Browsing by master's degree program "Magisterprogrammet i mikrobiologi och mikrobibioteknik"
Now showing items 41-47 of 47
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(2021)The Italian white truffle Tuber magnatum is globally one of the most sought after culinary delicacies with retail prices ranging up to 4000€/kg. The White truffles sold on the market are harvested from natural truffle forests as the successful cultivation of the species has to date not been reached. Great economical interest lies in better understanding the growth, life cycle and ascocarp production of T. magnatum, which is why researchers have focused on studying the interactions between the truffle and soil microorganisms. In this study, the bacterial genus Methylosinus and the phylum Firmicutes were found to be present at T. magnatum productive sites. It was also found that the diversity of soil bacteria changes when a) moving from one succession stage to another according to a truffle producing site’s life cycle and b) when the canopy cover of an area changes. These findings can help with better characterising and forecasting T. magnatum productive sites, yet further research is needed to confirm whether the bacteria found present in this study are a cause or effect of white truffle productivity.
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(2020)The use of antimicrobials in livestock production has shown to increase the abundance of antibiotic resistance genes (ARGs) in animal microbiomes. The use of manure as a fertilizer is essential in animal agriculture, however, manure application disseminates ARGs to the farm environment. In soil, the ARGs could be horizontally transferred to the environmental bacteria. Antimicrobial resistance is currently mitigated by limiting the use of antimicrobials for animals; thus, it is important to examine the ARG dynamics in countries where antimicrobial use is restricted. In addition to the antimicrobial use, also manure application rates are tightly regulated in Finnish dairy farming, offering suitable sites for examining the transmissions of ARGs in response to agricultural practices. The main aim of this study was to determine the host range of three antibiotic resistance genes by culture-independent epicPCR to understand the fate of antimicrobial resistance in agricultural environments. The cells were extracted from manure and soil samples taken from two Finnish dairy farms. Aminoglycoside (strB), beta-lactam (blaOXA-58) and tetracycline (tetM) resistance genes were linked with a phylogenetic marker gene to determine the host bacteria using epicPCR. Results were compared to the total bacterial community. In total, 664 OTU’s were linked to ARGs. Antibiotic resistance genes strB and tetM shared six host genera and three genera were found to carry all the studied genes. The most common host genera for tetM were Escherichia-Shigella, Sedimentibacter and Fibrobacter. For blaOXA-58, the most common hosts were Sphingobacterium and Acinetobacter. Acinetobacter, Pseudomonas and Psychrobacter carried strB genes in all studied samples. For the first time the host range of ARGs in manure and soil communities were determined by epicPCR, providing also valuable information for further improving comparatively new method.
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(2020)The emerging crisis of antimicrobial resistance is especially worrisome in low-income countries that lack controlled antibiotic policy and have poor infrastructure. Inadequate hygiene practices combined with ability of microbes to quickly evolve and adapt to changes rise the concern of resistance of infectious pathogens to many first-line antimicrobial drugs. Moreover, wastewaters that are widely used as irrigation water in urban gardening in sub-Saharan Africa, can function as vehicle for the dissemination of bacteria that carry antimicrobial resistance genes into the surrounding environment. In this study, eight anthropogenically impacted water samples were collected from Burkina Faso and Mali and differences in their microbial communities were evaluated by 16S rRNA gene sequencing. Also, the presence of antimicrobial resistance genes was examined with SmartChip qPCR. The bacterial host range of blaNDM, blaCTX-M, blaOXA and qacE∆1 was profiled using a novel culture- independent technique, Emulsion, Paired Isolation and Concatenation PCR (epicPCR). The presence of 202 genes associated with antimicrobial resistance were detected with SmartChip qPCR array analysis, including carbapenemase genes that can transfer horizontally. Worryingly, sixteen taxonomical units, including possible human pathogens Acinetobacter, Klebsiella, Escherichia and Pseudomonas, were found to carry all the four genes investigated with epicPCR. The most abundant genus Arcobacter along with Dechloromonas, Methylotenera, MM1 and Methylophilus were new discoveries as blaNDM hosts. Furthermore, a considerable number of blaOXA and clinical class 1 integron marker qacE∆1 gene hosts were discovered in every sample. Lastly, putative events of horizontal gene transfer in two WWTP samples were observed. Broad host range of blaOXA and qacE∆1 genes suggests a heavy antimicrobial resistance genes burden in West Africa and the results support the theory that environmental bacteria can function as resistance gene reservoirs. These results show occurrence of horizontally transferrable blaNDM and blaCTX-M genes in pathogens especially in hospital wastewater, and a threat of their spread into the environment and to the community. However, to decipher their role in the infectious disease burden in Africa, more research is needed.
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(2021)The human gastrointestinal track is populated by gut microbiota that consist of viruses, bacteria, archaea, fungi and micro-eukaryotes. The gut microbiota is beneficial for the host in many ways, including synthesis of short chain fatty acids and vitamins, and supporting the maturation and normal functions of the immune system. A healthy gut microbiota provides colonization resistance by preventing the attachment and growth of pathogenic microorganisms in the intestines. The use of antibiotics is a common cause of intestinal microbiota disturbation and weakened colonization resistance, which can lead to intestinal infection after antibiotic treatment. The leading cause of antibiotic-associated diarrhea is infection caused by bacterium Clostridioides difficile. C. difficile infection is generally treated with vancomycin or metronidazole, but in approximately 20 % of the patients the infection renewed. In these cases, the infection is referred to as a recurrent C. difficile infection. A recurrent C. difficile infection is treated with a repeated course of antibiotics or a fecal microbiota transplantation (FMT). FMT is a medical procedure in which feces of a healthy pre-screened donor is infused into a patient’s intestine in order to restore a healthy gut microbiota. FMT is an effective treatment for recurrent Clostridioides difficile infections, and its efficacy to treat other diseases linked with intestinal dysbiosis is being studied. Rectal bacteriotherapy, in which a transplant consists of specific intestinal bacterial strains, is used similarly to FMT, has been studied as a substitute for FMT to further reduce possible risks of fecal transplant such as transferring yet-unknown pathogens into patients. The gut microbiota is a favorable ecosystem for enrichment of antibiotic resistant genes through horizontal gene transfer between bacteria. From individuals carrying C. difficile bacteria not everyone will develop C. difficile infection after antibiotic treatment, and not all C. difficile infections lead to recurrent C. difficile infections. The reason for this might be the colonization resistance against C. difficile provided by the antibiotic resistant microbes that weren’t affected by the antibiotic treatment. Antibiotic resistant commensal bacteria living in the intestine may have an important role in maintaining the colonization resistance during and after antibiotic treatment. Since vancomycin is used as a treatment for C. difficile infections, introduction of non-pathogenic vancomycin resistant bacteria in form of bacteriotherapy could provide a better colonization resistance during the antibiotic treatment. The aim of this study was to examine the incidence of vancomycin resistance gene vanB in the microbiota of three FMT donors and 13 recipients, and to study culturable vancomycin resistant gut bacteria isolated from FMT donors. The vanB gene carriage of FMT donors and patients before and after FMT was studied. Furthermore, a total of 68 vancomycin resistant bacterial isolates were cultivated and purified from FMT donors, and the taxonomical identification of isolates was performed based on their 16S rRNA gene. Whether the vancomycin resistance of the isolates resulted from vanB gene was assessed using a PCR method. The results showed that all the FMT donors carried a vanB gene in their microbiota. The gene was present in the patients’ intestinal microbiota one month after FMT and half of the patients carried the gene still after 8 or 12 months after FMT. The vanB gene wasn’t found in the samples collected from patients before FMT. It is likely that the vanB gene had transferred from donors to patients via FMT. However, vanB gene couldn’t be detected in any of the cultivated bacterial isolates, and thus the bacterial strains carrying the gene were left unidentified. The isolates represented 21 different bacterial species. Donors differed from each other with respect to overall species distribution, which supports the previous findings of individual specific microbiotas. All three donors carried species from the genus Bacteroides and lately reclassified genus Lactobacillus, but none of the species was found in all three donors. The isolates found in this study might be candidate strains for rectal bacteriotherapy, but further studies are required to determine the effectiveness and safety of these isolates.
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(2020)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.
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(2021)Formation of methane as a biogas from acetate through methanogenesis could be very energy efficient and economically feasible. The acetate utilisation through aceticlastic pathway in methanogenic archaea Methanosarcina is well understood, yet the regulation of acetate utilisation is mainly unknown. The study focused on determination of the time frame of the initiation periods of protein syntheses via bioorthogonal non-canonical amino acid tagging (BONCAT) and copper catalysed click chemistry in Methanosarcina acetivorans and Methanosarcina barkeri, during a long lag phase (around 25 days) after growth substrate was shifted from methanol to acetate. In two experiments the proteins translated after the substrate shift (newly synthetised proteins) were labelled with methionine surrogates via BONCAT and tagged with fluorescent dye and a biotin tag via click chemistry for further detections. To have more complete understanding of the substrate shift acetate concentrations were observed via nuclear magnetic resonance and cell density was monitored via optical density measurements. Only rough time frames of the initiation periods of protein syntheses in both organisms could be estimated from the gel detection of fluorescent tagged proteins. The results indicate that acetate consumption and de-novo protein translation occurs early after substrate switch. In conclusion, the overall utilisation of BONCAT in the labelling of newly synthetised proteins to provide information about the beginning of protein synthesis after substrate shift in Methanosarcina was successful. I was able to detect newly synthetised proteins in both experiments and estimate time windows for beginning of protein syntheses. The information of time windows helps further research to identify the proteins in substrate shift and understand the regulation of substrate shift in Methanosarcina.
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(2021)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.
Now showing items 41-47 of 47