Broad-range amplification and sequencing of the 16S rRNA gene, directly from clinical samples, is a method that potentially allows detection of any cultivable or non-cultivable bacteria. However, the method is prone to false positive results due to PCR contamination. Another concern is the human DNA abundance compared to bacterial DNA in samples from sterile sites. Those factors may decrease the sensitivity and specificity of the assay and can complicate the analysis and interpretation of the results. The objective of this prospective study was to try to avoid the most common pitfalls, mentioned above, and develop a molecular 16S assay with a high clinical sensitivity and specificity. Fifty-six consecutive tissue samples from patients with suspected deep infections were extracted by 3 different DNA-extraction methods; two based on a principle of bacterial DNA enrichment, and one conventional DNA extraction method. We compared three primer pairs, including both conventional and DPO principle, targeting different variable regions of the 16S rRNA gene. Results from routine tissue culture were used as reference. Clinical data was recorded from patient charts and analyzed in parallel. Of a total of 56 samples, collected from 39 patients, 70% (39 samples) were assessed as true infections by analysis of clinical data. Bacterial enrichment extraction increased sensitivity from 54% to 72%. The 2 sets of primer pairs defining region V1-V3 and V3-V4, showed similar sensitivity, but DPO-primers resulted in better specificity, i.e. less contaminations. The primer pairs covering V1-V8 show significantly lower sensitivity (p < .001) than V1-V3 and V3-V4. Optimizing extraction protocols and choice of primers can increase the sensitivity and specificity of a molecular 16S-analysis, rendering a valuable complement to tissue culture.

Objective: Fecal microbiota transplantation (FMT) is a highly effective treatment forClostridioides difficileinfection (CDI). However, the fecal transplant’s causal components translating into clearance of the CDI are yet to be identified. The commensal bacteriaFaecalibacterium prausnitziimay be of great interest in this context, since it is one of the most common species of the healthy gut microbiota and produces metabolites with anti-inflammatory properties. Although there is mounting evidence thatF.prausnitziiis an important regulator of intestinal homeostasis, data about its role in CDI and FMT are relatively scarce.Methods: Stool samples from patients with recurrent CDI were collected to investigate the relative abundance ofF.prausnitziibefore and after FMT. Twenty-one patients provided fecal samples before the FMT procedure, at 2 weeks post-FMT, and at 2–4 months post-FMT. The relative abundance ofF.prausnitziiwas determined using quantitative polymerase chain reaction.Results: The abundance ofF.prausnitziiwas elevated in samples (N = 9) from donors compared to pre-FMT samples (N = 15) from patients (adjusted P<0.001). No significant difference in the abundance ofF.prausnitziibetween responders (N = 11) and non-responders (N = 4) was found before FMT (P = 0.85). In patients with CDI, the abundance ofF.prausnitziisignificantly increased in the 2 weeks post-FMT samples (N = 14) compared to the pre-FMT samples (N = 15, adjusted P<0.001). The increase persisted 2–4 months post-FMT (N = 15) compared to pre-FMT samples (N = 15) (adjusted P<0.001).Conclusions: FMT increases the relative abundance ofF.prausnitziiin patients with recurrent CDI, and this microbial shift remains several months later. The baseline abundance ofF.prausnitziiin donors or recipients was not associated with future treatment response, although a true predictive capacity cannot be excluded because of the limited sample size. Further studies are needed to discern whetherF.prausnitziiplays an active role in the resolution of CDI.

Background and Objectives: Lichen sclerosus (LSc) is a chronic inflammatory and sclerosing condition that primarily affects the genital region. In women, vulvar lichen sclerosus presents with a wide range of clinical features, including pruritus, erythema, burning, pain, dysuria, architectural distortion, scarring, and the formation of ivory-white atrophic plaques. While the precise etiology of LSc remains unclear, increasing evidence indicates that prolonged exposure to urine and occlusion may play a central role in its pathogenesis. Additionally, the role of the genital microbiome has garnered increasing attention, with dysbiosis—an imbalance in microbial communities—emerging as a potential contributing factor. Prior investigations have reported an increased abundance of Gardnerella in women with LSc, but findings regarding Streptococcus and Lactobacillus have been inconsistent. This study aimed to investigate the relationship between the vulvar microbiome and LSc. Materials and Methods: Forty-seven women with LSc and seven healthy controls were recruited from the Division of Dermatology, Ryhov Hospital, Jönköping. Vulvar swabs were obtained from affected skin areas. The 16S rRNA regions were amplified and sequenced using the Illumina MiSeq system. Differential abundance analyses were conducted using four regression methods, and genera were considered significant only if identified by at least two methods. Results: Compared to controls, LSc patients showed reduced abundance of Lactobacillus, Finegoldia, Lawsonella, Stapylococcus, Cutibacterium, and an unidentified genus belonging to the Actinomycetaceae family. In contrast, Porphyromonas and Ezekiella, were significantly increased. Beta diversity analysis revealed significant differences in microbiome composition between groups (p = 0.022). Conclusions: The vulvar microbiome of women with LSc differs significantly from that of healthy controls. These findings support a possible role of microbial dysbiosis in LSc pathogenesis, highlighting the need for further research into microbiome-targeted interventions.

Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder characterized by abdominal pain, altered bowel habits, and frequent comorbidity with anxiety and depression. The gut microbiota has been implicated in gut-brain axis (GBA) dysfunction, but consistent microbial signatures remain unclear. We performed whole metagenome shotgun sequencing of stool samples from 63 female patients with moderate to severe IBS and 34 female healthy controls and assessed microbial composition and functional pathways. Microbial richness and diversity were slightly reduced in IBS, though with high variability and no robust separation from controls. Differential abundance analyses revealed enrichment of Streptococcus sp. and the sulfate-reducing bacterium Desulfovibrio piger in IBS, alongside reductions in Bifidobacterium and Methanobrevibacter . Functional profiling identified 39 differentially abundant pathways: amino acid biosynthesis (e.g., L-isoleucine, L-threonine) was more prominent in IBS, while carbohydrate degradation pathways (e.g., galactose, stachyose) were enriched in healthy controls. These findings indicate modest but significant IBS-associated shifts in gut microbial composition and function that may contribute to IBS symptoms. However, high intra-group variability underscores the complexity of IBS and highlights the need for larger, multi-omics studies to define robust microbial markers. These results contribute to a growing body of evidence emphasizing the complexity of gut microbiota-host interactions and the need for high-resolution, systems-level approaches in microbiome-associated disorders.