Identification of a Novel Genomic Island Associated with vanD-Type Vancomycin Resistance in Six Dutch Vancomycin-Resistant Enterococcus faecium Isolates

Genomic comparison of the first six Dutch vanD-type vancomycin-resistant Enterococcus faecium (VRE) isolates with four vanD gene clusters from other enterococcal species and anaerobic gut commensals revealed that the vanD gene cluster was located on a genomic island of variable size. Phylogenetic inferences revealed that the Dutch VRE isolates were genetically not closely related and that genetic variation of the vanD-containing genomic island was not species specific, suggesting that this island is transferred horizontally between enterococci and anaerobic gut commensals.Peer reviewe

Quantifying effects of radiotherapy-induced microvascular injury; review of established and emerging brain MRI techniques

Microvascular changes are increasingly recognised not only as primary drivers of radiotherapy treatment response in brain tumours, but also as an important contributor to short- and long-term (cognitive) side effects arising from irradiation of otherwise healthy brain tissue. As overall survival of patients with brain tumours is increasing, monitoring long-term sequels of radiotherapy-induced microvascular changes in the context of their potential predictive power for outcome, such as cognitive disability, has become increasingly relevant. Ideally, radiotherapy-induced significant microvascular changes in otherwise healthy brain tissue should be identified as early as possible to facilitate adaptive radiotherapy and to proactively start treatment to minimise the influence on these side-effects on the final outcome. Although MRI is already known to be able to detect significant long-term radiotherapy induced microvascular effects, more recently advanced MR imaging biomarkers reflecting microvascular integrity and function have been reported and might provide a more accurate and earlier detection of microvascular changes. However, the use and validation of both established and new techniques in the context of monitoring early and late radiotherapy-induced microvascular changes in both target-tissue and healthy tissue currently are minimal at best. This review aims to summarise the performance and limitations of existing methods and future opportunities for detection and quantification of radiotherapy-induced microvascular changes, as well as the relation of these findings with key clinical parameters. (C) 2019 Elsevier B.V. All rights reserved

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

Medication management strategy for older people with polypharmacy in general practice: a qualitative study on prescribing behaviour in primary care

BACKGROUND: For older patients with polypharmacy, medication management is a process of careful deliberation that needs periodic adjustment based on treatment effects and changing conditions. Because of the heterogeneity of the patient group, and limited applicability of current guidelines, it is difficult for GPs to build up a routine. AIM: To gain insight into GPs’ medication management strategies for patients with polypharmacy, and to explore the GPs’ perspectives and needs on decision-making support to facilitate this medication management. DESIGN AND SETTING: Two focus group meetings with Dutch GPs, discussing four clinical vignettes of patients with multimorbidity and polypharmacy. METHOD: Questions about medication management of the vignettes were answered individually; the strategy chosen in each case was discussed in plenary. Analysis followed a Framework approach. RESULTS: In total, 12 GPs described a similar strategy regarding the patients’ medication management: defining treatment goals; determining primary goals; and adjusting medications based on the treatment effect, GPs’ and patients’ preferences, and patient characteristics. There was variation in the execution of this strategy between the GPs. The GPs would like to discuss their choices with other professionals and they valued structured medication reviews with the patient, as well as quick and practical support tools that work on demand. CONCLUSION: To facilitate decision making, a more extensive and structured collaboration between healthcare professionals is desired, as well as support to execute structured medication reviews with eligible patients, and some on-demand tools for individual consultations

Detection of bacteria in burn wounds with a novel handheld autofluorescence wound imaging device: A pilot study

Objective: To compare the detection of bacteria in burn wounds between an bacterial fluorescence imaging device MolecuLight i:X, (Canada), and standard microbiological swabs. Methods: Wounds were swabbed three times on one occasion; once with a standard swab, once with a high-fluorescent area swab, indicating a bacterial load >104 colony-forming units (CFU)/ gram and a finally with a non-fluorescent (nF) area swab. Proportion agreement of the microbiological results was calculated and the accuracy of the device to detect relevant bacteria was assessed. Results: A total of 14 patients with 20 wounds participated in the study. Median post-burn day at sampling time was 21 days. Of the 20 wounds, nine had a positive swab result in either of the three swabs, and 11 showed a highfluorescent area. Overall, positive and negative proportion agreement between standard swab and highfluorescent swab sample results were 100%. Sensitivity, specificity, positive and negative predictive values of presence of highfluorescence were 78%, 64%, 64%, and 78%, respectively. For Pseudomonas aeruginosa detection, these results were 100%, 70%, 44% and 100%, respectively. Conclusion: The diagnostic accuracy of the bacterial fluorescence imaging device to detect relevant bacteria in burn wounds was moderate and the reliability was equal to standard swabbing. Further research in larger sample sizes and on the relevance of minimal bacterial load and its potential to help with Pseudomonas aeruginosa management is needed. Declaration of interest: The authors have no conflicts of interests to declare. The authors gratefully acknowledge the contribution of MolecuLight Inc. and Jamie Joint; this included the use a MolecuLight device for two months at no charge

Indirect burst pressure measurements for the mechanical assessment of biological vessels

\u3cp\u3eIn the evaluation of tissue-engineered blood vessels (TEBVs), the utilization of the correct mechanical test to assess the burst pressure is pivotal for translation to a clinical setting. The ISO 7198 standard outlines various methods that may be implemented to evaluate the mechanical characteristics of vascular prosthetics. The gold standard is the direct measurement of the pressurized burst pressure. Two alternative indirect methods are the circumferential tensile strength (CTS) and the probe burst pressure. There are limited data validating the use of the indirect methods for their predictive capacity of the pressurized burst pressure in single biological vessel samples. We assess the two indirect methods compared with the direct pressurized burst pressure measurement, for their correlation within single biological samples, using methods presently used in literature and as they are proposed by the ISO 7198. The CTS, the probe burst pressure, and the pressurized burst pressure correlated very well (All R\u3csup\u3e2\u3c/sup\u3e > 0.89) when silicone samples were assessed, although the indirect methods resulted in a large overestimation of the burst pressure. The correlation between the three mechanical tests was poor (all R\u3csup\u3e2\u3c/sup\u3e <0.18) when arterial and venous samples were investigated. Freezing and subsequent thawing before testing had no impact on the mechanical properties of the vessels. Strain rates within the strain rate window provided by the ISO 7198 (50-200 mm/min), likewise, had no impact on the outcome of the tests. Neither the CTS nor the probe burst pressure is predictive of the pressurized burst pressure of the biological vascular tissue. Unless explicitly validated in a testing system on a range of biological tissues, the derived methods should not be utilized for the evaluation of the burst pressure of biological TEBVs for clinical purposes.\u3c/p\u3

Growth condition-dependent cell surface proteome analysis of Enterococcus faecium

The last 30 years Enterococcus faecium has become an important nosocomial pathogen in hospitals worldwide. The aim of this study was to obtain insight in the cell surface proteome of E. faecium when grown in laboratory and clinically relevant conditions. Enterococcus faecium E1162, a clinical blood stream isolate, was grown until mid-log phase in brain heart infusion medium (BHI) with, or without 0.02% bile salts, Tryptic Soy Broth with 1% glucose (TSBg) and urine, and its cell surface was "shaved" using immobilized trypsin. Peptides were identified using MS/MS. Mapping against the translated E1162 whole genome sequence identified 67 proteins that were differentially detected in different conditions. In urine, 14 proteins were significantly more and nine proteins less abundant relative to the other conditions. Growth in BHI-bile and TSBg, revealed four and six proteins, respectively, which were uniquely present in these conditions while two proteins were uniquely present in both conditions. Thus, proteolytic shaving of E. faecium cells identified differentially surface exposed proteins in different growth conditions. These proteins are of special interest as they provide more insight in the adaptive mechanisms and may serve as targets for the development of novel therapeutics against this multi-resistant emerging pathogen. All MS data have been deposited in the ProteomeXchange with identifier PXD002497 (http://proteomecentral.proteomexchange.org/dataset/PXD002497)

Growth condition-dependent cell surface proteome analysis of Enterococcus faecium

The last 30 years Enterococcus faecium has become an important nosocomial pathogen in hospitals worldwide. The aim of this study was to obtain insight in the cell surface proteome of E. faecium when grown in laboratory and clinically relevant conditions. Enterococcus faecium E1162, a clinical blood stream isolate, was grown until mid-log phase in brain heart infusion medium (BHI) with, or without 0.02% bile salts, Tryptic Soy Broth with 1% glucose (TSBg) and urine, and its cell surface was "shaved" using immobilized trypsin. Peptides were identified using MS/MS. Mapping against the translated E1162 whole genome sequence identified 67 proteins that were differentially detected in different conditions. In urine, 14 proteins were significantly more and nine proteins less abundant relative to the other conditions. Growth in BHI-bile and TSBg, revealed four and six proteins, respectively, which were uniquely present in these conditions while two proteins were uniquely present in both conditions. Thus, proteolytic shaving of E. faecium cells identified differentially surface exposed proteins in different growth conditions. These proteins are of special interest as they provide more insight in the adaptive mechanisms and may serve as targets for the development of novel therapeutics against this multi-resistant emerging pathogen. All MS data have been deposited in the ProteomeXchange with identifier PXD002497 (http://proteomecentral.proteomexchange.org/dataset/PXD002497)

The Recombinase IntA Is Required for Excision of esp-Containing ICEEfm1 in Enterococcus faecium▿

Comparative genome analysis of Enterococcus faecium recently revealed that a genomic island containing the esp gene, referred to as the esp-containing pathogenicity island (esp PAI), can be transferred by conjugation and contains a partial Tn916-like element and an integrase gene, intA. Here, we characterize the role of intA in the excision of the esp PAI. An intA insertion-deletion mutant in E. faecium E1162 (E1162ΔintA) was constructed and in trans complemented with wild-type intA (E1162ΔintA::pEF30). Circular intermediates (CI) of excised esp PAI were determined using inverse PCR analysis on purified chromosomal DNA from strains E1162, E1162Δesp, E1162ΔintA, and E1162ΔintA::pEF30. In E1162 and E1162Δesp, CI of the esp PAI were detected. No CI were detected in E1162ΔintA, while in the complemented strain E1162ΔintA::pEF30 CI formation was restored, indicating that intA is essential for excision and subsequent mobilization of the esp-containing genomic island in E. faecium. Based on the fact that this island can be mobilized and is self-transmissible, we propose to change the name of the esp PAI to ICEEfm1

Similar Sensitivity of SARS-CoV-2 Detection in Oropharyngeal/Nasopharyngeal and Saliva Samples on the Hologic Panther Platform

Background: Oropharyngeal (OP) and nasopharyngeal (NP) sampling has historically been considered the reference specimen type used for respiratory virus detection. Saliva could be a less invasive alternative for SARS-CoV-2 detection, but limited evidence is available. Methods: The technical and clinical performance of saliva was compared to OP/NP on the Hologic Panther platform with two Aptima assays, the End-Point Transcription-Mediated Amplification assay (EP-TMA) and Real-Time Transcription-Mediated Amplification assay (RT-TMA). The samples were collected at the Public Health Service Testing Site XL location in Schiphol Amsterdam Airport. At the site, the Regional Public Health Laboratory Kennemerland (RPHLK) has a fully equipped laboratory facility. Results: A total of 374 samples (187 OP/NP swabs and 187 saliva samples) were collected from 187 unique patients. The Real-Time Transcription-Mediated Amplification assay (RT-TMA) resulted in comparable sensitivities for the detection of SARS-CoV-2 in both the OP/NP swabs (88.3%; 113/128) and saliva samples (87.5%; 112/128). The End-Point Transcription-Mediated Amplification assay (EP-TMA) analyses showed a similar sensitivity (86.7%; 111/128) in the OP/NP swabs but a lower sensitivity in the saliva samples (80.5%; 103/128). Within the discordant analyses, we found no associations in the symptoms, earlier SARS-CoV-2 infections and eating, smoking, drinking and tooth brushing habits within one hour before testing. Conclusions: The Hologic Panther platform Real-Time Transcription-Mediated Amplification assay (RT-TMA) yields a sensitivity for the detection of SARS-CoV-2 in saliva that is comparable to the OP/NP swabs derived from participants presenting themselves at a public health testing facility with minimal or mild symptoms