Search

Objectives: Patients having previous contact with healthcare systems abroad are routinely screened for resistant bacteria on admission to hospitals in Copenhagen. This study aimed to present carriage prevalence and geographical risk stratification, as well as phenotypic and genotypic characterisation of resistant isolates. Methods: This study included screening samples analysed at one department of clinical microbiology in Copenhagen from 2016–2019. Patients who had previous contact with healthcare systems abroad within 6 months were screened at admission for methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE) and carbapenemase-producing organisms (CPO). Isolates were characterised phenotypically and by whole-genome sequencing. The relative frequency of positive findings stratified by geographical regions correlated with relative frequency of Danish residents’ travel destinations. Results: Of 2849 screening sets included in the study, 103 (3.6%) were positive. A total of 120 resistant isolates were detected (36 MRSA, 31 VRE and 53 CPO). The carrier prevalence for MRSA was 1.3%, 1.1% for VRE and 1.5% for CPO. Southern and Western Asia were overrepresented travel destinations in positive screening sets (41%). For VRE, 40% were related to Southern Europe, which also represented 35% of travel destinations. Genotypic characterisation confirmed a heterogenous genomic background reflecting global distribution of resistant clones. Conclusions: Exposure targeted screening identified a substantial number of asymptomatic carriers of MRSA, VRE and CPO with heterogenous genetic backgrounds. Although some geographical regions were overrepresented, the complex epidemiology of the different pathogens did not allow a restriction of the screening strategy to certain geographical regions.

Coral reefs worldwide are rapidly declining due to increasing anthropogenic stressors and environmental changes, with large-scale mortalities of coral reefs observed in many locations across the globe. It has become clear that the microbiome of corals is important in understanding the causes of coral infections, although its exact role is yet to be fully understood. Here, we characterize the bacteria and fungi associated with the non-lesional and lesional (identified by discoloration and tissue loss) tissues of coral species from Vietnam. Metabarcoding of the bacterial 16S rRNA gene and the fungal ITS rRNA gene region were performed. We sampled across two Porites species with potentially multiple causes of stresses, yet the bacterial compositions of lesional regions were consistently different from non-lesional areas of the same coral. These differences were driven by a considerable and significant increase in OTU richness within the lesional region compared to the non-lesional region. While no single OTU was consistently associated with lesional tissue, indicator analysis revealed that nine OTUs were significantly more persistent in the lesional regions that could represent useful bioindicators of stress. Meanwhile, there were no indicator OTUs in the non-lesional region. Further investigations are needed to determine whether changing bacterial communities play a mechanistic role in inducing lesioning, or are opportunistically colonizing stressed corals.

OBJECTIVES: Sample preparation for High-throughput sequencing (HTS) includes treatment with various laboratory components, potentially carrying viral nucleic acids, the extent of which has not been thoroughly investigated. Our aim was to systematically examine a diverse repertoire of laboratory components used to prepare samples for HTS in order to identify contaminating viral sequences.METHODS: A total of 322 samples of mainly human origin were analysed using eight protocols, applying a wide variety of laboratory components. Several samples (60% of human specimens) were processed by different protocols. In total 712 sequencing libraries were investigated for viral sequence contamination.RESULTS: Among sequences showing similarity to viruses, 493 were significantly associated to the use of laboratory components. Each of these viral sequences showed sporadic appearance, only being identified in a subset of the samples treated with the linked laboratory component, and some were not identified in the non-template control (NTC) samples. Remarkably, more than 65% of all viral sequences identified were within viral clusters linked to the use of laboratory components.CONCLUSIONS: We show that high prevalence of contaminating viral sequences can be expected in HTS-based virome data and provide an extensive list of novel contaminating viral sequences that can be used for evaluation of viral findings in future virome and metagenome studies. Moreover we show that detection can be problematic due to stochastic appearance and limited NTCs. Although the exact origin of these viral sequences requires further research, our results support laboratory component linked viral sequence contamination of both biological and synthetic origin.

Sample preparation for High-throughput sequencing (HTS) includes treatment with various laboratory components, potentially carrying viral nucleic acids, the extent of which has not been thoroughly investigated. Our aim was to systematically examine a diverse repertoire of laboratory components used to prepare samples for HTS in order to identify contaminating viral sequences. A total of 322 samples of mainly human origin were analysed using eight protocols, applying a wide variety of laboratory components. Several samples (60% of human specimens) were processed by different protocols. In total 712 sequencing libraries were investigated for viral sequence contamination. Among sequences showing similarity to viruses, 493 were significantly associated to the use of laboratory components. Each of these viral sequences showed sporadic appearance, only being identified in a subset of the samples treated with the linked laboratory component, and some were not identified in the non-template control (NTC) samples. Remarkably, more than 65% of all viral sequences identified were within viral clusters linked to the use of laboratory components. We show that high prevalence of contaminating viral sequences can be expected in HTS-based virome data and provide an extensive list of novel contaminating viral sequences that can be used for evaluation of viral findings in future virome and metagenome studies. Moreover we show that detection can be problematic due to stochastic appearance and limited NTCs. Although the exact origin of these viral sequences requires further research, our results support laboratory component linked viral sequence contamination of both biological and synthetic origin.

Viruses and other infectious agents cause more than 15% of human cancer cases. High-throughput sequencing-based studies of virus-cancer associations have mainly focused on cancer transcriptome data. Here we applied a diverse selection of pre-sequencing enrichment methods targeting all major viral groups, to characterise the viruses present in 197 samples from 18 sample types of cancerous origin. Using high-throughput sequencing we generated 710 datasets constituting 57 billion sequencing reads. Detailed in silico investigation of the viral content, including exclusion of viral artefacts, from de novo assembled contigs and individual sequencing reads yielded a map of the viruses detected. Our data reveals a virome dominated by papillomaviruses, anelloviruses, herpesviruses, and parvoviruses. More than half of the included samples contained one or more viruses, however, no link between specific viruses and cancer types were found. Our study sheds light on viral presence in cancers and provides highly relevant virome data for future reference.

Background: Primary screening for VRE with PCR directed against vanA allowed identification of vanA! samples from which VRE could not be isolated when selective culture methods were used. From such a sample a vancomycin-susceptible, vanA! Enterococcus faecium, Efm-V1511, was isolated, when vancomycin selection was not used during culture. Similar isolates with variable susceptibility to vancomycin were obtained in the following months. Objectives: To characterize Efm-V1511 and investigate the causes of variable susceptibility to vancomycin. Methods: All strains were sequenced using Illumina technology. Plasmids containing vanA were reconstructed by scaffolding to known plasmids or plasmids were sequenced using Oxford Nanopore MinION. Derived structures were verified by PCR and sequencing. Furthermore, selected vanA! vancomycin-susceptible isolates were passaged in the presence of vancomycin and vancomycin-resistant variants obtained were sequenced. Results: Efm-V1511 belonged to ST1421 and contained a 49 696 bp plasmid pHVH-V1511 carrying a Tn1546-derived genetic element. Within this element vanX was truncated by a 252 bp 30 deletion explaining the susceptibility of Efm-V1511. Between March 2016 and April 2017, 48 isolates containing pHVH-V1511 were identified. All were ST1421. In isolates resistant to vancomycin, resistance could be attributed to changes in ddl disrupting gene function sometimes accompanied by changes in vanS, increased pHVH-V1511 copy number or the existence of an additional vanA-containing plasmid encoding a functional vanX. Conclusions: E. faecium carrying pHVH-V1511 is capable of nosocomial transmission and may develop clinical resistance to vancomycin. Strains may not be detected using standard culture methods for VRE.

Propionibacterium acnesis the most abundant bacterium on human skin, particularly in sebaceous areas.P. acnesis suggested to be an opportunistic pathogen involved in the development of diverse medical conditions but is also a proven contaminant of human clinical samples and surgical wounds. Its significance as a pathogen is consequently a matter of debate. In the present study, we investigated the presence ofP. acnesDNA in 250 next-generation sequencing data sets generated from 180 samples of 20 different sample types, mostly of cancerous origin. The samples were subjected to either microbial enrichment, involving nuclease treatment to reduce the amount of host nucleic acids, or shotgun sequencing. We detected high proportions ofP. acnesDNA in enriched samples, particularly skin tissue-derived and other tissue samples, with the levels being higher in enriched samples than in shotgun-sequenced samples.P. acnesreads were detected in most samples analyzed, though the proportions in most shotgun-sequenced samples were low. Our results show thatP. acnescan be detected in practically all sample types when molecular methods, such as next-generation sequencing, are employed. The possibility of contamination from the patient or other sources, including laboratory reagents or environment, should therefore always be considered carefully whenP. acnesis detected in clinical samples. We advocate that detection ofP. acnesalways be accompanied by experiments validating the association between this bacterium and any clinical condition.

Virus discovery from high throughput sequencing data often follows a bottom-up approach where taxonomic annotation takes place prior to association to disease. Albeit effective in some cases, the approach fails to detect novel pathogens and remote variants not present in reference databases. We have developed a species independent pipeline that utilises sequence clustering for the identification of nucleotide sequences that co-occur across multiple sequencing data instances. We applied the workflow to 686 sequencing libraries from 252 cancer samples of different cancer and tissue types, 32 non-template controls, and 24 test samples. Recurrent sequences were statistically associated to biological, methodological or technical features with the aim to identify novel pathogens or plausible contaminants that may associate to a particular kit or method. We provide examples of identified inhabitants of the healthy tissue flora as well as experimental contaminants. Unmapped sequences that co-occur with high statistical significance potentially represent the unknown sequence space where novel pathogens can be identified.

Rattus norvegicus (R. norvegicus) are ubiquitous around areas populated by human and are known vectors of pathogens to humans. Therefore the surveillance of R. norvegicus is important if we want to understand which pathogens they spread. Metagenomics and second-generation sequencing are paving the way for increasing rates of pathogen discovery and identification, thereby enabling faster containment of wildlife vectors. In this thesis, I have used metagenomics to assess the virome and resistome of the wild urban R. norvegicus. Many new potential viruses are discovered through virome analyses; including the first known R. norvegicus associated polyomavirus, a novel papillomavirus, several circular ssDNA viruses and some cardioviruses. The resistome analyses on these samples reveals many shared as well as location-specific antibiotic resistance genes, but there is a clear selection for vancomycin resistance in the samples from a hospital environment. The work presented in this thesis characterizes some of the pathogens carried around by a small but ubiquitous creature. With this work, we emphasize the need for a frequent screening of animals like rats that are in contact with us so that we react to the emerging health threats more quickly and efficiently.