Browsing by Subject "kokogenomisekvensointi"
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(2019)The Master's thesis was completed as part of the joint project (Haittaeläin) by the Finnish Food Authority (Ruokavirasto) and Natural Resources Institute Finland (Luke). In the project, rodents and shrews caught on farm premises were investigated for zoonotic bacterial pathogens. This thesis covers the thermophilic cam-pylobacter findings in the caught pests in autumn 2017.Rodents and shrews caught on farm premises were investigated for zoonotic bacterial pathogens. This thesis deals with the result of thermophilic campylobac-ters isolated from the pests in the autumn 2017. Thermophilic campylobacters (Campylobacter jejuni ja Cam-pylobacter coli) cause gastroenteritis called campylobacteriosis in humans, which is one of the most common cause for human gastroenteritis in the world. The literature review discusses the characteristics of thermo-philic campylobacters and their epidemiology, which focused on aspects of a public health and risk factors for campylobacteriosis and the major sources of campylobacters. Moreover, a current knowledge of campylo-bacters’ occurrence in rodents and shrews were summarized in the literature review. The presence of thermophilic campylobacters was investigated from 227 pooled samples, which comprised of a total of 442 intestinal samples collected from 12 different species. Three species (yellow-necked mouse, house mouse and bank vole) covered 81,3 % of all caught pests. The numbers of caught pests were not signif-icantly different between the farms when compared to a geographical position (south or north) or a type of farm (pig or cattle). The numbers of captured yellow-necked mice were significantly higher in farms in the south. Especially, yellow-necked mice and bank voles were detected to be campylobacter positive. Other campylobacter positive species were harvest mice, rats, field voles and southern voles. A total of 93 samples were detected as campylobacter positive and all of them were identified as C. jejuni. There was not signifi-cant difference on the numbers of campylobacter positive samples between the farms when compared the geographical position or the type of farm. 46 isolates of all 96 campylobacter positive samples were selected for a whole genome sequencing. After the raw read data was assembled, the contigs were analysed with MLST and cgMLST typing schemes by Ridom SeqSphere+. 41 isolates contained 14 new sequence types (STs). Instead, 5 isolates contained previously described STs: ST-1304, ST-2219 and ST-4791. According to MLST typing isolates obtained from voles differed from isolates from mice and rats. Moreover, cgMLST typing supported the conclusion. In the cgMLST typing scheme vole’s isolates were found to have more missing loci than isolates from mice or rats. Therefore, all isolates from voles, apart from two bank vole isolates belonging to ST-1304, contained less than 90 % loci compared to a reference genome. Furthermore, these same isolates were poorly identified in MALDI TOF analysis unlike other isolates. One interpretation to the difference between isolates could be that isolates from voles belong to a new C. jejuni subspecies.
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(2022)Kampylobakteerit ovat yleisin bakteeriperäisen suolistotulehduksen aiheuttaja maailmanlaajuisesti. Ne aiheuttavat kuumeista ripulitautia, joka voi kestää jopa viikon. Ihmisten tautitapauksissa yleisimmin esiintyviä lajeja ovat termotolerantit Campylobacter jejuni ja Campylobacter coli. Kampylobakteeritartunta saadaan tyypillisimmin saastuneesta elintarvikkeesta tai juomavedestä. Kampylobakteerit ovat zoonoottisia eli ne voivat siirtyä eläimistä ihmisiin ja päinvastoin. Monet eläinlajit kantavat kampylobakteereita oireetta, jonka vuoksi bakteeri voi päätyä elintarvikkeisiin tartunnan saaneen eläimen välityksellä. Kampylobakteereiden esiintyvyyttä on tutkittu laajasti tuotantoeläimillä ja jonkin verran luonnonvaraisilla eläimillä, mutta rottien osalta tutkimustieto on edelleen puutteellista. Kaupunkirotat elävät ihmisasutuksen keskellä ja niillä on potentiaalia levittää erilaisia taudinaiheuttajia ihmisiin. Tämän alkuperäistutkimuksen sisältävän tutkielman tavoite oli selvittää, esiintyykö rotilla ihmisten terveyden kannalta merkityksellisiä kampylobakteerikantoja. Tutkimuksen hypoteesina oli, että ainakin osa rotilla esiintyvistä kampylobakteerikannoista on ihmisten terveyden kannalta merkityksellisiä. Tutkimusaineisto oli kerätty Helsingin kaupunkirottaprojektissa, joka on vuodesta 2018 lähtien käynnissä ollut, Tuomas Aivelon johtama monitieteinen tutkimushanke. Aineisto koostui 201 näytteestä, jotka oli kerätty Uudenmaan alueelta pyydetyistä rotista (Rattus norvegicus). Näytteitä oli otettu sekä kuolleista että elävistä eläimistä. Näytteet oli viljelty selektiivisille kasvatusmaljoille, jotka inkuboitiin termofiilisille kampylobakteereille sopivissa olosuhteissa. Kampylobakteereille positiivisista näytteistä oli tehty puhdasviljelmiä, joista bakteerien genominen DNA oli eristetty ja sekvensoitu Illuminalla. Tässä tutkielmassa isolaattien kokogenomisekvenssejä analysoitiin käyttäen vapaasti saatavilla olevia bioinformatiikan työkaluja: Microbial Genomes Atlas Online (MiGa), PubMLST Campylobacter jejuni/coli, ResFinder ja Comprehensive Antibiotic Resistance Database (CARD). Analyyseihin lukeutui lajintunnistus, MLST-tyypitys ja mahdollisten mikrobilääkeresistenssien selvitys. Tutkituista näytteistä oli eristetty 22 kampylobakteeri-isolaattia, joista 18 tunnistettiin Campylobacter jejuni-lajin edustajiksi. Muita neljää isolaattia ei kyetty tunnistamaan luotettavasti, ja ne todennäköisesti edustavat uusia aiemmin kuvaamattomia Campylobacterales-lahkon lajeja. MLST-tyypityksessä havaittiin, että Campylobacter jejuni-kantojen joukossa esiintyi sekvenssityyppejä ST-45 (ST-45 CC), ST-3904 (ST-45 CC), ST-11 (ST-45 CC) ja ST-9477. Lisäksi löydettiin uusi sekvenssityyppi, ST-11770. Näistä erityisesti ST-45 on aikaisemmin ollut yleisin siipikarjalla Suomessa tavattu sekvenssityyppi ja yleinen Suomessa myös ihmisten infektioissa. Mikrobilääkeresistenssiä koskevissa analyyseissa ei havaittu moniresistenssiä tai hankittua resistenssiä yhdelläkään kannalla. Tulokset ovat hypoteesin mukaisia ja osoittavat, että tutkituilla rotilla esiintyy erilaisia kampylobakteereita, joista osalla on potentiaalia tarttua ihmisiin ja tuotantoeläimiin. Rottien merkitystä kampylobakteerin leviämisessä tulisi kuitenkin tutkia vielä lisää ennen kuin voidaan tehdä johtopäätöksiä rottien roolista kampylobakteerin levittäjinä.
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(2020)Constantly increasing level of bacteria becoming resistant to clinically relevant antibiotics challenges the modern medical achievements made over the past century. In global scale, one of the most significant information gaps concerning the occurrence of resistant bacteria is located in West African countries. Klebsiella pneumoniae and Escherichia coli strains resistant to 3rd generation cephalosporins and carbapenems are a major risk to public health through infections with limited or no available treatment options. The resistance to these antibiotics among Enterobacteriaceae is mainly mediated by hydrolyzing enzymes such as extended-spectrum beta-lactamases (ESBL). The focus of this thesis is to study the genes encoding these enzymes and other resistance factors found in K. pneumoniae and E. coli isolated from human stool and waste water samples in Burkina Faso and Mali. Tree Enterobacteriaceae isolates were selected for whole genome sequence (WGS) analysis based on their phenotypic resistance profiles defined by disk diffusion method. Reads were assembled to draft genomes and the genomes were studied for their antibiotic resistance genes, virulence genes and their associations to mobile genetic elements found in these isolates’ genomes. Additionally a pan-genome was created to investigate species specific features of K. pneumoniae and their role in heavy load of antibiotic resistance genes among these isolates. Pan-genome consisted of two genomes sequenced in this study and 12 genomes from the publically available database. 16-month old Burkinabe child was a carrier of one ESBL-producing K. pneumoniae (isolate Burkina_1) and one ESBL-positive E. coli along with the resistance to multiple other antibiotics. With genome wide analysis the K. pneumoniae strain could be described as sequence type (ST) 45 representing, multidrug resistant and ESBL-gene CTX-M-15 carrying strain with highly similar virulence gene profile to strains previously described as pathogenic K. pneumoniae causing neonatal sepsis. K. pneumoniae isolated from the stool sample of an adult living in Burkina Faso was found to be multidrug resistant, though non-ESBL-producer strain (isolate Burkina_2). The isolate showed no similarity to any previously described sequence type. CTX-M-15 encoding E. coli of ST38 (isolate Mali_1) carried by Malian child showed resistance to five different classes of antibiotics in addition to the 3rd generation cephalosporins. At the same time the isolate showed hybrid virulence gene profile with virulence genes associated to many different E. coli pathotypes including neonatal meningitis causing E. coli (NMEC). The exceptional plasticity of K. pneumoniae genome could be recognized as one of the putative explanations for the high number of resistance genes found among the isolates studied in this work. Antibiotic resistance genes were found to be associated to mobile genetic elements (MGE) and as the genetic plasticity is caused by the acquisition of external genetic material via MGEs such as plasmids, this can lead to indirect accumulation of resistance genes in these genomes. The results in this thesis work show alarming examples of pathogens that potentially cause severe infections, have extremely narrow or no treatment options and are carried by infants. These findings are in line with the few data about the level of faecal carriage of ESBL-producing strains by people in Burkina Faso and Mali reported previously.
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(2019)Fecal microbiota transplantation (FMT) is a medical treatment procedure in which feces of a healthy donor are transplanted into a recipient in order to re-establish a healthy gut microbiota. Currently, FMT is used routinely to treat recurrent Clostridioides difficile infections, but it is being studied as a potential treatment for other diseases also resulting from a disbalance in the gut microbiota. FMT provides an excellent platform for studies addressing the determinants of a healthy gut microbiota, as it makes it possible to survey specific microbes involved in the process of re-establishment. In this sense, one particularly interesting group of gut microbes is the bifidobacteria. Although usually associated with commercial probiotics, bifidobacteria are actually one of first major colonizers of the human gut after birth, and they play a key role in the establishment and maintenance of a healthy gut microbiota. The aim of this study was to assess the long-term colonization of donor-derived bifidobacteria in recipients of FMT by using a combination of a culture-dependent method and molecular typing of strains. The culture-dependent method was used for the isolation of bifidobacterial strains from fecal samples of donors and recipients, whereas the molecular typing methods (rep-PCR and whole genome sequencing) were employed to differentiate the isolated strains. This study was based on the premise that in order to be of donor-origin, a bifidobacterial strain must occur in the recipient only after FMT and show close genetic relatedness to a strain isolated from the respective donor. Furthermore, in order to exhibit long-term colonization, a donor-derived strain must be present at later time points of the study’s follow-up period. The results showed that all the recipients had acquired some donor-like bifidobacteria after FMT. In addition, certain donor-derived strains of Bifidobacterium longum had persisted in some recipients throughout the follow-up period, indicating their successful colonization. This finding is of special interest, as extraneous bifidobacteria, such as probiotics, are typically lost without their continuous influx. Additionally, long-term colonization of bifidobacteria has not previously been documented in adults by a culture-dependent method. As efficient colonizers, the strains isolated in this study represent potential candidates for therapeutic agents.
Now showing items 1-4 of 4