Aptamer-assisted bioconjugation of tyrosine derivatives with hemin/G-quadruplex (hGQ) DNAzyme nucleoapzyme nanostructures

Hemin/G-quadruplex (hGQ) DNAzymes are horseradish peroxidase-mimicking catalysts capable of the oxidation of a variety of substrates. We now implement aptamer-functionalized hGQ DNAzymes, also known as nucleoapzymes, to achieve increased bioconjugation of N-methyl luminol to tyrosine-containing residues and peptides. We found that the presence of a tyrosinamide-binding aptamer leads to a 12-fold increase in the catalytic rate constant (k(cat)), and the saturation kinetics curves that were obtained provide evidence for the involvement of the substrate binding site in the reaction. The application of the best performing nucleoapzymes for the modification of Tyr-containing peptides reveals that (i) the aptamer also recognizes the ligand structure when this is embedded in a larger peptide structure, and (ii) distant residues in the peptide substrate can influence the conversion. As such, we show that nucleoapzymes display enzyme-like features and provide an additional tool in the toolbox of bioconjugation chemistry.FWN – Publicaties zonder aanstelling Universiteit Leide

Imipenem as mono therapy in the treatment of intensive care patients with severe infections

In an open study, 24 intensive care patients were treated with imipenem/cilastatin as monotherapy for serious bacterial infections. Twenty-one patients were treated for bronchopulmonary infection, two patients for septicaemia, and one patient for an empyema. Initially all strains were susceptible to imipenem. Gram-negative bacilli accounted for 80% of these isolates. The most frequently isolated species were Proteus mirabilis, Escherichia coli and Pseudomonas aeruginosa. All 24 patients were considered clinically cured. Sixteen of these patients (67%) were both clinically and microbiologically cured. In eight of the 24 patients (33%), the strains isolated initially persisted. In eight of the 24 patients (33%), colonization of the respiratory tract developed. Two of the five Ps. aeruginosa isolates developed resistance during therapy but in none of these patients was therapy considered to have failed. In 12 patients (50%), transient elevations in hepatic function tests were observed and these were probably drugrelated. The present study supports the view that imipenem/cilastatin may be useful as monotherapy in the treatment of severe infections in intensive care patients

Molecular characteristics of carbapenemase-producing Enterobacterales in the Netherlands; results of the 2014–2018 national laboratory surveillance

Objectives: Carbapenem resistance mediated by mobile genetic elements has emerged worldwide and has become a major public health threat. To gain insight into the molecular epidemiology of carbapenem resistance in The Netherlands, Dutch medical microbiology laboratories are requested to submit suspected carbapenemase-producing Enterobacterales (CPE) to the National Institute for Public Health and the Environment as part of a national surveillance system. Methods: Meropenem MICs and species identification were confirmed by E-test and MALDI-TOF and carbapenemase production was assessed by the Carbapenem Inactivation Method. Of all submitted CPE, one species/carbapenemase gene combination per person per year was subjected to next-generation sequencing (NGS). Results: In total, 1838 unique isolates were received between 2014 and 2018, of which 892 were unique CPE isolates with NGS data available. The predominant CPE species were Klebsiella pneumoniae (n = 388, 43%), Escherichia coli (n = 264, 30%) and Enterobacter cloacae complex (n = 116, 13%). Various carbapenemase alleles of the same carbapenemase gene resulted in different susceptibilities to meropenem and this effect varied between species. Analyses of NGS data showed variation of prevalence of carbapenemase alleles over time with blaOXA-48 being predominant (38%, 336/892), followed by blaNDM-1 (16%, 145/892). For the first time in the Netherlands, blaOXA-181, blaOXA-232 and blaVIM-4 were detected. The genetic background of K. pneumoniae and E. coli isolates was highly diverse. Conclusions: The CPE population in the Netherlands is diverse, suggesting multiple introductions. The predominant carbapenemase alleles are blaOXA-48 and blaNDM-1. There was a clear association between species, carbapenemase allele and susceptibility to meropenem

National laboratory-based surveillance system for antimicrobial resistance: a successful tool to support the control of antimicrobial resistance in the Netherlands

An important cornerstone in the control of antimicrobial resistance (AMR) is a well-designed quantitative system for the surveillance of spread and temporal trends in AMR. Since 2008, the Dutch national AMR surveillance system, based on routine data from medical microbiological laboratories (MMLs), has developed into a successful tool to support the control of AMR in the Netherlands. It provides background information for policy making in public health and healthcare services, supports development of empirical antibiotic therapy guidelines and facilitates in-depth research. In addition, participation of the MMLs in the national AMR surveillance network has contributed to sharing of knowledge and quality improvement. A future improvement will be the implementation of a new semantic standard together with standardised data transfer, which will reduce errors in data handling and enable a more real-time surveillance. Furthermore, the