<p><strong><p>The <em>Enterococcus genus consists of natural members of the gastrointestinal tract but they are also opportunistic pathogens. They are a common cause of urinary tract infections but can also cause sepsis and other infections. <em>Enterococcus faecalis and <em>Enterococcus faecium are the most abundant in clinical specimens. Enterococci are a leading cause of nosocomial infections and they have developed resistance against a number of antibiotics e.g. vancomycin. <em>E. faecalis V583 was the first vancomycin resistant isolate reported in the U.S. Movable genetic elements such as pathogenicity islands, PAI, are important for bacterial evolution. PAI:s are large chromosomal fragments mostly seen in pathogenic strains and carry regions such as transposons and insertion elements along with virulence factors and transfer genes. A PAI has been detected in the chromosome of <em>E. faecalis. Excision of PAI:s has been studied for uropathogenic <em>E. coli and frequencies of 10-5 and 10-6 have been reported. In this study the excision rate and excision patterns of <em>E. faecalis V583 was studied using the counterselectable marker <em>pheS*, causing <em>p-Cl-phe sensitivity, and a chloramphenicol resistance gene, <em>cat, inserted at two different positions of the PAI and selecting for excisions by growth on <em>p-Cl-phe. Excision rates of 10-6 and 10-8 were seen based on the <em>p-Cl-phe resistance and chloramphenicol sensitivity. Mutation rate in the <em>pheS* gene was high compared to excision rate which made the method difficult to work with. No obvious excision patterns were detected but the excisions seemed to be limited to the close surroundings of the <em>pheS*/cat insertion. </em></em></em></em></em></em></em></em></em></em></em></em></em></em></p></strong></p> / <p>Bakterier finns överallt i vår omgivning och hos oss människor, exempelvis på huden och i vår mag-tarmkanal. Flertalet av dessa är apatogena, d.v.s. orsakar inte sjukdom. Vissa normalt goda bakterier kan dock orsaka sjukdom när de hamnar på fel plats och får tillfälle att orsaka sjukdom s.k. opportunistiska patogener. Ett exempel på detta är bakterien</p><p>Genetiska egenskaper hos såväl människor som bakterier styrs av arvsmassan, DNA. Hos människor är arvsmassan samlad i 46 kromosomer medan bakterier har en. På senare år har vi lärt oss hur man kan klippa och klistra i exempelvis bakteriers DNA för att introducera egenskaper eller ta bort. Detta används inom forskning för att studera t.ex. bakteriers förmåga att orsaka sjukdom eller anpassning till sin omgivning. Bakterier är mycket duktiga på just anpassning vilket beror på deras förmåga att snabbt förändra sitt DNA ofta genom utbyte med andra bakterier, detta kan bl.a. leda till utveckling av antibiotikaresistens eller nyvunnen förmåga att orsaka sjukdom. Största delen av en bakteries arvsmassa består av konserverade regioner medan andra är mycket föränderliga exempelvis s.k. isertions element, tansposoner och patogenicitetsöar, som har visat sig kunna lämna kromosomen via excision. En patogenicitetsö har hittas hos</p><p>I den här studien klistras en gen in i patogenicitetsön hos</p><p>Bakteriekloner där excision förekommit erhölls och excisionsfrekvensen bestämdes till 10</p><p>bakterier. Inga kloner där hela patogenicitetsön lämnat kromosomen kunde detekteras, dock visade det sig att områden precis intill området där genen klistrats in hade försvunnit. Inga tydliga excisionsmönster kunde bestämmas. En hög frekvens av mutationer i den insatta genen gjorde metoden svår att arbeta med.</p><p><em>Enterococcus faecalis som finns i mag-tarmkanalen hos friska människor men som när den hamnar på fel plats kan orsaka bl.a. urinvägsinfektion, sårinfektioner och i svåra fall blodförgiftning, s.k. sepsis. <em>E. faecalis. Patogenicitetsöar är delar av kromosomen som ofta innehåller virulensfaktorer, som gör bakterien patogen och hos vissa bakterier har man påvisat gener för antibiotikaresistens på sådana öar. <em>E. faecalis som gör att bakterien inte kan växa på ett speciellt selektivt media. Detta gör det möjligt att välja bakterier där ön lämnat kromosomen för vidare studier och en excisionsfrekvens bestämmas. -6 till 10-8. Detta är låga frekvenser jämfört med vad man kommit fram till hos andra 3 </em></em></em></p>

Diss. (sammanfattning) Uppsala : Univ., 2007. / Härtill 5 uppsatser.

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

<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>

The human vaginal ecosystem is dominated by Lactobacillus species. An altered vaginal flora can result in symptomatic conditions such as bacterial vaginosis and vulvo-vaginal candidiasis, and urogenital colonisation by uropathogenic bacteria can cause urinary tract infection. The protective role of lactobacilli is gradually being accepted and clinical studies have been carried out in order to evaluate the use of promising probiotic bacteria, which are defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. This thesis includes an investigation into the ecological role of lactobacilli in the genital tract in healthy women, with respect to the relationship to other species and vaginal pH. Furthermore, in order to find different probiotic strains with promising probiotic qualities, Lactobacillus strains were screened in two diverse screening processes. The selected strains were further evaluated in clinical trials. The prevalence of group B streptococci (GBS) and yeast was significantly dependent on the number of vaginal lactobacilli among healthy women. GBS were less frequently found in women with high numbers of vaginal lactobacilli than in women with low numbers and the prevalence of yeast was significantly higher in women with 3-6.99 log10 lactobacilli sample-1 than in women with less than 3 or ≥7 log10 lactobacilli sample-1. Furthermore, the first screening made on 511 strains isolated from the female genital tract resulted in the final selection of a Lactobacillus plantarum, designated LB931. The screening showed that LB931 had a strong technical growth, survived through freeze-thawing, produced substances bactericidal to uropathogenic bacteria and was a rapid and strong producer of hydrogen peroxide. Further characterisation showed that LB931 possessed the properties required for probiotics with the capability to prevent urogenital infections. LB931 could be supplied to the genital tract through the usage of panty liners impregnated with the strain. In the second screening, Lactobacillus fermentum, designated Ess-1, was the only one out of 126 Lactobacillus strains with strong capacity to inhibit Candida albicans and Candida glabrata. Additional characterisation showed that L. fermentum Ess-1 had the properties that are needed to prevent over-growth of Candida in the vulvo-vaginal tract. The result of the case study showed that a high and frequent dosage of Ess-1 is needed and that improved vulvo-vaginal candidiasis specific diagnostic criteria are required. In conclusion, L. plantarum LB931 and L. fermentum Ess-1 are promising probiotic strains to be used in the prevention of recurrent urogenital infections in women and to enhance the normal flora in healthy women.

Francisella tularensis is a gram-negative bacterium that is the cause of the serious and sometimes fatal disease, tularemia, in a wide range of animal species and in humans. The response of cells of the mouse macrophage cell line J774 to infection with Francisella tularensis LVS was analyzed by means of a DNA microarray. It was observed that the infection conferred an oxidative stress upon the target cells and many of the host defense mechanisms appeared to be intended to counteract this stress. The infection was characterized by a very modest inflammatory response. Tularemia caused by inhalation of F. tularensis subspecies tularensis is one of the most aggressive infectious diseases known. We used the mouse model to examine in detail the host immune response in the lung. After an aerosol challenge all mice developed clinical signs of severe disease, showed weight loss by day four of infection, and died the next day. Gene transcriptional changes in the mouse lung samples were examined on day one, two, and four of infection. Genes preferentially involved in host immune responses were activated extensively on day four but on day one and two, only marginally or not at all. Several genes upregulated on day four are known to depend on IFN-gamma or TNF-alpha for their regulation. In keeping with this finding, TNF-alpha and IFN-gamma levels were found to be increased significantly in bronchoalveolar lavage on day four. We undertook an analysis of the transcriptional response in peripheral blood during the course of ulceroglandular tularemia by use of Affymetrix microarrays. Samples were obtained from seven individuals at five occasions during two weeks after the first hospital visit and convalescent samples three months later. In total 265 genes were differentially expressed. The most prominent changes were noted in samples drawn on days 2-3 and a considerable proportion of the upregulated genes appeared to represent an IFN-gamma-induced response and also a pro-apoptotic response. Genes involved in the generation of innate and acquired immune responses were found to be downregulated, presumably a pathogen-induced event. A logistic regression analysis revealed that seven genes were good predictors of the early phase of tularemia. Recently, a large number of methods for the analysis of microarray data have been proposed but there are few comparisons of their relative performances. We undertook a study to evaluate established and novel methods for filtration, background adjustment, scanning, and censoring. For all analyses, the sensitivities at low false positive rates were observed together with a bias measurement. In general, there was a trade off between the analyses ability to identify differentially expressed genes and their ability to obtain unbiased estimators of the desired ratios. A commonly used standard analysis using background adjustment performed poorly. Interestingly, the constrained model combining data from several scans resulted in high sensitivities. For experiments where only low false discovery rates are acceptable, the use of the constrained model or the novel partial filtration method are likely to perform better than some commonly used standard analyses.

A general aim was to combine molecular typing methods with clinical background information to increase epidemiological knowledge about Chlamydia trachomatis infections. An outbreak of Lymfogranuloma venereum (LGV), caused by a more invasive variant of C. trachomatis, was reported from the Netherlands in 2003 among men who have sex with men (MSM). All Chlamydia positive specimens from a venereal disease clinic for MSM in Stockholm during one year were genotyped. No spread of LGV was found, apart from three symptomatic cases. The same ompA genotypes were found among MSM in Melbourne, but the genotype distribution was different compared to findings among the heterosexual population in Sweden. The standard method for genotyping of Chlamydia is ompA-sequencing, but it has low resolution because one genotype predominates. A multilocus sequence typing (MLST) system based on five targets was developed. In a sample of 47 specimens, 32 variants were found with MLST, but only 12 variants with ompA-sequencing. The polymorphisms in the hctB gene, one MLST target, are caused by an element of 108 bp that is present in two to four repetitions and in different variants. Although the DNA-binding function of Hc2 that is encoded by hctB has been studied, our findings of a considerable size variation show that new studies are needed. In 2006, specimens with a 377 bp deletion in the cryptic plasmid covering the target region for diagnostic test systems from Abbott and Roche were discovered in Sweden. Applying MLST to these specimens indicated that there was a single clone, denoted nvCT. The proportion of nvCT in all detected Chlamydia cases was higher (20% to 65%) in counties using Abbott/Roche compared to counties using the BectonDickinson test system (7% to 20%). The proportions of nvCT converge in counties with high or low levels when detection systems were adjusted to detect nvCT.

The ecological niche of the vagina contains a large number of different microbes that are constantly interacting with each other and the host. Culture methods have not been sufficient in order to resolve the complexity of the normal vaginal flora. Further, the methods for delineating normal flora from not normal flora are not easily handled and are traditionally not based on culture but on microscopy of elements of the vaginal fluid. In the work presented in this thesis, an international collaboration was established that pin-pointed some of the difficulties in classifying vaginal floras, including staining, sampling, and discordance when lactobacilli are few in number, and that emphasized the importance of the size of the vision field in microscopes. As lactobacilli are prominent members of the normal vaginal flora they need to be carefully classified if further work towards more robust scoring tools is to be achieved. Phenotypic methods have not been able to separate the closely related Lactobacillus species of the vagina. Progress in molecular biology has provided possibilities to characterize these lactobacilli, which are mainly from the Lactobacillus acidophilus group. In this work a large number of strains collected by true random sampling were subjected to RAPD-PCR, TTGE and multiplex PCR for species identification. The major species found were L. crispatus, L. gasseri and L. jensenii and the recently described L. iners. The presence of L. iners has not been detected in previous studies due to its special nutrient requirements. Development of pyrosequencing technology also made it possible to match signatures of the two variable regions V1 and V3 of the 16S rRNA gene of the vaginal lactobacilli and identify them to the species level in a high throughput manner. The study confirmed that the dominating flora in women with normal vaginal flora comprises the four species mentioned previously. Repetitive sampling during IVF-treatment with highly varying oestrogen levels demonstrates changes that possibly occur during changes in the natural life cycle. Furthermore, L. iners was found to be the first species to be established after spontaneously resolved or treated Bacterial Vaginosis. These findings can be of help in developing new strategies for regaining and retaining the normal vaginal flora.

Francisella tularensis is a gram-negative, highly virulent, intracellular bacterium which causes the zoonotic disease tularemia. The subspecies tularensis and holarctica are clinically important, and the former is the more virulent. The intracellular lifestyle of F. tularensis is not completely understood, but after uptake in monocytes, the bacterium escapes from the phagosome within hours and replicates massively in the cytosol. The escape is dependent on factors encoded by the Intracellular Growth Locus (igl) operon, located in the Francisella Pathogenicity Island, FPI. The thesis was aimed to clarify and understand the interaction of F. tularensis strains with the endosomal pathway of monocytic cells in general and the roles of the Igl proteins and the global regulator MglA for this interaction in particular. A focus has also been to elucidate the roles of reactive oxygen and nitrogen species for the intracellular host-parasite interaction. We show that mutants in the IglB, IglC, or IglD proteins or their regulator MglA of the live vaccine strain, LVS (subspecies holarctica), all demonstrated reduced replication rates and lowered cytopathogenicity compared to the wild type in a J774 mouse macrophage cell model. Colocalization with LAMP-1 was significantly increased for the IglC, IglD and MglA mutants compared to LVS. This indicated an impaired ability to escape into the cytoplasm, while at the same time they, like LVS, partly prevented fusion with lysosomes. IFN-γ activation of the J774 host cells prior to infection had a bactericidal effect on LVS and all of the mutants, though the cidal effect was significantly more pronounced for the mutants. Following IFN-γ activation, a majority of the mutant-containing phagosomesfused with lysosomeswhile LVS remained localized in the cytosol without significantly increased interactions with the endosomal pathway. Previous studies have revealed that IFN-γ activation of F. tularensis-infected macrophages leads to control of infection but conclusions about the importance of reactive nitrogen and oxygen species on bacterial killing are inconsistent. We found that the growth inhibition resulting from IFN-γ activation could not be attributed to an increased oxidative burst since PMA-induced superoxide production was still inhibited by LVS to the same extent as in non-activated macrophages. On the other hand, reactive nitrogen species may in part have contributed to the cidal effect. To further assess the role of reactive nitrogen species to the killing of F. tularensis, nitric oxide was administrated exogenously to J774 cells infected with LVS. This led to significant killing of intracellular LVS with a concomitant increased phagosomal localization and downregulation of the virulence gene regulator mglA. These effects were reversed by addition of a peroxynitrite decomposition catalyst. A spontaneous avirulent mutant of subspecies tularensis, strain FSC043, was previously demonstrated to provide protective immunity in mice. Here, microscopic analyses of the strain revealed an unusual intracellular localization with a delayed phagosomal escape. This may account for the low virulence, while at the same time FSC043 remains immunogenic and thereby confers protection. The igl operon is intact in strain FCS043 and we hypothesize that a defect in the FPI gene pdpC contributed to the observed phenotype. Altogether, this thesis work demonstrates the importance of the mglA and igl genes for the virulence of F. tularensis and specifically their important roles for a functional phagosomal escape and inhibition of the host cell oxidative burst. Also, addition of exogenous nitric oxide likely leads to formation of peroxynitrite intracellularly, a reactive molecule which confines the bacterium to the phagosome and confers a significant bactericidal effect on intracellular F. tularensis.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 4 uppsatser.