Single gene-based distinction of individual microbial genomes from a mixed population of microbial cells

Background: Recent progress in environmental microbiology has revealed vast populations of microbes in any given habitat that cannot be detected by conventional culturing strategies. The use of sensitive genetic detection methods such as CARD-FISH and in situ PCR have been limited by the cell wall permeabilization requirement that cannot be performed similarly on all cell types without lysing some and leaving some unpermeabilized. Furthermore, the detection of low copy targets such as genes present in single copies in the microbial genomes, has remained problematic.

Methodology/Principal Findings: We describe an emulsion-based procedure to trap individual microbial cells into picoliter-volume polyacrylamide droplets that provide a support for genetic material and therefore allow degradation of cellular material to expose the individual genomes. The polyacrylamide droplets are subsequently converted into picoliter-scale reactors for genome amplification. The amplified genomes are labelled based on the presence of a target gene and differentiated from those that do not contain the gene by flow cytometry. Using the Escherichia coli strains XL1 and MC1061, which differ with respect to the presence (XL1) or absence (MC1061) of a single copy of a tetracycline resistance gene per genome, we demonstrate that XL1 genomes present at 0.01% of MC1061 genomes can be differentiated using this method. Using a spiked sediment microbial sample, we demonstrate that the method is applicable to complex environmental microbial communities as a target gene-based screen for individual microbes. 

Conclusions/Significance: The genomic support for complete cell degradation allows an exposure of individual genomes of environmental bacteria. The genome exposure followed by genome amplification and labelling combines the benefits of in situ-PCR and FISH methods and permits the detection cells with single copy chromosomal targets in complex mixtures of microbial cells. The method could be optimized for fluorescence-activated cell sorting to enrich genetic material of interest from complex environmental samples.
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Molecular characterization of sediment bacterial communities affected by fish farming

Fish farming introduces nutrients, microbes and a wide variety of chemicals such as heavy metals, antifoulants and antibiotics to the surrounding environment. Introduction of antibiotics has been linked with the increased incidence of antibiotic resistant pathogenic bacteria in the farm vicinities. In this thesis molecular methods such as quantitative PCR and DNA sequencing were applied to analyze bacterial communities in sediments from fish farms and pristine locations. Altogether four farms and four pristine sites were sampled in the Baltic Sea. Two farm and two pristine locations were sampled over a surveillance period of four years. Furthermore, a new methodology was developed as a part of the study that permits amplifying single microbial genomes and capturing them according to any genetic traits, including antibiotic resistance genes. The study revealed that several resistance genes for tetracycline were found at the sediment underneath the aquaculture farms. The copy number of these genes remained elevated even at a farm that had not used any antibiotics since year 2000, six years before this study started. Similarly, an increase in the amount of mercury resistance gene merA was observed at the aquaculture sediment. The persistence of the resistance genes in absence of any selection pressure from antibiotics or heavy metals suggests that the genes may be introduced to the sediment by the farming process. This is also supported by the diversity pattern of the merA gene between farm and pristine sediments. The bacterial community-level changes in response to fish farming were very complex and no single phylogenetic groups were found that would be typical to fish farm sediments. However, the community structures had some correlation with the exposure to fish farming. Our studies suggest that the established approaches to deal with antibiotic resistance at the aquaculture, such as antibiotic cycling, are fundamentally flawed because they cannot prevent the introduction of the resistance genes and resistant bacteria to the farm area by the farming process. Further studies are required to study the entire fish farming process to identify the sources of the resistance genes and the resistant bacteria. The results also suggest that in order to prevent major microbiological changes in the surrounding aquatic environment, the farms should not be founded in shallow water where currents do not transport sedimenting matter from the farms. Finally, the technique to amplify and select microbial genomes will potentially have a considerable impact in microbial ecology and genomics.Kalankasvatuksen yhteydessä meriympäristöön vapautuu ravinteita, mikrobeita, raskasmetalleja, pilaantumisenestoaineita sekä antibiootteja. Antibioottien sekä muiden edellä mainittujen aineiden yhteisvaikutuksesta kasvattamoiden lähiympäristössä on havaittu korkeita määriä antibiooteille vastustuskykyisiä bakteereita. Tässä väitöskirjatyössä sovellettiin molekyylimenetelmiä (kvantitatiivinen PCR ja DNA-sekvensointi) selvittämään kalankasvatuksen vaikutusta kasvattamoiden alla olevan sedimentin bakteeriyhteisöihin. Kaikkiaan neljältä kasvattamolta ja neljältä puhtaalta paikalta Itämeressä kerättiin sedimenttinäytteitä. Kahdelta kasvattamolta ja puhtaalta paikalta näytteitä kerättiin neljän peräkkäisen vuoden ajan. Lisäksi työn aikana kehitettiin uudenlainen menetelmä yksittäisten mikrobisolujen genomien monistamiseen ja seulomiseen geneettisten ominaisuuksien perusteella. Tutkimuksessa kasvattamoiden alla olevasta sedimentistä löydettiin kohonneita määriä useita geenejä, jotka antavat vastustuskyvyn tetrasykliini-antibiootille. Kyseisten geenien määrä pysyi jatkuvasti kohonneena jopa kasvattamolla, joka oli lopettanut kaikkien antibioottien käytön jo vuonna 2000; kuusi vuotta ennen tämän tutkimuksen alkua. Samankaltainen ilmiö havaittiin myös elohopealle sietokyvynkyvyn antavalla merA-geenillä, jota löytyi kalankasvatukselle altistuneista sedimenteistä enemmän kuin muista sedimenteistä. Geenien korkea määrä sekä merA-geenin DNA-sekvenssityyppien jakautuminen viittaa siihen, että geenit saattavat tulla kasvattamoiden sedimenttiin kasvatusprosessin seurauksena. Kalankasvatus ei aiheuttanut selvästi tyypillisiä muutoksia sedimentin bakteeriyhteisöissä. Yhteisön rakenteessa tapahtui kuitenkin tietynlaisia muutoksia, jotka olivat yhteydessä viljelyn suuruusluokkaan. Tutkimuksessa saadut tulokset viittaavat siihen, että kalankasvattamoilla perinteisesti käytetyt menetelmät, erityisesti antibioottikierto, ovat tehottomia antibiooteille vastustuskykyisten bakteerien määrän hallitsemiseksi, sillä ne eivät estä kalankasvatusprosessin aikaisemmista vaiheista tulevien resistenssigeenien ja resistenttien bakteereiden päätymistä kasvattamoille. Lisätutkimuksia tarvitaan selvittämään, mikä vaihe kalankasvatusprosessissa saa aikaan antibiooteille vastustuskyvyn antavien geenien leviämisen. Tulosten mukaan merkittävien mikrobiologisten yhteisömuutoksien välttämiseksi sedimentissä kalankasvattamoita ei tulisi perustaa matalaan veteen, missä virtaukset eivät pääse sekoittamaan sedimenttiä. Tutkimuksen yhteydessä kehitetyllä mikrobigenomien monistuksen ja seulomisen mahdollistavalla menetelmällä tulee mahdollisesti olemaan merkittävä vaikutus tuleviin mikrobiekologisiin ja genomisiin tutkimuksiin

Single gene-based distinction of individual microbial genomes from a mixed population of microbial cells

Recent progress in environmental microbiology has revealed vast populations of microbes in any given habitat that cannot be detected by conventional culturing strategies. The use of sensitive genetic detection methods such as CARD-FISH and in situ PCR have been limited by the cell wall permeabilization requirement that cannot be performed similarly on all cell types without lysing some and leaving some nonpermeabilized. Furthermore, the detection of low copy targets such as genes present in single copies in the microbial genomes, has remained problematic. We describe an emulsion-based procedure to trap individual microbial cells into picoliter-volume polyacrylamide droplets that provide a rigid support for genetic material and therefore allow complete degradation of cellular material to expose the individual genomes. The polyacrylamide droplets are subsequently converted into picoliter-scale reactors for genome amplification. The amplified genomes are labeled based on the presence of a target gene and differentiated from those that do not contain the gene by flow cytometry. Using the Escherichia coli strains XL1 and MC1061, which differ with respect to the presence (XL1), or absence (MC1061) of a single copy of a tetracycline resistance gene per genome, we demonstrate that XL1 genomes present at 0.1% of MC1061 genomes can be differentiated using this method. Using a spiked sediment microbial sample, we demonstrate that the method is applicable to highly complex environmental microbial communities as a target gene-based screen for individual microbes. The method provides a novel tool for enumerating functional cell populations in complex microbial communities. We envision that the method could be optimized for fluorescence-activated cell sorting to enrich genetic material of interest from complex environmental samples.Peer reviewe

Large-Scale Analysis of Plasmid Relationships through Gene-Sharing Networks

Plasmids are vessels of genetic exchange in microbial communities. They are known to transfer between different host organisms and acquire diverse genetic elements from chromosomes and/or other plasmids. Therefore, they constitute an important element in microbial evolution by rapidly disseminating various genetic properties among different communities. A paradigmatic example of this is the dissemination of antibiotic resistance (AR) genes that has resulted in the emergence of multiresistant pathogenic bacterial strains. To globally analyze the evolutionary dynamics of plasmids, we built a large graph in which 2,343 plasmids (nodes) are connected according to the proteins shared by each other. The analysis of this gene-sharing network revealed an overall coherence between network clustering and the phylogenetic classes of the corresponding microorganisms, likely resulting from genetic barriers to horizontal gene transfer between distant phylogenetic groups. Habitat was not a crucial factor in clustering as plasmids from organisms inhabiting different environments were often found embedded in the same cluster. Analyses of network metrics revealed a statistically significant correlation between plasmid mobility and their centrality within the network, providing support to the observation that mobile plasmids are particularly important in spreading genes in microbial communities. Finally, our study reveals an extensive (and previously undescribed) sharing of AR genes between Actinobacteria and Gammaproteobacteria, suggesting that the former might represent an important reservoir of AR genes for the latter

Quantitative online survey of self-perceived knowledge and knowledge gaps of medicines research and development among Finnish general public

Objectives This study explored self-reported knowledge and interest to learn more about medicines research, development and health technology assessment among Finnish general public. It also aimed to define possible knowledge gaps and needs for public education regarding these topics. Design Online survey with 503 participants. The questionnaire was originally developed as a part of the Needs Assessment Work Package of the European Patients' Academy on Therapeutic Innovation Project. The survey was carried out in Finland in 2019. Methods The survey was conducted as an online survey by Kantar TNS Gallup Forum online panel. The data were analysed by using the freely available programming language R. Relationships between the demographic characteristics (such as age, gender and education level) of respondents and their knowledge or interest in medicines research and development were determined using Pearson's chi(2) tests. Statistically significant responses of demographic characteristics in the respondents' knowledge or interest in medicines research were determined by logistic regression. Results Of the 503 respondents (age 16-64) only 12% reported having good or very good knowledge of medicines research and development in general. Regarding health technology assessment, pharmacoeconomics and regulation, the percentage of respondents reporting good or very good knowledge was as low as 8%. Respondents were most interested in learning more about predictive and personalised medicine (47%) and least interested in medicines regulation (30%) and pharmacoeconomics (31%). Conclusions Self-reported knowledge about medicines research and development and health technology assessment appears to be very low in Finland. Patient and public participation is recognised as an important and essential element in up-to-date medical research and assessment of new treatments. In order to participate as an active and equal partner in these processes, the public needs more information and education in these topics.Peer reviewe

Ecology determines how low antibiotic concentration impacts community composition and horizontal transfer of resistance genes

Peer reviewe

Host range of antibiotic resistance genes in wastewater treatment plant influent and effluent

10.1093/femsec/fiy038Wastewater 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.Peer reviewe

Prider: multiplexed primer design using linearly scaling approximation of set coverage

BackgroundDesigning oligonucleotide primers and probes is one of the key steps of various laboratory experiments such as multiplexed PCR or digital multiplexed ligation assays. When designing multiplexed primers and probes to complex, heterogeneous DNA data sets, an optimization problem can arise where the smallest number of oligonucleotides covering the largest diversity of the input dataset needs to be identified. Tools that provide this optimization in an efficient manner for large input data are currently lacking.ResultsHere we present Prider, an R package for designing primers and probes with a nearly optimal coverage for complex and large sequence sets. Prider initially prepares a full primer coverage of the input sequences, the complexity of which is subsequently reduced by removing components of high redundancy or narrow coverage. The primers from the resulting near-optimal coverage are easily accessible as data frames and their coverage across the input sequences can be visualised as heatmaps using Prider’s plotting function. Prider permits efficient design of primers to large DNA datasets by scaling linearly to increasing sequence data, regardless of the diversity of the dataset.ConclusionsPrider solves a recalcitrant problem in molecular diagnostics: how to cover a maximal sequence diversity with a minimal number of oligonucleotide primers or probes. The combination of Prider with highly scalable molecular quantification techniques will permit an unprecedented molecular screening capability with immediate applicability in fields such as clinical microbiology, epidemic virus surveillance or antimicrobial resistance surveillance.</p

Construction and characterization of synthetic bacterial community for experimental ecology and evolution

Experimental microbial ecology and evolution have yielded foundational insights into ecological and evolutionary processes using simple microcosm setups and phenotypic assays with one- or two-species model systems. The fields are now increasingly incorporating more complex systems and exploration of the molecular basis of observations. For this purpose, simplified, manageable and well-defined multispecies model systems are required that can be easily investigated using culturing and high-throughput sequencing approaches, bridging the gap between simpler and more complex synthetic or natural systems. Here we address this need by constructing a completely synthetic 33 bacterial strain community that can be cultured in simple laboratory conditions. We provide whole-genome data for all the strains as well as metadata about genomic features and phenotypic traits that allow resolving individual strains by amplicon sequencing and facilitate a variety of envisioned mechanistic studies. We further show that a large proportion of the strains exhibit coexistence in co-culture over serial transfer for 48 days in the absence of any experimental manipulation to maintain diversity. The constructed bacterial community can be a valuable resource in future experimental work.Peer reviewe

Quantitative online survey of self-perceived knowledge and knowledge gaps of medicines research and development among Finnish general public

Objectives This study explored self-reported knowledge and interest to learn more about medicines research, development and health technology assessment among Finnish general public. It also aimed to define possible knowledge gaps and needs for public education regarding these topics.Design Online survey with 503 participants. The questionnaire was originally developed as a part of the Needs Assessment Work Package of the European Patients’ Academy on Therapeutic Innovation Project. The survey was carried out in Finland in 2019.Methods The survey was conducted as an online survey by Kantar TNS Gallup Forum online panel. The data were analysed by using the freely available programming language R. Relationships between the demographic characteristics (such as age, gender and education level) of respondents and their knowledge or interest in medicines research and development were determined using Pearson’s χ2 tests. Statistically significant responses of demographic characteristics in the respondents’ knowledge or interest in medicines research were determined by logistic regression.Results Of the 503 respondents (age 16–64) only 12% reported having good or very good knowledge of medicines research and development in general. Regarding health technology assessment, pharmacoeconomics and regulation, the percentage of respondents reporting good or very good knowledge was as low as 8%. Respondents were most interested in learning more about predictive and personalised medicine (47%) and least interested in medicines regulation (30%) and pharmacoeconomics (31%).Conclusions Self-reported knowledge about medicines research and development and health technology assessment appears to be very low in Finland. Patient and public participation is recognised as an important and essential element in up-to-date medical research and assessment of new treatments. In order to participate as an active and equal partner in these processes, the public needs more information and education in these topics.</p