Candida proteinases in the degradation of oral mucosal tissue components associated with Candida invasion

The aim of this thesis was to compare the degradation of human oral epithelial proteins by proteinases of different Candida yeast species. We focused on proteins associated with Candida invasion in the cell-to-cell junction, the basement membrane zone, the extracellular matrix, and local tissue inflammatory regulators. Another main objective was to evaluate the effect of the yeast/hyphal transition and pH on the degradative capability of Candida. The enzymatic activity of the Candida proteinases was verified by gelatin zymography. Laminins-332 (Lm-322) and -511(Lm-511) produced by human oral keratinocytes were gathered from the growth media, and E-cadherin (E-Cad) was isolated from the cell membrane of the keratinocytes by immunoprecipitation. The proteins were incubated with Candida cells and cell-free fractions, and degradation was detected by fluorography. Fibronectin degradation was visualised by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). Matrix metalloproteinase-9 (MMP-9) activation and tissue inhibitor of metalloproteinase-1 (TIMP-1) fragmentation was detected by using the Western blot and enhanced chemoluminescence (ECL) techniques. Residual activity of TIMP-1 was evaluated by a casein degradation assay. A fluorimetric assay was used to detect and compare Candida proteinase activities with MMP-9. These studies showed that the ability of the different Candida yeast species to degrade human Lm-332, fibronectin, and E-Cad vary from strain to strain and that this degradation is pH-dependent. This indicates that local acidic pH in tissue may play a role in tissue destruction by activating Candida proteinases and aid invasion of Candida into deeper tissue. A potential correlation exists between the morphological form of the yeasts and the degradative ability; the C. albicans yeast form seems to be related to superficial infections, and hyphal forms can apparently invade deeper tissues between the epithelial cells by degradation of E-Cad. Basement membrane degradation is possible, especially in the junctional epithelium, which contains only Lm-332 as a structural component. Local tissue host inflammatory mediators, such as MMP-9, were activated, and TIMP-1 was degraded by certain Candida species, thus indicating the possibility of a weakened host tissue defence mechanism in vivo.

Stabilization of Phospholipid Coatings in Capillary Electrophoresis

The development of a simple method of coating a semi-permanent phospholipid layer onto a capillary for electrochromatography use was the focus of this study. The work involved finding good coating conditions, stabilizing the phospholipid coating, and examining the effect of adding divalent cations, cetyltrimethylammonium bromide, and polyethylene glycol (PEG)-lipids on the stability of the coating. Since a further purpose was to move toward more biological membrane coatings, the capillaries were also coated with cholesterol-containing liposomes and liposomes of red blood cell ghost lipids. Liposomes were prepared by extrusion, and large unilamellar vesicles with a diameter of about 100 nm were obtained. Zwitterionic phosphatidylcholine (PC) was used as a basic component, mainly 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) but also eggPC and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Different amounts of sphingomyelin, bovine brain phosphatidylserine, and cholesterol were added to the PC. The stability of the coating in 40 mM N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES) solution at pH 7.4 was studied by measuring the electroosmotic flow and by separating neutral steroids, basic proteins, and low-molar-mass drugs. The presence of PC in the coating solution was found to be essential to achieving a coating. The stability of the coating was improved by the addition of negative phosphatidylserine, cholesterol, divalent cations, or PEGylated lipids, and by working in the gel-state region of the phospholipid. Study of the effect on the PC coating of divalent metal ions calcium, magnesium, and zinc showed a molar ratio of 1:3 PC/Ca2+ or PC/Mg2+ to give increased rigidity to the membrane and the best coating stability. The PEGylated lipids used in the study were sterically stabilized commercial lipids with covalently attached PEG chains. The vesicle size generally decreased when PEGylated lipids of higher molar mass were present in the vesicle. The predominance of discoidal micelles over liposomes increased PEG chain length and the average size of the vesicles thus decreased. In the capillary electrophoresis (CE) measurements a highly stable electroosmotic flow was achieved with 20% PEGylated lipid in the POPC coating dispersion, the best results being obtained for disteroyl PEG (3000) conjugates. The results suggest that smaller particles (discoidal micelles) result in tighter packing and better shielding of silanol groups on the silica wall. The effect of temperature on the coating stability was investigated by using DPPC liposomes at temperatures above (45 C) and below (25 C) the main phase transition temperature. Better results were obtained with DPPC in the more rigid gel state than in the fluid state: the electroosmotic flow was heavily suppressed and the PC coating was stabilized. Also dispersions of DPPC with 0−30 mol% of cholesterol and sphingomyelin in different ratios, which more closely resemble natural membranes, resulted in stable coatings. Finally, the CE measurements revealed that a stable coating is formed when capillaries are coated with liposomes of red blood cell ghost lipids.

SU-8-Based Microchips for Capillary Electrophoresis and Electrospray Ionization Mass Spectrometry

Miniaturization of analytical instrumentation is attracting growing interest in response to the explosive demand for rapid, yet sensitive analytical methods and low-cost, highly automated instruments for pharmaceutical and bioanalyses and environmental monitoring. Microfabrication technology in particular, has enabled fabrication of low-cost microdevices with a high degree of integrated functions, such as sample preparation, chemical reaction, separation, and detection, on a single microchip. These miniaturized total chemical analysis systems (microTAS or lab-on-a-chip) can also be arrayed for parallel analyses in order to accelerate the sample throughput. Other motivations include reduced sample consumption and waste production as well as increased speed of analysis. One of the most promising hyphenated techniques in analytical chemistry is the combination of a microfluidic separation chip and mass spectrometer (MS). In this work, the emerging polymer microfabrication techniques, ultraviolet lithography in particular, were exploited to develop a capillary electrophoresis (CE) separation chip which incorporates a monolithically integrated electrospray ionization (ESI) emitter for efficient coupling with MS. An epoxy photoresist SU-8 was adopted as structural material and characterized with respect to its physicochemical properties relevant to chip-based CE and ESI/MS, namely surface charge, surface interactions, heat transfer, and solvent compatibility. As a result, SU-8 was found to be a favorable material to substitute for the more commonly used glass and silicon in microfluidic applications. In addition, an infrared (IR) thermography was introduced as direct, non-intrusive method to examine the heat transfer and thermal gradients during microchip-CE. The IR data was validated through numerical modeling. The analytical performance of SU-8-based microchips was established for qualitative and quantitative CE-ESI/MS analysis of small drug compounds, peptides, and proteins. The CE separation efficiency was found to be similar to that of commercial glass microchips and conventional CE systems. Typical analysis times were only 30-90 s per sample indicating feasibility for high-throughput analysis. Moreover, a mass detection limit at the low-attomole level, as low as 10E+5 molecules, was achieved utilizing MS detection. The SU-8 microchips developed in this work could also be mass produced at low cost and with nearly identical performance from chip to chip. Until this work, the attempts to combine CE separation with ESI in a chip-based system, amenable to batch fabrication and capable of high, reproducible analytical performance, have not been successful. Thus, the CE-ESI chip developed in this work is a substantial step toward lab-on-a-chip technology.

Development of microdevices for proteome research and diagnosis

The basic goal of a proteomic microchip is to achieve efficient and sensitive high throughput protein analyses, automatically carrying out several measurements in parallel. A protein microchip would either detect a single protein or a large set of proteins for diagnostic purposes, basic proteome or functional analysis. Such analyses would include e.g. interactomics, general protein expression studies, detecting structural alterations or secondary modifications. Visualization of the results may occur by simple immunoreactions, general or specific labelling, or mass spectrometry. For this purpose we have manufactured chip-based proteome analysis devices that utilize the classical polymer gel electrophoresis technology to run one and two-dimensional gel electrophoresis separations of proteins in just a smaller size. In total, we manufactured three functional prototypes of which one performed a miniaturized one-dimensional gel electrophoresis (1-DE) separation, the second and third preformed two-dimensional gel electrophoresis (2-DE) separations. These microchips were successfully used to separate and characterize a set of predefined standard proteins, cell and tissue samples. Also, the miniaturized 2-DE (ComPress-2DE) chip presents a novel way of combining the 1st and 2nd dimensional separations, thus avoiding manual handling of the gels, eliminate cross-contamination, and make analyses faster and repeatability better. They all showed the advantages of miniaturization over the commercial devices; such as fast analysis, low sample- and reagent consumption, high sensitivity, high repeatability and inexpensive performance. All these instruments have the potential to be fully automated due to their easy-to-use set-up.

Modern techniques in detection, identification and quantification of bacteria and peptides from foods

Standards have been placed to regulate the microbial and preservative contents to assure that foods are safe to the consumer. In a case of a food-related disease outbreak, it is crucial to be able to detect and identify quickly and accurately the cause of the disease. In addition, for every day control of food microbial and preservative contents, the detection methods must be easily performed for numerous food samples. In this present study, quicker alternative methods were studied for identification of bacteria by DNA fingerprinting. A flow cytometry method was developed as an alternative to pulsed-field gel electrophoresis, the golden method . DNA fragment sizing by an ultrasensitive flow cytometer was able to discriminate species and strains in a reproducible and comparable manner to pulsed-field gel electrophoresis. This new method was hundreds times faster and 200,000 times more sensitive. Additionally, another DNA fingerprinting identification method was developed based on single-enzyme amplified fragment length polymorphism (SE-AFLP). This method allowed the differentiation of genera, species, and strains of pathogenic bacteria of Bacilli, Staphylococci, Yersinia, and Escherichia coli. These fingerprinting patterns obtained by SE-AFLP were simpler and easier to analyze than those by the traditional amplified fragment length polymorphism by double enzyme digestion. Nisin (E234) is added as a preservative to different types of foods, especially dairy products, around the world. Various detection methods exist for nisin, but they lack in sensitivity, speed or specificity. In this present study, a sensitive nisin-induced green fluorescent protein (GFPuv) bioassay was developed using the Lactococcus lactis two-component signal system NisRK and the nisin-inducible nisA promoter. The bioassay was extremely sensitive with detection limit of 10 pg/ml in culture supernatant. In addition, it was compatible for quantification from various food matrices, such as milk, salad dressings, processed cheese, liquid eggs, and canned tomatoes. Wine has good antimicrobial properties due to its alcohol concentration, low pH, and organic content and therefore often assumed to be microbially safe to consume. Another aim of this thesis was to study the microbiota of wines returned by customers complaining of food-poisoning symptoms. By partial 16S rRNA gene sequence analysis, ribotyping, and boar spermatozoa motility assay, it was identified that one of the wines contained a Bacillus simplex BAC91, which produced a heat-stable substance toxic to the mitochondria of sperm cells. The antibacterial activity of wine was tested on the vegetative cells and spores of B. simplex BAC91, B. cereus type strain ATCC 14579 and cereulide-producing B. cereus F4810/72. Although the vegetative cells and spores of B. simplex BAC91 were sensitive to the antimicrobial effects of wine, the spores of B. cereus strains ATCC 14579 and F4810/72 stayed viable for at least 4 months. According to these results, Bacillus spp., more specifically spores, can be a possible risk to the wine consumer.

Biodiversity and Phylogeny of Planktic Cyanobacteria in Temperate Freshwater Lakes

Currently, the classification used for cyanobacteria is based mainly on morphology. In many cases the classification is known to be incongruent with the phylogeny of cyanobacteria. The evaluation of this classification is complicated by the fact that numerous strains are only described morphologically and have not been isolated. Moreover, the phenotype of many cyanobacterial strains alters during prolonged laboratory cultivation. In this thesis, cyanobacterial strains were isolated from lakes (mainly Lake Tuusulanjärvi) and both morphology and phylogeny of the isolates were investigated. The cyanobacterial community composition in Lake Tuusulanjärvi was followed for two years in order to relate the success of cyanobacterial phenotypes and genotypes to environmental conditions. In addition, molecular biological methods were compared with traditional microscopic enumeration and their ability and usefulness in describing the cyanobacterial diversity was evaluated. The Anabaena, Aphanizomenon, and Trichormus strains were genetically heterogeneous and polyphyletic. The phylogenetic relationships of the heterocytous cyanobacteria were not congruent with their classification. In contrast to heterocytous cyanobacteria, the phylogenetic relationships of the Snowella and Woronichinia strains, which had not been studied before this thesis, reflected the morphology of strains and followed their current classification. The Snowella strains formed a monophyletic cluster, which was most closely related to the Woronichinia strain. In addition, a new cluster of thin, filamentous cyanobacterial strains identified as Limnothrix redekei was revealed. This cluster was not closely related to any other known cyanobacteria. The cyanobacterial community composition in Lake Tuusulanjärvi was studied with molecular methods [denaturant gradient gel electrophoresis (DGGE) and cloning of the 16S rRNA gene], through enumerations of cyanobacteria under microscope, and by strain isolations. Microcystis, Anabaena/Aphanizomenon, and Synechococcus were the major groups in the cyanobacterial community in Lake Tuusulanjärvi during the two-year monitoring period. These groups showed seasonal succession, and their success was related to different environmental conditions. The major groups of the cyanobacterial community were detected by all used methods. However, cloning gave higher estimates than microscopy for the proportions of heterocytous cyanobacteria and Synechococcus. The differences were probably caused by the high 16S rRNA gene copy numbers in heterotrophic cyanobacteria and by problems in the identification and detection of unicellular cyanobacteria.

Methicillin-resistant Staphylococcus aureus in Finland: recent changes in the epidemiology, long-term facility aspects, and phenotypic and molecular detection of isolates

Staphylococcus aureus is one of the most important bacteria that cause disease in humans, and methicillin-resistant S. aureus (MRSA) has become the most commonly identified antibiotic-resistant pathogen in many parts of the world. MRSA rates have been stable for many years in the Nordic countries and the Netherlands with a low MRSA prevalence in Europe, but in the recent decades, MRSA rates have increased in those low-prevalence countries as well. MRSA has been established as a major hospital pathogen, but has also been found increasingly in long-term facilities (LTF) and in communities of persons with no connections to the health-care setting. In Finland, the annual number of MRSA isolates reported to the National Infectious Disease Register (NIDR) has constantly increased, especially outside the Helsinki metropolitan area. Molecular typing has revealed numerous outbreak strains of MRSA, some of which have previously been associated with community acquisition. In this work, data on MRSA cases notified to the NIDR and on MRSA strain types identified with pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and staphylococcal cassette chromosome mec (SCCmec) typing at the National Reference Laboratory (NRL) in Finland from 1997 to 2004 were analyzed. An increasing trend in MRSA incidence in Finland from 1997 to 2004 was shown. In addition, non-multi-drug resistant (NMDR) MRSA isolates, especially those resistant only to methicillin/oxacillin, showed an emerging trend. The predominant MRSA strains changed over time and place, but two internationally spread epidemic strains of MRSA, FIN-16 and FIN-21, were related to the increase detected most recently. Those strains were also one cause of the strikingly increasing invasive MRSA findings. The rise of MRSA strains with SCCmec types IV or V, possible community-acquired MRSA was also detected. With questionnaires, the diagnostic methods used for MRSA identification in Finnish microbiology laboratories and the number of MRSA screening specimens studied were reviewed. Surveys, which focused on the MRSA situation in long-term facilities in 2001 and on the background information of MRSA-positive persons in 2001-2003, were also carried out. The rates of MRSA and screening practices varied widely across geographic regions. Part of the NMDR MRSA strains could remain undetected in some laboratories because of insufficient diagnostic techniques used. The increasing proportion of elderly population carrying MRSA suggests that MRSA is an emerging problem in Finnish long-term facilities. Among the patients, 50% of the specimens were taken on a clinical basis, 43% on a screening basis after exposure to MRSA, 3% on a screening basis because of hospital contact abroad, and 4% for other reasons. In response to an outbreak of MRSA possessing a new genotype that occurred in a health care ward and in an associated nursing home of a small municipality in Northern Finland in autumn 2003, a point-prevalence survey was performed six months later. In the same study, the molecular epidemiology of MRSA and methicillin-sensitive S. aureus (MSSA) strains were also assessed, the results to the national strain collection compared, and the difficulties of MRSA screening with low-level oxacillin-resistant isolates encountered. The original MRSA outbreak in LTF, which consisted of isolates possessing a nationally new PFGE profile (FIN-22) and internationally rare MLST type (ST-27), was confined. Another previously unrecognized MRSA strain was found with additional screening, possibly indicating that current routine MRSA screening methods may be insufficiently sensitive for strains possessing low-level oxacillin resistance. Most of the MSSA strains found were genotypically related to the epidemic MRSA strains, but only a few of them had received the SCCmec element, and all those strains possessed the new SCCmec type V. In the second largest nursing home in Finland, the colonization of S. aureus and MRSA, and the role of screening sites along with broth enrichment culture on the sensitivity to detect S. aureus were studied. Combining the use of enrichment broth and perineal swabbing, in addition to nostrils and skin lesions swabbing, may be an alternative for throat swabs in the nursing home setting, especially when residents are uncooperative. Finally, in order to evaluate adequate phenotypic and genotypic methods needed for reliable laboratory diagnostics of MRSA, oxacillin disk diffusion and MIC tests to the cefoxitin disk diffusion method at both +35°C and +30°C, both with or without an addition of sodium chloride (NaCl) to the Müller Hinton test medium, and in-house PCR to two commercial molecular methods (the GenoType® MRSA test and the EVIGENETM MRSA Detection test) with different bacterial species in addition to S. aureus were compared. The cefoxitin disk diffusion method was superior to that of oxacillin disk diffusion and to the MIC tests in predicting mecA-mediated resistance in S. aureus when incubating at +35°C with or without the addition of NaCl to the test medium. Both the Geno Type® MRSA and EVIGENETM MRSA Detection tests are usable, accurate, cost-effective, and sufficiently fast methods for rapid MRSA confirmation from a pure culture.

Archaea, Bacteria, and methane production along environmental gradients in fens and bogs

Metanogeenit ovat hapettomissa oloissa eläviä arkkien pääryhmään kuuluvia mikrobeja, joiden ainutlaatuisen aineenvaihdunnan seurauksena syntyy metaania. Ilmakehässä metaani on voimakas kasvihuonekaasu. Yksi suurimmista luonnon metaanilähteistä ovat kosteikot. Pohjoisten soiden metaanipäästöt vaihtelevat voimakkaasti eri soiden välillä ja yhden suon sisälläkin, riippuen muun muassa vuodenajasta, suotyypistä ja kasvillisuudesta. Väitöskirjatyössä tutkittiin metaanipäästöjen vaihtelun mikrobiologista taustaa. Tutkimuksessa selvitettiin suotyypin, vuodenajan, tuhkalannoituksen ja turvesyvyyden vaikutusta metanogeeniyhteisöihin sekä metaanintuottoon kolmella suomalaisella suolla. Lisäksi tutkittiin ei-metanogeenisia arkkeja ja bakteereita, koska ne muodostavat metaanin tuoton lähtöaineet osana hapetonta hajotusta. Mikrobiyhteisöt analysoitiin DNA- ja RNA-lähtöisillä, polymeraasiketjureaktioon (PCR) perustuvilla menetelmillä. Merkkigeeneinä käytettiin metaanin tuottoon liittyvää mcrA-geeniä sekä arkkien ja bakteerien ribosomaalista 16S RNA-geeniä. Metanogeeniyhteisöt ja metaanintuotto erosivat huomattavasti happaman ja vähäravinteisen rahkasuon sekä ravinteikkaampien sarasoiden välillä. Rahkasuolta löytyi lähes yksinomaan Methanomicrobiales-lahkon metanogeeneja, jotka tuottavat metaania vedystä ja hiilidioksidista. Sarasoiden metanogeeniyhteisöt olivat monimuotoisempia, ja niillä esiintyi myös asetaattia käyttäviä metanogeeneja. Vuodenaika vaikutti merkittävästi metaanintuottoon. Talvella havaittiin odottamattoman suuri metaanintuottopotentiaali sekä viitteitä aktiivisista metanogeeneista. Arkkiyhteisön koostumus sen sijaan vaihteli vain vähän. Tuhkalannoitus, jonka tarkoituksena on edistää puiden kasvua ojitetuilla soilla, ei merkittävästi vaikuttanut metaanintuottoon tai -tuottajiin. Ojitetun suon yhteisöt kuitenkin muuttuivat turvesyvyyden mukaan. Vertailtaessa erilaisia PCR-menetelmiä todettiin, että kolmella mcrA-geeniin kohdistuvalla alukeparilla havaittiin pääosin samat ojitetun suon metanogeenit, mutta lajien runsaussuhteet riippuvat käytetyistä alukkeista. Soilla havaitut bakteerit kuuluivat pääjaksoihin Deltaproteobacteria, Acidobacteria ja Verrucomicrobia. Lisäksi löydettiin Crenarchaeota-pääjakson ryhmiin 1.1c ja 1.3 kuuluvia ei-metanogeenisia arkkeja. Tulokset ryhmien esiintymisestä hapettomassa turpeessa antavat lähtökohdan selvittää niiden mahdollisia vuorovaikutuksia metanogeenien kanssa. Tutkimuksen tulokset osoittivat, että metanogeeniyhteisön koostumus heijastaa metaanintuottoon vaikuttavia kemiallisia tai kasvillisuuden vaihteluita kuten suotyyppiä. Soiden metanogeenien ja niiden fysiologian parempi tuntemus voi auttaa ennustamaan ympäristömuutosten vaikutusta soiden metaanipäästöihin.

Microbial activities in boreal soils: Biodegradation of organic contaminants at low temperature and ammonia oxidation

This thesis deals with the response of biodegradation of selected anthropogenic organic contaminants and natural autochthonous organic matter to low temperature in boreal surface soils. Furthermore, the thesis describes activity, diversity and population size of autotrophic ammonia-oxidizing bacteria (AOB) in a boreal soil used for landfarming of oil-refinery wastes, and presents a new approach, in which the particular AOB were enriched and cultivated in situ from the landfarming soil onto cation exchange membranes. This thesis demonstrates that rhizosphere fraction of natural forest humus soil and agricultural clay loam soil from Helsinki Metropolitan area were capable of degrading of low to moderate concentrations (0.2 50 µg cm-3) of PCP, phenanthrene and 2,4,5-TCP at temperatures realistic to boreal climate (-2.5 to +15 °C). At the low temperatures, the biodegradation of PCP, phenanthrene and 2,4,5-TCP was more effective (Q10-values from 1.6 to 7.6) in the rhizosphere fraction of the forest soil than in the agricultural soil. Q10-values of endogenous soil respiration (carbon dioxide evolution) and selected hydrolytic enzyme activities (acetate-esterase, butyrate-esterase and β-glucosidase) in acid coniferous forest soil were 1.6 to 2.8 at temperatures from -3 to +30 °C. The results indicated that the temperature dependence of decomposition of natural autochthonous soil organic matter in the studied coniferous forest was only moderate. The numbers of AOB in the landfarming (sandy clay loam) soil were determined with quantitative polymerase chain reaction (real-time PCR) and with Most Probable Number (MPN) methods, and potential ammonium oxidation activity was measured with the chlorate inhibition technique. The results indicated presence of large and active AOB populations in the heavily oil-contaminated and urea-fertilised landfarming soil. Assessment of the populations of AOB with denaturing gradient gel electrophoresis (DGGE) profiling and sequence analysis of PCR-amplified 16S rRNA genes showed that Nitrosospira-like AOB in clusters 2 and 3 were predominant in the oily landfarming soil. This observation was supported by fluorescence in situ hybridization (FISH) analysis of the AOB grown on the soil-incubated cation-exchange membranes. The results of this thesis expand the suggested importance of Nitrosospira-like AOB in terrestrial environments to include chronically oil-contaminated soils.

Leuconostoc spoilage of refrigerated, packaged foods

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.

Finnish cattle as reservoir of Campylobacter spp.

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 studied bovine rectal faecal samples (n=952) and carcass surface samples (n=948) from twelve Finnish slaughterhouses from January to December 2003. The total prevalence of Campylobacter spp. in faecal samples was 31.1%, and in carcass samples 3.5%. Campylobacter jejuni, the most common species, was present in 19.5% of faecal samples and in 3.1% of carcasses. In addition to thermophilic Campylobacter spp., C. hyointestinalis ssp. hyointestinalis was present in bovine samples. The prevalence of campylobacters was higher among beef cattle than among dairy cattle. Using the enrichment method, the number of positive faecal samples was 7.5 times higher than that obtained by direct plating. The predominant serotypes of faecal C. jejuni, determined by serotyping with a set of 25 commercial antisera for heat-stable antigens (Penner), were Pen2 and Pen4-complex, which covered 52% of the samples. Genotyping with pulsed-field gel electrophoresis (PFGE) using SmaI restriction yielded a high diversity of C. jejuni subtypes in cattle. Determining the minimum inhibitory concentrations of ampicillin, enrofloxacin, erythromycin, gentamicin, nalidixic acid, and oxytetracycline among bovine C. jejuni isolates using a commercial broth microdilution method yielded 9% of isolates resistant to at least one of the antimicrobials examined. No multiresistant isolates were found among the bovine C. jejuni strains. The study of the shedding patterns of Campylobacter spp. among three Finnish dairy cattle herds included the examination of fresh faecal samples and tank milk samples taken five times, as well as samples from drinking troughs taken once during the one-year study. The semiquantitative enrichment method detected C. jejuni in 169 of the 340 faecal samples, mostly at low levels. In addition, C. jejuni was present in one drinking trough sample. The prevalence between herds and sampling occasions varied widely. PFGE, using SmaI as restriction enzyme, identified only a few subtypes in each herd. In two 2 of the herds, two subtypes persisted throughout the sampling. Individual animals presented various shedding patterns during the study. Comparison of C. jejuni isolates from humans, chickens and cattle included the design of primers for four new genetic markers selected from completely sequenced C. jejuni genomes 81-176, RM1221 and NCTC 11168, and the PCR examination of domestic human isolates from southern Finland in 1996, 2002 and 2003 (n=309), chicken isolates from 2003, 2006 and 2007 (n=205), and bovine isolates from 2003 (n=131). The results revealed that bovine isolates differed significantly from human and chicken isolates. In particular, the - glutamyl transpeptidase gene was uncommon among bovine isolates. The PFGE genotyping of C. jejuni isolates, using SmaI and KpnI restriction enzymes, included a geographically representative collection of isolates from domestic sporadic human infections, chicken slaughter batches, and cattle faeces and carcasses 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 notable reservoir of C. jejuni, the most important Campylobacter sp. 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.