Browsing by Subject "elintarvikehygienia"

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  • Keto-Timonen, Riikka (Helsingin yliopisto, 2008)
    In epidemiological studies, techniques that effectively discriminate between individual bacterial strains are essential. Recent developments in molecular techniques necessitate an ongoing need to tailor new genotyping methods for optimal characterization of different bacterial species and to evaluate their performance and suitability for research purposes. In this thesis amplified fragment length polymorphism (AFLP) analysis was tailored for optimal characterization of Listeria monocytogenes and Clostridium botulinum. The suitability of the developed AFLP protocol to type L. monocytogenes, C. botulinum and Clostridium perfringens at strain level was evaluated. AFLP proved to be a highly reproducible, easy-to-use, relatively fast and highly discriminative approach. When AFLP was applied to L. monocytogenes strains, its discriminatory power was shown to equal that of PFGE, which is considered the current gold standard for molecular fingerprinting of L. monocytogenes. These features make AFLP analysis a useful alternative to other genotyping methods in, for example, outbreak investigations and contamination route studies. Since phenotypic identification of Clostridium isolates is laborious, the suitability of AFLP for genomic species identification was assessed. The AFLP technique was applied to 129 strains representing 24 different Clostridium species. AFLP differentiated all species tested, except for Clostridium ramosum and Clostridium limosum. AFLP also differentiated between six different Listeria species. If AFLP profiles of well-defined strains are collected in identification libraries, the database can be a valuable additional tool for identification of Clostridium and Listeria species. Due to high throughput of samples, AFLP proved to be especially suitable for screening large numbers of isolates. AFLP was also used to trace contamination routes of L. monocytogenes in a chilled food processing plant producing ready-to-eat and ready-to-reheat meals during an 8-year period. Cleaning routines, product type and degree of compartmentalization seemed to have an influence on the contamination status in compartments that produced cooked meals. In addition, raw materials were shown to cause contamination of uncooked meals. Thus, special attention should be paid to quality control of raw ingredients when uncooked ready-to-eat meals are produced. This work also demonstrated that structural adjustments of a production line may facilitate the eradication of L. monocytogenes from the food processing environment. AFLP and PFGE analysis of sporadic L. monocytogenes strains and strains that cause persistent plant contamination revealed that persistent strains differ from sporadic strains. However, no specific evolutionary lineage of persistent strains was observed.
  • Olkkola, Satu (Helsingin yliopisto, 2016)
    Campylobacteriosis is the most common cause of human bacterial gastroenteritis in the developed world. The most often isolated causative agent from diseased humans is C. jejuni, but also C. coli and C. upsaliensis, common colonizers of pigs and dogs, respectively, are known to cause disease. Campylobacteriosis is usually self-limiting but antimicrobial treatment is warranted in severe cases, with macrolides and fluoroquinolones being the first and second options, respectively. Intravenous aminoglycosides are indicated in Campylobacter bacteraemia. However, high rates of fluoroquinolone-resistant Campylobacter spp. have emerged in many parts of the world. Also, in several studies, high proportions of streptomycin-resistant C. coli or C. upsaliensis, have been found. Yet, the mechanisms of STR resistance have been only partially characterized in C. jejuni and C. coli and completely ignored in C. upsaliensis. The primary aim of this thesis was to investigate the molecular mechanisms of STR resistance in porcine C. coli and canine C. upsaliensis isolates. We were able to associate high level of STR resistance in porcine C. coli to mutations in the rpsL gene. In C. upsaliensis, a mutation in rpsL was also noted in all the low- and high-level STR-resistant isolates. All highly STR-resistant C. upsaliensis isolates had, in addition to the rpsL mutation, significant truncation of rsmG, encoding a conserved methyltransferase responsible for methylation of the ribosomal STR binding site. Even though STR resistance conferring mutations in rpsL and rsmG have been well documented in other bacterial species, they were first time described in Campylobacter spp. in the present study. Further, using genomics and insertional mutagenesis, a novel STR resistance-conferring gene was identified in the intermediately STR-resistant C. coli isolates. This gene is homologous, albeit at a low level, to other previously described aminoglycoside 6-adenylyltransferase encoding genes, and does not appear to originate from Gram-positive bacterial species. Based on our findings, we hypothesize that this gene could have evolved from a proto-resistance element in Campylobacter spp. Altogether these results provide a significant advance in understanding the mechanisms of STR resistance in Campylobacter spp. and will aid in predicting the phenotypic resistance from genome data. Fluoroquinolone resistance-associated mutations in the DNA gyrase-encoding gene gyrA were characterized in porcine C. coli treated with danofloxacin as well as among canine C. upsaliensis. The commonly described C257T mutation was found in both species. In C. coli this caused the amino acid change T86I in DNA gyrase and high levels of ciprofloxacin resistance, while in C. upsaliensis the predicted amino acid change was T86M causing only minor increase in CIP MIC but a high level of nalidixic acid resistance. Therefore, danofloxacin does not seem to induce novel mutations in C. coli in vivo but the same mutation appears not to be sufficient to cause a high level of fluoroquinolone resistance in C. upsaliensis.
  • Hellström, Sanna (Helsingin yliopisto, 2011)
    Listeria monocytogenes is the causative agent of the severe foodborne infection listeriosis. The number of listeriosis cases in recent years has increased in many European countries, including Finland. Contamination of the pathogen needs to be minimized and growth to high numbers in foods prevented in order to reduce the incidence of human cases. The aim of this study was to evaluate contamination routes of L. monocytogenes in the food chain and to investigate methods for control of the pathogen in food processing. L. monocytogenes was commonly found in wild birds, the pig production chain and in pork production plants. It was found most frequently in birds feeding at landfill site, organic farms, tonsil samples, and sites associated with brining. L. monococytogenes in birds, farms, food processing plant or foods did not form distinct genetic groups, but populations overlapped. The majority of genotypes recovered from birds were also detected in foods, food processing environments and other animal species and birds may disseminate L. monocytogenes into food chain. Similar genotypes were found in different pigs on the same farm, as well as in pigs on farms and later in the slaughterhouse. L. monocytogenes contamination spreads at farm level and may be a contamination source into slaughterhouses and further into meat. Incoming raw pork in the processing plant was frequently contaminated with L. monocytogenes and genotypes in raw meat were also found in processing environment and in RTE products. Thus, raw material seems to be a considerable source of contamination into processing facilities. In the pork processing plant, the prevalence of L. monocytogenes increased in the brining area, showing that the brining was an important contamination site. Recovery of the inoculated L. monocytogenes strains showed that there were strain-specific differences in the ability to survive in lettuce and dry sausage. The ability of some L. monocytogenes strains to survive well in food production raises a challenge for industry, because these strains can be especially difficult to remove from the products and raises a need to use an appropriate hurdle concept to control most resistant strains. Control of L. monocytogenes can be implemented throughout the food chain. Farm-specific factors affected the prevalence of L. monocytogenes and good farm-level practices can therefore be utilized to reduce the prevalence of this pathogen on the farm and possibly further in the food chain. Well separated areas in a pork production plant had low prevalences of L. monocytogenes, thus showing that compartmentalization controls the pathogen in the processing line. The food processing plant, especially the brining area, should be subjected to disassembling, extensive cleaning and disinfection to eliminate persistent contamination by L. monocytogenes, and replacing brining with dry-salting should be considered. All of the evaluated washing solutions decreased the populations of L. monocytogenes on precut lettuce, but did not eliminate the pathogen. Thus, the safety of fresh-cut produce cannot rely on washing with disinfectants, and high-quality raw material and good manufacturing practices remain important. L. monocytogenes was detected in higher levels in sausages without the protective culture than in sausages with this protective strain, although numbers of L. monocytogenes by the end of the ripening decreased to the level of < 100 MPN/g in all sausages. Protective starter cultures provide an appealing hurdle in dry sausage processing and assist in the control of L. monocytogenes.
  • Tolvanen, Riina (Helsingin yliopisto, 2016)
    In this thesis contamination routes of L. monocytogenes were examined in a food establishment, the survival of L. monocytogenes strains was studied in dry-fermented sausages prepared using two different starter cultures, the acid and heat tolerance of L. monocytogenes strains were studied, and efficacy of ultrasonic cleaning was tested on conveyor belts contaminated with L. monocytogenes. Contamination routes of L. monocytogenes were examined during an 8-year period in a chilled food-processing establishment that produced ready-to-eat meals using amplified fragment length polymorphism (AFLP) analysis. Compartment I of the establishment, producing cooked meals, was heavily contaminated with three persistent AFLP types, and compartment II, producing uncooked chilled foods, was contaminated with persistent and non-persistent AFLP types. L. monocytogenes was isolated only once from compartment III. The persistent contamination appears to be influenced by the cleaning routines, product types and lack of compartmentalisation in facilities producing cooked meals. The reconstruction of the production line in compartment II resulted in the elimination of two persistent AFLP types. The survival of five L. monocytogenes strains was studied in dry-fermented sausages prepared using two different starter cultures with or without a bacteriocin-producing Lactobacillus plantarum DDEN 2205 strain. L. monocytogenes was detected throughout the ripening process in sausages containing no bacteriocin-producing strain. The use of both starters with bacteriocin-producing culture resulted in L. monocytogenes-negative sausages after ripening. Two of the L. monocytogenes strains survived in sausages with bacteriocin-producing cultures better than the other strains. Bacteriocin-producing strains provide an appealing hurdle in dry sausage processing, but differences in survival of L. monocytogenes strains require the use of other hurdles as well. The acid and heat tolerance of persistent and non-persistent L. monocytogenes strains were studied. L. monocytogenes strains exhibited large variation in both acid and heat tolerance. The persistent strains exhibited higher tolerance to acidic conditions than the non-persistent strains, but significant differences in heat tolerance between persistent and non-persistent strains were not detected. Due to the great differences in acid and heat tolerances between L. monocytogenes strains, preventive measures should be designed to be effective against the most tolerant strains. Ultrasonic cleaning was tested on three conveyor belt materials contaminated with L. monocytogenes strains. The ultrasonic cleaning was efficient for all materials, but the reduction of L. monocytogenes was significantly greater in stainless steel than in plastic materials. The ultrasonic cleaning was further studied by building a pilot-scale conveyor with an ultrasonic cleaning bath. The detachment of L. monocytogenes from the stainless steel conveyor belt caused by the ultrasonic treatment was significantly greater than without ultrasound. In both studies, lengthening of the treatment time did not significantly increase the detachment of L. monocytogenes. However, an increase in temperature improved the effect of the ultrasonic treatment, and 10 s at 50 °C reduced L. monocytogenes counts by more than 5 log units. These results indicate that the ultrasonic cleaning of conveyor belts is effective even with short treatment times.
  • Pöntinen, Anna (Helsingin yliopisto, 2019)
    Listeria monocytogenes is a remarkable bacterium, as it is able to shift from a capable environmental saprophyte into a severe intracellular pathogen. As a strictly foodborne pathogen, L. monocytogenes poses a notable risk, particularly to those consumers among the risk groups for whom invasive listeriosis is potentially fatal. Furthermore, modern consumption habits and increasingly favoured ready-to-eat foods, often consumed without proper heating, increase the risk of acquiring the foodborne disease. The aim of this study was to investigate the genetic mechanisms conferring wide-ranging stress tolerance in L. monocytogenes. Two-component systems, comprising a sensor histidine kinase and a cognate response regulator, aid bacteria in sensing and adapting to changes in both surrounding environmental as well as intracellular conditions. The histidine kinases, in particular, have lacked comprehensive studies on their roles in the stress tolerance of L. monocytogenes. Thus, histidine kinases were studied by expressional analyses under cold conditions and by mutationally disrupting each histidine kinase-encoding gene in a parental model strain, L. monocytogenes EGD-e. The modified strains were individually challenged at high (42.5 degrees C) and low (3.0 degrees C) temperatures, high (9.4) and low (5.6) pH levels, and high salt (6% NaCl), ethanol (3.5 vol%) and hydrogen peroxide (5 mM) concentrations. Expressional studies and growth experiments on genetically modified strains proved lisK and yycG to respectively play central roles in the acclimation and immediate growth of L. monocytogenes at low temperatures. The most substantial increase in gene expression under cold conditions was that of the chemotaxis gene cheY with 236-fold upregulation at 3 degrees C. The disrupted ΔliaS strain displayed impaired growth in response to all the other stresses, particularly at a high temperature and under osmotic stress. These studies demonstrated the prominent importance of the histidine kinase-encoding genes yycG and lisK to cold tolerance and liaS, with roles in the growth of L. monocytogenes under multiple stresses. To shed light on the accessory genetic mechanisms that cause large strain variation in L. monocytogenes in withstanding heat treatments, heat resistance-conferring traits were further investigated by means of whole-genome sequencing. Comparing the complete genomes of heat-resistant L. monocytogenes AT3E and -sensitive AL4E strains revealed the heat-resistant strain to harbour a novel 58-kb plasmid, pLM58, which was absent in the sensitive strain. Furthermore, curing of the plasmid in AT3E produced a marked decrease in heat resistance from virtually no reduction to a 1.1 cfu/ml log10 reduction at 55.0 degrees C. In pLM58, a 2,155-bp open reading frame annotated as an ATP-dependent ClpL protease-encoding gene was identified. Conjugation of the coding sequence and the putative promoter of the clpL gene into a natively heat-sensitive L. monocytogenes 10403S strain, in turn, enhanced the survival of the strain from a 1.2 cfu/ml log10 reduction to a 0.4 cfu/ml log10 reduction in heat challenge at 55.0 degrees C. In this study, we presented the first evidence of plasmid-mediated heat resistance in L. monocytogenes and identified the protease ClpL to be a novel plasmid-borne heat-resistance mediator. The emerging resistance of L. monocytogenes to benzalkonium chloride, a quaternary ammonium compound widely used as a detergent in food-processing facilities, is a significant concern for food safety and public health. The resistance of 392 L. monocytogenes isolates from Finland (n = 197) and Switzerland (n = 195) to benzalkonium chloride was assessed. A minimal inhibitory concentration of 20 µg/ml was defined. Altogether, 11.5% of the strains proved to be resistant to benzalkonium chloride. Serotype 1/2c harboured the highest prevalence, 32.4% (11/34), of benzalkonium chloride-resistant strains, while in total, most of the resistant strains belonged to serotype 1/2a. Altogether, 68.9% of the resistant strains harboured at least one of the efflux pump system-encoding genes, bcrABC, emrE or qacH, known to confer benzalkonium chloride resistance in L. monocytogenes. We found resistant strains with partially or completely efflux pump-dependent benzalkonium chloride resistance, with the exception of the known resistance-mediating efflux pumps, suggesting the existence of other resistance-contributing efflux pump systems. The lacking of known efflux pump system-encoding genes in addition to efflux pump-independent benzalkonium chloride resistance, in turn, indicates the contribution of completely novel benzalkonium chloride resistance mechanisms. The aim of these studies was to shed light on the genes contributing to the versatile stress tolerance abilities and strain variation of the severe foodborne pathogen, L. monocytogenes. Knowledge of such traits may aid in developing targeted strategies and measures to identify and control the contamination and risks caused, in particular, by stress-tolerant L. monocytogenes strains.
  • Tuominen, Pirkko (Helsingin yliopisto, 2009)
    The drive for risk-based food safety management, systems and control has spread world-wide in recent decades. Since the term is still internationally undefined, its use and implementation vary, producing different realizations. In this Ph.D. thesis, microbiological risk assessment (MRA) was investigated as a basis for risk-based food safety management, which was defined as ‘food safety management based on risk assessment in order to achieve an appropriate level of protection (ALOP)’. Governments are responsible for commissioning MRAs and also for setting food safety targets up to a certain point, but the practical management measures that need to be in place in order to achieve the targets are to be addressed by the operators. On the plant level, food safety is usually managed through regulation, quality assurance systems and a hazard analysis and critical control point (HACCP) programme with its prerequisites. In Finland, food safety management on the food plant level is implemented through an HACCP-like regulated system termed an own-checking (OC) programme. A quantitative microbiological risk assessment (QMRA) was conducted on salmonella in the beef production chain according to the official standards of the Codex Alimentarius Commission (Codex Alimentarius), and utilized in determining the food safety metrics for beef production. The Finnish Salmonella Control Programme (FSCP) and the main official interventions due to it were examined in the light of risk-based food safety management. The targets set for beef processing plants by the government were converted into quantitative limits, and the results of salmonella monitoring included in the FSCP were examined by the QMRA. The goal of the FSCP was declared in 1994 to ‘maintain the present salmonella situation’, which was considered to refer to the salmonella incidence in humans at that time, and also the de facto ALOP. The requirement for a maximum salmonella prevalence of 1% at defined stages of the beef production chain was embodied in the FSCP. This statement was considered to convey performance objectives (PO) for the aforementioned stages. According to the QMRA, the de facto ALOP was achieved in the referred year 1999, and even the true prevalence levels in the FSCP were estimated to be clearly under the set PO limits with 95% credibility. However, the PO limits were set too high for the de facto ALOP to be maintained in practice. If the salmonella prevalence reached the PO limit of 1% or values near it, the public health risk would increase and overrun the de facto ALOP. The QMRA produced in this work has for the first time provided the possibility to quantitatively asses the relationships between targets set in the FSCP and their impact on public health. At present, imports of beef and beef-derived foods may impose on Finnish consumers a significantly greater exposure than domestic products. If their salmonella prevalence or their share of the foods consumed in Finland increase, the number of human cases could rapidly rise. The models for the QMRA were mainly Bayesian hierarchical models using Markov chain Monte Carlo (MCMC) techniques, which was found to be a flexible and appropriate method for this type of complex modelling. The resulting distributions were also regarded as an advantage compared to the results from models developed with the deterministic approach, because the presentation of results included the extent of the uncertainty, and also in this manner better illustrated the actual operational environment. Based on an inquiry, the personnel in food processing plants had a positive attitude towards food safety management systems, but the knowledge, training and involvement of those employees directly operating on the site with these systems were discovered to be deficient. Therefore, a generic semi-quantitative hygiene risk assessment model, Hygram®, was developed for small and medium-sized food enterprises to offer assistance in understanding, training, and, first of all, detecting the critical steps of the processes, and thereby to contribute to the development of their own-checking systems towards risk-based food safety management. Hygram® was not considered a risk-based tool as such, but whenever the critical limits of the process have been defined as equal to a risk assessment, Hygram® can be used as a risk-based management tool. It can also serve as a tool for systematic hazard analysis and CCP detection when establishing a food safety management system. To conclude, the development of risk-based food safety management is a process in which risk assessment is an essential tool. Scientific, technical, psychological and resource-bound barriers need to be overcome in order to put risk-based management systems into practice. This study showed that QMRA can be valuable in national risk management decision making, although few QMRAs are currently available. Appropriate tools for practical risk management decision making on the industrial level, such as Hygram®, need to be further developed.
  • Laukkanen, Riikka (Helsingin yliopisto, 2010)
    Enteropathogenic Yersinia, that is pathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis, are zoonotic pathogens causing yersiniosis, the third most frequently reported zoonosis in the EU. Enteropathogenic Yersinia are frequently isolated from the tonsils and intestinal contents of pigs. Similar Y. enterocolitica genotypes have been identified both in pig and human strains and human yersiniosis has been statistically associated with the consumption of pork products in case-control studies, indicating pigs and pork products as an important source of human Y. enterocolitica infections. The link between pathogenic Y. pseudotuberculosis and pork is less clear; however, Y. pseudotuberculosis has also been isolated from carcasses and pork, indicating a possible route from pigs to humans. This work aimed at clarifing the transmission of enteropathogenic Yersinia from farm to slaughterhouse, determining factors affecting the prevalence of enteropathogenic Yersinia on pig farms, and test bagging as an intervention at the slaughterhouse. In addition, methods for the isolation of enteropathogenic Yersinia were evaluated. Isolation of enteropathogenic Yersinia from samples of animal origin is difficult and time-consuming. However, in many cases such as in outbreak investigations, isolates are needed for further typing. Of the isolation methods used, cold enrichment was efficient at isolating enteropathogenic Yersinia, whereas the sensitivity of other methods, such as direct isolation and selective irgasan-ticarcillin-potassium chlorate enrichment, for the isolation of enteropathogenic Yersinia was low. However, none of the isolation methods tested detected all the enteropathogenic Yersinia-positive samples and new isolation methods need to be developed. The transmission of enteropathogenic Yersinia from pigs to carcasses and pluck sets was investigated by collecting samples from individual ear-tagged pigs on the farm and at the slaughterhouse and by analyzing the isolated strains using pulsed-field gel electrophoresis (PFGE). Since the same PFGE types can be isolated from pigs and their subsequent pluck sets and carcasses, the main contamination source of pluck sets and carcasses at the slaughterhouse appears to be pigs that carry enteropathogenic Yersinia from farms to the slaughterhouse. However, since non related genotypes could also be isolated from carcasses and pluck sets, the slaughterhouse environment and tools can also contaminate carcasses and pluck sets. The high prevalence of enteropathogenic Yersinia in pigs results in high contamination rates of pluck sets and carcasses. Therefore, interventions at the farm level can decrease the transmission of Yersinia from pigs to pluck sets and carcasses. Farm factors such as production capacity and type may affect the prevalence of enteropathogenic Yersinia on farms. Since the prevalence of pathogenic Y. enterocolitica and Y. pseudotuberculosis varies among farms, within-farm factors can affect how enteropathogenic Yersinia spreads in pigs on farms. In statistical studies, factors affecting Y. pseudotuberculosis included organic production, contacts with pest animals, and the outside environment, whereas the high prevalence of pathogenic Y. enterocolitica was associated with factors such as high production capacity and conventional production. The epidemiology of pathogenic Y. enterocolitica and Y. pseudotuberculosis appears to be different on pig farms and this difference needs to be addressed if interventions on pig farms are considered. However, further information on the factors affecting the prevalence of enteropathogenic Yersinia on pig farms is needed before interventions at the farm level can be used. The effect of bagging of the rectum was studied by sampling tonsils, intestinal content, and carcasses with and without bagging of the rectum and constructing a Bayesian hidden variable model. According to the model, bagging of the rectum reduced significantly the contamination of carcasses at the slaughterhouse. However, since after bagging the prevalence of pathogenic Y. enterocolitica in different parts of the carcass was relatively high, 4 14%, other interventions are also needed. Most of the positive carcass samples were head and chest swabs, indicating that tonsils may be the contamination source.
  • Hakkinen, Marjaana (Helsingin yliopisto, 2010)
    The reported incidence of human campylobacteriosis in Finland is higher than in most other European countries. A high annual percentage of sporadic infections is of foreign origin, although a notable proportion of summer infections is domestically acquired. While chickens appear to be a major source of campylobacters for humans in most countries, the prevalence of campylobacters is very low in chicken slaughter batches in Finland. Data on other potential animal reservoirs of human pathogenic campylobacters in Finland are scarce. Consequently, this study aimed to investigate the status of Finnish cattle as a potential source of thermophilic Campylobacter spp. and antibiotic-resistant Campylobacter jejuni for human sporadic campylobacter infections of domestic origin. A survey of the prevalence of thermophilic Campylobacter spp. in Finnish cattle at twelve Finnish slaughterhouses from January to December 2003 yielded total campylobacter prevalences of 31.1% in faecal samples and 3.5% in carcass samples. Campylobacter jejuni was the most common species present in bovine samples. The prevalence of campylobacters was higher among beef cattle than among dairy cattle. Two predominant serotypes of faecal C. jejuni covered 52% of the samples. Genotyping with pulsed-field gel electrophoresis (PFGE) restriction yielded a high diversity of C. jejuni subtypes in cattle. Determining MICs of six antimicrobials among C. jejuni isolates yielded 9% of isolates resistant to at least one of the antimicrobials examined. No multiresistant isolates were found among the bovine C. jejuni strains. In the one-year study of the occurrence of Campylobacter spp. among three Finnish dairy cattle herds diverse shedding patterns occurred among both cattle herds and individual animals. The level of excretion was usually low. The same few subtypes of C. jejuni were able to persist in a herd over the sampling period. The faecal colonisation of water troughs can maintain the colonisation during indoor housing, whereas at pasture, preventing access to natural waters can limit colonisation. Comparison of C. jejuni isolates from humans, chickens and cattle included the design of primers for four new genetic markers, and the PCR examination of domestic human isolates from southern Finland in 1996, 2002 and 2003, chicken isolates from 2003, 2006 and 2007, and bovine isolates from 2003. The results revealed that bovine isolates differed significantly from human and chicken isolates. The PFGE genotyping of C. jejuni isolates included a geographically representative collection of isolates from domestic sporadic human infections, chicken slaughter batches, and cattle during the seasonal peak of campylobacteriosis in the summer of 2003. The study determined that 55.4% of human isolates were indistinguishable from those of chickens and cattle. Temporal association between isolates from humans and chickens was possible in 31.4% of human infections. Approximately 19% of the human infections may have been associated with cattle. However, isolates from bovine carcasses and human cases represented different PFGE subtypes. In conclusion, this study suggests that Finnish cattle is a constant reservoir of C. jejuni, the most important Campylobacter spp. in human enteric infections. Although the concentration of these organisms in bovine faeces appeared to be low, excretion can be persistent. The genetic diversity and presence or absence of marker genes support previous suggestions of host-adapted C. jejuni strains, and may indicate variations in virulence between strains from different hosts. In addition to chickens, Finnish cattle appeared to be an important reservoir and possible source of C. jejuni in domestic sporadic human infections. However, sources of campylobacters may differ between rural and urban areas in Finland, and in general, the transmission of C. jejuni of bovine origin probably occurs via other routes than food.
  • Dahlsten, Elias (Helsingin yliopisto, 2013)
    Clostridium botulinum presents a significant hazard to the food processing industry. However, the cellular mechanisms and factors that contribute to their regulation utilized by this feared foodborne pathogen in response and adaptation to food processing and storage-induced stress are poorly characterized. Another major aspect of C. botulinum presenting serious implications on food safety is its capability to produce heat-resistant endospores. Nevertheless, the sporulation cascade of C. botulinum has not been characterized. This study sought to investigate the effects of temperature downshift on the global gene expression pattern of Group I C. botulinum type strain ATCC 3502, and further characterize the roles of regulatory mechanisms identified as cold tolerance-related. Furthermore, the role of a major regulatory element, the alternative sigma factor SigK, in the sporulation cascade of C. botulinum was determined. Additionally, its putative function in stress tolerance was investigated. Transcriptomic analysis of the foodborne pathogen C. botulinum ATCC 3502 upon temperature downshift revealed the induction of several mechanisms previously identified as cold-related in other bacteria, thus suggesting that also C. botulinum utilizes these mechanisms in cold tolerance. Mechanisms with hitherto uncharacterized functions in cold tolerance were also found. The results suggested that secondary oxidative stress was present as a component of cold stress. Additionally, two previously uncharacterized putative DNA-binding regulatory proteins CBO0477 and CBO0558A were shown to play a role in cold tolerance of C. botulinum ATCC 3502. The two-component system (TCS) CBO0366/CBO0365 was shown to be important in the cold tolerance of C. botulinum ATCC 3502. Expression of this TCS was induced upon temperature downshift, but not under optimal temperature and growth conditions. Disruption of either of the TCS components resulted in deteriorated cold tolerance, whereas over-expression of cbo0366 in a wild-type strain resulted in an increase of growth rate at low temperature. Inactivation of the TCS response regulator-encoding cbo0365 markedly altered the transcriptome of C. botulinum ATCC 3502. Totals of 150 and 141 chromosomal coding sequences (CDS) were significantly differently expressed in the cbo0365 mutant at either 37 °C or 15 °C, respectively. There was an overlap of 141 common CDSs between the two temperatures. The genes differentially expressed included ones related to acetone-butanol-ethanol (ABE) fermantion, arsenic resistance, phosphate uptake and flagellar rotation. The involvement of CBO0365-regulated metabolic pathways in cold tolerance was demonstrated by the deteriorated cold tolerance of mutants of the respective pathways. Cold-sensitive phenotypes were observed for mutants of the acetone-butanol-ethanol fermentation pathway components bcd, crt, bdh and ctfA, the arsenic detoxifying machinery components arsC and arsR, and the phosphate uptake mechanism component phoT. Electrophoretic mobility shift assays confirmed transcriptional activation or repression as a means for CBO0365 in regulating itself, and the crt, ars, and pho operons. A dual role for the alternative sigma factor SigK in sporulation and in stress tolerance of C. botulinum ATCC 3502 was demonstrated. Disruption of SigK halted sporulation at an early stage but the phenotype was restored by in trans complementation of the mutation. The expression of sigK was induced upon exposure to low temperature and high salinity, but not upon a downshift in pH. A deteriorated tolerance to low temperature and to high salinity was observed for the sigK mutant strains.
  • Jaakkonen, Anniina (Helsingin yliopisto, 2020)
    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.
  • Honkanen, Elina (Helsingin yliopisto, 2019)
    Hepatiitti E -virus (HEV) on yksijuosteinen RNA-virus. Virus kuuluu Hepeviridae heimoon ja Orthohepevirus A -lajiin. Lajin seitsemästä genotyypistä neljä kykenee infektoimaan ihmisiä. HEV-1 ja HEV-2 ovat ihmisspesifisiä, kun taas HEV-3 ja HEV-4 zoonoottisia. Zoonoottisen HEV-3:n määrä on lisääntynyt Euroopassa. Pääreservuaarina toimivat siat, ja ihmiset saavat tartunnan yleensä syömällä raakaa tai huonosti kypsennettyä sianlihaa tai -maksaa. Suomalaisilla tuotantosioilla tiedetään esiintyvän hepatiitti E -infektioita yleisesti. Vasta-aineita on löydetty yli 80 %:lla teurastusikäisistä sioista. Sioilla tauti on oireeton, ja useimmiten siat saavat tartunnan 2–4 kuukauden iässä. Perusterveillä ihmisillä infektio on yleensä oireeton tai lieväoireinen. Jos immuniteetti on heikentynyt, voi infektio johtaa krooniseen maksatulehdukseen ja vakavaan maksan vajaatoimintaan. Viruksen esiintyvyyttä on selvitetty teurastusikäisillä sioilla, koska viruksella kontaminoitunut elintarvike toimii tartunnan lähteenä ihmisille. Euroopassa hepatiitti E -viruksen esiintyvyys sian maksoissa vaihtelee 3 %:n ja 75 %:n välillä. Tutkimuksen tavoitteena oli selvittää hepatiitti E -viruksen esiintyvyys suomalaisilla teurasikäisillä sioilla osoittamalla viruksen nukleiinihappo maksakudoksesta reaaliaikaisella käänteiskopiointi (RT) -PCR menetelmällä. RT PCR monistaa spesifisesti viruksen RNA:sta käänteiskopioitua kohdesekvenssiä. Oletuksena oli, että viruksen esiintyvyys on Suomessa alhaisempi muihin Euroopan maihin verrattuna. Tutkimuksessa analysoitiin 47 teurastamolta saatua sianmaksaa. Maksat pyrittiin keräämään eri lihasikaloista tulleilta sioilta. Analysoinnissa kontrollina toimi sisäinen mengovirus. Maksakudos (100mg) homogenisoitiin maksasolujen hajottamiseksi ja viruksen vapauttamiseksi. Homogenaatille suoritettiin RNA eristys, jossa viruksen proteiinikuori hajoaa, RNA vapautuu ja sitä puhdistetaan. Lisäpuhdistus tehtiin inhibitoristen aineiden poistolla. Näytteestä osoitettiin HEV:n ja kontrolliviruksen perimä spesifisiä alukkeita ja koetinta käyttäen. Kahdessa maksassa esiintyi hepatiitti E -viruksen RNA:ta (esiintyvyys 4,3 %). Tulosten perusteella Suomessa teurasikäisillä sioilla esiintyy hepatiitti E-infektioita ja esiintyvyys on eurooppalaista keskivertoa pienempi. Virus leviää yksilöstä toiseen muun muassa ulostekontaminaation kautta, joten sikojen kanssa työskentelevät sikatilalliset, eläinlääkärit ja teurastamotyöntekijät kuuluvat tartunnan riskiryhmään. Tartunnan välttämiseksi käsihygienia nousee avainasemaan. Tulokset viittaavat siihen, että hepatiitti E - viruksella kontaminoituneet sianmaksaa tai -lihaa sisältävät elintarvikkeet ovat harvinaisia, mutta tartunnan mahdollisuus on todellinen. Kuluttajien kannalta hyvä keittiöhygienia ja riittävä lihan kypsennys ovat tärkeitä tekijöitä tartunnan ehkäisemisessä. Henkilöiden, joiden immuniteetti on heikentynyt, tulisi välttää elintarvikkeita, joissa sianlihaa ei ole käsitelty riittävällä kuumennuksella. Tuoreen tutkimuksen mukaan vähintään yhden minuutin kuumennusjakso yli 80 °C:ssa inaktivoi viruksen, joten kyseisen lämpötilan saavuttaminen kaikkialla lihassa on suositeltavaa. Jatkotutkimuksia tarvitaan hepatiitti E -viruksen esiintyvyyden selvittämiseksi sianlihassa.
  • Mitronen, Aino; Mitronen, Aino (Helsingin yliopisto, 2021)
    Hepatiitti E -virus (HEV) aiheuttaa ihmisille maksatulehdusta eli hepatiittia. HEV kuuluu Orthohepevirus A-lajiin, joka on osa Orthohepevirus-sukua. HEV:tä esiintyy ympäri maailmaa, ollen yleisin virusperäisen hepatiitin aiheuttaja. HEV tarttuu pääasiassa ulosteen saastuttaman ravinnon tai veden välityksellä. HEV voi tarttua myös äidiltä sikiölle, veren- tai elinsiirtojen yhteydessä sekä elintarvikkeiden välityksellä. HEV-tartunta aiheuttaa yleensä vain lieviä yleisoireita, mutta myös vakavat tai kuolemaan johtavat komplikaatiot ovat mahdollisia. Tartunta on vaarallinen etenkin immuunipuutteisille ja kroonisesti maksasairaille. HEV:stä esiintyy tiettävästi kahdeksaa eri genotyyppiä, joista ihmisiä yleisimmin infektoivat neljä ensimmäistä. Genotyyppejä 1 ja 2 esiintyy lämpimissä, kehittyvissä maissa aiheuttaen vesivälitteisiä epidemioita. Genotyypit 3 ja 4 ovat zoonoottisia, ja niitä esiintyy ihmisten lisäksi niitä useissa eläinlajeissa. Euroopassa kotoperäisenä esiintyy lähinnä genotyyppiä 3. Euroopassa HEV-tartuntojen määrä on ollut kasvussa 2000-luvun alusta alkaen. Eurooppalaiset tartunnat ovat pääasiassa kotoperäisiä ja genotyypin 3 viruksen aiheuttamia, mutta tartuntojen lähteet ovat epäselviä. HEV:n genotyyppi 3 on hyvin yleinen sikojen keskuudessa. Yleensä siat saavat tartunnan jo pikkuporsaana, mutta tartunta voi tapahtua myös vanhemmalla iällä. Jos sialla on teurastettaessa virusta elimistössään, sitä voi päätyä elintarvikkeisiin. Kuluttaja voi saada tartunnan puutteellisesti kuumennetuista elintarvikkeista. HEV:tä on havaittu mm. sianlihassa, sian maksassa ja erilaisissa makkaroissa. Sioista peräisin olevien elintarvikkeiden uskotaan olevan pääasiallinen tartuntareitti Euroopassa, vaikka vahvistettuja tapauksia on vähän. Sianlihaa kulutetaan paljon Euroopassa, minkä vuoksi sianlihan turvallisuutta HEV:n suhteen pitäisi tutkia enemmän. Tartuntalähteiden ja -reittien selvittäminen olisi välttämätöntä tartuntojen ehkäisemiseksi. Tutkimuksen tavoitteena oli kartoittaa HEV:n esiintyvyyttä suomalaisessa teurassianlihassa reaaliaikaisen, käänteiskopiointiin perustuvan polymeraasiketjureaktion (RT-PCR) avulla. Tutkimusmateriaalina oli 60 sian pallealihasta. Tutkimushypoteesina oletettiin enintään kahden näytteen sisältävän HEV:n RNA:ta. Lihanäytteet pakastettiin ja sulatettiin, minkä jälkeen lihaneste kerättin talteen. Lihanesteestä eristettiin viruksen RNA. Eristetty RNA lisäpuhdistettiin PCR-inhibitorisuuden vähentämiseksi. RNA:n eristäminen ja lisäpuhdistus suoritettiin kaupallisilla kiteillä. Näytteistä analysoitiin HEV:n perimä reaaliaikaisella RT-PCR menetelmällä. Tuloksena kaikki 60 näytettä olivat HEV:n perimän suhteen negatiivisia. Saatu tulos oli tutkimushypoteesin mukainen ja tukee muita kansainvälisiä tutkimuksia. Tuloksen perusteella HEV:n esiintyvyys suomalaisessa sianlihassa on matala (esiintyvyyden luottamusväli on 0–6%). Tutkimuksen perusteella ei kuitenkaan voida pitää suomalaista sianlihaa täysin HEV-vapaana. Tutkimuksen avulla saatiin ensimmäistä kertaa tietoa HEV:n esiintyvyydestä suomalaisessa sianlihassa. Riski HEV-tartunnan saamiseen suomalaisesta sianlihasta on pieni, mutta tartunnan ehkäisemiseksi sianliha tulisi kypsentää huolella.
  • Säde, Elina (Helsingin yliopisto, 2011)
    Leuconostoc spp. are lactic acid bacteria (LAB) implicated in food spoilage, especially on refrigerated, modified atmosphere packaged (MAP) meats. The overall aim of this thesis was to learn more about Leuconostoc spp. as food spoilage organisms with a focus on commercial products where LAB spoilage is considered a problem and the main factor limiting shelf-life. Therefore, we aimed to identify Leuconostoc spp. involved in food spoilage, as well as to characterise the spoilage reactions they caused and their contamination sources during poultry meat processing. In addition, we examined the distribution of strains of Leuconostoc gasicomitatum in different food commodities. Finally, we analysed the genome content of L. gasicomitatum LMG 18811 with a special focus on metabolic pathways related to food spoilage. The findings show that Leuconostoc gelidum and L.gasicomitatum were responsible for the discoloration and off-odours developed in beef steaks. Together with Leuconostoc mesenteroides, these Leuconostoc spp., also cause spoilage of vegetable sausages. In contrast, we showed that Leuconostoc spp. are not important for the shelf-life or quality of non-marinated broiler products although, in marinated broiler fillet products, Leuconostoc spp., L.gasicomitatum in particular, are considered spoilage organisms. Furthermore, the findings of the contamination survey we carried out in a poultry processing plant indicated that spoilage Leuconostoc spp. are derived from the processing environment rather than from the broilers, and that air movement distributes psychrotrophic spoilage LAB, including leuconostocs, and has an important role in meat contamination during poultry processing. Pulsed-field gel electrophoresis (PFGE) based genotyping of L. gasicomitatum strains demonstrated that certain genotypes are common in various meat products. In contrast, genotypes associated with meat were not recovered in vegetable-based sources. This suggests that these two food categories either become contaminated with, or favour the growth of different genotypes. Furthermore, the results indicated that the meat processing environment contributes to L. gasicomitatum contamination as certain genotypes were repeatedly identified from products of the same processing plant. Finally, the sequenced and annotated genome of L.gasicomitatum LMG 18811 allowed us to identify the metabolic pathways and reactions resulting in food spoilage.
  • Verkola, Marie (Helsingin yliopisto, 2022)
    Antimicrobial resistance is a major global concern driven especially by the misuse and overuse of antimicrobials both in humans and in animals. Methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE) are major causes of nosocomial infections. As these multiresistant bacteria are increasingly found in livestock, the role of livestock as a reservoir for human infections deserves attention. MRSA has been causing healthcare-related infections since the 1960s and community-associated infections since the 1990s. In 2004, new MRSA strains were detected in humans in Europe that were shown to be related to contact with pigs. These strains were later shown to belong to MRSA clonal complex 398 and are called livestock-associated MRSA (LA-MRSA). Prevalence of LA-MRSA in Europe is highest in pigs and veal calves, but it has also been found in other livestock, horses and companion animals. From asymptomatic, colonized animals, LA-MRSA may be transmitted especially to people working in close contact with livestock such as farmers and veterinarians, who in turn may transmit it further to healthcare facilities. ESBL-PE species are found in livestock, horses and companion animals. In Europe, prevalence is highest in pigs and broilers. Evidence on the zoonotic transmission of ESBL-PE is contradictory. People working on European pig and broiler farms have in some studies been shown to have higher ESBL-/AmpC-producing Escherichia coli prevalence rates than the general population. Infection prevention and control (IPC) practices including proper hand hygiene and the use of personal protective equipment are important to prevent the spread of multiresistant bacteria and other pathogens in healthcare settings. Studies on IPC practices in the veterinary setting have mostly concentrated on companion animal clinics. Ambulatory veterinarians work on several farms a day and transport their equipment and medications from farm to farm. Hand hygiene facilities are offered by the farm or stable owner. There are several studies on the eradication of LA-MRSA from pig farms. A less studied approach is the use of bacteriophages, viruses that infect bacteria, which have been studied as an alternative to antimicrobial agents. Studies on bacteriophage treatment against S. aureus infections in livestock are few and the results have been inconclusive. The aim of this study was to study 1) the carriage of multiresistant zoonotic bacteria in veterinarians in Finland, 2) LA-MRSA colonization and genomic diversity in Finnish pigs, 3) the practices of ambulatory livestock and equine veterinarians to prevent zoonotic transmission, and 4) bacteriophage treatment as a possible way to eradicate LA-MRSA from colonized pigs. MRSA and ESBL-PE prevalence was studied in veterinarians in Finland using both phenotypic and genotypic methods to characterize the isolates. A questionnaire was used to collect important background information on risk factors. To study the adherence of ambulatory livestock and/or equine veterinarians to IPC measures, the replies to the questionnaire of a subset of the respondents were analysed using statistical methods. Quantity and colonization patterns of LA-MRSA in Finnish pigs were studied on two farms – farm 1 and farm 2 (10 pigs per farm) – and in a controlled facility (9 pigs). The strains were further compared with sequences from Finnish Food Authority surveillance and infection samples using core genome multilocus sequence typing (cgMLST). The suitability of bacteriophages for the eradication of LA-MRSA from healthy carrier pigs was investigated in an open, controlled study. A cocktail containing three Staphylococcus bacteriophages was administered to the study group pigs (n=10) and a placebo solution to the control group pigs (n=9) three times. Phage and MRSA levels were observed for 22 days. In addition, phage antibodies in pig sera were monitored. Prevalence of LA-MRSA in veterinarians in Finland was 0.3% (1/320) and the strain found belonged to spa type t011 and multilocus sequence type (ST)398. Virulence genes and resistance genes were typical for LA-MRSA strains found in Finnish pigs. ESBL-producing E. coli were carried by 3.0% of veterinarians (9/297) and one of the strains was also an AmpC producer. Several STs, blaESBL/AmpC genes and plasmid types were found, with ST131 being the most prevalent ST. Travelling and use of antibiotics during the past 12 months was as common as among the average Finnish population. Ambulatory livestock and equine veterinarians (n=129) did not conform adequately to hygiene guidelines. Handwashing facilities were often adequate on farms according to 66.9% of the veterinarians but in stables only according to 21.4% (p<.001). When moving to the next farm or stable, 75.0% always washed their hands or used hand sanitizer in livestock practice but only 42.5% in equine practice (p<.001). In livestock practice a protective coat or overalls were always used by 91.6%, whereas in equine practice only by 27.7% (p<0.001). Altogether, 30.0% of the veterinarians reported cleaning their stethoscope less frequently than once a week. Quantities of LA-MRSA in pigs were low. On farm 1, MRSA was detected in all three samplings in the nares of all 10 pigs. On farm 2, MRSA was detected only in one sampling in three pigs. Quantities in the nasal samples were between 101 and 103 CFU/swab and in the skin samples between 101 and 102 CFU/swab. In the controlled facility, the nasal samples of all nine pigs were MRSA-positive at least once, and the trend was declining. Persistent low levels were detected on the skin. The strains and all sequences from the Finnish Food Authority but one clustered into one cluster in the cgMLST analysis. Bacteriophage treatment did not reduce LA-MRSA levels in healthy carrier pigs. Phages were detected in the samples the day following application. No side effects were observed in the pigs, and no bacteriophage antibodies were detected in the pig sera. In conclusion, prevalence of MRSA and ESBL-PE in veterinarians was low despite frequent animal contact. However, LA-MRSA was detected for the first time in a veterinarian in Finland, which, regarding the high prevalence in Finnish pigs, underlines the importance of protecting people working in close contact with pigs also in Finland. Ambulatory livestock and equine veterinarians’ adherence to IPC guidelines varied significantly between practice types. This may be partly explained by the poor availability of proper handwashing facilities in stables compared with farms as reported by the veterinarians. In addition, further education of both veterinarians and farm or stable owners is necessary. Low levels of LA-MRSA were observed in pigs on two farms and in the controlled facility. The declining trend in nasal samples in the clean environment of the controlled facility may indicate that air contamination could have been an important factor upholding the nasal carriage of LA-MRSA in pigs. The close relatedness of LA-MRSA t034 strains at the national level raises the question of which factors facilitate the spread of this successful clone. Possibly, due to the low levels of LA-MRSA in the pigs in the bacteriophage treatment trial, eradication of LA-MRSA using bacteriophages did not succeed. Further studies on the LA-MRSA t034 clone circulating in Finnish pigs as well as factors related to pig management that may explain the lower LA-MRSA prevalence in alternative farming systems may help to find ways to curb the spread of LA-MRSA in Finnish pigs.
  • Woivalin, Emma (Helsingin yliopisto, 2020)
    Työn tavoitteena oli selvittää metsästetyn sorsanlihan elintarvikehygieenistä laatua mikrobien kokonaispesäkeluvun ja Escherichia coli -bakteerimäärän avulla, sekä kartoittaa sorsissa esiintyvien elintarvikevälitteisten patogeenien esiintyvyyttä. Kirjallisuuskatsaus tutustuu sinisorsaan lintulajina sekä sorsan metsästykseen yleisellä tasolla. Kokeellisessa osassa tutkittiin metsästettyjen sorsien pintasivelynäytteitä. Ruhonäytteistä todettiin aerobisten bakteerien kokonaismäärän keskiarvo 3,5 log10 pmy/cm2 ja E. colin 1,2 log10 pmy/cm2. Tutkimuksessa eristettiin Listeria monocytogenes bakteeria 13 %:ssa näytteistä ja Yersinia enterocoliticabakteeria 3 %:ssa näytteistä. Näytteistä 9 % oli PCR:llä Campylobakteeri-positiivisia ja kaikki olivat Salmonella negatiivisia. Sorsanlihan hygieeninen laatu osoittautui hyväksi tai kohtalaiseksi. Näytteiden välillä esiintyi vaihtelua. Tutkimus osoittaa, että sorsanliha voi sisältää myös ruokamyrkytyksiä aiheuttavia bakteereja, mistä syystä ruhon käsittelyn ja lihan valmistuksen hyvät hygieniakäytännöt tulisi muistaa sekä ammatti- että kotikeittiöissä.
  • Berzins, Aivars (Helsingin yliopisto, 2010)
    The prevalence, contamination and heat resistance of Listeria monocytogenes were investigated in meat products and meat-processing plants. Moreover, trends of human listeriosis in Latvia were studied over a 10-year period from 1998 to 2007. A high prevalence (40%) of L. monocytogenes was found in cold-smoked meat products compared with other heat-treated ready-to-eat meat products (0.7%) available in retail markets in Latvia. Pulsed-field gel electrophoresis (PFGE) and serotyping were applied to analyse the diversity of L. monocytogenes isolates present in ready-to-eat (RTE) meat products and meat-processing plants. A high genetic diversity was seen among L. monocytogenes isolates from cold-smoked meat products, suggesting the existence of various sources of contamination at different production stages in the meat-processing environment. The manufacture of cold-smoked meat products involves no processing steps to eliminate L. monocytogenes, thus, contamination of the raw meat and contamination during processing can both contribute to L. monocytogenes in the finished product. Logistic multivariable regression model was successfully applied to identify the main factors associated with L. monocytogenes contamination during the manufacturing of cold-smoked pork products. Meat brining by injections was a significant factor (odds ratio 10.66; P<0.05) for contamination of products with L. monocytogenes. Of the cold-smoked meat-processing plant environmental samples, most contaminated sites were associated with brining machine and brining area. Long cold-smoking times (≥ 12 h) also had a significant predictive value (odds ratio 24.38; P<0.014) for a sample testing positive for L. monocytogenes. A genetically diverse population of L. monocytogenes entered the meat-processing plant, where only some of the strains colonized and established a persistent microbial community within the plant over a 5-year period. L. monocytogenes PFGE types belonging to serotypes 1/2a and 4b were isolated from imported, defrosted, raw pork from Germany and Belgium in meat-processing plant B. In total, two L. monocytogenes PFGE types originating from raw meat were found also in finished RTE cold-smoked pork products, whereas one PFGE type was recovered later only from the meat-processing environment. One of the L. monocytogenes PFGE types, belonging to serotype 1/2c, was isolated from RTE cold-smoked meat products and from the feeding teeth of the brining machine, thus showing that improper cleaning, disinfection and poor hygiene design of the brining machine may cause L. monocytogenes contamination over time. Post-package pasteurization of high- and low-fat content cooked sausages at temperatures higher than 55°C was found to be an effective method of post-process thermal treatment to reduce contamination of L. monocytogenes. However, heating to 55°C, 60°C and 62.5°C may not be practical in the meat industry because the process takes too long to reach a 3-log reduction. The formulation of high-fat content RTE cooked sausages may require modification to maintain product quality. During the 10-year study period the overall incidence of listeriosis in Latvia was 0.4 per 100 000 population, with the highest incidences recorded in 2000 and 2002 (1.5 and 0.7 per 100 000 population, respectively). The highest incidence of listeriosis in Latvia was observed in 2000, which significantly exceeded incidence levels in all Baltic and Nordic countries, and was the highest among all EU member states during the same period. A marked clustering of human listeriosis cases was observed from September to December 2000, possibly indicating one large outbreak. The lack of serotyping and molecular typing methods for subtyping of L. monocytogenes isolates in the present surveillance system is one of the main reasons why there have been no officially documented listeriosis outbreaks in Latvia to date. Measures to allow the application of appropriate actions at the food industry level need to be implemented to prevent or significantly reduce the real burden of foodborne listeriosis in Latvia.
  • Koort, Joanna (Helsingin yliopisto, 2006)
    Systematiikka on tieteenala, joka tutkii eliöiden, esimerkiksi bakteereiden, sukulaisuussuhteita, määrittelyä ja luokittelua. Taksonomian voidaan ajatella olevan tämän tieteenalan käytännön sovellus: taksonomian tavoitteena on jakaa eliöt niiden sukulaisuussuhteita kuvaaviin, järkeviin ryhmiin, antaa jokaiselle ryhmälle sitä kuvaava nimi sekä löytää ne ryhmän ominaispiirteet, joiden avulla kyseinen ryhmä voidaan jatkossakin vaikeuksitta tunnistaa uudelleen. Taksonomia siis tarjoaa tutkijoille yhteisen kielen, jotta kaikki puhuisivat asioista niiden "oikeilla nimillä" ja vältyttäisiin väärinkäsityksiltä. Taksonomisten joukkojen perusyksikkö on laji. Suvullisesti lisääntyvillä eliöillä voidaan ajatella, että samaan lajiin kuuluvat ne eliöt, jotka pystyisivät keskenään tuottamaan lisääntymiskykyisiä jälkeläisiä. Asia tosin ei läheskään aina ole näin yksinkertainen. Suvullisen lisääntymisen puuttuessa bakteereiden perhesuhteita on vieläkin vaikeampi määritellä, ja siksi tiedon lisääntyessä aiempaan luokitukseen tulee jatkuvasti muutoksia. Jotta luokittelusta saataisiin mahdollisimman pysyvä ja pitkäkestoinen, on bakteereiden luokittelussa ryhdytty käyttämään niin sanottua polyfaasista (monivaiheista) taksonomiaa, menetelmää, jossa bakteerijoukon ominaisuuksia tutkitaan mahdollisimman monella tavalla ja lopuksi yhdistetään kaikki saatu tieto. Tässä tutkimuksessa polyfaasista taksonomiaa käytettiin hyväksi viiden broilerinlihavalmistesiin liittyvän maitohappobakteeriryhmän tunnistuksessa. Samalla arvioitiin polyfaasisen taksonomian soveltuvuutta maitohappobakteereiden luokittelussa. Tutkimuksessa käytettiin sekä fenotyyppisä (ilmiasuun perustuvia), genotyyppisiä (perimään perustuvia) että fylogeneettisiä (kehityshistoriaan eli evoluutioon perustuvia) menetelmiä. Lopuksi kunkin bakteeriryhmän tulokset koottiin yhteen. Useissa aiemmissa tutkimuksissa oli saatu viitteitä siitä, että Lactobacillus curvatus ja Lactobacillus sakei -lajien taksonomiassa on epäselvyyttä. Tämän tutkimuksen tulosten perusteella luokittelua korjattiin siirtämällä Lactobacillus curvatus- bakteerin melibiosus- alalaji osaksi Lactobacillus sakei- lajia, sen alalajiin carnosus. Polyfaasista lähestymistapaa sovellettiin myös neljän tuntemattoman bakteeriryhmän tunnistamisessa. Tulosten perusteella havaittiin että kaksi näistä, kokkimainen koiran nielurisoista ja suojakaasupakatusta broilerista eristetty ryhmä ja sauvamainen, pääosin pilaantuneesta, marinoimattomasta, suojakaasupakatusta broilerinlihavalmisteesta eristetty ryhmä, oli aiemmin kuvaamattomia lajeja. Molemmat uudet lajit kuvattiin, eli niille annettiin nimet ja niiden ominaisuudet listattiin. Kokkibakteeri sai nimekseen Enterococcus hermanniensis, eläinlääketieteellisen tiedekunnan Helsingin Hermannin kaupunginosassa sijainneen vanhan kampuksen mukaan, ja sauvabakteeri Lactobacillus oligofermentans, sen energiankäytön mukaan (bakteeri pystyy käyttämään eli fermentoimaan vain harvoja = oligo sokereita). Kaksi muuta ryhmää sen sijaan tunnistettiin jo olemassa oleviksi lajeiksi. Kokkimainen bakteeri, joka oli eristetty suojakaasupakatusta, marinoidusta ja marinoimattomasta broilerin koipireisivalmisteesta ja broilerinlihanleikkaamon ilmasta tunnistettiin Streptococcus parauberis- bakteeriksi. Osalla näistä bakteereista oli kuitenkin kaksi ominaisuutta, joiden perusteella ne erosivat muista samaan lajiin kuuluvista kannoista: ne eivät kyenneet hyödyntämään laktoosia energianlähteenään, ja galaktoosiakin vain huonosti. Poikkeuksellisiksi nämä ominaisuudet tekee se, että tätä lajia on pidetty lähinnä lehmän utaretulehdusta aiheuttavana bakteerina, ja siten olisi ollut odotettavissa että kaikki sen jäsenet pystyisivät käyttämään laktoosia ja galaktoosia, maidon yleisimpiä energianlähteitä. Toinen, sauvamainen, espanjalaisesta, vakuumi- ja suojakaasupakatusta "Morcilla de Burgos" verimakkarasta eristetty ryhmä tunnistettiin Weissella viridescens- bakteeriksi. Polyfaasinen lähestymistapa sopi hyvin näille maitohappobakteereille. Yksittäisten testien perusteella ei kestävää lajimääritystä olisi saatu aikaan. Vaikka fenotyyppisten testien tulokset voivat ollakin kirjavia, ne kuuluvat kuitenkin uuden lajin kuvaukseen uusien genotyyppisten menetelmien rinnalle. Tutkimuksessa havaittiin myös, että erilaisten testien tuloksista luodut kirjastot ovat luotettavia apuvälineitä bakteereiden tunnistuksessa, kunhan niitä päivitetään jatkuvasti.
  • Piilola, Piela (Helsingin yliopisto, 2021)
    Kananmuna on suosittu ja terveellisenä pidetty elintarvike sekä Suomessa että muualla maailmassa. Kananmunissa piilee myös ruokamyrkytysvaara: kananmunien on todettu olevan merkittävin yksittäinen lähde Salmonella-bakteerien aiheuttamille ruokamyrkytyksille maailmalaajuisesti. Kananmuna voi saastua salmonellabakteereilla jo alkutuotannossa, tai myöhemmin elintarvikeketjun aikana. Salmonellavalvonta ja -torjunta on tärkeä osa koko kananmunan matkaa alkaen jo munintakanan vanhemmista, päättyen vasta kuluttajan vatsaan. Salmonella on maailmanlaajuisesti merkittävä ruokamyrkytyksiä aiheuttava bakteeri, arvion mukaan jopa 78 miljoonaa ihmistä sairastuu salmonelloosiin vuosittain. Salmonelloosin oireita voi olla esimerkiksi kuume, ripuli, pahoinvointi ja oksentelu. Immuunivajeesta kärsiville henkilöille, kuten vanhuksille, salmonelloosi voi olla tappava. Salmonellan torjuntaan on tartuttu eri puolilla maailmaa erilaisilla tavoilla. Suomalainen ja eurooppalainen pyrkimys on tuottaa mahdollisimman puhdas kananmuna. Suomessa on muihin EU-maihin verrattuna tiukka salmonellavalvontaohjelma, johon kuuluvilla näytteillä pyritään estämään salmonellalla saastuneiden kananmunien päätyminen kuluttajille. Tässä on onnistuttu hyvin, Suomessa salmonellaa on munintakanaloissa vain vähän: Suomessa todetaan 0-4 salmonellapositiivista kanalaa vuosittain, EU:n laajuisessa tutkimuksessa esiintyvyys suomalaisissa munintakanaloissa oli n. 0,4%, kun EU:n keskiarvo oli 30%. Yhdysvalloissa ja esimerkiksi Japanissa on toisenlainen lähestymistapa: jokainen kuluttajalle kuorellisena myytävä kananmuna pestään pesuaineilla sekä käsitellään esimerkiksi desinfiointiaineella tai öljyllä. Sekä kananmunien pesemisellä että pesemättä jättämisellä pyritään takaamaan kuluttajalle mahdollisimman turvallinen ja puhdas kokonainen kananmuna. Pestyt kananmunat ovat tutkimusten mukaan pinnaltaan puhtaampia sekä silmämääräisesti että mikrobiologisesti, mikäli pesuprosessi onnistuu. Pesun epäonnistuessa puhtaatkin kananmunat saattavat saastua salmonellalla. Myös hyvin onnistunut pesuprosessi saattaa vaurioittaa kananmunan luontaista puolustusmekanismia, johon kuuluu esimerkiksi kuoren uloin, vahamainen kerros kutikula. Vaurioitunut kananmuna on herkempi kontaminoitumaan bakteereilla. Tutkimusta kananmunien mikrobiologisesta laadusta ja siihen vaikuttavista tekijöistä on tehty paljon. Pesuprosessien haittoja sekä hyötyjä on tutkittu, ja tiedetään millaisia hyötyjä sekä riskejä kananmunien käsittelyyn tai käsittelemättä jättämiseen liittyy. Kuitenkaan ei ole saatavilla selvää vertailevaa tutkimusta siitä, aiheuttavatko pestyt ja käsitellyt vai käsittelemättömät kananmunat enemmän ruokamyrkytyksiä väestössä, vaan päätökset kananmunien pesemisestä tai pesemättömyydestä on tehty pitkälti sen mukaan, mihin toimintatapaan maassa on aiemmin totuttu. Suomessa alkutuotannon salmonellavalvonta on todistetusti onnistunutta, sen ansiosta esimerkiksi vuonna 2021 tehtiin takaisinveto, kun yhdessä munintakanalassa todettiin salmonellapositiivinen näyte. Suomessa salmonellaa esiintyy niin vähän, ettei kananmunien pesulle ole tarvetta. Turha käsittely rasittaa sekä luontoa että kananmunatuotannon taloutta, eikä siksi ole tarkoituksenmukaista.
  • Mattila, Mirjami (Helsingin yliopisto, 2020)
    Listeria monocytogenes is the causative agent of serious food-borne illness, listeriosis. The ability of L monocytogenes to survive and proliferate over a wide range of environmental conditions allows it to overcome various food preservation and safety barriers. To be able to control the risk of L. monocytogenes in the entire food chain, it is important to understand the mechanisms behind the stress tolerance of this pathogen. The aim of this study was to investigate the stress response mechanisms in L. monocytogenes strain EGD-e. Various cellular processes needed for growth and survival under unfavourable conditions are regulated through alternative sigma factors σB and σL. To gather deeper understanding about the role of these sigma factors at low temperatures, this study investigated the regulons of σB and σL, and the expression profile of the ΔsigBL double-mutant strain with a whole-genome expression analysis at 3°C and 37°C. This analysis revealed 198 and 86 genes positively regulated by σB during exponential growth at 3°C and 37°C, respectively. Altogether 29 genes were found to be under positive σB transcriptional regulation during exponential growth at both temperatures. At 3°C, 237 genes were detected to be under positive σL dependent transcriptional control, and at 37°C the number of down-regulated genes was 203. Only 47 of these genes exhibited positive σL transcriptional regulation at both temperatures. Of the 254 genes down-regulated in ΔsigBL at 3°C, 38% were detected also in ΔsigB or ΔsigL, and 62% were present only in ΔsigBL. At 37°C, 87% of the 139 down-regulated genes were present in either ΔsigB or ΔsigL and 13% were detected only in ΔsigBL. It thus appears that the growth at low temperature expands the specialized expression profile of ΔsigBL, but at optimal growth temperature the ΔsigBL expression profile resembles more the expression profiles of ΔsigB and ΔsigL. The phenotypic evaluation of the ∆sigB, ∆sigL, and ∆sigBL mutants revealed impaired growth in the presence of ethanol and organic acids at low growth temperature. The growth of the ΔsigB mutant was compromised in the presence of lactic acid, acetic acid, and ethanol, while both the ∆sigL and ∆sigBL mutants showed impaired growth in the presence of lactic acid, acetic acid, citric acid, and ethanol relative to the wild-type strain. The growth of the ∆sigL and ∆sigBL mutants was also slower at low temperature (4°C) in defined medium relative to the parental strain. In addition, phenotypic microarray analysis exposed significant differences in growth of the wild-type strain and the ∆sigB, ∆sigL, and ∆sigBL mutant strains on different carbon sources and in the presence of various chemical compounds, such as antibiotics targeting cell wall, membrane, DNA, or protein synthesis. Apparently, the deletion in both sigB and sigL increases the sensitivity of the L. monocytogenes towards different stress conditions and chemical compounds and exposes phenotypic characteristics absent from ∆sigB or ∆sigL mutant strains. The stress response mechanisms positively regulated by both sigB and sigL might thus be more relevant than has been previously suspected. Overall, this study provided an expanded insight into σB and σL phenotypic roles and functional interactions in L. monocytogenes. DEAD-box proteins are conserved RNA helicases that can be found in most living organisms. They are needed in RNA metabolism and other metabolic processes and have been linked with cold stress tolerance in L. monocytogenes and many other bacteria. To investigate the role of DEAD-box RNA helicase encoding genes under low growth temperature, transcriptomic and phenotypic analyses of lmo0866, lmo1246, lmo1450, and lmo1722 deletion mutants were performed at 3°C, 25°C, and 37°C. The relative expression levels of lmo0866, lmo1246, lmo1450, and lmo1722 were significantly higher during growth at 3°C than at 37°C and also 30 min, 3 h, and 7 h after cold shock from 37°C to 5°C. At 3°C, the growth of ∆lmo0866 and ∆lmo1722 was totally impaired, and ∆lmo1450 showed only slight growth. The minimum growth temperatures of these mutants were significantly higher compared to the wild-type strain. The results suggest that lmo0866, lmo1450, and lmo1722 play an important role in the cold stress tolerance of L. monocytogenes. The temperature-dependent induction of flagella is a well-characterized phenomenon in L. monocytogenes. However, the essentiality of increased flagellum production during growth at low temperatures is still unclear. The role of flagella synthesis and motility genes flhA and motA in the cold stress tolerance of L. monocytogenes was studied at 3°C, 25°C, and 37°C. The relative expression levels of flhA and motA were found to be higher at 3°C compared to both 25°C and 37°C. The growth of the ΔflhA and ΔmotA deletion mutant strains was compromised at 3°C relative to the wild-type strain. These results illustrate that flhA and motA have a role in the cold stress tolerance of L. monocytogenes, yet the exact functions and regulation of these genes at low growth temperatures remains unknown. Deletion mutant ∆sigL was completely non-motile at 3°C and 10°C, and un-flagellated at 3°C. The ∆sigB and ∆sigBL mutants showed similar swarming pattern compared to the wild-type at 3°C, but the flagella formation of ∆sigBL was slightly compromised at 3°C compared to the wild-type. The cold-sensitive deletion mutant strains ∆lmo0866 and ∆lmo1450 were completely non-motile in BHI at 3°C, while the swarming pattern of the ∆lmo1246 was similar to the wild-type strain. The deletion mutant strains ΔflhA and ΔmotA were completely non-motile at 3°C and 25°C, and flagella formation was deficient. The growth of these strains was also significantly compromised when grown in BHI at 3°C. These findings suggest that the motility and flagella formation may play a role in the cold response of L. monocytogenes.
  • Rahkila, Riitta (Helsingin yliopisto, 2015)
    Ks. RahkilaAbstract.docx