Characterization of Tularemia Cases in Slovenia with Multiple-Locus Variable-Number Tandem Repeat Analysis.

Francisella tularensis is the etiologic agent of tularemia, a bacterial zoonotic disease. The genome of F. tularensis shows a recent evolutionary change, especially in reservoirs. Variable number of tandem repeats (VNTR) is described as a high-speed molecular clock and can thus be used as a high-resolution typing system. The main objective of our study was to investigate the molecular diversity of F. tularensis strains and reveal possible sources of infection. Using real-time PCR targeting the ISFtu2 region, we successfully amplified targeted DNA in 13/31 Slovenian patients with a clinical diagnosis of tularemia, and with PCR targeting the fopA gene, we obtained 11/13 PCR products. Sequencing revealed that all samples were identified as F. tularensis subsp. holarctica. We successfully obtained one F. tularensis isolate from a lymph node aspirate by culture on chocolate agar. Our isolate was clustered into major clade B12 (subclade B43). We optimized VNTR typing to be used directly on clinical samples. Multiple-locus VNTR analysis (MLVA) revealed five unique MLVA types; 45.5% samples had the same MLVA type, another 27.3% shared a different MLVA type, and each of the remaining had a unique MLVA type. Most samples differed at only two VNTR markers (Ft-M03 and Ft-M06). Additionally, we investigated samples from small mammals (n = 532) and Ixodes ricinus ticks (n = 232) captured in the same geographical area in which patients with tularemia were found. No F. tularensis DNA was detected in samples of small mammals or I. ricinus ticks. The diversity of MLVA types in Slovenia was high, despite the small region, but most of the samples from the same region shared the same MLVA type. Our results suggest that MLVA is a useful tool for quick molecular characterization of F. tularensis directly from patient samples, especially when investigating geographically localized outbreaks.

Clostridioides difficile in bat guano.

Bats are associated with the emergence of several mammalian diseases. Their sessional migration, and tendency to form large colonies in close proximity to human habitats enables effective intra- and inter-species transmission of pathogens. Clostridioides difficile is an important enteric pathogen in humans and animals; however, the source of its dissemination in the population is unknown. The purpose of this study was to determine the prevalence of C. difficile in bats, and to characterize C. difficile isolates. Feces (n = 93) was sampled from bats during their migration across Europe. Eighteen samples (19.4%) were positive for C. difficile; ribotypes 078, 056, and a new ribotype CDB3 were identified. Clostridioides difficile ribotypes 078 and 056 are associated with human and animal diseases. The C. difficile prevalence and ribotypes in this study do not necessarily identify bats as a significant source, but more likely as an indicator of C. difficile perpetuation in the environment.

The fecal bacterial microbiota of bats; Slovenia.

METHODS: Fecal samples were collected from 92 bats in Slovenia, consisting of 12 different species, and the bacterial microbiota was assessed via next generation sequencing of the 16S rRNA gene V4 region. RESULTS: Sequences were assigned to 28 different phyla, but only Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria accounted for ≥1% of sequences. One phylum (Proteobacteria), one class (Gammaproteobacteria), three orders (Pseudomonadales, Lactobacillales, Bacillales), four families (Enterobacteriaceae, Pseudomonadaceae, Staphylococcaceae, Carnobacteriaceae), and five genera (Pseudomonas, Staphylococcus, Carnobacterium, an unclassified Enterobacteriaceae, Acinetobacter) accounted for 50% of sequences. There were no significant differences in the relative abundances of any phyla between bat species, but various differences were noted at lower taxonomic levels, such as Enterobacteriaceae (P = 0.007, most abundant in M. blythii), Pseudomonadaceae (P = 0.007, most abundant in Rhinolophus hipposideros) and Chlamydiaceae (P = 0.04, most abundant in Myotis myotis). There were significant differences in richness between species in both adults and juveniles/subadults, but there was no impact of sex on any alpha diversity index. When only adults are considered, there were significant differences in community membership between M. blythii and M. emarginatus (P = 0.011), and M. blythii and R. hipposideros (P = 0.004). There were also significant differences in community structure between M. blythii and M. emarginatus (P = 0.025), and M. blythii and R. hipposideros (P = 0.026). When adults of the four main species were compared, 14 OTUs were identified as differentially abundant using LEfSe. Only one difference was identified when comparing R. hipposideros adults and juvenile/subadults, with Klebsiella over-represented in the younger bats. CONCLUSIONS: Bats have a complex and diverse microbiota with a high relative abundance of Proteobacteria. The relevance of this difference is unclear and requires further study. Differences in the microbiota were observed between bat species, perhaps reflecting different diets and environmental exposures.

Morphologically Cryptic Amphipod Species Are "Ecological Clones" at Regional but Not at Local Scale: A Case Study of Four Niphargus Species.

Recent studies indicate that morphologically cryptic species may be ecologically more different than would be predicted from their morphological similarity and phylogenetic relatedness. However, in biodiversity research it often remains unclear whether cryptic species should be treated as ecologically equivalent, or whether detected differences have ecological significance. In this study, we assessed the ecological equivalence of four morphologically cryptic species of the amphipod genus Niphargus. All species live in a small, isolated area on the Istrian Peninsula in the NW Balkans. The distributional ranges of the species are partially overlapping and all species are living in springs. We reconstructed their ecological niches using morphological traits related to feeding, bioclimatic niche envelope and species' preference for epi-hypogean habitats. The ecological meaning of differences in niches was evaluated using distributional data and co-occurrence frequencies. We show that the species comprise two pairs of sister species. All species differ from each other and the degree of differentiation is not related to phylogenetic relatedness. Moreover, low co-occurrence frequencies in sympatric zones imply present or past interspecific competition. This pattern suggests that species are not differentiated enough to reduce interspecific competition, nor ecologically equivalent to co-exist via neutral dynamics. We tentatively conclude that the question of ecological equivalence relates to the scale of the study: at a fine scale, species' differences may influence dynamics in a local community, whereas at the regional level these species likely play roughly similar ecological roles.

Phylogeographic diversity of pathogenic and non-pathogenic hantaviruses in slovenia.

Slovenia is a very diverse country from a natural geography point of view, with many different habitats within a relatively small area, in addition to major geological and climatic differences. It is therefore not surprising that several small mammal species have been confirmed to harbour hantaviruses: A. flavicollis (Dobrava virus), A. agrarius (Dobrava virus-Kurkino), M. glareolus (Puumala virus), S. areanus (Seewis virus),M. agrestis, M. arvalis and M. subterraneus (Tula virus). Three of the viruses, namely the Dobrava, Dobrava-Kurkino and Puumala viruses, cause disease in humans, with significant differences in the severity of symptoms. Due to changes in haemorrhagic fever with renal syndrome cases (HFRS) epidemiology, a detailed study on phylogenetic diversity and molecular epidemiology of pathogenic and non-pathogenic hantaviruses circulating in ecologically diverse endemic regions was performed. The study presents one of the largest collections of hantavirus L, M and S sequences obtained from hosts and patients within a single country. Several genetic lineages were determined for each hantavirus species, with higher diversity among non-pathogenic compared to pathogenic viruses. For pathogenic hantaviruses, a significant geographic clustering of human- and rodent-derived sequences was confirmed. Several geographic and ecological factors were recognized as influencing and limiting the formation of endemic areas.