Umeå University's logo

Recent studies have revealed that the long-recognized link between the historically defined Fusobacterium nucleatum group and colorectal cancer is largely driven by Fusobacterium animalis. This species, along with two others (Fusobacterium polymorphum and Fusobacterium vincentii), was recently reclassified as distinct from F. nucleatum, highlighting functional divergence within this group. Due to their close genetic relatedness, traditional partial 16S rRNA gene sequencing lacks the resolution to reliably distinguish these species. Nevertheless, accurate species-level identification remains essential in cancer-associated microbiome research. Here, we demonstrate that full-length 16S rRNA sequencing using Oxford Nanopore Technology, combined with a novel custom demultiplexing software, enables robust species-level discrimination within the Fusobacterium genus. Our approach accurately classified clinically relevant Fusobacterium species and recovered their expected proportions from whole cells, DNA mixtures, and clinical CRC specimens. This method provides high-resolution profiling to elucidate species-specific roles of Fusobacterium in colorectal cancer.

Orthohantaviruses, transmitted primarily by rodents, cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus pulmonary syndrome in the Americas. These viruses, with documented human-to-human transmission, exhibit a wide case-fatality rate, 0.5–40 %, depending on the virus species, and no vaccine or effective treatment for severe Orthohantavirus infections exists. In Europe, the Puumala virus (PUUV), carried by the bank vole Myodes glareolus, causes a milder form of HFRS. Despite the reliance on serology and PCR for diagnosis, the three genomic segments of Swedish wild-type PUUV have yet to be completely sequenced.

We have developed a targeted hybrid-capture method aimed at comprehensive genomic sequencing of wild-type PUUV isolates and the identification of other Orthohantaviruses. Our custom-designed panel includes >11,200 probes covering the entire Orthohantavirus genus. Using this panel, we sequenced complete viral genomes from bank vole lung tissue, human plasma samples, and cell-cultured reference strains. Analysis revealed that Swedish PUUV isolates belong to the Northern Scandinavian lineage, with nucleotide diversity ranging from 2.8 % to 3.7 % among them. Notably, no significant genotypic differences were observed between the viral sequences from reservoirs and human cases except in the nonstructural protein.

Despite the high endemicity of PUUV in Northern Sweden, these are the first complete Swedish wild-type PUUV genomes and substantially increase our understanding of PUUV evolution and epidemiology. The panel's sensitivity enables genomic sequencing of human samples with viral RNA levels reflecting the natural progression of infection and underscores our panel's diagnostic value, and could help to uncover novel Orthohantavirus transmission routes.

Background: Mounting evidence suggests a significant role of the gut microbiota in the development and progression of colorectal cancer (CRC). In particular, an over-representation of oral pathogens has been linked to CRC. The aim of this study was to further investigate the faecal microbial landscape of CRC patients, with a focus on the oral pathogens Parvimonas micra and Fusobacterium nucleatum.

Methods: In this study, 16S rRNA sequencing was conducted using faecal samples from CRC patients (n = 275) and controls without pathological findings (n = 95).

Results: We discovered a significant difference in microbial composition depending on tumour location and microsatellite instability (MSI) status, with P. micra, F. nucleatum, and Peptostreptococcus stomatis found to be more abundant in patients with MSI tumours. Moreover, P. micra and F. nucleatum were associated with a cluster of CRC-related bacteria including Bacteroides fragilis as well as with other oral pathogens such as P. stomatis and various Porphyromonas species. This cluster was distinctly different in the control group, suggesting its potential linkage with CRC.

Conclusions: Our results suggest a similar distribution of several CRC-associated bacteria within CRC patients, underscoring the importance of considering the concomitant presence of bacterial species in studies investigating the mechanisms of CRC development and progression.

The importance of the tumour microbiome in different aspects of colorectal cancer (CRC) has been increasingly recognised, but many questions remain. The aim of this study was to explore the effect of specific CRC associated microbes on the tumour immune response, which has a considerable prognostic value in CRC. We applied specific qPCR to detect Parvimonas micra and Fusobacterium nucleatum in tumour tissues from an immunologically well-characterised cohort of 69 CRC patients. This cohort included detailed analyses of immune profiles based on flow cytometry and transcriptomics in tumour tissue and blood, along with comprehensive analyses of molecular subtypes. P. micra and F. nucleatum were detected in 24% and 64% of tumour tissues, respectively. We found a significant association of P. micra with high-grade tumours and tumours of CMS1 subtype. F. nucleatum was significantly associated with right-sided tumours, microsatellite instability, and CMS1 tumours. The immunological analyses revealed significant associations of P. micra with activated CD69+ T lymphocytes and increased antigen-presenting HLA-DR+ B lymphocytes. P. micra was also positively associated with M1 and M2 macrophage traits. The impact of P. micra tumour colonisation on the immune response was further assessed using transcriptomics in validation of our findings. No associations were found between F. nucleatum and immune profiles in this study. Our findings support novel associations between P. micra and the immune response in CRC. A better understanding of these interactions might help to identify important predictive and prognostic tools as well as new targets for therapy.

Colorectal cancer (CRC) is a heterogeneous disease with different genetic and molecular backgrounds, leading to a diverse patient prognosis and treatment response. Four consensus molecular subtypes (CMS 1–4) have recently been proposed based on transcriptome profiling. A clinically practical immunohistochemistry (IHC) based CMS classifier consisting of the four markers FRMD6, ZEB1, HTR2B, and CDX2 was then demonstrated. However, the IHC-CMS classifier did not distinguish between CMS2 and CMS3 tumours. In this study, we have applied the proposed transcriptome based and IHC-based CMS classifiers in a CRC cohort of 65 patients and found a concordance of 77.5 %. Further, we modified the IHC-CMS classifier by analysing the differentially expressed genes between CMS2 and CMS3 tumours using RNA-sequencing data from the TCGA dataset. The result showed that WNT signalling was among the most upregulated pathways in CMS2 tumours, and the expression level of CTNNB1 (encoding β-catenin), a WNT pathway hallmark, was significantly upregulated (P = 1.15 × 10−6). We therefore introduced nuclear β-catenin staining to the IHC-CMS classifier. Using the modified classifier in our cohort, we found a 71.4 % concordance between the IHC and RNA-sequencing based CMS classifiers. Moreover, β-catenin staining could classify 16 out of the 19 CMS2/3 tumours into CMS2 or CMS3, thereby showing an 84.2 % concordance with the RNA-sequencing-based classifier. In conclusion, we evaluated CMS classifiers based on transcriptome and IHC analysis. We present a modified IHC panel that categorizes CRC tumours into the four CMS groups. To our knowledge, this is the first study using IHC to identify all four CMS groups.

Colorectal cancer (CRC) is a heterogeneous disease, with varying clinical presentations and patient prognosis. Different molecular subgroups of CRC should be treated differently and therefore, must be better characterized. Organoid culture has recently been suggested as a good model to reflect the heterogeneous nature of CRC. However, organoid cultures cannot be established from all CRC tumors. The study examines which CRC tumors are more likely to generate organoids and thus benefit from ex vivo organoid drug testing. Long-term organoid cultures from 22 out of 40 CRC tumor specimens were established. It was found that organoid cultures were more difficult to establish from tumors characterized as microsatellite instable (MSI), BRAF-mutated, poorly differentiated and/or of a mucinous type. This suggests that patients with such tumors are less likely to benefit from ex vivo organoid drug testing, but it may also suggest biological difference in tumor growth. RNA sequencing analysis of tumor sections revealed that the in vivo maintenance of these non-organoid-forming tumors depends on factors related to inflammation and pathogen exposure. Furthermore, using TCGA data we could show a trend towards a worse prognosis for patients with organoid-forming tumors, suggesting also clinical differences. Results suggest that organoids are more difficult to establish from tumors characterized as MSI, BRAF-mutated, poorly differentiated and/or of a mucinous type. We further suggest that the maintenance of cell growth of these tumors in vivo may be promoted by immune-related factors and other stromal components within the tumor microenvironment.

The use of faecal microbial markers as non-invasive biomarkers for colorectal cancer (CRC) has been suggested, but not fully elucidated. Here, we have evaluated the importance of Parvimonas micra as a potential non-invasive faecal biomarker in CRC and its relation to other microbial biomarkers. The levels of P. micra, F. nucleatum and clbA+bacteria were quantified using qPCR in faecal samples from a population-based cohort of patients undergoing colonoscopy due to symptoms from the large bowel. The study included 38 CRC patients, 128 patients with dysplasia and 63 controls. The results were validated in a second consecutive CRC cohort including faecal samples from 238 CRC patients and 94 controls. We found significantly higher levels of P. micra in faecal samples from CRC patients compared to controls. A test for P. micra could detect CRC with a specificity of 87.3% and a sensitivity of 60.5%. In addition, we found that combining P. micra with other microbial markers, could further enhance test sensitivity. Our findings support the potential use of P. micra as a non-invasive biomarker for CRC. Together with other microbial faecal markers, P. micra may identify patients with "high risk" microbial patterns, indicating increased risk and incidence of cancer.

Francisella noatunensis is a fastidious facultative intracellular bacterial pathogen that causes 'piscine francisellosis', a serious disease affecting both marine and fresh water farmed and wild fish worldwide. Currently two F. noatunensis subspecies are recognized, i.e. F. noatunensis subsp. noatunensis and F. noatunensis subsp. orientalis. In the present study, the taxonomy of F noatunensis was revisited using a polyphasic approach, including whole genome derived parameters such as digital DNA-DNA hybridization, whole genome average nucleotide identity (wg-ANlm), whole genome phylogenetic analysis, whole genome G+C content, metabolic fingerprinting and chemotaxonomic analyses. The results indicated that isolates belonging to F. noatunensis subsp. orientalis represent a phenotypically and genetically homogenous taxon, clearly distinguishable from F. noatunensis subsp. noatunensis that fulfils requirements for separate species status. We propose, therefore, elevation of F. noatunensis subsp. orientalis to the species rank as Francisella orientalis sp. nov. with the type strain remaining as Ehime-1T (DSM 21254(T)=LMG 24544(T)). Furthermore, we identified sufficient phenotypic and genetic differences between F. noatunensis subsp. noatunensis recovered from diseased farmed Atlantic salmon in Chile and those isolated from wild and farmed Atlantic cod in Northern Europe to warrant proposal of the Chilean as a novel F. noatunensis subspecies, i.e. Francisella noatunensis subsp. chilensis subsp. nov. with strain PQ1106(T) (CECT 9798(T)=NCTC14375(T)) as the type strain. Finally, we emend the description of F. noatunensis by including further metabolic information and the description of atypical strains.