Browsing by Subject "epicPCR"

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  • Puhakainen, Kai (Helsingfors universitet, 2016)
    Bakteereita ja eukaryootteja esiintyy erittäin runsaasti jätevedessä, jossa ne voivat muodostaa fyysisiä vuorovaikutuksia toistensa kanssa. Fyysisiä vuorovaikutuksia bakteerien ja eukaryoottien välille voi muodostua symbioosien tai saalis-saalistaja suhteen välityksellä. Bakteerit ja eukaryootit osallistuvat myös jäteveden puhdistusprosesseihin, joten näillä molemmilla on iso merkitys puhdistusprosessissa. EpicPCR-menetelmän avulla voidaan tutkia isoja mikrobipopulaatioita sekvensoimalla genomin alueita suuresta määrästä yksittäisiä soluja. Aiemmin menetelmää on käytetty selvittämään sulfaatin pelkistämiseen osallistuvien bakteerien fylogeneettista diversiteettiä järven sedimenteissä. Tämän työn tarkoituksena oli tutkia bakteerien ja eukaryoottien fyysisiä vuorovaikutuksia jätevedessä käyttäen apuna epicPCR-menetelmää. Tutkimuksessa yhdistettiin tiiviissä fyysisessä yhteydessä olleen bakteerin 16S rRNA- ja eukaryootin 18S rRNA -geeni. Samalla selvitettiin miten epicPCR-menetelmä soveltuu käytettäväksi kahden fylogeneettisen 16S rRNA- ja 18S rRNA -geenin yhdistämiseen ja miten jätevesi soveltuu näytteeksi. Tulosten perusteella pohdittiin millaisia voisivat olla bakteerien ja eukaryoottien fyysiset vuorovaikutukset ja mitä ne merkitsevät kummallekin osapuolelle. Fyysisiä yhteisvaikutuksia tutkittiin käyttämällä yksinkertaiseen teknologiaan perustuvaa epicPCR-menetelmää ja sekvensointi suoritettiin Illumina MiSeq -menetelmällä. Sekvenssien käsittely ja analyysi tehtiin käyttäen Unixin Bash -komentorivikieltä ja R-ohjelmointikieltä CSC:n Taito-laskentaklusterilla käyttäen apuna Qiime-työkalupakettia. Tulosten perusteella bakteerien ja eukaryoottien välillä havaittiin fyysisiä yhteyksiä, jotka jakaantuivat kahdella eri tavalla. Jätevedestä löytyi eukaryoottiryhmiä, jotka muodostivat yhteyden useamman bakteeriryhmän kanssa ja eukaryoottiryhmiä, jotka muodostivat yhteyden vain tietyn bakteeriryhmän kanssa. Saalistukseen pystyvät eukaryoottiryhmät muodostivat yhteyksiä useamman bakteeriryhmän kanssa kuten myös määrällisesti enemmän kuin fotosynteettiset eukaryoottiryhmät.
  • Roman, Veronica L.; Merlin, Christophe; Virta, Marko P. J.; Bellanger, Xavier (2021)
    EpicPCR (Emulsion, Paired Isolation and Concatenation PCR) is a recent single-cell genomic method based on a fusion-PCR allowing us to link a functional sequence of interest to a 16S rRNA gene fragment and use the mass sequencing of the resulting amplicons for taxonomic assignment of the functional sequence-carrying bacteria. Although it is interesting because it presents the highest efficiency for assigning a bacterial host to a marker, epicPCR remains a complex multistage procedure with technical difficulties that may easily impair the approach depth and quality. Here, we described how to adapt epicPCR to new gene targets and environmental matrices while identifying the natural host range of SXT/R391 integrative and conjugative elements in water microbial communities from the Meurthe River (France). We notably show that adding a supplementary PCR step allowed us to increase the amplicon yield and thus the number of reads obtained after sequencing. A comparison of operational taxonomic unit (OTU) identification approaches when using biological and technical replicates demonstrated that, although OTUs can be validated when obtained from three out of three technical replicates, up to now, results obtained from two or three biological replicates give a similar and even a better confidence level in OTU identification, while allowing us to detect poorly represented SXT/R391 hosts in microbial communities.
  • Pezzutto, Denise (Helsingin yliopisto, 2019)
    Antimicrobial resistance is an emerging concern at the global scale, threatening the effectiveness of antibiotics in treating bacterial infections. Among anthropogenically impacted environments, wastewater treatment plants have been indicated as possible reservoirs of antibiotic resistance genes, putative hotspots for their horizontal gene transfer, and a source of their dissemination to the environment. Generally, the abundance of antibiotic resistance genes is reduced during the wastewater treatment process. However, some genes were shown to be enriched in purified effluent water and dried sludge, which are then released to the environment, compared to influent water. Also, the taxonomy of the hosts carrying antibiotic resistance genes could change as a result of horizontal gene transfer events. The aim of this study was to analyse and compare the host range of a series of antibiotic resistance genes in influent water, effluent water and dried sludge collected from the Viikinmäki wastewater treatment plant in Helsinki, Finland, by applying Emulsion, Paired Isolation and Concatenation PCR (epicPCR). EpicPCR is a method that can link a gene of interest to the 16S rRNA gene from the genome of the host bacterium, without any cultivation step. The abundance of the hosts was also evaluated by sequencing the 16S rRNA gene from the whole bacterial community. In several cases, the target antibiotic resistance genes (blaIMP, blaNDM, ermB, ermF, sul1 and strB) were carried in effluent water and dried sludge by taxa that were not hosting them in influent water, suggesting that horizontal gene transfer events might have occurred during the treatment. All the examined genes were detected both in abundant and in rare taxa, including genera that also comprise pathogenic species, such as Arcobacter and Acinetobacter. Some of the detected hosts were not previously known to show resistant phenotypes, namely members of the family Methylophilaceae. These results corroborate the idea that wastewater treatment plants might be hotspots for the horizontal gene transfer of resistance determinants, and potentially disseminate antibiotic resistant pathogens to the environment. However, in order to ensure the accuracy of the results, the limits of epicPCR as a method need to be identified and addressed.
  • Hultman, Jenni; Tamminen, Manu; Pärnänen, Katariina; Cairns, Johannes; Karkman, Antti; Virta, Marko (2018)
    Wastewater treatment plants (WWTPs) collect wastewater from various sources for a multi-step treatment process. By mixing a large variety of bacteria and promoting their proximity, WWTPs constitute potential hotspots for the emergence of antibiotic resistant bacteria. Concerns have been expressed regarding the potential of WWTPs to spread antibiotic resistance genes (ARGs) from environmental reservoirs to human pathogens. We utilized epicPCR (Emulsion, Paired Isolation and Concatenation PCR) to detect the bacterial hosts of ARGs in two WWTPs. We identified the host distribution of four resistance-associated genes (tetM, int1, qacE Delta 1 and bla(OXA-58)) in influent and effluent. The bacterial hosts of these resistance genes varied between the WWTP influent and effluent, with a generally decreasing host range in the effluent. Through 16S rRNA gene sequencing, it was determined that the resistance gene carrying bacteria include both abundant and rare taxa. Our results suggest that the studied WWTPs mostly succeed in decreasing the host range of the resistance genes during the treatment process. Still, there were instances where effluent contained resistance genes in bacterial groups not carrying these genes in the influent. By permitting exhaustive profiling of resistance-associated gene hosts in WWTP bacterial communities, the application of epicPCR provides a new level of precision to our resistance gene risk estimates.
  • Sarekoski, Anniina Karoliina (Helsingin yliopisto, 2020)
    The emerging crisis of antimicrobial resistance is especially worrisome in low-income countries that lack controlled antibiotic policy and have poor infrastructure. Inadequate hygiene practices combined with ability of microbes to quickly evolve and adapt to changes rise the concern of resistance of infectious pathogens to many first-line antimicrobial drugs. Moreover, wastewaters that are widely used as irrigation water in urban gardening in sub-Saharan Africa, can function as vehicle for the dissemination of bacteria that carry antimicrobial resistance genes into the surrounding environment. In this study, eight anthropogenically impacted water samples were collected from Burkina Faso and Mali and differences in their microbial communities were evaluated by 16S rRNA gene sequencing. Also, the presence of antimicrobial resistance genes was examined with SmartChip qPCR. The bacterial host range of blaNDM, blaCTX-M, blaOXA and qacE∆1 was profiled using a novel culture- independent technique, Emulsion, Paired Isolation and Concatenation PCR (epicPCR). The presence of 202 genes associated with antimicrobial resistance were detected with SmartChip qPCR array analysis, including carbapenemase genes that can transfer horizontally. Worryingly, sixteen taxonomical units, including possible human pathogens Acinetobacter, Klebsiella, Escherichia and Pseudomonas, were found to carry all the four genes investigated with epicPCR. The most abundant genus Arcobacter along with Dechloromonas, Methylotenera, MM1 and Methylophilus were new discoveries as blaNDM hosts. Furthermore, a considerable number of blaOXA and clinical class 1 integron marker qacE∆1 gene hosts were discovered in every sample. Lastly, putative events of horizontal gene transfer in two WWTP samples were observed. Broad host range of blaOXA and qacE∆1 genes suggests a heavy antimicrobial resistance genes burden in West Africa and the results support the theory that environmental bacteria can function as resistance gene reservoirs. These results show occurrence of horizontally transferrable blaNDM and blaCTX-M genes in pathogens especially in hospital wastewater, and a threat of their spread into the environment and to the community. However, to decipher their role in the infectious disease burden in Africa, more research is needed.
  • Karnola, Laura (Helsingin yliopisto, 2020)
    Antimicrobial resistance (AMR) is an emerging global health threat with the growing number of antibiotic-resistant bacteria (ARB) having the alarming potential to return humanity to the pre-antibiotic era. Intensive animal production is globally one of the biggest sectors using antibiotics. It has been studied that fertilizing fields with animal manure spreads antimicrobial resistance genes (ARGs) in natural environments. The aim of this study was to determine the host range of three ARGs tetM, strB and qacE∆1 in soil and manure samples collected from a Finnish swine farm. In addition, the microbial communities in the same soil and manure samples were studied and compared. Six different sample types were taken, four from soil and two from manure. Soil samples included unfertilized soil, fertilized soil, soil two weeks after fertilization and soil six weeks after fertilization. Manure samples were taken from fresh and stored manure. Host range analysis was done by using Emulsion, Paired Isolation and Concatenation PCR (epicPCR). EpicPCR enables to link a gene of interest to the 16S rRNA gene of the bacterium that carries the gene in its genome. Microbial communities in soil and manure were analyzed and compared with the traditional 16S rRNA gene sequencing. Host range analysis with epicPCR revealed various bacterial genera as carriers for studied ARGs. Fertilized soil had the highest number of genera carrying the ARGs. This indicates that land application with animal manure increases the ARG load in soil. Microbial communities were found significantly different in soil and manure according to the 16S rRNA gene sequences. The results of epicPCR indicate that epicPCR has also potential for solid samples such as soil and manure as according to publications it has been mainly used for different water samples e.g., wastewaters. As a method epicPCR still requires optimization if applied for these sample materials in the future. A clear reduction in the number of genera carrying the ARGs was observed in six weeks after fertilization. Therefore, fertilizing fields only before cropping season, instead of fertilizing the fields year-round, might be one solution for reducing the ARG dissemination in soil in countries with high antibiotic consumption.
  • Qin, Huayu; Wang, Shang; Feng, Kai; He, Zhili; Virta, Marko P J; Hou, Weiguo; Dong, Hailiang; Deng, Ye (BioMed Central, 2019)
    Abstract Sulfate reduction is an important biogeochemical process in the ecosphere; however, the major taxa of sulfate reducers have not been fully identified. Here, we used epicPCR (Emulsion, Paired Isolation, and Concatenation PCR) technology to identify the phylogeny of sulfate-reducing prokaryotes (SRP) in sediments from Tibetan Plateau saline lakes. A total of 12,519 OTUs and 883 SRP-OTUs were detected in ten lakes by sequencing of 16S rRNA gene PCR amplicons and epicPCR products of fused 16S rRNA plus dsrB gene, respectively, with Proteobacteria, Firmicutes, and Bacteroidetes being the dominant phyla in both datasets. The 120 highly abundant SRP-OTUs (> 1% in at least one sample) were affiliated with 17 described phyla, only 7 of which are widely recognized as SRP phyla. The majority of OTUs from both the whole microbial communities and the SRPs were not detected in more than one specific lake, suggesting high levels of endemism. The α-diversity of the entire microbial community and SRP sub-community showed significant positive correlations. The pH value and mean water temperature of the month prior to sampling were the environmental determinants for the whole microbial community, while the mean water temperature and total nitrogen were the major environmental drivers for the SRP sub-community. This study revealed there are still many undocumented SRP in Tibetan saline lakes, many of which could be endemic and adapted to specific environmental conditions.
  • Qin, Huayu; Wang, Shang; Feng, Kai; He, Zhili; Virta, Marko P. J.; Hou, Weiguo; Dong, Hailiang; Deng, Ye (2019)
    Sulfate reduction is an important biogeochemical process in the ecosphere; however, the major taxa of sulfate reducers have not been fully identified. Here, we used epicPCR (Emulsion, Paired Isolation, and Concatenation PCR) technology to identify the phylogeny of sulfate-reducing prokaryotes (SRP) in sediments from Tibetan Plateau saline lakes. A total of 12,519 OTUs and 883 SRP-OTUs were detected in ten lakes by sequencing of 16S rRNA gene PCR amplicons and epicPCR products of fused 16S rRNA plus dsrB gene, respectively, with Proteobacteria, Firmicutes, and Bacteroidetes being the dominant phyla in both datasets. The 120 highly abundant SRP-OTUs (>1% in at least one sample) were affiliated with 17 described phyla, only 7 of which are widely recognized as SRP phyla. The majority of OTUs from both the whole microbial communities and the SRPs were not detected in more than one specific lake, suggesting high levels of endemism. The -diversity of the entire microbial community and SRP sub-community showed significant positive correlations. The pH value and mean water temperature of the month prior to sampling were the environmental determinants for the whole microbial community, while the mean water temperature and total nitrogen were the major environmental drivers for the SRP sub-community. This study revealed there are still many undocumented SRP in Tibetan saline lakes, many of which could be endemic and adapted to specific environmental conditions.