Surveillance of vancomycin-resistant enterococci reveals shift in dominating clusters from vanA to vanB Enterococcus faecium clusters, Denmark, 2015 to 2022.

BackgroundVancomycin-resistant enterococci (VRE) are increasing in Denmark and Europe. Linezolid and vancomycin-resistant enterococci (LVRE) are of concern, as treatment options are limited. Vancomycin-variable enterococci (VVE) harbour the vanA gene complex but are phenotypically vancomycin-susceptible.AimThe aim was to describe clonal shifts for VRE and VVE in Denmark between 2015 and 2022 and to investigate genotypic linezolid resistance among the VRE and VVE.MethodsFrom 2015 to 2022, 4,090 Danish clinical VRE and VVE isolates were whole genome sequenced. We extracted vancomycin resistance genes and sequence types (STs) from the sequencing data and performed core genome multilocus sequence typing (cgMLST) analysis for Enterococcus faecium. All isolates were tested for the presence of mutations or genes encoding linezolid resistance.ResultsIn total 99% of the VRE and VVE isolates were E. faecium. From 2015 through 2019, 91.1% of the VRE and VVE were vanA E. faecium. During 2020, to the number of vanB E. faecium increased to 254 of 509 VRE and VVE isolates. Between 2015 and 2022, seven E. faecium clusters dominated: ST80-CT14 vanA, ST117-CT24 vanA, ST203-CT859 vanA, ST1421-CT1134 vanA (VVE cluster), ST80-CT1064 vanA/vanB, ST117-CT36 vanB and ST80-CT2406 vanB. We detected 35 linezolid vancomycin-resistant E. faecium and eight linezolid-resistant VVEfm.ConclusionFrom 2015 to 2022, the numbers of VRE and VVE increased. The spread of the VVE cluster ST1421-CT1134 vanA E. faecium in Denmark is a concern, especially since VVE diagnostics are challenging. The finding of LVRE, although in small numbers, ia also a concern, as treatment options are limited.

Three cases of Cutibacterium avidum prosthetic valve infective endocarditis at a single heart center.

OBJECTIVE: To resolve an exceptional clustering of Cutibacterium avidum prosthetic valve infective endocarditis at a single heart center. METHODS: During a period of 21 months, three patients experienced C. avidum bacteraemia 24-128 days after aortic valve replacement. Operative procedures and electronic prescriptions of antimicrobials were surveyed, and bacterial isolates were genome sequenced. RESULTS: The prosthetic valves were inserted by separate surgical teams. In one case, echocardiographic confirmation of infective endocarditis was not achieved until four months after the first positive blood culture, but the causative agents were irrefutably documented in all cases by culture, or amplification of bacterial DNA, from removed prosthetic material. Whole genome sequencing clustered isolates to a distinctive subgroup of the species, but did not suggest inter-patient transmission of isolates. CONCLUSIONS: Despite vigorous sampling of blood and tissue, detection of C. avidum was not unconditional, neither by culture nor PCR. The causative agent is likely underreported and should be meticulously searched for in culture-negative prosthetic valve endocarditis.

Gonorrhoea on the rise in Denmark since 2022: distinct clones drive increase in heterosexual individuals.

A surge in gonorrhoea in Denmark has occurred since 2022, a 46% increase from 2021. National surveillance, leveraging mandatory reporting and epidemiological data, highlights three distinct clades linked to heterosexual transmission. Despite the rise, these exhibit high susceptibility, contrasting MSM-associated strains. Geographical hotspots and age-specific patterns further illuminate transmission dynamics. The combination of genomic and epidemiological data provides novel insights into the evolving landscape of gonorrhoea, indicating potential shifts in infection dynamics and transmissibility.

Increase in invasive group A streptococcal infections and emergence of novel, rapidly expanding sub-lineage of the virulent Streptococcus pyogenes M1 clone, Denmark, 2023.

A highly virulent sub-lineage of the Streptococcus pyogenes M1 clone has been rapidly expanding throughout Denmark since late 2022 and now accounts for 30% of the new invasive group A streptococcal infections. We aimed to investigate whether a shift in variant composition can account for the high incidence rates observed over winter 2022/23, or if these are better explained by the impact of COVID-19-related restrictions on population immunity and carriage of group A Streptococcus.

Characterisation of carbapenemase-producing Acinetobacter baumannii isolates from danish patients 2014-2021: detection of a new international clone - IC11.

OBJECTIVES: This study aimed to characterise carbapenemase-producing Acinetobacter baumannii (A. baumannii) isolates from Danish patients using whole genome sequencing (WGS). It also compared typing and epidemiological data for further investigation of the spread and origin of the carbapenemase-producing A. baumannii isolates. METHODS: From 1 January 2014 to 30 September 2021, 141 carbapenemase-producing A. baumannii isolates, received at the national reference laboratory at Statens Serum Institut, were investigated using WGS. Multilocus sequence typing (MLST) and cgMLST data, obtained by SeqSphere+ software, were linked to data related to source of isolation, patient age and sex, hospital admission and travel history. RESULTS: Most of the carbapenemase-producing A. baumannii isolates were from males (n = 100, 71%). Most patients (n = 88, 63%) had travelled outside Scandinavia before admission to a Danish hospital. The most prevalent carbapenemase gene was blaOXA-23 (n = 124). Isolates belonging to the dominating international clone IC2 accounted for 78% of all isolates. A new international ST164/OXA-91 clone, proposed to be named IC11, was recognised and described. The cgMLST analysis revealed 17 clusters, reflecting both sporadic travel to similar geographical areas and confirmed outbreaks in Danish hospitals. CONCLUSIONS: The occurrence of carbapenemase-producing A. baumannii in Denmark was still low; however, isolates belonging to major international clones with a high potential to spread within hospitals, mainly IC2, dominated. OXA-23 was by far the most prevalent carbapenemase detected. Sporadic and travel-related introductions to Danish hospitals, also intra-hospital transmission, could be confirmed, emphasising the need for continuing vigilance.

Azole resistance in Aspergillus fumigatus. The first 2-year's Data from the Danish National Surveillance Study, 2018-2020.

BACKGROUND: Azole resistance complicates treatment of patients with invasive aspergillosis with an increased mortality. Azole resistance in Aspergillus fumigatus is a growing problem and associated with human and environmental azole use. Denmark has a considerable and highly efficient agricultural sector. Following reports on environmental azole resistance in A. fumigatus from Danish patients, the ministry of health requested a prospective national surveillance of azole-resistant A. fumigatus and particularly that of environmental origin. OBJECTIVES: To present the data from the first 2 years of the surveillance programme. METHODS: Unique isolates regarded as clinically relevant and any A. fumigatus isolated on a preferred weekday (background samples) were included. EUCAST susceptibility testing was performed and azole-resistant isolates underwent cyp51A gene sequencing. RESULTS: The azole resistance prevalence was 6.1% (66/1083) at patient level. The TR34 /L98H prevalence was 3.6% (39/1083) and included the variants TR34 /L98H, TR34 3 /L98H and TR34 /L98H/S297T/F495I. Resistance caused by other Cyp51A variants accounted for 1.3% (14/1083) and included G54R, P216S, F219L, G54W, M220I, M220K, M220R, G432S, G448S and Y121F alterations. Non-Cyp51A-mediated resistance accounted for 1.2% (13/1083). Proportionally, TR34 /L98H, other Cyp51A variants and non-Cyp51A-mediated resistance accounted for 59.1% (39/66), 21.2% (14/66) and 19.7% (13/66), respectively, of all resistance. Azole resistance was detected in all five regions in Denmark, and TR34 /L98H specifically, in four of five regions during the surveillance period. CONCLUSION: The azole resistance prevalence does not lead to a change in the initial treatment of aspergillosis at this point, but causes concern and leads to therapeutic challenges in the affected patients.

Update 2016-2018 of the Nationwide Danish Fungaemia Surveillance Study: Epidemiologic Changes in a 15-Year Perspective.

As part of a national surveillance programme initiated in 2004, fungal blood isolates from 2016-2018 underwent species identification and EUCAST susceptibility testing. The epidemiology was described and compared to data from previous years. In 2016-2018, 1454 unique isolates were included. The fungaemia rate was 8.13/100,000 inhabitants compared to 8.64, 9.03, and 8.38 in 2004-2007, 2008-2011, and 2012-2015, respectively. Half of the cases (52.8%) involved patients 60-79 years old and the incidence was highest in males ≥70 years old. Candida albicans accounted for 42.1% of all isolates and Candida glabrata for 32.1%. C. albicans was more frequent in males (p = 0.03) and C. glabrata in females (p = 0.03). During the four periods, the proportion of C. albicans decreased (p < 0.001), and C. glabrata increased (p < 0.001). Consequently, fluconazole susceptibility gradually decreased from 68.5% to 59.0% (p < 0.001). Acquired fluconazole resistance was found in 4.6% Candida isolates in 2016-2018. Acquired echinocandin resistance increased during the four periods 0.0%, 0.6%, 1.7% to 1.5% (p < 0.0001). Sixteen echinocandin-resistant isolates from 2016-2018 harboured well-known FKS resistance-mutations and one echinocandin-resistant C. albicans had an FKS mutation outside the hotspot (P1354P/S) of unknown importance. In C. glabrata specifically, echinocandin resistance was detected in 12/460 (2.6%) in 2016-2018 whereas multidrug-class resistance was rare (1/460 isolates (0.2%)). Since the increase in incidence during 2004-2011, the incidence has stabilised. In contrast, the species distribution has changed gradually over the 15 years, with increased C. glabrata at the expense of C. albicans. The consequent decreased fluconazole susceptibility and the emergence of acquired echinocandin resistance complicates the management of fungaemia and calls for antifungal drug development.

Molecular characterization of Danish ESBL/AmpC-producing Klebsiella pneumoniae from bloodstream infections, 2018.

OBJECTIVES: The aim of the study was to molecularly characterize third-generation cephalosporin-resistant Klebsiella pneumoniae isolated from bloodstream infections in Denmark in 2018 using whole-genome sequencing (WGS) data, and to compare these isolates to the most common clones detected in 2006 and 2008. METHODS: Sixty-two extended-spectrum beta-lactamase (ESBL)/AmpC-producing K. pneumoniae isolates from Danish blood cultures from 2018 were analysed using WGS to obtain multilocus sequence typing (MLST), core genome MLST (cgMLST), resistance profile and phylogeny. These were compared to the most common ESBL K. pneumoniae clones detected in 2006 and 2008. RESULTS: The most common ESBL clone was ST15 CTX-M-15, the DHA-1 enzyme was the most common in AmpC isolates, and the OXA-48-like group was the most common carbapenemase. Thirty-nine different sequence types (STs) were found, with the most frequent being ST14, ST15 and ST37, accounting for 24% of the isolates. The isolates were subdivided into 55 complex types (CTs) of which 49 were singletons, with the most frequent being ST14-CT2080. Two of the CTX-M-15-producing isolates from 2018 belonged to the ST15-CT105/CT3078 clone, which was first detected in 2006. CONCLUSIONS: The ESBL/AmpC K. pneumoniae isolates detected in Danish blood cultures belonged to many different types. No dominant clones were circulating in Danish hospitals, but the ST15-CT105/CT3078 CTX-M-15 K. pneumoniae clone was seen 13 years after its first detection.

Investigation of possible clonal transmission of carbapenemase-producing Klebsiella pneumoniae complex member isolates in Denmark using core genome MLST and National Patient Registry Data.

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 blaOXA-48, followed by blaNDM-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.

Community-acquired meningitis caused by beta-haemolytic streptococci in adults: a nationwide population-based cohort study.

The objective of this study was to examine the clinical presentation of community-acquired beta-haemolytic streptococcal (BHS) meningitis in adults. This is a nationwide population-based cohort study of adults (≥ 16 years) with BHS meningitis verified by culture or polymerase chain reaction of the cerebrospinal fluid (CSF) from 1993 to 2005. We retrospectively evaluated clinical and laboratory features and assessed outcome by Glasgow Outcome Scale (GOS). We identified 54 adults (58% female) with a median age of 65 years (IQR 55-73). Mean incidence rate was 0.7 cases per 1,000,000 person-years. Alcohol abuse was noted among 11 (20%) patients. Group A streptococci (GAS) were found in 17 (32%) patients, group B (GBS) in 18 (34%), group C (GCS) in four (8%) and group G (GGS) in 14 (26%). Patients with GAS meningitis often had concomitant otitis media (47%) and mastoiditis (30%). Among patients with GBS, GCS or GGS meningitis, the most frequent concomitant focal infections were bone and soft tissue infections (19%) and endocarditis (16%). In-hospital mortality was 31% (95% CI 19-45), and 63% (95% CI 49-76) had an unfavourable outcome at discharge (GOS < 5). BHS meningitis in adults is primarily observed among the elderly and has a poor prognosis. GAS meningitis is primarily associated with concomitant ear-nose-throat infection.

Treatment of candidemia in a nationwide setting: increased survival with primary echinocandin treatment.

BACKGROUND: In accordance with international guidelines, primary antifungal treatment (AFT) of candidemia with echinocandins has been nationally recommended in Denmark since 2009. Our nationwide cohort study describes the management of candidemia treatment focusing on the impact of prophylactic AFT on species distribution, the rate of adherence to the recommended national guidelines for AFT, and the effect of AFT on patient outcomes. MATERIALS AND METHODS: Incident candidemia cases from a 2-year period, 2010-2011, were included. Information on AFT was retrospectively collected from patient charts. Vital status was obtained from the Danish Civil Registration System. HRs of mortality were reported with 95% CIs using Cox regression. RESULTS: A total of 841 candidemia patients was identified. Prior to candidemia diagnosis, 19.3% of patients received AFT (162/841). The risk of non-albicans candidemia increased after prior AFT (59.3% vs 45.5% among nontreated). Echinocandins as primary AFT were given for 44.2% (302/683) of patients. Primary treatment with echinocandins resulted in adequate treatment in a higher proportion of patients (97.7% vs 72.1%) and was associated with lower 0- to 14-day mortality compared with azole treatment (adj. HR 0.76, 95% CI: 0.55-1.06). Significantly lower 0- to 14-day mortality was observed for patients with Candida glabrata and Candida krusei with echinocandin treatment compared with azole treatment (adj. HR 0.50, 95% CI: 0.28-0.89), but not for patients with Candida albicans or Candida tropicalis. CONCLUSION: The association shown between prior AFT and non-albicans species underlines the importance of treatment history when selecting treatment for candidemia. Compliance with national recommendations was low, but similar to previously reported international rates. Primary treatment of candidemia with echinocandins compared with azoles yielded both a higher proportion of adequately treated patients and improved mortality rates. This real-life setting supports guidelines recommendation, and further focus on compliance with these seems warranted.

Incidence of HACEK bacteraemia in Denmark: A 6-year population-based study.

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 100000 population. The annual incidence for males was 0.56 per 100000 and for females was 0.31 per 100000. 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.

Emergence of vanA Enterococcus faecium in Denmark, 2005-15.

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 <50 isolates/year until 2012 to > 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.

WGS-based surveillance of third-generation cephalosporin-resistant Escherichia coli from bloodstream infections in Denmark.

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.

Multiple hospital outbreaks of vanA Enterococcus faecium in Denmark, 2012-13, investigated by WGS, MLST and PFGE.

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 <17 SNPs) were identified using WGS. Direct or indirect transmission of VREfm between patients and intra- and inter-regional spreading clones was observed. We identified 10 STs. PFGE identified four major clusters (13-43 isolates) and seven minor clusters (two to three isolates). The results from the typing methods were highly concordant. However, WGS-based typing had the highest discriminatory power. 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.

Performance of matrix-assisted laser desorption-time of flight mass spectrometry for identification of clinical yeast isolates.

Accurate and fast yeast identification is important when treating patients with invasive fungal disease as susceptibility to antifungal agents is highly species related. Matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF-MS) provides a powerful tool with a clear potential to improve current diagnostic practice. Two MALDI-TOF-MS-systems (BioTyper/Bruker and Saramis/AXIMA) were evaluated using: (i) A collection of 102 archived, well characterised yeast isolates representing 14 different species and (ii) Prospectively collected isolates obtained from clinical samples at two participating laboratories. Of the 102 archived isolates, 81 (79%) and 92 (90%) were correctly identified by Saramis/AXIMA and BioTyper/Bruker respectively. Saramis/AXIMA was unable to separate Candida albicans, C. africana and C. dubliniensis in 13 of 32 isolates. After manual interpretation of the mass spectra output, all 13 isolates were correctly identified, resulting in an overall identification performance of 92%. No misidentifications occurred with the two systems. Of the routine isolates one laboratory identified 99/99 (100%) and 90/99 (91%) to species level by Saramis/Axima and conventional identification, respectively, whereas the other laboratory identified 83/98 (85%) to species level by both BioTyper/Bruker and conventional identification. Both MALDI-TOF-MS systems are fast, have built-in databases that cover the majority of clinically relevant Candida species, and have an accuracy that outperforms our conventional identification systems.