| 1. |
- Cumlin, Tomas, et al.
(author)
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From SARS-CoV-2 to Global Preparedness : A Graphical Interface for Standardised High-Throughput Bioinformatics Analysis in Pandemic Scenarios and Surveillance of Drug Resistance
- 2024
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In: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 25:12
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Journal article (peer-reviewed)abstract
- The COVID-19 pandemic highlighted the need for a rapid, convenient, and scalable diagnostic method for detecting a novel pathogen amidst a global pandemic. While command-line interface tools offer automation for SARS-CoV-2 Oxford Nanopore Technology sequencing data analysis, they are inapplicable to users with limited programming skills. A solution is to establish such automated workflows within a graphical user interface software. We developed two workflows in the software Geneious Prime 2022.1.1, adapted for data obtained from the Midnight and Artic's nCoV-2019 sequencing protocols. Both workflows perform trimming, read mapping, consensus generation, and annotation on SARS-CoV-2 Nanopore sequencing data. Additionally, one workflow includes phylogenetic assignment using the bioinformatic tools pangolin and Nextclade as plugins. The basic workflow was validated in 2020, adhering to the requirements of the European Centre for Disease Prevention and Control for SARS-CoV-2 sequencing and analysis. The enhanced workflow, providing phylogenetic assignment, underwent validation at Uppsala University Hospital by analysing 96 clinical samples. It provided accurate diagnoses matching the original results of the basic workflow while also reducing manual clicks and analysis time. These bioinformatic workflows streamline SARS-CoV-2 Nanopore data analysis in Geneious Prime, saving time and manual work for operators lacking programming knowledge.
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| 2. |
- Davies, Emma, et al.
(author)
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Genomic and Phenotypic Characterisation of Campylobacter jejuni Isolates From a Waterborne Outbreak
- 2020
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In: Frontiers in Cellular and Infection Microbiology. - : FRONTIERS MEDIA SA. - 2235-2988. ; 10
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Journal article (peer-reviewed)abstract
- Campylobacter infections are the leading cause of bacterial gastroenteritis. In Europe, over 246,000 cases are confirmed annually. Infections are often transmitted via contaminated food, such as poultry products, but water may be the source of infection as well. The aim of this study was to characterise a selection of Campylobacter jejuni human isolates, together with a water isolate, from a waterborne outbreak in Norway in 2019, including human isolates from early, mid-, and late epidemic. The isolates were characterised with whole-genome sequencing, analysing the expression of putative virulence genes and demonstrating the pathogenic potential in an in vitro adhesion model using HT-29 cells. All isolates belonged to the multilocus sequence type 1701 and ST45 clonal complex. In the genomic analysis, the water isolate clustered somewhat separately from the human isolates. There was some variation between the human isolates, but the water isolate seemed to display the greatest pathogenic potential, demonstrated by the highest levels of virulence gene expression, adhesion to epithelial cells and IL-8 induction. These results suggest that the water isolate of the study has potential to cause human infections, and that some bacterial changes due to host or environmental adaptation, may occur during a waterborne Campylobacter epidemic. This is, to the best of our knowledge, the first study on C. jejuni isolates from a waterborne outbreak, including both human isolates and a water isolate, characterised with genomic and phenotypic approaches.
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| 3. |
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| 4. |
- Haars, Jonathan, et al.
(author)
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Prevalence of SARS-CoV-2 Omicron Sublineages and Spike Protein Mutations Conferring Resistance against Monoclonal Antibodies in a Swedish Cohort during 2022–2023
- 2023
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In: Microorganisms. - : MDPI. - 2076-2607. ; 11:10
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Journal article (peer-reviewed)abstract
- Monoclonal antibodies (mAbs) are an important treatment option for COVID-19 caused by SARS-CoV-2, especially in immunosuppressed patients. However, this treatment option can become ineffective due to mutations in the SARS-CoV-2 genome, mainly in the receptor binding domain (RBD) of the spike (S) protein. In the present study, 7950 SARS-CoV-2 positive samples from the Uppsala and Örebro regions of central Sweden, collected between March 2022 and May 2023, were whole-genome sequenced using amplicon-based sequencing methods on Oxford Nanopore GridION, Illumina MiSeq, Illumina HiSeq, or MGI DNBSEQ-G400 instruments. Pango lineages were determined and all single nucleotide polymorphism (SNP) mutations that occurred in these samples were identified. We found that the dominant sublineages changed over time, and mutations conferring resistance to currently available mAbs became common. Notable ones are R346T and K444T mutations in the RBD that confer significant resistance against tixagevimab and cilgavimab mAbs. Further, mutations conferring a high-fold resistance to bebtelovimab, such as the K444T and V445P mutations, were also observed in the samples. This study highlights that resistance mutations have over time rendered currently available mAbs ineffective against SARS-CoV-2 in most patients. Therefore, there is a need for continued surveillance of resistance mutations and the development of new mAbs that target more conserved regions of the RBD.
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| 5. |
- Herrmann, Björn, 1955-, et al.
(author)
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SNP-based high-resolution typing of Chlamydia psittaci from humans and wild birds in Sweden : circulation of the Mat116 genotype reveals the transmission mode to humans
- 2024
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In: Microbes and infection. - : Elsevier. - 1286-4579 .- 1769-714X. ; 26:3
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Journal article (peer-reviewed)abstract
- The incidence of Chlamydia psittaci respiratory tract infections in humans has increased in Sweden in recent years. This study aimed to identify the transmission route by genotyping C. psittaci from infected humans and birds.42 human C. psittaci samples and 5 samples from C. psittaci-infected birds were collected. Genotyping was performed using ompA sequencing, Multi-locus sequence typing, and/or SNP-based high-resolution melting-PCR. Epidemiological data was also collected, and a phylogenetic analysis was conducted.Analysis of ompA provided limited resolution, while the SNP-based PCR analysis successfully detected the Mat116 genotype in 3/5 passerine birds and in 26/29 human cases, indicating a high prevalence of this genotype in the human population. These cases were associated with contact with wild birds, mainly through bird feeding during winter or other outdoor exposure. Human cases caused by other genotypes (psittacine and pigeon) were less common and were linked to exposure to caged birds or pigeons.The SNP-genotype Mat116 is rare, but predominated in this study. The use of SNP-based PCR provided a better understanding of the C. psittaci transmission from birds to humans compared to ompA analysis. In Sweden, human psittacosis appears mainly to be transmitted from garden birds during bird feeding in the winter season.
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| 6. |
- Johansson, Cecilia, et al.
(author)
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Campylobacter coli clade 3 isolates induce rapid cell death in vitro
- 2019
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In: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 85:5
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Journal article (peer-reviewed)abstract
- Campylobacter are major human enteropathogens. C. coli show less genetic diversity than C. jejuni and cluster into three clades, of which clade 1 includes most human and farm animal isolates while environmental C. coli mainly belong to clades 2 and 3. Recently, we whole genome-sequenced eight C. coli clade 2 and 3 isolates cultivated from water, and here we studied their interaction with human HT-29 colon cancer cells compared to clinical clade 1 isolates. All C. coli clade 3 isolates caused cell necrosis already 1-2 hours after inoculation, whereas none of the clade 1 and 2 isolates analyzed induced cell death. Isolates from clades 2 and 3 adhered better than clade 1 isolates to epithelial cells but all isolates induced similar levels of IL-8. Alignment and phylogenetic analysis of translated putative virulence genes cadF, flpA, iamA, ciaB and ceuE revealed clade-specific protein sequence variations with clade 1 and 2 sequences more closely related and clade 3 sequences further apart in general.Moreover, when RNA levels were measured, clade 3 isolates showed a significantly lower expression of cadF, iamA and ceuE than clade 2 isolates, while flpA levels were higher in clade 3 isolates. The cytolethal distending toxin genes were also expressed in clades 2 and 3 although there was no difference between clades. Our findings demonstrate differences between effects of C. coli clade 1, 2 and 3 isolates on human cells and suggest that C. coli clade 3 might be more virulent than clade 2 due to the observed cytotoxicity.IMPORTANCECampylobacter coli is a common zoonotic cause of gastroenteritis in humans worldwide. The majority of infections are caused by C. coli clade 1 isolates, whereas infections due to clade 2 and 3 isolates are rare. Whether this depends on a low prevalence of clade 2 and 3 isolates in reservoirs important for human infections or their lower ability to cause human disease is unknown. Here, we studied the effects of C. coli clade 2 and 3 isolates on a human cell line. These isolates adhered to human cells to a higher degree than clinical clade 1 isolates. Furthermore, we could show that C. coli clade 3 isolates rapidly induced cell death suggesting differences in the virulence of C. coli The exact mechanism of cell death remains to be revealed but selected genes showed interesting clade-specific expression patterns.
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| 7. |
- Johansson, Cecilia, et al.
(author)
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Differences in virulence gene expression between human blood and stool Campylobacter coli clade 1 ST828CC isolates
- 2019
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In: Gut Pathogens. - : BioMed Central. - 1757-4749. ; 11:1
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Journal article (peer-reviewed)abstract
- Background: Campylobacter colonise the gastrointestinal tract of warm-blooded animals and are major enteropathogens in humans. C. coli is less common than C. jejuni and accounts for about 10% of the total number of Campylobacter infections although the two species seem to share many virulence determinants. Campylobacter bacteraemia is rare, estimated to occur in less than 1% of the infections, and the exact mechanisms regulating the progression of the infection from the gastrointestinal tract to the blood stream are unclear. Here, we looked at the contribution of C. coli to Campylobacter infections and further compared various virulence traits in C. coli clade 1 blood and stool isolates. Results: We assessed the numbers of C. jejuni and C. coli among typed isolates in the PubMLST database and found that C. coli accounted for 25.9% of blood isolates, but only 8.9% of the stool isolates. Phylogenetic analysis of 128 C. coli clade 1 whole genome sequences deposited to NCBI revealed no specific clustering of the human blood, stool or animal isolates. Of the six C. coli isolates chosen for phenotypic analyses, stool isolates adhered significantly better to human HT-29 colon cancer cells than the blood isolates, while there was no difference in induced IL-8 levels between the isolates. Furthermore, the stool isolates had two-to fourfold higher RNA expression levels of the flpA, ciaB, iamA and cdt virulence genes than the blood isolates. Finally, we looked at the gene structure of the cdtA, B and C toxin genes and found numerous nucleotide additions and deletions disrupting the open reading frames. In contrast to 58% isolates of animal origin, only 38% and 32% of human blood and stool isolates, respectively, had all three cdt genes intact, a prerequisite to produce functional toxins. Conclusions: This study reveals interesting differences between C. coli clade 1 isolates of human and animal origin on one hand, and also between human blood and stool isolates, on the other. The results suggest that C. coli might downregulate and/or inactivate various virulence determinants as the isolates pass from the animal host to the human gastrointestinal tract and enter the human blood stream.
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| 8. |
- Johansson, Cecilia, et al.
(author)
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Staphylococcus argenteus and Staphylococcus schweitzeri are cytotoxic to human cells in vitro due to high expression of alpha-hemolysin Hla
- 2019
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In: Virulence. - : Taylor & Francis. - 2150-5594 .- 2150-5608. ; 10:1, s. 502-510
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Journal article (peer-reviewed)abstract
- Staphylococcus argenteus and Staphylococcus schweitzeri are newly identified species of the S. aureus-related complex. S. argenteus, as occurring globally and showing significant prevalence and comparable infection and morbidity rates compared to S. aureus, is becoming clinically important. Whole genome sequencing has revealed the presence of several virulence genes but the molecular mechanisms of S. argenteus infection and virulence are largely unknown. Here, we studied the effect of a previously characterized clinical S. argenteus isolate on human cells in vitro. The clinical isolate, together with the S. argenteus type strain MSHR1132T and the S. schweitzeri type strain FSA084T, had a cytotoxic effect on the cells, which showed necrotic cell death after a few hours of treatment. The protein causing the cytotoxic effect was purified and identified by mass spectrometry as alpha-hemolysin, Hla, which is awell-known pore-forming toxin in S.aureus. The cytotoxic effect could be blocked with an antibody against Hla. S.argenteus showed 12-15 fold higher expression levels of hla at the RNA level and 4-6 fold higher expression levels at the protein level compared to S.aureus. The higher expression levels of hla were supported by higher RNA levels of the regulatory factors sarA and saeR. Also, the RNAIII component of the accessory gene regulator (agr) quorum sensing system was 8,000-10,000 fold higher in the S.argenteus isolates compared to S.aureus. This is the first study on the effect of S.argenteus on ahuman cell line and strengthens the idea of significant virulence of S.argenteus.
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| 9. |
- Kaden, René, 1975-, et al.
(author)
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A novel real-time PCR assay for specific detection of Brucella melitensis
- 2017
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In: BMC Infectious Diseases. - : BIOMED CENTRAL LTD. - 1471-2334. ; 17
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Journal article (peer-reviewed)abstract
- Background: Brucellosis is a zoonosis that occurs worldwide. The disease has been completely eradicated in livestock in Sweden in 1994, and all cases of confirmed human brucellosis are imported into Sweden from other countries. However, due to an increase in the number of refugees and asylum seekers from the middle-east to Sweden, there is a need to improve the current diagnostic methodology for Brucella melitensis. Whilst culture of Brucella species can be used as a diagnostic tool, real-time PCR approaches provide a much faster result. The aim of this study was to set up a species-specific real-time PCR for the detection of all biovars of Brucella melitensis, which could be used routinely in diagnostic laboratories. Methods: A Brucella melitensis real-time PCR assay was designed using all available genomes in the public database of Brucella (N=96) including all complete genomes of Brucella melitensis (N=17). The assay was validated with a collection of 37 Brucella species reference strains, 120 Brucella melitensis human clinical isolates, and 45 clinically relevant non-Brucella melitensis strains. Results: In this study we developed a single real-time PCR for the specific detection of all biovars of Brucella melitensis. Conclusions: This new real-time PCR method shows a high specificity (100%) and a high sensitivity ( 1.25 GE/mu l) and has been implemented in the laboratories of four governmental authorities across Sweden.
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| 10. |
- Kaden, Rene, et al.
(author)
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Application of the Dynamic Cultivation System for Microorganisms – a new way to culture the unculturables
- 2014
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In: Clays and clay minerals. - 0009-8604 .- 1552-8367. ; 62:3-4, s. 203-210
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Journal article (peer-reviewed)abstract
- To date, ~1% of all bacteria that occur in environmental ecosystems such as soil, sedimentary rocks, and groundwater have been described. Comprehensive explanation of ecological interactions on a microscale level is thus almost impossible. The Dynamic Cultivation System (DCS) was developed in order to detect more microbial taxa than with common cultivation approaches, as well as previously undescribed bacterial species. The DCS is a quick and easy in situ method for the cultivation of numerous bacterial taxa in support of the description of microbial colonized ecosystems. To investigate the bacterial populations within a clay-maturation process after mining the raw material, the DCS was used to increase the microbial biomass for further molecular analysis. Two different methods were applied to extract the bacteria from the DCS and these were compared in terms of efficiency at detection of large numbers of different taxa and in terms of applicability to the detection of previously undescribed species in raw clays. A collection of different undescribed species was detected with sequencing. While direct picking of bacterial colonies leads to the detection of different genera, species mainly of the genus Arthobacter were proved in the phosphate-buffered saline-suspended biomass. Thus, a combination of the approaches mentioned above is recommended to increase the number of detectable species. The DCS will help to describe better the microbial content of ecosystems, especially soils that contain charged particles.
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