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Background Since 2008, Danish national surveillance of Clostridioides difficile has focused on binary toxin-positive strains in order to monitor epidemic types such as PCR ribotype (RT) 027 and 078. Additional surveillance is needed to provide a more unbiased representation of all strains from the clinical reservoir. Aim Setting up a new sentinel surveillance scheme for an improved understanding of type distribution relative to time, geography and epidemiology, here presenting data from 2016 to 2019. Methods For 2─4 weeks in spring and autumn each year between 2016 and 2019, all 10 Danish Departments of Clinical Microbiology collected faecal samples containing toxigenic C. difficile. Isolates were typed at the national reference laboratory at Statens Serum Institut. The typing method in 2016–17 used tandem-repeat-sequence typing, while the typing method in 2018–19 was whole genome sequencing. Results During the study period, the sentinel surveillance scheme included ca 14–15% of all Danish cases of C. difficile infections. Binary toxin-negative strains accounted for 75% and 16 of the 20 most prevalent types. The most common sequence types (ST) were ST2/13 (RT014/020) (19.5%), ST1 (RT027) (10.8%), ST11 (RT078) (6.7%), ST8 (RT002) (6.6%) and ST6 (RT005/117) (5.1%). The data also highlighted geographical differences, mostly related to ST1 and temporal decline of ST1 (p = 0.0008) and the increase of ST103 (p = 0.002), ST17 (p = 0.004) and ST37 (p = 0.003), the latter three binary toxin-negative. Conclusion Sentinel surveillance allowed nationwide monitoring of geographical differences and temporal changes in C. difficile infections in Denmark, including emerging types, regardless of binary toxin status.

Background Carbapenemase-producing Escherichia coli are increasing worldwide. In recent years, an increase in OXA-244-producing E. coli isolates has been seen in the national surveillance of carbapenemase-producing organisms in Denmark. Aim Molecular characterisation and epidemiological investigation of OXA-244-producing E. coli isolates from January 2016 to August 2019. Methods For the epidemiological investigation, data from the Danish National Patient Registry and the Danish register of civil registration were used together with data from phone interviews with patients. Isolates were characterised by analysing whole genome sequences for resistance genes, MLST and core genome MLST (cgMLST). Results In total, 24 OXA-244-producing E. coli isolates were obtained from 23 patients. Among the 23 patients, 13 reported travelling before detection of the E. coli isolates, with seven having visited countries in Northern Africa. Fifteen isolates also carried an extended-spectrum beta-lactamase gene and one had a plasmid-encoded AmpC gene. The most common detected sequence type (ST) was ST38, followed by ST69, ST167, ST10, ST361 and ST3268. Three clonal clusters were detected by cgMLST, but none of these clusters seemed to reflect nosocomial transmission in Denmark. Conclusion Import of OXA-244 E. coli isolates from travelling abroad seems likely for the majority of cases. Community sources were also possible, as many of the patients had no history of hospitalisation and many of the E. coli isolates belonged to STs that are present in the community. It was not possible to point at a single country or a community source as risk factor for acquiring OXA-244-producing E. coli.

Background Carbapenemase-producing Escherichia coli are increasing worldwide. In recent years, an increase in OXA-244-producing E. coli isolates has been seen in the national surveillance of carbapenemase-producing organisms in Denmark. Aim Molecular characterisation and epidemiological investigation of OXA-244-producing E. coli isolates from January 2016 to August 2019. Methods For the epidemiological investigation, data from the Danish National Patient Registry and the Danish register of civil registration were used together with data from phone interviews with patients. Isolates were characterised by analysing whole genome sequences for resistance genes, MLST and core genome MLST (cgMLST). Results In total, 24 OXA-244-producing E. coli isolates were obtained from 23 patients. Among the 23 patients, 13 reported travelling before detection of the E. coli isolates, with seven having visited countries in Northern Africa. Fifteen isolates also carried an extended-spectrum beta-lactamase gene and one had a plasmid-encoded AmpC gene. The most common detected sequence type (ST) was ST38, followed by ST69, ST167, ST10, ST361 and ST3268. Three clonal clusters were detected by cgMLST, but none of these clusters seemed to reflect nosocomial transmission in Denmark. Conclusion Import of OXA-244 E. coli isolates from travelling abroad seems likely for the majority of cases. Community sources were also possible, as many of the patients had no history of hospitalisation and many of the E. coli isolates belonged to STs that are present in the community. It was not possible to point at a single country or a community source as risk factor for acquiring OXA-244-producing E. coli.

Objectives: To evaluate a genome-based surveillance of all Danish third-generation cephalosporin-resistant Escherichia coli (3GC-R Ec ) from bloodstream infections between 2014 and 2015, focusing on horizontally transferable resistance mechanisms.Methods: A collection of 552 3GC-R Ec isolates were whole-genome sequenced and characterized by using the batch uploader from the Center for Genomic Epidemiology (CGE) and automatically analysed using the CGE tools according to resistance profile, MLST, serotype and fimH subtype. Additionally, the phylogenetic relationship of the isolates was analysed by SNP analysis.Results: The majority of the 552 isolates were ESBL producers (89%), with bla CTX-M-15 being the most prevalent (50%) gene, followed by bla CTX-M-14 (14%), bla CTX-M-27 (11%) and bla CTX-M-101 (5%). ST131 was detected in 50% of the E. coli isolates, with the remaining isolates belonging to 73 other STs, including globally disseminated STs (e.g. ST10, ST38, ST58, ST69 and ST410). Five of the bloodstream isolates were carbapenemase producers, carrying bla OXA-181 (3) and bla OXA-48 (2). Phylogenetic analysis revealed 15 possible national outbreaks during the 2 year period, one caused by a novel ST131/ bla CTX-M-101 clone, here observed for the first time in Denmark. Additionally, the analysis revealed three individual cases with possible persistence of closely related clones collected more than 13 months apart.Conclusions: Continuous WGS-based national surveillance of 3GC-R Ec , in combination with more detailed epidemiological information, can improve the ability to follow the population dynamics of 3GC-R Ec , thus allowing for the detection of potential outbreaks and the effects of changing treatment regimens in the future.

We describe clonal shifts in vanA Enterococcus faecium isolates from clinical samples obtained from patients in Denmark from 2015 to the first quarter (Q1) of 2019. During Q1 2019, the vancomycin-variable enterococci (VVE) ST1421-CT1134 vanA E. faecium became the most dominant vanA E. faecium clone and has spread to all five regions in Denmark. Among 174 E. faecium isolates with vanA, vanB or vanA/vanB genes in Q1 2019, 44% belonged to this type. We describe the clonal shift for vanA Enterococcus faecium during the last 4 years and the national spread of a vancomycin-variable vanA E. faecium ST1421-CT1134 clone in Denmark. The aim is to highlight the importance of using molecular methods for detecting vancomycin-variable enterococci (VVE), and to alert other countries about this emerging nosocomial clone.

Objectives: The aim of this study was to identify clonally-related carbapenemase-producing Klebsiella pneumoniae complex members that could be involved in outbreaks among hospitalized patients in Denmark, and to identify possible epidemiological links. Methods: From January 2014 to June 2018, 103 isolates belonging to the K. pneumoniae complex were collected from 102 patients. From the whole-genome sequencing (WGS) data, presence of genes encoding carbapenemase and multilocal sequence typing (MLST) data were extracted. Core genome MLST (cgMLST) cluster analysis was performed. Using data from the Danish National Patient Registry (DNPR) and reported travel history, presumptive outbreaks were investigated for possible epidemiological links. Results: The most common detected carbapenemase gene was bla OXA-48, followed by bla NDM-1. The 103 K. pneumoniae complex isolates belonged to 47 sequence types (STs) and cgMLST subdivided the isolates into 80 different complex types. cgMLST identified 13 clusters with 2-4 isolates per cluster. For five of the 13 clusters, a direct link (the patients stayed at the same ward on the same day) could be detected between at least some of the patients. In two clusters, the patients resided simultaneously at the same hospital, but not the same ward. A possible link (same ward within 1-13 days) was detected for the patients in one cluster. For five clusters detected by cgMLST, no epidemiological link could be detected using data from DNPR. Conclusion: In this study, cgMLST combined with patient hospital admission data and travel information was found to be a reliable and detailed approach to detect possible clonal transmission of carbapenemase-producing K. pneumoniae complex members.

OBJECTIVES: To investigate the usefulness of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) typing as a first-line epidemiological tool in a nosocomial outbreak of vancomycin-resistant Enterococcus faecium (VREfm).METHODS: Fifty-five VREfm isolates, previously characterized by whole-genome sequencing (WGS), were included and analysed by MALDI-TOF MS. To take peak reproducibility into account, ethanol/formic acid extraction and other steps of the protocol were conducted in triplicate. Twenty-seven spectra were generated per isolate, and spectra were visually inspected to determine discriminatory peaks. The presence or absence of these was recorded in a peak scheme.RESULTS: Nine discriminatory peaks were identified. A characteristic pattern of these could distinguish between the three major WGS groups: WGS I, WGS II and WGS III. Only one of 38 isolates belonging to WGS I, WGS II or WGS III was misclassified. However, ten of the 17 isolates not belonging to WGS I, II or III displayed peak patterns indistinguishable from those of the outbreak strain.CONCLUSIONS: Using visual inspection of spectra, MALDI-TOF MS typing proved to be useful in differentiating three VREfm outbreak clones from each other. However, as non-outbreak isolates could not be reliably differentiated from outbreak clones, the practical value of this typing method for VREfm outbreak management was limited in our setting.

Objectives: Bacteria with common microbiological and clinical characteristics are often recognized as a particular group. The acronym HACEK stands for five fastidious genera associated with infective endocarditis (Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella, and Kingella). Data on the epidemiology of HACEK are sparse. This article reports a 6-year nationwide study of HACEK bacteraemia in Denmark. Methods: Cases of HACEK bacteraemia occurring during the years 2010–2015 were retrieved from the national Danish microbiology database, covering an average surveillance population of 5.6 million per year. Results: A total of 147 cases of HACEK bacteraemia were identified, corresponding to an annual incidence of 0.44 per 100 000 population. The annual incidence for males was 0.56 per 100 000 and for females was 0.31 per 100 000. The median age was 56 years (range 0–97 years), with variation among the genera. One hundred and forty-three isolates were identified to the species level and six to the genus level: Haemophilus spp, n = 55; Aggregatibacter spp, n = 37; Cardiobacterium spp, n = 9; Eikenella corrodens n = 21; and Kingella spp, n = 27. Conclusions: This is the first study on the incidence of HACEK bacteraemia in a large surveillance population and may inspire further studies on the HACEK group. Haemophilus spp other than Haemophilus influenzae accounted for most cases of HACEK bacteraemia in Denmark, with Aggregatibacter spp in second place. Keywords: Epidemiology, Incidence, Age, Sex, Haemophilus, Aggregatibacter

Objectives: To describe the changing epidemiology of vancomycin-resistant Enterococcus faecium and Enterococcus faecalis in clinical samples in Denmark 2005-15 according to species and van type, and, furthermore, to investigate the genetic relatedness of the clinical E. faecium isolates from 2015.Methods: During 2005-14, all clinical VRE isolates were tested for the presence of vanA/B/C genes by PCR. In 2015, all clinical VRE isolates were whole-genome sequenced. From the WGS data, the presence of van genes and MLST STs were extracted in silico . Core-genome MLST (cgMLST) analysis was performed for the vancomycin-resistant E. faecium isolates.Results: During 2005-15, 1043 vanA E. faecium , 25 vanB E. faecium , 4 vanA E. faecalis and 28 vanB E. faecalis were detected. The number of VRE was 200 isolates/year in 2013-15. In 2015, 368 vanA E. faecium and 1 vanB E. faecium were detected along with 1 vanA E. faecalis and 1 vanB E. faecalis . cgMLST subdivided the 368 vanA E. faecium isolates into 33 cluster types (CTs), whereas the vanB E. faecium isolate belonged to a different CT. ST203-CT859 was most prevalent (51%), followed by ST80-CT14 (22%), ST117-CT24 (6%), ST80-CT866 (4%) and ST80-CT860 (2%). Comparison with the cgMLST.org database, previous studies and personal communications with neighbouring countries revealed that the novel cluster ST203-CT859 emerged in December 2014 and spread to the south of Sweden and the Faroe Islands during 2015.Conclusions: VRE increased in Denmark during 2005-15 due to the emergence of several vanA E. faecium clones.

OBJECTIVES: In Denmark, the incidence of vancomycin-resistant Enterococcus faecium (VREfm) has increased since 2012. The aim of this study was to investigate the epidemiology and clonal relatedness of VREfm isolates in Danish hospitals in 2012-13 using WGS. The second aim was to evaluate if WGS-based typing could replace PFGE for typing of VREfm.METHODS: A population-based study was conducted including all VREfm isolates submitted for national surveillance from January 2012 to April 2013. All isolates were investigated by WGS, MLST and PFGE.RESULTS: One-hundred and thirty-two isolates were included. The majority of the isolates were from clinical samples (77%). Gastroenterology/abdominal surgery (29%) and ICUs (29%) were the predominant departments with VREfm. Genomics revealed a polyclonal structure of the VREfm outbreak. Seven subgroups of 3-44 genetically closely related isolates (separated by CONCLUSIONS: This study emphasizes the importance of infection control measures to limit transmission of VREfm between patients. However, the diversity of the VREfm isolates points to the fact that other important factors may also affect the VREfm increase in Denmark. Finally, WGS is suitable for typing of VREfm and has replaced PFGE for typing of VREfm in Denmark.