Characterisation of antibiotic-resistant psychrotrophic bacteria in raw milk

Dynamics of raw milk associated bacteria during cold storage of raw milk and their antibiotic resistance was reviewed, with focus on psychrotrophic bacteria. This study aimed to investigate the significance of cold storage of raw milk on antibiotic-resistant bacterial population and analyse the antibiotic resistance of the Gram-negative antibiotic-resistant psychrotrophic bacteria isolated from the cold-stored raw milk samples. Twenty-four raw milk samples, six at a time, were obtained from lorries that collected milk from Finnish farms and were stored at 4°C/4 d, 6°C/3 d and 6°C/4 d. Antibiotics representing four classes of antibiotics (gentamicin, ceftazidime, levofloxacin and trimethoprim-sulfamethoxazole) were used to determine the antibiotic resistance of mesophilic and psychrotrophic bacteria during the storage period. A representative number of antibiotic-resistant Gram-negative isolates retrieved from the cold-stored raw milk samples were identified by the phenotypic API 20 NE system and a few isolates by the 16S rDNA gene sequencing. Some of the isolates were further evaluated for their antibiotic resistance by the ATB PSE 5 and HiComb system. The initial average mesophilic counts were found below 105 CFU/mL, suggesting that the raw milk samples were of good quality. However, the mesophilic and psychrotrophic population increased when stored at 4°C/4 d, 6°C/3 d and 6°C/4 d. Gentamicin- and levofloxacin-resistant bacteria increased moderately (P < 0.05) while there was a considerable rise (P < 0.05) of ceftazidime- and trimethoprim-sulfamethoxazole-resistant population during the cold storage. Of the 50.9 % (28) of resistant isolates (total 55) identified by API 20 NE, the majority were Sphingomonas paucimobilis (8), Pseudomonas putida (5), Sphingobacterium spiritivorum (3) and Acinetobacter baumanii (2). The analysis by ATB PSE 5 system suggested that 57.1% of the isolates (total 49) were multiresistant. This study showed that the dairy environment harbours multidrug-resistant Gramnegative psychrotrophic bacteria and the cold chain of raw milk storage amplifies the antibioticresistant psychrotrophic bacterial population.

Bacteria colonizing paper machines

Bacteria growing in paper machines can cause several problems. Biofilms detaching from paper machine surfaces may lead to holes and spots in the end product or even break the paper web leading to expensive delays in production. Heat stable endospores will remain viable through the drying section of paper machine, increasing the microbial contamination of paper and board. Of the bacterial species regularly found in the end products, Bacillus cereus is the only one classified as a pathogen. Certain B. cereus strains produce cereulide, the toxin that causes vomiting disease in food poisonings connected to B. cereus. The first aim of this thesis was to identify harmful bacterial species colonizing paper machines and to assess the role of bacteria in the formation of end product defects. We developed quantitative PCR methods for detecting Meiothermus spp. and Pseudoxanthomonas taiwanensis. Using these methods I showed that Meiothermus spp. and Psx. taiwanensis are major biofoulers in paper machines. I was the first to be able to show the connection between end product defects and biofilms in the wet-end of paper machines. I isolated 48 strains of primary-biofilm forming bacteria from paper machines. Based on one of them, strain K4.1T, I described a novel bacterial genus Deinobacterium with Deinobacterium chartae as the type species. I measured the transfer of Bacillus cereus spores from packaging paper into food. To do this, we constructed a green fluorescent protein (GFP) labelled derivative of Bacillus thuringiensis and prepared paper containing spores of this strain. Chocolate and rice were the recipient foods when transfer of the labelled spores from the packaging paper to food was examined. I showed that only minority of the Bacillus cereus spores transferred into food from packaging paper and that this amount is very low compared to the amount of B. cereus naturally occurring in foods. Thus the microbiological risk caused by packaging papers is very low. Until now, the biological function of cereulide for the producer cell has remained unknown. I showed that B. cereus can use cereulide to take up K+ from environment where K+ is scarce: cereulide binds K+ ions outside the cell with high affinity and transports these ions across cell membrane into the cytoplasm. Externally added cereulide increased the growth rate of cereulide producing strains in medium where potassium was growth limiting. In addition, cereulide producing strains outcompeted cereulide non-producing B. cereus in potassium deficient environment, but not when the potassium concentration was high. I also showed that cereulide enhances biofilm formation of B. cereus.

Root system traits of Norway spruce, Scots pine, and silver birch in mixed boreal forests: an analysis of root architecture, morphology, and anatomy

The aim of this thesis was to unravel the functional-structural characteristics of root systems of Betula pendula Roth., Picea abies (L.) Karst., and Pinus sylvestris L. in mixed boreal forest stands differing in their developmental stage and site fertility. The root systems of these species had similar structural regularities: horizontally-oriented shallow roots defined the horizontal area of influence, and within this area, each species placed fine roots in the uppermost soil layers, while sinker roots defined the maximum rooting depth. Large radial spread and high ramification of coarse roots, and the high specific root length (SRL) and root length density (RLD) of fine roots indicated the high belowground competitiveness and root plasticity of B. pendula. Smaller radial root spread and sparser branching of coarse roots, and low SRL and RLD of fine roots of the conifers could indicate their more conservative resource use and high association with and dependence on ectomycorrhiza-forming fungi. The vertical fine root distributions of the species were mostly overlapping, implying the possibility for intense belowground competition for nutrients. In each species, conduits tapered and their frequency increased from distal roots to the stem, from the stem to the branches, and to leaf petioles in B. pendula. Conduit tapering was organ-specific in each species violating the assumptions of the general vascular scaling model (WBE). This reflects the hierarchical organization of a tree and differences between organs in the relative importance of transport, safety, and mechanical demands. The applied root model was capable of depicting the mass, length and spread of coarse roots of B. pendula and P. abies, and to the lesser extent in P. sylvestris. The roots did not follow self-similar fractal branching, because the parameter values varied within the root systems. Model parameters indicate differences in rooting behavior, and therefore different ecophysiological adaptations between species.

Does the age of fine root carbon indicate the age of fine roots in boreal forests?

To test the reliability of the radiocarbon method for determining root age, we analyzed fine roots (originating from the years 1985 to 1993) from ingrowth cores with known maximum root age (1 to 6 years old). For this purpose, three Scots pine (Pinus sylvestris L.) stands were selected from boreal forests in Finland. We analyzed root 14C age by the radiocarbon method and compared it with the above-mentioned known maximum fine root age. In general, ages determined by the two methods (root 14C age and ingrowth core root maximum age) were in agreement with each other for roots of small diameter (<0.5mm). By contrast, in most of the samples of fine roots of larger diameter (1.5-2mm), the 14C age of root samples of 1987-89 exceeded the ingrowth core root maximum age by 1-10 years. This shows that these roots had received a large amount of older stored carbon from unknown sources in addition to atmospheric CO2 directly from photosynthesis. We conclude that the 14C signature of fine roots, especially those of larger diameter, may not always be indicative of root age, and that further studies are needed concerning the extent of possible root uptake of older carbon and its residence time in roots. Keywords: fine root age, Pinus sylvestris, radiocarbon, root carbon, ingrowth cores, tree ring

Resistance of Norway spruce (Picea abies) to root and butt rot (Heterobasidion parviporum) in peatland and mineral soil

Root and butt rot is the most harmful fungal disease affecting Norway spruce in southern Finland. In approximately 90 % of cases the causal agent is Heterobasidion parviporum. Root and butt rot infections have not been reported in Finnish peatlands. However, the increase in logging operations in peatlands means there is a risk that the fungus will eventually spread to these areas. The aim of this study was to find out the impact of growing site on the resistance of Norway spruce to Heterobasidion parviporum infections. This was investigated by artificially inoculating H. parviporum to spruce trees in pristine mire, drained peatland and mineral soil and comparing the defence reactions. Additionally, the effect of genotype on resistance was studied by comparing the responses of spruce clones representing different geographic origins. The roots and stems of the trees to be sampled were wounded and inoculated with wood dowels pre-colonised by H. parviporum hyphae. The resulting necrosis around the point of inoculation was observed. It was presumed that increased length of necrosis indicates high susceptibility of the tree to the disease. The relationship between growth rate and host resistance was also studied. The results indicated that growing site does not have a statistically significant effect on host resistance. The average length of necrosis around the point of inoculation was 35 mm in pristine mire, 37 mm in drained peatland and 40 mm in mineral soil. It was observed that growth rate does not affect resistance, but that the genotype of the tree does have an effect. The most resistant spruce clone was the one with Russian origin. The results suggest that the spruce stands in peatlands are not more resistant to root and butt rot infections than those in mineral soil. These findings should be taken into consideration when logging peatland forests.