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

The objective of the study was to demonstrate proof-of-concept for ResistApp – a newly developed digital platform for antibiotic resistance monitoring in hospital wastewater. ResistApp combines culture-independent, high throughput gene quantification with automated data analysis to synthesise and visualise monitoring data in an interactive dashboard. To do this, wastewater of two hospitals in Helsinki, Finland (HUS1 and HUS2) were monitored for over nine weeks (weeks 25-33 in 2020) for a total of 216 antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), integrons, and taxonomy of bacteria, including bacteria causing hospital acquired infections, and the 16S rRNA gene using high-throughput quantitative PCR. The data from HT-qPCR was analysed and visualised using ResistApp. A higher number of ARGs and MGEs were detected at both hospitals in weeks 27-30 compared to other sampling weeks, with weeks 27-30 grouped separately from other sampling weeks by non-metric multidimensional scaling (NMDS)-ordination analysis. The NMDS ordination also indicated that the two hospitals, which use different amounts of antibiotics, had distinct resistance profiles. The study found that blaGES was the most abundant and prevalent carbapenem resistance gene in both hospitals throughout the sampling period. Low abundances of HAI-bacteria were detected in both hospitals. A correlation analysis was done, which revealed a positive association between blaGES and MGEs in both hospitals. Moreover, substantially more positive associations between carbapenem resistance genes and MGEs were found in HUS1 than HUS2, as well as a strong positive association between blaKPC and Klebsiella pneumoniae in the wastewater of HUS1. Wastewater monitoring with high-throughput qPCR is a promising tool for wastewater-based epidemiology, and it has been successfully used for the surveillance of SARS-CoV-2 -virus. Routine monitoring using ResistApp can capture both the impact of antibiotic use on resistance profiles and the dynamics of these profiles in hospital wastewater. In addition, ResistApp can simplify the analysis of HT-qPCR data considerably, compared to processing large amounts of raw data by hand.

The brown rat (Rattus norvegicus) is a common rodent species found worldwide, especially in urban areas where they cohabit very closely with humans. Known as reservoirs for several zoonotic pathogens and parasites, rats present a significant risk to public health. Despite the concern, studies on rat-borne pathogens in Finland have been limited. This thesis aims to address the gap by focusing on two major zoonotic pathogens in rats, Leptospira spp. and hepatitis E virus. Leptospirosis is a zoonotic disease caused by a spirochete bacterium of the genus Leptospira. Hepatitis E virus is a common cause of acute hepatitis in tropical and subtropical countries, and rat specific hepatitis E virus, Rocahepevirus ratti, is a potential emerging zoonotic pathogen. The aim of the study was to discover if the zoonotic pathogens Leptospira spp. and hepatitis E virus are present in urban rat populations in Helsinki. This was achieved by molecular detection of Leptospira and hepatitis E virus from rat tissue samples. The rat carcasses in this study were received from pest management operators and citizens of Helsinki between 2018 and 2023. Rat antibodies against hepatitis E virus were also attempted to study with commercial ELISA. The PCR positive samples were studied further with either Sanger sequencing or Next-generation sequencing (NGS). Both pathogens were observed in this study. Leptospira spp. was found in 1.23 % of the rat samples studied. Sequence analysis of the Sanger sequenced PCR product suggested that the species was Leptospira interrogans. Rat hepatitis E virus, Rocahepevirus ratti, was observed for the first time in Finland, in 1.79 % of the studied rats. NGS confirmed the virus and phylogenetic analysis showed high homology with sequences of Rocahepevirus ratti genotype C1 from around the world. Even though both pathogens were found in lower quantities than expected, further research and surveillance is needed to estimate the real prevalence and the potential risk for human and animal health.

Brewer's yeasts metabolize sugars and produce ethanol and CO2. This study aimed to investigate the relation between the assimilation of sugars in all-malt wort and isotopic signature of carbon and oxygen in the evolved CO2 from brewery fermentations. The isotopic composition of CO2 was measured by a tunable diode laser absorption spectrometer. The isotopic data obtained with automatic sampling, on-line, and in real-time. Wort samples were collected with 3 h intervals to quantify the residual sugars by high-performance liquid chromatography. Patterns of changes in δ13C and δ18O values were unique to experiments with each yeast type. The common overall ascending trend in δ13C and δ18O values in all experiments can be described by kinetic fractionation of isotopes, which explains that in a bioreaction the lighter isotopes participate in the reaction more readily than the heavier ones. Therefore, the early emergence of light isotopologues of CO2 may be a consequence of the fermentation of light isotopologues of sugars. A sudden decrease and then increase in delta values were observed in all experiments before the residual concentrations of glucose and fructose reach their lowest levels. This can be an indicator of the complete assimilation of monosaccharides by yeast. In the fermentations that yeast was able to consume maltose, δ13C and δ18O values raised considerably in a short period. Concurrently, maltose approached its eventual residual concentrations indicating an endpoint for its utilization by yeast. Our results confirm the hypothesis of a connection between the assimilation of sugars and the isotopic signature of evolved CO2 during brewery fermentations. The findings support the potential of off-gas isotopic analysis to monitor sugar assimilation in brewery fermentations.

Rift Valley fever virus (RVFV) is a mosquito-borne virus of the order Bunyavirales with a tripartite (-)ssRNA genome. It infects humans and cattle, causing a febrile disease with symptoms ranging from mild to severe. Safe and efficient human vaccines have not yet been developed, which underlines the importance of gaining a clear understanding of the viral antigenic surface. One significant challenge for RVFV research is posed by the costly and time-consuming biosafety precautions warranted by the pathogenicity of the virus. The surface of RVFV, formed by the two viral envelope glycoproteins Gn and Gc, could also be studied with a non-pathogenic model, such as a virus-like particle (VLP). A VLP is a macromolecular complex that resembles the virus, especially with respect to its outer structure, but lacks the viral genome. In this work, RVFV VLPs were produced by transient transfection of mammalian cells with genes encoding RVFV glycoproteins Gn and Gc. The objective was to design an optimized production and purification pipeline for RVFV VLPs to elucidate their structure by cryo-electron microscopy. To optimize the VLP production and purification, the effect of sample harvest times and DNA-to-cell ratios of transfection on RVFV glycoprotein expression was examined. Several methods were tested for VLP sample concentration and purification. VLPs were successfully detected in the purified samples by immuno-electron microscopy. Despite some challenges related to sample purity and scarcity of VLPs in samples, which prevented analyses by cryo-electron microscopy, the expression system described in this thesis has great potential to streamline RVFV VLP sample preparation for electron microscopy and to accelerate vital research into the structural properties of this emerging pathogen.

The emergence of zoonotic viruses has been a great concern and threat for global public health. It is important to be aware of the persistence and circulation of zoonoses and their natural hosts in their habitants where wildlife-human interaction can more likely occur. Not many studies have been published regarding the situation in Ireland. In this study, we concentrated in three viral zoonoses, poxviruses, hantaviruses, and arenaviruses, that were screened and detected from Irish rodents specifically bank voles (Myodes glareolus) and wood mice (Apodemus sylvaticus) collected in spring and fall 2021 and spring 2022. By using serological approach such as indirect immunofluorescence assay (IFA), 683 rodent heart samples were screened. Based on serological screening, molecular assays were performed to detect each virus from tissue samples. As a result of the serological screening, 34 antibody-positives either for pox- (CPXV), hanta- (PUUV, DOBV), and arenaviruses (LCMV) were found from which 15 were CXPV, five PUUV, 11 DOBV and three LCMV. The seroprevalence was ranging from 0.4% to 2.2%. When it comes to molecular detection, no viral genome was identified in any of the tissue samples. Although the seroprevalences were low, this study presents the first evidence of CXPV, PUUV, DOBV and LCMV exposures found in Ireland. These findings suggest that there is a possibility of circulation of these viruses among wild rodents and therefore novel risks for viral transmission should be considered. Further research requires increased effort to monitor these viruses and their wildlife hosts in Ireland.

Rhinovirus infections cause the common cold, for which there is no cure. The consequences of the disease are mainly seen as absence from school or work, as the disease is usually mild, self-limiting and symptoms disappear in a few days, but infections can also lead to severe asthma exacerbations and hospitalization. This research is based on earlier findings of benzene sulfonamide derivative compounds that inhibited a broad range of enteroviruses by binding to a newly identified capsid pocket. Here, four promising compounds were tested against four different rhinoviruses belonging to Rhinovirus A and B species in a cell-based inhibition assay. After identification of a potential inhibitory compound against rhinovirus A9 and rhinovirus B14, a rhinovirus A9 homology model was generated, and then the binding was predicted with computational analysis using the rhinovirus A9 homology model and the published structure for rhinovirus B14. As the same compound has previously been shown to bind and inhibit coxsackievirus B4, this virus was used a control. Thermal stability testing of rhinovirus A9 revealed that the virus survives heat treatment up to at least 58°C, in contrast to previously published results. An efficient rhinovirus A9 purification protocol was established and high- quality cryo electron microscopy data were collected for structure determination of rhinovirus A9, which resulted in a 3.1 Å resolution map. The map was used for building the first atomic model of rhinovirus A9. The atomic model revealed that rhinovirus A9 has the expected T=1, pseudo T=3 capsid structure composed primarily of the three β-barrel proteins VP1, VP2 and VP3, with VP4 between the inside of the capsid and the RNA genome. The model confirmed the presence of the interprotomer pocket and VP1 pocket, although density for lipid factor was not detected. The final atomic model was compared to the homology model and used for comparative docking of the inhibitory compound to the pocket. The results revealed that empirical structure determination is still more accurate for large macromolecule complexes than modelling and that empirical data of the binding is required for reliable computational work.

The objective of this thesis was to isolate and characterized phages from Beninese wastewater samples against clinical Acinetobacter baumannii strains for phage therapy use. A. baumannii is one of the most threatening nosocomial bacteria because most of the strains are resistant towards all commonly used antibiotics. One promising alternative treatment method could be phage therapy that utilizes lytic phages to dispose of specific bacteria. In this thesis, seven phages infecting clinical A. baumannii strains were isolated and two of them were characterized more in detail. Phages vB_AbaA_fBenAci001 (fBen-Aci001) and vB_Aba_fBenAci002 (fBen-Aci002) were members of the Friunavirus genus of the Autographiviridae family. In addition, they were the only phages characterised from their respective species to date. The genome analysis revealed 82.2% identity between the phages. No genes indicating lysogenic lifecycle, or genes encoding bacterial toxins or antibiotic resistance were identified from either of them. Phage fBen-Aci001 were infecting 4% and fBen-Aci002 were infecting 9% of tested 23 clinical A. baumannii isolates. Phylogenetic tree which was constructed based on whole genome sequences was compared to the trees that were made using tailspike proteins and capsid proteins. No correlation between genome-wide tree and trees built based on single genes were seen. In conclusion, the Beninese hospital wastewater appeared to be a good source for A. baumannii phages, as several phages were isolated and they were infecting clinical multidrug resistant strains isolated from Finnish patients. Phages fBen-Aci001 and fBen-Aci002 were concluded to be potential candidates to be used in the phage therapy though the narrow host range might negatively affect their usability.

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.

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.

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.