Browsing by Subject "MLST"

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  • Meier, Anja B.; Guldimann, Claudia; Markkula, Annukka; Pöntinen, Anna; Korkeala, Hannu; Tasara, Taurai (2017)
    Reduced susceptibility of Listeria monocytogenes to benzalkonium chloride (BC), a quaternary ammonium compound widely used in food processing and hospital environments, is a growing public health and food safety concern. The minimal inhibitory concentration of BC on 392 L. monocytogenes strains from Switzerland (CH) and Finland (FIN) was determined. Within this strain collection, benzalkonium chloride resistance was observed in 12.3% (24/195) of Swiss and 10.6% (21/197) of Finnish strains. In both countries, the highest prevalence of BC-resistant strains (CH: 29.4%; FIN: 38.9%) was detected among serotype 1/2c strains. Based on PCR analysis, genes coding for the qacH efflux pump system were detected for most of the BC-resistant strains ( CH: 62.5%; FIN: 52.4%). Some Swiss BC-resistant strains harbored genes coding for the bcrABC(16.7%) efflux pump system, while one Finnish BC-resistant strain harbored the emrE gene previously only described among BC-resistant L. monocytogenes strains from Canada. Interestingly, a subset of BC-resistant strains (CH: 5/24, 20.8%; FIN: 9/21, 42.8%) lacked genes for efflux pumps currently known to confer BC resistance in L. monocytogenes. BC resistance analysis in presence of reserpine showed that the resistance was completely or partially efflux pump dependent in 10 out of the 14 strains lacking the known BC resistance genes. Sequence types 155 and ST403 were over-representated among these strains suggesting that these strains might share similar but yet unknown mechanisms of BC resistance.
  • Mohan, Vathsala; Cruz, Cristina D.; van Vliet, Arnoud H. M.; Pitman, Andrew R.; Visnovsky, Sandra B.; Rivas, Lucia; Gilpin, Brent; Fletcher, Graham C. (2021)
    Listeria monocytogenes is a foodborne human pathogen that causes systemic infection, fetal-placental infection in pregnant women causing abortion and stillbirth and meningoencephalitis in elderly and immunocompromised individuals. This study aimed to analyse L. monocytogenes from different sources from New Zealand (NZ) and to compare them with international strains. We used pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and whole-genome single nucleotide polymorphisms (SNP) to study the population structure of the NZ L. monocytogenes isolates and their relationship with the international strains. The NZ isolates formed unique clusters in PFGE, MLST and whole-genome SNP comparisons compared to the international isolates for which data were available. PFGE identified 31 AscI and 29 ApaI PFGE patterns with indistinguishable pulsotypes being present in seafood, horticultural products and environmental samples. Apart from the Asc0002:Apa0002 pulsotype which was distributed across different sources, other pulsotypes were site or factory associated. Wholegenome analysis of 200 randomly selected L. monocytogenes isolates revealed that lineage II dominated the NZ L. monocytogenes populations. MLST comparison of international and NZ isolates with lineage II accounted for 89% (177 of 200) of the total L. monocytogenes population, while the international representation was 45.3% (1674 of 3473). Rarefaction analysis showed that sequence type richness was greater in NZ isolates compared to international trend, however, it should be noted that NZ isolates predominantly came from seafood, horticulture and their respective processing environments or factories, unlike international isolates where there was a good mixture of clinical, food and environmental isolates.
  • Tikkanen, Jouni (Helsingin yliopisto, 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.
  • Sauvala, Mikaela; Woivalin, Emma; Kivistö, Rauni; Laukkanen-Ninios, Riikka; Laaksonen, Sauli; Stephan, Roger; Fredriksson-Ahomaa, Maria (2021)
    Game birds may carry zoonotic bacteria in their intestines and transmit them to hunters through bird handling or through the handling and consumption of contaminated meat. In this study, the prevalence of foodborne bacteria was screened from game bird faeces and mallard breast meat using PCR. The sampling occurred in southern Finland from August to December during the hunting season. Isolates were characterized by multi-locus sequence typing. Mesophilic aerobic bacteria and Escherichia coli counts were used to assess the microbial contamination of mallard meat. In total, 100 woodpigeon (Columba palumbus), 101 pheasants (Phasianus colchicus), 110 mallards (Anas platyrhynchos), and 30 teals (Anas crecca) were screened during the hunting season. Additionally, 100 mallard breast meat samples were collected. Campylobacter and Listeria were commonly detected in the faeces and Listeria on mallard meat. L. monocytogenes of sequence types associated with human listeriosis were frequently found in game bird faeces and on mallard meat. Good hygiene during game bird handling, storing the game bird meat frozen, and proper heat treatment are important measures to minimize the health risk for hunters and consumers.
  • Bozcal, Elif; Eldem, Vahap; Aydemir, Sohret; Skurnik, Mikael (2018)
    Background. Extraintestinal pathogenic Escherichia coli (ExPEC) is an important bacterium and responsible for many bloodstream infections, including urinary tract infections and even fatal bacteremia. The aim of this research was to investigate whether ExPEC strains isolated from Turkish blood cultures have a relationship between 16S rRNA based phylogenetic clusters and antibiotic resistance profiles, virulence factors or clonal lineages. Methods. Phenotypically identified ExPEC blood culture isolates (n = 104) were included in this study. The 16S rRNA partial sequence analysis was performed for genotypic identification of ExPEC isolates. Antibiotic susceptibility and Extended Spectrum beta-Lactamase testing of isolates were performed. Phylogenetic classification (A, B1, B2 and D), Multi Locus Sequence Typing analysis and virulence-associated genes were investigated. Results. Based on 16S rRNA partial sequence analysis, 97 out of 104 (93.26%) ExPEC isolates were confirmed as E. coli. Ampicillin (74.22%) and cefuroxime axetil (65.97%) resistances had the highest frequencies among the ExPEC isolates. In terms of phylogenetic classification of ExPEC, D (38.14%, 37/97) was the most prevalent group after A (29.89%, 29/97), B2 (20.61%, 20/97), and B1 (11.34%, 11/97). The sequence types of the 20 ExPEC isolates belonging to the B2 phylogenetic group were analyzed by Multi Locus Sequence Typing. Ten isolates out of 20 (50.0%) were identified as ST131. The other STs were ST95 (n=1), ST14 (n=1), ST10 (n=1), ST69 (n=1), ST1722 (n=2), ST141 (n=1), ST88 (n=1), ST80 (n=1), and ST998 (n=1). Of the ST131 strains, six (60%, 6/10) represented serogroup O25. The most common virulence factor genes were serum resistance factor gene, traT (55.7%) aerobactin siderophore receptor and yersiniabactin encoding genes iutA (45.3%) and fyuA (50.5%), respectively. In addition, PAI (41.2%), iroN (23.7%), hlyA (15.4%), kpsII (13.4%), ompT (13.4%), papG (12.4%), iss (9.3%), cnf1 (7.2%), ibeA (2.06%), and sfaS (2.06%) genes were present in the ExPEC isolates. Conclusion. The 16S rRNA-based phylogenetic relationship tree analysis showed that a large cluster was present among 97 ExPEC isolates along with related reference strains. There were 21 main clusters with 32 closely related subclusters. Based on our findings, different clonal lineages of ExPEC can display different antibiotic susceptibilities and virulence properties. We also concluded that virulence factors were not distributed depending on phylogenetic groups (A, B1, B2, and D). The ExPEC isolates belonging to the same phylogenetic group and sequence type could display different resistance and virulence characteristics.
  • Kovanen, Sara; Kivisto, Rauni; Llarena, Ann-Katrin; Zhang, Ji; Karkkainen, Ulla-Maija; Tuuminen, Tamara; Uksila, Jaakko; Hakkinen, Marjaana; Rossi, Mirko; Hanninen, Marja-Liisa (2016)
    Campylobacter jejuni is the leading cause of bacterial gastroenteritis and chicken is considered a major reservoir and source of human campylobacteriosis. In this study, we investigated temporally related Finnish human (n = 95), chicken (n = 83) and swimming water (n = 20) C. jejuni isolates collected during the seasonal peak in 2012 using multilocus sequence typing (MIST) and whole-genome MIST (wgMLST). Our objective was to trace domestic human C jejuni infections to C jejuni isolates from chicken slaughter batches and swimming water. At MIST level, 79% of the sequence types (STs) of the human isolates overlapped with chicken STs suggesting chicken as an important reservoir. Four STs, the ST-45, ST-230, ST-267 and ST-677, covered 75% of the human and 64% of the chicken isolates. In addition, 50% of the swimming water isolates comprised ST-45, ST-230 and ST-677. Further wgMLST analysis of the isolates within STs, accounting their temporal relationship, revealed that 22 of the human isolates (24%) were traceable back to C jejuni positive chicken slaughter batches. None of the human isolates were traced back to swimming water, which was rather sporadically sampled. The highly discriminatory wgMLST, together with the patient background information and temporal relationship data with possible sources, offers a new, accurate approach to trace back the origin of domestic campylobacteriosis. Our results suggest that potentially a substantial proportion of campylobacteriosis cases during the seasonal peak most probably are due to other sources than chicken meat consumption. These findings warrant further wgMLST-based studies to reassess the role of other reservoirs in the Campylobacter epidemiology both in Finland and elsewhere. (C) 2016 Elsevier B.V. All rights reserved.
  • Päivärinta, M.; Latvio, S.; Fredriksson-Ahomaa, M.; Heikinheimo, A. (2020)
    Plasmid-encoded extended-spectrum β-lactamase and AmpC gene-carrying Escherichia coli (ESBL/AmpC E. coli) is an increasing cause of human infections worldwide. Increasing carbapenem and colistin resistance further complicate treatment of these infections. The aim of this study was to assess the occurrence of ESBL/AmpC E. coli in different broiler flocks and farms, as well as in broiler meat, in a country with no antimicrobial usage in broiler production. An additional goal was to assess the genetic characteristics of ESBL/AmpC E. coli isolates by using whole genome sequencing (WGS). Altogether 520 caecal swabs and 85 vacuum-packed broiler meat samples were investigated at the slaughterhouse level. WGS of the bacterial isolates revealed acquired antimicrobial resistance (AMR) genes, multilocus sequence types (MLST) and plasmid sequences. ESBL/AmpC E. coli was identified in 92 (18%) of the caecum and 27 (32%) of the meat samples. ESBL/AmpC E. coli-carrying birds derived from six (33%) out of 18 farms. Of the two blaESBL/AmpC genes detected by PCR, blaCMY-2 (96%) was predominant over blaCTX-M-1 (4%). Furthermore, WGS revealed an additional AMR gene sul2. Carbapenemase, colistin, and other AMR genes were not detected from the isolates of either the caecal or meat samples. Altogether seven MLSTs (ST101, ST117, ST212, ST351, ST373, ST1594 and an unknown ST) and a variety of different plasmid sequences (IncB/O/K/Z, IncI1, IncFII, IncII, IncFIB, IncFIC, IncX1 and an additional set of Col-plasmids) were detected. This is the first study on genomic epidemiology of ESBL/AmpC E. coli on broiler farms and flocks with no antimicrobial usage, by using WGS analysis. Results show that ESBL/AmpC E. coli occurrence is common both in the caecum and in the packaged meat. However, compared to other European countries, the occurrence is low and the presence of AMR genes other than blaCMY-2 and blaCTX-M-1 is rare. More studies are needed to understand the ESBL/AmpC E. coli occurrence in broiler production to prevent the meat from contamination during slaughter and processing, thereby also preventing zoonotic transmission of ESBL/AmpC E. coli. Additionally, more studies are needed to understand the ecology and fitness cost of Enterobacteriaceae plasmids in animal production in order to prevent their acquisition of plasmid-encoded antimicrobial resistance genes such as carbapenem and colistin resistance genes, as this would pose a great hazard to food safety.
  • Mäkelä, Anu (Helsingfors universitet, 2017)
    Viime vuosina Yersinia enterocolitican bioserotyypin 2/O:9 esiintyminen on lisääntynyt Euroopassa. Bioserotyyppi 4/O:3 on pitkään ollut yleisin ihmisten yersinioosia aiheuttava kanta, mutta myös bioserotyypin 2/O:9 on todettu olevan patogeeninen ihmisille. Bioserotyypin kantoja on eristetty sioista, lampaista ja naudoista, mutta ne eivät pääosin aiheuta tautia eläimillä. Suomessa tätä bioserotyyppiä on erittäin harvoin eristetty eläimistä. Y. enterocolitican tunnistus on pitkään tapahtunut pääosin bio- ja serotyypityksen avulla. Näiden menetelmien heikkous on kuitenkin herkkyys ympäristön vaikutuksille. Multilocus sequence typing (MLST) – menetelmän avulla bakteereita voidaan tunnistaa sekvensoimalla bakteerin ylläpitogeenejä. Tämän tutkimuksen tavoitteena oli tutkia eri lähteistä peräisin olevia Y. enterocolitica - kantoja MLST-menetelmällä, joka perustuu seitsemään ylläpitogeeniin (Hall ym. 2015), ja tämän avulla vertailla suomalaisten ja eurooppalaisten kantojen eroavaisuuksia. Lisäksi tavoitteena oli pystyttää toimiva seitsemään ylläpitogeeniin perustuva Yersinia MLST-menetelmä Elintarvikehygienian- ja ympäristöterveyden osastolle. Yhteensä tutkittiin 10 Y. enterocolitica –kantaa, joiden oletettiin alustavien bioserotyypitystulosten perusteella kuuluvan biotyppiin 2 ja serotyyppiin O:9. Suomalaiset kannat oli eristetty lampaiden (4/10), myyrän (1/10) ja ihmisen ulosteesta (1/10). Suomalaisia kantoja verrattiin Sveitsissä ja Saksassa eristettyihin kantoihin, jotka oli eristetty sian nielurisasta (1/10), villisian nielurisasta (1/10) ja ihmisen ulosteesta (2/10). Kantojen puhtaus varmistettiin veri- ja selektiivilevyllä, ja tyypitettiin kaupallisella testillä sekä bio- ja serotyypitysmenetelmillä. MLST-menetelmää varten tutkittavien kantojen DNA eristettiin kaupallisella menetelmällä ja lisäksi DNA:n laatu ja määrä optimoitiin. Kannoista monistettiin MLST-karakterisointia varten PCR:n avulla seitsemän ylläpitogeeniä (aarf, dfp, galR, glnS, hemA, rfaE ja speA). Ennen tutkimusta, PCR-ajo optimoitiin kaikille geeneille. Saadut PCR-tuoteet sekvensoitiin ja sekvenssejä vertailtiin Yersinia MLST-tietokantaan sekä aiempiin tutkittuihin sekvenssityyppeihin. Kaikkien suomalaisista lampaista eristettyjen kantojen MLST-sekvenssityypit muistuttivat toisiaan. Yksi ulkomaalaisista kannoista osoittautui tyypityksessä bioserotyypiksi 1A/O:5, joka näkyi myös poikkeavana MLST-sekvenssityyppinä. Suomalaisesta myyrästä eristetty kanta muistutti profiililtaan lampaista eristettyjen kantojen MLST-tyyppejä. Pääosin kaikki yhdeksän bioserotyypin 2/O:9 kantaa muistuttivat huomattavasti toisiaan. Eroavaisuudet olivat pääosin ihmisistä eristettyjen kantojen välillä. Myös MLST-profiilit vastasivat tietokannan O:9 -serotyypin kantoja. Seitsemästä tutkitusta geeneistä geenin rfaE osalta emme saaneet luotettavia tuloksia. Geenin galR kohdalla saimme laadukasta sekvenssiä vain yhteen suuntaan sekvensoidessa. Muiden geenien kohdalla MLSTmenetelmän pystyttäminen onnistui odotetusti. Suomesta eläimistä eristettyjen kantojen profiilit olivat hyvin samankaltaisia, viitaten niiden olevan läheistä alkuperää. Tämä on tärkeä tekijä selvitettäessä bioserotyypin leviämisreittejä Suomessa. Villieläinten on epäilty olevan bioserotyypin 2/O:9 reservuaari. Tutkimuksemme lampaiden, villisian ja myyrän samankaltaiset MLST-profiilit tukevat tätä hypoteesia edelleen. Tuloksia arvioitaessa tulee myös huomioida MLST-menetelmän mahdollinen heikko kyky erottaa geneettisesti läheisiä kantoja.