| 1. |
- Bergman, Jessica M.
(författare)
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Genetics and Growth Regulation in Salmonella enterica
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Most free-living bacteria will encounter different environments and it is therefore critical to be able to rapidly adjust to new growth conditions in order to be competitively successful. Responding to changes requires efficient gene regulation in terms of transcription, RNA stability, translation and post-translational modifications.Studies of an extremely slow-growing mutant of Salmonella enterica, with a Glu125Arg mutant version of EF-Tu, revealed it to be trapped in a stringent response. The perceived starvation was demonstrated to be the result of increased mRNA cleavage of aminoacyl-tRNA synthetase genes leading to lower prolyl-tRNA levels. The mutant EF-Tu caused an uncoupling of transcription and translation, leading to increased turnover of mRNA, which trapped the mutant in a futile stringent response.To examine the essentiality of RNase E, we selected and mapped three classes of extragenic suppressors of a ts RNase E phenotype. The ts RNase E mutants were defective in the degradation of mRNA and in the processing of tRNA and rRNA. Only the degradation of mRNA was suppressed by the compensatory mutations. We therefore suggest that degradation of at least a subset of cellular mRNAs is an essential function of RNase E.Bioinformatically, we discovered that the mRNA of tufB, one of the two genes encoding EF-Tu, could form a stable structure masking the ribosomal binding site. This, together with previous studies that suggested that the level of EF-Tu protein could affect the expression of tufB, led us to propose three models for how this could occur. The stability of the tufB RNA structure could be affected by the elongation rate of tufB-translating ribosomes, possibly influenced by the presence of rare codons early in the in tufB mRNA.Using proteomic and genetic assays we concluded that two previously isolated RNAP mutants, each with a growth advantage when present as subpopulations on aging wild-type colonies, were dependent on the utilization of acetate for this phenotype. Increased growth of a subpopulation of wild-type cells on a colony unable to re-assimilate acetate demonstrated that in aging colonies, acetate is available in levels sufficient to sustain the growth of at least a small subpopulation of bacteria.
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| 2. |
- Ancillotti, Mirko, 1981-
(författare)
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Antibiotic Resistance: A Multimethod Investigation of Individual Responsibility and Behaviour
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The rapid development of antibiotic resistance is directly related to how antibiotics are used in society. The international effort to decrease and optimise the use of antibiotics should be sustained by the development of policies that are sensitive to social and cultural contexts.The overarching aim of the thesis was to explore and discuss the Swedish public’s beliefs, values and preferences influencing engagement in judicious antibiotic behaviour.Study I explored through focus group discussions lay people’s perceptions and beliefs about antibiotics and antibiotic resistance. The Health Belief Model was used to identify factors that could promote or hinder engagement in judicious antibiotic behaviour. Participants found antibiotic resistance to be a serious problem but were not equally worried about being affected by it. There was a tension between individual and collective reasons for engaging in judicious behaviour.Study II explored lay people’s views on the moral challenges posed by antibiotic resistance through focus group discussions. Participants identified in the decreasing availability of effective antibiotics a problem of justice, which involves individual as well as collective moral responsibility. Different levels of policy demandingness were discussed in light of these results.Study III investigated, through an online Discrete Choice Experiment, public preferences regarding antibiotic treatment and the relative weight of antibiotic resistance in decision-making. Public behaviour may be influenced by concerns over the rise of antibiotic resistance. Therefore, stressing individual responsibility for antibiotic resistance in clinical and societal communication may affect personal decision-making.Study IV clarified the notions of collective and individual moral responsibility for antibiotic resistance and suggested a virtue-based account thereof. While everyone is morally responsible for minimising his/her own contribution to antibiotic resistance, individuals do or do not engage in judicious antibiotic behaviour with different degrees of voluntariness.The findings suggest that people could change their behaviour due to concerns over their own contribution to antibiotic resistance. Effective health communication should be developed from an appraisal of people’s attitudes, beliefs and social norms that influence antibiotic resistance related behaviours. Policy demandingness should take into account socioeconomic factors characterising local realities.
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| 3. |
- Andersson, Dan, 1967-
(författare)
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Exploring Perceptions of Route Environments in Relation to Walking
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: Every walk takes place in a route environment, which can play an important role in deterring or facilitating walking. The focus of this thesis is on the perceptions of environmental variables, and how they relate to appraisals of route environments as hindering – stimulating for walking and unsafe – safe for reasons of traffic, in two metropolitan environments. Another focus is to expand the state of knowledge concerning the criterion-related validity of the Active Commuting Route Environment Scale (ACRES).Methods: Commuting pedestrians in Greater Stockholm, Sweden, were recruited via advertisements. From the inner urban area there were 294 participants (77% women), aged 49.5 years, and from the suburban areas there were 233 participants (82% women), aged 50.0 years. 77 individuals walked in both areas. The participants evaluated their commuting route environments using the ACRES. Correlation, multiple regression, and mediation analyses were used to explore the relationships between the variables. Comparisons of environmental ratings between groups and settings were performed with t-tests. Studies 1 and 2 focused on the inner urban area and studies 3 and 4 on the suburban areas. Studies 1 and 3 focused exclusively on the relations between the four motorized traffic variables (vehicle speed, vehicle flow, noise, and exhaust fumes), and their relations to the outcome variables (hinders – stimulates walking and unsafe – safe traffic). Studies 2 and 4 utilised proxies from studies 1 and 3 and combined them with other environmental variables to further the understanding of route environmental variables in relation to walking.Results: In both areas, aesthetics and greenery were positively related to stimulating walking, whereas noise, a proxy for motorized traffic, was negatively related. Aesthetics was also positively related to unsafe – safe traffic in the inner urban area, whereas greenery had the corresponding role in suburbia. Another important finding was that greenery also influenced aesthetics positively in both areas. Thus, greenery had both a direct and an indirect positive effect. On the other hand, noise influenced aesthetics negatively in the inner urban area, whereas vehicle flow had the corresponding role in suburbia. A number of variables conjointly influenced the outcome unsafe – safe traffic negatively in both areas (speeds of motor vehicles, noise, conflicts, congestion: pedestrians, red lights, and course of the route). The route environment profiles differed distinctly between the two areas.Conclusions: Several route environmental variables appear to be particularly influential in relation to pedestrian commuting, e.g., aesthetics, greenery, and noise. An important finding is that both positive and negative interactions, between certain predictor variables, were disclosed. The contrasting route environment profiles in the different settings strengthen the criterion-related validity of the ACRES. The findings expands the state of knowledge concerning the relations between the environment and walking. If implemented, these findings can influence public health positively.
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| 4. |
- Garbom, Sara, 1975-
(författare)
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A strategy to identify novel antimicrobial compounds : a bioinformatics and HTS approach
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bacterial infections are again becoming difficult to treat because the microbes are growing increasingly resistant to the antibiotics in use today. The need for novel antimicrobial compounds is urgent and to achieve this new targets are crucial. In this thesis we present a strategy for identification of such targets via a bioinformatics approach. In our first study we compared proteins with unknown and hypothetical function of the spirochete Treponema pallidum to five other pathogens also causing chronic or persistent infections in humans (Yersinia pestis, Neisseria gonorrhoeae, Helicobacter pylori, Borrelia burgdorferi and Streptococcus pneumoniae). T. pallidum was used as a starting point for the comparisons since this organism has a condensed genome (1.1 Mb). As we aimed at identifying conserved proteins important for in vivo survival or virulence of the pathogens we reasoned that T. pallidum would have deleted genes not important in the human host. This comparison yielded 17 ORFs conserved in all six pathogens, these were deleted in our model organism, Yersinia pseudotuberculosis, and the virulence of these mutant strains was evaluated in a mouse model of infection. Five genes were found to be essential for virulence and thus constitute possible antimicrobial drug targets.We have studied one of these virulence associated genes (vags), vagH, in more detail. Functional and phenotypic analysis revealed that VagH is an S-adenosyl-methionine dependent methyltransferase targeting Release factor 1 and 2 (RF1 and RF2). The analysis also showed that very few genes and proteins were differentially expressed in the vagH mutant compared to wild-type Yersinia. One major finding was that expression of the Type III secretion system effectors, the Yops, were down regulated in a vagH mutant. We dissected this phenotype further and found that the down regulation was due to lowered amounts of the positive regulator LcrF. This can be suppressed either by a deletion of yopD or by over expression of the Ribosomal Recycling Factor (RRF). These results indicate that YopD in addition to its role in translational regulation of the Yops also plays a part in the regulation of LcrF translation. We suggest also that the translation of LcrF is particularly sensitive to the amount of translation competent ribosomes and that one effect of a vagH mutation in Y. pseudotuberculosis is that the number of free ribosomes is reduced; this in turn reduces the amount of LcrF produced thereby causing a down regulation of the T3SS. This down regulation is likely the cause of the attenuated virulence of the vagH mutant.Finally, we set up a high throughput screening assay to screen a library of small molecules for compounds with inhibiting the VagH methyltransferase activity. Five such compounds were identified and two were found to inhibit VagH also in bacterial culture. Furthermore, analogues to one of the compounds showed improved inhibitory properties and inhibited the T3SS-dependent cytotoxic response induced by Y. pseudotuberculosis on HeLa cells.We have successfully identified five novel targets for antimicrobial compounds and in addition we have discovered a new class of molecules with antimicrobial properties.
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| 5. |
- Lind, Peter A, 1980-
(författare)
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Evolutionary Dynamics of Mutation and Gene Transfer in Bacteria
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The study of bacterial evolution is fundamental for addressing current problems of antibiotic resistance and emerging infectious diseases and lays a solid foundation for successful and rational design in biotechnology and synthetic biology. The main aim of this thesis is to test evolutionary hypotheses, largely based on theoretical considerations and sequence analysis, by designing scenarios in a laboratory setting to obtain experimental data. Paper I examines how genomic GC-content can be reduced following a change in mutation rate and spectrum. Transcription-related biases in mutation location were found, but no replicative bias was detected. Paper II explores the distribution of fitness effects of random substitutions in two ribosomal protein genes using a highly sensitive fitness assay. The substitutions had a weakly deleterious effect, with low frequencies of both neutral and inactivating mutations. The surprising finding that synonymous and non-synonymous substitutions have very similar distribution of fitness effects suggests that, at least for these genes, fitness constraints are present mainly on the level of mRNA instead of protein. Paper III examines selective barriers to inter-species gene transfer by constructing mutants with a native gene replaced by an orthologue from another species. Results suggest that the fitness costs of these gene replacements are large enough to provide a barrier to this kind of horizontal gene transfer in nature. The paper also examines possible compensatory mechanisms that can reduce the cost of the poorly functioning alien genes and found that gene amplification acts as a first step to improve the selective contribution after transfer. Paper IV investigates the fitness constraints on horizontal gene transfer by inserting DNA from other species into the Salmonella chromosome. Results suggest that insertion of foreign DNA often is neutral and the manuscript provides new experimental data for theoretical analysis of interspecies genome variation and horizontal gene transfer between species.
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| 6. |
- Lofton Tomenius, Hava, 1976-
(författare)
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Mechanisms and Biological Costs of Bacterial Resistance to Antimicrobial Peptides
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The global increasing problem of antibiotic resistance necessarily drives the pursuit and discovery of new antimicrobial agents. Antimicrobial peptides (AMPs) initially seemed like promising new drug candidates. Already members of the innate immune system, it was assumed that they would be bioactive and non-toxic. Their common trait for fundamental, non-specific mode of action also seemed likely to reduce resistance development.In this thesis, we demonstrate the ease with which two species of pathogenic bacteria, the gram-negative Salmonella typhimurium (S. typhimurium), and the gram-positive Staphylococcus aureus (S. aureus), can gain increased tolerance and stable resistance to various AMPs. By serially passaging each bacterial species separately under increasing AMP selection pressure we observed increasing AMP tolerance. Resulting in independent bacterial lineages exposed to four different AMPs (including a two-AMP combination) that exhibited 2 to 16-fold increases in MIC. Substantial cross-resistance between the AMPs was observed. Additionally, the S. aureus mutants were found to be cross-resistant to human beta-defensins 1, 2, 3, and 4.The LPS molecule, with mutations in the waaY, pmrB and phoP genes, was the principal target for S. typhimurium resistance development. The main target for S. aureus remained elusive. Reduced membrane potential was a common change for two of the mutants, but not for the others. All sequenced mutants had one or more mutations in various stress response pathways.Fitness of the resistant mutants was assayed by growth rate analysis and in vitro virulence factor testing (e.g. survival response to bile, superoxide, acidic pH). Furthermore an in vivo survival/virulence test involving a mouse competition experiment (S. typhimurium) and sepsis model (S. aureus) was performed. In the absence of AMPs there was often little or no fitness reduction in the mutants. Our results suggest that AMP resistance mechanisms do not irrevocably weaken either species with regard to virulence characteristics or survival within the host.In light of these findings, we suggest that the progression of therapeutic use of AMPs should proceed with great caution since otherwise we might select for AMP resistant mutants that are more resistant to our innate host defenses and thereby potentially more virulent.
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| 7. |
- Lytsy, Birgitta, 1968-
(författare)
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Enterobacteriaceae Producing Extended-Spectrum Beta-Lactamases : Aspects of Detection, Epidemiology and Control
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Enterobacteriaceae belong to the normal enteric flora in humans and may cause infections. Escherichia coli is the leading urinary tract pathogen with septicaemic potential, whereas Klebsiella pneumoniae causes opportunistic infections and often outbreaks in hospital settings. Beta-lactams are the first choice for treatment of infections caused by Enterobacteriaceae, and might be destroyed by extended-spectrum beta-lactamases, ESBLs. ESBLs hydrolyse all beta-lactams except cephamycin and carbapenems, and constitute a large heterogeneous group of enzymes with different origins. The phenotypic and molecular characteristics of a K. pneumoniae strain causing a major outbreak at Uppsala University Hospital between 2005 and 2008 were described. The strain was multiresistant and produced CTM-M-15, a common ESBL type in Europe. Due to the lack of obvious epidemiological links between patients, a case-control study was performed, which identified risk factors for the acquisition of the outbreak strain in urine cultures. The complex chain of transmission facilitated by patient overcrowding and the interventions applied to curb the outbreak, was revealed in the subsequent study. In the final study, the genetic background of the observed increase in ESBL-producing E. coli isolates during the K. pneumoniae outbreak was explored. The utility of six typing methods in epidemiological investigations of a local outbreak with ESBL-producing E. coli was compared. The increase of ESBL-producing E. coli isolates was not secondary to the K. pneumoniae outbreak. Twentytwo per cent belonged to the epidemic O25b-ST131 clone and only a limited number of infections were caused by nosocomial transmission. ESBL-producing Enterobacteriaceae are a challenge to clinical microbiology laboratories and infection control teams. To investigate their dissemination, typing methods need to be continuously adapted to the current situation. Proper hand disinfection and structural key problems such as over-crowding, under-staffing, lack of single rooms and bathrooms must be adressed to limit transmission.
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| 8. |
- Norrmén, Gunilla, 1944-
(författare)
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To be or not to be Sick Certified with Special Reference to Physician and Patient Related Factors
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Objectives The aim of this thesis was to assess the importance of general practitioners (GP) and patient related factors for the GPs’ decision to sick certify or not to sick certify the patients. Study population and methods The data were obtained from a cross-sectional questionnaire study of GP-patient consultations. 65 GPs responded to one questionnaire about themselves and one questionnaire about each of the altogether 642 consultations. The patients responded to a questionnaire about themselves and the consultation, altogether 521 consultations. Various combinations of the three questionnaires were used in the four papers on which this thesis is based. Results Among GPs, long experience of family medicine and working part-time were significant determinants for issuing more sick leave certificates. Complaints perceived as clearly somatic by the physician decreased the chance of sick certifications, and complaints resulting in severe limitation of occupational work capacity, as assessed by the patient as well as the physician, increased the chance of sick certification, as did appointments for loco-motor complaints. Among work related factors, high ‘authority over decisions’ and high ‘social support’ were associated with reduced sickness certification probability. Worrying about illness or injury risks from work increased sickness certification. GPs and their patients took a fairly similar view to statements on health related and insurance system related matters. GPs’ opinions seem to have a greater impact than patients’ on the GPs’ decision to sickness certify a patient or not. Conclusions A number of patient and GP related factors were associated with the probability of getting sick certified. The patient’s own judgement of impaired work ability was important for sickness certification, but a shared judgement and decision between the GP and the patient appears probable in most cases.
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| 9. |
- Thulin, Elisabeth, 1984-
(författare)
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Mechanisms and Dynamics of Mecillinam Resistance in Escherichia coli
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The introduction of antibiotics in healthcare is one of the most important medical achievements with regard to reducing human morbidity and mortality. However, bacterial pathogens have acquired antibiotic resistance at an increasing rate, and due to a high prevalence of resistance to some antibiotics they can no longer be used therapeutically. The antibiotic mecillinam, which inhibits the penicillin-binding protein PBP2, however, is an exception since mecillinam resistance (MecR) prevalence has remained low. This is particularly interesting since laboratory experiments have shown that bacteria can rapidly acquire MecR mutations by a multitude of different types of mutations.In this thesis, I examined mechanisms and dynamics of mecillinam resistance in clinical and laboratory isolates of Escherichia coli. Only one type of MecR mutations (cysB) was found in the clinical strains, even though laboratory experiments demonstrate that more than 100 genes can confer resistance Fitness assays showed that cysB mutants have higher fitness than most other MecR mutants, which is likely to contribute to their dominance in clinical settings.To determine if the mecillinam resistant strains could compensate for their fitness cost, six different MecR mutants (cysB, mrdA, spoT, ppa, aspS and ubiE) were evolved for 200-400 generations. All evolved mutants showed increased fitness, but the compensation was associated with loss of resistance in the majority of cases. This will also contribute to the rarity of clinical MecR isolates with chromosomal resistance mutations.How MecR is mediated by cysB mutations was previously unclear, but in this thesis I propose and test a model for the mechanism of resistance. Thus, inactivation of CysB results in cellular depletion of cysteine that triggers an oxidative stress response. The response alters the intracellular levels of 450 proteins, and MecR is achieved by the increase of two of these, the LpoB and PBP1B proteins, which rescue the cells with a mecillinam-inhibited PBP2.Mecillinam is used for UTI treatments and to investigate mecillinam resistance in a more host-like milieu, MecR strains were grown in urine and resistance was examined. Interestingly, this study showed that neither laboratory, nor clinical cysB mutants are resistant in urine, most likely because the cysteine present in the urine phenotypically reverts the bacteria to susceptibility. These findings suggest that mecillinam can be used to treat also those clinical strains that are identified as MecR in standard laboratory tests, and that testing of mecillinam susceptibility in the laboratory ought to be performed in media that mimics urine to obtain clinically relevant results.In summary, the work described in this thesis has increased ourgeneral knowledge of mecillinam resistance and its evolution. Hopefully this knowledge can be put to good use in clinical settings to reduce the negative impact of antibiotic resistance.
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| 10. |
- Tobin, Christina, 1980-
(författare)
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Removal and Replacement of Ribosomal Proteins : Effects on Bacterial Fitness and Ribosome Function
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein synthesis is a complex process performed by sophisticated cellular particles known as ribosomes. Although RNA constitutes the major structural and functional component, ribosomes from all kingdoms contain an extensive array of proteins with largely undefined functional roles. The work presented in this thesis addresses ribosomal complexity using mutants of Salmonella typhimurium to examine the physiological effects of ribosomal protein (r-protein) removal and orthologous replacement on bacterial fitness and ribosome function. The results of paper I demonstrate that removal of small subunit protein S20 conferred two independent translation initiation defects: (i) a significant reduction in the rate and extent of mRNA binding and (ii) a drastic decrease in the yield of 70S complexes caused by an impairment in subunit association. The topographical location of S20 in mature 30S subunits suggests that these perturbations are the result of improper orientation of helix 44 of the 16S rRNA when S20 is absent. In paper II we show that the major functional impairment associated with loss of large subunit protein L1 manifested as an increase in free ribosomal subunits at the expense of translationally active 70S particles. Furthermore, the formation of free ribosomal subunits was imbalanced suggesting that L1 is required to suppress degradation or promote formation of 30S subunits. Compensatory evolution revealed that mutations in other large subunit proteins mitigate the cost of L1 removal, in one case seemingly via an increase in 70S complex formation. As shown in paper III, the large fitness costs associated with complete removal of r-proteins is in contrast to the generally mild costs of orthologous protein replacement, even in the absence of a high degree of homology to the native protein. This clearly demonstrates the robustness and plasticity of the ribosome and protein synthesis in general and it also implies that functional constraints are highly conserved between these proteins. The findings of paper III also allowed us to examine the barriers that constrain horizontal gene transfer and we find that increased gene dosage of the sub-optimal heterologous protein may be an initial response to stabilize deleterious transfer events. Overall the results highlight the requirement of r-proteins for the maintenance of ribosomal structural integrity.
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