Browsing by Subject "typpikaasukäsittely"
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(2017)Small colony variants are exceptional form from the wild type bacteria. Most specific feature for small colony variants is significantly smaller colony that can be 1/10 of the size of the original colony. Other features are slow growth and weakened pigmentation. Small colony variants are usually more resistant to antibiotics. Small colony variants have been discovered for many different species, from which S. aureus is the most investigated one. For Bacillus species small colony variants have been discovered for B. weihenstephanensis, B. licheniformis, B. cereus and B. subtilis -bacteria. B. weihenstephanensis is a spore forming and psychotropic bacteria that has been shown to grow in milk and dominate milks microbiota during long cold storage. B. weihenstephanensis bacteria have also been shown to resist nitrogen flushing that has been investigates to longen milks shelf-life. Purpose of this study was to compare the original strain of B. weihenstephanensis to its small colony variants that were discovered during flushing with nitrogen gas. In this study the original strains growth in different temperatures, utilization of carbohydrates and antibiotic resistance was compared to its small colony variants. In this study multiple strains of B. weihenstephanensis were analyzed, other were the original strains and others their small colony variant. Original strains were flushed in nitrogen gas in 15 °C temperature in milk, to see if small colonies would appear, but no stable small colonies appeared during the treatment. Nitrogen gas flushing inhibited the bacterial growth. Bacterial strains in this thesis grew well in 12, 30 and 37 °C temperature and some strains even grew in 43 °C temperature, but no great difference in bacterial growth between two growth media, UHT -milk and BHI -broth were detected. Differences in carbohydrate metabolism were identified with API 50 CH and microplate PM1 methods. According to ATB VET method there was a difference in antibiotic resistance between original strains and their small colony variants, but no difference was identified with microplate PM15. According to this master thesis there were differences between parental strain and its small colony variant in growth, carbohydrate metabolism and antibiotic resistance. As there phenotypic methods results were not compatible with each other, to confirm the discoveries repeats of these analyses are recommended in addition to genotypic methods.
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(2016)The cold storage, utilized to increase the shelf life of raw milk, favors the growth of psychrotrophic bacteria. Psychrotrophs are considered as problematic because they produce heat-stable spoilage enzymes. In addition, some pathogenic bacteria are capable of growing at cold storage temperatures and it has been observed in previous studies that the psychrotrophs in raw milk can be resistant to several antibiotics. In the literature review section of this study, the microbial composition of raw milk and the effect of cold storage, nitrogen gas (N2) treatment and activation of the lactoperoxidase system (LPS) on the raw milk bacteria was reviewed; moreover, the evolution, spread and mechanisms of bacterial antibiotic resistance and the antibiotic resistance on dairy farms and of raw milk bacteria was reviewed. Also the methods to study raw milk bacteria and their antibiotic resistance were documented. The aim of the experimental part was to study the effects of cold storage, N2 gas treatment and LPS treatment on the levels of antibiotic resistant bacteria in raw milk. Untreated, N2-treated and LPS-treated raw milk samples were stored at 6 °C and the counts of bacteria resistant to gentamicin, ceftatzidime, levofloxacin and trimethoprim-sulfamethoxazole were determined in the beginning of the experiments and after three and seven days of cold storage. The antibiotic resistance and spoilage features of bacterial isolates selected from different plates were compared. In addition, DNA fingerprints of selected isolates were obtained by rep-PCR method and for a couple of isolates 16S rRNA gene partial sequencing was performed. The presence of certain antibiotic resistance genes for the isolates partially identified was investigated using a PCR-based method. The length of cold storage affected the proportions of antibiotic resistant bacteria in raw milk. The proportions of antibiotic resistant bacteria in the control milk sample were generally, with the exception of TS-resistant bacteria in some cases, at their lowest after seven days of cold storage when the milk was, though, microbiologically unacceptable. There was a lot of variation in the prevalence of bacterial antibiotic resistance between the milk samples treated differently. The proportions of resistant bacteria were on average clearly higher in LPS samples than in control samples, whereas the effect of the N2 treatment on the proportions of resistant bacteria varied between the experiments. The bacterial isolates resistant to all the considered antibiotics produced less frequently protease and phospholipase than the isolates resistant to fewer antibiotics. The isolates from LPS samples were more frequently resistant to all the antibiotics studied and produced less frequently protease and phospholipase than the isolates from control and N2-treated samples. On the basis of the rep-PCR fingerprints, there were both similar and different strains among the bacterial isolates. Based on the 16S rRNA partial gene sequence, two isolates were identified as Pseudomonas spp. and two isolates as Stenotrophomonas spp. The targeted antibiotic resistance genes were not detected among the considered isolates. Possible reasons can be variations in the gene sequences, suboptimal PCR conditions or that the isolates lacked the genes studied. A PCR product was obtained for one Pseudomonas isolate using mexA-F/R primers, but according to the database the gene sequence did not show homology with antibiotic resistance genes. Further analyses would be required to confirm if the gene studied is linked to antibiotic resistance.
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