Abstract Knowledge about diversity and taxonomic structure of the microbial population present in traditional fermented foods plays a key role in starter culture selection, safety improvement and quality enhancement of the end product. Aim of this study was to investigate microbial consortia composition in Slovak bryndza cheese. For this purpose, we used culture-independent approach based on 16S rDNA amplicon sequencing using next generation sequencing platform. Results obtained by the analysis of three commercial (produced on industrial scale in winter season) and one traditional (artisanal, most valued, produced in May) Slovak bryndza cheese sample were compared. A diverse prokaryotic microflora composed mostly of the genera Lactococcus, Streptococcus, Lactobacillus, and Enterococcus was identified. Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris were the dominant taxons in all tested samples. Second most abundant species, detected in all bryndza cheeses, were Lactococcus fujiensis and Lactococcus taiwanensis, independently by two different approaches, using different reference 16S rRNA genes databases (Greengenes and NCBI respectively). They have been detected in bryndza cheese samples in substantial amount for the first time. The narrowest microbial diversity was observed in a sample made with a starter culture from pasteurised milk. Metagenomic analysis by high-throughput sequencing using 16S rRNA genes seems to be a powerful tool for studying the structure of the microbial population in cheeses.
The complete genome sequence of a Slovak SL-1 isolate of Tomato mosaic virus (ToMV) was determined from the next generation sequencing (NGS) data, further confirming a limited sequence divergence in this tobamovirus species. Tomato genotypes Monalbo, Mobaci and Moperou, respectively carrying the susceptible tm-2 allele or the Tm-1 and Tm-2 resistant alleles, were tested for their susceptibility to ToMV SL-1. Although the three tomato genotypes accumulated ToMV SL-1 to similar amounts as judged by semi-quantitative DAS-ELISA, they showed variations in the rate of infection and symptomatology. Possible differences in the intra-isolate variability and polymorphism between viral populations propagating in these tomato genotypes were evaluated by analysis of the capsid protein (CP) encoding region. Irrespective of genotype infected, the intra-isolate haplotype structure showed the presence of the same highly dominant CP sequence and the low level of population diversity (0.08-0.19%). Our results suggest that ToMV CP encoding sequence is relatively stable in the viral population during its replication in vivo and provides further demonstration that RNA viruses may show high sequence stability, probably as a result of purifying selection. Keywords: tobamovirus, diversity, haplotype, resistance gene
Aim.The study of minimal ter operon as a determinant of tellurium resistance (Te R ) is important for the purpose of confirming the relationship of these genes to the pathogenicity of microorganisms.The ter operon is widespread among bacterial species and pathogens, implicated also in phage inhibition, oxidative stress and colicin resistance.So far, there is no experimental evidence for the role of the Escherichia coli (E.coli) minimal ter operon in ultraviolet C (UVC) resistance, biofilm formation and autoaggregation.To identify connection with UVC resistance of the minimal ter operon, matched pairs of Terpositive and -negative E. coli cells were stressed and differences in survival and whole genome sequence analysis were performed.This study was aimed also to identify differences in phenotype of cells induced by environmental stress.Methods.In the current study, a minimal ter operon (terBCDE∆F) originating from the uropathogenic strain E. coli KL53 was used.Clonogenic assay was the method of choice to determine cell reproductive death after treatment with UVC irradiation at certain time intervals.Bacterial suspensions were irradiated with 254 nm UVC-light (germicidal lamp in biological safety cabinet) in vitro.UVC irradiance output was 2.5 mW/cm 2 (calculated at the UVC device aperture) and plate-lamp distance of 60 cm.DNA damage analysis was performed using shot-gun sequencing on Illumina MiSeq platform.Biofilm formation was measured by a crystal violet retention assay.Auto-aggregation assay was performed according to the Ghane et al. (2020). Results.A large fraction of Ter-positive E. coli cells survived treatment with 120-s UVC light (300 mJ/cm 2 ) compared to matched Ter-negative cells; ~ 5-fold higher resistance of Ter-positive cells to UVC dose (p = 0.0007).Moreover, UVC surviving Ter-positive cells showed smaller mutation rate as Ternegative cells.The study demonstrated that a 1200-s exposure to UVC (3000 mJ/cm 2 ) was sufficient for 100% inhibition of growth for all the Ter-positive and -negative E. coli cells.The Ter-positive strain exhibited of 26 % higher auto-aggregation activities and was able to inhibit biofilm formation over than Ter-negative strain (**** P ˂ 0.0001). Conclusion.Our study shows that Ter-positive cells display lower sensitivity to UVC radiation, corresponding to a presence in minimal ter operon.In addition, our study suggests that also autoaggregation ability is related to minimal ter operon.The role of the minimal ter operon (terBCDE∆F) in resistance behavior of E. coli under environmental stress is evident.
Abstract Rationale Severe alcohol-associated hepatitis (SAH) is the most critical, acute, inflammatory phenotype within the alcohol-associated liver disease (ALD) spectrum, characterized by high 30- and 90-day mortality. Since several decades, corticosteroids (CS) are the only approved pharmacotherapy offering highly limited survival benefits. Contextually, there is an evident demand for 3PM innovation in the area meeting patients’ needs and improving individual outcomes. Fecal microbiota transplantation (FMT) has emerged as one of the new potential therapeutic options. In this study, we aimed to address the crucial 3PM domains in order to assess (i) the impact of FMT on mortality in SAH patients beyond CS, (ii) to identify factors associated with the outcome to be improved (iii) the prediction of futility, (iv) prevention of suboptimal individual outcomes linked to increased mortality, and (v) personalized allocation of therapy. Methods We conducted a prospective study (NCT04758806) in adult patients with SAH who were non-responders (NR) to or non-eligible (NE) for CS between January 2018 and August 2022. The intervention consisted of five 100 ml of FMT, prepared from 30 g stool from an unrelated healthy donor and frozen at − 80 °C, administered daily to the upper gastrointestinal (GI) tract. We evaluated the impact of FMT on 30- and 90-day mortality which we compared to the control group selected by the propensity score matching and treated by the standard of care; the control group was derived from the RH7 registry of patients hospitalized at the liver unit (NCT04767945). We have also scrutinized the FMT outcome against established and potential prognostic factors for SAH — such as the model for end-stage liver disease (MELD), Maddrey Discriminant Function (MDF), acute-on-chronic liver failure (ACLF), Liver Frailty Index (LFI), hepatic venous-portal pressure gradient (HVPG) and Alcoholic Hepatitis Histologic Score (AHHS) — to see if the 3PM method assigns them a new dimension in predicting response to therapy, prevention of suboptimal individual outcomes, and personalized patient management. Results We enrolled 44 patients with SAH (NR or NE) on an intention-to-treat basis; we analyzed 33 patients per protocol for associated factors (after an additional 11 being excluded for receiving less than 5 doses of FMT), and 31 patients by propensity score matching for corresponding individual outcomes, respectively. The mean age was 49.6 years, 11 patients (33.3%) were females. The median MELD score was 29, and ACLF of any degree had 27 patients (81.8%). FMT improved 30-day mortality ( p = 0.0204) and non-significantly improved 90-day mortality ( p = 0.4386). Univariate analysis identified MELD ≥ 30, MDF ≥ 90, and ACLF grade > 1 as significant predictors of 30-day mortality, ( p = 0.031; p = 0.014; p = 0.034). Survival was not associated with baseline LFI, HVPG, or AHHS. Conclusions and recommendations in the framework of 3PM In the most difficult-to-treat sub-cohort of patients with SAH (i.e., NR/NE), FMT improved 30-day mortality. Factors associated with benefit included MELD ≤ 30, MDF ≤ 90, and ACLF < 2. These results support the potential of gut microbiome as a therapeutic target in the context of 3PM research and vice versa — to use 3PM methodology as the expedient unifying template for microbiome research. The results allow for immediate impact on the innovative concepts of (i) personalized phenotyping and stratification of the disease for the clinical research and practice, (ii) multilevel predictive diagnosis related to personalized/precise treatment allocation including evidence-based (ii) prevention of futile and sub-optimally effective therapy , as well as (iii) targeted prevention of poor individual outcomes in patients with SAH. Moreover, our results add to the existing evidence with the potential to generate new research along the SAH’s pathogenetic pathways such as diverse individual susceptibility to alcohol toxicity, host-specific mitochondrial function and systemic inflammation, and the role of gut dysbiosis thereof.
Aim.The study of minimal ter operon as a determinant of tellurium resistance (Te R ) is important for the purpose of confirming the relationship of these genes to the pathogenicity of microorganisms.The ter operon is widespread among bacterial species and pathogens, implicated also in phage inhibition, oxidative stress and colicin resistance.So far, there is no experimental evidence for the role of the Escherichia coli (E.coli) minimal ter operon in ultraviolet C (UVC) resistance, biofilm formation and autoaggregation.To identify connection with UVC resistance of the minimal ter operon, matched pairs of Terpositive and -negative E. coli cells were stressed and differences in survival and whole genome sequence analysis were performed.This study was aimed also to identify differences in phenotype of cells induced by environmental stress.Methods.In the current study, a minimal ter operon (terBCDE∆F) originating from the uropathogenic strain E. coli KL53 was used.Clonogenic assay was the method of choice to determine cell reproductive death after treatment with UVC irradiation at certain time intervals.Bacterial suspensions were irradiated with 254 nm UVC-light (germicidal lamp in biological safety cabinet) in vitro.UVC irradiance output was 2.5 mW/cm 2 (calculated at the UVC device aperture) and plate-lamp distance of 60 cm.DNA damage analysis was performed using shot-gun sequencing on Illumina MiSeq platform.Biofilm formation was measured by a crystal violet retention assay.Auto-aggregation assay was performed according to the Ghane et al. (2020). Results.A large fraction of Ter-positive E. coli cells survived treatment with 120-s UVC light (300 mJ/cm 2 ) compared to matched Ter-negative cells; ~ 5-fold higher resistance of Ter-positive cells to UVC dose (p = 0.0007).Moreover, UVC surviving Ter-positive cells showed smaller mutation rate as Ternegative cells.The study demonstrated that a 1200-s exposure to UVC (3000 mJ/cm 2 ) was sufficient for 100% inhibition of growth for all the Ter-positive and -negative E. coli cells.The Ter-positive strain exhibited of 26 % higher auto-aggregation activities and was able to inhibit biofilm formation over than Ter-negative strain (**** P ˂ 0.0001). Conclusion.Our study shows that Ter-positive cells display lower sensitivity to UVC radiation, corresponding to a presence in minimal ter operon.In addition, our study suggests that also autoaggregation ability is related to minimal ter operon.The role of the minimal ter operon (terBCDE∆F) in resistance behavior of E. coli under environmental stress is evident.
Abstract The genus Elaphe Fitzinger, 1833 includes 17 species of charismatic, large-sized, non-venomous, Eurasian snakes. In the Western Palearctic, the genus is represented by three species from the Elaphe quatuorlineata group ranging from the Apennine peninsula to Central Asia. The southernmost population of this group is distributed in the mountains of the Southern Levant, with more than 400 km gap to other Elaphe populations. This population has been known to science for only 50 years and is virtually unstudied due to its extreme rarity. We studied these snakes’ morphological and genetic variation from the three countries where they are known to occur, i.e., Israel (Hermon, the Israeli-controlled Golan Heights), Lebanon, and Syria. We used nine mitochondrial and nuclear genes, complete mitogenome sequences, and a comprehensive morphological examination including published data, our own field observations, and museum specimens, to study its relationship to other species in the group. The three currently recognized species of the group ( E . quatuorlineata , E . sauromates , E . urartica ), and the Levant population, form four deeply divergent, strongly supported clades. Three of these clades correspond to the abovementioned species while the Southern Levant clade, which is genetically and morphologically distinct from all named congeners, is described here as a new species, Elaphe druzei sp. nov. The basal divergence of this group is estimated to be the Late Miocene with subsequent radiation from 5.1 to 3.9 Mya. The revealed biogeography of the E . quatuorlineata group supports the importance of the Levant as a major center of endemism and diversity of biota in Eurasia. The new species is large-sized and is one of the rarest snakes in the Western Palearctic. Because of its small mountain distribution range, in an area affected by land use and climate change, the new Elaphe urgently needs strict protection. Despite political issues, we hope this will be based on the cooperation of all countries where the new species occurs.
I. ricinus is an obligate hematophagous parasitic arthropod that is responsible for the transmission of a wide range of zoonotic pathogens including spirochetes of the genus Borrelia, Rickettsia spp., C. burnetii , Anaplasma phagocytophilum and Francisella tularensis , which are part the tick´s microbiome. Most of the studies focus on “pathogens” and only very few elucidate the role of “non-pathogenic” symbiotic microorganisms in I. ricinus . While most of the members of the microbiome are leading an intracellular lifestyle, they are able to complement tick´s nutrition and stress response having a great impact on tick´s survival and transmission of pathogens. The composition of the tick´s microbiome is not consistent and can be tied to the environment, tick species, developmental stage, or specific organ or tissue. Ovarian tissue harbors a stable microbiome consisting mainly but not exclusively of endosymbiotic bacteria, while the microbiome of the digestive system is rather unstable, and together with salivary glands, is mostly comprised of pathogens. The most prevalent endosymbionts found in ticks are Rickettsia spp., Ricketsiella spp., Coxiella -like and Francisella -like endosymbionts, Spiroplasma spp. and Candidatus Midichloria spp. Since microorganisms can modify ticks’ behavior, such as mobility, feeding or saliva production, which results in increased survival rates, we aimed to elucidate the potential, tight relationship, and interaction between bacteria of the I. ricinus microbiome. Here we show that endosymbionts including Coxiella -like spp., can provide I. ricinus with different types of vitamin B (B2, B6, B7, B9) essential for eukaryotic organisms. Furthermore, we hypothesize that survival of Wolbachia spp., or the bacterial pathogen A. phagocytophilum can be supported by the tick itself since coinfection with symbiotic Spiroplasma ixodetis provides I. ricinus with complete metabolic pathway of folate biosynthesis necessary for DNA synthesis and cell division. Manipulation of tick´s endosymbiotic microbiome could present a perspective way of I. ricinus control and regulation of spread of emerging bacterial pathogens.
Total RNAs from a symptomless tomato plant were subjected to next-generation sequencing (NGS) analysis, revealing the presence of a single viral agent -potato virus Y (PVY).The analysis of determined full-length genome sequence assigned the PVY SL16 isolate to the recombinant PVY-N-Wi strain group.A series of primers targeting the four main recombinant junction (RJ) sites were used for characterization of additional 5 tomato PVY isolates recovered in Western Slovakia.Based on the partial sequences, the isolates could be classified as belonging to PVY-N-Wi and PVY-NTNa strain groups.Interestingly, both these distinct recombinant PVY types were identified in mixed infection in one tomato sample (SL31).Our results further reinforce the data on the complexity of PVY infection and confirm the recombination as a significant evolutionary process shaping the PVY diversity.
