Browsing by Subject "FIELD GEL-ELECTROPHORESIS"

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  • Lienemann, Taru; Kyyhkynen, Aino; Halkilahti, Jani; Haukka, Kaisa; Siitonen, Anja (2015)
    Background: Salmonella enterica spp. enterica serotype Typhimurium (STM) is the most common agent of domestically acquired salmonellosis in Finland. Subtyping methods which allow the characterization of STM are essential for effective laboratory-based STM surveillance and for recognition of outbreaks. This study describes the diversity of Finnish STM isolates using phage typing, antimicrobial susceptible testing, pulsed-field gel electrophoresis (PFGE) and multilocus variable-number tandem repeat analysis (MLVA), and compares the discriminatory power and the concordance of these methods. Results: A total of 375 sporadic STM isolates were analysed. The isolates were divided into 31 definite phage (DT) types, dominated by DT1 (47 % of the isolates), U277 (9 % of the isolates) and DT104 (8 % of the isolates). Of all the isolates, 62 % were susceptible to all the 12 antimicrobials tested and 11 % were multidrug resistant. Subtyping resulted in 83 different XbaI-PFGE profiles and 111 MLVA types. The three most common XbaI-PFGE profiles (STYM1, STYM7 and STYM8) and one MLVA profile with three single locus variants accounted for 56 % and 49 % of the STM isolates, respectively. The studied isolates showed a genetic similarity of more than 70 % by XbaI-PFGE. In MLVA, 71 % of the isolates lacked STTR6 and 77 % missed STTR10p loci. Nevertheless, the calculated Simpson's diversity index for XbaI-PFGE was 0.829 (95 % CI 0.792-0.865) and for MLVA 0.867 (95 % CI 0.835-0.898). However, the discriminatory power of the 5-loci MLVA varied among the phage types. The highest concordance of the results was found between XbaI-PFGE and phage typing (adjusted Wallace coefficient was 0.833 and adjusted Rand coefficient was 0.627). Conclusions: In general, the calculated discriminatory power was higher for genotyping methods (MLVA and XbaI-PFGE) than for phenotyping methods (phage typing). Overall, comparable diversity indices were calculated for PFGE and MLVA (both DI > 0.8). However, MLVA was phage type dependent providing better discrimination of the most common phage types. Furthermore, 5-loci MLVA was a less laborious method and easier to interpret than XbaI-PFGE. Thus, the laboratory-based surveillance of the Finnish human STM infections has been conducted with a combination of phage typing, antimicrobial susceptibility testing and 5-loci MLVA since January 2014.
  • Åvall-Jääskeläinen, Silja Tuulia; Taponen, Suvi Sinikka; Kant, Ravi; Paulin, Lars Göran; Blom, Jochen; Palva, Airi Marjatta; Koort, Joanna Maria Kataliina (2018)
    Non-aureus staphylococci (NAS) are most commonly isolated from subclinical mastitis. Different NAS species may, however, have diverse effects on the inflammatory response in the udder. We determined the genome sequences of 20 staphylococcal isolates from clinical or subclinical bovine mastitis, belonging to the NAS species Staphylococcus agnetis S. chromogenes, and S. simulans, and focused on the putative virulence factor genes present in the genomes. For comparison we used our previously published genome sequences of four S. aureus isolates from bovine mastitis. The pan-genome and core genomes of the non-aureus isolates were characterized. After that, putative virulence factor orthologues were searched in silico. We compared the presence of putative virulence factors in the NAS species and S. aureus and evaluated the potential association between bacterial genotype and type of mastitis (clinical vs. subclinical). The NAS isolates had much less virulence gene orthologues than the S. aureus isolates. One third of the virulence genes were detected only in S. aureus. About 100 virulence genes were present in all S. aureus isolates, compared to about 40 to 50 in each NAS isolate. S. simulans differed the most. Several of the virulence genes detected among NAS were harbored only by S. simulans, but it also lacked a number of genes present both in S. agnetis and S. chromogenes. The type of mastitis was not associated with any specific virulence gene profile. It seems that the virulence gene profiles or cumulative number of different virulence genes are not directly associated with the type of mastitis (clinical or)subclinical), indicating that host derived factors such as the immune status play a pivotal role in the manifestation of mastitis.
  • Williamson, Charles H. D.; Sahl, Jason W.; Smith, Theresa J.; Xie, Gary; Foley, Brian T.; Smith, Leonard A.; Fernandez, Rafael A.; Lindström, Miia; Korkeala, Hannu; Keim, Paul; Foster, Jeffrey; Hill, Karen (2016)
    Background: Clostridium botulinum is a diverse group of bacteria characterized by the production of botulinum neurotoxin. Botulinum neurotoxins are classified into serotypes (BoNT/A-G), which are produced by six species/Groups of Clostridia, but the genetic background of the bacteria remains poorly understood. The purpose of this study was to use comparative genomics to provide insights into the genetic diversity and evolutionary history of bacteria that produce the potent botulinum neurotoxin. Results: Comparative genomic analyses of over 170 Clostridia genomes, including our draft genome assemblies for 59 newly sequenced Clostridia strains from six continents and publicly available genomic data, provided in-depth insights into the diversity and distribution of BoNT-producing bacteria. These newly sequenced strains included Group I and II strains that express BoNT/A,/B,/E, or/F as well as bivalent strains. BoNT-producing Clostridia and closely related Clostridia species were delineated with a variety of methods including 16S rRNA gene, concatenated marker genes, core genome and concatenated multi-locus sequencing typing (MLST) gene phylogenies that related whole genome sequenced strains to publicly available strains and sequence types. These analyses illustrated the phylogenetic diversity in each Group and the diversity of genomic backgrounds that express the same toxin type or subtype. Comparisons of the botulinum neurotoxin genes did not identify novel toxin types or variants. Conclusions: This study represents one of the most comprehensive analyses of whole genome sequence data for Group I and II BoNT-producing strains. Read data and draft genome assemblies generated for 59 isolates will be a resource to the research community. Core genome phylogenies proved to be a powerful tool for differentiating BoNT-producing strains and can provide a framework for the study of these bacteria. Comparative genomic analyses of Clostridia species illustrate the diversity of botulinum-neurotoxin-producing strains and the plasticity of the genomic backgrounds in which bont genes are found.
  • Skarp, C. P. A.; Akinrinade, O.; Nilsson, A. J. E.; Ellstrom, P.; Myllykangas, S.; Rautelin, H. (2015)
    Campylobacter jejuni is a major pathogen in bacterial gastroenteritis worldwide and can cause bacteremia in severe cases. C. jejuni is highly structured into clonal lineages of which the ST677CC lineage has been overrepresented among C. jejuni isolates derived from blood. In this study, we characterized the genomes of 31 C. jejuni blood isolates and 24 faecal isolates belonging to ST677CC in order to study the genome biology related to C. jejuni invasiveness. We combined the genome analyses with phenotypical evidence on serum resistance which was associated with phase variation of wcbK; a GDP-mannose 4,6-dehydratase involved in capsular biosynthesis. We also describe the finding of a Type III restriction-modification system unique to the ST-794 sublineage. However, features previously considered to be related to pathogenesis of C. jejuni were either absent or disrupted among our strains. Our results refine the role of capsule features associated with invasive disease and accentuate the possibility of methylation and restriction enzymes in the potential of C. jejuni to establish invasive infections. Our findings underline the importance of studying clinically relevant well-characterized bacterial strains in order to understand pathogenesis mechanisms important in human infections.
  • Revez, Joana; Llarena, Ann-Katrin; Schott, Thomas; Kuusi, Markku; Hakkinen, Marjaana; Kivistö, Rauni; Hänninen, Marja-Liisa; Rossi, Mirko (2014)
  • 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.
  • Grönthal, Thomas; Moodley, Arshnee; Nykäsenoja, Suvi; Junnila, Jouni; Guardabassi, Luca; Thomson, Katariina; Rantala, Merja (2014)
  • Jalava, Katri; Rintala, Hanna; Ollgren, Jukka; Maunula, Leena; Gomez-Alvarez, Vicente; Revez, Joana; Palander, Marja; Antikainen, Jenni; Kauppinen, Ari; Räsänen, Pia; Siponen, Sallamaari; Nyholm, Outi; Kyyhkynen, Aino; Hakkarainen, Sirpa; Merentie, Juhani; Pärnänen, Martti; Loginov, Raisa; Ryu, Hodon; Kuusi, Markku; Siitonen, Anja; Miettinen, Ilkka; Domingo, Jorge W. Santo; Hänninen, Marja-Liisa; Pitkänen, Tarja (2014)
  • Castro, Hanna; Ruusunen, Marjo; Lindström, Miia (2017)
    The increased availability of packaged raw drinking milk necessitates the investigation of the occurrence and growth of Listeria monocytogenes in raw milk during distribution and storage. The occurrence of L. monocytogenes in 105 retailed raw milk bottles, 115 bulk tank milk samples, 23 in-line milk filter socks and in 50 environmental samples collected from an on-farm dairy establishment were investigated. Growth of inoculated low-level L. monocytogenes contamination was also investigated in two types of raw milk packaging, namely in 1-litre plastic bottles and 3-litre bag-in-boxes, both stored at three different storage temperatures of 6, 8 and 10 degrees C. The occurrence of L. monocytogenes was higher (4.8%) in bottled raw milk stored until the use-by-date of the package compared to fresh bulk tank milk (1.7%). L. monocytogenes counts were 5 13 CFU/ml in bottled raw milk and 5 1 CFU/ml in bulk tank milk. L. monocytogenes was not detected in the packaging facility, but occurred very frequently (39%) in the milk filter socks. Subtyping of L. monocytogenes isolates using pulsed -field gel-electrophoresis revealed seven pulsotypes, of which two occurred in multiple samples. Targeted inoculum levels of 1-2 CFU/ml yielded L. monocytogenes counts 100 CFU/ml within seven days of storage in 22% of the raw milk packages stored at 6 degrees C, and in all of the raw milk packages stored at 8 degrees C. The frequent occurrence of L. monocytogenes in raw milk and the ability of a low-level L. monocytogenes contamination to grow at refrigeration temperatures highlight the importance of consumer education regarding the appropriate raw milk storage and handling.
  • Aalto-Araneda, Mariella; Korkeala, Hannu; Lundén, Janne (2018)
    Vacuum-packaged cold-salted and cold-smoked fish products are considered typical vehicles for Listeria monocytogenes, the causative agent of the food-borne disease listeriosis, which is increasingly prevalent in the European Union. Efficacy of both the fish processing plant self-checking system and official food control conducted by authorities are crucial for L. monocytogenes prevention in the processing of these risky products. However, the impact of official control on L. monocytogenes prevention in the processing of fish products has not been extensively studied. We investigated the occurrence, control measures, and correction of non-compliances predisposing to L. monocytogenes in Finnish fish processing plants. The following features were associated with L. monocytogenes occurrence: (a) frequency of non-compliances concerning processing machinery, (b) recurrence of non-compliances, and (c) frequency of non-compliances for which official control measures were requested by inspecting authorities. Official control of fish processing plants had focused on risky areas, but non-compliances were common and their correction exhibited deficiencies. We conclude that L. monocytogenes prevention in fish processing can be enhanced by strengthening official food control measures and processing plant compliance. In particular, timely correction of all food safety violations must be improved.