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.

Contaminated dicloxacillin capsules as the source of an NDM-5/OXA-48-producing Enterobacter hormaechei ST79 outbreak, Denmark and Iceland, 2022 and 2023.

From October 2022 through January 2023, nine patients with NDM-5/OXA-48-carbapenemase-producing Enterobacter hormaechei ST79 were detected in Denmark and subsequently one patient in Iceland. There were no nosocomial links between patients, but they had all been treated with dicloxacillin capsules. An NDM-5/OXA-48-carbapenemase-producing E. hormaechei ST79, identical to patient isolates, was cultured from the surface of dicloxacillin capsules in Denmark, strongly implicating them as the source of the outbreak. Special attention is required to detect the outbreak strain in the microbiology laboratory.

Bloodstream Infections in Children With Cancer: Pathogen Distribution and Antimicrobial Susceptibility Patterns Over a 10-Year Period.

Bloodstream infections (BSIs) adversely affect clinical outcome in children with cancer. Over 1 decade, this retrospective cohort study describes pathogen distribution in BSIs and antimicrobial susceptibility against empirical antibiotics frequently prescribed in children with cancer. The antibiotic efficacy was evaluated through the determination of minimal inhibitory concentrations for piperacillin-tazobactam and meropenem and by disk diffusion for remaining antibiotics. From 2004 to 2013, 398 BSIs occurred in 196 children with cancer (median age: 5.4 y), resulting in 457 bacteria. Overall, 266 (58.2%) were Gram-positive, and 191 (41.8%) were Gram-negative with a significant Gram-positive increase over time (P=0.032). Coagulase-negative staphylococci (74, 16.2%), viridans group streptococci (67, 14.7%), Escherichia coli (52, 11.4%), and Staphylococcus aureus (39, 8.5%) were the most common pathogens. Susceptibility to piperacillin-tazobactam (95.9%, P=0.419) and meropenem (98.9%, P=0.752) was stable over time, and resistance was observed among viridans group streptococci against piperacillin-tazobactam (18%) and meropenem (7%) and among Enterobacterales against piperacillin-tazobactam (3%). Vancomycin showed 98% Gram-positive activity, gentamicin 82% Gram-negative activity and ampicillin, cefotaxime, and cefuroxime were active in 50%, 72%, and 69% of pathogens, respectively, and BSI-related mortality was 0%. In conclusion, over 1 decade, we report an increase in Gram-positive BSIs, and stable, low-resistance rates against currently recommended empirical antibiotics, piperacillin-tazobactam and meropenem.

Faecal microbiota transplantation for eradication of co-infection with Clostridioides difficile and extensively drug-resistant KPC-producing Klebsiella pneumoniae.

Clostridioides difficile infection may be complicated by co-infection with other pathogens. We here describe the successful use of faecal microbiota transplantation to eradicate concomitant C. difficile and extensively drug-resistant (XDR) KPC-producing Klebsiella pneumoniae. Donor microbiota efficiently engrafted in the patient, and a donor-like microbial assemblage persisted in the patient during six months follow-up. The report explores the potential for the donor microbiota to eradicate and replace multi-resistant microorganisms.

Meropenem to Children With Febrile Neutropenia Induces Monoresistant Pseudomonas aeruginosa.

Antimicrobial resistance in Pseudomonas aeruginosa is a threat to children with cancer. We explored the association between P. aeruginosa resistance and previous antibiotic exposure. All children with cancer and P. aeruginosa bacteremia in 2007 to 2016 in Denmark, a country with an overall resistance rate of ∼3%, were included. Twenty percent (10/49) of isolates from children previously exposed to meropenem were meropenem nonsusceptible. The only significant risk factor of meropenem nonsusceptibility was previous meropenem therapy (P=0.03). On the basis of these results, we suggest that meropenem should be reserved as a last resort for children with febrile neutropenia in countries with low antimicrobial resistance.

Surveillance of OXA-244-producing Escherichia coli and epidemiologic investigation of cases, Denmark, January 2016 to August 2019.

BackgroundCarbapenemase-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.AimMolecular characterisation and epidemiological investigation of OXA-244-producing E. coli isolates from January 2016 to August 2019.MethodsFor 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).ResultsIn 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.ConclusionImport 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.

Population pharmacokinetics of piperacillin in febrile children receiving cancer chemotherapy: the impact of body weight and target on an optimal dosing regimen.

BACKGROUND: The ß-lactam antibiotic piperacillin (in combination with tazobactam) is commonly chosen for empirical treatment of suspected bacterial infections. However, pharmacokinetic variability among patient populations and across ages leads to uncertainty when selecting a dosing regimen to achieve an appropriate pharmacodynamic target. OBJECTIVES: To guide dosing by establishing a population pharmacokinetic model for unbound piperacillin in febrile children receiving cancer chemotherapy, and to assess pharmacokinetic/pharmacodynamic target attainment (100% fT > 1×MIC and 50% fT > 4×MIC) and resultant exposure, across body weights. METHODS: Forty-three children admitted for 89 febrile episodes contributed 482 samples to the pharmacokinetic analysis. The typical doses required for target attainment were compared for various dosing regimens, in particular prolonged infusions, across MICs and body weights. RESULTS: A two-compartment model with inter-fever-episode variability in CL, and body weight included through allometry, described the data. A high CL of 15.4 L/h (70 kg) combined with high glomerular filtration rate (GFR) values indicated rapid elimination and hyperfiltration. The target of 50% fT > 4×MIC was achieved for an MIC of 4.0 mg/L in a typical patient with extended infusions of 2-3 (q6h) or 3-4 (q8h) h, at or below the standard adult dose (75 and 100 mg/kg/dose for q6h and q8h, respectively). Higher doses or continuous infusion were needed to achieve 100% fT > 1×MIC due to the rapid piperacillin elimination. CONCLUSIONS: The licensed dose for children with febrile neutropenia (80 mg/kg q6h as a 30 min infusion) performs poorly for attainment of fT>MIC pharmacokinetic/pharmacodynamic targets. Given the population pharmacokinetic profile, feasible dosing regimens with reasonable exposure are continuous infusion (100% fT > 1×MIC) or prolonged infusions (50% fT > 4×MIC).

LRE-Finder, a Web tool for detection of the 23S rRNA mutations and the optrA, cfr, cfr(B) and poxtA genes encoding linezolid resistance in enterococci from whole-genome sequences.

OBJECTIVES: In enterococci, resistance to linezolid is often mediated by mutations in the V domain of the 23S rRNA gene (G2576T or G2505A). Furthermore, four genes [optrA, cfr, cfr(B) and poxtA] encode linezolid resistance in enterococci. We aimed to develop a Web tool for detection of the two mutations and the four genes encoding linezolid resistance in enterococci from whole-genome sequence data. METHODS: LRE-Finder (where LRE stands for linezolid-resistant enterococci) detected the fraction of Ts in position 2576 and the fraction of As in position 2505 of the 23S rRNA and the cfr, cfr(B), optrA and poxtA genes by aligning raw sequencing reads (fastq format) with k-mer alignment. For evaluation, fastq files from 21 LRE isolates were submitted to LRE-Finder. As negative controls, fastq files from 1473 non-LRE isolates were submitted to LRE-Finder. The MICs of linezolid were determined for the 21 LRE isolates. As LRE-negative controls, 26 VRE isolates were additionally selected for linezolid MIC determination. RESULTS: LRE-Finder was validated and showed 100% concordance with phenotypic susceptibility testing. A cut-off of 10% mutations in position 2576 and/or position 2505 was set in LRE-Finder for predicting a linezolid resistance phenotype. This cut-off allows for detection of a single mutated 23S allele in both Enterococcus faecalis and Enterococcus faecium, while ignoring low-level sequencing noise. CONCLUSIONS: A Web tool for detection of the 23S rRNA mutations (G2576T and G2505A) and the optrA, cfr, cfr(B) and poxtA genes from whole-genome sequences from enterococci is now available online.

Piperacillin pharmacokinetics and target attainment in children with cancer and fever: Can we optimize our dosing strategy?

BACKGROUND: Data on piperacillin-tazobactam pharmacokinetics and optimal dosing in children with cancer and fever are limited. Our objective was to investigate piperacillin pharmacokinetics and the probability of target attainment (PTA) with standard intermittent administration (IA), and to simulate PTA in other dosing regimens. PROCEDURE: This prospective pharmacokinetic study was conducted from April 2016 to January 2018. Children with cancer receiving empiric piperacillin-tazobactam to treat infections were included. Piperacillin-tazobactam 100 mg/kg was infused over 5 min every 8 hours (IA). An optimized sample schedule provided six blood samples per subject for piperacillin concentration determination. The evaluated targets included: (1) 100% time of free piperacillin concentration above the minimum inhibitory concentration (fT > MIC) and (2) 50% fT > 4× MIC. MIC50 and MIC90 were defined based on an intrainstitutional MIC range. RESULTS: A total of 482 piperacillin concentrations were obtained from 43 children (aged 1-18 years) during 89 fever episodes. Standard IA resulted in insufficient target attainment, with significant differences in piperacillin pharmacokinetics for different body weights. Median fT > MIC was 61.2%, 53.5%, and 36.3% for MIC50 (2.0 mg/L), MIC90 (4.0 mg/L), and breakpoint for Pseudomonas aeruginosa (16.0 mg/L), respectively. Correspondingly, the median fT > 4× MIC was 43%, 36.3%, and 20.1%. Simulations showed that only continuous infusion reached a PTA of 95% for MIC = 16.0 mg/L, while extended infusion lasting half of the dosing interval reached a PTA of 95% for MIC ≤ 8 mg/L. CONCLUSIONS: Our data revealed insufficient PTA with standard IA of piperacillin-tazobactam in children with cancer and fever. Alternative dosing strategies, preferably continuous infusion, are required to ensure adequate PTA.

Performance of the EUCAST disc diffusion method and two MIC methods in detection of Enterobacteriaceae with reduced susceptibility to meropenem: the NordicAST CPE study.

Objectives: To examine performance of EUCAST disc diffusion and supplementary MIC methods for detection of Enterobacteriaceae with reduced susceptibility to meropenem using EUCAST screening recommendations. Methods: Sixty-one Nordic laboratories delivered data on EUCAST disc diffusion (n = 61), semi-automated meropenem MIC (n = 23; VITEK2, n = 20 and Phoenix, n = 3) and gradient meropenem MIC (n = 58) methods. The strains (n = 27) included the major carbapenemase classes (A, n = 4; B, n = 9; D, n = 6) involved in the global spread of carbapenemase-producing Enterobacteriaceae (CPE) and non-CPE strains (n = 8) covering a range of broth microdilution (BMD) meropenem MICs. Results: A triplicate Klebsiella variicola (meropenem MIC 0.5 mg/L) harbouring OXA-48 and Escherichia coli ATCC 25922 showed an overall good precision. Meropenem zone diameters below the EUCAST screening cut-off (<27 mm) were reported for strains with MIC ≥1 mg/L (n = 21), irrespective of resistance mechanism. For three strains (MIC = 0.5 mg/L) with OXA-48/-181, eight laboratories provided meropenem zone diameters above the screening cut-off. Very major errors (VMEs) were not observed. The overall distributions of major errors (MEs) and minor errors (mEs) were 9% and 36% (disc diffusion), 26% and 18% (VITEK2) and 7% and 20% (gradient MIC), respectively. Differences in ME and mE distributions between disc diffusion and MIC gradient tests compared with semi-automated methods were significant (P < 0.0001), using BMD MICs as a reference for categorization. Conclusions: The EUCAST disc diffusion method is a robust method to screen for CPE but isolates with meropenem MICs <1 mg/L pose challenges. The high ME rate in semi-automated methods might deter appropriate use of carbapenems in CPE infections with limited therapeutic options.

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.

Automated image analysis for quantification of filamentous bacteria.

BACKGROUND: Antibiotics of the ß-lactam group are able to alter the shape of the bacterial cell wall, e.g. filamentation or a spheroplast formation. Early determination of antimicrobial susceptibility may be complicated by filamentation of bacteria as this can be falsely interpreted as growth in systems relying on colorimetry or turbidometry (such as Vitek-2, Phoenix, MicroScan WalkAway). The objective was to examine an automated image analysis algorithm for quantification of filamentous bacteria using the 3D digital microscopy imaging system, oCelloScope. RESULTS: Three E. coli strains displaying different resistant profiles and differences in filamentation kinetics were used to study a novel image analysis algorithm to quantify length of bacteria and bacterial filamentation. A total of 12 ß-lactam antibiotics or ß-lactam-ß-lactamase inhibitor combinations were analyzed for their ability to induce filamentation. Filamentation peaked at approximately 120 min with an average cell length of 30 µm. CONCLUSION: The automated image analysis algorithm showed a clear ability to rapidly detect and quantify ß-lactam-induced filamentation in E. coli. This rapid determination of ß-lactam-mediated morphological alterations may facilitate future development of fast and accurate AST systems, which in turn will enable early targeted antimicrobial therapy. Therefore, rapid detection of ß-lactam-mediated morphological changes may have important clinical implications.

Identification of antimicrobial resistance genes in multidrug-resistant clinical Bacteroides fragilis isolates by whole genome shotgun sequencing.

Bacteroides fragilis constitutes the most frequent anaerobic bacterium causing bacteremia in humans. The genetic background for antimicrobial resistance in B. fragilis is diverse with some genes requiring insertion sequence (IS) elements inserted upstream for increased expression. To evaluate whole genome shotgun sequencing as a method for predicting antimicrobial resistance properties, one meropenem resistant and five multidrug-resistant blood culture isolates were sequenced and antimicrobial resistance genes and IS elements identified using ResFinder 2.1 (http://cge.cbs.dtu.dk/services/ResFinder/) and a custom BLAST database. Combinations of cfxA, cepA, cfiA, nimA, nimD, nimE, nimJ, tetQ, ermB, ermF, bexB, linAn2 and mefEn2 genes were identified in the six isolates. blaOXA-347, an open reading frame predicted to be a ß-lactamase (Cheng et al., 2012), was identified in one strain. Full length IS elements were identified directly upstream of four genes, but in most cases contigs terminated 100-150 bases upstream of the gene in question. Even though partial IS elements were identified in these short sequences, certain identification could not be ascertained. Full antiobiograms for B. fragilis from genetic data will most likely require complete or nearly complete genomes. Current approaches to this are laborious and/or costly. Emerging technologies such as nanopore based single DNA strand sensing could perhaps provide a solution in the future.

Bacteria of the genus Dyella can chronically colonise the airways of patients with cystic fibrosis and elicit a pronounced antibody response.

A patient with cystic fibrosis became chronically colonised with an unusual non-fermenting Gram-negative rod that could be cultured on Burkholderia cepacia selective agar. Phenotypic characterisation by VITEK-2 suggested identification as Elizabethkingia meningoseptica, however 16S rRNA gene sequencing revealed it belonged to a putative novel species of genus Dyella. Thirty months after the initial detection, the patient produced a high level of precipitating antibodies against the bacterium.

Nine different carbapenemase-producing organisms in a Ukrainian war-injured patient.

This is a case report of a Ukrainian war-injured patient who was colonised/infected with nine different carbapenemase-producing organisms (CPO). The patient was initially treated in Ukraine. After two months he was admitted to a Danish hospital where he underwent extensive surgery and received broad-spectrum antibiotics. In screening and clinical samples, nine different CPO were cultured which in combination were untreatable with antibiotics. To our knowledge, this is the first patient in Denmark with such a high number of different CPO. This may be a sign that we are entering a postantibiotic era.

Polyether Ionophore Antibiotics Target Drug-Resistant Clinical Isolates, Persister Cells, and Biofilms.

Polyether ionophores are complex natural products known to transport various cations across biological membranes. While several members of this family are used in agriculture (e.g., as anti-coccidiostats) and have potent antibacterial activity, they are not currently being pursued as antibiotics for human use. Polyether ionophores are typically grouped as having similar functions, despite the fact that they significantly differ in structure; for this reason, how their structure and activity are related remains unclear. To determine whether certain members of the family constitute particularly interesting springboards for in-depth investigations and future synthetic optimization, we conducted a systematic comparative study of eight different polyether ionophores for their potential as antibiotics. This includes clinical isolates from bloodstream infections and studies of the compounds' effects on bacterial biofilms and persister cells. We uncover distinct differences within the compound class and identify the compounds lasalocid, calcimycin, and nanchangmycin as having particularly interesting activity profiles for further development. IMPORTANCE Polyether ionophores are complex natural products used in agriculture as anti-coccidiostats in poultry and as growth promoters in cattle, although their precise mechanism is not understood. They are widely regarded as antimicrobials against Gram-positive bacteria and protozoa, but fear of toxicity has so far prevented their use in humans. We show that ionophores generally have very different effects on Staphylococcus aureus, both in standard assays and in more complex systems such as bacterial biofilms and persister cell populations. This will allow us to focus on the most interesting compounds for future in-depth investigations and synthetic optimizations.

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.

Multilocus sequence analysis of isolates of Achromobacter from patients with cystic fibrosis reveals infecting species other than Achromobacter xylosoxidans.

A multilocus sequence analysis (MLSA) scheme was developed for characterization of strains and species from the genus Achromobacter, which are increasingly recovered from patients with cystic fibrosis (CF). Five conserved housekeeping genes were selected for the MLSA, which was applied to a diverse collection of 77 strains originating from Europe, Asia, and South America and including type strains of the seven recognized Achromobacter species, six environmental strains, eight non-CF clinical strains, and 56 CF clinical strains. The discriminatory power of MLSA, based on 2,098 nucleotides (nt), was much superior to a 16S rRNA gene comparison based on 1,309 nt. Congruence was observed between single-gene trees and a concatenated gene tree. MLSA differentiated all seven current Achromobacter species and also demonstrated the presence of at least four novel potential species within the genus. CF isolates were predominantly Achromobacter xylosoxidans (64%), an undescribed Achromobacter species (18%), and Achromobacter ruhlandii (7%). A clone of Achromobacter, which has spread among patients from Danish CF centers in Aarhus and Copenhagen, was identified as Achromobacter ruhlandii. MLSA facilitates the specific identification of isolates of Achromobacter necessary for describing their role in clinical infections.