M., Posteraro, B., Fiori, B., Citton, R., D'Inzeo, T., Fadda, G., Cauda, R., Spanu, T., 2007. Predictors of mortality in patients with bloodstream infections caused by extended-spectrum-betalactamase-producing Enterobacteriaceae: importance of inadequate initial antimicrobial treatment. Antimicrob Agents Chemother 51, 1987-1994. Tumbarello, M., Spanu, T., Sanguinetti, M., Citton, R., Montuori, E., Leone, F., Fadda, G., Cauda, R., 2006. Bloodstream infections caused by extended-spectrumbeta-lactamase-producing Klebsiella pneumoniae: risk factors, molecular epidemiology, and clinical outcome. Antimicrob Agents Chemother 50, 498-504. Valentin, L., Sharp, H., Hille, K., Seibt, U., Fischer, J., Pfeifer, Y., Michael, G.B., Nickel, S., Schmiedel, J., Falgenhauer, L., Friese, A., Bauerfeind, R., Roesler, U., Imirzalioglu, C., Chakraborty, T., Helmuth, R., Valenza, G., Werner, G., Schwarz, S.,

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Guerra, B., Appel, B., Kreienbrock, L., Kasbohrer, A., 2014. Subgrouping of ESBLproducing Escherichia coli from animal and human sources: An approach to quantify the distribution of ESBL types between different reservoirs. Int J Med Microbiol. Vehreschild, M.J.G., Peterson, L., Schubert, S., Vogel, W., Peter, S., Schafhausen, P., Rohde, H., von Lilienfeld-Toal, M., Bekeredjian-Ding, I., Cornely, O.A., Seifert, H., 2014. A multicenter cohort study on colonization and infection with extendedspectrum beta-lactamase producing Enterobacteriaceae (ESBL-E) in high-risk patients with hematological malignancies. Oncol Res Treat 37, 121-122. Yagupsky, P., Nolte, F.S., 1990. Quantitative aspects of septicemia. Clin Microbiol Rev 3, 269-279.

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Tables and Figures

us

619 620

Table 1. Bacterial strains used in this study.

622

Table 2. Determination of detection limits using TEMPUSTM technology for RNA isolation directly from EDTA blood

623

Table 3. Comparison between TempusTM RNA Tubes isolation and Trizol/RNeasy method for positive blood cultures.

624

Table 4. Comparison of methods for ESBL detection directly from EDTA blood or from positive blood cultures.

M an

621

ed

625

Fig. 1. Calibration of RT and PCR reactions. A) Calibration of the qPCR by pCTXM15 DNA: Threshold 0.1, efficiency=0.92, R2=

627

0.99908. B) Agarose gel electrophoresis to analyse the specificity of standards and RT-qPCR reactions, lane 1: 100 bp DNA ladder,

628

lanes 2 to 6: CTX-M-15 standard DNA (1 pg, 0.1 pg, 0.01 pg, 1 fg, and 0.1 fg), lanes 7 to 10: CTX-M-1 (RKI 26/08) with decreasing

629

primer concentrations (1.92 M, 0.96 M, 0.48 M, 0.24 M). C) Amplification of the cDNA derived from spiked blood samples with

630

different primer concentrations (as indicated in the figure) at RT. D) Melting curves of PCR products amplified in (C) (Tm of the

631

specific product derived from the pCTX-M was 91 °C). The black curves within picture C and D replace the pCTXM15 standard

632

DNA in a concentration of 1 pg.

Ac

633

ce pt

626

634

Fig. 2. Detection limits of the RNA-based RT-qPCR-pyrosequencing assay. A) Melting curves depending on bacterial load in the

635

EDTA blood sample. The specific 268 bp fragment melted at 92 °C. B) 2 % agarose gel of the PCR products, lane 1: 100 bp DNA 22 Page 22 of 30

ip t cr

ladder (Fermentas), lane 2: CTX-M-15 standard DNA 1 pg, lanes 3 to 6: CTX-M-9 (RKI 428/08) (104 CFU/mL, 103 CFU/mL, 102

637

CFU/mL, 101 CFU/mL). C) Corresponding real-time amplification curves. D) Pyrogram for decreasing template concentrations.

us

636

M an

638

Fig. 3. CTX-M specificity of the RT-qPCR by degenerate primers. Lane 1: 100 bp DNA ladder (Fermentas), lane 2: CTX-M-15

640

standard DNA 1 pg, lanes 3 to 6: CTX-M-1 (RKI 26/08) (104 CFU/mL, 103 CFU/mL, 102 CFU/mL, 101 CFU/mL), lanes 7 to 10: K.

641

pneumoniae SHV-4 (RKI 45/08) (104 CFU/mL, 103 CFU/mL, 102 CFU/mL, 101 CFU/mL), lane 11: pure blood, lanes 12 to 15: E. coli

642

K12J53 (104 CFU/mL, 103 CFU/mL, 102 CFU/mL, 101 CFU/mL), lane 16: no-template control (NTC).

ed

639

Ac

ce pt

643

23 Page 23 of 30

cr

ip t Strain

us

Table 1. Bacterial strains used in this study. CTX-M variant

Other beta-lactamases

RKI 26/08*

CTX-M-1

/

RKI 25/08*

CTX-M-15

/

RKI 427/08*

CTX-M-27

TEM-1

RKI 443/08*

CTX-M-14

/

RKI 128/08*

CTX-M-2

/

RKI 2/10*

CTX-M-15

TEM-1, OXA-1, OXA-2, NDM-1

K12J53**

/

/

RKI 209/10*

CTX-M-8

K. pneumoniae

ed

S. enterica

M an

E. coli

/

RKI 45/08*

/

RKI 93/07*

CTX-M-15

RKI 346/12*

CTX-M-15

TEM-1, SHV-1, OXA-1, OXA-9, OXA-48

CTX-M-25

/

RKI 428/08*

CTX-M-9

/

RKI 1/10*

CTX-M-15

TEM-1, OXA-1, OXA-48, AmpC

E. cloacae

TEM-1, SHV-28, OXA-1, OXA-9, NDM-1, CMY-like

ce pt

RKI VW823*

Ac

Enterob.

SHV-4

*Reference: Stein et al. 2013; **Reference: DSMZ = Leibniz Institute DSMZ-German Collection of Microogranisms an Cell Cultures, Braunschweig (Germany)

24 Page 24 of 30

ip t cr

CTX-M

Strain

RNA

variant c

°C

CFU/mL  SD

30

91

20  12

7

33

89

351  331

2.13

8.5

32

92

90  34

RIN

CT value

2.05

8

2.09

RKI 25/08

CTX-M-15

45.7  10.3

RKI 427/08

CTX-M-27

55.7  4.3

RKI 443/08

CTX-M-14

60.7  36.7

2.04

8.2

27

92

23  12

RKI 128/08

CTX-M-2

48.6  12.3

2.06

8

31

90

447  351

CTX-M-15

43.1  29.3

2.11

n.d.

30

89

212  275

/

58.1  9.8

2.07

7.6

/

/

/

CTX-M-8

57.1  6.6

2.06

8.7

24

92

114  109

RKI 45/08

/

33.9  23.8

1.81

7.3

38

unspec.

/

RKI 93/07

CTX-M-15

50.4  7.1

2.05

n.d.

33

89

1065  92

RKI 346/12

CTX-M-15

57.8  13.6

1.99

n.d.

36

89

5500  2970

Ac

RKI 209/10

ed

46  23

K12J53

K. pneumoniae

LOD

ratio260/280

CTX-M-1

RKI 2/10

S. enterica

colony count

RKI 26/08

ce pt

E. coli

PCR

Tm

M an

ng/L ± SD

us

Table 2. Determination of detection limits using TEMPUSTM technology for RNA isolation directly from EDTA blood

Enterob.

RKI VW823

CTX-M-25

48.6  6.4

2.12

n.d.

33

91

333  247

E. cloacae

RKI 428/08

CTX-M-9

38.7  16.7

1.96

8.5

32

92

573  634

CTX-M-15

55  12.4

2.07

n.d.

33

89

630  468

RKI 1/10

LOD = Limit of detection; n.d. = not detected; unspec. = unspecific product; SD = standard deviation; RIN = RNA integrity number

25 Page 25 of 30

ip t cr

TEMPUS RNA Tubes volume BC in mL

c [ng/µL]

ratio260/280

Trizol/ RNeasy

CT value

c [ng/µL]

ratio260/280

CT value

M an

Sample

us

Table 3. Comparison between TempusTM RNA Tubes isolation and Trizol/RNeasy method for positive blood cultures.

0.5

40.5

1.16

n.d.

158.3

1.97

16.7

1

135.9

1.40

n.d.

280.9

2.01

16.68

2

267.0

0.99

n.d.

449.3

2.11

17.57

Ac

ce pt

ed

BC = blood culture

26 Page 26 of 30

ip t cr

Time-to-result Template

LOD

direct

BC

RT-qPCR-

mRNA

20-447

6-7

5

a

pyrosequencing

Discriminative performance

Reference

M an

Method

us

Table 4. Comparison of methods for ESBL detection directly from EDTA blood or from positive blood cultures.

CTX-M on subgroup

this work

level, extensible due to custom primer

Check-Points

DNA

8

10

/

~4-6*

Commercial kit, probes predefined

(Fishbain et al., 2012)

ed

without subgroup discrimination

Microchip gel

DNA

5

10 -10

9b

/

electrophoresis Enzymatic activity HMRZ-86

Possible, due to

(Fujita et al., 2011)

custom primer

Enzymatic activity

ce pt

ESBL NDP

1.5

10 -10

7

9b

/

ca. 2

ESBL

(Nordmann et al., 2012)

7

9b

/

2.5-4

ESBL

(Jain et al., 2007)

10 -10

The time-to-positivity of the blood culture (BC) is not included in the time-to-result and might differ depending on the species and bacterial load of the specimen;

b

Estimated value, since no declaration was done in the original work; * Time to result depends on the DNA-preparation method;

LOD = limit of detection

Ac

a

27 Page 27 of 30

Ac

ce

pt

ed

M

an

us

cr

i

Figure 1

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Ac

ce

pt

ed

M

an

us

cr

i

Figure 2

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Ac

ce

pt

ed

M

an

us

cr

i

Figure 3

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