A genetic cluster of MDR Enterobacter cloacae complex ST78 harbouring a plasmid containing blaVIM-1 and mcr-9 in the Netherlands
Authors: Hendrickx Antoni P A , Debast Sylvia , Pérez-Vázquez María , Schoffelen Annelot F , Notermans Daan W , Landman Fabian , Wielders Cornelia C H , Cañada Garcia Javier E , Flipse Jacky , de Haan Angela , Witteveen Sandra , van Santen-Verheuvel Marga , de Greeff Sabine C , Kuijper Ed , Schouls Leo M ,   Dutch CPE surveillance Study Group, Maijer-Reuwer A , Leversteijn-Van Hall M A , Kluytmans J A J W , Spijkerman I J B , Van Dijk K , Halaby T , Zwart B , Diederen B M W , Voss A , Dorigo-Zetsma J W , Ott A , Oudbier J H , Van der Vusse M , Vlek A L M , Buiting A G M , Bode L , Paltansing S , Van Griethuysen A J , Den Reijer M , Van Trijp M , Van Elzakker E P M , Muller A E , Van der Linden M P M , Van Rijn M , Wolfhagen M J H M , Waar K , Kolwijck E , Silvis W , Schulin T , Damen M , Dinant S , Van Mens S P , Melles D C , Cohen Stuart J W T , Van Ogtrop M L , Overdevest I T M A , Van Dam A P , Wertheim H , Frénay H M E , Sinnige J C , Mattsson E E , Bosboom R W , Stam A , De Jong E , Roescher N , Heikens E , Steingrover R , Troelstra A , Bathoorn E , Trienekens T A M , Van Dam D W , De Brauwer E I G B , Stals F S 
Published: March 2021

Abstract

Background
Carbapenemases produced by Enterobacterales are often encoded by genes on transferable plasmids and represent a major healthcare problem, especially if the plasmids contain additional antibiotic resistance genes. As part of Dutch national surveillance, 50 medical microbiological laboratories submit their Enterobacterales isolates suspected of carbapenemase production to the National Institute for Public Health and the Environment for characterization. All isolates for which carbapenemase production is confirmed are subjected to next-generation sequencing.

Objectives
To study the molecular characteristics of a genetic cluster of Enterobacter cloacae complex isolates collected in Dutch national surveillance in the period 2015–20 in the Netherlands.

Methods
Short- and long-read genome sequencing was used in combination with MLST and pan-genome MLST (pgMLST) analyses. Automated antimicrobial susceptibility testing (AST), the Etest for meropenem and the broth microdilution test for colistin were performed. The carbapenem inactivation method was used to assess carbapenemase production.

Results
pgMLST revealed that nine E. cloacae complex isolates from three different hospitals in the Netherlands differed by &lt;20 alleles and grouped in a genetic cluster termed EclCluster-013. Seven isolates were submitted by one hospital in 2016–20. EclCluster-013 isolates produced carbapenemase and were from ST78, a globally disseminated lineage. EclCluster-013 isolates harboured a 316 078 bp IncH12 plasmid carrying the blaVIM-1 carbapenemase and the novel mcr-9 colistin resistance gene along with genes encoding resistance to different antibiotic classes. AST showed that EclCluster-013 isolates were MDR, but susceptible to meropenem (&lt;2 mg/L) and colistin (&lt;2 mg/L).

Conclusions
The EclCluster-013 reported here represents an MDR E. cloacae complex ST78 strain containing an IncH12 plasmid carrying both the blaVIM-1 carbapenemase and the mcr-9 colistin resistance gene.