This thesis is about analyzing genetic differences among isolates of Francisella tularensis – the tularemia-causing bacterium. To elucidate how these bacterial isolates are related, and their geographical and genetic origins, I have developed typing assays for Francisella and used them to study the epidemiology of tularemia. Tularemia is an infectious disease of humans and other mammals found throughout the Northern Hemisphere. The severity of the disease depends on the type of F. tularensis causing the infection. In Sweden, as in other countries of Europe and Eurasia, tularemia is caused by F. tularensis subsp. holarctica, while other varieties of the bacterium occur in Middle Asia and North America. It is important to identify a tularemia infection promptly in order to initiate the correct antibiotic treatment. A rapid identification of the causative F. tularensis variety gives additional clinical information. In recent years, several genomes of various Francisella strains have been sequenced, and in this thesis, I have utilized these genomes to identify genetic markers. In studies reported in the first paper (I) appended to the thesis, we identified and analyzed insertion/deletion mutations (INDELs) inferred to have resulted from a sequence repeat-mediated excision mechanism. We found eight new Regions of Difference (RDs) among Francisella strains. Using RDs together with single nucleotide polymorphisms (SNPs), we were able to predict an evolutionary scenario for F. tularensis in which Francisella novicida was the oldest variety while F. tularensis subsp. holarctica was the youngest. We also found that all virulence-attenuated isolates analyzed had deletions at two specific genetic regions - denoted RD18 and RD19 – suggesting that repeat-mediated excision is a mechanism of attenuation in F. tularensis. In subsequent studies (presented in paper II), we developed a combined analysis of INDELs lacking flanking repeats and variable number of tandem repeats (VNTRs). Both markers could be assayed using the same analytical equipment. The inclusion of INDELs provided increased phylogenetic robustness compared with the use of VNTRs alone, while still maintaining a high level of genetic resolution. In analyses described in the next paper (III), we selected INDELs from paper (II) and discovered novel SNPs by DNA comparisons of multiple Francisella strains. Thirty-four phylogenetically informative genetic markers were included in a hierarchical real-time PCR array for rapid and robust characterization of Francisella. We successfully used the assay to genotype 14 F. tularensis isolates from tularemia patients and DNA in six clinical ulcer specimens. Finally, in paper (IV) we demonstrated a strategy to enhance epidemiological investigations of tularemia by combining GIS-mapping of disease-transmission place collected from patient interviews, with high-resolution genotyping of F. tularensis subsp. holarctica isolates recovered from tularemia patients. We found the geographic distributions of specific F. tularensis subsp. holarctica sub-populations to be highly localized during outbreaks (infections by some genotypes being restricted to areas as small as 2 km2), indicative of a landscape epidemiology of tularemia with distinct point sources of infection. In conclusion, the results acquired during the studies underlying this thesis contribute to our understanding of the genetic genealogy of tularemia at both global and local outbreak scales.

<p>Characteristically, in infections that are caused by the zoonotic pathogen <i>Bartonella</i> naturally infected reservoir hosts are asymptomatic, where infected incidental, non-natural, hosts develop symptomatic disease. Cat-scratch disease (CSD) is a well known example. <i>Bartonella </i>infections in humans may be self-limiting or fulminant and affect different organ systems. </p><p>The objectives of the present thesis were to (1) identify and characterise <i>Bartonella </i>infection cases in Sweden, (2) to investigate certain human populations regarding <i>Bartonella </i>infections, and (3) compare natural populations of different <i>Bartonella </i>species.</p><p>Cases with typical and atypical CSD were recognised by using a combination of PCR and serology. Gene sequence comparisons of different genes in <i>B. henselae</i> isolates from the United States and Europe showed that<i> fts</i>Z gene variation is a useful tool for <i>Bartonella</i> genotyping. </p><p>Myocarditis was a common finding among Swedish elite orienteers succumbing to sudden unexpected cardiac death (SUCD). The natural cycle of <i>Bartonella</i> spp., the life style of orienteers, elevated antibody titres to <i>Bartonella</i> antigens, <i>Bartonella</i> DNA amplified from myocardium and the lack of another feasible explanation make <i>Bartonella</i> a plausible aetiological factor.</p><p>The first reported case of <i>Bartonella</i> endocarditis (<i>B. quintana</i>) was identified in an immunocompromised patient who underwent heart valve replacement. The patient had been body louse-infested during his childhood. It is hypothesised that a chronic <i>B. quintana</i> infection was activated by the immunosuppression.</p><p>There was no evidence of an ongoing trench fever (TF) epidemic in a Swedish homeless population, although an increased risk for exposure to <i>Bartonella</i> antigens was demonstrated. The lack of louse infestation might explain the absence of <i>B. quintana</i> bacteremia and low <i>B. quintana</i> antibody titres. </p><p>Comparisons of genetic loci and the whole genomes of environmental <i>B. grahamii</i> isolates from the Uppsala region, Sweden displayed variants that were not related to specific host species but to geographic locality. Natural boundaries seemed to restrict gene flow.</p>

Characteristically, in infections that are caused by the zoonotic pathogen Bartonella naturally infected reservoir hosts are asymptomatic, where infected incidental, non-natural, hosts develop symptomatic disease. Cat-scratch disease (CSD) is a well known example. Bartonella infections in humans may be self-limiting or fulminant and affect different organ systems. The objectives of the present thesis were to (1) identify and characterise Bartonella infection cases in Sweden, (2) to investigate certain human populations regarding Bartonella infections, and (3) compare natural populations of different Bartonella species. Cases with typical and atypical CSD were recognised by using a combination of PCR and serology. Gene sequence comparisons of different genes in B. henselae isolates from the United States and Europe showed that ftsZ gene variation is a useful tool for Bartonella genotyping. Myocarditis was a common finding among Swedish elite orienteers succumbing to sudden unexpected cardiac death (SUCD). The natural cycle of Bartonella spp., the life style of orienteers, elevated antibody titres to Bartonella antigens, Bartonella DNA amplified from myocardium and the lack of another feasible explanation make Bartonella a plausible aetiological factor. The first reported case of Bartonella endocarditis (B. quintana) was identified in an immunocompromised patient who underwent heart valve replacement. The patient had been body louse-infested during his childhood. It is hypothesised that a chronic B. quintana infection was activated by the immunosuppression. There was no evidence of an ongoing trench fever (TF) epidemic in a Swedish homeless population, although an increased risk for exposure to Bartonella antigens was demonstrated. The lack of louse infestation might explain the absence of B. quintana bacteremia and low B. quintana antibody titres. Comparisons of genetic loci and the whole genomes of environmental B. grahamii isolates from the Uppsala region, Sweden displayed variants that were not related to specific host species but to geographic locality. Natural boundaries seemed to restrict gene flow.

No description available.

Drug resistance is a major obstacle to management and control of malaria and currently progressing at a rapid rate across Africa. This thesis has examined factors influencing evolution of resistant P. falciparum at two sites in Africa, including parasite migration, cross mating and fitness cost of resistance. In Asar village, eastern Sudan, the frequencies of drug sensitive and resistant parasites were monitored throughout the dry season in the absence of anti-malarial drug usage to examine whether persistence of resistant parasites is reduced in the absence of drug pressure. Two cohorts of P. falciparum infected patients were treated with chloroquine in the transmission season (Oct-Dec), and followed monthly in the dry season into the next transmission season. A large proportion of the cohort maintained sub-patent asymptomatic P. falciparum infections throughout the entire study period. Alleles of the chloroquine resistance transporter (Pfcrt) and multi-drug resistance protein (Pfmdr1) were examined. Mutant alleles of Pfcrt reached fixation following CQ treatment and remained high in the transmission season. However, at the start of the dry season, wild type alleles of both genes started to emerge and increased significantly in frequency as the season progressed. The mutant Pfcrt haplotype was invariably CVIET, indicating migration of CQ resistant parasites into an area; otherwise the CVMNK haplotype is normal. In addition, microsatellite haplotypes of dihydrofolate reductase (dhfr) gene and dihydropteroate synthase (dhps) genes, which control the parasite response to pyrimethamine and sulfadoxine respectively, were characterized. One major dhfr haplotype with double dhfr mutations and two major mutant dhps haplotypes were seen in eastern Sudan. These haplotypes are distinct from those prevailing in other African countries, suggesting the likely local origin of dhfr and dhps haplotypes conferring drug resistance. Transmission capacities of different P. falciparum clones within a single infection in The Gambia have a high ability to produce gametocytes and infect Anopheles mosquitoes even when they exist at levels not detectable by microscopy and PCR. These findings emphasize the crucial role of gametocyte complexity and infectivity in generating the remarkable diversity of P. falciparum genotypes seen in infected people. Parasites with different resistant dihydrofolate reductase (dhfr) haplotypes have the ability to infect Anopheles mosquitoes following drug treatment, and cross-mating between parasites with different dhfr haplotypes was detected. Our results showed that the major dhfr haplotype in the Gambia is similar to the common one seen in other African countries, suggesting that parasite migration plays a major role in spread of resistance. Indeed, the dominant resistant haplotype seen in infected patients was readily transmitted to infect mosquitoes.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 6 uppsatser.

No description available.

<p>The obligate intracellular bacterium <i>Chlamydia pneumoniae (Cp</i>), a common human pathogen, has been associated with atherosclerotic cardiovascular disease. The aetiology of non-rheumatic aortic valve sclerosis has, however, not been clarified. In two prospective studies of 42 and 46 patients undergoing surgical valve replacement because of aortic valve stenosis, the presence of <i>Cp </i>DNA could be demonstrated by polymerase chain reaction (PCR) in 49% and 35% of the sclerotic valves as compared to 9 % and 0%, respectively, of valves from forensic control cases with no heart valve disease. Some inflammatory and infectious diseases are associated with trace element changes. Eleven of 15 trace elements showed changed concentrations in sclerotic valve tissue compared to control valves in support of an active process in the sclerotic valves. Notable was an increased iron concentration in the patients´ valves suggesting a possible link to <i>Cp</i>. Furthermore, a disturbed trace element balance existed in the patients´ sera, the pattern of which was compatible with ongoing infection. In a prospective study of 38 patients operated on for thoracic aortic aneurysm or dissection, <i>Cp</i> DNA <i>w</i>as detected byPCR in 12 % of the aneurysms and the result was confirmed byelectron microscopy(EM<i>).</i> In none of the dissection patients could <i>Cp </i>be demonstratedin the removed tissues. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values for doxycycline and azithromycin increased with longer <i>Cp </i>preincubation times when tested in vitro<i>.</i> EMwas performed to visualise the inactivation at a cellular level.Thus, the results demonstrate <i>Cp </i>in the tissues in non-rheumatic aortic valve sclerosis and in thoracic aortic aneurysm but not in aortic dissection.</p>

<p>The first hospital outbreak of vancomycin-resistant enterococci (VRE) and carriage rates of VRE and ampicillin-resistant enterococci (ARE) in Sweden were investigated. Clonal relationships and mutations in fluoroquinolone resistance determining regions among ARE collected nation-wide were studied. Risk factors for ARE infection, shedding of ARE and the presence of the virulence gene <i>esp</i> in ARE isolates and patients on a hematology unit and other units at Uppsala University Hospital were further investigated. </p><p>The first Swedish hospital VRE outbreak was due to clonal spread of <i>E. faecium, vanA</i>. The nation wide carriage rates of ARE and VRE were 21.5% / 1% and 6% / 0%, among hospitalized patients and non-hospitalized individuals respectively. All ARE and VRE were <i>E. faecium</i> and >90% resistant to ciprofloxacin. All VRE carried<i> vanB</i>. Carriage of ARE was independently associated with >5 days of antibiotic treatment. Phenotypic and genetic typing showed a significantly higher homogeneity among ARE compared to matched ASE <i>E. faecium</i> isolates. Mutations conferring high-level ciprofloxacin resistance were found only in ARE. Risk factors for ARE infection included long duration of hospital stay and exposure to antibiotics. Skin carriage was associated with ARE shedding. ARE bacteremia was independently associated with prior ARE colonization and hematopoietic stem cell transplantation. Death was more common in ARE septicemia cases compared to controls. <i>Esp</i> was significantly more common in ARE surveillance compared to ARE blood isolates from patients on the hematology ward.</p><p>In conclusion, VRE were rare but clonally related multi-resistant ARE <i>E. faecium</i> were highly prevalent in Swedish hospitals. Spread of ARE in hospitals during the 1990s is suggested to be the main explanation for the emergence of ARE in Sweden. Spread was facilitated by use of antibiotics and probably by the presence of virulence genes in<i> E. faecium</i> isolates.</p>

<p>Toxic products, such as endotoxin from the gram-negative and exotoxin from the gram-positive bacteria, are the most important initiators of the inflammatory host response in sepsis. In addition to antibacterial treatment, numerous attempts have been made to interfere with the exaggerated proinflammatory cascade initiated by the toxins. As most antitoxic and anti-inflammatory agents have shown no clear efficacy, an attractive alternative has been to prevent or minimise their release. Therefore, it was of interest to further study the antibiotic-induced release of toxins after exposure to antibiotics used for the treatment of the most severe infections, especially if protein synthesis inhibitors could reduce the release induced by PBP 3-specific β-lactam antibiotics.</p><p>There were significant reductions in endotoxin release from gram-negative bacteria when the combination of the PBP 3-specific β-lactam antibiotic, cefuroxime, and the protein synthesis inhibitor, tobramycin, was compared with cefuroxime alone. Increasing doses of tobramycin reduced endotoxin release and increased the killing rate. In a kinetic <i>in vitro</i> model the endotoxin release from <i>E.coli</i> was higher after the second dose of cefuroxime. Nevertheless, it was reduced after addition of tobramycin.</p><p>No binding of tobramycin to endotoxin was observed, either <i>in vivo</i> or <i>in vitro</i>. In a porcine sepsis model, a possible anti-inflammatory effect of ceftazidime and tobramycin, expressed as late cytokine inhibition, was seen.</p><p>The protein synthesis inhibitor, clindamycin, released less streptococcal pyrogenic exotoxin A (SpeA) from a group A streptococcus strain than penicillin, and addition of clindamycin to penicillin resulted in less toxin production than penicillin alone. The SpeA production was dependent on the bacterial number at the start of treatment. Higher doses of penicillin also led to less SpeA. </p><p>The choice of antibiotic class and dose may be important in the severely ill septic patient in whom an additional toxin release could be deleterious. A combination of a β-lactam antibiotic and a protein synthesis inhibitor seems beneficial but further investigations are needed.</p>