Browsing by Subject "Listeria monocytogenes"

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  • Keto-Timonen, R.; Tolvanen, R; Lundén, J.; Korkeala, H. (2007)
    Contamination routes of Listeria monocytogenes were examined in a chilled food processing plant that produced ready-to-eat and ready-to-reheat meals during an 8-year period by amplified fragment length polymorphism (AFLP) analysis. A total of 319 L. monocytogenes isolates were recovered from raw materials (n=18), the environment (n=77), equipment (n=193), and products (n=31), and 18 different AFLP types were identified, five of which were repeatedly found to be persistent types. The three compartments (I to III) of the plant showed markedly different contamination statuses. Compartment I, which produced cooked meals, was heavily contaminated with three persistent AFLP types. AFLP type A1 dominated, and it comprised 93% of the isolates of the compartment. Compartment II, which produced uncooked chilled food, was contaminated with four persistent and five nonpersistent AFLP types. The equipment of compartment III, which produced cooked ready-to-reheat meals, was free of contamination. In compartments that produced cooked meals, the cleaning routines, product types, and lack of compartmentalization seemed to predispose production lines to persistent contamination. The most contaminated lines harbored L. monocytogenes in coolers, conveyors, and packing machines. Good compartmentalization limited the flow of L. monocytogenes into the postheat -treatment area and prevented the undesired movement of equipment and personnel, thus protecting the production lines from contamination. In compartment II, grated cheese was shown to cause product contamination. Therefore, special attention should be paid to continuous quality control of raw ingredients when uncooked ready-to-eat foods are produced. In compartment II, reconstruction of the production line resulted in reduced prevalence rates of L. monocytogenes and elimination of two persistent AFLP types.
  • Nikparvar, Bahareh; Andreevskaya, Margarita; Duru, Ilhan C.; Bucur, Florentina I.; Grigore-Gurgu, Leontina; Borda, Daniela; Nicolau, Anca I.; Riedel, Christian U.; Auvinen, Petri; Bar, Nadav (2021)
    Background The pathogen Listeria (L.) monocytogenes is known to survive heat, cold, high pressure, and other extreme conditions. Although the response of this pathogen to pH, osmotic, temperature, and oxidative stress has been studied extensively, its reaction to the stress produced by high pressure processing HPP (which is a preservation method in the food industry), and the activated gene regulatory network (GRN) in response to this stress is still largely unknown. Results We used RNA sequencing transcriptome data of L. monocytogenes (ScottA) treated at 400 MPa and 8(circle)C, for 8 min and combined it with current information in the literature to create a transcriptional regulation database, depicting the relationship between transcription factors (TFs) and their target genes (TGs) in L. monocytogenes. We then applied network component analysis (NCA), a matrix decomposition method, to reconstruct the activities of the TFs over time. According to our findings, L. monocytogenes responded to the stress applied during HPP by three statistically different gene regulation modes: survival mode during the first 10 min post-treatment, repair mode during 1 h post-treatment, and re-growth mode beyond 6 h after HPP. We identified the TFs and their TGs that were responsible for each of the modes. We developed a plausible model that could explain the regulatory mechanism that L. monocytogenes activated through the well-studied CIRCE operon via the regulator HrcA during the survival mode. Conclusions Our findings suggest that the timely activation of TFs associated with an immediate stress response, followed by the expression of genes for repair purposes, and then re-growth and metabolism, could be a strategy of L. monocytogenes to survive and recover extreme HPP conditions. We believe that our results give a better understanding of L. monocytogenes behavior after exposure to high pressure that may lead to the design of a specific knock-out process to target the genes or mechanisms. The results can help the food industry select appropriate HPP conditions to prevent L. monocytogenes recovery during food storage.
  • Nikparvar, Bahareh; Andreevskaya, Margarita; Duru, Ilhan C; Bucur, Florentina I; Grigore-Gurgu, Leontina; Borda, Daniela; Nicolau, Anca I; Riedel, Christian U; Auvinen, Petri; Bar, Nadav (BioMed Central, 2021)
    Abstract Background The pathogen Listeria (L.) monocytogenes is known to survive heat, cold, high pressure, and other extreme conditions. Although the response of this pathogen to pH, osmotic, temperature, and oxidative stress has been studied extensively, its reaction to the stress produced by high pressure processing HPP (which is a preservation method in the food industry), and the activated gene regulatory network (GRN) in response to this stress is still largely unknown. Results We used RNA sequencing transcriptome data of L. monocytogenes (ScottA) treated at 400 MPa and 8∘C, for 8 min and combined it with current information in the literature to create a transcriptional regulation database, depicting the relationship between transcription factors (TFs) and their target genes (TGs) in L. monocytogenes. We then applied network component analysis (NCA), a matrix decomposition method, to reconstruct the activities of the TFs over time. According to our findings, L. monocytogenes responded to the stress applied during HPP by three statistically different gene regulation modes: survival mode during the first 10 min post-treatment, repair mode during 1 h post-treatment, and re-growth mode beyond 6 h after HPP. We identified the TFs and their TGs that were responsible for each of the modes. We developed a plausible model that could explain the regulatory mechanism that L. monocytogenes activated through the well-studied CIRCE operon via the regulator HrcA during the survival mode. Conclusions Our findings suggest that the timely activation of TFs associated with an immediate stress response, followed by the expression of genes for repair purposes, and then re-growth and metabolism, could be a strategy of L. monocytogenes to survive and recover extreme HPP conditions. We believe that our results give a better understanding of L. monocytogenes behavior after exposure to high pressure that may lead to the design of a specific knock-out process to target the genes or mechanisms. The results can help the food industry select appropriate HPP conditions to prevent L. monocytogenes recovery during food storage.
  • Nowak, Jessika; Visnovsky, Sandra B.; Pitman, Andrew R.; Cruz, Cristina D.; Palmer, Jon; Fletcher, Graham C.; Flint, Steve (2021)
    Listeria monocytogenes is a ubiquitous foodborne pathogen that results in a high rate of mortality in sensitive and immunocompromised people. Contamination of food with L. monocytogenes is thought to occur during food processing, most often as a result of the pathogen producing a biofilm that persists in the environment and acting as the source for subsequent dispersal of cells onto food. A survey of seafoodprocessing plants in New Zealand identified the persistent strain 15G01, which has a high capacity to form biofilms. In this study, a transposon library of L. monocytogenes 15G01 was screened for mutants with altered biofilm formation, assessed by a crystal violet assay, to identify genes involved in biofilm formation. This screen identified 36 transposants that showed a significant change in biofilm formation compared to the wild type. The insertion sites were in 27 genes, 20 of which led to decreased biofilm formation and seven to an increase. Two insertions were in intergenic regions. Annotation of the genes suggested that they are involved in diverse cellular processes, including stress response, autolysis, transporter systems, and cell wall/membrane synthesis. Analysis of the biofilms produced by the transposants using scanning electron microscopy and fluorescence microscopy showed notable differences in the structure of the biofilms compared to the wild type. In particular, inactivation of uvrB and mltD produced coccoid-shaped cells and elongated cells in long chains, respectively, and the mgtB mutant produced a unique biofilm with a sandwich structure which was reversed to the wild-type level upon magnesium addition. The mltD transposant was successfully complemented with the wild-type gene, whereas the phenotypes were not or only partially restored for the remaining mutants. IMPORTANCE The major source of contamination of food with Listeria monocytogenes is thought to be due to biofilm formation and/or persistence in food-processing plants. By establishing as a biofilm, L. monocytogenes cells become harder to eradicate due to their increased resistance to environmental threats. Understanding the genes involved in biofilm formation and their influence on biofilm structure will help identify new ways to eliminate harmful biofilms in food processing environments. To date, multiple genes have been identified as being involved in biofilm formation by L. monocytogenes; however, the exact mechanism remains unclear. This study identified four genes associated with biofilm formation by a persistent strain. Extensive microscopic analysis illustrated the effect of the disruption of mgtB, clsA, uvrB, and mltD and the influence of magnesium on the biofilm structure. The results strongly suggest an involvement in biofilm formation for the four genes and provide a basis for further studies to analyze gene regulation to assess the specific role of these biofilm-associated genes.
  • 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.
  • Nowak, J.; Visnovsky, S. B.; Cruz, C. D.; Fletcher, G. C.; van Vliet, A. H. M.; Hedderley, D.; Butler, R.; Flint, S.; Palmer, J.; Pitman, A. R. (2021)
    Aims To understand the genetics involved in surface attachment and biofilm formation ofListeria monocytogenes. Methods and Results Anin vitroscreen of a Himar1 transposon library ofL. monocytogenesstrain 15G01 identified three transposants that produced significantly different biofilm levels when compared to the wild-type strain; two mutants exhibited enhanced biofilm formation and one produced less biofilm biomass than the wild-type. The mutant 15G01mprF::Himar1, which had a transposon insertion in themprFgene, was selected for further analysis. The mutant produced a more densely populated biofilm on solid surfaces such as stainless steel and polystyrene, as determined using scanning electron and light microscopy. The 15G01mprF::Himar1 mutant remained viable in biofilms, but showed an increase in sensitivity to the cationic antimicrobial gallidermin. The mutant also displayed reduced invasiveness in CaCo-2 intestinal cells, suggesting virulence properties are compromised by the inactivation ofmprF. Conclusions Biofilm formation and gallidermin resistance ofL. monocytogenesis influenced bymprF, but this trait is associated with a compromise in invasiveness. Significance and Impact of the Study The presence of pathogenic microorganisms in the food processing environment can cause a significant problem, especially when these microorganisms are established as biofilms. This study shows that the inactivation of themprFgene results in enhanced biofilm formation and abiotic surface attachment ofL. monocytogenes.
  • Miinalainen, Paula (Helsingin yliopisto, 2022)
    Kuivatut siankorvat ovat suosittuja koirien herkkuja. Siankorvien mikrobiologiseen laatuun vaikuttaa moni tekijä, kuten teurastettavien sikojen puhtaus ja siankorvien valmistuksen prosessihygienia. Vaikka kuivattujen siankorvien valmistusketjussa on monia bakteereita tuhoavia tai poistavia vaiheita, ei kaikkia bakteereita saada poistettua. Näin ollen kuluttajalle päätyessään kuivatut siankorvat voivat olla myös patogeenisilla eli tautia aiheuttavilla sekä antibioottiresistenteillä bakteereilla saastuneita. Tämä altistaa sekä koirat että niiden omistajat tartunnoille. Erityisesti pienet lapset, iäkkäät, immuunipuutteiset ja raskaana olevat henkilöt ovat alttiita saastuneiden lemmikinruokien tai infektoituneiden lemmikkien välittämille tartunnoille. Saastuneet siankorvat ovat aiheuttaneet ihmisten salmonellaepidemioita. Epidemioiden seurauksena siankorvien mikrobiologista laatua on tutkittu eri maissa erityisesti Salmonella-bakteerien osalta. Useassa tutkimuksessa on havaittu salmonellan eri serotyyppejä kuivatuissa siankorvissa. Eristettyjen salmonellakantojen joukossa on ollut myös antibioottiresistenttejä kantoja. Ruotsalaisessa tutkimuksessa havaittiin salmonellaa ja ESBL-entsyymiä tuottavia resistenttejä bakteereita kuivatuissa siankorvissa. Suomessa myytävistä siankorvista ei ole julkaistu vastaavia tutkimuksia. Sen vuoksi suomalaiselle siankorvien mikrobiologista laatua selvittävälle tutkimukselle oli tarvetta. Tämän tutkielman tutkimusosiossa oli tavoitteena tutkia salmonellan, resistenttien ESBL/AmpC-entsyymiä tuottavien Escherichia coli -bakteerien ja tuotantolaitoksissa ympäristökontaminanttina esiintyvän patogeenisen Listeria monocytogenes -bakteerin esiintymistä sekä hygieniaindikaattoreina toimivien kokonais- ja enterobakteerien määriä kuivatuissa siankorvissa. Lisäksi tavoitteena oli vertailla kotimaisten ja ulkomaisten siankorvien mikrobiologista laatua keskenään ja selvittää, onko mikrobiologisen laadun kannalta merkitystä, ostetaanko siankorvat pakattuina vai irtotuotteina. Hypoteesina oli, että ulkomaisissa siankorvissa on enemmän bakteereita kuin kotimaisissa, ja että pakatuissa ja irtomyydyissä siankorvissa ei ole eroa bakteereiden osalta. Tutkimusosiossa näytteenä oli yhteensä 50 Suomesta tai ulkomaisista verkkokaupoista ostettua kuivattua siankorvaa. Näytteistä 31 oli alkuperältään kotimaisia ja loput ulkomaisia. Kotimaisista näytteistä 16 oli irtotuotteita ja muut näytteet olivat pakattuja. Näytteiden kokonais- ja enterobakteerien määriä tutkittiin pintaviljelymenetelmällä. ESBL/AmpC-E. colin esiintymistä tutkittiin kefotaksiimi-pitoisella valikoivalla maljalla sekä varmistustesteillä. Salmonellan ja listerian esiintymistä kartoitettiin aluksi PCR-menetelmällä. Positiivisen PCR-tuloksen saaneille näytteille tehtiin näiden bakteerien osalta viljelyt valikoivilla maljoilla sekä varmistustestejä puhdasviljelmistä. Ulkomaisista näytteistä löytyi sekä salmonellaa että ESBL/AmpC-E. colia. Kotimaisista näytteistä löytyi listeriaa, mutta vain PCR-menetelmällä. Kokonais- ja enterobakteerimäärät olivat suurimpia ulkomaisissa pakatuissa näytteissä. Tämä tutkimus osoitti, että koirille tarkoitettujen kuivattujen siankorvien mikrobiologinen laatu vaihtelee huomattavasti, ja että ulkomaisissa siankorvissa voi esiintyä ihmisten terveydelle haitallisia patogeenisia ja/tai resistenttejä bakteereita. Pakatut siankorvat eivät ole pakkaamattomia turvallisempia. Tulosten perusteella kuivattuja siankorvia käsiteltäessä tulee kiinnittää huomiota hyvään hygieniaan.
  • Antila, Emmi (Helsingin yliopisto, 2021)
    Listeria monocytogenes on maailmanlaajuisesti yksi merkittävimmistä elintarvikevälitteistä tautia aiheuttavista bakteereista. L. monocytogenes -bakteeria esiintyy erityisesti raaoissa liha- ja kalavalmisteissa, pastöroimattomissa maitotuotteissa sekä tuoreissa kasviksissa. L. monocytogenes on ongelma elintarvikkeiden lisäksi myös tuotantolaitoksissa, joiden pinnoille muodostamissaan biofilmeissä bakteeria voi esiintyä saneerauksesta huolimatta jopa vuosia. Leuconostoc gelidum -maitohappobakteerit ovat ympäristössä esiintyviä bakteereja, jotka ovat yleisiä kylmäsäilytettyjen suojakaasu- ja tyhjiöpakattujen elintarvikkeiden valtalajeja viimeisenä käyttöpäivänä. Jotkut L. gelidum -kannat tuottavat pieniä antimikrobisia peptidejä, bakteriosiineja, joilla on osoitettu olevan L. monocytogenes -bakteerin kasvua estävä vaikutus. Tämän työn tavoitteena oli tutkia bakteriosiinia tuottavan ja tuottamattoman L. gelidum -kannan vaikutusta L. monocytogenes -bakteerin kasvuun kylmäsäilytetyissä elintarvikkeissa tarkkaillen samalla kantojen läsnäolon vaikutusta elintarvikkeen happamuuteen. Tutkimuksen hypoteesina oli, että I) L. gelidum- kantojen läsnäololla on L. monocytogenes -bakteerin kasvua estävä vaikutus ja II) bakteriosiinia tuottavan kannan estovaikutus on voimakkaampi. Estovaikutusta tutkittiin elintarvikkeissa (6 ºC), joihin oli siirrostettu L. monocytogenes ATCC 7644ᵀ -kantaa. Elintarvikkeista määritettiin L. monocytogenes -pesäkemäärä (pmy/g) useassa aikapisteessä L. monocytogenes -kannan kasvaessa yksin, yhdessä bakteriosiinia tuottavan L. gelidum Vvan 9 -kannan kanssa ja yhdessä bakteriosiinia tuottamattoman L. gelidum LMG 18811ᵀ -kannan kanssa. Kokeen aikana seurattiin myös mahdollisia muutoksia elintarvikkeiden pH-arvossa. Tulosten perusteella molemmilla L. gelidum -kannoilla oli L. monocytogenes ATCC 7644ᵀ -kannan kasvua hidastava vaikutus. Bakteriosiinia tuottavan Vvan 9 -kannan estovaikutuksen havaittiin kuitenkin olevan huomattavasti voimakkaampi kuin bakteriosiinia tuottamattoman LMG 18811ᵀ -kannan estovaikutuksen. Tämä viittaisi siihen, että hypoteesin mukaisesti Vvan 9 -kannan tuottamat bakteriosiinin kaltaiset yhdisteet tehostivat kannan estovaikutusta. L. gelidum -kantojen läsnäolo laski pH-arvoa matriiseissa, joissa ei ollut runsasta luonnollista mikrobistoa. Työ osoittaa, että L. gelidum Vvan 9 -kanta ja sen tuottamat bakteriosiinit ovat potentiaalinen keino torjua L. monocytogenes -bakteerin kasvua tyhjiö- ja suojakaasupakatuissa kylmäsäilytetyissä elintarvikkeissa. Kyseinen kanta sekä muut bakteriosiineja tuottavat L. gelidum -kannat tarjoavatkin jatkossa mielenkiintoisen tutkimuskohteen.
  • Roiha, Ella (Helsingin yliopisto, 2021)
    Kylmäsäilytetyissä elintarvikkeissa esiintyy yleisesti Leuconostoc gelidum -maitohappobakteereja, jotka on perinteisesti yhdistetty elintarvikkeiden pilaantumiseen. Joidenkin L. gelidum -kantojen on lisäksi todettu tuottavan bakteriosiineja, pieniä antimikrobisia peptidiyhdisteitä, jotka estävät tehokkaasti Listeria monocytogenesin sekä joidenkin maitohappobakteerien kasvua. Tämän tutkimuksen tarkoituksena oli seuloa bakteriosiineja tuottavia L. gelidum -kantoja, jotka estävät L. monocytogenesin kasvua kylmäsäilytetyissä elintarvikkeissa. Ensin estovaikutusta tutkittiin yksinkertaisella agardiffuusiokokeella. Kokeeseen valittiin neljä suomalaisista elintarvikkeista eristettyä L. gelidum -kantaa, joiden genomista oli aiemmin BAGEL3- tietokoneohjelmalla tunnistettu bakteriosiinien tuotantoon viittaava operoni. Agardiffuusiokokeet tehtiin L. gelidum -kantojen soluvapaalla pintaliuoksella. Estovaikutuksen varmistettiin johtuvan bakteriosiinin kaltaisesta yhdisteestä käsittelemällä pintaliuoksia peptidiyhdisteitä pilkkovalla proteinaasilla. Jatkokokeisiin valittiin tehokkaimmin L. monocytogenesta estävä kanta, jonka estovaikutusta tutkittiin yhteisviljelmässä, jossa bakteriosiinia tuottavan kannan ja L. monocytogenesin kasvua seurattiin kasvatusliemessä (20 °C). Lopuksi koe toistettiin kasvis-broilersoseessa jääkaappilämpötilassa (5 °C). Tulosten perusteella kolme L. gelidum -kantaa tuotti kasvatusliemeensä L. monocytogenesta ja useaa maitohappobakteerikantaa estävää bakteriosiinin kaltaista yhdistettä. Estovaikutus hävisi proteinaasin lisäämisen jälkeen, eikä johtunut pintaliuosten happamuudesta. Kantojen tuottamat bakteriosiinit estivät tehokkaasti L. monocytogenesin kasvua pidentäen sen viivevaihetta ja rajoittaen lopullista solutiheyttä kasvatusliemessä. Yhteisviljelmäkokeisiin valittu L. gelidum -kanta rajoitti L. monocytogenesin kasvua kasvatusliemessä (20 °C) ja jääkaappilämpötilassa säilytetyssä kasvis-broilersoseessa. Kyseisen L. gelidum -kannan läsnä ollessa L. monocytogenesin lopullinen solutiheys jäi kummassakin tapauksessa 100-krt pienemmäksi kuin yksin kasvatettuna. Tämä työ osoittaa, että kylmäsäilytettävissä elintarvikkeissa yleisesti esiintyvät L. gelidum -kannat voivat rajoittaa L. monocytogenesin kasvua kasvispohjaisessa elintarvikkeessa. Estovaikutteisiksi tunnistetut L. gelidum -kannat ja niiden tuottamat antilisteriset bakteriosiinit tarjoavat kiinnostavan jatkotutkimuskohteen pohdittaessa listerian torjuntakeinoja ruokaketjussa.
  • Holm, Jenny (Helsingfors universitet, 2013)
    Listeria monocytogenes on elintarvikevälitteinen, opportunistinen patogeeni, joka voi aiheuttaa ihmiselle listerioosin. Kannat sietävät hyvin jääkaappilämpötiloja ja sopeutuvat erilaisiin elinympäristöihin. Elintarvikelaitoksissa listeriat ovat usein pysyviä laitoskantoja, jotka kontaminoivat tuotteita usein pitkällä aikavälillä. Tämän maisterin tutkielman laboratorio-osuus tehtiin syyslukukauden 2012 aikana Eläinlääketieteellisen tiedekunnan elintarvikehygienian ja ympäristöterveyden osastolla, mikrobiologisen elintarviketurvallisuuden huippuyksikössä. Tarkoituksena oli tutkia DEAD-Box RNA helikaasien vaikutusta L. monocytogenes EGD-e –kannan flagellan ilmenemiseen ja kiinnittymiseen. Kirjallisuuden mukaan L. monocytogenes –kantojen kiinnittymiseen vaikuttavat suuresti mahdollisesti adhesiivisina toimivien flagellojen läsnäolo sekä solujen liikkuvuus. L. monocytogenes EGD-e–kannalla on neljä DEAD-Box RNA helikaasigeeniä (lmo0866, lmo1246, lmo1450 ja lmo1722), joista osa vaikuttaa kasvuun ja liikkuvuuteen. Kaksi geeneistä vaikuttaa lisäksi positiivisesti bakteerin selviämiseen kylmän, etanolin ja emäksisen tai hapettavan kasvuympäristön aiheuttamassa stressitilanteessa. L. monocytogenes EGD-e mutanttikannoista ?lmo0866 ja ?lmo1450 on todettu liikkumattomiksi ja ?lmo1722 osittain liikkuvaksi. Nämä mutanttikannat ovat lisäksi kylmänherkkiä DEAD-Box RNA helikaasimutanttikannat ja villityyppi kuvattiin TEM-tekniikalla ja kahden flagellageenin (flhA ja motA) ilmentymistä mitattiin qRT-PCR:lla. Lopuksi kantojen kiinnittymistä tutkittiin mikrotiitterilevymenetelmällä. Kaikki tutkimukset tehtiin kahdessa lämpötilassa (+25 ja +37°C:ssa). Elektronimikroskooppikuvat ja qRT-PCR –tulokset olivat toisiaan tukevia. Mutanttikanta ?lmo0866 ei tuottanut flagelloja lainkaan, kun taas kannalla ?lmo1450 flagellojen tuotto oli heikompaa villityyppiin verrattuna +25°C:ssa. Kannat ?lmo1246 ja ?lmo1722 vastasivat villityyppiä flagellan esiintymisen suhteen. Mutanttikannoista ?lmo0866 ilmensi +25°C:ssa molempia tutkittuja flagellageenejä yli 20-kertaa vähemmän kuin villityyppi. Myös geenin lmo1450 poistaminen johti flagellageenien ilmentymisen vähenemiseen. Mutanttikannan ?lmo1722 osalta ilmentyminen oli alentunut vain toisen tutkitun flagellageenin (motA) osalta. Geenin lmo1246 deleetio ei vaikuttanut flagellageenien ilmentymiseen. Kiinnittymisessä eroja ei havaittu helikaasimutanttien ja villityypin välillä kummassakaan tutkitussa lämpötilassa, joten yksittäiset DEAD-Box RNA helikaasigeenit eivät vaikuta L. monocytogenes EGD-e –kannan kiinnittymiseen. L. monocytogenes –bakteerin kiinnittymiseen polystyreenipinnalle vaikuttavat kirjallisuuden mukaan useat eri tekijät, kuten inkubointilämpötila, -aika, ravinteet ja kanta. Flagellan ja liikkuvuuden merkitys kiinnittymiseen ei ole mahdollisesti niin suuri kuin on aiemmin oletettu.
  • Koskensalo, Sirja (Helsingin yliopisto, 2018)
    Listeria monocytogenes is a gram-positive, non-sporeforming, rod-shaped bacterium, a human and animal pathogen and a food-borne pathogen/agent. Listeriosis, caused by L. monocytogenes, can be a serious disease and even cause death. L. monocytogenes is a major risk factor for the microbiological quality of foodstuffs, and a pathogen especially for those in risk groups. The aim of this study was to investigate sporadic and persistent strains of L. monocytogenes, isolated in a milk-production environment, and their growth in acidic and alkaline environments, as well as in environments containing the detergents Basix and Cidmax, and to investigate eventual differences between these strains. The features and occurrence of L. monocytogenes, its capability to resist stress and listeriosis are discussed in the literature part of this thesis. In this study it was found that bacteria were able to grow in the environments tested, in the concentrations used. No statistically significant differences between the strains were discovered. All investigated L. monocytogenes strains grew in pH 5.6, pH 9.0 and in liquids containing the detergents Cidmax and Basix. In acidic conditions, the majority of the strains grew similarly to reference strain EGD-e. Strain E3 started its exponential growth faster than the other strains. In acidic conditions strain D3 had the highest growth rate (30.06). The strains that grew the most weakly in acidic conditions were B3 and reference strain ATCC 19115. In alkaline conditions and in Cidmax-BHI liquid, the most of L. monocytogenes strains grow similarly to reference strain EGD-e. Strains E3 and D3 grew faster than other strains in alkaline conditions and Cidmax-BHI liquid and strains B3 and ATCC 19115 grew slower than the other strains. In Basix-BHI liquid, the strains of L. monocytogenes grew mainly similarly to each other, and these strains grew a little faster than reference strain EGD-e. Compared to the other strains, strain E3 grew the fastest and strains B3 and ATCC 19115 grew slower than the other strains. In this study, differences in stress tolerance between L. monocytogenes strains were found. In future, the results of this study may be utilized in genomic association research where geno- and phenotypes are compared to each other. This could produce new information on the development of stress tolerance of L. monocytogenes strains with different genomes and phenotypes.
  • Laaksonen, Sauli; Oksanen, Antti; Julmi, Jerome; Zweifel, Claudio; Fredriksson-Ahomaa, Maria; Stephan, Roger (2017)
    Background: Various food-producing animals were recognized in recent years as healthy carriers of bacterial pathogens causing human illness. In northern Fennoscandia, the husbandry of semi-domesticated reindeer (Rangifer tarandus tarandus) is a traditional livelihood and meat is the main product. This study determined the presence of selected foodborne pathogens, methicillin-resistant Staphylococcus aureus (MRSA), and extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in healthy semi-domesticated reindeer at slaughter in northern Finland and Norway. Results: All 470 reindeer fecal samples tested negative for Salmonella spp., whereas L. monocytogenes was detected in 3%, Yersinia spp. in 10%, and Shiga toxins genes (stx1 and/or stx2) in 33% of the samples. Listeria monocytogenes isolates belonged to the serotype 1/2a (14/15) and 4b, Yersinia spp. were identified mainly as Y. kristensenii (30/46) and Y. enterocolitica (8/46), and stx2 predominated among the Shiga toxin genes (stx2 alone or in combination with stx1 was found in 25% of the samples). With regard to the frequency and distribution of stx1/stx2, striking differences were evident among the 10 different areas of origin. Hence, reindeer could constitute a reservoir for Shiga toxin-producing E. coli (STEC), but strain isolation and characterization is required for verification purposes and to assess the potential human pathogenicity of strains. On the other hand, the favorable antibiotic resistance profiles (only 5% of 95 E. coli isolates were resistant to one or more of the tested antibiotics) and the absence of MRSA and ESBL-producing Enterobacteriaceae (when applying selective methods) suggest only a limited risk of transmission to humans. Conclusions: Healthy semi-domesticated reindeer in northern Finland and Norway can be carriers of certain bacterial foodborne pathogens. Strict compliance with good hygiene practices during any step of slaughter (in particular during dehiding and evisceration) is therefore of central importance to avoid carcass contamination and to prevent foodborne pathogens from entering the food chain.
  • Laaksonen, Sauli; Oksanen, Antti; Julmi, Jérôme; Zweifel, Claudio; Fredriksson-Ahomaa, Maria; Stephan, Roger (BioMed Central, 2017)
    Abstract Background Various food-producing animals were recognized in recent years as healthy carriers of bacterial pathogens causing human illness. In northern Fennoscandia, the husbandry of semi-domesticated reindeer (Rangifer tarandus tarandus) is a traditional livelihood and meat is the main product. This study determined the presence of selected foodborne pathogens, methicillin-resistant Staphylococcus aureus (MRSA), and extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae in healthy semi-domesticated reindeer at slaughter in northern Finland and Norway. Results All 470 reindeer fecal samples tested negative for Salmonella spp., whereas L. monocytogenes was detected in 3%, Yersinia spp. in 10%, and Shiga toxins genes (stx1 and/or stx2) in 33% of the samples. Listeria monocytogenes isolates belonged to the serotype 1/2a (14/15) and 4b, Yersinia spp. were identified mainly as Y. kristensenii (30/46) and Y. enterocolitica (8/46), and stx2 predominated among the Shiga toxin genes (stx2 alone or in combination with stx1 was found in 25% of the samples). With regard to the frequency and distribution of stx1/stx2, striking differences were evident among the 10 different areas of origin. Hence, reindeer could constitute a reservoir for Shiga toxin-producing E. coli (STEC), but strain isolation and characterization is required for verification purposes and to assess the potential human pathogenicity of strains. On the other hand, the favorable antibiotic resistance profiles (only 5% of 95 E. coli isolates were resistant to one or more of the tested antibiotics) and the absence of MRSA and ESBL-producing Enterobacteriaceae (when applying selective methods) suggest only a limited risk of transmission to humans. Conclusions Healthy semi-domesticated reindeer in northern Finland and Norway can be carriers of certain bacterial foodborne pathogens. Strict compliance with good hygiene practices during any step of slaughter (in particular during dehiding and evisceration) is therefore of central importance to avoid carcass contamination and to prevent foodborne pathogens from entering the food chain.
  • Seyed Hameed, Ahkam Saddam (Helsingin yliopisto, 2015)
    Increased consumption of raw milk has been observed among the urban population in Europe along with an increasing consumer interest in foods that are less processed and locally produced. Raw milk and raw milk products have been associated with listeriosis caused by Listeria monocytogenes. These new trends in consumer behavior increase the need for more effective surveillance methods for L. monocytogenes at farm level. In Finland, raw milk sales are allowed directly from the farms to the consumer. The renewed Finnish national legislation stipulates that screening of L. monocytogenes on farms that sell raw milk includes five replicates of 25-ml bulk tank milk (BTM) samples. Due to dilution to the high volume of BTM, it is unlikely to detect low levels of bacterial contamination from the milk samples. Testing milk samples is also expensive and logistically difficult for continuous monitoring of L. monocytogenes at the farms. An alternative approach could be the testing of milk filters. Theoretically, milk filters may be more sensitive than BTM as a sampling material since all of the milk in the bulk tank has to pass through the filter. The objective of this longitudinal study is to compare milk filters and BTM for the best sampling material suitable for routine monitoring of L. monocytogenes in the dairy farms selling raw milk. Five BTM samples and a milk filter sample from two farms were tested for L. monocytogenes each week, for a period of 10 months, using NMLK 136:2010, ISO 11290-1:1996 and ISO 11290-2:1998 methods. The isolation of L. monocytogenes and other Listeria species were identified by API biochemical assay and multiplex PCR. The results indicate that milk filters are ideal as a sampling material in detecting L. monocytogenes than BTM samples. However, the presence of L. monocytogenes in milk filters was not associated to the presence of L. monocytogenes in BTM. Thus, milk filters could be used as indicators of the farm’s contamination status. Along with these findings, seasonal variation in detection, and advantages of identification methods were also discussed in this thesis.
  • Pöntinen, Anna; Lindström, Miia; Skurnik, Mikael; Korkeala, Hannu (2017)
    To study the role of each two-component system (TCS) histidine kinase (HK) in stress tolerance of Listeria monocytogenes EGD-e, we monitored the growth of individual HIC deletion mutant strains under heat (42.5 degrees C), acid (pH 5.6), alkali (pH 9.4), osmotic (6% NaCl), ethanol (3.5 vol%), and oxidative (5 mM H2O2) stresses. The growth of Delta liaS (Delta lmo1021) strain was impaired under each stress, with the most notable decrease under heat and osmotic stresses. The Delta ivirS (Delta lmo1741) strain showed nearly completely restricted growth at high temperature and impaired growth in ethanol. The growth of Delta agrC (Delta lmo0050) strain was impaired under osmotic stress and slightly under oxidative stress. We successfully complemented the HIC mutations using a novel allelic exchange based approach. This approach avoided the copy-number problems associated with in trans complementation from a plasmid. The mutant phenotypes were restored to the wild-type level in the complemented strains. This study reveals novel knowledge on the HKs needed for growth of L monocytogenes EGD-e under abovementioned stress conditions, with LiaS playing multiple roles in stress tolerance of L monocytogenes EGD-e. (C) 2017 Elsevier Ltd. All rights reserved.
  • Kinnunen, Susanna; Karhapää, Pauli; Juutilainen, Auni; Finne, Patrik; Helanterä, Ilkka (2018)
    Background and objectives Infections are the most common noncardiovascular causes of death after kidney transplantation. We analyzed the current infection-related mortality among kidney transplant recipients in a nationwide cohort in Finland. Design, setting, participants, & measurements Altogether, 3249 adult recipients of a first kidney transplant from 1990 to 2012 were included. Infectious causes of death were analyzed, and the mortality rates for infections were compared between two eras (1990-1999 and 2000-2012). Risk factors for infectious deaths were analyzed with Cox regression and competing risk analyses. Results Altogether, 953 patients (29%) died during the follow-up, with 204 infection-related deaths. Mortality rate (per 1000 patient-years) due to infections was lower in the more recent cohort (4.6; 95% confidence interval, 3.5 to 6.1) compared with the older cohort (9.1; 95% confidence interval, 7.6 to 10.7); the incidence rate ratio of infectious mortality was 0.51 (95% confidence interval, 0.30 to 0.68). The main causes of infectious deaths were common bacterial infections: septicemia in 38% and pulmonary infections in 45%. Viral and fungal infections caused only 2% and 3% of infectious deaths, respectively (such as individual patients with Cytomegalovirus pneumonia, Herpes simplex virus meningoencephalitis, Varicella zoster virus encephalitis, and Pneumocystis jirovecii infection). Similarly, opportunistic bacterial infections rarely caused death; only one deathwas caused by Listeria monocytogenes, and two were caused by Mycobacterium tuberculosis. Only 23 (11%) of infection-related deaths occurred during the first post-transplant year. Older recipient age, higher plasma creatinine concentration at the end of the first post-transplant year, diabetes as a cause of ESKD, longer pretransplant dialysis duration, acute rejection, low albumin level, and earlier era of transplantation were associated with increased risk of infectious death in multivariable analysis. Conclusions The risk of death due to infectious causes after kidney transplantation in Finland dropped by one half since the 1990s. Common bacterial infections remained the most frequent cause of infection-related mortality, whereas opportunistic viral, fungal, or unconventional bacterial infections rarely caused deaths after kidney transplantation.
  • Keto-Timonen, Riikka; Markkula, Annukka; Halkilahti, Jani; Huttunen, Reetta; Räsänen, Sirpa; Salmenlinna , Saara; Heikkilä, Anne; Puisto, Mia; Närhinen, Maria; Hakkinen, Marjaana; Korkeala, Hannu; Jalava, Katri (2019)
    In November 2016, an elderly patient was diagnosed with Listeria monocytogenes bacteremia in Finland. Grocery store loyalty card records and microbiological investigation of foods found in the home fridge and freezer of the patient revealed commercial, modified-atmosphere packaged meatballs as the source of the infection. Investigation of the meatball production plant revealed that the floor drain samples were contaminated with the same L. monocytogenes strain as those isolated from the patient and meatballs. Ready-to-eat meatballs were likely contaminated after heat treatment from the production environment before packaging. Long-term cold storage, modified-atmosphere conditions, and the absence of competing bacteria presumably enhanced the growth of L. monocytogenes. We recommend that collection of shopping details and home fridge and freezer sampling should be part of surveillance of all cases of L. monocytogenes infections to complement information obtained from in-depth interviews.
  • Wambui, Joseph; Eshwar, Athmanya K.; Aalto-Araneda, Mariella; Pöntinen, Anna; Stevens, Marc J. A.; Njage, Patrick M. K.; Tasara, Taurai (2020)
    Nisin is a commonly used bacteriocin for controlling spoilage and pathogenic bacteria in food products. Strains possessing high natural nisin resistance that reduce or increase the potency of this bacteriocin against Listeria monocytogenes have been described. Our study sought to gather more insights into nisin resistance mechanisms in natural L. monocytogenes populations by examining a collection of 356 field strains that were isolated from different foods, food production environments, animals and human infections. A growth curve analysis-based approach was used to access nisin inhibition levels and assign the L. monocytogenes strains into three nisin response phenotypic categories; resistant (66%), intermediate (26%), and sensitive (8%). Using this categorization isolation source, serotype, genetic lineage, clonal complex (CC) and strain-dependent natural variation in nisin phenotypic resistance among L. monocytogenes field strains was revealed. Whole genome sequence analysis and comparison of high nisin resistant and sensitive strains led to the identification of new naturally occurring mutations in nisin response genes associated with increased nisin resistance and sensitivity in this bacterium. Increased nisin resistance was detected in strains harboring RsbUG77S and PBPB3V240F amino acid substitution mutations, which also showed increased detergent stress resistance as well as increased virulence in a zebra fish infection model. On the other hand, increased natural nisin sensitivity was detected among strains with mutations in sigB, vir, and dlt operons that also showed increased lysozyme sensitivity and lower virulence. Overall, our study identified naturally selected mutations involving pbpB3 (lm0441) as well as sigB, vir, and dlt operon genes that are associated with intrinsic nisin resistance in L. monocytogenes field strains recovered from various food and human associated sources. Finally, we show that combining growth parameter-based phenotypic analysis and genome sequencing is an effective approach that can be useful for the identification of novel nisin response associated genetic variants among L. monocytogenes field strains.
  • Mattila, Mirjami; Somervuo, Panu; Korkeala, Hannu; Stephan, Roger; Tasara, Taurai (2020)
    Numerous gene expression and stress adaptation responses in L. monocytogenes are regulated through alternative sigma factors sigma(B) and sigma(L). Stress response phenotypes and transcriptomes were compared between L. monocytogenes EGD-e and its Delta sigB and Delta sigBL mutants. Targeted growth phenotypic analysis revealed that the Delta sigB and Delta sigBL mutants are impaired during growth under cold and organic-acid stress conditions. Phenotypic microarrays revealed increased sensitivity in both mutants to various antimicrobial compounds. Genes de-regulated in these two mutants were identified by genome-wide transcriptome analysis during exponential growth in BHI. The Delta sigB and Delta sigBL strains repressed 198 and 254 genes, respectively, compared to the parent EGD-e strain at 3 degrees C, whereas 86 and 139 genes, respectively, were repressed in these mutants during growth at 37 degrees C. Genes repressed in these mutants are involved in various cellular functions including transcription regulation, energy metabolism and nutrient transport functions, and viral-associated processes. Exposure to cold stress induced a significant increase in sigma(B) and sigma(L) co-dependent genes of L. monocytogenes EGD-e since most (62%) of the down-regulated genes uncovered at 3 degrees C were detected in the Delta sigBL double-deletion mutant but not in Delta sigB or Delta sigL single-deletion mutants. Overall, the current study provides an expanded insight into sigma(B) and sigma(L) phenotypic roles and functional interactions in L. monocytogenes. Besides previously known sigma(B)- and sigma(L)-dependent genes, the transcriptomes defined in Delta sigB and Delta sigBL mutants reveal several new genes that are positively regulated by sigma(B) alone, as well as those co-regulated through sigma(B)- and sigma(L)-dependent mechanisms during L. monocytogenes growth under optimal and cold-stress temperature conditions.