Introduction

Methods

Results

Conclusions

Objectives

P-151DiagnosticMethods

EvaluationofAmplidiag CarbaR kitforthedetectionofcarbapenemase-producingbacteria

Saoussen Oueslati,DelphineGirlich,LaurentDortet andThierryNaas

- EA7361“Structure,dynamic,functionandexpressionofbroadspectrumb-lactamases”,Paris-Sud University,LabEx Lermit,FacultyofMedecine,LeKremlin-Bicêtre,France,- AssociatedFrenchNationalReferenceCenter forAntibioticResistance:Carbapenemase-producingEnterobacteriaceae,LeKremlin-Bicêtre,France,- EERAUnit« EvolutionandEcologyofResistancetoAntibiotics »,Institut Pasteur-APHP-Université ParisSud,Paris,Franceoueslati.saoussen@gmail.com

Carbapenemase-producing Enterobacteriaceae (CPE)and carbapenemase-producing non-fermenters (CPNF;Pseudomonadaceae and Acinetobacter sp.) have beenincreasingly reported worldwide. Therefore, reliabledetection of carbapenemase production is essentialfor the prompt implementation of infection controlmeasures in order to prevent clonal expansion ortransfer of carbapenemase genes.We evaluated the performance of Amplidiag®CarbaR+VRE (Mobidiag Ltd, Espoo, Finland), aqualitative multiplexed nucleic acid-based in vitrodiagnostic test on cultured bacteria.

Qualitative multiplexed nucleic acid-based diagnostictest intended for the detection of carbapenemase:targeted genes are blaKPC, blaNDM, blaVIM,blaIMP,blaOXA-48-like, Acinetobacter OXA genes includingblaOXA-23, blaOXA-24/-40, blaOXA-58, and blaOXA-51with upstream inserted ISAba1. PCR was performed onQiamp purified total DNA and directly on colonies.

The Amplidiag® CarbaR+VRE was able to detect alltargeted genes including all their variants.As claimed by the manufacturer, other carbapenemasessuch as GES-like carbapenemases (GES-2, GES-5 in P.aeruginosa, GES-14 in A. baumannii), GIM-1, AIM-1,SPM-1, DIM-1 or OXA-198 in P. aeruginosa, or OXA-143-like in A. baumannii were not detected.The Amplidiag® CarbaR+VRE assay is well adapted tothe French epidemiology with a good sensitivity(100%) and specificity (96% ).The main advantage of this test is that it contains alarge panel of targeted resistance determinants.Interestingly, this assay is able to detect A. baumanniicarbapenemases that have recently been also detectedin Enterobacteriaceae, such as OXA-58-producing P.mirabilis isolates (Girlich et al., AAC, 2016).

Strains b-Lactamase contenta MIC (µg/ml) b Amplidiag® Carba-R+VREc

IMP ETP MEM KPC NDM VIM IMP OXA-48-like AcOXA

EnterobacteriaceaeNon carbapenemase producersOverexpressed AmpC/ ESBLe

E. coli ìììCased 16 > 32 2 - - - - - -E. cloacae ììì Case 0.12 1 0.12 - - - - - -M. morgannii ììì Case 1.5 0.02 0.12 - - - - - -

ESBL + impermeability

E. coli CTX-M-15 2 4 1 - - - - - -K. pneumoniae CTX-M-15 + SHV-1 1 >32 4 - - - - - -

Overexpressed AmpC+ ESBL + impermeability

E. cloacae ììì Case +CTX-M-15 1.5 6 1 - - - - - -C. freundii ììì Case +TEM-3 1 8 1 - - - - - -

Extended spectrum oxacillinase

K. pneumoniae OXA-163 0.5 0.38 0.12 - - - - - -S. marcescens OXA-405 0.5 0.75 0.19 - - - - + +

Carbapenemase producersKPC-typeE. coli KPC-2 1 >32 3 - - - - -+E. coli KPC-2 + TEM-1 + OXA-9 2 1.5 1 + - - - - -E. coli KPC-2 + CTX-M-9 + TEM-1 4 4 2 + - - - - -

K. pneumoniae KPC-2 + SHV-11 + TEM-1 + CTX-M-2

16 24 32+ - - - - -

K. pneumoniae KPC-2 + SHV-11 + TEM-1 + CTX-M-2 + OXA-9

>32 >32 >32+ - - - - -

K. ozoenae KPC-3 + OXA-9 + TEM-1 >32 >32 2 + - - - - -E. cloacae KPC-2 + TEM-1 + SHV-11 2 4 1.5 + - - - - -C. freundii KPC-2 + TEM-1 8 1.5 3 + - - - - -S. marcescens KPC-2 + TEM-1 + SHV-12 >32 >32 >32 + - - - - -NDM-typeE. coli NDM-1 + OXA-1 + OXA-10

+ CMY-16 + TEM-11 3 1 - + - - - -

E. coli NDM-4 + CTX-M-15 + OXA-1 >32 >32 >32

- + - - - -E. coli NDM-5 + TEM-1 + CTX-M-

15 >32 >32 >32- + - - - -

E. coli NDM-6 + CTX-M-15 + OXA-1 6 32 8

- + - - - -E. coli NDM-7 4 16 3 - + - - - -VIM-typeE. coli VIM-1 + CTX-M-3 1.5 0.38 0.5 - - - - -+

E. cloacaeVIM-4 + CTX-M-15 + TEM-1 + SHV-31 3 2 1 - - + - - -

K. pneumoniae VIM-19 + CTX-M-3 + TEM-1 + SHV-1 8 16 4

- - + - - -C. freundii VIM-2 + TEM-1 + OXA-9 +

OXA-10 1.5 4 0.5- - + - - -

IMP-typeE. coli IMP-1 0.5 3 0.5 - - - - -+E. cloacae IMP-8 1.5 1 1 - - - + - -S. marcescens IMP-11 8 >32 2 - - - + - -OXA-48-type

OXA-48 + CTX-M-15 3 16 1 - - - - -E. coli +E. coli OXA-48 + CTX-M-15 0.5 0.75 0.12 - - - - +K. pneumoniae OXA-48 0.5 2 0.5 - - - - + -K. pneumoniae OXA-48 + CTX-M-15 2 3 2 - - - - + -

E. cloacaeOXA-48 + TEM-1 + CTX-M-15 + OXA-1

1 16 1.5 - - - - + -

C. koseri OXA-48 + TEM-1 0.75 2 0.38 - - - - + -

K. pneumoniae OXA-162 + TEM-1 + SHV-11

4 8 1- - - - + -

K. pneumoniae OXA-204 + CMY-4 0.5 2 0.5 - - - - + -E. coli OXA-181 0.38 0.19 1 - - - - + -E. coli OXA-181 0.38 0.25 0.06 - - - - + -

K. pneumoniae OXA-181 + SHV-11 + CTXM-15 + OXA-1

0.5 2 0.5 - - - - + -

E. coliOXA-232 + CTX-M-15 + OXA-1

>32 >32 >32- - - - + -

K. pneumoniae OXA-232 +SHV-1 + TEM-1 + CTX-M-15 + OXA-1

3 >32 12 - - - - + -

E. coliOXA-244 + TEM-1 + CMY-2

0.5 2 0.5- - - - + -

E. coliOXA-244 + TEM-1 + CMY-2

0.5 1.5 0.5- - - - + -

Multiples carbapenemasesE. coli NDM-1 + OXA-48 1.5 >32 3 - + - - + -K. pneumoniae NDM-1 + OXA-232 4 >32 8 - + - - + -K. pneumoniae NDM-5 + OXA-232 >32 >32 >32 - + - - + -E. coli NDM-1 + VIM-2 0.75 1 0.38 - + + - - -E. cloacae VIM-4 + OXA-48 1 3 0.75 - - + - + -

Table.Results oftheAmplidiag Carba-R+VREonacollectionofcarbapenemase andnon-carbapenemase producing organisms.

a ììì Case, overexpression of a likely chromosome-encoded cephalosporinase, b MICs were determined using Etest method (bioMérieux, Marcy-l’Etoile, France) according to themanufacturer’s instructions. c + (grey box) indicates a positive result and – indicates a negative result , d Case, cephalosporinase, e ESBL, extended spectrum β-lactamase

Strains b-Lactamase contenta MIC (µg/ml) b Amplidiag® Carba-R+VREc

IMP ETP MEM KPC NDM VIM IMP OXA-48-like AcOXA

PseudomonadaceaeNon carbapenemase producersEfflux/OprD deficiency/AmpC /+ other b-lactamases

P. aeruginosa Mex C/D-OprJ >32 4 - - - - - -

P. aeruginosa Mex A/B-OprM 1.5 1.5 - - - - - -

P. aeruginosa OprD deficient 4 4 - - - - - -

P. aeruginosaOprD deficient + AmpC + MexA/B-OprM

32 32- - - - - -

P. aeruginosa OprD deficient + AmpC + MexA/B-OprM + MexX/Y-OprM

64 64- - - - - -

P. aeruginosa OprD deficient + AmpC + MexA/B-OprM + MexC/D-OprJ

16 8- - - - - -

P. aeruginosa GES-9 2 1 - - - - - -P. aeruginosa PER-1 >32 12 - - - - - -P. aeruginosa VEB-1 2 1.5 - - - - - -

P. aeruginosa OXA-32 >32 12 - - - - - -

Carbapenemase producersKPC-typeP. aeruginosa KPC-2 >32 >32 - - - - -+P. aeruginosa KPC-2 >32 >32 + - - - - -GES-typeP. aeruginosa GES-2 3 1 - - - - - -P. aeruginosa GES-5 >32 >32 - - - - - -VIM-typeA. xylosoxydans VIM-1 >32 >32 - - - - -+P. aeruginosa VIM-1 >32 >32 - - + - - -P. fluorescens VIM-2 >32 >32 - - + - - -P. aeruginosa VIM-2 >32 >32 - - + - - -P. putida VIM-2 >32 >32 - - + - - -P. aeruginosa VIM-4 >32 >32 - - + - - -P. aeruginosa VIM-4 >32 >32 - - + - - -IMP-typeP. aeruginosa IMP-1 12 >32 - - - - -+P. putida IMP-1 1 0.19 - - - + - -P. aeruginosa IMP-2 >32 >32 - - - + - -P. aeruginosa IMP-13 >32 >32 - - - + - -Other typesP. aeruginosa GIM-1 >32 >32 - - - - - -P. aeruginosa AIM-1 >32 >32 - - - - - -P. aeruginosa SPM-1 >32 >32 - - - - - -P. stutzeri DIM-1 >32 >32 - - - - - -P. aeruginosa OXA-198 >32 8 - - - - - -

Acinetobacter sp.Non carbapenemase producersA. baumannii OXA-21 0.75 0.5 - - - - - -A. baumannii PER-1 1.5 1.5 - - - - - -

Carbapenemase producersOverexpressed chromosome-encoded OXA-51-like carbapenemaseA. baumannii GES-12 + OXA-51+

ISAba132 32

- - - - - +A. baumannii SHV-5 + OXA-51+ ISAba1 6 4 - - - - - +A. baumannii OXA-51+ ISAba1 3 3 - - - - - +A. baumannii OXA-66 + ISAba1 4 6 - - - - - +OXA-23-typeA. baumannii OXA-23 >32 >32 - - - - - +A. baumannii OXA-23 >32 >32 - - - - - +A. baumannii OXA-23 >32 >32 - - - - - +OXA-24/-40-typeA. baumannii OXA-24/-40 >32 >32 - - - - - +A. baumannii OXA-72 >32 >32 - - - - - +OXA-58-typeA. baumannii OXA-58 >32 16 - - - - - +A. baumannii OXA-58 + VEB-1a >32 16 - - - - - +OXA-143-typeA. baumannii OXA-143 >32 >32 - - - - - -A. baumannii OXA-253 >32 >32 - - - - - -Other typesA. baumannii NDM-1 >32 >32 - - - -+ +

A. baumannii NDM-1 + OXA-23 >32 >32 - + - - - +A. baumannii IMP-1 4 6 - - - + - -A. genomospecies16 VIM-4

>32 >32- - + - - +

A. baumannii GES-14 24 26 - - - - - -

The Amplidiag® CarbaR+VRE has been tested on acollection of 100 well-characterized isolates with areduced susceptibility to carbapenems, and 200isolates collected at the National Reference Center forAntibiotic Resistance between the 20st January to10th February 2016.

TheperformancesoftheAmplidiag® CarbaR+VRE ontheprospectivestudywereof100%and96%sensitivityandspecificity,respectively.

Ø Results ofthe100well-characterizedisolates

Ø Results ofthe200isolatescollectedattheNationalReferenceCenter forAntibioticResistance