Optimal detection of extended-spectrum β-lactamase producers, carbapenemase producers, polymyxin-resistant Enterobacterales, and vancomycin-resistant enterococci from stools

Journal Pre-proof Optimal detection of extended-spectrum ß-lactamase producers, carbapenemase producers, polymyxin-resistant Enterobacterales, and vancomycin-resistant enterococci from stools

Mustafa Sadek, Laurent Poirel, Patrice Nordmann PII:

S0732-8893(19)30929-0

DOI:

https://doi.org/10.1016/j.diagmicrobio.2019.114919

Reference:

DMB 114919

To appear in:

Diagnostic Microbiology & Infectious Disease

Received date:

12 September 2019

Revised date:

8 October 2019

Accepted date:

16 October 2019

Please cite this article as: M. Sadek, L. Poirel and P. Nordmann, Optimal detection of extended-spectrum ß-lactamase producers, carbapenemase producers, polymyxin-resistant Enterobacterales, and vancomycin-resistant enterococci from stools, Diagnostic Microbiology & Infectious Disease(2019), https://doi.org/10.1016/ j.diagmicrobio.2019.114919

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© 2019 Published by Elsevier.

Journal Pre-proof

Optimal detection of extended-spectrum ß-lactamase producers, carbapenemase producers, polymyxin-resistant Enterobacterales, and vancomycin-resistant enterococci from stools Mustafa Sadek,1 Laurent Poirel,1,2,3* and Patrice Nordmann1,2,3,4 1

Medical and Molecular Microbiology Unit, Faculty of Science and Medicine, University of Fribourg, Fribourg, Switzerland; 2INSERM European Unit (IAME, France), University of Fribourg, Fribourg; 3Swiss National

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Reference Center for Emerging Antibiotic Resistance (NARA), University of Fribourg, Fribourg; and 4Institute

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for Microbiology, University of Lausanne and University Hospital Centre, Lausanne, Switzerland

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Running title: Optimal detection of MDR bacteria

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*Corresponding author. Mailing address: Medical and Molecular Microbiology Unit, Department of Medicine,
Faculty of Science,
University of Fribourg, Chemin du Musée 18, 
CH-1700 Fribourg, Switzerland. Phone: 41-26-300-9581. E-mail: [email protected]

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ABSTRACT

This study compared the impact of different approaches, namely non-enrichment, non-selective

producing Enterobacterales

(ESBL-E), carbapenemase-producing

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spectrum ß-lactamase

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enrichment, and selective (antibiotic-containing) enrichment steps, for detecting extended-

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Enterobacterales (CPE), polymyxin-resistant Enterobacterales (PMR-E), and vancomycin-

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resistant enterococci (VRE) from spiked stools. The use of a non-selective 18-h enrichment broth

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culture significantly improved the recovery rate of all types of resistant bacteria after their

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plating onto selective media. In addition, the detection of ESBL-E, CPE, PMR-E and VRE was

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further improved when using an enrichment step using antibiotic-supplemented broths,

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respectively supplemented with cefotaxime (0.1 µg/ml), ertapenem (0.1 µg/ml), colistin (0.5 µg/ml),

and vancomycin (1 µg/ml). Therefore, we showed here that a screening strategy based on a

selective broth enrichment step significantly contributes to an increased rate of detection of

multidrug-resistant bacteria, that may be crucial in term of improvement of infection control.

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INTRODUCTION

Infections caused by multidrug-resistant (MDR) bacteria are associated with increased

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morbidity and mortality due to the limited treatment options (Cerceo et al., 2016). Therefore, the

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early detection of patients colonized with MDR bacteria is becoming fundamental for the prompt

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implementation of optimal infection control strategies (Fang et al., 2012). In many institutions and

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hospitals, screening of patients colonized by MDR bacteria is performed as part of the infection

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control measures. However, limited data exist regarding the optimal screening strategy for MDR

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bacteria. For vancomycin-resistant enterococci (VRE), use of a selective culture medium containing

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vancomycin is known to improve their detection from stool samples (Cuzon et al., 2008; Nordmann

et al., 2012). Although it is clear that the use of chromogenic and antibiotic-containing media is

useful for improving the identification and recovery of MDR bacteria from stool samples (Cerceo et

al., 2016; Fang et al., 2012; Cuzon et al., 2008; Nordmann et al., 2012; Poirel et al., 2017), the use of

an enrichment step for their detection is not a common practice and the added-value of this approach

remains unclear.

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Journal Pre-proof Extended-spectrum ß-lactamase producing Enterobacterales (ESBL-E) are increasingly

reported worldwide. Gastrointestinal colonization with ESBL-E is associated with an increased risk

of bacteremia (Cornejo-Juárez et al., 2016). Detection of carbapenemase-producing Enterobacterales

(CPE) is also challenging since the level of resistance to carbapenems may vary significantly

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depending on the nature of the carbapenemase itself, on the level of expression of the

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carbapenemase gene, and also on the resistance background of the strain (variability of the outer

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membrane permeability). Finally, polymyxins are now considered as last-resort antibiotics against

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MDR Enterobacterales in regions endemic for CPE, such as Italy and Greece (Poirel et al., 2017).

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Concomitantly, resistance to polymyxins in Gram-negative bacteria is now increasingly reported

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(Poirel et al., 2017). Therefore, rapid and early identification of patients colonized with polymyxin-

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resistant Enterobacterales (PMR-E) is also important.

The aim of our study was to determine whether an enrichment step, with and without

antibiotics, prior to inoculation on selective agar plates increased the sensitivity of MDR screening.

Our evaluation of different screening strategies focused on four clinically-relevant MDR bacteria, i.e.

VRE, ESBL-E, CPE, and PMR-E.

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MATERIAL AND METHODS

Bacterial strains. This study was carried out with a well-characterized collection (16S rRNA

sequencing) of multidrug bacterial isolates from the Medical and Molecular Microbiology Unit,

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Faculty of Science and Medicine, University of Fribourg, Switzerland (Tables 1 to 4). Isolates were

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obtained from various clinical sources and from various countries and continents. Resistance

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mechanisms to the selected antibiotics were previously determined at the molecular level, using

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PCR approaches.

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Susceptibility testing. MIC values of antibiotics except for colistin were determined using

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ETEST strips (bioMérieux, La Balme-les-Grottes, France) on Mueller-Hinton agar plates at 37°C,

and the results were interpreted according to the latest EUCAST breakpoints (www.eucast.org).

Broth microdilution was performed for determination of MICs of colistin in cation-adjusted

Mueller-Hinton broth (Bio-Rad, Marnes-La-Coquette, France) and results were interpreted

according to the EUCAST/CLSI joint guidelines (www.eucast.org) (resistance, >2 µg/ml for

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Journal Pre-proof Enterobacterales). E. coli ATCC 25922 and E. faecalis ATCC 29212 were used as quality control

strains for susceptibility testing of Enterobacterales and enterococci, respectively.

Spiking experiments. Serial 10-fold dilutions were made in 0.85% saline solution with

bacterial suspensions of MDR bacteria starting with an optical density at a 0.5 McFarland standard

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(inoculum of ~ 1.5×108 CFU/ml). To quantify the viable bacteria in each dilution step, tryptic soy

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(TS) agar plates were inoculated with 100 μl of each suspension and incubated overnight at 37°C.

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The number of colonies that grew was counted the following day. Then, spiked fecal samples were

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made by adding 100 μl of serial-fold bacterial dilutions to 900 μl of stool suspension. Stool

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suspensions were obtained by suspending 6 g of pooled feces from healthy volunteers in 60 ml of

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distilled water. Healthy volontees were previously screened to be negative for the VRE, ESBL-E,

controls.

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CPE, and PMR-E. Stool suspensions without addition of bacterial strains were used as negative

For all the enrichment strategies, two procedures were compared, namely a 18 h-enrichment

step in TS broth without antibiotic supplementation, and an enrichment step using TS broth

containing a given concentration of antibiotic. Several antibiotic concentrations were tested,

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Journal Pre-proof remaining below the breakpoints of resistance of each tested antibiotic, according to EUCAST

(www.eucast.org) and CLSI (https://clsi.org/2018/) guidelines.

Aliquots of 100 μl of the stool suspension spiked with ESBL-E were inoculated either a)

directly onto chromID ESBL agar (bioMérieux), or b) into 5 ml of non-selective TS broth, or c) into

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5 ml of TS broth with cefotaxime (Acros Organics, Chemie Brunschwig AG, Switzerland) at 0.1 or

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0.5 µg/ml. Aliquot of 100 μl of the fecal suspension without the addition of ESBL-E directly spread

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onto chromID ESBL agar (bioMérieux) was used as a negative control. Aliquot of 100 μl of the

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fecal suspension without the addition of ESBL-E inoculated into 5 ml of TSB was used as a negative

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control. Aliquot of 100 μl of the fecal suspension without the addition of ESBL-E inoculated into 5

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ml of TS broth with cefotaxime was used as negative control. Agar plates and enrichment broths

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were incubated overnight at 37°C. The following day, aliquots of 100 μl of non-selective TS broth

and selective TS broth were inoculated onto selective chromID ESBL agar.

A similar procedure was followed for screening CPE and aliquots of 100 μl of spiked stool

suspension were inoculated either: i) directly on SuperCarba agar (CHROMAgar Ltd, Paris, France)

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Journal Pre-proof (Nordmann et al., 2012)), ii) in 5 ml of non-selective TS broth, or iii) in 5 ml of TS broth with

ertapenem (0.1 and 0.5 µg/ml) (Acros Organics).

For PMR-E, aliquots of 100 μl of spiked stool suspensions were inoculated either: i) directly

on SuperPolymyxin agar (ELITech Microbiology, Puteaux, France) (Nordmann et al., 2016; Jayol et

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al., 2018), ii) in 5 ml of non-selective TS broth, or iii) in 5 ml of TS broth with colistin (0.5 and 2

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µg/ml) (Sigma Aldrich Chemie GmbH, Buchs, Switzerland).

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For VRE, aliquots of 100 μl of the spiked stool suspension were inoculated either i) directly

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on ChromID VRE agar (bioMérieux), ii) in 5 ml of non-selective TS broth, or iii) in 5 ml of TSB

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with vancomycin (1 and 3 µg/ml) (Duchefa Biochemie, Haarlem, The Netherlands).

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Colony forming units were counted after 18 h of incubation at 37°C. All experiments were

performed in duplicate. Data were statistically analyzed with SPSS using Wilcoxon signed rank test.

RESULTS AND DISCUSSION

The impact of direct plating, non-selective broth enrichment, and selective broth enrichment

for the detection of ESBL-E, CPE, PMR-E, and VRE revealed remarkable differences. Following

enrichment in TS broth, many more ESBL-E were recovered when compared to direct plating onto 8

Journal Pre-proof agar plates (p >0.05) (Table 1). Furthermore, the use of selective enrichment with cefotaxime -

regardless of the concentration - further improved the detection of ESBL-E compared to nonselective enrichment step (p >0.05). However, no significant difference was observed when

comparing enrichment steps using any of the tested concentrations of cefotaxime (0.1 versus 0.5

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µg/ml) (data not shown). Consequently, data presented in Table 1 refer to a single concentration of

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antibiotic for each enrichment procedure, but same results would apply for the second concentration

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tested.

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It was previously shown that an overnight pre-enrichment step in TS broth improves the

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detection of ESBL-E and allows an earlier recognition of patients that are ESBL-E carriers (Murk et

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al., 2010). Likewise, Jazmati et al. (2017) showed the added value of an enrichment step for the

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screening of broad-spectrum cephalosporins-resistant Enterobacterales. In another study, an

enrichment step was shown to increase the sensitivity of detection of ESBL-E from 75% to 97% in

stool samples (Blane et al., 2016).

Detection of CPE was also improved by the use of a similar enrichment step when compared to direct plating on selective medium (p >0.05) (Table 2). Moreover, the use of selective broth

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Journal Pre-proof enrichment containing ertapenem (0.1 or 0.5 µg/ml) further improved the detection of CPE

compared to non-selective broth (p>0.05) regardless of the ertapenem MIC (Table 2). Two new

methods of CPE detection from stool samples have been recently proposed: an automated broth

medium method (Arena et al., 2018) and a temocillin-containing broth to improve detection of a

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specific group of CPE, namely the OXA-48 producers (Cieselinczuk et al., 2018). The use of

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temocillin as an enrichment factor is related to the high resistance level to this antibiotic conferred

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by OXA-48-like. Based on our results, specific enrichment of OXA-48-like producers with

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temocillin would not be necessary since all tested strains including OXA-48 and OXA-181

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producers were well detected after the ertapenem-based enrichment step. Similarly, Glaser et al.

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(2015) report that a growth step in a meropenem-containing broth may enhance the selection of CPE.

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On the opposite, by using clinical rectal swabs, Simner et al. (2016) did not evidence higher CPE

recovery by using an ertapenem-containing broth.

For detection of carbapenemase-producing Klebsiella spp. and E. coli, the Centers for Disease

Control and Prevention (CDC) recommendation includes an overnight selective broth enrichment culture of the rectal swab in 5 ml of TS broth supplemented with a 10-μg ertapenem or 5-μg

meropenem disk, followed by a subculture onto MacConkey agar plates (CDC, 2009; Viau et al., 10

Journal Pre-proof 2016). The addition of ertapenem at a given concentration that we propose is following the same

principle of selection. However our strategy is not only based on screening of carbapenem resistant

Enterobacterales but concomitantly of other MDR bacteria.

A significant benefit of an enrichment step was also observed for PMR-E detection, regardless

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of the type of polymyxin-resistance mechanism (Table 3). A higher detection yield was achieved by

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using a selective enrichment TS broth containing colistin (at 0.5 or 2 µg/ml) prior to plating on

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selective agar (Table 3). To our knowledge, this is the first study evaluating the impact of direct

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plating, broth enrichment, and selective broth enrichment for an optimal recovery of PMR-E. In a

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significant.

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context of growing polymyxin resistance worldwide, this detection strategy may be clinically

The same increase in sensitivity of the screening test was observed for VRE when using a

non-selective enrichment step (p>0.05) (Table 4). However, a selective enrichment step with

addition of vancomycin resulted in a further increase of recovery of VRE compared to the

nonselective enrichment, although these differences were not found statistically significant (p<0.05).

This result is consistent with previous studies indicating that a pre-culture step in broth with or

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Journal Pre-proof without vancomycin (15 µg/ml) enhances the recovery of VRE from feces (Palladino et al., 2003;

Novicki et al., 2004).

CONCLUSION

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The use of selective enrichment broths prior to direct inoculation onto selective culture media

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resulted in significant increases of recovery of the MDR bacteria tested. Although such enrichment

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steps require an additional overnight culture, consequently delaying turnaround time, the present

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data demonstrate the increased sensitivity of an enrichment procedure. In clinical practice, use of an

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enrichment procedure may be particularly important in outbreak situations to decrease false negative

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results. This enrichment procedure shall be performed in addition to direct plating of stools on

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screening culture media. From a practical point of view, antibiotic concentrations may be prepared

in advance as a set and kept at -20°C to be used on demand.

Noteworthy, our study showed that the detection rate of MDR bacteria may be improved

thanks to a pre-enrichment step, especially in the case of patients with low inoculum in their stools.

Even though these patients may represent a low risk of dissemination and generate rare secondary cases (D’Agata et al., 2002), our clinical experience indicates us that those patients with low

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Journal Pre-proof inoculum shall be identified as early as possible to prevent an hidden dissemination of MDR

bacteria.

Noticeably, we did not identify any difference of MDR recovery when using two different

antibiotic concentrations in the selective enrichment strategies, showing that the presence rather than

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the absolute concentration of selective antibiotics is indeed crucial. We retained antibiotic

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concentrations 0.1 µg/ml of cefotaxime, 0.1 µg/ml of ertapenem, 0.5 µg/ml of colistin, and 1 µg/ml

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of vancomycin for optimal detection of ESBL-E, CPE, PMR-E, and VRE, respectively. Those

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antibiotic concentrations were defined for the first time by precisely quantifying the bacterial

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inocula in stools, the antibiotic concentrations and the resistant bacteria type. To our knowledge, this

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is the first study to show that an enrichment step containing a defined concentration of colistin

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improves the detection of PMR-E.

We believe that this screening protocol should be repeated with a larger and more diverse

collection of isolates including the producers of other types of carbapenemases, particularly those

conferring unusual and difficult-to-detect susceptibility profiles, such as OXA-244 known to be

difficult to detect when relying only on phenotypic approaches (Potron et al., 2016; Hoyos-Mallecot

et al., 2017). Also, there is need for additional experiments to test other MDR organisms and 13

Journal Pre-proof different marketed screening agar plates. Furthermore, testing other specimen such as skin or throat

samples through the same approach would significantly add to the knowledge in the field.

A limitation of our study was the use of spiked stools rather than stools recovered from

colonized patients. Therefore, clinical evaluation of the proposed procedures may be interesting as

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well as the evaluation of a similar screening strategy for identification of colonization by MDR

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Acinetobacter baumannii and Pseudomonas aeruginosa. Finally, our proposed MDR screening

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technique may be a relatively low cost, easy to implement strategy to perform extended surveys

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either in humans, animals or environmental settings.

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ACKNOWLEDGMENTS

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This work was financed by the University of Fribourg, Switzerland. It has also been funded by the

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Swiss National Science Foundation (project FNS-407240_177382). We warmly thank L.S. Munoz-

Price for critical reading of our manuscript.

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3- Centers for Disease Control and Prevention. 2009. Laboratory protocol for detection of carbapenem-resistant or carbapenemase-producing, Klebsiella spp. and E. coli from rectal

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4- Cerceo E, Deitelzweig SB, Sherman BM, Amin AN. Multidrug-resistant gram-negative

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bacterial infections in the hospital setting: overview, implications for clinical practice, and emerging treatment options. Microb Drug Resist 2016;22:412-31. 5- Cieselinczuk H, Phee LM, Dolphin H, Wilks M, Cherian BP, Wareham DW. Optimal detection of carbapenemase-producing Enterobacteriaceae from rectal samples: a role for enrichment. J Hosp Infect 2018;98:270-4. 6- Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; 28th informational supplement (M100–S28). Wayne (PA): The Institute; 2018.

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11- Glaser L, Andreacchio K, Lyons M, Alby K. Improved surveillance for carbapenem resistant

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Enterobacteriaceae using chromogenic media with a broth enrichment. Diagn Microbiol Infect

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12- Hoyos-Mallecot Y, Naas T, Bonnin RA, Patino R, Glaser P, Fortineau N, et al. OXA-244producing Escherichia coli isolates, a challenge for clinical microbiology laboratories. Antimicrob Agents Chemother 2017;61:pii: e00818-17. 13- Jayol A, Poirel L, André C, Dubois V, Nordmann P. Detection of colistin-resistant Gramnegative rods by using the SuperPolymyxin medium. Diagn Microbiol Infect Dis 2018;92:95101. 14- Jazmati N, Jazmati T, Hamprecht A. Importance of pre-enrichment for detection of thirdgeneration cephalosporin-resistant Enterobacteriaceae (3GCREB) from rectal swabs. Eur J Clin Microbiol Infect Dis 2017;36:1847-51. 16

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Gram-negative isolates. J Clin Microbiol 2016;54:1395-9.

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Convenient selective differential broth for isolation of broth of vancomycin-resistant enterococcus from fecal material. J Clin Microbiol 2004;42:1637-40.

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19- Palladino S, Kay ID, Flexman JP, Boehm I, Costa AM, Lambert EJ, et al. Rapid detection of of

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vanA and vanB genes directly from clinical specimens and enrichment broths by real-time multiplex assay J Clin Microbiol 2003;41:2483-6.

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20- Poirel L, Jayol A, Nordmann P. Polymyxins: antibacterial activity, susceptibility testing, and

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resistance mechanisms encoded by plasmids or chromosomes. Clin Microbiol Rev 2017;30:557-96.

21- Potron A, Poirel L, Nordmann P. Characterization of OXA-244, a chromosomally-encoded OXA-48 like ß-lactamase from Escherichia coli. Int J Antimicrob Agents 2016;47:102-3. 22- Simner P, Martin I, Opene B, Tamma PD, Karoll KC, Milstone AM. Evaluation of multiple methods for detection of gastro-intestinal colonization of carbapenem-resistant organisms from rectal swabs. J Clin Microbiol 2016;54:1664-7.

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ESBL enzymes are boldened

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Table 1. Evaluation of direct plating on ChromID ESBL agar of spiked stools, with and without a 18 h enrichment step in TSB and TSB+cefotaxime (CTX) at 0.1 µg/ml for the detection of ESBL-E. Enrichment broth MIC of ChromID (CFU/ml) Strain Species β-Lactamase contenta cefotaxime ESBL agar TSB TSB + CTX (µg/ml) (CFU/ml) R889 K. pneumoniae 16 101 3.1×104 5.5×107 CTX-M-15 R1003 E. coli CTX-M-15+SHV-1 16 4×101 1.93×104 2.75×107 1 R1063 K. pneumoniae CTX-M-3 + TEM-1+ SHV-11 16 3×10 1.2× 106 1.2× 109 R1907 E coli CTX-M-3 + TEM-1 16 5×101 2.7×104 5.8×108 R985 E. coli CTX-M-9 + TEM-1 32 1×102 4.5×104 3×107 2 5 R1095 K. pneumoniae 8 1×10 8×10 1.77×109 SHV-5 R1097 K. pneumoniae 2 6×101 2.4×106 6.9×108 SHV-12 R1099 K. pneumoniae TEM-3 + SHV-28 4 9×101 3×105 1.21×109 1 6 R1101 K. pneumoniae TEM-52 + SHV-28 >32 4×10 2.2×10 8.2×108 R 301 K. pneumoniae SHV-11 + KPC-2 >32 1×102 2.8×106 3.1×108 1 5 R3092 K. pneumoniae CTX-M-55 + TEM-1 >32 3×10 1.5×10 4.5×108

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Table 2. Evaluation of direct plating on SUPERCARBA agar of spiked stools , with and without a 18 h enrichment step in TSB and in TSB+ ertapenem (ETP) at 0.1 µg/ml for the detection of CPE. Enrichment broth (CFU/ml) Strain Species β-Lactamase contenta MIC of SUPERCARBA TSB + ETP ertapenem agar TSB (µg/ml) (CFU/ml) R293 E. coli KPC- 2 + TEM-1 + OXA-1 >32 2.4×102 8×106 5×107 R300 K. KPC-2 + TEM-1 + OXA-9 >32 4.4×102 6×106 45×107 pneumoniae R24 K. 0.38 6×101 3.9×105 9×107 NDM-1 pneumoniae R61 E. coli 0.5 7×101 8×105 3×107 VIM-1 R68 K. IMP-1 + SHV-12 0.25 3×101 4.5×105 4×106 pneumoniae R83 K. 0.25 5×101 2.2×105 4×108 KPC-3 pneumoniae R721 E. coli OXA-48 + TEM-1 0.12 5×101 1×107 4.8×108 R753 K. OXA-48 + SHV-1 >32 6×101 4×106 7×107 pneumoniae R2815 E. coli 0.5 1.3×102 3×106 3×107 OXA-181 1 6 R2854 K. >32 8×10 1×10 3.2×108 OXA-181 pneumoniae a Carbapenemase names are boldened

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Table 3. Evaluation of direct plating on SuperPolymyxin agar plates of spiked stools, with and without an 18henrichment step in TSB, and selective enrichment in TSB + colistin (CST) at 0.5 µg/ml for the detection of PMR-E. SuperPolymyxin Enrichment broth Strain Species Resistance MIC of Agar (CFU/ml) mechanism colistin (CFU/ml) TSB TSB + CST (µg/ml) R2739 E. coli MCR-1 4 4.1×102 3.5×106 4.3×108 R3330 E. coli MCR-1 8 6×101 4×106 3.5×108 2 7 IS1R into mgrB prom -45/-46 R3193 K pneumoniae 64 3×10 4.38×10 1.2×109 2 6 R3218 K. pneumoniae G53S PmrA 128 1×10 2.5×10 3×108 1 5 R3210 K. pneumoniae R16C in PhoQ 128 2×10 2×10 1.4×108 R3222 K. pneumoniae T157P substitution in PmrB 32 4×101 7×105 5×107

VRE Agar (CFU/ml)

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VanB VanA VanB VanA VanB VanA

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Enterococcus faecalis Enterococcus faecium Enterococcus faecalis Enterococcus faecalis Enterococcus faecium Enterococcus faecium

MIC of vancomycin (µg/ml) 64 256 96 256 12 256

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Resistance mechanism

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R2953 R2961 R2147 R2148 R2150 R2152

Species

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Strain

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Table 4. Evaluation of direct plating on VRE agar plates of spiked stools, with and without a 18 h enrichment step in TSB, and selective enrichment in TSB+ vancomycin (VAN) at 1 µg/ml for the detection of VRE.

20

4.7×102 5.1×102 7×101 5×101 8×101 1×102

Enrichment brotha (CFU/ml) TSB + VAN TSB 4.85×107 1.2×108 7×105 2.6×107 1.8×107 3.1×106

5.9×107 1.77×108 1×107 1×107 3×108 2×107