The adhesion of bacteria to uroepithelial cells or urinary catheters is the first step in the development of biofilm formation and urinary tract infections (UTIs). Previous research has suggested that consumption of cranberry juice can prevent the recurrence of UTIs by decreasing bacterial adhesion since isolated compounds in cranberries, known as A-type proanthocyanidins (PACs), change the conformation of proteinaceous fimbriae that help attach bacteria to epithelial cell receptors. Most clinical and laboratory studies have shown the effects of cranberry juice cocktail (CJC) on large communities of bacteria; however, very few studies have evaluated how cranberry affects the adhesion forces of a single bacterium as well as effects on cellular composition and biofilm formation. We used atomic force microscopy (AFM) to investigate the effects of CJC and PACs on the adhesion forces between E. coli and a silicon nitride tip. Bacterial cultures were grown in tryptic soy broth (TSB), supplemented with 0 and 10 wt.% light cranberry juice cocktail (L-CJC) or 128 µg/mL PACs. E. coli bacteria were continuously cultured in the presence of cranberry products up to twelve times. Experiments were conducted at different scales to test bacterial attachment and adhesion forces. At the macroscale, bacteria were incubated with uroepithelial cells and the number of bacteria attached per uroepithelial cell was determined. In nanoscale experiments, the forces of adhesion between E. coli and a silicon nitride AFM tip were probed for bacteria grown in L-CJC or PACs for different numbers of culture times. Successive replacement of media and continued culture in L-CJC and PACs resulted in a significant decrease in adhesion forces for E. coli strains. Finally, during the continuous exposure of L-CJC to bacteria we examined the growth, morphology, and ability to form biofilms of E. coli. We found a decrease in growth rates related to changes in Gram staining with increasing number of cultures in L-CJC. Growth of bacteria in L-CJC or PACs inhibited the development of biofilms on polyvinyl-chloride, which can model biofilm formation on urinary catheters. We also determined that growth of E. coli in L-CJC results in prevention of the expression of indole which can be linked to the inhibition of biofilm formation. Our results help support the molecular mechanisms for the role of cranberry in preventing the adhesion of E. coli to biotic and abiotic surfaces, thus helping to scientifically validate the use of cranberry juice as a prophylactic treatment for the prevention of UTIs.

Escherichia coli patogênica extra intestinal (ExPEC) constitui um importante patotipo de Escherichia coli (E. coli), sendo responsável por causar infecções em seres humanos e animais. Estudos sobre as relações filogenéticas entre isolados NMEC, UPEC e APEC enfatizam as semelhanças genéticas entre estes grupos de E. coli, sugerindo que as estirpes aviárias possam representar um potencial risco zoonótico. Um dos grandes problemas da infecção por UPEC é o fato de que a colonização da bexiga favorece a formação de biofilmes. Diante da grande incidência de infecções do trato urinário causadas por UPEC, este trabalho buscou avaliar a relação dos grupos filogenéticos de patogenicidade com a formação de biofilmes em amostras de Escherichia coli uropatogênicas em duas diferentes temperaturas. Em estudo prévio, as cepas de Escherichia coli, isoladas de pacientes hospitalizados com infecção urinária, foram classificadas em grupos filogenéticos. Os grupos designados B2 e D são conhecidos por possuírem patogenicidade. B2 está associado a amostras com alta patogenicidade e D, com amostras de patogenicidade intermediária. As amostras pertencentes aos grupos A e B1 são consideradas não patogênicas ou comensais. Com base neste trabalho, todas as 215 cepas de E. coli uropatogênica do presente estudo são oriundas do Hospital de Clinicas de Porto Alegre coletadas entre os anos de 2012 a 2014. Dentre estas 52 cepas de UPEC foram classificadas como pertencentes ao grupo filogenético A (24,2%), 21 amostras ao grupo B1 (9,8%), 69 amostras ao grupo B2 (32%) e 73 amostras ao grupo D (34%). A formação de biofilme foi testada nas temperaturas de 25ºC e 37ºC. Quanto a formação de biofilme em placas de poliestireno na temperatura de 25±1ºC observamos que 51 (23,6%) cepas foram não produtoras de biofilme e 165 (76,4%) foram produtoras de biofilme; na temperatura de 37ºC±1 encontramos apenas 18 (8,3%) isolados não formadores de biofilme e 198 (91,7%) que formaram biofilme. A análise de correlação entre a produção de biofilme in vitro e a classificação dos grupos filogenéticos não apresenta uma diferença significativa (p<0,05), ou seja, a produção de biofilme em poliestireno não pode ser relacionada com a patogenicidade da bactéria em questão, conforme as condições do estudo. / Extra intestinal Pathogenic Escherichia coli (Expectations) is an important pathotype of Escherichia coli (E. coli), being responsible for causing infections in humans and animals. Studies on the phylogenetic relationships among isolates NMEC, UPEC and APEC emphasize the genetic similarities between these groups of E. coli, suggesting that avian strains may represent a potential zoonotic risk. One of the major problems of the UPEC infection is the fact that the colonization of the bladder encourages the formation of biofilms. Given the high incidence of urinary tract infections caused by UPEC, this study sought to assess the relationship of pathogenicity phylogenetic groups with the biofilm formation of Escherichia coli samples uropatogênicas at two different temperatures. In preliminary study, E. coli strains, isolated from hospitalized patients with urinary tract infection, were classified in phylogenetic groups. The groups designated B2 and D are known to possess pathogenicity. B2 is associated with the samples with high pathogenicity and D, with samples of intermediate pathogenicity. The samples belonging to the groups A and B1 are considered non-pathogenic or Diners. Based on this work, all 215 E. coli uropatogênica strains in this study are from the Clinical Hospital Porto Alegre collected from 2012 to 2014. Among these 52 UPEC strains were classified as belonging to the phylogenetic Group (24.2%), 21 samples to group B1 (9.8%), 69 samples the Group B2 (32%) and 73 samples to Group D (34%). Biofilm formation was tested at temperatures of 25° C and 37° c. As the formation of biofilms on polystyrene plates in the temperature of 25 ± 1° C we observe that 51 (23.6%) strains were not producing biofilm and 165 (76.4%) were biofilm producers; at the temperature of 37° C ± 1 we found only 18 (8.3%) isolates not trainers of biofilm and 198 (91.7%) who formed biofilms. The analysis of correlation between in vitro biofilm production and classification of phylogenetic groups does not present a significant difference (p < 0.05), i.e. the production of polystyrene biofilm can not be linked to the pathogenicity of the bacteria in question, as the conditions of the study.

The aim of this study was to determine the prevalence of extended-spectrum b-lactamase (ESBL)- producing Klebsiella pneumoniae isolated from urinary tract and bloodstream infections in a rural hospital in Manhic¸a, Mozambique. ESBLs were investigated among ceftriaxone-non-susceptible K. pneumoniae clinical isolates recovered between 2004 and 2009. Characterisation of blaCTX-M, blaSHV, blaOXA and blaTEM genes was performed by PCR and sequencing. Epidemiological relationships were established by phylogenetic analysis, repetitive extragenic palindromic PCR (REP-PCR), pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), whilst plasmid transferability was evaluated by conjugation. In addition,the presence of class 1 and 2 integrons was studied.A total of 19 K. pneumoniae were analysed. The blaCTX-M-15 gene was found in all strains. Other ESBL genes were found concomitantly, including blaSHV-5, blaSHV-2, blaSHV-2A, blaSHV-12 and blaSHV-38. In addition, other b-lactamases such as blaTEM-1 and blaOXA-30 were also detected. REP-PCR identified 15 different epidemiological profiles. MLST analysis also showed great variability of sequence types. The blaCTX-M-15 gene showed a high transfer capacity. The presence of class 1 integrons was high. High levels of multidrug resistance were also found. In conclusion, these data show the dominance of the CTX-M-type ESBL, particularly CTX-M-15, supporting its worldwide dissemination, including in areas with limited access to third-generation cephalosporins. This finding is a matter of concern for clinical management as third-generation cephalosporins are an alternative for treating severe cases of multidrug-resistant infections in this community. / Revisión por pares

In the United States, home health care (HHC) is the most frequently used form of post-acute care services. Majority of the HHC patients are elderly and have known activities of daily living (ADL) dependencies. The role of HHC as a post-acute care provider has been emphasized under the Affordable Care Act (ACA) as it is expected that HHC services will help patients stay in the community and reduce acute care hospitalization. Urinary tract infection (UTI) -related hospitalization is an adverse patient outcome that affects elderly patients in the HHC setting. Studies examining the ADLs of HHC patients are limited. Although dependence in ADLs is a known risk factor for hospitalization, no study has assessed the relationship between ADL dependency and UTI-related hospitalization among HHC patients. This dissertation describes the ADLs of elderly patients receiving HHC services, and examines risk of UTI-related hospitalization among this population, specifically the potential risk of ADL dependency. In Chapter One, the problems of UTI-related hospitalization and ADL dependency are introduced and their significance is described. In Chapter Two, an integrative review of the literature describing methods of assessing ADLs in skilled nursing facilities (SNF) and HHC are described. In Chapter Three, a cross-sectional study elucidating the risk factors for severe ADL dependency and predictors of ADL improvement among HHC patients is reported. In Chapter Four, the risk factors for UTI-related hospitalization among HHC patients is reported. In Chapter Five, findings of the three studies are summarized and conclusions are provided including strengths, limitations, and implications for practice and policy. Andersen’s Behavioral Model was the theoretical framework used for this study. The Andersen model posits that health care utilization is a function of patients predisposing (e.g. age, gender, race/ethnicity), enabling (e.g. living alone, insurance status, living condition, primary care giver) and need factors (e.g. ADL dependency level, comorbidity, impaired decision making). This model fits this dissertation because evidence shows that health care utilization (UTI-related hospitalizations) depends on predisposing, enabling and need factors. This was a retrospective cohort research design study based on secondary analysis of the Outcome and Assessment Information Set (OASIS) data set of 154,801 beneficiaries who received home health care services in 2013. Descriptive statistics, bivariate analysis, and multivariable logistic regression analyses were conducted to examine the effect of each individual variable on the outcomes of interest (severe ADL dependency, ADL improvement and UTI-related hospitalizations). The study population was elderly (mean age 77 years), mostly female (65%) and white (79.8%). Key findings indicated that, (a) over 60% of patients had severe ADL dependency, and impaired decision making is a strong predictor of severe ADL dependency, (b) Overall, patients experienced ADL improvement from admission to discharge. However, blacks experienced significantly less ADL improvement compared to Whites. Longer HHC length of stay was also associated with ADL improvement, and (c) For the UTI-related hospitalization outcome model, multivariable analysis showed that Medicaid insurance, severe ADL dependency and impaired decision making was associated with increased risk for UTI-related hospitalization

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.

Seasonality, or a cycling of high and low incidence, of infectious diseases has long been recognized but remains little understood. For many diseases, even major ones such as influenza, our knowledge of the seasonal drivers is very limited. One proposed driver of seasonality for many diseases is weather, especially temperature and humidity. I studied how likely an admission to a hospital was to be diagnosed with a UTI or pneumonia caused by Legionella across the US under various climates and weather conditions. I found that patients were 10–20% more likely to have a UTI when the monthly mean temperature was between 65–85°F compared to under 40°F. This may be due to slightly lower levels of hydration at warm temperatures reducing protection against UTIs. Pneumonia caused by Legionella was more common in warm (60–80°F) months than in cool or hot months. Within warm months, when humidity was above 60% there was a doubling in the odds of Legionella pneumonia. When the humidity was above 65%, the odds were quadrupled. Understanding why some diseases are seasonal and what role weather plays in this seasonality is important for both daily practice (e.g., recent weather can help diagnosis Legionella versus a more typical cause of pneumonia) and for larger policy adapting to changing weather and climate.

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