Browsing by master's degree program "Master's Programme in Microbiology and Microbial Biotechnology"

Antimicrobial resistance (AMR) is an emerging global health threat with the growing number of antibiotic-resistant bacteria (ARB) having the alarming potential to return humanity to the pre-antibiotic era. Intensive animal production is globally one of the biggest sectors using antibiotics. It has been studied that fertilizing fields with animal manure spreads antimicrobial resistance genes (ARGs) in natural environments. The aim of this study was to determine the host range of three ARGs tetM, strB and qacE∆1 in soil and manure samples collected from a Finnish swine farm. In addition, the microbial communities in the same soil and manure samples were studied and compared. Six different sample types were taken, four from soil and two from manure. Soil samples included unfertilized soil, fertilized soil, soil two weeks after fertilization and soil six weeks after fertilization. Manure samples were taken from fresh and stored manure. Host range analysis was done by using Emulsion, Paired Isolation and Concatenation PCR (epicPCR). EpicPCR enables to link a gene of interest to the 16S rRNA gene of the bacterium that carries the gene in its genome. Microbial communities in soil and manure were analyzed and compared with the traditional 16S rRNA gene sequencing. Host range analysis with epicPCR revealed various bacterial genera as carriers for studied ARGs. Fertilized soil had the highest number of genera carrying the ARGs. This indicates that land application with animal manure increases the ARG load in soil. Microbial communities were found significantly different in soil and manure according to the 16S rRNA gene sequences. The results of epicPCR indicate that epicPCR has also potential for solid samples such as soil and manure as according to publications it has been mainly used for different water samples e.g., wastewaters. As a method epicPCR still requires optimization if applied for these sample materials in the future. A clear reduction in the number of genera carrying the ARGs was observed in six weeks after fertilization. Therefore, fertilizing fields only before cropping season, instead of fertilizing the fields year-round, might be one solution for reducing the ARG dissemination in soil in countries with high antibiotic consumption.

Time-kill assays are commonly used for determining an antibiotic’s activity against a bacterial strain over time. The assay is carried out by adding an antibiotic to media containing bacteria, and the colony-forming units/ml is determined at different timepoints using the plate count method. Time-kill assays are commonly used for determining synergism between two or more antibiotics, and for determining if an antibiotic has a time- or concentration dependent effect. The aim of this study was to validate a modified time-kill assay. The modified assay is high-throughput screening-compatible, and less reagent- and labour-intensive compared to the traditional method. In this modified assay, the number of viable bacteria is evaluated with resazurin reduction. Metabolically active bacteria reduce resazurin (non-fluorescent) to resorufin (fluorescent) during growth. Resazurin to resorufin reduction occurs faster with increasing amounts of bacteria. The resazurin reduction was measured by two alternative methods: with a spectrophotometer measuring fluorescence and with OmniLog®, an imaging incubator that measures light transmission through the wells of microplates. First, the applicability of the assay to different bacterial species was tested by comparing optical density- and fluorescence-based readings. Then, cold tolerance and plate uniformity assays were carried out, and the minimum inhibitory concentrations of the selected antibiotics were then determined. The traditional method and the resazurin-reduction assay were then performed in parallel. The results showed that the spectrophotometric and OmniLog® measurements worked similarly for Enterococcus faecalis and Klebsiella pneumoniae. The cold treatment did not affect the viability of neither E. faecalis nor K. pneumoniae. With K. pneumoniae, the plates were uniform at both high and low bacterial concentrations, whereas the variability increased at lower bacterial concentrations with E. faecalis. The colony counting assay and the resazurin reduction assay correlated well for five out of the seven antibiotics against E. faecalis. These include the cell wall-active antibiotics ampicillin and vancomycin. When the translation inhibitors tetracycline and chloramphenicol were used, the results obtained from the two methods differed markedly. In conclusion, the correlation between the two methods was dependent on the antibiotics’ mechanism of action.

Tick-borne diseases pose a major One Health threat in Finland. Tick-borne pathogens are transmitted through a tick-bite, impacting human, companion animal, livestock, and wildlife health. The geographical distributions of many tick species are shifting as a result of climate change. In northern Europe, tick distributions are expanding northward due to previously inhospitable climates becoming warmer. This has led to enhanced contact between ticks, humans and companion animals – increasing the incidence of tick-borne disease transmission. Established tick-borne pathogens such as TBEV and Borrelia spp. are considered endemic across Finland. However, the diversity of viruses found within ticks in Finland remains largely unexplored. Thus, here we aimed to characterize the viromes of the dominant tick species found in Finland. We used Next-generation sequencing methods to characterize the viral components of Ixodes Ricinus and Ixodes persulcatus ticks associated with companion dogs across Finland. In total, 4 different pools consisting of 36 ticks were sequenced. Our results identified 9 unique viruses belonging to the families Nairoviridae, Phenuiviridae, Iflaviridae and Partitiviridae. We identified numerous recently characterized nairo-like viruses, including Pustyn virus and Gakugsa tick virus. We were able to bridge the gap on the geographical spread of many of these viruses, including reporting Sulina virus and Norway nairovirus 1 for the first time after their discovery. Phylogenetic analyses revealed close relationships between the recently characterized RNA viruses, and a high degree of conservation across wide geographical ranges. As previously highlighted, the pathogenicity of these newly identified viruses is not yet established. However, our results indicate many of the viruses identified are closely related to viruses associated with acute febrile illness in humans. Due to our small sample size, we are unable to imply the virome composition for all ticks in Finland. However, we highlight a high degree of viral diversity even within a small sample size of ticks. Further research is urgently required to establish whether these recently characterised viruses pose a threat to human or animal health in Finland.