Urinalysis is a common laboratory test used for diagnosis of a variety of systemic and genitourinary diseases. Although, collection of sample for urinalysis is extremely easy, when performed during an office visit, in pediatric and geriatric populations, who use diaper, such collection is not trivial and can result in missing important diagnostic information. For example, urinary tract infections (UTIs), are a major source of morbidity in incontinent elderly with dementia who cannot communicate their symptom to their caregivers. Although most UTIs are easily treatable with antibiotics, if not identified and treated timely, they can cause ascending infection, loss of kidney function, sepsis, and possible death. Deployment of smart, autonomous, diaper-embedded systems that can detect early signs of urinary dysfunction can have a significant impact on healthcare of our rapidly aging population. In this dissertation, I propose a diaper-embedded, low-cost, and disposable sensing platform comprising of a urine-activated battery and sensors for detection of nitrite (a surrogate for UTI), red blood cells (hematuria), and protein (proteinuria). I will first discuss my efforts to develop an optical/colorimetric nitrite sensor and a urine-activated power source, all fabricated on a hydrophobic paper/polymeric substrate through laser-assisted machining and lamination-assembly. The system stays in a dormant state until wetted by urine, after which the on-board power source is activated, awakening the rest of the measurement system (i.e., a light emitting diode, a photodetector, interface electronics, and a low-power Bluetooth module) and transmitting the presence or absence of nitrite in the urine to vicinal caregivers in a point-of-care and autonomous fashion. Thorough characterization of the performance and reliability analysis of the platform are also presented to envision its use as an end product. Afterwards, I will discuss the characterization of sensors, based on similar principle, for detecting red blood cells (hematuria) and protein (proteinuria), and the extendibility of the proposed platform for a multi-parameter system measuring nitrite, blood, and protein in the urine. Finally, I will conclude with other possible applications besides urinalysis for the proposed system.

Klebsiella pneumoniae is a Gram negative, enteric bacterium that frequently causes disease in immunocompromised individuals. These types of infections are often associated with the presence of indwelling medical devices, which provide a site for the organism to attach and subsequently form a biofilm. A key component in K. pneumoniae biofilm formation in vitro is type 3 fimbriae. The two main components of this project have been to determine if type 3 fimbriae are an in vivo virulence factor using a mouse model of catheter associated urinary tract infection (CAUTI) and to examine the mechanism by which the production of type 3 fimbriae are regulated. Using a mouse model in which a silicone tube is implanted into the bladder of mice, mimicking the effects of catheterization, we have been able to show that type 3 fimbriae are required for colonization and persistence. Using different time points and conditions, we demonstrated that there are conditions when type 3 fimbriae alone are sufficient for colonization and other conditions where both type 1 and type 3 fimbriae have unique roles in colonization and persistence. Additionally, competition experiments showed that neither fimbrial mutant alone, or a double mutant in type 1 and type 3 fimbriae could compete with wildtype K. pneumoniae. In most animals, only wild-type bacteria were recovered by 24 hours post-inoculation. This work reinforced the role of type 1 fimbriae in pathogenesis and showed, for the first time, a role for type 3 fimbriae using an in vivo model. Our early work has indicated that type 3 fimbriae are regulated at least in part by the intracellular levels of the secondary messenger molecule cyclic di-GMP. Downstream from the type 3 fimbrial operon a gene encoding a phosphodiesterase is present; the product of this gene breaks down cyclic di-GMP. In the absence of this gene the levels of type 3 fimbrial expression are increased. Also adjacent to the mrk operon is a two-gene operon containing the determinants we have named mrkH and mrkI. mrkH encodes a PilZ domain containing protein, which we have shown binds cyclic di-GMP. Using a transcriptional fusion we have shown that the mrk gene promoter is activated modestly in the presence of MrkH, but when MrkH and MrkI are both present the activity is increased 100-fold. This has lead to the hypothesis that MrkH and MrkI interact, which we have been able to demonstrate using copurification procedures. This interaction appears to occur in a cyclic di-GMP dependent manner with the resulting protein complex binding to the mrk promoter region and activating the expression of type 3 fimbriae.

L’infection urinaire (IU) est un problème majeur de Santé publique. La cystite aiguë touchant principalement les femmes est la plus fréquente des IU. La bactérie la plus fréquemment isolée au cours de ces IU est Escherichia coli. Une des particularités de la cystite est sa propension à récidiver. Le traitement préconisé pour ces infections est la prise d’antibiotiques, qui peut être fréquente en cas de cystites récidivantes. C’est dans ce contexte que de nouvelles stratégies doivent être développées afin de prévenir et traiter les IU récidivantes. Parmi ces différentes stratégies, l’utilisation de produits naturels tels que la canneberge (Vaccinium macrocarpon) apparaît comme prometteuse. En effet, des études précédentes ont montré que la canneberge a un effet négatif sur l’adhésion des bactéries aux cellules superficielles de l’épithélium vésical facilitant l’élimination des bactéries par le flux urinaire. Cette activité est portée par la proanthocyanidine de type A (PAC-A). D’autre part, une étude menée par notre équipe a montré que l’effet de la canneberge sur l’adhésion et la virulence de souches d’E. coli uropathogènes pouvait être potentialisé par l’ajout d’un autre composé naturel : la propolis. Depuis l’Antiquité ses propriétés anti-bactériennes sont reconnues et des études plus récentes ont démontré son impact sur des bactéries à Gram positif mais également sur deux bactéries à Gram négatif : E. coli et Pseudomonas aeruginosa. Ce travail de thèse a permis : i) de décrire l’impact de la canneberge, de la propolis et de leur association sur le transcriptome d’une souche clinique d’E. coli uropathogène (G50). Cette analyse transcriptomique a montré que la canneberge entrainait une sous-expression de gènes liés à l’adhésion, mais également de gènes liés à la mobilité et à la formation de biofilm. En revanche, la canneberge augmentait l’expression des gènes liés au métabolisme du fer ainsi qu’à la réponse au stress. Ces effets étaient potentialisés par l’ajout de la propolis. En parallèle, des tests phénotypiques menés sur une collection de souches d’E. coli uropathogènes sur la mobilité et la formation de biofilm ont confirmé les résultats précédents ; ii) de développer un test, basé sur les précédents travaux de transcriptomique, permettant une évaluation standardisée de l’effet de la PAC-A sur E. coli, indépendamment de sa concentration car il n’existe pas de techniques standardisées pour doser cette molécule. C’est ainsi que 4 gènes (tsr, ftnA, fecB, feoB) ont été sélectionnés, le suivi de leur expression permettant une mesure de l’activité anti-bactérienne de la canneberge; iii) de mesurer l’effet potentialisateur de la propolis sur l’activité des antibiotiques utilisés dans le traitement des IU. C’est ainsi qu’il a été montré que l’ajout de la propolis permettait d’augmenter l’activité bactéricide des antibiotiques testés et de diminuer les concentrations minimales inhibitrices de ces antibiotiques. / Urinary Tract Infection (UTI) is a major problem of public health. Acute cystitis which touches mostly women is the most common form of UTI. The bacteria which is mostly isolated in an UTI is Escherichia coli. A particularity of cystitis is to come back. In this context news strategies have to be developed to prevent and cure recurrent UTI. One of these strategies is the utilization of natural products like the cranberry (Vaccinium macrocarpon) which is promising. Indeed, previous studies showed the negative impact of cranberry on the adhesion of bacteria on the superficial cells of bladder which help the elimination of bacteria by the urinary flux. This activity is carried by the type A proanthocyanidin (PAC-A). Moreover, a study lead by our team has demonstrated an improvement of the activity of cranberry on the adhesion and the virulence of uropathogenic E. coli (UPEC) by another natural product: the propolis. Since Antiquity its antibacterial activities have been recognize and more recent studies have demonstrated its impact of Gram positive bacteria and also on two Gram negative bacteria: E. coli and Pseudomonas aeruginosa. This thesis has allowed for: i) the description of the impact of cranberry, propolis and its association on the transcriptome of a clinical strain of UPEC (G50). This transcriptomic analyze have shown that the cranberry down regulated genes linked to the adhesion and also genes linked to the motility and biofilm formation. However the cranberry up regulated genes linked to the iron metabolism and the stress response. These effects are improve by the addition of propolis. Concurrently phenotypics tests have been conducted on a collection of UPEC on the motility and the biofilm formation and they confirmed the previous results; ii) the development of a test, based on our transcriptomic results, enable to performed a standardized evaluation of the impact of PAC-A on E. coli, independently of its concentration. Indeed, this molecule cannot be measure in a standard way. Four genes have been selected (tsr, ftnA, fecB, feoB), the monitoring of their expression allow us to measure the anti-bacterial efficiency of the cranberry; iii) the measurement of the potential effect of the propolis of the antibiotic’s activities used to treat UTIs. Thus it have been observed that the addition of propolis improve the bactericide activity of the antibiotics tested and reduces the minimal inhibitory concentration of these antibiotics.

Urinary tract infections are diagnosed in female populations primarily in ambulatory care settings in the United States. Yet, published evidence documents that many of the antibiotics prescribed in these settings are unnecessary, erroneous, or, inappropriately prescribed. Improper management of uncomplicated urinary tract infections in nonpregnant women has resulted in higher morbidity rates due to antibiotic resistance. The purpose of this retrospective observational cohort study was to explore a current national database for associations between nonpregnant American female patients who were exposed to poverty and at risk for urinary tract infection antibiotic resistance in an ambulatory care setting. Krieger's ecosocial theory was utilized as the study's theoretical foundation to complement current public health social change priorities. Data from the National Ambulatory Medical Care Survey were analyzed to explore potential associations with urinary tract infections and antibiotic resistance. The sample consisted of ambulatory patients with urinary tract infection symptoms (n=45). The independent variables selected were antibiotics prescribed initially in 3 months or less after the onset of urinary tract infection symptoms, the continuation of antibiotics prescribed in 12 months or less after recurrence, and three classes of antibiotics prescribed for urinary tract infection symptoms known as broad-spectrum, narrow-spectrum, and combined broad- and narrow-spectrum antibiotics, while the dependent variable was urinary tract infection antibiotic resistance. Relationships between the variables were analyzed using binary logistic regression, however, there were no statistically significant outcomes. Promoting antibiotic stewardship programs in all health care settings in the U.S. can effect positive social changes that will prevent further antibiotic resistance.

Orientador: Cristiano Gallina Moreira / Resumo: As infecções do trato urinário são frequentes no mundo todo, sendo a Escherichia coli Uropatogênica (UPEC) o patógeno responsável pela maior parte dos casos de cistite e pielonefrite aguda. A patogenicidade das UPECs está relacionada a expressão de diversos fatores de virulência, sendo a regulação da expressão desses fatores mediada por moléculas sinalizadoras químicas que permitem a comunicação célula-célula inter e intra-reinos, o que facilita o processo de colonização e estabelecimento da patogênese. Um dos sistemas responsáveis por essas cascatas de sinalização é composto por uma proteína sensora de membrana (QseC) e outra reguladora de resposta citoplasmática (QseB), constituindo o sistema de dois componentes QseBC, capaz de reconhecer sinais produzidos pelo hospedeiro e por outras bactérias, levando a regulação da expressão de genes de virulência do patógeno. Estudos realizados pelo nosso grupo, evidenciaram atenuação da virulência de patógenos Gram-negativos na ausência do gene qseC, levando ao desenvolvimento de moléculas que atuassem inibindo essa via como o LED209. O objetivo do presente trabalho foi caracterizar cepas multi-droga resistentes (MDR) de UPECs obtidas a partir de isolados clínicos, além de investigar, in vitro e in vivo, a participação da via QseBC na patogênese e na virulência de cepas de UPECs e isolados clínicos MDR, com o uso do composto LED209 na atenuação da virulência frente a esses patógenos. Também constituiu o objetivo, o uso do ácido 3,4-di-... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Urinary tract infections are found commonly worldwide, whereas Uropathogenic Escherichia coli (UPEC) is the most prevalent pathogen, responsible for utmost cases of cystitis and acute pyelonephritis. The pathogenicity of UPECs is related to the expression of several virulence factors, and the regulation of the expression of these factors is mediated by chemical signaling molecules. The communication allow inter-intra-kingdom and cell-to-cell facilitates the process of colonization and establishment of pathogenesis. QseBC two-component system is capable of recognizing signals produced by the host leading to regulation of pathogen virulence gene expression. This system consists of membrane sensing protein (QseC) and a cytoplasmic response regulator (QseB) that mediate the entire cascade of virulence genes. Studies conducted by our group showed attenuation of the virulence of Gram-negative pathogens in the absence of the qseC gene, leading to the development of molecules that act by inhibiting this pathway such as LED209. The aim of the present study was to characterize UPEC multidrug-resistant strains (MDR) from clinical isolates. Investigate, in vitro and in vivo, the involvement of the QseBC pathway in the pathogenesis and virulence of UPEC strains and clinical isolates. The use of LED209 and the 3,4-dihydroximandelic acid (DHMA), norepinephrine intermediate metabolite, as chemoattractant, were employed to attenuate of virulence against these pathogens. The results showed a h... (Complete abstract click electronic access below) / Mestre

<p>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. </p><p>This thesis presents studies examining whether antibiotic dosing at high levels can prevent the selection of fluoroquinolone-resistant mutants in <i>Escherichia coli</i>. It also addresses the genetics of fluoroquinolone resistance in <i>E. coli</i> in relation to fitness costs for the resistant bacteria, and the evolution of <i>E. coli</i> to reduce the costs of resistance.</p><p>The mutant prevention concentration (MPC) of ciprofloxacin was measured for a set of clinical urinary tract infection <i>E. coli</i> strains showing that MPC could not be predicted from the minimum inhibitory concentration (MIC). Results from an <i>in vitro</i> kinetic model showed that an AUC/MPC >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. </p><p>Step-wise selection of fluoroquinolone resistance showed that the accumulation of mutations usually reduced bacterial fitness<i> in vitro</i> and <i>in vivo</i>. 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. </p><p>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.</p>

<p>The emergence of multidrug resistant bacteria world wide is a serious problem, and very few new drugs are under development. The selection of resistant bacteria is affected by factors such as mutation rate, biological fitness cost and the rate of fitness compensation. This thesis is focused on how mutation rate affects resistance to fluoroquinolones and on exploring a dosing strategy that might slow resistance development. </p><p>In a set of urinary tract <i>Escherichia coli</i> isolates MIC values above the breakpoint for the fluoroquinolones norfloxacin and ciprofloxacin carried at least three resistance-associated mutations. In these isolates the number of resistance mutations correlated with the mutation rate. During step-wise selection for decreased susceptibility to fluoroquinolones, the accumulation of mutations in <i>E. coli</i> was associated with an increasing biological cost both <i>in vitro</i> and <i>in vivo</i>. However, in some lineages an additional selection step for resistance was associated with a partial restoration of fitness. During step-wise selections we found, as expected, that reduced ciprofloxacin susceptibility frequently hitchhiked with a strong mutator phenotype. More surprisingly, we also found that reduced susceptibility was frequently associated with the emergence of rifampicin-resistant populations. We hypothesise that this correlation reflects selection for fitness-compensating mutations in RNA polymerase.</p><p>Mutant prevention concentration (MPC) dosing has been proposed as a strategy to reduce the selection of resistant bacterial populations. Based on limited data it had been thought that MPC might be a simple multiple of MIC, which can easily be determined. However, we showed for a collection of susceptible urinary tract <i>E. coli </i>that MPC could not be predicted from MIC and must be measured directly for relevant populations. Using an <i>in vitro</i> kinetic model we also showed that the pharmacodynamic index that best predicted prevention of resistance development in wild type <i>E. coli</i> was an AUC/MPC of > 22 for ciprofloxacin.</p>

The emergence of multidrug resistant bacteria world wide is a serious problem, and very few new drugs are under development. The selection of resistant bacteria is affected by factors such as mutation rate, biological fitness cost and the rate of fitness compensation. This thesis is focused on how mutation rate affects resistance to fluoroquinolones and on exploring a dosing strategy that might slow resistance development. In a set of urinary tract Escherichia coli isolates MIC values above the breakpoint for the fluoroquinolones norfloxacin and ciprofloxacin carried at least three resistance-associated mutations. In these isolates the number of resistance mutations correlated with the mutation rate. During step-wise selection for decreased susceptibility to fluoroquinolones, the accumulation of mutations in E. coli was associated with an increasing biological cost both in vitro and in vivo. However, in some lineages an additional selection step for resistance was associated with a partial restoration of fitness. During step-wise selections we found, as expected, that reduced ciprofloxacin susceptibility frequently hitchhiked with a strong mutator phenotype. More surprisingly, we also found that reduced susceptibility was frequently associated with the emergence of rifampicin-resistant populations. We hypothesise that this correlation reflects selection for fitness-compensating mutations in RNA polymerase. Mutant prevention concentration (MPC) dosing has been proposed as a strategy to reduce the selection of resistant bacterial populations. Based on limited data it had been thought that MPC might be a simple multiple of MIC, which can easily be determined. However, we showed for a collection of susceptible urinary tract E. coli that MPC could not be predicted from MIC and must be measured directly for relevant populations. Using an in vitro kinetic model we also showed that the pharmacodynamic index that best predicted prevention of resistance development in wild type E. coli was an AUC/MPC of &gt; 22 for ciprofloxacin.

Herbal teas and fruit juices used in our regular diet may have importance in the protective treatment of some infectious diseases. In this study, selected dietary beverages were investigated for their antioxidant capacities and antimicrobial activities against Proteus mirabilis, a well known bacteria in urinary tract infections. Herbal teas / sage (Salvia fruticosa Mill), anise (Pimpinella anisum L.), rosehip (Rosa canina L.), camomile (Anthemis arvensis L.) and fruit juices / grape (Vitis vinifera L.), orange (Citrus sinensis L.), peach (Prunus persica L.), and pomegranate (Punica granatum L.) were chosen as samples of regular diets. Selected fruit juices and aqueous infusion tea extracts, lyophilised to dryness, were used throughout this study. Antioxidant capacities of the extracts were carried out by using 2,2&rsquo / -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging (ABTS) and 2,2-diphenyl-1-picrylhydrazyl radical scavenging (DPPH) methods along with the determination of total phenolic compounds in the extracts. Antimicrobial activities of extracts were determined by disc diffusion test, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) methods. Among the herbal teas, sage infusion extract has displayed the highest radical scavenging capacity with ABTS EC50 value of 5.152 mg/mL, DPPH EC50 value of 0.072 mg/mL and with its high phenolic content of 0.411 mg/mg gallic acid equivalence. Among the fruit juices pomegranate has revealed significantly high DPPH EC50 and TEAC values 0.924 mg/mL and 0.552 mmol/g, respectively. Peach juice has been found with the highest total phenolic amount of 0.067 mg/mg gallic acid equivalent. Antimicrobial activities of herbal teas were correlating with antioxidant capacity studies, whereas sage infusion tea extract exhibited 3 mg/mL of minimum inhibitory concentration (MIC) and 6 mg/mL of minimum bactericidal concentration (MBC). Rosehip was also found as an effective antimicrobial agent with a minimum inhibitory concentration value of 3 mg/mL. In the meantime, there was no significant difference in the zone inhibition of herbal tea infusion extracts. In case of fruit juices grape and pomegranate may be effective antimicrobials in P. mirabilis infections with 0.75 mg/mL MIC and 6 mg/mL MBC, respectively at the same time both juices revealed significantly high inhibition zones with 11 mm.

Šlapimo organų infekcija (ŠOI) vaikams yra antra pagal dažnį po kvėpavimo organų infekcijų. Tai dažniausia vaikų nefrologinė liga. ŠOI iki 3–6 mėn. amžiaus dažniau serga berniukai, vyresniame amžiuje – mergaitės. Priešmokykliniame amžiuje mergaitės simptomine šlapimo organų infekcija serga 6–20 kartų dažniau nei berniukai. Beveik kas trečia moteris iki 24 metų ŠOI yra sirgusi bent vieną kartą, o per gyvenimą ŠOI yra sirgusi beveik kas antra moteris. ŠOI linkusi pasikartoti. Kartojantis ŠOI, liga gali įgauti lėtinę eigą, sukelti inkstų randėjimą, lėtinį inkstų funkcijos nepakankamumą (IFN), nulemti hipertenzijos atsiradimą, o moterims – nėštumo komplikacijas. 29 % vaikų, kuriems buvo atliktos inkstų transplantacijos, inkstų pažeidimas buvo sąlygotas pielonefrito ar intersticinio nefrito. Lietuvoje vaikų lėtinio IFN priežastis 31,7 proc. obstrukcinė nefropatija ir lėtinis pielonefritas. Kol nebuvo taikomas profilaktinis gydymas, 60 proc. mergaičių ir 20 proc. berniukų ŠOI pasikartodavo jau pirmaisiais metais po pirmos ŠOI. Taikant profilaktinį gydymą, 1 m. laikotarpyje po persirgtos ŠOI. infekcijos pasikartojimas sumažėjo iki 15 proc. Mažo amžiaus vaikams ŠOI pasikartojimą dažniausiai lemia įgimtos šlapimo organų anomalijos. Dauguma jaunesnio mokyklinio amžiaus vaikų, kuriems yra pasikartojanti ŠOI, turi organiškai nepakitusius šlapimo organus. Pastaruoju metu atliekamos studijos, kurių tikslas nustatyti elgesio ir funkcinių sutrikimų vertę ŠOI pasikartojimui, tačiau duomenys... [toliau žr. visą tekstą] / Urinary tract infection (UTI) is one of the most common bacterial diseases in childhood. Recurrent UTI occurs in 20 – 86% of children. Recurrent UTI is relatively frequent in girls. At the age of 7, the prevalence of recurrent UTI in boys population is 1%, in girls population – 5%. Nearly one of three women will have at least one episode of UTI requiring antimicrobial therapy by the age of 24 years. Almost half of all women will experience one UTI during their lifetime. It is known, that in the group of young children the most common reason of recurrent UTI is anatomic abnormalities, such as vesicoureteral reflux (VUR), hydronephrosis. However, not all recurrent UTI can be explained by anatomic abnormalities. The vast majority of school age children with recurrent UTI have anatomically normal urinary tract. We found changes in urodynamic investigation for 91.4% of children with recurrent urinary tract infection at the age of 5–18. Having a history of previous recurrent UTI is a strong risk factor for having subsequent UTI. Antibacterial characteristics of urine and other host defence mechanisms may be important signs associated with UTI risk, but have not been clearly shown to be associated with UTI in healthy persons. Recent studies discuss about the role of behavioural and functional abnormalities (inadequate fluid intake, stool retention, infrequent voiding, etc.) that can predispose recurrent urinary tract infections. Influence of some these abnormalities for recurrent... [to full text]