Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 5 uppsatser.

<p>Aim Because of the major problems that certain Vibrio specie, especially Vibrio harveyi, can cause the aquaculture industries a rapid method to identify Vibrio isolates is required. Early diagnosis of a V. harveyi infection could facilitate disease surveillance, treatment and prevention in cultured marine animals. Therefore, the use of PCR to aid in the identification of Vibrio is increasing and a way of extracting DNA in a cheap, fast and easy way is also of an important requirement to facilitate rapid diagnosis.</p><p>Methods This report comprises biochemical profiling and PCR methods in the characterisation of four isolates of V. harveyi and single isolates of V. tubiashii, V. alginolyticus, V. anguillarum, V. splendidus, V. tapetis and V. parahaemolyticus.</p><p>Strains were examined for adherence to a Hep-2 cell line. Four different DNA extraction methods were evaluated and compared. The detection limits and the analytical limits of two PCR methods for Vibrio were determined.</p><p>Results The overall findings were that the use of a greater range of biochemical substrates than are in the API 20E is necessary to identify Vibrio strains, and that none of the strains tested adhered to Hep-2 cells. All extraction methods successfully produced DNA with the kit method giving the purest samples. RNA was a contaminant of the other techniques but this could be overcome by treating extracts with RNase. The rapid microwave extraction method gave appropriate PCR amplicons when tested.</p><p>Conclusion PCR determination of the VH-sequence in combination with VHA and a distinguishable colonial morphology may be a good choice for the identifying of Vibrio harveyi.</p>

<p>The purpose of the study was to set up a method for cultivation of C. pneumoniae from infected mouse tissue in Hep2 cells. We also evaluated a new reagent, Chlamatis, which is considered to increase the detection sensitivity of the bacterium with both PCR and cultivation. All 10 serum samples treated with Chlamatis were negative for C. pneumoniae in PCR. The cultivation of tissue was found to work without problems. The yield of the bacteria was highest at the speed of 30 Hz using homogenization with TissueLyser. Mice infected with C. pneumoniae were killed at days 4, 8, 20 and 40. The highest yields of C. pneumoniae were found at days 4 and 8 using PCR with all infected mice. The results obtained with PCR and cultivation confirmed each other to a large extent. For heart tissue, PCR positive mice were found only at days 4 and 8. The number of PCR positive lung samples confirmed to a large extent the number found by cultivation, except for mice killed after 4 days and 40 days where the results differed slightly. This indicated that the optimization of the cultivation method was successful</p>

Only two out of more than 200 V. cholerae serogroups, classified on the basis of LPS structure, are associated with epidemic or pandemic cholera. These toxigenic serogroups carry phage-derived pathogenicity islands coding for the main virulence factors for establishment of cholera disease – cholera toxin (CTX) and toxin-coregulated pilus (TCP). The latter also serves as a bacterial surface receptor for the CTXΦ – the filamentous phage which carries the cholera toxin genes into otherwise harmless to human, environmental bacterium V. cholerae. In its natural aquatic habitat V. cholerae is subject to predator grazing, bacteriophage killing, temperature and pH changes, seasonality of plankton blooms and other environmental factors. Therefore understanding V. cholerae pathogenic and virulence potential requires the knowledge of its interaction not only with human host but also members of aquatic environment and environmental factors. V. cholerae is capable of killing the nematode Caenorhabditis elegans. Using a reverse genetics approach, we demonstrated that the quorum sensing regulated protease PrtV is essential for this killing. Other proteases did not seem to contribute to virulence in this model. The data from this study suggest that the PrtV could be important to V. cholerae in its natural niche for its resistance to the grazing predators. The PrtV protease belongs to an M6 family of metallopeptidases which is represented by an Immune Inhibitor A protease from the insect killing bacterium Bacillus thuringiensis. To characterize the protease in more detail, the PrtV was cloned, overexpressed in V. cholerae and purified from the culture supernatant. The enzyme was calcium stabilized and inhibited by metal ion chelators. In tests with in vitro cultured cells of the human intestinal cell line HCT8, the PrtV protein showed a cytotoxic effect leading to cell detachment and death. Using human blood plasma as a source of potential substrates, and by tests with purified candidate substrate proteins, we have identified fibrinogen (all α, β and γ chains), fibronectin and plasminogen to be degraded by the protease. Additionally, PrtV was found to alter the stability of V. cholerae cytolysin implicating its role in modulation of the reactogenicity of V. cholerae secreted factors. Pigmentation has been considered to be important in microbial pathogenesis because it has been associated with virulence in many microorganisms. Using transposon mutagenesis we identified the mutated locus of a pigment producing V. cholerae strain to encode a gene of a tyrosine catabolic pathway. The mutation in a putative homogentisate 1,2-dioxigenase gene lead to accumulation of homogentisic acid, its spontaneous oxidation and formation of a dark pigment. The pigment producing strain was altered in its ability to survive UV exposure and H2O2 stress, and was more efficient in colonizing the suckling mouse intestine compared to the wild type strain. Under the in vitro growth conditions the major virulence factor TcpA and CT expression was found to be somewhat enhanced too.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 5 uppsatser.

<p>Apoptosis, or programmed cell death, is a controlled process by which aged or damages cells are eliminated in multicellular organisms. Neutrophils, short-lived phagocytes of the innate immune system, are highly equipped effectors that can sense, locate, ingest and kill bacterial pathogens. Inflammatory mediators and the presence of bacterial products at the foci of infection regulate the function and life span of these cells. Modulation of neutrophil apoptosis and the subsequent clearance by scavenger cells, such as macrophages, is part of a balanced inflammatory process leading to resolution of inflammation. Many pathogens are capable of modulating host cell apoptosis, and thereby influence the progression of disease. Hence, this thesis was aiming at elucidating mechanisms involved in pathogen- and host-modulated apoptosis and its contribution to the inflammatory process.</p><p>We found that different routes of bacterial entry, i.e. through invasion or by receptor-mediated phagocytosis, triggered different signaling pathways within phagocytes. Invasion of virulent Salmonella caused apoptosis, a process requiring activation of the Rho GTPases Rac1 and Cdc42. On the other hand, phagocytosis of the non-invasive Salmonella inhibited apoptosis despite similar intracellular survival as the invasive bacteria. Protection against phagocytosis-induced apoptosis was regulated by tyrosine- and PI3-kinase-dependent activation of AKT (also called PKB for protein kinase B). Furthermore, inhibiting the intraphagosomal production of reactive oxygen species (ROS) in neutrophils during phagocytosis of <em>E. coli</em> decreased apoptosis below spontaneous apoptosis, further indicating that both pro- and anti-apoptotic pathways are triggered by receptor-mediated phagocytosis.</p><p>Type 1 fimbria-expressing <em>E. coli </em>adhering to neutrophils resisted ingestion, and induced a ROS-dependent apoptosis by a cooperative effect of the FimH adhesin and LPS. To explore how compartmentalization of ROS during neutrophil activation was involved in modulating apoptosis, we evaluated the stability of lysosomes. In contrast to phagocytosis of <em>E. coli</em>, the adhesive strain induced intracellular non-phagosomal ROS production which triggered early permeabilization and release of lysosomal enzymes to the cytosol. Cathepsin B and/or L were responsible for targeting of the pro-apoptotic Bcl-2 protein Bid, thereby inducing mitochondrial damage, and apoptosis. These data propose a novel pathway for ROS-induced apoptosis in human neutrophils, where the location of the ROS rather than production <em>per se</em> is important.</p><p>Moreover, we found that pathogen-induced apoptotic neutrophils, in contrast to uninfected apoptotic neutrophils, activated blood-monocyte derived macrophages to increase their FcγRI surface expression and to produce large quantities of the pro-inflammatory cytokine TNF-α. This demonstrates that during the early phase of infection, pathogen-induced neutrophil apoptosis will help local macrophages to gain control over the microbes. Furthermore, we suggest that heat shock protein 60 and 70 represent a stress signal that enables macrophages to distinguish between, and react differently to, uninfected and inflammatory apoptotic neutrophils.</p>

<p>Chlamydia and gonorrhea are major causes of sexually transmitted infections (STI) in adolescents worldwide. The infections are caused by <em>Chlamydia trachomatis</em> or <em>Neisseria gonorrhoeae, </em>bacteria with clinical manifestations such as urethritis, prostatitis and epididymitis among men, and urethritis, cervicitis and upper genital tract infection (i.e. pelvic inflammatory disease) among women. However, in many cases of genital tract infection, the etiology remains uncertain. In light of this, <em>Mycoplasma genitalium</em> was somewhat accidentally isolated in 1980 after prolonged incubation of urogenital specimens from men with non-gonococcal urethritis. Following the initial isolation in 1980, repeated attempts have been made to recover the extremely fastidious organism from clinical samples by culture techniques, but isolates have been rare and difficult to obtain. With the development of PCR methods in the early 1990s, detection of <em>M. genitalium</em> infection became more feasible.</p><p>The aim in paper <strong>I</strong> was to compare three different PCR assays (conventional and real-time 16S rRNA gene PCR as well as real-time <em>Mycoplasma genitalium</em> adhesin protein (MgPa) gene PCR) for detection of <em>M. genitalium</em>. The study also determined the prevalence of <em>M. genitalium</em>. Clinical specimens collected from STI attendees, 381 men and 298 women, were used to determine the prevalence of <em>M. genitalium</em> and 213 of these specimens were used in the PCR comparative study. The prevalence of <em>M. genitalium</em> infection in men and women was 27/381 (7.1 %) and 23/298 (7.7 %) respectively. In the PCR comparative study, <em>M. genitalium </em>DNA were detected in 61/76 (80.3 %) of true-positive specimen by conventional 16S rRNA gene PCR, in 52/76 (68.4 %) by real-time 16S rRNA gene PCR and in 74/76 (97.4 %) by real-time MgPa gene PCR. Hence, real-time MgPa gene PCR is well suited for clinical diagnosis of <em>M. genitalium</em> in urogenital specimens from men and women.</p><p>The aim in paper <strong>II</strong> was to determine whether a patients’ endocervical swab specimen can be transported in first void urine (FVU) as combined specimens in detection of <em>Mycoplasma genitalium </em>by real-time PCR. The study also compared two different DNA extraction methods (manual Chelex DNA extraction and automated BioRobot M48 DNA extraction) for observation of possible PCR inhibition. Clinical specimens collected from 329 women attending a STI clinic were used in the study. A total of 100 endocervical swab specimens transported in FVU was used in the PCR inhibition analysis. <em>M. genitalium</em> was detected in 25/329 (7.6 %) women. Endocervical swab specimens transported in FVU demonstrate higher sensitivity compared to both FVU alone and specimens transported in 2-SP medium detecting 24/25 (96 %), 22/25 (88 %) and 17/25 (68 %) of <em>M. genitalium</em> positive women, respectively. Automated BioRobot M48 DNA extraction was shown to be superior to manual Chelex extraction leaving no PCR inhibition and slightly higher DNA yield and/or better sensitivity. The results from these two studies are important knowledge in establishing the future diagnostic level of this STI in our county and also nationally.</p>