<p>Urinary tract infections (UTIs) are the second most common type of infection identified in the clinical setting and disproportionately afflict women. UTIs most frequently manifest in the form of infection of the lower urinary tract, involving the bladder. Uropathogens, particularly uropathogenic E. coli, progressively colonize the urethra and ascend to the bladder, where they initiate cystitis. In some cases, infection further ascends through the ureters and reaches the kidneys, where it causes pyelonephritis. Infection of both the upper and lower urinary tract can have serious ramifications for the host, and this is in large part due not to infection itself but to host-directed responses to bacterial insults. </p><p> In this thesis, I will describe and discuss two distinct aspects of UTIs. In the first study, in vivo work in a mouse model of urinary tract infection revealed a novel role for mast cells, which are tissue-resident granulated innate immune cells, in directing the detachment and death of epithelial cells during cystitis, facilitating the clearance of bacteria from the bladder. An ex vivo porcine bladder infection model suggested a specific role for mast cell granules and the proteases contained therein, which was corroborated with in vitro experiments utlizing isolated mast cell granules and human epithelial cells to demonstrate granule-induced exfoliation and cell death. From this work, it is clear that mast cells play a highly targeted role in modulating urothelial integrity during bladder infection by mediating host-directed epithelial loss.</p><p> In the second study described in this dissertation, the synergistic roles of both pyelonephritis and vesico-ureteric reflux (VUR), a congenital urinary tract defect that results in the improper backflow of urine from the bladder to the kidney, in the development of reflux nephropathy, a fibrotic host response characterized by renal scar formation, were elucidated in a series of in vivo experiments. Specifically, the C3H mouse, which is naturally susceptible to VUR, was utilized to characterize the dynamics of kidney infection and the onset of reflux nephropathy. Renal scarring was dependent on the presence of sustained kidney infection and the accompanying inflammatory response due to VUR, while neither transient infection nor reflux alone were sufficient to provoke nephropathy. Thus, the development of reflux nephropathy is dependent upon the confluence of both infection and VUR. </p><p> This body of work reveals the double-edged sword of the host inflammatory response to urinary tract infection. In the bladder, mast cell activation and degranulation leads to granule-induced epithelial exfoliation and consequently a reduction in the bacterial burden in the bladder. However, the sustained inflammatory response that accompanies pyelonephritis in vesico-ureteric reflux-affected individuals results in significant damage to the kidney without any accompanying reduction in infection. These findings highlight the dueling roles of the host inflammatory response to infection in the upper and lower urinary tract and strongly suggest that differential clinical approaches to cystitis and pyelonephritis are necessary to promote an effective mast cell in the bladder in the former and facilitate the clearance of renal infection while mitigating tissue damage in the latter.</p> / Dissertation

<p>Due to its close proximity to the gastrointestinal tract, the human urinary tract is</p><p>subjected to constant barrage by gut-­associated bacteria. However, for the most part, this tract has resisted infection by various microbes. The impregnability of the urinary tract to microbial colonization is attributable to the ability of the bladder to promptly sense and mount robust responses to microbial challenge. A powerful mechanism for the elimination of invading bacteria was recently described in bladder epithelial cells, involving non-­lytic ejection of intracellular bacteria back into the extracellular milieu. In spite of the effectiveness of this defense strategy, much of the underlying mechanisms surrounding how this powerful cellular defense activity detects intracellular UPEC and shuttles them from their intracellular location to the plasma membrane of BECs to be exported remains largely a mystery.</p><p> Here, we describe uropathogenic E.coli (UPEC) expelled from infected bladder</p><p>epithelium cells (BECs) within membrane-­bound vesicles as a distinct cellular defense</p><p>response. Examination of the intracellular UPEC revealed that intracellular bacteria were</p><p>initially processed via autophagy, the conventional degradative pathway, then delivered</p><p>into multivesicular bodies (MVBs) and encapsulated in nascent intraluminal vesicle membrane. We further show the bacterial expulsion is triggered when intracellular UPEC follow the natural degradative trafficking pathway and reach lysosomes and attempt to neutralize its pH to avoid degradation. This pathogen-­mediated activity is detected by mucolipin TRP channel 3 (TRPML3), a transient receptor potential cation channel localized on lysosomes, which spontaneously initiates lysosome exocytosis resulting in expulsion of exosome-­encased bacteria. These studies reveal a cellular default system for lysosome homeostasis and also, how it is coopted by the autonomous defense program to clear recalcitrant pathogens.</p> / Dissertation

Urinary tract infection (UTI) is a common bacterial infection in women of all ages but the incidence and prevalence increase with age. Despite the high incidence of UTI, little is known about its impact on morale or subjective wellbeing and daily life in old women. UTI in older people can be a complex problem in terms of approach to diagnosis, treatment and prevention because in these patients it frequently presents with a range of atypical symptoms such as delirium, gastrointestinal signs and falls. Even if UTI has been shown to be associated with delirium it has frequently been questioned whether UTI can cause delirium or if it is only accidentally detected when people with delirium are assessed. The main purpose of this thesis was to describe the prevalence of UTI, to identify factors associated with UTI among very old women and to illuminate the impact of a UTI on old women’s health and wellbeing.  This thesis is based on two main studies, the GErontological Regional DAtabase (GERDA) a cross-sectional, population-based study carried out in the northern parts of Sweden and Finland during 2005-2007 and a qualitative interview study in western Sweden 2008-2009. Data were collected from structured interviews and assessments made during home visits, from medical records, care givers and relatives. UTI was diagnosed if the person had a documented symptomatic UTI, with either short- or long-term ongoing treatment with antibiotics, or symptoms and laboratory tests judged to indicate the presence of UTI by the responsible physician or the assessor. One hundred and seventeen out of 395 women (29.6%) were diagnosed as having suffered from at least one UTI during the preceding year and 233 of these 395 (60%) had had at least one diagnosed UTI during the preceding 5 years. These old women with UTI were more dependent in their activities of daily living, and had poorer cognition and nutrition. In these women, UTI during the preceding year was associated with vertebral fractures, urinary incontinence, inflammatory rheumatic disease and multi-infarct dementia. Eighty-seven of 504 women (17.3%), were diagnosed as having a UTI with or without ongoing treatment when they were assessed, and almost half (44.8%) were diagnosed as delirious or having had episodes of delirium during the past month. In all, 137 of the 504 women (27.2%) were delirious or had had episodes of delirium during the past month and 39 (28.5%) of them were diagnosed as having a UTI. Delirium was associated with Alzheimer’s disease, multi-infarct dementia, depression, heart failure and UTI. Forty-six out of 319 women (14.4%) were diagnosed as having had a UTI with or without ongoing treatment and these had a significantly lower score on the Philadelphia Geriatric Center Morale Scale (PGCMS), (10.4 vs 11.9, p=0.003) than those without UTI, indicating a significant impact on morale or subjective wellbeing among very old women. The medical diagnoses significantly and independently associated with low morale were depression, UTI and constipation. The experience of suffering from repeated UTI was described in interviews conducted with 20 old women. The interviews were analysed using qualitative content analysis. The participants described living with repeated UTI as being in a state of manageable suffering and being dependent on alleviation. Being in a state of manageable suffering was described in terms of experiencing physical and psychological inconveniences, struggling to deal with the illness and being restricted regarding daily life. Being dependent on alleviation was illustrated in terms of having access to relief but also experiencing receiving inadequate care. In conclusion, UTI is very common among old and very old women and is a serious health problem. UTI seems to be associated with delirium and to have a significant impact on the morale or subjective wellbeing of old women and those affected suffer both physically and psychologically and their social life is limited. UTI was also associated with vertebral fractures, urinary incontinence, inflammatory rheumatic disease and multi-infarct dementia which might raise the suspicion that UTI can have serious medical effects on health in old women. / Embargo t o m 2011-11-11

<p>The mammalian urinary bladder is a highly specialized organ that must be able to withstand considerable amounts of osmotic pressure at its mucosal surface, in addition to maintaining an impenetrable barrier against potential pathogens. The lower urinary tract's virtually inevitable exposure to external microbial pathogens warrants efficient tissue-specialized defenses to maintain sterility. The observation that the bladder can become chronically infected with uropathogenic E.coli (UPEC) in combination with clinical observations that antibody responses following bladder infections are not detectable, suggest defects in the formation of adaptive immunity and immunological memory. We have identified a broadly immunosuppressive transcriptional program specific to the bladder, but not the kidney, during infection of the urinary tract that is dependent on tissue-resident mast cells. This mast cell-dependent phenomenon involves localized production of IL-10 and results in suppressed humoral and cell-mediated responses and bacterial persistence. Therefore, in addition to the previously described role of mast cells orchestrating the early innate immune responses in the bladder during infection, they subsequently play a tissue-specific immunosuppressive role. These findings may explain the prevalent recurrence of bladder infections and suggest the bladder as a site exhibiting an intrinsic degree of mast cell-maintained immune privilege.</p><p> Interestingly, though the bladder is not capable of initiating an effective adaptive immune response during bladder infections, we have generated data showing that it was possible to circumvent the immune limitations of the bladder to provoke a strong adaptive and protective immune response by vaccinating against UPEC at an alternate mucosal site. We reasoned that by immunizing the nasal regions of mice with a vaccine formulation comprising of FimH adhesin, a highly conserved adhesive moiety of type 1 fimbriae expressed on UPEC, and an effective mucosal adjuvant we would evoke protective immunity against UPEC infections. We found that a FimH vaccine coupled with either a mast cell activating adjuvant c48/80 or CpG oligodeoxynucleotide, a TLR9 agonist, evoked high levels of FimH specific IgG antibody in the serum and IgA in the urine of immunized mice. We also observed that following UPEC challenge, these FimH/adjuvant immunized mice exhibited significantly reduced bacterial load in the bladders compared to mice challenged with just FimH. These studies reveal that immunization of nasal regions with a FimH vaccine is an effective strategy to overcome the limitation in adaptive immunity observed in the bladder.</p> / Dissertation

Urine culture, the current gold standard for urinary tract infection (UTI) diagnosis, does not produce results in an acceptable length of time. An ultra-sensitive, cost-effective electrochemical biosensing platform with nanostructured microelectrodes was designed to address the need for a rapid, point-of-care (PoC) test that could achieve a sample-to-answer time in less than an hour. Printed circuit boards and metallized glass slides were processed using various techniques and then tested for their ability to form nanostructured microelectrodes. Peptide nucleic acid probes for the bacteria and yeast as well as ten probes for antibiotic resistance genes were designed and synthesized for use with the new platform. Validation of the sensor's specificity was performed using high concentrations (100nM) of synthetic DNA oligomers. Furthermore, a clinically relevant sensitivity of 103 cfu/mL was demonstrated by detecting 4 pathogen lysates (Staphylococcus saprophyticus, Pseudomonas aeruginosa, Enterococcus faecalis and Klebsiella pneumoniae) in a buffered solution.

Urine culture, the current gold standard for urinary tract infection (UTI) diagnosis, does not produce results in an acceptable length of time. An ultra-sensitive, cost-effective electrochemical biosensing platform with nanostructured microelectrodes was designed to address the need for a rapid, point-of-care (PoC) test that could achieve a sample-to-answer time in less than an hour. Printed circuit boards and metallized glass slides were processed using various techniques and then tested for their ability to form nanostructured microelectrodes. Peptide nucleic acid probes for the bacteria and yeast as well as ten probes for antibiotic resistance genes were designed and synthesized for use with the new platform. Validation of the sensor's specificity was performed using high concentrations (100nM) of synthetic DNA oligomers. Furthermore, a clinically relevant sensitivity of 103 cfu/mL was demonstrated by detecting 4 pathogen lysates (Staphylococcus saprophyticus, Pseudomonas aeruginosa, Enterococcus faecalis and Klebsiella pneumoniae) in a buffered solution.

Antibiotic-resistant bacteria are increasingly a major healthcare problem but very few new classes of antibiotics have been discovered or launched in recent decades. Approaches to dealing with the problem include learning how bacteria evolve to resistance and improving dosing regimens with current antibiotics so as to reduce the selection of resistant bacteria. This thesis presents studies examining whether antibiotic dosing at high levels can prevent the selection of fluoroquinolone-resistant mutants in Escherichia coli. It also addresses the genetics of fluoroquinolone resistance in E. coli in relation to fitness costs for the resistant bacteria, and the evolution of E. coli to reduce the costs of resistance. The mutant prevention concentration (MPC) of ciprofloxacin was measured for a set of clinical urinary tract infection E. coli strains showing that MPC could not be predicted from the minimum inhibitory concentration (MIC). Results from an in vitro kinetic model showed that an AUC/MPC &gt;22 for ciprofloxacin was the single best pharmacodynamic index that predicted prevention of resistance emergence in the wild-type. Simulating currently approved dosing regimens for three different fluoroquinolones it was found that only a few were effective in preventing the selection of a small sub-population of pre-existing mutants. Step-wise selection of fluoroquinolone resistance showed that the accumulation of mutations usually reduced bacterial fitness in vitro and in vivo. Systematic construction of isogenic resistant strains confirmed this result and revealed that some combinations of resistance mutations mutually compensate and increase both resistance and fitness. It was discovered that mutations altering RNA polymerase could ameliorate the fitness costs of fluoroquinolone resistance. Thus, the major fitness cost of fluoroquinolone resistance is due to defective transcription. The finding that fluoroquinolone resistance mutations can increase resistance while mutually compensating their fitness costs, shows that resistance to fluoroquinolones can continue to evolve in the absence of antibiotic selection.

abstract: In this thesis, I present a lab-on-a-chip (LOC) that can separate and detect Escherichia Coli (E. coli) in simulated urine samples for Urinary Tract Infection (UTI) diagnosis. The LOC consists of two (concentration and sensing) chambers connected in series and an integrated impedance detector. The two-chamber approach is designed to reduce the non-specific absorption of proteins, e.g. albumin, that potentially co-exist with E. coli in urine. I directly separate E. coli K-12 from a urine cocktail in a concentration chamber containing micro-sized magnetic beads (5 µm in diameter) conjugated with anti-E. coli antibodies. The immobilized E. coli are transferred to a sensing chamber for the impedance measurement. The measurement at the concentration chamber suffers from non-specific absorption of albumin on the gold electrode, which may lead to a false positive response. By contrast, the measured impedance at the sensing chamber shows ~60 kÙ impedance change between 6.4x104 and 6.4x105 CFU/mL, covering the threshold of UTI (105 CFU/mL). The sensitivity of the LOC for detecting E. coli is characterized to be at least 3.4x104 CFU/mL. I also characterized the LOC for different age groups and white blood cell spiked samples. These preliminary data show promising potential for application in portable LOC devices for UTI detection. / Dissertation/Thesis / M.S. Electrical Engineering 2011

Introdução: As Infecções no trato urinário (ITU) estão entre as doenças infecciosas mais comumente diagnosticadas e são responsáveis por uma grande proporção do consumo de agentes antimicrobianos, sendo a Escherichia coli o microorganismo mais freqüentemente envolvido. Os antimicrobianos utilizados no tratamento das ITU sofrem concentração no processo de eliminação, ocasionando altos níveis de antimicrobiano na urina. No entanto, os testes que avaliam a susceptibilidade aos antimicrobianos são baseados em concentrações séricas. Assim, é de interesse a padronização de um teste que utilize níveis urinários (discos de alta potência) dos antimicrobianos frente aos uropatógenos. Objetivo: padronizar teste de susceptibilidade aos antimicrobianos para E. coli de ITU, com concentrações de antimicrobianos atingidas na urina. Métodos: foram utilizados 204 isolados bacterianos resistentes a pelo menos um dos seguintes antimicrobianos: ampicilina, cefalotina, ciprofloxacino, gentamicina, sulfametoxazol-trimetoprima(cotrimoxazol) para os quais foi determinada a MIC através de diluição em agar. Através da análise das MIC obtidas e das concentrações que os antimicrobianos atingem na urina, foram estabelecidas as concentrações dos discos de alta concentração. As seguintes concentrações de disco foram utilizadas: ampicilina (500, 400, 300 μg), cefalotina (200, 100 μg) e ciprofloxacino (200, 150 μg). Resultados: a ampicilina apresentou MIC ≥ 512μg/mL para cerca de 50% dos isolados. Dos discos testados, o de 500 μg mostrou melhor coeficiente de correlação de Spearman (rs: -0,659) e foi escolhido para a determinação do ponto de corte através de curva ROC (receiver operating characteristic). Pela curva, um diâmetro de halo de 9 mm separaria as amostras resistentes (R) das susceptíveis (S) a altos níveis de ampicilina com 100% de sensibilidade e especificidade de 50%. A cefalotina apresentou MIC inferior a 256μg/mL para 78% dos isolados sendo que o “rs” para os discos de 100 e 200 μg foi o mesmo: -0,744. Pela curva ROC, halos de 17mm (disco de 200μg) e 13mm (disco de 100μg) discriminariam amostras R de S com 100% de sensibilidade e 96,9% de especificidade. O ciprofloxacino apresentou MIC ≤ 128 μg/mL para mais de 50% dos isolados, sendo que o “rs” foi melhor com o disco de 200μg (rs:-0,916). Um halo de 21mm separaria as amostras S e R com 100% de sensibilidade e 78,9% de especificidade. Para o cotrimoxazol, 95% das amostras se mostraram resistentes a altos níveis e os testes com disco de alta potência não foram realizados. Para a gentamicina o baixo nº de amostras (n=22) prejudicou a análise, mas cerca de 63% dos isolados seriam susceptíveis a altos níveis. Conclusão: O presente estudo demonstrou que o uso de discos de alta concentração pode ser útil para avaliar a susceptibilidade de isolados urinários. Isto tem importância em laboratórios clínicos onde, embora haja dificuldade na realização da MIC, testes de disco-difusão estão incorporados na rotina. / Introduction: The urinary tract infections (UTI) are among the most common infectious diseases and are responsible for a high antimicrobial usage. The Escherichia coli is the main microorganism involved in UTI. The antimicrobials used for treatment of UTI concentrate in the urinary tract and this leads to high levels of them in the urine. However, the susceptibility tests are based on the serum levels of the antimicrobials. Therefore, it is of interest to develop a test which correlates the urinary levels of antibiotics (discs with high levels) with the uropathogens. Objective: to propose a susceptibility test of E. coli from UTI using doses of the antimicrobials reached in the urine. Methods: a total of 204 clinical isolates of E.coli resistant, according to conventional antimicrobial susceptibility test to, at least, one of the following antimicrobials: ampicillin, cefalotin, ciprofloxacin, gentamicin and trimetoprima- sulfametoxazol. The MIC was determined by the agar dilution technique. According to the results of the MICs and the urinary levels of the antimicrobials it was possible to establish the concentration of the high potency discs. The following concentration of discs were used: ampicilina (500, 400, 300 μg), cefalotin (200, 100 μg) and ciprofloxacin (200, 150 μg). Results: ampicillin presented MIC ≥ 512μg/mL for around 50% of E. coli isolates. The disc containing 500 μg of ampicillin displayed the best coeficient of correlation (rs: -0,659) and it was chosen to establish diameter breakpoint according to the ROC curve. A diameter of 9 mm would discriminate high level ampicillin resistant isolates with 100% and 50% of sensitivity and specificity, respectively. Cefalotin presented MIC below 256 μg/mL for 78% of E. coli isolates. Its “rs” for discs containing 100 and 200μg was the same: - 0,744. According to the ROC curve zone diameters of 17mm (disk of 200μg) e 13mm (disk of 100μg) were able to discriminate isolates resistant with 100% and 96.9% of sensitivity and specificity, respectively. Ciprofloxacin presented MIC ≤ 128 μg/mL for more than 50% of isolates and the best “rs” was achieved with the 200μg (r:-0,916) disk. A zone diameter of 21mm would discriminate susceptible isolates with 100% and 78.9% of sensitivity and specificity. respectively. A total of 95% of E. coli isolates proved to be resistant to high levels of cotrimoxazole and, therefore, tests with high potency disks were not performed. Due to the small numbers of isolates resistant to gentamycin (n=22) we were not able to perform further analysis although 63% of E coli isolates proved to be susceptible to high levels of this antimicrobial. Conclusion: This study indicated that the susceptibility test with disks containing high levels of antimicrobials may be useful to evaluate the susceptibility of urinary tract isolates This may be of importance as the clinical laboratory may not be able to perform the MIC test for routine isolates.

Master of Science / Department of Industrial & Manufacturing Systems Engineering / Malgorzata J. Rys / According to the Centers for Disease Control and Prevention (2015), urinary tract infections (UTIs) are the most commonly reported healthcare-associated infection (HAI), of which approximately 75% of infections are attributed to the presence of a urinary catheter. Urinary catheters are commonplace within hospitals as approximately 15-25% of patients receive a urinary catheter during their hospitalization, introducing the risk of a catheter associated urinary tract infection (CAUTI) during their stay (CDC, 2015). In recent years there have been efforts to reduce CAUTI in U.S. hospitals; however, despite these efforts, CAUTI rates indicate the need to continue prevention efforts. Researchers have investigated the use of human reliability analysis (HRA) techniques to predict and prevent CAUTI (Griebel, 2016), and this research builds on that topic by applying the Human Error Assessment and Reduction Technique (HEART) to develop a model for a patient’s probability of CAUTI. HEART considers 40 different error-producing conditions (EPCs) present while performing a task, and evaluates the extent to which each EPC affects the probability of an error. This research considers the task of inserting a Foley catheter, where an error in the process could potentially lead to a CAUTI. Significant patient factors that increase a patient’s probability of CAUTI (diabetes, female gender, and catheter days) are also considered, along with obesity which is examined from a process reliability perspective. Under the HEART process, human reliability knowledge and the knowledge of eight expert healthcare professionals are combined to evaluate the probability that a patient will acquire a CAUTI. In addition to predicting the probability of CAUTI, HEART also provides a systematic way to prioritize patient safety improvement efforts by examining the most significant EPCs or process steps. The proposed CAUTI model suggests that 7 of the 26 steps in the catheter insertion process contribute to 95% of the unreliability of the process. Three of the steps are related to cleaning the patient prior to inserting the catheter, two of the steps are directly related to actually inserting the catheter, and two steps are related to maintaining the collection bag below the patient’s bladder. An analysis of the EPCs evaluated also revealed that the most significant factors affecting the process are unfamiliarity, or the possibility of novel events, personal psychological factors, shortage of time, and inexperience. By targeting reliability improvements in these steps and factors, healthcare organizations can have the greatest impact on preventing CAUTI.