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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Antibiotic resistance in the wild: an eco-evolutionary perspective

The legacy of the use and misuse of antibiotics in recent decades has left us with a global public health crisis: antibiotic-resistant bacteria are on the rise, making it harder to treat infections. At the same time, evolution of antibiotic resistance is probably the best-documented case of contemporary evolution. To date, research on antibiotic resistance has largely ignored the complexity of interactions that bacteria engage in. However, in natural populations, bacteria interact with other species; for example, competition and grazing are import interactions influencing bacterial population dynamics. Furthermore, antibiotic leakage to natural environments can radically alter bacterial communities. Overall, we argue that eco-evolutionary feedback loops in microbial communities can be modified by residual antibiotics and evolution of antibiotic resistance. The aim of this review is to connect some of the well-established key concepts in evolutionary biology and recent advances in the study of eco-evolutionary dynamics to research on antibiotic resistance. We also identify some key knowledge gaps related to eco-evolutionary dynamics of antibiotic resistance, and review some of the recent technical advantages in molecular microbiology that offer new opportunities for tackling these questions. Finally, we argue that using the full potential of evolutionary theory and active communication across the different fields is needed for solving this global crisis more efficiently. This article is part of the themed issue 'Human influences on evolution, and the ecological and societal consequences'.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

The spread of the plasmid RP4 in a synthetic bacterial community is dependent on the particular donor strain

The rapid spread of antibiotic resistance challenges modern medicine. So far, mechanistic and quantitative knowledge concerning the spread of resistance genes mainly relies on laboratory experiments with simplified setups, e.g. two strain communities. Thus, the transferability of the obtained process rates might be limited. To investigate the role of a diverse community concerning the dissemination of the multidrug resistance plasmid RP4, an Escherichia coli harboring RP4 invaded a microbial community consisting of 21 species. Changes in the community composition as well as plasmid uptake by community members were monitored for 22 days. Special focus was laid on the question of whether the observed changes were dependent on the actual invading donor isolate and the ambient antibiotic concentration. In our microcosm experiment, the community composition was primarily influenced by the given environmental variables and only secondarily by the particular invader E. coli. The establishment of resistance within the community, however, was directly dependent on the donor identity. The extent to which ambient conditions influence the spread of RP4 depended on the E. coli donor strain. These results emphasize that even within one species there are great differences in the ability to conquer an ecological niche and to spread antibiotic resistance.Peer reviewe

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

Ympäristön vaikutus mikrobilääkeresistenssiin

English summaryPeer reviewe

Tutkimus suomalaisten erityisluokanopettajien työuupumuksen tilasta : "siis pidän työstäni, enkä haluaisi muuta tehdä, mutta olen ihan loppu"

Tutkimuksessa selvitettiin, kokevatko suomalaiset erityisluokanopettajat työuupumuksen tunnetta. Lisäksi selvitettiin, onko sukupuolella, työkokemuksella ja työpaikan sijainnilla yhteyttä työuupumuksen tunteen kokemiseen. Tutkimuksessa saatiin myös selville työhön liittyviä uuvuttavia tekijöitä erityisluokanopettajan ammatissa. Tutkimus on kvantitatiivinen eli määrällinen tutkimus. Aineisto analysoitiin IMB SPSS Statistic 26-ohjelmalla. Työuupumusta mitattiin MBI (Maslach Burnout Inventory) -mittarin avulla. Tutkimus toteutettiin Webropol-kyselynä tammikuussa 2020. Kyselylomakkeet lähetettiin sähköpostitse OAJ:n aluetoimijoiden puheenjohtajille sekä SEL.ry:n puheenjohtajalle. Heitä pyydettiin jakamaan kyselyä eteenpäin jäsenistölleen. Lisäksi kysely jaettiin ”erkkamaikat” ja ”erityisopettajat” Facebook-ryhmiin. Kyselyyn vastasi 151 erityisluokanopettajaa, joista kymmenen oli miehiä ja 141 oli naisia. Vastauksia kyselyyn saatiin jokaisesta Suomen maakunnasta. Tutkimustulokset osoittivat, että erityisluokanopettajat ovat uupuneita. Kyselyyn vastanneista erityisluokanopettajista joka toinen koki uupumuksen tunnetta ja joka kolmas oli uupunut. Sukupuolten välinen vertailu työuupumuksen tunteen kokemisessa ei ollut kannattavaa epätasaisen sukupuolijakauman vuoksi. Lisäksi työkokemuksella eikä työpaikan sijainnilla ollut tilastollista merkitsevyyttä työuupumuksen tunteen kokemisessa. Työkokemuksella ei ollut tilastollista merkitsevyyttä, sillä kaikissa ”työkokemusjoukoissa” koettiin uupumuksen tunnetta. Tutkimuksessa selvisi, että erityisluokanopettajia uuvuttavia tekijöitä ovat kollegoiden ja esimiehen tuen puute, haastavat oppilaat sekä muut lisääntyneet asiat työssä, jotka vievät aikaa opettamiselta. Tutkimustuloksen mukaan erityisluokanopettajien tilanne Suomessa on huolestuttava. Erityisluokanopettajat ovat uupuneita, joten toimenpiteitä tilanteen parantamiseen olisi syytä selvittää ja heidän työssä jaksamista tulisi edistää kaikin mahdollisin keinoin. Koulutuksesta ei tulisi enää leikata, vaan resursseja tulisi lisätä kouluihin. Erityisluokanopettajilla tulisi olla mahdollisuus vaikuttaa, kun erityisopetukseen liittyviä muutoksia tehdään. Asiasanat: erityisluokanopettaja, erityisopetus, työuupumus, stressi, työhyvinvoint

Profiles of environmental antibiotic resistomes in the urban aquatic recipients of Sweden using high-throughput quantitative PCR analysis

Antibiotic resistance in aquatic ecosystems presents an environmental health issue worldwide. Urban recipient water quality is susceptible to effluent discharges with antibiotic resistance contaminants and needs to be protected, particularly for those as sources of drinking water production. Knowledge on aquatic resistome profiles in downstream of wastewater treatment plants allows a better understanding of the extent to which antibiotic resistance contaminants emerge and spread in recipient waters, but such information remains very limited in Sweden. The key objective of this study was to determine the resistome profiles of numerous antibiotic resistance genes (ARGs), mobile genetic elements (MGEs) and other genes in urban recipient water systems connected to Sweden's major drinking water reservoir. This was achieved through analysis of surface water samples for 296 genes using high-throughput quantitative PCR arrays. A total of 167 genes were detected in at least one of the samples, including 150 ARGs conferring resistance to 11 classes of antibiotics, 7 integrase MGEs and 9 other genes. There was a spatial difference in the resistome profiles with the greatest average relative abundance of resistance genes observed in the water body of Vasteras followed by Uppsala, Stockholm and Eskilstuna, as similar to the general pattern of the antibiotic sales for these regions. ARGs against beta-lactams and sulfonamides showed the highest average relative abundance in the studied water bodies, while vancomycin resistance genes were only found in the Uppsala water environment. Generally, the recipient water bodies were detected with higher numbers of genes and greater relative abundances as compared to the upstream sites. Anthropogenic pollution, i.e., wastewater discharge, in the recipient water was also reflected by the finding of intI, sul1 and crAssphage. Overall, this study provided the first quantitative assessment of aquatic environmental resistomes in Sweden, highlighting the widespread of antibiotic resistance contaminants in urban recipient waters.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