Next-generation sequencing (NGS) and metagenomics revolutionized our capacity for analysis and identification of the microbial communities in complex samples. The existence of a blood microbiome in healthy individuals has been confirmed by sequencing, but some researchers suspect that this is a cell-free circulating DNA in blood, while others have had isolated a limited number of bacterial and fungal species by culture. It is not clear what part of the blood microbiota could be resuscitated and cultured. Here, we quantitatively measured the culturable part of blood microbiota of healthy individuals by testing a medium supplemented with a high concentration of vitamin K (1 mg/mL) and culturing at 43 °C for 24 h. We applied targeted sequencing of 16S rDNA and internal transcribed spacer (ITS) markers on cultured and non-cultured blood samples from 28 healthy individuals. Dominant bacterial phyla among non-cultured samples were Proteobacteria 92.97%, Firmicutes 2.18%, Actinobacteria 1.74% and Planctomycetes 1.55%, while among cultured samples Proteobacteria were 47.83%, Firmicutes 25.85%, Actinobacteria 16.42%, Bacteroidetes 3.48%, Cyanobacteria 2.74%, and Fusobacteria 1.53%. Fungi phyla Basidiomycota, Ascomycota, and unidentified fungi were 65.08%, 17.72%, and 17.2% respectively among non-cultured samples, while among cultured samples they were 58.08%, 21.72%, and 20.2% respectively. In cultured and non-cultured samples we identified 241 OTUs belonging to 40 bacterial orders comprising 66 families and 105 genera. Fungal biodiversity accounted for 272 OTUs distributed in 61 orders, 105 families, and 133 genera. Bacterial orders that remained non-cultured, compared to blood microbiota isolated from fresh blood collection, were Sphingomonadales, Rhizobiales, and Rhodospirillales. Species of orders Bacillales, Lactobacillales, and Corynebacteriales showed the best cultivability. Fungi orders Tremellales, Polyporales, and Filobasidiales were mostly unculturable. Species of fungi orders Pleosporales, Saccharomycetales, and Helotiales were among the culturable ones. In this study, we quantified the capacity of a specific medium applied for culturing of blood microbiota in healthy individuals. Other culturing conditions and media should be tested for optimization and better characterization of blood microbiota in healthy and diseased individuals.

Mycobacterium bovis bacillus Calmette–Guérin (BCG) is the only live attenuated vaccine available against tuberculosis. The first BCG vaccination was done exactly 100 years ago, in 1921. The BCG vaccine strains used worldwide represent a family of daughter sub-strains with distinct genotypic characteristics. BCG SL222 Sofia is a seed lot sub-strain descending from the Russian BCG-I (seed lot 374a) strain and has been used for vaccine production in Bulgaria since 1972. Here, we report the assembled circular genome sequence of Mycobacterium bovis BCG SL222 Sofia and phylogeny analysis with the most closely related BCG sub-strains. The full circular genome of BCG SL222 Sofia had a length of 4,370,706 bp with an average GC content of 65.60%. After 49 years of in vitro evolution in a freeze-dried condition, we identified four SNP mutations as compared to the reference BCG-I (Russia-368) sequence. BCG vaccination is of central importance for the TB elimination programs in many countries. Since 1991, almost 40 million vaccine doses of the BCG SL222 Sofia have been distributed annually through the United Nations Children’s Fund (UNICEF) and the Pan American Health Organization (PAHO) to approximately 120 countries. The availability of the complete reference genome sequence for M. bovis BCG SL222 Sofia, a WHO reference reagent for the Russian BCG-I sub-strain, will facilitate the identity assurance of the genomic stability, will contribute to more consistent manufacturing, and has an important value in standardization and differentiation of sub-strains used in vaccine production. We propose to rename the sub-strain BCG SL222 Sofia to BCG-Sofia for practical and common use.

Veryovkina Cave is the world’s deepest known cave (2212 m deep). It is located in the Arabika Massif of Gagra Mountain in the Western Caucasus. Its microbiome remains unknown because of difficulties in access. Ten sediment samples were collected at vertical depths ranging from 300 m to 2204 m; they varied by substrate type, moisture content, and visitor accessibility. Total microbial DNA was isolated, and 16S ribosomal gene metabarcoding was applied for taxonomic identification. Seven samples showed reliable content, whereas three samples indicated no recoverable reads. Proteobacteria, Acidobacteria, and Actinobacteria were the most abundant phyla in total. Depth stratification of microbiota showed that (1) shallow wet clays were dominated by Acidimicrobia and Actinobacteria; (2) mid-depth wet clays showed the highest abundance of Nitrospira, Betaproteobacteria, and Vicinamibacter; and (3) deep, dry substrates were dominated by Thermoleophilia and Rubrobacteria. Multivariate analyses showed that substrate type and moisture tended to explain more variation in microbial abundance than depth or human activity. We demonstrate the presence of distinct ecological niches within the cave ecosystem, which emphasizes the role of local conditions in shaping microbial diversity.

Background: Bats are important reservoir hosts for numerous emerging pathogens with zoonotic potential. In Europe, 47 bat species have been recorded, 33 of which occur in Bulgaria. Previous research has shown that bats harbor several dangerous viruses, including SARS, Ebola, Nipah, Hendra, Marburg, and MERS, as well as various bacterial, fungal, and protozoan pathogens. Despite these findings, information about the composition of the bat blood microbiome remains limited. The present study aimed to characterize the microbial diversity present in the blood of bats inhabiting caves in Bulgaria. Materials and Methods: Blood samples were collected from bat species belonging to the families Vespertilionidae and Rhinolophidae inhabiting three caves in Bulgaria. Between 10 and 30 µL of blood was obtained from each individual. Samples from 122 bats were combined into 14 pooled samples. Additionally, heart tissue and two spleens were collected. DNA extraction was performed, followed by metagenomic sequencing using Illumina shotgun technology with a sequencing depth of 6 Gb. Taxonomic classification of quality-filtered reads was conducted using the Kraken2 tool with the RefSeq NCBI bacterial database within the Galaxy platform (www.usegalaxy.eu). Operational taxonomic unit (OTU) tables were generated using Bracken and visualized with Pavian software. Results: Metagenomic analysis revealed the presence of numerous microbial taxa in bat blood samples. A notable abundance of potentially pathogenic bacteria was detected, with sequencing reads exceeding 10000 for species belonging to the genera Mycobacterium, Mycoplasma, and Bartonella. Conclusions: The results indicate that bats in Bulgaria may serve as hidden or potential reservoirs of bacterial pathogens with zoonotic relevance. These findings highlight the importance of continued surveillance of bat-associated microbiota to better understand potential public health risks.

Background: Following the global resurgence of cholera in 2022, 51 cholera cases were reported by European countries. We previously characterised 49 Vibrio cholerae O1 isolates from these cases using whole genome sequencing. In 2023 and 2024, 59 additional cholera cases were reported by 11 European countries. Aim: We aimed to confirm that V. cholerae O1 isolates associated with cholera cases reported in Europe in 2023–2024 belonged to the epidemic seventh pandemic El Tor (7PET) lineage rather than to non-epidemic V. cholerae O1 lineages, and to characterise their virulence, antimicrobial resistance (AMR) determinants and phylogenetic relationships. Methods: Fifty V. cholerae O1 isolates were available for whole genome sequencing, of which 49 yielded genomes of sufficient quality for analysis. Genomes were analysed together with more than 1,500 publicly available 7PET genomes to place the European isolates into a global phylogenetic context. Results: All 49 genomes belonged to the 7PET lineage and Wave 3. Four sub-lineages were identified: BD1.2 (2/49), AFR12 (6/49), AFR13 (8/49) and Pre-AFR15 (33/49). The predominance of Pre-AFR15 confirms its continued contribution to the ongoing global cholera resurgence. In contrast to our 2022 survey, eight highly drug-resistant AFR13 isolates were detected, documenting the international spread of this highly drug-resistant clone resistant to multiple first-line antimicrobial agents. Conclusion: Whole genome sequencing should be routinely used by reference laboratories to distinguish epidemic 7PET from non-epidemic V. cholerae O1 lineages and to monitor the emergence and international spread of AMR clones. Continued collaborative genomic surveillance of travel-associated cholera cases can provide early insights into the emergence, international spread and antimicrobial resistance of 7PET sub-lineages.

Antarctica peninsula periphery islands undergo one of the most dramatic ecological changes due to ongoing global warming. The front fields of the Antarctic glaciers are extreme environments and pioneering sites for ecological succession. Rising temperatures lead to deglaciation in the Antarctic habitats, and the new terrain is subjected to the process of soil formation and microbial colonization. In the present study, we investigated the formation of pathogenic fungal soil microbiomes as an effect of forefield deglaciation. Soil samples were taken from two different forefields, one formed several years ago and the other freshly uncovered. Both habitats were in the vicinity of the Perunika Glacier situated in the northeastern direction of Hurd Peninsula, Livingston Island, the second largest island from the South Shetland Archipelago, about 100 km north of the Antarctic Peninsula. Total DNA was extracted and targeted ITS amplicon sequencing was applied. The ITS marker sequences were then taxonomically identified. The abundance of the fungal taxa was calculated. Alpha and Beta diversity analyses to obtain fungal richness in samples were performed. Our results showed that soil habitat formation, initiated by deglaciation, was such that: In the newly deglaciated forefield, there was almost no fungal DNA, which prevented further analysis; At older drier fields metagenome content was much higher; Further analysis showed that the most abundant genera were Pseudogymnoascus, Simplicillium, Hanseniaspora, Mycothermus, and Malassezia. In the newly deglaciated forefield, there was almost no fungal DNA, which prevented further analysis; At older drier fields metagenome content was much higher; Further analysis showed that the most abundant genera were Pseudogymnoascus, Simplicillium, Hanseniaspora, Mycothermus, and Malassezia. It is known that Pseudogymnoascus and Malassezia species have pathogenic keratinolytic activity. In conclusion, the phylum Ascomycota, which dominated the core microbiome showed much higher ecological diversity and abundance, i.e. potential for colonization of the glacier forefields. In contrast, the phylum Basidiomycota appeared to be less fit for these conditions.