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Antibiotic Perturbations in Experimental Microbial Community

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Antibiotic Perturbations in Experimental Microbial Community Antibiotic perturbations in experimental microbial community Roosa Jokela Master’s thesis University of Helsinki Department of Microbiology Microbiology March 2019 Tiedekunta/Osasto Fakultet/Sektion – Faculty Laitos/Institution– Department Faculty of Agriculture and Forestry Department of Microbiology Tekijä/Författare – Author Roosa Jokela Työn nimi / Arbetets titel – Title Antibiotic perturbations in experimental microbial community Oppiaine /Läroämne – Subject Microbiology Työn laji/Arbetets art – Level Aika/Datum – Month and year Sivumäärä/ Sidoantal – Number of pages Master’s Thesis April 2019 53 + appendices (14) Tiivistelmä/Referat – Abstract The purpose of this thesis was to study the antibiotic perturbations in bacterial populations. Perturbations are common in ecosystems and they can change the composition and functionality of a community substantially. The response of a community is governed by ecological and evolutionary factors: perturbations change the competitive ability of species in the community, but rapid evolution can further affect species fate. This thesis focuses more on the ecological effects. Understanding the community response to a disturbance would be interesting both from a general point of view and from the more practical approach of understanding natural communities under perturbations caused by antibiotics but also, for example, by climate change or chemicals. Thus far, most studies have been performed in one- or two-species systems, not taking into account the effects communities have on the fate of a single species. To study antibiotic perturbations, a multi-species bacterial community was exposed to a streptomycin pulse of three different levels concentrations. Changes in community composition were studied in the end of the pulse (ecological resistance) and after a recovery period (resilience) from the antibiotic perturbation comparing to the pre-perturbed communities. Further, the presence of species flow was manipulated to examine if it could enhance community resistance and resilience. Based on the analysis, even low antibiotic concentrations can have a long-lasting effect on community composition, but the magnitude of the effect is dependent on the concentration. Community diversity was recovered better than the composition, especially after the weaker perturbations. Species flow aids in community recovery but does not affect resistance. The results were relatively reproducible between replicate communities, and species traits steered the species fate in, pointing to deterministic ecological processes driving the community response. However, repeatability decreased in communities perturbed with the highest antibiotic concentration, which could point to evolution. Avainsanat – Nyckelord – Keywords Antibiotic perturbations, community perturbations, community shifts, regime shift Säilytyspaikka – Förvaringställe – Where deposited HELDA - Digital Repository of the University of Helsinki, ethesis.helsinki.fi Muita tietoja – Övriga uppgifter – Additional information Supervisors Dr. Johannes Cairns and PI Dr. Teppo Hiltunen Tiedekunta/Osasto Fakultet/Sektion – Faculty Laitos/Institution– Department Maatalous-metsätieteellinen tiedekunta Mikrobiologian osasto Tekijä/Författare – Author Roosa Jokela Työn nimi / Arbetets titel – Title Antibioottihäiriöt kokeellisessa mikrobiyhteisössä Oppiaine /Läroämne – Subject Mikrobiologia Työn laji/Arbetets art – Level Aika/Datum – Month and year Sivumäärä/ Sidoantal – Number of pages Pro gradu Huhtikuu 2019 53 + liitteet (14) Tiivistelmä/Referat – Abstract Tässä tutkimuksessa tarkasteltiin antibiootin aiheuttamia häiriöitä bakteeripopulaati- oissa. Häiriöt ovat yleisiä ekosysteemeissä ja ne voivat aiheuttaa huomattavia muutok- sia yhteisökoostumukseen ja yhteisön toimintaan. Yhteisön vaste määräytyy ekologis- ten ja evolutiivisten tekijöiden perusteella: häiriöt muuttavat lajien kilpailukykyä yh- teisössä, mutta lajien nopea evoluutio voi vaikuttaa lajien kohtaloon. Tässä tutkiel- massa keskityttiin pääasiassa häiriöiden ekologisiin vaikutuksiin. Häiriön yhteisövas- teen parempi ymmärtäminen on tärkeää yleisesti sekä käytännön kysymysten kannalta niin antibioottihäiriöiden osalta kuin esimerkiksi yhteisöissä, jotka ovat häiriintyneet ilmastonmuutoksen tai erilaisten kemikaalien takia. Tähän asti suurin osa tutki- muksista on käyttänyt yksi- tai kaksilajisia yhteisöjä, jolloin mahdolliset yhteisövai- kutukset yhden lajin kohtaloon jäävät huomioimatta. Antibiootti häiriöitä tutkittiin altistamalla monilajinen bakteeriyhteisö kolmelle erita- soiselle streptomysiinipulssille. Yhteisökoostumuksen muutoksia tutkittiin pulssin lo- pussa (ekologinen vastustuskyky) ja antibioottipulssin päättymisen ja yhteisön toipu- misen jälkeen (palautumiskyky) verraten häiriötä edeltäneeseen yhteisökoostumuk- seen. Yhteisöön saapuvaa lajivirtaa manipuloimalla tutkittiin parantaisiko se yhteisön vastustus- ja palautumiskykyä. Analyysien perusteella jopa matalilla antibioottikon- sentraatioilla voi olla pitkäaikaisia vaikutuksia yhteisökoostumukseen, joskin vaiku- tuksen suuruusluokka riippui antibioottipitoisuudesta. Yhteisöjen monimuotoisuus pa- lautui koostumusta paremmin etenkin heikompien häiriöiden jälkeen. Lajivirta edes- auttoi yhteisöjen palautumista, mutta ei vaikuttanut niiden ekologiseen vastustusky- kyyn. Tulokset olivat suhteellisen toistettavia rinnakkaisyhteisöjen välillä, ja lajien ominaisuudet ohjasivat lajien kohtaloa, mikä viittaa determinististen ekologisten pro- sessien ohjaavan yhteisövastetta. Kuitenkin toistettavuus väheni yhteisöissä, jotka oli altistettu korkeimman konsentraation antibioottihäiriölle, mikä puolestaan voi viitata evoluutioon. Avainsanat – Nyckelord – Keywords Antibioottihäiriöt, yhteisö häiriöt, yhteisön muutokset Säilytyspaikka – Förvaringställe – Where deposited Helsingin yliopiston kirjasto – Helda / E-thesis (opinnäytteet), ethesis.helsinki.fi Muita tietoja – Övriga uppgifter – Additional information Ohjaajina tohtori Johannes Cairns ja ryhmän johtajaTeppo Hiltunen 1 Table of contents 1 Table of contents ................................................................................................... 4 2 Abbreviations........................................................................................................ 6 3 Introduction .......................................................................................................... 7 3.1 Experimental microbial community ecology ................................................... 8 3.2 Multi-species experimental communities .......................................................10 3.3 Perturbations and regime shift .......................................................................12 3.4 Resistance and resilience ...............................................................................13 3.5 Immigration ...................................................................................................16 3.6 Antibiotics .....................................................................................................17 3.6.1 Antibiotic resistance ...............................................................................17 3.6.2 Impact of antibiotics on microbial communities ......................................18 3.6.3 Streptomycin ..........................................................................................21 4 Research objectives ..............................................................................................22 5 Materials and methods .........................................................................................23 5.1 Strains and culture conditions ........................................................................23 5.2 Serial passage antibiotic pulse experiment .....................................................26 5.3 DNA extraction and sequencing for 16S rRNA amplicon analysis .................27 5.4 Pheno- and genotypic data collected for community members .......................28 5.5 Statistical analysis .........................................................................................29 6 Results .................................................................................................................31 6.1 General features of communities ....................................................................31 6.2 Community resistance ...................................................................................33 6.3 Community resilience ....................................................................................35 6.4 Role of phenotypic traits ................................................................................38 7 Discussion ...........................................................................................................40 7.1 Streptomycin concentration determines the level of perturbation ...................40 7.2 Even low concentrations cause long-lasting composition shifts ......................40 7.3 General dynamics and future prospects ..........................................................42 8 Conclusions .........................................................................................................44 9 Acknowledgements ..............................................................................................45 10 List of references .................................................................................................46 Appendix 1 ..................................................................................................................54
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