Genomic epidemiology of Shiga toxin-producing Escherichia coli and Campylobacter jejuni on dairy farms and in raw milk

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http://urn.fi/URN:ISBN:978-951-51-6027-0
Title: Genomic epidemiology of Shiga toxin-producing Escherichia coli and Campylobacter jejuni on dairy farms and in raw milk
Author: Jaakkonen, Anniina
Contributor: University of Helsinki, Faculty of Veterinary Sciences, Department of Food Hygiene and Environmental Health
Doctoral Programme in Food Chain and Health
Finnish Food Authority, Laboratory and Research Division, Microbiology Unit
Publisher: Helsingin yliopisto
Date: 2020-06-02
Belongs to series: Dissertationes Schola Doctoralis Scientiae Circumiectalis, Alimentariae, Biologicae - URN:ISSN:2342-5431
URI: http://urn.fi/URN:ISBN:978-951-51-6027-0
http://hdl.handle.net/10138/315061
Thesis level: Doctoral dissertation (article-based)
Abstract: Cattle are commonly asymptomatic carriers of Shiga toxin-producing Escherichia coli (STEC) and Campylobacter jejuni, which cause gastroenteritis in humans. Especially STEC infections may lead to severe or fatal consequences. Both STEC and C. jejuni are intermittently shed in cattle feces and can contaminate bulk tank milk via fecal contamination during milking. These bacteria are effectively eliminated from milk by pasteurization, but the consumption of unpasteurized milk, or raw milk, poses a risk of infection. In recent years, the consumption of raw milk has become more popular, with public demand to relax legislation that restricts sales of raw milk. However, on-farm epidemiology of these pathogens have warranted further investigation to support the development of on-farm risk management practices and pathogen monitoring of dairy farms that sell raw milk to consumers. These studies investigated a milkborne outbreak caused by STEC (Study I) and obtained longitudinal data on the contamination of bulk tank milk by STEC and C. jejuni, and explored on-farm contamination routes of these pathogens (Study II). Furthermore, the studies revealed strain characteristics of C. jejuni that may affect survival and persistence of this pathogen in milk (Study III). The occurrence of STEC and C. jejuni, or C. jejuni alone, was determined in bulk tank milk, in-line milk filters of the milking machine, cattle feces, and the farm environment on four dairy farms (STEC and C. jejuni in Studies I and II) or one dairy farm (C. jejuni in Study III). STEC and C. jejuni isolates from the dairy farms were further subjected to phenotypic characterization and whole-genome sequencing, followed by comparative genomic analyses to explore gene contents and phylogenetic relationships between the isolates. Furthermore, questionnaire data were collected to trace back the outbreak source (Study I) and to determine on-farm risk factors associated with milk contamination using a logistic regression model (Study II). Ultimately, the results contributed to the revision of the Finnish legislation that restrict the sales of raw milk in 2017 (Study II). Study I elucidated the reservoirs and transmission routes of atypical, sorbitol-fermenting (SF) STEC O157, which have largely been unknown. The study presents microbiological and epidemiologic evidence that an outbreak of SF STEC O157 with 11 cases originated from a recreational farm housing dairy cattle and was transmitted via the consumption of raw milk. Thus, these results strongly support bovine origin of SF STEC O157. In longitudinal monitoring (Study II), one clone of STEC O157:H7, which represented a bovine-associated lineage, was simultaneously isolated on each of the three dairy farms. STEC O157:H7 persisted in two herds for up to 12 months, and a similar but distinct clone was reintroduced in one herd 2.5 years after the previous detection. These results support evidence that few STEC O157:H7 clones persist on-farm simultaneously. Unlike STEC, both persistent and numerous sporadic C. jejuni strains appeared simultaneously on dairy farms (Studies II and III). Persistence for 11 months or longer was associated with a few C. jejuni genotypes, especially the host generalist sequence type (ST) ST-883. C. jejuni of ST-883 outperformed other STs in environmental fitness, representing the only ST that could be isolated from bulk tank milk and milk filters and the dominant ST found from environmental samples. Therefore, ST-883 imposes a higher contamination pressure on milk than other STs among the farm isolates and represents a candidate for on-farm risk-based monitoring. In the longitudinal monitoring, STEC was rarely isolated from bulk tank milk and milk filters and only simultaneously with fecal isolation. Higher detection rates were obtained from milk filters than milk by both culture methods and real-time PCR. Therefore, milk filters are more reliable sampling targets for monitoring of STEC than milk. Isolation of C. jejuni from milk and milk filters was associated with C. jejuni clone rather than sample material, but the isolation rates of C. jejuni appeared generally poor. To enhance the isolation rates for monitoring purposes, the sampling regime also warrants further consideration. Reduced milk contamination by STEC was associated with on-farm practices: pasturing and culling of dairy cows and rigorous cleansing in the barn. Higher outdoor temperatures were associated with increased milk contamination. In Study III, C. jejuni of ST-883 persistently contaminated bulk tank milk of a dairy farm for seven months or longer after having caused a milkborne outbreak. Although ST-883 survived in refrigerated raw milk longer than other STs from the same farm, the persistence of ST-883 in bulk tank milk was likely affected by other phenotypic traits such as biofilm formation. Outbreak strain of ST-883 reversibly adapted to survival in bulk tank milk, showing biofilm formation in an on/off manner among replicate cultures and cellular heterogeneity by phase variation in genes related to capsule and oxidative stress response. Furthermore, the outbreak strain harbored a pTet-like genomic element, which may have contributed to higher biofilm quantities. This study identified candidate phenotypic and genotypic mechanisms affecting survival and persistence of C. jejuni in milk. Taken together, STEC and C. jejuni can persist on dairy farms for months or longer and contaminate bulk tank milk despite stringent on-farm hygiene measures. Although these measures cannot totally prevent milk contamination, they likely reduce the contamination pressure on milk. Therefore, cost-effective hygiene measures should be applied on all farms that sell raw drinking milk to consumers. Detection of pathogens from milk may be challenging, and milk may also be contaminated by highly virulent STEC and C. jejuni strains that show atypical phenotype, increasing their environmental endurance or hampering their detection. Therefore, only heat treatment of raw milk before consumption can adequately assure its food safety.Shigatoksiinia tuottavat Escherichia coli (STEC) ja Campylobacter jejuni -bakteerit aiheuttavat ihmisille suolistotulehduksia. Erityisesti STEC-infektio voi olla vakava. Vakaville seurauksille alttiita riskiryhmiä ovat lapset, vanhukset ja immuunipuutteiset. STEC ja C. jejuni -bakteereita esiintyy oireettomien nautojen suolistossa, mistä ne voivat levitä ulosteen välityksellä navettaympäristöön ja lypsyn yhteydessä tankkimaitoon. Maidon pastörointi tuhoaa nämä ja muut taudinaiheuttajat, mikä takaa maitotuotteiden turvallisuuden. Kuluttajaturvallisuus poikkeaa kuitenkin raakamaidon, ternimaidon ja niistä valmistettujen tuotteiden (etupäässä juustojen) osalta. Siksi raakamaidon ja ternimaidon tuotantoa ja luovutusta rajoitetaan Suomessa lainsäädännöllä. Raakamaidon kuluttajasuosio on kuitenkin kasvanut viime vuosina luoden painetta lainsäädännön uudistamiselle. STECin ja C. jejunin esiintymisestä ja leviämisreiteistä navettaympäristössä tarvitaan lisätietoa, jotta voidaan kehittää tilatason riskinhallintaa ja taudinaiheuttajien seurantaa raakamaitoa luovuttavilla tiloilla. Tässä väitöstutkimuksessa osoitettiin, että epätyypillinen STEC O157 voi tarttua nautakarjasta ihmiseen raakamaidon välityksellä aiheuttaen vakavan taudin (osatutkimus I). Tämän STEC-bakteerin tartuntalähteitä ei ole aiemmin tunnettu, ja sitä on todettu muualla maailmassa naudoista vain harvoin. Väitöstutkimuksessa saatiin pitkäaikaisella ja tiheällä seurannalla uutta tietoa STECin ja C. jejunin esiintymisestä tankkimaidossa ja lypsylaitteiston maitosuodattimissa tiloilla, missä karja erittää näitä bakteereita ulosteisiinsa (osatutkimus II). Lisäksi selvitettiin tankkimaidon bakteerisaastumiseen vaikuttavia tekijöitä. Väitöstutkimuksessa havaittiin bakteerikantakohtaisia ominaisuuksia, mitkä saattoivat edesauttaa tankkimaidon pitkäkestoista saastumista yhdellä C. jejuni ST-883 -kannalla (osatutkimus III). Väitöstutkimus osoitti, että STEC ja C. jejuni -kannat voivat säilyä tiloilla kuukausia tai pidempään ja saastuttaa tankkimaidon tehostetuista hygieniakäytännöistä huolimatta. Vaikka maidon saastumista ei voitu täysin estää hygieniakäytännöillä, tilatoimenpiteiden havaittiin vähentävän maidon saastumista. Kustannustehokkaita hygieniakäytäntöjä suositellaan siksi kaikille raakamaitoa luovuttaville tiloille. Maito voi saastua STEC- ja C. jejuni -kannoilla, jotka ovat erittäin taudinaiheuttamiskykyisiä, selviävät navettaympäristössä muita kantoja paremmin tai ovat hankalia todeta laboratorioanalyyseillä. Siksi ainoastaan kuumennuskäsittely takaa raakamaidon kuluttajaturvallisuuden. Tutkimustuloksia hyödynnettiin raakamaitolainsäädännön uudistuksessa 2017.
Subject: elintarvikehygienia
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