Nationwide, population-based observational study of the molecular epidemiology and temporal trend of carbapenemase-producing Enterobacterales in Norway, 2015 to 2021

Introduction National and regional carbapenemase-producing Enterobacterales (CPE) surveillance is essential to understand the burden of antimicrobial resistance, elucidate outbreaks, and develop infection-control or antimicrobial-treatment recommendations. Aim This study aimed to describe CPE and their epidemiology in Norway from 2015 to 2021. Methods A nationwide, population-based observational study of all verified clinical and carriage CPE isolates submitted to the national reference laboratory was conducted. Isolates were characterised by antimicrobial susceptibility testing, whole genome sequencing (WGS) and basic metadata. Annual CPE incidences were also estimated. Results A total of 389 CPE isolates were identified from 332 patients of 63 years median age (range: 0–98). These corresponded to 341 cases, 184 (54%) being male. Between 2015 and 2021, the annual incidence of CPE cases increased from 0.6 to 1.1 per 100,000 person-years. For CPE-isolates with available data on colonisation/infection, 58% (226/389) were associated with colonisation and 38% (149/389) with clinical infections. WGS revealed a predominance of OXA-48-like (51%; 198/389) and NDM (34%; 134/389) carbapenemases in a diversified population of Escherichia coli and Klebsiella pneumoniae, including high-risk clones also detected globally. Most CPE isolates were travel-related (63%; 245/389). Although local outbreaks and healthcare-associated transmission occurred, no interregional spread was detected. Nevertheless, 18% (70/389) of isolates not directly related to import points towards potentially unidentified transmission routes. A decline in travel-associated cases was observed during the COVID-19 pandemic. Conclusions The close-to-doubling of CPE case incidence between 2015 and 2021 was associated with foreign travel and genomic diversity. To limit further transmission and outbreaks, continued screening and monitoring is essential.


Introduction
Carbapenemase production is a mechanism, which allows bacteria (including Enterobacterales) to develop resistance to carbapenems, a group of antibiotics used to treat serious infections. Worldwide, a dramatic increase in infections caused by carbapenemaseproducing Enterobacterales (CPE) has been observed since the early 2000s [1]. This development, which is a concern, is characterised by the occurrence of difficult-to-treat multidrug-resistant (MDR) Escherichia coli and Klebsiella pneumoniae associated with significant morbidity and mortality [2,3]. Consequently, carbapenem-resistant Enterobacterales constitute a major threat to modern healthcare and are listed at the top of the World Health Organization (WHO) priority pathogen list for discovery, research and development of new antibiotics [4]. Fortunately, the development of new β-lactam-β-lactamase inhibitor combinations and cefiderocol has provided alternative treatment options [5,6].
Carbapenemases can be grouped into Ambler class A (e.g. Klebsiella pneumoniae carbapenemase (KPC)), class B (e.g. Verona integron-encoded metallo-βlactamase (VIM), New Delhi metallo-β-lactamase (NDM) and imipenemase (IMP)) and class D (e.g. oxacillinase-48 (OXA-48)) [7]. The relative proportions of carbapenemases show considerable geographical variation across the globe [8]. The genes encoding carbapenemases are most often carried by mobile genetic elements (e.g. plasmids) that can spread horizontally. For CPE, the dissemination mode is complex and comprises expansion of MDR high-risk clones, epidemic plasmids, and transient associations of carbapenemase genes with a diversity of plasmids and clones [9]. In terms of transmission of CPE, this is mainly impelled by healthcare-associated transmission of a small number of clonal lineages [10].
The prevalence of CPE has been low in the Nordic countries and mainly attributed to imported cases of CPE [11,12]. Nonetheless, as an increased prevalence of CPE in clinical samples throughout Europe has been observed since the early 21 st century, we expect a similar development in Norway [13,14]. Thus, it is essential to monitor the national epidemiology of CPE to understand the burden of MDR pathogens as well as to support outbreak surveillance and recommendations for antimicrobial treatment and infection control [15,16].
We have previously described the molecular epidemiology of CPE in Norway during the 2007 to 2014 period [17]. In this study we present essential molecular and epidemiological characteristics of CPE in Norway between 2015 and 2021 including potential inter-and intra-regional transmission.

Study setting and data collection
This is a nationwide, population-based observational study, covering the four health regions (North, Central, West and South-East) of Norway. All confirmed CPE isolates submitted to the Norwegian National Advisory Unit on Detection of Antimicrobial Resistance, which is the national reference laboratory (NRL) of CPE in Norway, during 2015-2021 were included. Private and public clinical microbiology laboratories in Norway are required to submit Enterobacterales to the NRL for confirmation that they are CPE and for surveillance. Submission of isolates is based on the Nordic Committee on Antimicrobial Susceptibility Testing (NordicAST) (www.nordicast.org) CPE algorithm for analysis of carbapenemase-production and includes isolates with a meropenem minimum inhibitory concentration (MIC) of > 0.125 mg/L or disc diffusion zone diameter of < 27 mm (2015-2017)/ < 28 mm (2018-2021). Criteria for CPE screening are described in a national infection control guidance document (https://www. fhi.no/nettpub/smittevernveilederen/) and include (i) screening of patients admitted to healthcare institutions outside the Nordic countries or in institutions in Norway or Nordic countries with an ongoing outbreak in the past 12 months, (ii) previous identification of CPE or living with a person with identified CPE infection/carriage, and (iii) admission to departments (e.g. intensive care unit (ICU), burn-units etc.) with particularly vulnerable patients after local considerations. After CPE-verification, the Norwegian Surveillance System for Communicable Diseases (MSIS) and the referring laboratory are notified, generating a request to the responsible physician to provide clinical patient data to MSIS. For this study, clinical data regarding age, sex (female, male, unknown), foreign travel and clinical infection/screening were retrieved from laboratory requisition forms and MSIS.
In accordance with our previous publication, multiple isolates from the same patient were included if they were (i) of different species, (ii) the same species, but harboured a different carbapenemase gene or sequence type (ST), (iii) or if the isolates were of the same species, ST and harboured the same carbapenemase gene, but were identified more than 1 year apart [17]. A case was defined as a patient positive for one or more CPE within 12 months according to criteria set up by MSIS [18]. Thus, the same patient could represent two cases if the CPE isolate was detected > 12 months from the previous last CPE isolate.

Phenotypic analysis
Species identification was performed using matrixassisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS; Bruker Daltonik GmbH, Bremen, Germany) in combination with whole genome sequencing (WGS). Antimicrobial susceptibility testing (AST) was performed by broth microdilution (BMD) using in-house designed premade Sensititre microtitre plates (TREK Diagnostic Systems/Thermo Fisher Scientific, East Grinstead, United Kingdom). Interpretation was based on European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical MIC breakpoints version 13.0 [19]. MIC data for ceftazidime-avibactam were acquired from 2017 to 2021.

Statistical analysis
Categorical data were summarised as proportions. Incidence rates were determined by dividing the number of annual CPE cases with the population of Norway. For categorical variables, the chi-squared test was used to detect statistical significance. A p value < 0.05 was considered statistically significant. Population data were accessed from Statistics Norway [33].

Carbapenemase-producing Enterobacterales' incidence during 2015-2021
In total, 341 CPE cases in 332 patients were identified. The incidence of CPE cases increased between 2015 to 2021, from 0.6 to 1.1 per 100,000 person-years with a peak of 1.4 in 2019 ( Figure 1A). The incidence also markedly increased compared with previous surveillance period (2007-2014) when the incidence ranged from 0.04 to 0.28 per 100,000 person-years [17]. The mean CPE incidence between the periods increased from 0.13 in 2007-2014 to 0.91 in 2015-2021.
A high genomic diversity was also observed for other Enterobacterales species (Supplementary Table A1) and no indication of clonal spread. Fourteen different STs were found among the 19 Enterobacter spp. isolates with a defined ST (4 isolates non-typable). ST114 was found in three isolates and two isolates each belonged to ST171, ST418 and ST78. Among Citrobacter spp. isolates (n = 10) none of the isolates were of the same ST.

Outbreaks and onward transmission
The genomic data revealed two outbreaks (with 19 cases overall) and cases of onward transmission (including a total of 9 cases). Twelve E. coli ST38-bla OXA-244 isolates represented a clonal intraregional outbreak involving three hospitals in the western region in 2020 [36]. There were no indications that the index case had acquired the outbreak strain from abroad. Six cases were related to infection. Excluding the isolates linked to the outbreak, only 28% (9/32) of the ST38 isolates were associated with import compared with 63% (245/389) overall. However, no clear genetic and epidemiological link could be established between ST38 isolates where 'no import' was reported, as well as no link to and between ST38 isolates related to import or with missing import information. Another, single hospital clonal outbreak of K. pneumoniae ST22 bla OXA-181 (n = 7) was identified in south-east Norway in 2021. Also, in this instance, there was no clear indication that the index case had acquired the outbreak strain outside of Norway and the outbreak continued into 2022. Five cases were associated with infection. Detailed investigations of these outbreaks are ongoing.
In addition, minor clusters of carbapenemaseproducing K. pneumoniae were identified. Two clusters of ST147 isolates: one cluster of two ST147bla NDM-1 isolates identified in 2019 previously linked to nosocomial transmission within Norway not linked to import [37] and one cluster identified in 2021 of three isolates harbouring bla NDM-1 (n = 2) or bla NDM-1 + bla KPC-2 without a clear epidemiological link and no association to travel or missing import information. Four out of the five ST147 cases were cases of infection. The genomic and epidemiological data also indicated two separate cases of possible single case onward transmission of K. pneumoniae ST392-bla OXA-48 imported from Spain. These were identified between 2018 and 2019 and in 2020. One of the four ST392 cases was infected.

Discussion
In this nationwide study we observed an increasing incidence of CPE cases in Norway with cases near doubling during the study period. The mean incidence rate during 2015-2021 (0.91 per 100,000 person-years) is seven-times that of the 2007-2014 period (0.13 per 100,000 person-years) ( Figure 1A) [17]. The increase is concerning and most likely would have been higher if not for the COVID-19-pandemic. Less travel related screening during the pandemic explains the reduction in number of CPE isolates associated with colonisation ( Figure 1C). The basis behind the increase in number of CPE isolates associated with infection in 2020 and 2021 is unclear but is in part due to the two outbreaks. During the pandemic an overall decline in numbers of reported cases of notifiable infectious diseases in Norway has been noticed [38]. Few studies report national or population-based incidence of CPE cases, hindering comparisons between countries. In an international context, the mean CPE-incidence rate of 0.91 cases per 100,000 inhabitants in Norway is likely low and reflects the Nordic situation. Sweden reports a mean incidence of 1.4 cases per 100,000 inhabitants in 2015-2020 [39]. In the Survey on Carbapenemaseproducing Enterobacteriaceae in Europe (EuSCAPE) study, incidence estimates of carbapenemase-producing K. pneumoniae/E. coli per 10,000 hospital admissions were 0.2 in Norway in contrast to > 5 in southern European countries such as Greece and Italy [11]. The relatively low proportion of CPE associated with clinical infections (38%; 149/389) and the identification of only nine bloodstream related infections shows that the burden of invasive CPE infections in Norway is still low. However, the MDR profile (Table) challenges treatment options in each case.
We have previously reported that the CPE epidemiology in Norway is characterised by sporadic cases [40]. Although no interregional spread was observed, cases of healthcare-associated transmission as well as two hospital-related clonal outbreaks indicate a change in the epidemiology. Noteworthy, the proportion of isolates (18%; 70/389) reported as not associated with import were represented by a diversity of genetic backgrounds and carbapenemase variants intermingled in the overall CPE population.
In 2007-2014 only 15% of CPE isolates were identified through screening [17]. The large proportion of CPE identified as colonisation (58%; 226/389) in 2015-2021 is likely due to introduction of revised recommendations for screening of CPE in August 2015. Further, increased awareness and establishment of appropriate diagnostics at the laboratories has likely also contributed. The results support the current broad guidelines for screening before admissions to hospitals. Overall, this underlines the importance of continued vigilance, and that targeted screening is crucial in limiting the spread of CPE.
The high proportion of CPE isolates associated with import (63%; 245/389) and the large diversity of species, carbapenemases and STs likely illustrate the global epidemiology of CPE. OXA-48-like and/or NDMproducing E. coli/K. pneumoniae are the dominant combinations observed in Asia and Europe [11,41] and 90% (220/245) of the isolates associated to import were linked to travel to those continents. A high proportion of CPE in Norway is represented either by specific carbapenemase-genetic background associations (e.g. OXA-244-producing E. coli ST38 and OXA-48-producing K. pneumoniae ST392) or emergence of successful carbapenemase-variants such as NDM-5 in a diversity of genetic backgrounds including global highrisk ExPEC and K. pneumoniae clones [34,35]. Genomic surveillance is important to track these and decrease the possibility for clonal expansion in Norway.
The emergence of OXA-244 in Norway and the high proportion of OXA-48 variants also challenge current diagnostic screening algorithms in CPE detection [42]. In our study, CPE isolates with OXA-244 and other OXA-48-variants had the lowest MIC for meropenem compared with the other groups of carbapenemases ( Figure 3). Most laboratories use disc diffusion as primary AST method. The higher sensitivity of the meropenem disc diffusion breakpoint is likely the reason for identification of OXA-48-like-producers with meropenem MIC below the current MIC breakpoint [43]. Thus, there is also grounds for monitoring the genetic determinants of carbapenemase-production and their phenotypic expression per se to optimise diagnostic CPE-screening.
The study has some limitations, such as the lack of antimicrobial susceptibility data on new antimicrobial agents such as cefiderocol, and the β-lactam-β-lactamase inhibitor combinations meropenem-vaborbactam and imipenem-relebactam. In addition, the susceptibility data were acquired by a broth microdilution method that deviates slightly from the International Organization for Standardization (ISO) standard [44]. Moreover, the travel parameter is associated with uncertainties since there are no clear guidelines for reporting this parameter. The travel parameter includes both travel with and without connection to a healthcare institution abroad and we do not have data on the timeline with respect to the positive CPE sample. The lack of available travel information on 18% of isolates also limits identification of possible sources and transmission chains.
A strength of this study, however, is the national requirement of mandatory reporting of CPE-cases including patient characteristics and submission of suspected CPE-isolates to a reference laboratory from all clinical microbiological laboratories. This ensures national population-based data of confirmed CPEcases. Nevertheless, the observed incidences should be interpreted with caution as there are relatively few CPE cases.

Conclusion
The near doubling of CPE-case incidence rates in Norway is associated with multiple factors including travel abroad and import of epidemiological successful high-risk clones and carbapenemase-variants. The relative high proportion of cases (18%; 70/389) not directly related to import underlines the potential importance of unidentified transmission routes. The single hospital and intraregional outbreaks are of concern. The downward incidence rate and travel-associated cases during the COVID-19 pandemic is likely to be reversed as international travel increases. Generic infection control measures and adherence to rectal screening recommendations as well as close monitoring of the molecular CPE epidemiology is of particular importance to avoid further transmission, outbreaks and establishment of CPE in Norway.

Ethical statement
The study was approved by the Regional Committee for Medical and Health Research Ethics (reference no. 29292).

Funding statement
This work was performed as part of national surveillance obligations. OL is supported by funding from the Tegger and Stig and Ragna Gorthon foundations.

Data availability
Sequence data are deposited in the European Nucleotide Archive under Bioproject PRJEB56146.