Molecular and epidemiological characterization of carbapenemase-producing Enterobacteriaceae in Norway, 2007 to 2014

Abstract

The prevalence of carbapenemase-producing Enterobacteriaceae (CPE) is increasing worldwide. Here we present associated patient data and molecular, epidemiological and phenotypic characteristics of all CPE isolates in Norway from 2007 to 2014 confirmed at the Norwegian National Advisory Unit on Detection of Antimicrobial Resistance. All confirmed CPE isolates were characterized pheno- and genotypically, including by whole genome sequencing (WGS). Patient data were reviewed retrospectively. In total 59 CPE isolates were identified from 53 patients. Urine was the dominant clinical sample source (37%) and only 15% of the isolates were obtained from faecal screening. The majority of cases (62%) were directly associated with travel or hospitalization abroad, but both intra-hospital transmission and one inter-hospital outbreak were observed. The number of CPE cases/year was low (2–14 cases/year), but an increasing trend was observed. Klebsiella spp. (n = 38) and E. coli (n = 14) were the dominant species and bla_KPC (n = 20), bla_NDM (n = 19), bla_OXA-48-like (n = 12) and bla_VIM (n = 7) were the dominant carbapenemase gene families. The CPE isolates were genetically diverse except for K. pneumoniae where clonal group 258 associated with bla_KPC dominated. All isolates were multidrug-resistant and a significant proportion (21%) were resistant to colistin. Interestingly, all bla_OXA-48-like, and a large proportion of bla_NDM-positive Klebsiella spp. (89%) and E. coli (83%) isolates were susceptible in vitro to mecillinam. Thus, mecillinam could have a role in the treatment of uncomplicated urinary tract infections caused by OXA-48- or NDM-producing E. coli or K. pneumoniae. In conclusion, the impact of CPE in Norway is still limited and mainly associated with travel abroad, reflected in the diversity of clones and carbapenemase genes.

Introduction

Carbapenemase-producing Enterobacteriaceae (CPE) have emerged as a global public health concern during the last two decades [1, 2]. CPE isolates are usually multidrug-resistant (MDR) or even extensively- or pandrug-resistant (XDR/PDR), resulting in limited antibiotic treatment options [1, 3, 4]. Due to the lack of effective therapy, CPE infections have been associated with high mortality rates [5, 6]. Currently, colistin and various combination regimens are generally used for treatment of CPE infections. However, the clinical evidence is mainly based on case reports and observational retrospective studies [1, 4]. Worryingly, high rates of colistin resistance among CPE have been observed in certain regions [7, 8]. Although colistin resistance is often mutation-based, plasmid-mediated colistin resistance has now also been described [9–14], and observed in CPE isolates [11, 15–17].

The main carbapenemases among Enterobacteriaceae include KPC (Ambler class A), the metallo-β-lactamases NDM, VIM and IMP (Ambler class B), and OXA-48-like enzymes (Ambler class D) [1]. Certain carbapenemases dominate in specific regions and countries, i.e. NDM in the Indian subcontinent, KPC in Italy, Portugal, Israel, Greece and the US, and OXA-48-like in many Mediterranean (e.g. Turkey and Malta) and North African countries as well as some other European countries (e.g. Belgium, France, Germany and Spain) [7, 18–20]. Specific clones or clonal groups (CG) are often associated with specific carbapenemases, while other carbapenemases show a more broad diversity with respect to host genetic backgrounds [2, 21]. The global spread of KPC has mainly been associated with Klebsiella pneumoniae sequence type (ST) 258 or CG 258 [2, 21, 22]. In contrast, NDM and OXA-48-like enzymes are broadly distributed in various genetic backgrounds of K. pneumoniae and Escherichia coli and for bla_NDM there is no clear link to a specific plasmid backbone [2, 21]. For bla_OXA-48-like there is molecular evidence supporting an association with a specific internationally epidemic IncL plasmid backbone [23–25].

The emergence of CPE in the Nordic countries has mainly been associated with single sporadic cases associated with import [26–36], and the prevalence is low compared with other European countries [7, 19]. However, there are indications of local dissemination unrelated to travel in Denmark [37, 38].

The aim of this study was to analyse the epidemiological, phenotypic and molecular characteristics of CPE isolated in Norway from 2007 to 2014 to understand the molecular epidemiology associated with the emergence of CPE in Norway.

Materials and methods

Bacterial strains and demographic data

The study collection consisted of 59 CPE isolates genetically-verified at the Norwegian National Advisory Unit on Detection of Antimicrobial Resistance from 2007–2014. The criteria for submitting isolates to the Unit included reduced susceptibility to carbapenems according to the Norwegian Working Group for Antibiotics (AFA, https://unn.no/fag-og-forskning/arbeidsgruppen-for-antibiotikasporsmal-og-metoder-for-resistensbestemmelse-afa)/Nordic Committee on Antimicrobial Susceptibility Testing (NordicAST) guidelines (www.nordicast.org). In 2012 mandatory reporting of confirmed CPE cases to the Norwegian Surveillance System for Communicable Diseases (MSIS) was established. After confirmation at the Advisory Unit, MSIS and the primary lab are notified. The primary laboratory subsequently notifies the responsible clinician, who also reports data to MSIS. Clinical data were collected from the laboratory requisition. Multiple isolates from the same patient were included in the analysis if they were (i) of different species, (ii) the same species, but harboured a different carbapenemase gene or (iii) if the isolates were of the same species and harboured the same carbapenemase gene, but were identified >1 year apart.

Phenotypic analysis

Species identification was performed using MALDI-TOF MS (Bruker Daltonik GmbH, Bremen, Germany). MIC profiling was performed using gradient strips (Liofilchem, Roseto degli Abruzzi, Italy/bioMérieux, Marcy-l’Étolie, France) and broth microdilution for colistin using in-house designed premade Sensititre microtiter plates (TREK Diagnostic Systems/Thermo Fisher Scientific, East Grinstead, UK). Interpretation was according to EUCAST clinical breakpoints version 6.0 (www.eucast.org). Non-susceptibility included both the intermediate and resistant categories. The AmpC Confirm kit (ROSCO Diagnostica, Taastrup, Denmark), ESBL combination discs (Becton-Dickinson, Franklin Lakes, NJ, USA), KPC, MBL and OXA-48 Confirm kit (ROSCO Diagnostica) and the in-house version of Carba NP test were used for phenotypic typing of β-lactamases [39, 40].

Molecular analysis

The presence of carbapenemase genes was initially determined by various PCRs for bla_KPC, bla_IMI, bla_VIM, bla_NDM, bla_IMP, bla_GIM, bla_SPM, bla_SIM and bla_OXA-48-like [41–44]. WGS was performed on all isolates using the MiSeq platform (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, genomic DNA was purified using the GenElute bacterial genomic DNA kit (Sigma-Aldrich, St. Louis, MO, USA). DNA libraries were prepared using Nextera/Nextera XT kits (Illumina) followed by paired-end sequencing. Contigs were assembled using SPAdes [45] through the iMetAMOS extension [46] of the MetAMOS package [47]. The presence of resistance genes/mutations, carbapenemase genes and single nucleotide polymorphisms (SNP) variations were determined using a customised algorithm that uses Bowtie 2 to map reads against a locally curated reference database and assembled from publically accessible databases. The database comprised sequences for all reported carbapenemase variants. Samtools was used to generate an mpileup file [48] which was then parsed based on read depth (> 10 reads per base) and base-call agreement (> 90%) to determine the base type at each nucleotide position relative to the closest reference sequence. Presence of reported carbapenemase variants were defined based on 100% identity across the whole length of the corresponding reference gene.

STs of Klebsiella spp., E. coli and Enterobacter cloacae complex were determined from WGS data using the Klebsiella MLST database (http://bigsdb.pasteur.fr/klebsiella/klebsiella.html), EnteroBase (http://enterobase.warwick.ac.uk/species/index/ecoli) for E. coli, and the E. cloacae MLST database (http://pubmlst.org/ecloacae). Core genome MLST (cgMLST) was performed on K. pneumoniae isolates using 694 loci as previously described [22]. A phylogenetic tree was constructed based on the concatenated sequence alignments using RAxML [49] and FigTree (http://tree.bio.ed.ac.uk/software/figtree/).

Genbank accession numbers

WGS data have been deposited at the National Center for Biotechnology Information (NCBI) under BioProject PRJNA295003.

Ethical considerations

The study was reviewed and approved by the Regional Committee for Medical and Health Research Ethics North (reference no. 2016/2122/REK Nord and 2017/146/REK Nord) and the Data Protection Officer at the University Hospital of North Norway (reference no. 2017/1562). The need for patient consent was waived by the Regional Committee for Medical and Health Research Ethics North (reference no. 2017/146/REK nord)

Results

Bacterial isolates

In total 59 CPE were identified from 53 patients of which 44 were hospitalized patients. Samples from eight patients were taken at general practitioners or in other health care institutions (e.g. elderly care homes). For one patient no information was obtained. Of the 53 patients, four had multiple CPE isolates belonging to different species or different STs. One patient had four bla_NDM-1-positive strains of different species (Proteus mirabilis, Providencia stuartii, Citrobacter sp. and K. pneumoniae) isolated within a four-month period. Another had bla_KPC-2-positive K. pneumoniae and Enterobacter cloacae complex isolates in the same faecal screening sample. A third had bla_NDM-1-positive E. coli and E. cloacae complex isolates identified in two different specimens (wound secretion and urine, respectively) within a one-month period. The fourth patient yielded two bla_NDM-1-positive K. pneumoniae strains with unrelated STs from specimens taken 21 months apart.

Increasing number of CPE identified during the study period from a high proportion of clinical isolates

CPE isolates were identified in 14 of 22 clinical microbiology laboratories representing all health regions in Norway. The number of CPE cases per year, diversity of carbapenemase variants and species increased during the study period (Table 1), but with a trend towards dominance of NDM and OXA-48-like carbapenemase variants and increasing number of carbapenemase-producing E. coli. Fifty-six percent of the patients were male. The patient age ranged from 3–96 years (mean 63 and median 66 years). The majority of CPE were isolated from urine (n = 22, 37%), blood culture (n = 9, 15%) and faecal screening (n = 9, 15%).

Table 1. Time-line and distribution of identified CPEs and carbapenemase variants.

No. of isolates in parenthesis.

Year	No. of isolates	No. of casesa	Klebsiella sp.	E. coli	Other Enterobacteriaceae
2007	3	3	KPC-2 (1), VIM-1 (2)
2008	6	6	KPC-2 (6)
2009	2	2	KPC-2 (2)
2010	8	7	KPC-2 (2), KPC-3 (1), VIM-27 (2), NDM-1 (1)	NDM-1 (1)	KPC-2 (1)
2011	4	4	KPC-2 (2), NDM-1+OXA-181 (1), OXA-48 (1)
2012	16	14	KPC-2 (1), VIM-1 (1), NDM-1 (2), NDM-7 (1), OXA-245 (1)	VIM-29 (1), NDM-1 (1), NDM-5 (1), NDM-7 (1), OXA-48 (2)	NDM-1 (3), IMI-9 (1)
2013	8	7	KPC-3 (1), NDM-1 (2), OXA-48 (1), OXA-245 (1)	NDM-1 (1),OXA-48 (2)
2014	12	10	KPC-2 (2), NDM-1 (2), OXA-48 (1), OXA-162 (1)	VIM-4 (1), NDM-1 (1), IMP-26 (1), OXA-181 (1)	KPC-2 (1), NDM-1 (1)
Total 2007–2014	59	53	KPC-2 (16), KPC-3 (2), VIM-1 (3), VIM-27 (2), NDM-1 (7), NDM-7 (1), NDM-1+OXA-181 (1), OXA-48 (3), OXA-162 (1), OXA-245 (2)	VIM-4 (1), VIM-29 (1), NDM-1 (4), NDM-4 (1), NDM-7 (1), IMP-26 (1), OXA-48 (4), OXA-181 (1)	KPC-2 (2), IMI-9 (1), NDM-1 (4)

^a Patients identified with multiple CPE defined as a single case.

Association with travel or hospitalization abroad

Thirty-three patients (62%) had a known history of travel and/or hospitalization abroad (Table 2). Sixteen patients (30%) reported no travel or hospitalization abroad and for four patients (8%), no information was obtained. With respect to the non-direct import cases, eight cases were associated with secondary spread from imported cases. This included six cases associated with a previously described, small but long-term outbreak of bla_KPC-2-positive K. pneumoniae/E. cloacae complex in 2007–2010 [50]. In addition, two other intra-hospital transmissions of bla_KPC-2-positive K. pneumoniae [28] and bla_VIM-27-positive K. pneumoniae were observed involving one additional patient in each case.

Table 2. Distribution of isolates according to association with importation.

Country	No. of isolates	Species	Sequence type (ST)	Carbapenemase
Greece	7	K. pneumoniae	ST258	KPC-2
	1	K. pneumoniae	ST147	VIM-27
India	1	K. pneumoniae	ST11	NDM-1
	1	K. pneumoniae	ST17	NDM-1
	1a	K. pneumoniae	ST147	NDM-1
	1	E. coli	ST101	NDM-7
	1	E. coli	ST131	NDM-1
	1	E. coli	ST410	NDM-1
Turkey	1	K. pneumoniae	ST273	VIM-1
	1	K. variicola	ST981	OXA-48
	1	E. coli	ST38	OXA-48
Serbiab	1	K. pneumoniae	ST17	NDM-1
	1	P. stuartii	-	NDM-1
	1	P. mirabilis	-	NDM-1
	1	Citrobacter sp.	-	NDM-1
Spain	1	K. pneumoniae	ST11	OXA-245
	1	K. quasipneumoniae	ST1466	VIM-1
	1	E. cloacae complex	ST635	IMI-9
Morocco	1	K. pneumoniae	ST405	OXA-48
	1	K. pneumoniae	ST11	OXA-245
Thailand	1	E. coli	ST405	OXA-48
	1	E. coli	ST6355	VIM-29
Brazil	1	K. pneumoniae	ST855	KPC-2
United Arab Emirates	1	K. pneumoniae	ST336	NDM-7
Syria/Jordan	1	E. coli	ST410	VIM-4
Jamaica	1	E. cloacae complex	ST456	KPC-2
Pakistan	1	E. coli	ST617	NDM-1
Romania	1	K. pneumoniae	ST525	NDM-1+OXA-181
Sri Lanka	1	K. pneumoniae	ST101	NDM-1
USA	1	K. pneumoniae	ST258	KPC-3
Unknown	1	K. pneumoniae	ST187	OXA-48
	2	E. coli	ST38	OXA-48
	1	E. coli	ST95	IMP-26
Norway (no reported overseas travel)	9c, d	K. pneumoniae	ST258	KPC-2
	1	K. pneumoniae	ST14	OXA-162
	1a	K. pneumoniae	ST37	NDM-1
	1e	K. pneumoniae	ST147	VIM-27
	1c	K. pneumoniae	ST461	KPC-2
	1	K. pneumoniae	ST2134	VIM-1
	1	E. coli	ST410	OXA-181
	1	E. coli	ST636	NDM-5
	1f	E. coli	ST681	NDM-1
	1f	E. cloacae complex	ST92	NDM-1
	1c	E. cloacae complex	ST484	KPC-2

^a Two bla_NDM-1-positive K. pneumoniae isolates, one ST147 and one ST37, were isolated from the same patient. The isolates were identified 21 months apart where the first detection was associated with importation, but not for the second detection.

^b All four bla_NDM-1-positive isolates were isolated from the same patient.

^c Six K. pneumoniae ST258, one K. pneumoniae ST461 and one E. cloacae complex ST484, all bla_KPC-2-positive, were associated with a long-term outbreak [50]. The first case (K. pneumoniae ST258 with bla_KPC-2) of the outbreak were associated with import from Greece.

^d One bla_KPC-2-positive K. pneumoniae ST258 associated with intra-hospital transmission (first case associated with import from Greece)[28].

^e The bla_VIM-27-positive isolate were associated with a case of intra-hospital transmission (first case associated with import from Greece).

^f Both isolates identified from the same patient.

Bacterial species and carbapenemase diversity

Overall Klebsiella spp. (K. pneumoniae, n = 36; Klebsiella variicola n = 1; Klebsiella quasipneumoniae n = 1) were dominant, followed by E. coli (n = 14), E. cloacae complex (n = 4) and single isolates of P. stuartii, P. mirabilis and Citrobacter sp. (Table 1 and S1 Table). The most dominant carbapenemase gene family was bla_KPC, found in K. pneumoniae (n = 18) and E. cloacae complex (n = 2), followed by bla_NDM identified in K. pneumoniae (n = 8), E. coli (n = 6), E. cloacae complex (n = 1), P. stuartii (n = 1), P. mirabilis (n = 1) and Citrobacter sp. (n = 1). bla_VIM was identified in K. pneumoniae (n = 4), E. coli (n = 2) and K. quasipneumoniae (n = 1) while bla_OXA-48-like was identified in K. pneumoniae (n = 5), E. coli (n = 5) and K. variicola (n = 1). In addition, we identified one K. pneumoniae isolate harbouring both bla_NDM and bla_OXA-48-like and single isolates with bla_IMI (E. cloacae complex) and bla_IMP (E. coli). With respect to KPC, KPC-2 (n = 18) was the most predominant allele with the closest KPC-3 (n = 2) variant detected in only two isolates. The remaining carbapenemase genes encoded three different variants of NDM (NDM-1, n = 16; NDM-7, n = 2; and NDM-5, n = 1), four OXA-48-like (OXA-48, n = 7; OXA-181, n = 2; OXA-245, n = 2 and OXA-162, n = 1) and four VIM (VIM-1, n = 3; VIM-27, n = 2; VIM-4, n = 1; and VIM-29, n = 1). The single isolates with bla_IMI and bla_IMP encoded IMI-9 and IMP-26, respectively.

Bacterial population structure and linkage to specific carbapenemase alleles

MLST and cgMLST (Fig 1) showed that K. pneumoniae was dominated by KPC-producing clonal group (CG) 258, more specifically ST258 (n = 15) and its single locus variants (SLV) ST855 (n = 1) and ST340 (n = 1). The CG258 cluster comprised 21 isolates and included nearly all KPC-producers (n = 17) in addition to four ST11 isolates carrying bla_NDM-1 (n = 2) or bla_OXA-245 (n = 2) genes. Outside CG258, bla_KPC was only identified in one isolate belonging to ST461. Among the K. pneumoniae isolates cgMLST identified two other clusters represented by more than one isolate: one representing CG147 and including ST147 with bla_VIM-27 (n = 2) or bla_NDM-1 (n = 1) and ST273 with bla_VIM-1 (n = 1), and one representing CG17 including ST17 with bla_NDM-1 (n = 2) and ST336 with bla_NDM-7 (n = 1). The remaining K. pneumoniae isolates represented genetically diverse single strains harbouring bla_NDM-1 (ST37 and ST101), bla_NDM-1 + bla_OXA-181 (ST525), bla_OXA-48 (ST187 and ST405), bla_OXA-162 (ST14) and bla_VIM-1 (ST2134). The K. quasipneumoniae isolate carrying bla_VIM-1 belonged to ST1466 and the K. variicola with bla_OXA-48 belonged to ST981.

Fig 1. Phylogenetic tree of K. pneumoniae isolates based on alignment of concatenated sequences of the 694 cgMLST scheme of K. pneumoniae [22].

The tree was constructed in RAxML [49] and visualized using FigTree (http://tree.bio.ed.ac.uk/software/figtree/). Clonal groups with >1 isolates are boxed. Sequence type (ST), carbapenemase gene and year of isolation is indicated for each isolate. Isolates associated with the long-term outbreak [50] and the two occurrences of intra-hospital transmission are labelled *, # and ¤, respectively.

Ten diverse genetic backgrounds were identified among the E. coli isolates (n = 14). None of the STs were SLVs or double locus variants (DLVs) of any other. Only ST38 (n = 3) and ST410 (n = 3) were represented by >1 isolate. All three ST38 isolates carried bla_OXA-48, while the three ST410 strains harboured each a different carbapenemase gene (bla_NDM-1, bla_VIM-4 or bla_OXA-181). The remaining strains were genetically diverse and carried various carbapenemase genes/variants: bla_NDM-1 (ST131, ST617 and ST681), bla_NDM-5 (ST636), bla_NDM-7 (ST101), bla_OXA-48 (ST405), bla_VIM-29 (ST6355) and bla_IMP-26 (ST95).

The four carbapenemase-producing E. cloacae complex isolates were all of different STs: ST456 and ST484 both with bla_KPC-2, ST92 with bla_NDM-1 and ST635 with bla_IMI-9. All STs were defined as singletons (no SLVs) by BURST analysis of the E. cloacae MLST database (http://pubmlst.org/ecloacae/, last accessed 24.06.2016).

Antimicrobial susceptibility profile and performance of phenotypic methods for detection of CPE

All isolates were multidrug-resistant (MDR) according to the definitions by Magiorakos et al. [51]. (Table 3 and S1 Table). One isolate, a bla_NDM-1-positive P. stuartii was non-susceptible to all relevant antimicrobial agents tested. Overall fosfomycin and colistin were the most active antimicrobial agents with 85% and 79% of the isolates being susceptible when excluding P. mirabilis and P. stuartii isolates which are intrinsically resistant to colistin [52] (Table 3). Seven of the twelve colistin-resistant isolates were K. pneumoniae ST258 with bla_KPC-2 (n = 6) or bla_KPC-3 (n = 1). The other colistin-resistant isolates included K. pneumoniae ST525 with bla_NDM-1 + bla_OXA-181, K. pneumoniae ST147 with bla_NDM-1, K. pneumoniae ST336 with bla_NDM-7, E. cloacae complex ST635 with bla_IMI-9 and E. cloacae complex ST456 with bla_KPC-2.

Table 3. Antimicrobial resistance profiles of CPE isolates according to species and carbapenemase variant.

		Percent non-susceptible (I+R)a
Species	Carbapenemase	TZP	MEC	CXM	CTX	CAZ	ATM	MEM	ETP	IPM	GEN	AMK	TOB	CIP	TGC	SXT	CST	FOS
Klebsiella spp.	KPC (n = 18)	100	100	100	100	100	100	83	100	50	28	78	83	94	83	72	39	6
	VIM (n = 5)	100	100	100	100	100	40	60	100	80	0	40	100	100	40	80	0	0
	NDM (n = 9)b	100	11	100	100	100	100	89	100	67	78	89	100	89	56	78	33	11
	OXA-48-like (n = 6)	100	0	67	50	67	50	17	100	33	17	0	50	83	83	33	0	33
E. coli	VIM/IMP (n = 3)	67	67	100	100	100	100	33	67	67	100	100	100	33	33	100	0	33
	NDM (n = 6)	100	17	100	100	100	83	33	100	83	83	83	83	67	0	33	0	0
	OXA-48-like (n = 5)	100	0	100	100	100	100	20	100	20	80	0	80	60	20	80	0	0
E. cloacae complex	KPC (n = 2)	100	-c	100	100	100	100	100	100	100	50	50	50	100	50	50	50	100
	NDM (n = 1)	100	-	100	100	100	100	100	100	100	100	100	100	100	100	100	0	100
	IMI (n = 1)	0	-	0	0	0	100	0	100	100	0	0	0	0	100	0	100	0
P. stuartii	NDM (n = 1)	100	-	-	100	100	100	100	100	100	100	100	100	100	-	100	-	100
P. mirabilis	NDM (n = 1)	0	100	100	100	100	100	0	0	100	100	100	100	100	-	100	-	0
Citrobacter spp.	NDM (n = 1)	100	-	100	100	100	100	0	100	100	100	100	100	100	100	100	0	0
Totald		95	53	95	93	95	88	59	97	61	51	63	83	83	58	68	21	15

^a according to EUCAST clinical breakpoint table v. 6.0. TZP: piperacillin-tazobactam; MEC: mecillinam; CXM: cefuroxime; CTX: cefotaxime; CAZ: ceftazidime; AZT: aztreonam; MEM: meropenem; ETP: ertapenem; IPM: imipenem; GEN: gentamicin; AMK: amikacin; TOB: tobramycin; CIP: ciprofloxacin; TGC: tigecycline; SXT: trimethoprim-sulfamethoxazole; CST: colistin; FOS: fosfomycin.

^b includes one isolate co-harboring bla_NDM-1 and bla_OXA-181.

^C “-”indicates lack of clinical breakpoint or intrinsic resistance according to EUCAST Expert Rules on Intrinsic Resistance and Exceptional Phenotypes v.3.1 (http://www.eucast.org/).

^d calculations excludes species/antibiotic combinations with intrinsic resistance.

High levels of non-susceptibility were observed to aminoglycosides (gentamicin, 51%; amikacin, 63%; and tobramycin, 83%), tigecycline (58%) and ciprofloxacin (83%).

With respect to the carbapenems, 41% were susceptible to meropenem, 39% to imipenem and 3% to ertapenem. All isolates had meropenem and ertapenem MIC values above the EUCAST screening breakpoint for carbapenemase detection (http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Resistance_mechanisms/EUCAST_detection_of_resistance_mechanisms_v1.0_20131211.pdf) (S1 Table). For imipenem nine isolates had MIC values below the screening breakpoint. There was no clear correlation between carbapenemase variant and susceptibility to meropenem and imipenem with the exception that among the isolates harbouring bla_OXA-48-like (excluding the strain with both bla_NDM-1 and bla_OXA-181) 9/11 and 8/11 were susceptible to meropenem and imipenem, respectively. As expected, a high level of resistance was observed against other β-lactams (Table 3 and S1 Table). Three isolates: one K. pneumoniae (bla_OXA-48), one K. variicola (bla_OXA-48) and the bla_IMI-9-positive E. cloacae complex isolate were susceptible to extended-spectrum cephalosporins (cefotaxime, ceftazidime and cefuroxime) and aztreonam. Interestingly, all OXA-48-like-positive E. coli and Klebsiella spp. as well as 83% and 89% of NDM-positive E. coli and Klebsiella spp. isolates, respectively were susceptible to mecillinam. Nine (15%) of the isolates tested negative for carbapenemase-production with the in-house Carba NP test (S1 Table), including six bla_NDM-1-positive isolates (E. coli n = 2, P. stuartii, P. mirabilis, Citrobacter sp. and K. pneumoniae), two bla_OXA-48-like-positive isolates (E. coli and K. pneumoniae) and one E. cloacae complex isolate (bla_IMI-9). The KPC, MBL and OXA-48 confirm kit correctly identified the presence of either an MBL or KPC in all relevant isolates except for one bla_NDM-1-positive P. mirabilis strain (S1 Table). The single bla_IMI-9-positive E. cloacae complex isolate also showed significant synergy with boronic acid only. With the exception of the isolate harbouring both bla_NDM-1 and bla_OXA-181, where synergy was observed between meropenem and dipicolinic acid, no synergy was observed with the β-lactamase inhibitors for all bla_OXA-48-like-positive isolates. Moreover, with the exception of two isolates, all bla_{OXA-48–like}-positive isolates showed no zones of inhibition around the temocillin tablet, which may indicate the presence of OXA-48-like carbapenemases according to the manufacturer’s guidelines. The meropenem-meropenem/EDTA gradient strip correctly identified all MBL-positive isolates, with the exception of the K. pneumoniae strain positive for both bla_NDM-1 and bla_OXA-181 where the test was inconclusive (S1 Table).

Association with other antibiotic resistance determinants

Bla_CTX-M and specifically bla_CTX-M-15 were the most common ESBL variants identified and were mainly associated with K. pneumoniae and E. coli isolates with bla_NDM (10/15 isolates) or bla_OXA-48-like (8/11 isolates) and E. coli isolates with bla_VIM (2/2 isolates) (S1 Table). Bla_CTX-M were not identified in bla_KPC-positive K. pneumoniae isolates. One E. coli isolate with bla_OXA-48 harboured both bla_CTX-M-14 and bla_CTX-M-15. bla_CTX-M-15 was also identified in one bla_KPC-2- and one bla_NDM-1-positive E. cloacae complex. Bla_CMY (n = 12) were the most common plasmid-mediated AmpC variants identified with bla_CMY-6 particularly associated with bla_NDM (n = 9). The two bla_OXA-48-like-positive Klebsiella spp. isolates that were susceptible to extended-spectrum cephalosporins and aztreonam were negative for ESBL and plasmid-mediated AmpC genes.

In addition to various genes encoding aminoglycoside-modifying enzymes, the 16S rRNA methylase genes rmtC and armA, were identified in eight and five isolates, respectively (S1 Table). With the exception of the single isolate of E. coli with bla_IMP-26, armA and rmtC were only associated with isolates harbouring bla_NDM-1. In Klebsiella spp. insertional disruption of mgrB [53] associated with colistin resistance was identified in seven K. pneumoniae isolates (S1 Table). Insertional disruption of mgrB was also observed in two clinically colistin susceptible (MIC = 1 mg/L) K. pneumoniae isolates. One K. pneumoniae isolate with a disrupted mgrB also carried a nonsense mutation in pmrB leading to a truncated PmrB. Two colistin-resistant K. pneumoniae isolates had mutations in pmrA resulting in amino acid substitutions of G53C and D86E in one, and G53C in the other. In one colistin-resistant Klebsiella spp. isolate (MIC >8 mg/L) no previously described colistin resistance determinants were identified. The strain had mutations in pmrA (PmrA E57G) and pmrB (PmrB T246A) compared with the colistin-susceptible K. pneumoniae strain MGH 78578 [54], but neither mutation has been linked with colistin resistance and PmrB T246A is commonly found in K. pneumoniae [54]. No mutations were identified in phoP, phoQ or the mgrB promoter for this isolate. The plasmid-mediated colistin resistance genes mcr-1 [9], mcr-2 [10], mcr-3 [12], mcr-4 [13] and mcr-5 [14] were not detected.

All E. coli, K. pneumoniae and E. cloacae complex isolates with high-level ciprofloxacin resistance (MIC ≥32 mg/L) harboured mutations in both gyrA and parC (S1 Table). In addition, various plasmid-mediated quinolone resistance determinants were identified, including aac(6’)-Ib-cr (n = 24), qnrB1 (n = 8), qnrB4 (n = 1), qnrB19 (n = 2), qnrD (n = 1) and qnrS1 (n = 8).

Discussion

The main objective of this study was to gain a better understanding of the molecular epidemiology associated with the emergence of CPE in Norway. As observed in other Nordic countries [26, 27, 32–36] the emergence of CPE in Norway is also mainly associated with importation, highlighting the importance of targeted screening of patients hospitalized abroad and patients with a recent travel history to a country with a high prevalence of CPE. A relatively low number of cases (15%) were identified through faecal screening in contrast to Sweden (74,5%) and France (59.8%) [26, 55]. This difference is most likely due to dissimilarities in the use of targeted screening and that CPE screening in Norway was not fully implemented in the study period. This could also explain why a higher proportion of CPE cases in Sweden (81%) were associated with import [26]. Revised recommendations for infection prevention and control, including indications for screening for CPE, were introduced in Norway in August 2015 and in the first six months of 2016, 63% of CPE cases were identified through faecal screening. The occurrence of one long-term outbreak and two separate incidences of secondary transmission further highlights the importance of rapid implementation of infection prevention and control measures before confirmation of CPE if patients have risk factors (e.g. hospitalization abroad) or when an MDR isolate is identified.

The diversity of species and genetic backgrounds observed is probably due to the high degree of importation from a variety of countries (Table 2). Several studies have shown that the dissemination of resistance genes among clinical strains of Enterobacteriaceae is often associated with high-risk clones and the linkage between specific genetic backgrounds and resistance genes [2, 21, 56]. The cgMLST analysis of K. pneumoniae isolates showed that the observed epidemiology reflects the current global epidemiology (Fig 1), where bla_KPC-2/-3 spread is primarily driven by strains associated with CG258 (and more specifically, ST258). In contrast, ST11 (a member of CG258, and a single locus variant of ST258) has been shown to be associated with a diversity of carbapenemase genes including bla_KPC, bla_NDM, bla_VIM and bla_OXA-48-like in different geographical regions [2, 57, 58]. Accordingly, the four ST11 strains in this study harboured either bla_NDM-1 (n = 2) or bla_OXA-245 (n = 2). Notably, cgMLST has shown that ST11 and ST340 represent a genetic sublineage within CG258 [22]. Isolates with bla_NDM and bla_VIM belonging to two other globally dispersed high-risk CGs like CG17 and CG147 [2] were also identified. The identification of bla_VIM-1 and bla_OXA-48 in K. quasipneumoniae and K. variicola, respectively shows that these Klebsiella species also contribute to the dissemination of carbapenemase genes and infections as both isolates were associated with infection. K. variicola have been shown to be frequently associated with bloodstream infections and associated with higher mortality than K. pneumoniae [59].

All three E. coli ST38 isolates harboured bla_OXA-48, which is consistent with previous observations showing a prevalent linkage of ST38 to bla_OXA-48 in a large collection of clinical isolates from European and North-African countries [23]. In contrast, the three E. coli isolates belonging to ST410 were associated with different carbapenemase genes (bla_NDM-1, bla_VIM-4 or bla_OXA-181) indicating the ability of this genetic background to maintain different plasmids and resistance genes. ST410 E. coli isolates have also previously been identified harbouring bla_KPC-2 [60]. The global dissemination of bla_NDM has so far not been linked to specific high-risk clones or epidemic plasmids [21] and this is also reflected among the five bla_NDM-positive E. coli isolates, which belonged to five different genetic backgrounds. However, one strain belonged to the international high-risk clone ST131 [21] and another to ST101, which has previously been found to be associated with bla_NDM and other carbapenemases in several countries (e.g. Bangladesh [61], USA [62], Canada [63, 64] and Bulgaria [65]).

CPE frequently exhibit MDR or XDR phenotypes, limiting treatment options [1, 4]. This was also observed in our strain collection (Table 2 and S1 Table) due to the association with a wide variety of other acquired resistance genes, including 16S rRNA methylase genes conferring high-level broad-spectrum aminoglycoside resistance [66] and chromosomal mutations/insertions resulting in ciprofloxacin and colistin resistance (S1 Table). The mechanism(s) behind colistin resistance in one K. pneumoniae strain and the colistin-resistant E. cloacae isolates remains to be determined. Interestingly, a high prevalence of susceptibility to mecillinam among OXA-48- and NDM-producing E. coli and K. pneumoniae isolates was observed. Marrs et al. also showed high levels of in vitro susceptibility to mecillinam among NDM-producing E. coli and K. pneumoniae isolates from Pakistan [67], suggesting that mecillinam could have a role in the treatment of uncomplicated urinary tract infections caused by OXA-48- or NDM-producing E. coli or K. pneumoniae [68].

Rapid identification of CPE is essential for timely implementation of enhanced infection control measures to reduce transmission of CPE and prevent infections [3]. As observed in previous studies [69, 70] false-negative results (15%) for carbapenemase production were observed with the in-house version of the Carba NP test, particularly with NDM- and OXA-48-like-producing isolates. Identification of OXA-48-like-producers can be particularly challenging due to their relatively low level of activity against carbapenems and the lack of specific inhibitors [71]. The relatively high number of false-negative Carba NP results could also be due to the media used. In our study, colonies for the Carba NP test were harvested from MH agar and Literacka et al have recently reported that MH agar from different companies were associated with false-negative results for MBL-producers [72]. High-level resistance to temocillin is a sensitive and specific indicator for the presence of OXA-48-like enzymes [73]. All bla_OXA-48-like–positive isolates in our collection showed high-level resistance (MIC>128 mg/L) to temocillin, but several isolates harboring bla_VIM and bla_NDM also had temocillin MIC >128mg/L showing that testing for synergy with metal chelators (e.g. EDTA or dipicolinic acid) is necessary to discriminate between isolates with OXA-48 and MBLs.

Conclusions

The low prevalence of clinical CPE in Norway is consistent with the general low level of antimicrobial resistance compared with other countries. The relatively low level of antibiotic consumption and the use of narrow spectrum antibiotics [74] have probably contributed to this situation. The low prevalence is also reflected in the epidemiology of Norwegian CPE; mainly associated with importation, exhibiting a broad diversity of genetic backgrounds and carbapenemase variants that mirror the global epidemiology. Only a few cases of secondary spread also support this notion. In order to limit the infection pressure brought by increasing travel and globalization, continued emphasis must be put on diagnostic capabilities, surveillance and infection control.

Supporting information

S1 Table. Strain collection and associated phenotypic and molecular data.

(XLSX)

Data Availability

Whole genome sequence data have been deposited at the National Center for Biotechnology Information (NCBI) under BioProject PRJNA295003. Original data regarding patient details have been aggregated to secure anonymity as approved by the Regional Ethical Committee and the Data Protection Officer at the University Hospital of North Norway. Complete data are available after consideration by the Regional Ethical Committee (rek-nord@asp.uit.no)/Data Protection Officer (personvernombudet@unn.no) for researchers who meet the criteria for access to confidential data. All other data contained within the paper and Supporting Information.

Funding Statement

The study was performed as part of our routine work and the authors received no specific funding for this study. There was no additional external funding received for this study.