Determinants of Listeria monocytogenes stress responses

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dc.contributor Helsingin yliopisto, eläinlääketieteellinen tiedekunta fi
dc.contributor Helsingfors universitet, veterinärmedicinska fakulteten sv
dc.contributor University of Helsinki, Faculty of Veterinary Sciences, Elintarvikehygienian ja ympäristöterveyden osasto en
dc.contributor Ruokaketjun ja terveyden tohtoriohjelma fi
dc.contributor Doktorandprogrammet i livsmedelskedjan och hälsa sv
dc.contributor Doctoral Programme in Food Chain and Health en
dc.contributor.author Pöntinen, Anna
dc.date.accessioned 2019-06-06T05:29:54Z
dc.date.available 2019-06-04
dc.date.available 2019-06-06T05:29:54Z
dc.date.issued 2019-06-14
dc.identifier.uri URN:ISBN:978-951-51-5293-0
dc.identifier.uri http://hdl.handle.net/10138/302554
dc.description.abstract Listeria monocytogenes is a remarkable bacterium, as it is able to shift from a capable environmental saprophyte into a severe intracellular pathogen. As a strictly foodborne pathogen, L. monocytogenes poses a notable risk, particularly to those consumers among the risk groups for whom invasive listeriosis is potentially fatal. Furthermore, modern consumption habits and increasingly favoured ready-to-eat foods, often consumed without proper heating, increase the risk of acquiring the foodborne disease. The aim of this study was to investigate the genetic mechanisms conferring wide-ranging stress tolerance in L. monocytogenes. Two-component systems, comprising a sensor histidine kinase and a cognate response regulator, aid bacteria in sensing and adapting to changes in both surrounding environmental as well as intracellular conditions. The histidine kinases, in particular, have lacked comprehensive studies on their roles in the stress tolerance of L. monocytogenes. Thus, histidine kinases were studied by expressional analyses under cold conditions and by mutationally disrupting each histidine kinase-encoding gene in a parental model strain, L. monocytogenes EGD-e. The modified strains were individually challenged at high (42.5 degrees C) and low (3.0 degrees C) temperatures, high (9.4) and low (5.6) pH levels, and high salt (6% NaCl), ethanol (3.5 vol%) and hydrogen peroxide (5 mM) concentrations. Expressional studies and growth experiments on genetically modified strains proved lisK and yycG to respectively play central roles in the acclimation and immediate growth of L. monocytogenes at low temperatures. The most substantial increase in gene expression under cold conditions was that of the chemotaxis gene cheY with 236-fold upregulation at 3 degrees C. The disrupted ΔliaS strain displayed impaired growth in response to all the other stresses, particularly at a high temperature and under osmotic stress. These studies demonstrated the prominent importance of the histidine kinase-encoding genes yycG and lisK to cold tolerance and liaS, with roles in the growth of L. monocytogenes under multiple stresses. To shed light on the accessory genetic mechanisms that cause large strain variation in L. monocytogenes in withstanding heat treatments, heat resistance-conferring traits were further investigated by means of whole-genome sequencing. Comparing the complete genomes of heat-resistant L. monocytogenes AT3E and -sensitive AL4E strains revealed the heat-resistant strain to harbour a novel 58-kb plasmid, pLM58, which was absent in the sensitive strain. Furthermore, curing of the plasmid in AT3E produced a marked decrease in heat resistance from virtually no reduction to a 1.1 cfu/ml log10 reduction at 55.0 degrees C. In pLM58, a 2,155-bp open reading frame annotated as an ATP-dependent ClpL protease-encoding gene was identified. Conjugation of the coding sequence and the putative promoter of the clpL gene into a natively heat-sensitive L. monocytogenes 10403S strain, in turn, enhanced the survival of the strain from a 1.2 cfu/ml log10 reduction to a 0.4 cfu/ml log10 reduction in heat challenge at 55.0 degrees C. In this study, we presented the first evidence of plasmid-mediated heat resistance in L. monocytogenes and identified the protease ClpL to be a novel plasmid-borne heat-resistance mediator. The emerging resistance of L. monocytogenes to benzalkonium chloride, a quaternary ammonium compound widely used as a detergent in food-processing facilities, is a significant concern for food safety and public health. The resistance of 392 L. monocytogenes isolates from Finland (n = 197) and Switzerland (n = 195) to benzalkonium chloride was assessed. A minimal inhibitory concentration of 20 µg/ml was defined. Altogether, 11.5% of the strains proved to be resistant to benzalkonium chloride. Serotype 1/2c harboured the highest prevalence, 32.4% (11/34), of benzalkonium chloride-resistant strains, while in total, most of the resistant strains belonged to serotype 1/2a. Altogether, 68.9% of the resistant strains harboured at least one of the efflux pump system-encoding genes, bcrABC, emrE or qacH, known to confer benzalkonium chloride resistance in L. monocytogenes. We found resistant strains with partially or completely efflux pump-dependent benzalkonium chloride resistance, with the exception of the known resistance-mediating efflux pumps, suggesting the existence of other resistance-contributing efflux pump systems. The lacking of known efflux pump system-encoding genes in addition to efflux pump-independent benzalkonium chloride resistance, in turn, indicates the contribution of completely novel benzalkonium chloride resistance mechanisms. The aim of these studies was to shed light on the genes contributing to the versatile stress tolerance abilities and strain variation of the severe foodborne pathogen, L. monocytogenes. Knowledge of such traits may aid in developing targeted strategies and measures to identify and control the contamination and risks caused, in particular, by stress-tolerant L. monocytogenes strains. en
dc.description.abstract Listeria monocytogenes on elintarvikevälitteinen, listerioosia aiheuttava bakteeri, joka kykenee lisääntymään vaativissa stressiolosuhteissa. Monet elintarvikeketjussa käytettävistä hallintamenetelmistä, kuten matala lämpötila ja korkea suolapitoisuus, eivät estä sen kasvua. L. monocytogenes on vakava taudinaiheuttaja, sillä invasiivisen listerioosin aiheuttama kuolleisuus on jopa 20–30 %. Viime vuosina listerioositapausten määrä on erityisesti Suomessa ollut tavallista suurempi Euroopan keskiarvoon nähden. L. monocytogeneksen monipuoliseen stressikestävyyteen johtavia geneettisiä tekijöitä ei vielä tunneta kattavasti. Lisäksi L. monocytogenes -kantojen välillä esiintyy eroja niiden kyvyssä sietää eri stressitekijöitä ja osa kannoista on siten erityisen stressikestäviä. Väitöskirjan kokonaistavoitteena oli selvittää erilaisia geneettisiä tekijöitä, jotka vaikuttavat L. monocytogeneksen stressinsietokykyyn ja kantakohtaisiin eroihin elintarvikehygieenisesti merkittävissä stressiolosuhteissa (kylmä, lämpö, happamuus, emäksisyys, korkea suola- ja etanolipitoisuus, oksidatiivinen stressi ja desinfektioaine benzalkoniumkloridi). Mutaatio- ja geeni-ilmentymismenetelmin todettiin useiden kaksoiskomponenttisäätelijöihin kuuluvien histidiinikinaasien edistävän L. monocytogeneksen kasvua eri stressiolosuhteissa. Genomisekvensoinnin avulla puolestaan tunnistettiin uusi lämpöresistenssiä lisäävä plasmidi. Suurella kantamäärällä voitiin erottaa selkeää kantakohtaista vaihtelua L. monocytogeneksen benzalkoniumkloridiresistenssissä ja todettiin osan resistenssistä johtuvan mahdollisesti täysin uusista resistenssimekanismeista. Tuntemalla paremmin L. monocytogeneksen stressinsiedon geneettisiä mekanismeja voidaan entistä tarkemmin suunnitella ja kohdentaa listerian torjuntatoimenpiteitä elintarvikeketjussa. fi
dc.format.mimetype application/pdf
dc.language.iso en
dc.publisher Helsingin yliopisto fi
dc.publisher Helsingfors universitet sv
dc.publisher University of Helsinki en
dc.relation.isformatof URN:ISBN:978-951-51-5292-3
dc.relation.isformatof Helsinki: Unigrafia, 2019
dc.rights Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty. fi
dc.rights This publication is copyrighted. You may download, display and print it for Your own personal use. Commercial use is prohibited. en
dc.rights Publikationen är skyddad av upphovsrätten. Den får läsas och skrivas ut för personligt bruk. Användning i kommersiellt syfte är förbjuden. sv
dc.subject Elintarvikehygienia
dc.title Determinants of Listeria monocytogenes stress responses en
dc.type.ontasot Väitöskirja (artikkeli) fi
dc.type.ontasot Doctoral dissertation (article-based) en
dc.type.ontasot Doktorsavhandling (sammanläggning) sv
dc.ths Korkeala, Hannu
dc.ths Lindström, Miia
dc.opn von Wright, Atte
dc.type.dcmitype Text

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