Elucidating vancomycin-resistant Enterococcus faecium outbreaks:the role of clonal spread and movement of mobile genetic elements

Background: Vancomycin-resistant Enterococcus faecium (VREfm) has emerged as a nosocomial pathogen worldwide. The dissemination of VREfm is due to both clonal spread and spread of mobile genetic elements (MGEs) such as transposons.Objectives: We aimed to combine vanB-carrying transposon data with core-genome MLST (cgMLST) typing and epidemiological data to understand the pathways of transmission in nosocomial outbreaks.Methods: Retrospectively, 36 VREfm isolates obtained from 34 patients from seven VREfm outbreak investigations in 2014 were analysed. Isolates were sequenced on a MiSeq and a MinION instrument. De novo assembly was performed in CLC Genomics Workbench and the hybrid assemblies were obtained through Unicycler v0.4.1. Ridom SeqSphere+ was used to extract MLST and cgMLST data. Detailed analysis of each transposon and their integration points was performed using the Artemis Comparison Tool (ACT) and multiple blast analyses.Results: Four different vanB transposons were found among the isolates. cgMLST divided ST80 isolates into three cluster types (CTs); CT16, CT104 and CT106. ST117 isolates were divided into CT24, CT103 and CT105. Within VREfm isolates belonging to CT103, two different vanB transposons were found. In contrast, VREfm isolates belonging to CT104 and CT106 harboured an identical vanB transposon.Conclusions: cgMLST provides a high discriminatory power for the epidemiological analysis of VREfm. However, additional transposon analysis is needed to detect horizontal gene transfer. Combining these two methods allows investigation of both clonal spread as well as the spread of MGEs. This leads to new insights and thereby better understanding of the complex transmission routes in VREfm outbreaks.</p

Passive Tracer Visualization to Simulate Aerodynamic Virus Transport in Noninvasive Respiratory Support Methods

BACKGROUND: Various forms of noninvasive respiratory support methods are used in the treatment of hypoxemic CO­VID-19 patients, but limited data are available about the corresponding respiratory droplet dispersion. OBJECTIVES: The aim of this study was to estimate the potential spread of infectious diseases for a broad selection of oxygen and respiratory support methods by revealing the therapy-induced aerodynamics and respiratory droplet dispersion. METHODS: The exhaled air-smoke plume from a 3D-printed upper airway geometry was visualized by recording light reflection during simulated spontaneous breathing, standard oxygen mask application, nasal high-flow therapy (NHFT), continuous positive airway pressure (CPAP), and bilevel positive airway pressure (BiPAP). The dispersion of 100 μm particles was estimated from the initial velocity of exhaled air and the theoretical terminal velocity. RESULTS: Estimated droplet dispersion was 16 cm for unassisted breathing, 10 cm for Venturi masks, 13 cm for the nebulizer, and 14 cm for the nonrebreathing mask. Estimated droplet spread increased up to 34 cm in NHFT, 57 cm in BiPAP, and 69 cm in CPAP. A nonsurgical face mask over the NHFT interface reduced estimated droplet dispersion. CONCLUSIONS: During NHFT and CPAP/BiPAP with vented masks, extensive jets with relatively high jet velocities were observed, indicating increased droplet spread and an increased risk of droplet-driven virus transmission. For the Venturi masks, a nonrebreathing mask, and a nebulizer, estimated jet velocities are comparable to unassisted breathing. Aerosols are transported unboundedly in all these unfiltered therapies. The adequate use of protective measures is of vital importance when using noninvasive unfiltered therapies in infectious respiratory diseases

Viral load dynamics in intubated patients with COVID-19 admitted to the intensive care unit

Background: Prolonged viral RNA detection in respiratory samples from patients with COVID-19 has been described, but the clinical relevance remains unclear. We studied the dynamics of SARS-CoV-2 on a group and individual level in intubated ICU patients. Methods: In a cohort of 86 patients, we analysed SARS-CoV-2 RT-PCR results on nasopharyngeal and sputum samples (obtained as part of clinical care twice a week) according to time after intubation. Subsequently, we performed survival analyses. Results: 870 samples were tested by RT-PCR. Overall viral load was highest in the first week (median nasopharynx 3.5. IQR 1.5-4.3; median sputum 4.3. IQR 3.3-5.6) and decreased over time. In 20% of patients a relapsing pattern was observed. Nasopharyngeal and sputum PCR status on day 14 was not significantly associated with survival up to day 60 in this small cohort. Conclusion: In general SARS-CoV-2 RNA levels in respiratory samples in patients with severe COVID-19 decease alter the first week after intubation, but individual SARS-CoV-2 RNA levels can show a relapsing pattern. Larger studies are needed to address the association of clearance of SARS-CoV-2 RNA from respiratory samples with survival, because we observed a trend towards better survival in patients with early clearance from sputum. (C) 2021 The Authors. Published by Elsevier Inc

Molecular Characterisation of Vancomycin-Resistant Enterococcus faecium Isolates Belonging to the Lineage ST117/CT24 Causing Hospital Outbreaks

Background: Vancomycin-resistant Enterococcus faecium (VREfm) is a successful nosocomial pathogen. The current molecular method recommended in the Netherlands for VREfm typing is based on core genome Multilocus sequence typing (cgMLST), however, the rapid emergence of specific VREfm lineages challenges distinguishing outbreak isolates solely based on their core genome. Here, we explored if a detailed molecular characterisation of mobile genetic elements (MGEs) and accessory genes could support and expand the current molecular typing of VREfm isolates sharing the same genetic background, enhancing the discriminatory power of the analysis. Materials/Methods: The genomes of 39 VREfm and three vancomycin-susceptible E. faecium (VSEfm) isolates belonging to ST117/CT24, as assessed by cgMLST, were retrospectively analysed. The isolates were collected from patients and environmental samples from 2011 to 2017, and their genomes were analysed using short-read sequencing. Pangenome analysis was performed on de novo assemblies, which were also screened for known predicted virulence factors, antimicrobial resistance genes, bacteriocins, and prophages. Two representative isolates were also sequenced using long-read sequencing, which allowed a detailed analysis of their plasmid content. Results: The cgMLST analysis showed that the isolates were closely related, with a minimal allelic difference of 10 between each cluster's closest related isolates. The vanB-carrying transposon Tn1549 was present in all VREfm isolates. However, in our data, we observed independent acquisitions of this transposon. The pangenome analysis revealed differences in the accessory genes related to prophages and bacteriocins content, whilst a similar profile was observed for known predicted virulence and resistance genes. Conclusion: In the case of closely related isolates sharing a similar genetic background, a detailed analysis of MGEs and the integration point of the vanB-carrying transposon allow to increase the discriminatory power compared to the use of cgMLST alone. Thus, enabling the identification of epidemiological links amongst hospitalised patients

Long-read sequencing-based in silico phage typing of vancomycin-resistant Enterococcus faecium

Abstract Background Vancomycin-resistant enterococci (VRE) are successful nosocomial pathogens able to cause hospital outbreaks. In the Netherlands, core-genome MLST (cgMLST) based on short-read sequencing is often used for molecular typing. Long-read sequencing is more rapid and provides useful information about the genome’s structural composition but lacks the precision required for SNP-based typing and cgMLST. Here we compared prophages among 50 complete E. faecium genomes belonging to different lineages to explore whether a phage signature would be usable for typing and identifying an outbreak caused by VRE. As a proof of principle, we investigated if long-read sequencing data would allow for identifying phage signatures and thereby outbreak-related isolates. Results Analysis of complete genome sequences of publicly available isolates showed variation in phage content among different lineages defined by MLST. We identified phage present in multiple STs as well as phages uniquely detected within a single lineage. Next, in silico phage typing was applied to twelve MinION sequenced isolates belonging to two different genetic backgrounds, namely ST117/CT24 and ST80/CT16. Genomic comparisons of the long-read-based assemblies allowed us to correctly identify isolates of the same complex type based on global genome architecture and specific phage signature similarity. Conclusions For rapid identification of related VRE isolates, phage content analysis in long-read sequencing data is possible. This allows software development for real-time typing analysis of long-read sequencing data, which will generate results within several hours. Future studies are required to assess the discriminatory power of this method in the investigation of ongoing outbreaks over a longer time period

Cost analysis of outbreaks with Methicillin-resistant Staphylococcus aureus (MRSA) in Dutch long-term care facilities (LTCF)

Objectives Highly resistant microorganisms (HRMOs) are of high concern worldwide and are becoming increasingly less susceptible for antibiotics. To study the cost effectiveness of infection prevention measures in long-term care, it is essential to first fully understand the impact of HRMOs. The objective of this study is to identify the costs associated with outbreaks caused by Methicillin-resistant Staphylococcus aureus (MRSA) in Dutch long-term care facilities (LTCF). Methods After an outbreak of MRSA, Dutch LTCF can submit a reimbursement form to the Dutch Healthcare Authority ("Nederlandse Zorgautoriteit"; NZa) to get a part of the total costs reimbursed. In this study, we requested NZa forms for financial impact analysis. Details regarding the costs of the outbreak have been extracted from these forms and additionally specific LTCF have been visited in person to validate the data. Results 34 complete reimbursement forms from the period between 2011 and 2016 were received from the NZa and have been included. The median cost per patient per day was estimated at (sic)83.80, varying between (sic)16.89 and (sic)1,820.09. We validated five reimbursement forms by visiting the facility and recalculating the costs. We found a non-significant positive difference of (sic)26.07 compared with the original data (p = 0.068). Conclusions This study is to our knowledge the first to give a national overview of total costs associated with an MRSA outbreak in LTCF in the Netherlands. Overall, costs per patient per day seem lower than in a hospital setting, although total costs are much higher due to the long term of care

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

Mammographic density and ageing:A collaborative pooled analysis of cross-sectional data from 22 countries worldwide

BACKGROUND: Mammographic density (MD) is one of the strongest breast cancer risk factors. Its age-related characteristics have been studied in women in western countries, but whether these associations apply to women worldwide is not known. METHODS AND FINDINGS: We examined cross-sectional differences in MD by age and menopausal status in over 11,000 breast-cancer-free women aged 35-85 years, from 40 ethnicity- and location-specific population groups across 22 countries in the International Consortium on Mammographic Density (ICMD). MD was read centrally using a quantitative method (Cumulus) and its square-root metrics were analysed using meta-analysis of group-level estimates and linear regression models of pooled data, adjusted for body mass index, reproductive factors, mammogram view, image type, and reader. In all, 4,534 women were premenopausal, and 6,481 postmenopausal, at the time of mammography. A large age-adjusted difference in percent MD (PD) between post- and premenopausal women was apparent (-0.46 cm [95% CI: -0.53, -0.39]) and appeared greater in women with lower breast cancer risk profiles; variation across population groups due to heterogeneity (I2) was 16.5%. Among premenopausal women, the √PD difference per 10-year increase in age was -0.24 cm (95% CI: -0.34, -0.14; I2 = 30%), reflecting a compositional change (lower dense area and higher non-dense area, with no difference in breast area). In postmenopausal women, the corresponding difference in √PD (-0.38 cm [95% CI: -0.44, -0.33]; I2 = 30%) was additionally driven by increasing breast area. The study is limited by different mammography systems and its cross-sectional rather than longitudinal nature. CONCLUSIONS: Declines in MD with increasing age are present premenopausally, continue postmenopausally, and are most pronounced over the menopausal transition. These effects were highly consistent across diverse groups of women worldwide, suggesting that they result from an intrinsic biological, likely hormonal, mechanism common to women. If cumulative breast density is a key determinant of breast cancer risk, younger ages may be the more critical periods for lifestyle modifications aimed at breast density and breast cancer risk reduction