Browsing by Subject "16S rRNA"

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  • Tasiyana, Diana Tendai (Helsingin yliopisto, 2015)
    The shelf life of ESL milks and pasteurized creams is limited by bacterial spoilage. The current state of knowledge with respect to the diversity and activities of the spoilage microflora in these products is still limited. To date, studies on this subject are yet to be carried out in Finland. The aim of this study was to characterize the spoilage microorganisms found in ESL milks and pasteurised creams mainly produced in Finland. 10 ESL milks and 8 pasteurised cream packages were obtained from the Finnish retail market and were stored at 8?C, 15?C, room temperature and 30?C, respectively, until expiration. Total bacterial counts were evaluated and 31 bacterial isolates were selected. An attempt was made to characterize the bacterial isolates using rpoB and 16S rRNA partial gene sequence analyses. Psychrotrophic, spore-forming and spoilage features were also determined. Strain diversity was determined by rep-PCR profiling. Five selected isolates were subjected to metabolic profiling using the API50 CHB test. When growth was detected, total bacterial counts ranged from 2.67 to 3.74 and 2.54 to 8.98 log units in cream and ESL milk samples, respectively. Both ESL milks and cream samples were dominated by heat-resistant, spore-forming species of the Bacillus genera: the isolates were related to B. licheniformis. B. weihenstephanensis, B. safensis, B. kochii, B. pumilus, B. subtilis and B. anthracis. Intraspecies and intrasample strain diversity was very high. Isolates mainly displayed proteolytic and lipolytic activity. Psychrotrophic activity was high in the cream samples while most ESL milk isolates were mostly mesophilic. Tested isolates were non-lactose fermenters and had varying metabolic profiles. The study revealed that Finnish ESL milks and pasteurized creams are prone to spoilage by species of the Bacillus genera that have potential for lipolytic and proteolytic spoilage and some of which are considered toxigenic. There is wide intraspecies strain diversity responsible for the wide variability in metabolic, psychrotrophic and spoilage features. Phenotypic tests based on API50 CHB cannot be relied upon to make conclusions on taxonomy. Combining various methods is important for the comprehensive characterization of the bacterial isolates. Strategies to eliminate bacterial species of the Bacillus genera are important to prolong shelf life. However, intervention should be strain-specific to be effective. Large scale studies are required to confirm findings from this study.
  • Lammel, Daniel R; Barth, Gabriel; Ovaskainen, Otso; Cruz, Leonardo M; Zanatta, Josileia A; Ryo, Masahiro; de Souza, Emanuel M; Pedrosa, Fábio O (BioMed Central, 2018)
    Abstract Background pH is frequently reported as the main driver for prokaryotic community structure in soils. However, pH changes are also linked to “spillover effects” on other chemical parameters (e.g., availability of Al, Fe, Mn, Zn, and Cu) and plant growth, but these indirect effects on the microbial communities are rarely investigated. Usually, pH also co-varies with some confounding factors, such as land use, soil management (e.g., tillage and chemical inputs), plant cover, and/or edapho-climatic conditions. So, a more comprehensive analysis of the direct and indirect effects of pH brings a better understanding of the mechanisms driving prokaryotic (archaeal and bacterial) community structures. Results We evaluated an agricultural soil pH gradient (from 4 to 6, the typical range for tropical farms), in a liming gradient with confounding factors minimized, investigating relationships between prokaryotic communities (16S rRNA) and physical–chemical parameters (indirect effects). Correlations, hierarchical modeling of species communities (HMSC), and random forest (RF) modeling indicated that both direct and indirect effects of the pH gradient affected the prokaryotic communities. Some OTUs were more affected by the pH changes (e.g., some Actinobacteria), while others were more affected by the indirect pH effects (e.g., some Proteobacteria). HMSC detected a phylogenetic signal related to the effects. Both HMSC and RF indicated that the main indirect effect was the pH changes on the availability of some elements (e.g., Al, Fe, and Cu), and secondarily, effects on plant growth and nutrient cycling also affected the OTUs. Additionally, we found that some of the OTUs that responded to pH also correlated with CO2, CH4, and N2O greenhouse gas fluxes. Conclusions Our results indicate that there are two distinct pH-related mechanisms driving prokaryotic community structures, the direct effect and “spillover effects” of pH (indirect effects). Moreover, the indirect effects are highly relevant for some OTUs and consequently for the community structure; therefore, it is a mechanism that should be further investigated in microbial ecology.
  • Lammel, Daniel R.; Barth, Gabriel; Ovaskainen, Otso; Cruz, Leonardo M.; Zanatta, Josileia A.; Ryo, Masahiro; de Souza, Emanuel M.; Pedrosa, Fabio O. (2018)
    Background: pH is frequently reported as the main driver for prokaryotic community structure in soils. However, pH changes are also linked to "spillover effects" on other chemical parameters (e.g., availability of Al, Fe, Mn, Zn, and Cu) and plant growth, but these indirect effects on the microbial communities are rarely investigated. Usually, pH also co-varies with some confounding factors, such as land use, soil management (e.g., tillage and chemical inputs), plant cover, and/or edapho-climatic conditions. So, a more comprehensive analysis of the direct and indirect effects of pH brings a better understanding of the mechanisms driving prokaryotic (archaeal and bacterial) community structures. Results: We evaluated an agricultural soil pH gradient (from 4 to 6, the typical range for tropical farms), in a liming gradient with confounding factors minimized, investigating relationships between prokaryotic communities (16S rRNA) and physical-chemical parameters (indirect effects). Correlations, hierarchical modeling of species communities (HMSC), and random forest (RF) modeling indicated that both direct and indirect effects of the pH gradient affected the prokaryotic communities. Some OTUs were more affected by the pH changes (e.g., some Actinobacteria), while others were more affected by the indirect pH effects (e.g., some Proteobacteria). HMSC detected a phylogenetic signal related to the effects. Both HMSC and RF indicated that the main indirect effect was the pH changes on the availability of some elements (e.g., Al, Fe, and Cu), and secondarily, effects on plant growth and nutrient cycling also affected the OTUs. Additionally, we found that some of the OTUs that responded to pH also correlated with CO2, CH4, and N2O greenhouse gas fluxes. Conclusions: Our results indicate that there are two distinct pH-related mechanisms driving prokaryotic community structures, the direct effect and "spillover effects" of pH (indirect effects). Moreover, the indirect effects are highly relevant for some OTUs and consequently for the community structure; therefore, it is a mechanism that should be further investigated in microbial ecology.
  • Bi, Qing-Fang; Jin, Bing-Jie; Zhu, Dong; Jiang, Yu-Gen; Zheng, Bang-Xiao; O'Connor, Patrick; Yang, Xiao-Ru; Richter, Andreas; Lin, Xian-Yong; Zhu, Yong-Guan (2021)
    The positive roles of earthworms on soil functionality has been extensively documented. The capacity of the earthworm gut microbiota on decomposition and nutrient cycling under long-term fertilization in field conditions has rarely been studied. Here, we report the structural, taxonomic, and functional responses of Eisenia foetida and Pheretima guillelmi gut microbiota to different fertilization regimes and durations using 16S rRNA gene-based Illumina sequencing and high-throughput quantitative PCR techniques. Our results revealed that the core gut microbiota, especially the fermentative bacteria were mainly sourced from the soil, but strongly stimulated with species-specificity, potential benefits for the host and soil health. The functional compositions of gut microbiota were altered by fertilization with fertilization duration being more influential than fertilization regimes. Moreover, the combination of organic and inorganic fertilization with the longer duration resulted in a higher richness and connectivity in the gut microbiota, and also their functional potential related to carbon (C), nitrogen, and phosphorus cycling, particularly the labile C decomposition, denitrification, and phosphate mobilization. We also found that long-term inorganic fertilization increased the abundance of pathogenic bacteria in the P. guillelmi gut. This study demonstrates that understanding earthworm gut microbiota can provide insights into how agricultural practices can potentially alter soil ecosystem functions through the interactions between soil and earthworm gut microbiotas.
  • Hynönen, Ulla; Zoetendal, Erwin G.; Virtala, Anna-Maija K.; Shetty, Sudarshan; Hasan, Shah; Jakava-Viljanen, Miia; de Vos, Willem M.; Palva, Airi (2020)
    In our previous studies on irritable bowel syndrome (IBS) –associated microbiota by molecular methods, we demonstrated that a particular 16S rRNA gene amplicon was more abundant in the feces of healthy subjects or mixed type IBS (IBS-M) –sufferers than in the feces of individuals with diarrhea-type IBS (IBS-D). In the current study, we demonstrated that this, so called Ct85-amplicon, consists of a cluster of very heterogeneous 16S rRNA gene sequences, and defined six 16S rRNA gene types, a to f, within this cluster, each representing a novel species-, genus- or family level taxon. We then designed specific PCR primers for these sequence types, mapped the distribution of the Ct85-cluster sequences and that of the newly defined sequence types in several animal species and compared the sequence types present in the feces of healthy individuals and IBS sufferers using two IBS study cohorts, Finnish and Dutch. Various Ct85-cluster sequence types were detected in the fecal samples of several companion and production animal species with remarkably differing prevalences and abundances. The Ct85 sequence type composition of swine closely resembled that of humans. One of the five types (d) shared between humans and swine was not present in any other animals tested, while one sequence type (b) was found only in human samples. In both IBS study cohorts, one type (e) was more prevalent in healthy individuals than in the IBS-M group. By revealing various sequence types in the widespread Ct85-cluster and their distribution, the results improve our understanding of these uncultured bacteria, which is essential for future efforts to cultivate representatives of the Ct85-cluster and reveal their roles in IBS.
  • Hintikka, Tuomas P. (Helsingin yliopisto, 2020)
    Nautojen (Bos taurus) sorkka-alueen ihotulehdus (DD) on ympäri maailman levinnyt sairaus, jota on myös havaittu muilla sorkkaeläimillä. Nykytiedon mukaan nautojen DD on bakteeri-, ei virus- tai sienitauti. Se aiheuttaa haavoja, vaurioita ja känsiä. DD leviää helposti ja aiheuttaa eläimelle myös kipua, heikentää sen yleiskuntoa ja hyvinvointia sekä aiheuttaa tuotantotappioita. Keski-Euroopassa ja Yhdysvalloissa DD:n on havaittu leviävän lähes epidemialuonteisesti, kun taas Pohjoismaissa sitä on tavattu yksittäisissä karjoissa. Taudin etiologiasta, esiintyvyydestä ja parhaasta mahdollisesta hoitomuodosta on eri teorioita. Nykytiedon ja tieteellisen kirjallisuuden mukaan merkittävimpinä DD:n aiheuttajabakteereina pidetään eri Treponema-bakteerilajeja. Uusimpien tutkimusten perusteella taudin vakavuuteen vaikuttaa eri Treponema-lajien yhteisvaikutus keskenään tai muiden bakteereiden kanssa. Tämän tutkielman tavoite oli pystyttää bakteerien 16S rRNA-geenin rinnakkaissekvensointiin perustuva metataksonomiatyövuo bakteereiden tunnistamiseksi. Tutkielmassa vertailtiin kahta eri DNA-eristysmenetelmää, kahta sekvensointityövuota ja kahta bioinformatiikan analyysimenetelmää. Näytemateriaalina oli suomalaisten nautojen sorkka-alueen ihosta otetut biopsianäytteet (n=10). Viisi näytteistä oli terveestä M0-naudasta ja viisi M2-naudasta. Tuloksena havaittiin pieniä määriä Spirochaetaceae-bakteeriperheen sekvenssejä kahdesta M0 -diagnosoduista naudasta. Kaikista M2 - diagnoosin naudoista havaittiin runsaasti Spirochaetaceae - sekvenssejä. Lisäselvityksiä Treponema-bakteerien luotettavasta lajitason tunnistamisesta sekä niiden roolista taudissa tarvitaan. Bovine (Bos taurus) digital dermatitis (DD) of the foot is a widespead disease around the world, and it has also been diagnosed with several other hoofed ruminants. According to current knowledge, bovine digital dermatitis is a bacterial disease, not a viral or fungal disease. It causes ulcers, lesions, and raised calluses. DD spreads easily, and it can cause pain to the animal, weaken its overall health and well-being, as well as cause loss of production. DD has been almost epidemic in Central Europe, as well as in the United States of America, whereas in Scandinavia it has only been encountered in individual herds. According to current knowledge and the scientific literature, the most important bacteria responsible for DD are Treponema species. Recent studies suggest that the severity of the disease is affected by the interaction of different Treponema species with each other or with other bacteria. The aim of this thesis was to set up a metataxonomic pipeline for the parallel sequencing of the 16S rRNA bacterial gene, in order to identify the bacteria. Two different DNA extraction methods, two different sequencing pipelines, and two different bioinformatics pipelines were evaluated in this thesis. Sample material was biopsy samples of the skin of the Finnish bovine hoof (n=10). Five of the samples were from healthy M0 cattle and five from M2 cattle (acute active ulcerative lesions). As a result, small amounts of Spirochaetaceae bacterial family sequences were detected in two M0 diagnostic cattle . Abundant Spirochaetaceae sequences were found in all bovine diagnosed with M2. Further studies on the reliable species identification of Treponema bacteria and their role in the disease are needed.
  • Bozcal, Elif; Eldem, Vahap; Aydemir, Sohret; Skurnik, Mikael (2018)
    Background. Extraintestinal pathogenic Escherichia coli (ExPEC) is an important bacterium and responsible for many bloodstream infections, including urinary tract infections and even fatal bacteremia. The aim of this research was to investigate whether ExPEC strains isolated from Turkish blood cultures have a relationship between 16S rRNA based phylogenetic clusters and antibiotic resistance profiles, virulence factors or clonal lineages. Methods. Phenotypically identified ExPEC blood culture isolates (n = 104) were included in this study. The 16S rRNA partial sequence analysis was performed for genotypic identification of ExPEC isolates. Antibiotic susceptibility and Extended Spectrum beta-Lactamase testing of isolates were performed. Phylogenetic classification (A, B1, B2 and D), Multi Locus Sequence Typing analysis and virulence-associated genes were investigated. Results. Based on 16S rRNA partial sequence analysis, 97 out of 104 (93.26%) ExPEC isolates were confirmed as E. coli. Ampicillin (74.22%) and cefuroxime axetil (65.97%) resistances had the highest frequencies among the ExPEC isolates. In terms of phylogenetic classification of ExPEC, D (38.14%, 37/97) was the most prevalent group after A (29.89%, 29/97), B2 (20.61%, 20/97), and B1 (11.34%, 11/97). The sequence types of the 20 ExPEC isolates belonging to the B2 phylogenetic group were analyzed by Multi Locus Sequence Typing. Ten isolates out of 20 (50.0%) were identified as ST131. The other STs were ST95 (n=1), ST14 (n=1), ST10 (n=1), ST69 (n=1), ST1722 (n=2), ST141 (n=1), ST88 (n=1), ST80 (n=1), and ST998 (n=1). Of the ST131 strains, six (60%, 6/10) represented serogroup O25. The most common virulence factor genes were serum resistance factor gene, traT (55.7%) aerobactin siderophore receptor and yersiniabactin encoding genes iutA (45.3%) and fyuA (50.5%), respectively. In addition, PAI (41.2%), iroN (23.7%), hlyA (15.4%), kpsII (13.4%), ompT (13.4%), papG (12.4%), iss (9.3%), cnf1 (7.2%), ibeA (2.06%), and sfaS (2.06%) genes were present in the ExPEC isolates. Conclusion. The 16S rRNA-based phylogenetic relationship tree analysis showed that a large cluster was present among 97 ExPEC isolates along with related reference strains. There were 21 main clusters with 32 closely related subclusters. Based on our findings, different clonal lineages of ExPEC can display different antibiotic susceptibilities and virulence properties. We also concluded that virulence factors were not distributed depending on phylogenetic groups (A, B1, B2, and D). The ExPEC isolates belonging to the same phylogenetic group and sequence type could display different resistance and virulence characteristics.
  • Rissanen, Antti J.; Saarela, Taija; Jäntti, Helena; Buck, Moritz; Peura, Sari; Aalto, Sanni L; Ojala, Anne; Pumpanen, Jukka; Tiirola, Marja; Elvert, Marcus; Nykänen, Hannu (2021)
    The vertical structuring of methanotrophic communities and its genetic controllers remain understudied in the water columns of oxygen-stratified lakes. Therefore, we used 16S rRNA gene sequencing to study the vertical stratification patterns of methanotrophs in two boreal lakes, Lake Kuivajarvi and Lake Lovojarvi. Furthermore, metagenomic analyses were performed to assess the genomic characteristics of methanotrophs in Lovojarvi and the previously studied Lake Alinen Mustajarvi. The methanotroph communities were vertically structured along the oxygen gradient. Alphaproteobacterial methanotrophs preferred oxic water layers, while Methylococcales methanotrophs, consisting of putative novel genera and species, thrived, especially at and below the oxic-anoxic interface and showed distinct depth variation patterns, which were not completely predictable by their taxonomic classification. Instead, genomic differences among Methylococcales methanotrophs explained their variable vertical depth patterns. Genes in clusters of orthologous groups (COG) categories L (replication, recombination and repair) and S (function unknown) were relatively high in metagenome-assembled genomes representing Methylococcales clearly thriving below the oxic-anoxic interface, suggesting genetic adaptations for increased stress tolerance enabling living in the hypoxic/anoxic conditions. By contrast, genes in COG category N (cell motility) were relatively high in metagenome-assembled genomes of Methylococcales thriving at the oxic-anoxic interface, which suggests genetic adaptations for increased motility at the vertically fluctuating oxic-anoxic interface.