Foodborne human isolates of Salmonella and Shiga toxin -producing Escherichia coli of domestic origin in Finland

Salmonella is one of the most commonly reported foodborne pathogens and enterohaemorrhagic Escherichia coli (EHEC) is one of the most dangerous. They both spread by zoonotic and person-to-person transmission routes. Most Salmonella infections are characterized by mild-to-moderate self-limited diarrhea but also serious disease resulting in death has been reported.Bloody diarrhea is a common symptom of human EHEC infection and the infection may lead to severe post-infection disease such as hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP) and even death. The purpose of this thesis was to investigate the diversity of Salmonella and EHEC strains isolated from domestically acquired infections using several pheno- and genotyping methods for surveillance and outbreak investigation purposes as well as evaluate certain epidemiological typing methods in Finland. All Salmonella and EHEC isolates of domestic origin during 2007-2014 in Finland were studied. Serotyping, phage typing, antimicrobial susceptibility testing, phenotype microarray, pulsed-field gel electrophoresis (PFGE) and multilocus variable-number tandem repeat analysis (MLVA) were applied as epidemiological typing tools for Salmonella isolates. EHEC isolates were analyzed using serotyping, phage typing, antimicrobial susceptibility testing, virulence gene detection (stx1, stx2, eae, hlyA and saa) and PFGE. During the study period, the number of domestically acquired Salmonella infections has decreased about one fifth compared to the previous decade whereas the number of domestic EHEC infections have increased about one third.Th e incidence of Salmonella infections was highest in 2012 (7.5/100,000 population) and lowest in 2014 (5.4/100,000 population). The incidence of EHEC infections was highest in 2013 (0.33/100,000 population) and lowest in 2008 (0.07/100,000 population). 15% of all Salmonella strains and 70% of all EHEC strains were considered domestically acquired. A total of 131 different Salmonella serovars were detected. The most common serovars were Typhimurium (32%), Enteritidis (15%) and group B (6%). Among Typhimurium strains, phage types DT1 (37%), RDNC (18%) and DT104 (9%) were the most common ones. The most frequently detected phage types among the domestically acquired S. Enteritidis infections were PT8 (17%), PT1B (14%) and PT4 (13%). Th e majority of domestic Typhimurium and Enteritidis (60%) strains were susceptible to tested antimicrobials. During the study period, a total of 188 infections caused by EHEC were detected. Most of them were caused by serotype O157:H7 (60%). The majority of O157 strains (63%) were unable to ferment sorbitol. PT8 was the most common phage type among the sorbitol-negative and PT88 among sorbitol positive O157 strains. Among non-O157, 22 distinct O:H serotypes were detected. The most common ones were O26:H11, O103:H2 and O145:H-. The majority of domestic EHEC strains (81%) were susceptible to all tested antimicrobials: 96% of O157:H7/H- and 60% of non-O157 strains. All O157 strains carried stx2 (40% in combination with stx1), eae and hlyA genes. In contrast, 55% of non-O157 stains had stx1 gene and 76% carried eae and hlyA genes. The MLVA method was found to be a powerful epidemiological tool for S. Typhimurium with discriminatory power similar to PFGE. In addition, MLVA was faster, cheaper and the results were easier to compare between laboratories. The domestic S. Typhimurium strains were divided into 170 distinct MLVA types (diversity index 0.891). In MLVA, the three most common profiles (3-16-NA-NA-0311, 3-15-NA-NA-0311 and 3-17-NA-NA-0311) counted for 47% of the strains showing that the lack of locus STTR6 and locus STTR10p was characteristic for domestic S. Typhimurium. However, XbaI-PFGE remains a useful genotyping method for investigations of other Salmonella serovars and EHEC strains. The interpretation of XbaI-PFGE profiles can be challenging as demonstrated by a Finnish nationwide outbreak caused by S. Newport and S. Reading -contaminated iceberg lettuce. The S. Reading strains had four diff erent XbaI-PFGE profiles. Based on epidemiological information, all these different variants of the outbreak causing strains were considered as outbreak-related. The sources of the most EHEC outbreaks remained undetermined. In one out four EHEC O157 outbreaks, unpasteurized milk was found as the source of the infections. Although 40% of the domestic EHEC strains were non-O157, only strains of serogroup O157 caused outbreaks in Finland. However, non-O157 strains caused several family clusters and were linked with HUS. In 2009, a sorbitol-fermenting EHEC O78:H-:stx1c:hlyA was detected in blood and fecal samples of a neonate. This EHEC serotype had not been seen in Finland prior to this family-related outbreak and bacteremia caused by EHEC is exceptionally rare. Taken together, Salmonella and EHEC infections are a major public health concern. This thesis provides new information about the characteristics of Salmonella and EHEC strains isolated from domestically acquired infections in Finland and evidence that effective surveillance is needed for early detection and prevention of the spread of Salmonella and EHEC infections. In particular, typing methods used should be internationally harmonized and the results made comparable.Salmonella on yksi yleisimmistä elintarvikevälitteisistä bakteeripatogeeneistä, enterohemorraaginen Escherichia coli (EHEC) yksi vaarallisimmista. Molemmat bakteerit ovat eläimen ja ihmisen välityksellä leviäviä ja tarttuvat myös henkilöstä toiseen. Useimmat salmonellojen aiheuttamat infektiot ovat lieviä, itsestään parantuvia suolistoinfektioita, mutta myös vakavia, kuolemaan johtavia tartuntoja todetaan. Veriripuli on yleinen oire erityisesti pikkulasten EHEC-infektiossa. Infektio saattaa johtaa myös vakaviin jälkitauteihin kuten hemolyyttisureemiseen oireyhtymään (HUS), tromboottiseen trombosytopeeniseen purppuraan (TTP) ja jopa kuolemaan. Tässä työssä tutkittiin salmonella- ja EHEC-kantojen ominaisuuksia useilla ilmiasuun (fenotyyppi) ja perimään (genotyyppi) perustuvilla menetelmillä. Lisäksi selvitettiin tiettyjen genotyypitysmenetelmien soveltuvuutta epidemiologiseen seurantaan ja tartuntalähteiden jäljittämiseen. Erityisesti oltiin kiinnostuneita niistä salmonella- ja EHEC-kannoista, joiden tartunta oli saatu Suomessa. Tutkimus käsitti kaikki kotimaiset vuosina 2007-2014 eristetyt salmonella- ja EHEC-kannat. Salmonella-kantojen epidemiologiseen tyypittämiseen käytettiin neljää fenotyypitysmenetelmää, serotyypitystä, faagityypitystä, mikrobilääkeherkkyysmääritystä ja mikroarray testiä sekä kahta genotyypitysmenetelmää, pulssikenttägeelielektroforeesiä (PFGE) ja multilocus variable-number tandem repeat analysis (MLVA) testiä. EHEC-kantojen tyypitykseen käytettiin O:H-serotyypitystä, faagityypitystä, mikrobilääkeherkkyysmääritystä, virulenssigeenien määritystä (stx1, stx2, eae, hlyA and saa) ja PFGE -menetelmää. Tutkimusaikavälillä kotimaassa saatujen salmonellainfektioiden määrä laski noin viidenneksen verrattuna edelliseen vuosikymmeneen kun taas EHEC tartuntojen lukumäärä nousi noin kolmanneksen. Kotimaisten salmonellatartuntojen ilmaantuvuus oli korkeimmillaan vuonna 2012 (7.5/100 000 asukasta) ja alimmillaan vuonna 2014 (5.4/100 000 asukasta). Kotimaisten EHECinfektioiden ilmaantuvuus oli korkein vuonna 2013 (0.33/100 000 asukasta) ja matalin vuonna 2008 (0.07/100 000 asukasta). Kaikista salmonellatartunnoista 15 % ja EHEC-tartunnoista 70 % oli kotimaista alkuperää. Kotimaisia salmonellatartuntoja aiheutti yhteensä 131 eri serotyyppiä. Niistä yleisimmät olivat Typhimurium (32 %), Enteritidis (15 %) ja ryhmä B (6 %). Kotimaisten S. Typhimurium -kantojen yleisimmät faagityypit olivat FT1 (37 %), NST (18 %) ja FT104 (9 %). Kotimaista alkuperää olevien S. Enteritidis -kantojen yleisimmät faagityypit olivat FT8 (17 %), FT1B (14 %) ja FT4 (13 %). Suurin osa kotimaisista Typhimurium- (60 %) ja Enteritidiskannoista (60 %) olivat herkkiä testatuille mikrobilääkkeille. Tutkimusaikavälillä todettiin 188 EHEC-tartuntaa, joista valtaosan aiheutti serotyyppi O157:H7 (60 %). Suurin osa EHEC O157 -kannoista (63 %) oli sorbitoli-negatiivisia. Faagityyppi FT8 oli yleisin sorbitoli-negatiivisten ja faagityyppi FT88 sorbitoli-positiivisten O157 kantojen keskuudessa. Non-O157 kannat tyypittyivät yhteensä 22 eri O:H serotyyppiin. Yleisimmät non-O157 serotyypit olivat O26:H11, O103:H2 ja O145:H-. Suurin osa kotimaista alkuperää olevista EHEC-kannoista (81 %) oli herkkiä testatuille mikrobilääkkeille: 96 % kaikista O157:H7 ja 60 % non-O157 kannoista. Kaikki O157 -kannat kantoivat geenejä stx2 (40 %:lla kannoista oli myös stx1 geeni), eae ja hlyA kun taas valtaosa non-O157 kannoista kantoi geeniä stx1 (55 %) ja 76 %:lla kannoista oli geenit eae ja hlyA. MLVA -menetelmä osoittautui toimivaksi epidemiologiseksi tyypitysmenetelmäksi, ja sen erottelukyky oli yhtä hyvä kuin aiemmin käytetyn PFGE -menetelmän. Lisäksi MLVA oli nopeampi ja halvempi kuin PGFE menetelmä ja tulokset olivat helpommin kansainvälisesti vertailtavissa. Kotimaista alkuperää olevat S. Typhimurium -kannat jakaantuivat 170 eri MLVAtyyppiin (diversiteetti-indeksi 0,891). Noin puolet (47 %) kannoista kuului kolmeen yleisimpään MLVA -tyyppiin (3-16-NA-NA-0311, 3-15-NA-NA-0311 ja 3-17-NA-NA-0311). Lokuksien STTR6 ja STTR10p puuttuminen oli tyypillistä kotoperäisille S. Typhimurium -kannoille. XbaI-PFGE oli kuitenkin hyödyllinen menetelmä muiden salmonellan serotyyppien ja EHEC-kantojen genotyypittämiseen. XbaI-PFGE tulosten tulkitseminen saattaa olla haastavaa, minkä myös maanlaajuinen S. Newport/S. Reading -epidemia vuonna 2009 havainnollisti. Kyseisessä epidemiassa S. Reading kannat jakaantuivat neljään eri PFGE -tyyppiin, joiden kuitenkin katsottiin epidemiologiseen tietoon perustuen liittyvän samaan epidemiaan. Useimpien EHEC-epidemioiden lähteet jäivät tuntemattomiksi. Tutkimusaikavälillä havaittiin neljä EHEC -bakteerin aiheuttamaa epidemiaa ja useita perheensisäisiä rypäitä, joista yhdessä lähteeksi todettiin pastöroimaton maito. Vaikka 40 % kaikista kotoperäisistä EHEC -infektioista oli non-O157 serotyyppien aiheuttamia, vain serotyyppi O157 aiheutti epidemioita Suomessa. Non-O157 kannat aiheuttivat kuitenkin perheensisäisiä tartuntoja ja yhdistettiin vakaviin jälkitauteihin (HUS). Vuonna 2009 sorbitoli-positiivinen EHEC O78:H-:stx1c:hlyA eristettiin vastasyntyneen verestä ja ulosteesta. Kyseistä serotyyppiä ei ollut havaittu Suomessa aikaisemmin ja EHEC -bakteerin aiheuttamat verenmyrkytykset ovat kansainvälisestikin erittäin harvinaisia. Salmonella ja EHEC aiheuttavat merkittäviä kansanterveysongelmia. Tässä työssä saatiin merkittävää uutta tietoa Suomessa todettujen kotimaista alkuperää olevien salmonella- ja EHEC-infektioiden aiheuttajabakteereiden ominaisuuksista. Tehokas salmonella- ja EHEC-seuranta auttaa havaitsemaan alkavat epidemiat ja estämään niiden leviämisen. Erityisesti uusia tutkimusmenetelmiä kehitettäessä tulisi huomioida, että tyypitysmenetelmä on kansainvälisesti harmonisoitu ja tulokset vertailukelpoisia

Osteomyelitis in a slaughter turkey flock caused by Yersinia pseudotuberculosis sequence type ST42

A Yersinia pseudotuberculosis outbreak was diagnosed in a male turkey flock in Finland. Y. pseudotuberculosis is a quite rare zoonotic bacterium, which typically causes enteritis in humans and sudden death in animals. In this study, osteomyelitis was diagnosed in small, lame, 11-to 12-wk-old male turkeys. Lameness and slower growth among the turkeys was observed on the farm. During pathological examination, multiple lesions were found in the metaphyseal and physeal areas of the femurs, tibiotarsi, and tarsometatarsi, with multifocal to coalescing mixed heterophilic/granulomatous necrotizing osteomyelitis. Y. pseudotuberculosis was isolated from the femoral and tibiotarsal bones or from the joints of six lame turkeys sent for necropsy. The isolation required homogenizing of lesion tissue in phosphate-mannitol-peptone broth, which was cultured directly -and, if needed, after cold enrichment - on selective cefsulodin-irgasan-novobiocin agar. Whole-genome sequencing was used for identification and typing. All isolates belonged to bio/serotype 1/O:1a and sequence type ST42 (Achtman scheme), which is commonly reported in both human and animal Y. pseudotuberculosis infections in Europe. The isolates from all six turkeys showed only one to two allele differences in the core genome comparison, indicating a common source of infection. All asymptomatic turkeys were slaughtered at the age of 17 weeks. Whole and partial carcass condemnation rates at the slaughterhouse were high, but no macroscopic changes in the skeletal system were found, showing that food chain information is essential. This study confirms earlier findings that Y. pseudotuberculosis can cause osteomyelitis in fattening turkeys, leading to lameness. Food chain information is essential for slaughterhouse operations, to protect the workers and emphasize good working hygiene during slaughter.Peer reviewe

Characterization of Salmonella Typhimurium isolates from domestically acquired infections in Finland by phage typing, antimicrobial susceptibility testing, PFGE and MLVA

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.Peer reviewe

Genotyping and phylogenetic placement of Bacillus anthracis isolates from Finland, a country with rare anthrax cases

Anthrax, the zoonotic disease caused by the gram-positive bacterium Bacillus anthracis, is nowadays rare in northern parts of Europe including Finland and Scandinavia. Only two minor outbreaks of anthrax in 1988 and in 2004 and one sporadic infection in 2008 have been detected in animals in Finland since the 1970’s. Here, we report on two Finnish B. anthracis strains that were isolated from spleen and liver of a diseased calf related to the outbreak in 1988 (strain HKI4363/88) and from a local scrotum and testicle infection of a bull in 2008 (strain BA2968). These infections occurred in two rural Finnish regions, i.e., Ostrobothnia in western Finland and Päijänne Tavastia in southern Finland, respectively

Prevalence and characterization of Salmonella enterica from the feces of cattle, poultry, swine and hedgehogs in Burkina Faso and their comparison to human Salmonella isolates

Peer reviewe

Salmonellan leviäminen suomalaisille sika- ja nautatiloille

Hankkeen tavoitteina oli tunnistaa nauta- ja sikatilojen salmonellatartuntojen lähteitä sekä toimintatapoja, joilla saneeraus ja tartuntojen ehkäisy tiloilla parhaiten onnistuu. Lukuisista tartuntalähteistä tärkeimmiksi arvioimme yhdyskuntalinnut ja muut haittaeläimet sekä turkistuotanto mahdollisena alkulähteenä haittaeläinten välittämille tartunnoille. Viime vuosina nauta- ja sikatiloille tehtyjen saneerausten aineistosta arvioimme seikkoja, jotka johtivat saneerauksen onnistumiseen tai pitkittymiseen tai tartunnan uusimiseen. Saneeraus pitkittyi, jos tartunta oli alkutilanteessa levinnyt laajalle, tartuntalähdettä ei saatu heti selville, salmonellapositiivisia eläimiä ei poistettu ajoissa, puhdistus-, pesu- ja desinfiointitoimet eivät olleet riittäviä tai käytettävissä oleva työpanos ei riittänyt. Saneerauksen onnistumista edistivät hyvä työnjohto, systemaattisuus, saneeraussuunnitelma sekä suunnitelman ja muiden ohjeiden noudattaminen. Tarkastelimme tilan salmonellatartunnan ja sen saneerauksen vaatimuksia myös työturvallisuuden suhteen. Salmonellatartunnan ehkäisy edellyttää tilatason tautisuojausta, jonka on oltava jokapäiväistä ja kohdistuttava tilan koko toimintaan ottaen huomioon haittaeläimistä aiheutuva tartuntavaara. Hankkeessa syntyi tietopääomaa salmonellan epidemiologiseen seurantaan, saneerausneuvontaan ja suosituksia tukemaan salmonellatorjuntaa.Tämä julkaisu on toteutettu osana valtioneuvoston selvitys- ja tutkimussuunnitelman toimeenpanoa. (tietokayttoon.fi) Julkaisun sisällöstä vastaavat tiedon tuottajat, eikä tekstisisältö välttämättä edusta valtioneuvoston näkemystä

Whole genome sequencing of multidrug-resistant Salmonella enterica serovar Typhimurium isolated from humans and poultry in Burkina Faso

Abstract Background Multidrug-resistant Salmonella is an important cause of morbidity and mortality in developing countries. The aim of this study was to characterize and compare multidrug-resistant Salmonella enterica serovar Typhimurium isolates from patients and poultry feces. Methods Salmonella strains were isolated from poultry and patients using standard bacteriological methods described in previous studies. The strains were serotype according to Kaufmann-White scheme and tested for antibiotic susceptibility to 12 different antimicrobial agents using the disk diffusion method. The whole genome of the S. Typhimurium isolates was analyzed using Illumina technology and compared with 20 isolates of S. Typhimurium for which the ST has been deposited in a global MLST database.The ResFinder Web server was used to find the antibiotic resistance genes from whole genome sequencing (WGS) data. For comparative genomics, publicly available complete and draft genomes of different S. Typhimurium laboratory-adapted strains were downloaded from GenBank. Results All the tested Salmonella serotype Typhimurium were multiresistant to five commonly used antibiotics (ampicillin, chloramphenicol, streptomycin, sulfonamide, and trimethoprim). The multilocus sequence type ST313 was detected from all the strains. Our sequences were very similar to S. Typhimurium ST313 strain D23580 isolated from a patient with invasive non-typhoid Salmonella (NTS) infection in Malawi, also located in sub-Saharan Africa. The use of ResFinder web server on the whole genome of the strains showed a resistance to aminoglycoside associated with carriage of the following resistances genes: strA, strB, and aadA1; resistance to β-lactams associated with carriage of a bla TEM-1B genes; resistance to phenicol associated with carriage of catA1 gene; resistance to sulfonamide associated with carriage of sul1 and sul2 genes; resistance to tetracycline associated with carriage of tet B gene; and resistance to trimethoprim associated to dfrA1 gene for all the isolates. Conclusion The poultry and human isolates were genetically similar showing a potential food safety risk for consumers. Our finding of multidrug-resistant S. Typhimurium ST313 in poultry feces calls for further studies to clarify the potential reservoirs of this emerging pathogen

Genotyping and phylogenetic placement of Bacillus anthracis isolates from Finland, a country with rare anthrax cases

Abstract Background Anthrax, the zoonotic disease caused by the gram-positive bacterium Bacillus anthracis, is nowadays rare in northern parts of Europe including Finland and Scandinavia. Only two minor outbreaks of anthrax in 1988 and in 2004 and one sporadic infection in 2008 have been detected in animals in Finland since the 1970’s. Here, we report on two Finnish B. anthracis strains that were isolated from spleen and liver of a diseased calf related to the outbreak in 1988 (strain HKI4363/88) and from a local scrotum and testicle infection of a bull in 2008 (strain BA2968). These infections occurred in two rural Finnish regions, i.e., Ostrobothnia in western Finland and Päijänne Tavastia in southern Finland, respectively. Results The isolates were genetically characterized by PCR-based methods such as multilocus variable number of tandem repeat analysis (MLVA) and whole genome-sequence analysis (WGS). Phylogenetic comparison of the two strains HKI4363/88 and BA2968 by chromosomal single nucleotide polymorphism (SNP) analysis grouped these organisms within their relatives of the minor canonical A-branch canSNP-group A.Br.003/004 (A.Br.V770) or canonical B-branch B.Br.001/002, respectively. Strain HKI4363/88 clustered relatively closely with other members of the A.Br.003/004 lineage from Europe, South Africa, and South America. In contrast, strain BA2968 clearly constituted a new sublineage within B.Br.001/002 with its closest relative being HYO01 from South Korea. Conclusions Our results suggest that Finland harbors both unique (autochthonous) and more widely distributed, common clades of B. anthracis. We suspect that members of the common clades such as strains HKI4363/88 have been introduced only recently by anthropogenic activities involving importation of contaminated animal products. On the other hand, autochthonous strains such as isolate BA2968 probably have an older history of their introduction into Finland as evidenced by a high number of single nucleotide variant sites in their genomes