Clostridium difficile is a leading cause of nosocomial infection in North America and a considerable challenge to healthcare professionals in hospitals and nursing homes. The Gram-positive bacterium produces two exotoxins, toxin A (TcdA) and toxin B (TcdB), which are the major virulence factors responsible for C. difficile-associated disease (CDAD) and are targets for CDAD therapy. In this work, recombinant single-domain antibody fragments (VHHs) which target the cell receptor binding domains of TcdA or TcdB were isolated from an immune, llama phage display library and characterized. Four VHHs (A4.2, A5.1, A20.1, and A26.8) were potent neutralizers of the cytopathic effects of TcdA in an in vitro assay and the neutralizing potency was enhanced when VHHs were administered in combinations. Epitope mapping experiments revealed that some synergistic combinations consisted of VHHs recognizing overlapping epitopes, an indication that factors other than mere epitope blocking are responsible for the increased neutralization. Binding assays revealed TcdA-specific VHHs neutralized TcdA by binding to sites other than the carbohydrate binding pocket of the toxin. The TcdB-specific VHHs failed to neutralize TcdB, as did a panel of human VL antibodies isolated from a synthetic library. To enhance the stability of the C. difficile TcdA-specific VHHs for oral therapeutic applications, the VHHs were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. The mutant VHHs were found to be well expressed, were non-aggregating monomers, retained low nM affinity for TcdA, and were capable of in vitro TcdA neutralization. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants without compromising inherent VHH trypsin resistance. Collectively, the second disulfide not only increased VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance. These are all desirable characteristics for the design of protein-based oral therapeutics. In conclusion, llama VHHs represent a class of novel, non-antibiotic inhibitors of infectious disease virulence factors such as C. difficile toxins.

Thesis (M.S.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 89 pages. Includes bibliographical references.

SANTOS, A. A. Q. A. Papel do receptor P2x7 no efeito da toxina a do Clostridium difficile em modelo de alça ileal em camundongos. 2017. 100 f. Tese (Doutorado em Ciências Médicas) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2017. / Submitted by Erika Fernandes (erikaleitefernandes@gmail.com) on 2017-01-30T16:00:58Z No. of bitstreams: 1 2017_tese_aaqasantos.pdf: 2658637 bytes, checksum: 8c134a5e88286c7d898fc1d1a091d893 (MD5) / Approved for entry into archive by Erika Fernandes (erikaleitefernandes@gmail.com) on 2017-01-30T16:01:09Z (GMT) No. of bitstreams: 1 2017_tese_aaqasantos.pdf: 2658637 bytes, checksum: 8c134a5e88286c7d898fc1d1a091d893 (MD5) / Made available in DSpace on 2017-01-30T16:01:09Z (GMT). No. of bitstreams: 1 2017_tese_aaqasantos.pdf: 2658637 bytes, checksum: 8c134a5e88286c7d898fc1d1a091d893 (MD5) Previous issue date: 2017-01-16 / Clostridium difficile (C. difficile) is the major cause of colitis associated with the use of antibiotics, with significant morbidity and mortality. The enteric nervous system (ENS) is located in the gastrointestinal tract (GIT) and has an important function of regulating the digestive system. The P2X7 receptor participates in the regulation of cellular permeability, cytokine release and apoptosis, processes that are involved in the pathogenesis of C. difficile induced disease.The aim of this study was to analyze the role of the P2X7 receptor in enteritis induced by C. difficile toxin A (TcdA). The mouse ileal loop was injected with saline (Sodium Chloride 0.9% - Control) or TcdA (50 μg / loop) in mice previously treated with the inhibitors: BBG (50 mg/kg) or A438079 (10 μM) i.p.injection 1 h prior to TcdA (TcdA + BBG; TcdA + A438079) or saline injection. The animals were euthanized after 4h latter and the ileal loop collected for the following analyzes: immunofluorescence for ChAT, NOS, P2X7 and calretinin in membrane preparations, immunohistochemistry for GFAP, HuC / D, S-100β, TUNEL and PCR for P2X7. The results demonstrated that TcdA significantly reduced myenteric plexus neurons in 66.42% when compared to the control, while in the membrane preparations a significant reduction of the immunoreactive neurons to ChAT was observed in 23.21% and Calretinin 28.17%. However, there was a 21.23% increase in labeling for the P2X7 receptor over the control. In the ileal tissue there was an increase of 411.95% and 22.40% respectively in the P2X7 positive area and in the gene expression. In relation to the glial markers, an increase of 125.71% and 144.30% was observed for GFAP and S-100β, respectively. In animals treated with inhibitors P2X7; BBG and A438079, as observed an improvement in histological parameters . In addition , a significant reduction in the number of apoptotic cells decrease in GFAP and S-100β-labeled cells, and cytokines ( TNF-α, IL-1β, IL -6 and IL-8) levels, compared to the control. This was associated with a significant increase in the HuC/D positive area . From these results, it is concluded that TcdA promotes changes in the enteric nervous system, increasing neuronal death and activating glial cells, and that the P2X7 receptor plays an important role in the changes induced by C. difficile TcdA. / O Clostridium difficile (C. difficile) é a maior causa de colite associada ao uso de antibióticos, com significante morbidade e mortalidade. O sistema nervoso entérico (SNE) está localizado no trato gastrointestinal (TGI) e possui importante função de regulação do sistema digestório. O receptor P2X7 presente nas células gliais entéricas (CGE) e no SNE participa na regulação da permeabilidade celular, liberação de citocinas e apoptose, processos que estam envolvidos na patogênese do C. difficile. O objetivo deste estudo foi analisar o papel do receptor P2X7 na enterite induzida pela toxina A (TcdA) do C. difficile. A alça ileal dos camundongos foi injetada com salina (Cloreto de Sódio 0,9%- Controle) ou TcdA (50 μg/alça) em camundongos previamente tratados com os inibidores: BBG (50 mg/kg) ou A438079 (10 μM) via intraperitoneal 1h antes da injeção da TcdA ou salina. Os animais foram eutanaziados após 4h da injeção e a alça ileal coletada para as seguintes analises: imunofluorescência para ChAT, NOS, P2X7 e calretinina nos preparados de membrana, imunohistoquímica para GFAP, HuC/D, S-100β, TUNEL e PCR para P2X7. Os resultados demonstraram que a TcdA reduziu significativamente em 66,42% os neurônios do plexo mioentérico quando comparado ao controle, enquanto nos preparados de membrana observou-se uma redução significativa dos neurônios imunorreativos a ChAT em 23,21% e Calretinina 28,17%, contudo, houve um aumento de 21,23% da marcação para o receptor P2X7 em relação ao controle. No tecido ileal verificou-se um aumento de 411,95% e 22,40% respectivamente na área positiva para P2X7 e na expressão gênica. Em relação aos marcadores gliais, observou-se um aumento de 125,71% e 144,30% para GFAP e S-100β, respectivamente. Nos animais tratados com os inibidores de P2X7: BBG e o A438079 observou-se uma melhora dos parâmetros histológicos, uma redução significativa do número de células apoptóticas, das células marcadas para GFAP e S-100β, das citocinas: TNF-α, IL-1β, IL-6 e IL-8 com relação ao controle e um aumento significativo na área positiva para HuC/D em relação a TcdA. A partir desses resultados, concluiu-se que a TcdA promoveu alterações no sistema nervoso entérico, aumentando a morte neuronal e ativando as células da glia, e que o receptor P2X7 tem participação importante nas alterações induzidas pela TcdA do C. difficile.

nÃo hà / Diarreia nosocomial (DN) à uma infecÃÃo relacionada à assistÃncia à saÃde (IRAS) com incidÃncia e severidade crescentes. PropÃe-se determinar a incidÃncia da DN, os fatores de risco e a incidÃncia da doenÃa associada a Clostridium difficile (C. difficile). Para isso, um estudo caso â controle, pareando pacientes por idade, sexo, perÃodo de admissÃo, clÃnica e diagnÃstico, foi conduzido, de 06/ fev/12 a 05/fev/13, no Hospital UniversitÃrio da UFC. Casos ̶ pacientes com DN e Controles ̶ pacientes sem DN. Definiu-se DN como fezes lÃquidas, trÃs ou mais vezes em 24 horas, com duraÃÃo superior a 12 horas, sem outras causas inflamatÃrias ou procedimentos diagnÃsticos. DN foi detectada mediante busca ativa, visitando-se os pacientes das Unidades de Hematologia, Transplante HepÃtico e Renal. O teste ELISA TOX A/B II foi utilizado para detectar as toxinas A e/ou B e diagnosticar doenÃa associada ao C. difficile. Demais IRAS foram investigadas por intermÃdio de fichas de notificaÃÃo de infecÃÃo hospitalar (IH). O Ãndice geral de IH foi de 7,17%. A incidÃncia da DN nas enfermarias de Hematologia, Transplante HepÃtico e Renal foi 4,80% (44/925) e da DN associada ao C. difficile 0,12% (01/925). Detectaram-se toxinas A/B de C. difficile em caso [1/43 (2,32%)] e controles [3/72 (4,17%)]. DN foi significantemente associada ao uso prÃvio > 6 antimicrobianos por paciente, alÃm do uso prÃvio de ciprofloxacina, metronidazol, polimixina B e dieta enteral (p≤ 0,05). Pacientes com DN permaneceram mais tempo internados, tiveram mais vÃmitos, cÃlicas e febre, verificando-se alta significÃncia estatÃstica (p≤ 0,05). Outras IRAS identificadas, nos casos e controles, foi infeÃÃo do trato urinÃrio 54% (15/28), seguida da corrente sanguÃnea 32% (8/28), do sÃtio cirÃrgico 11% (3/28) e de infecÃÃo de partes moles 4% (1/28). DN impÃe riscos aos pacientes jà debilitados. Os dados demonstram a presenÃa endÃmica do C. difficile. A atualizaÃÃo da epidemiologia local orienta medidas de controle da IH, como uso judicioso de antibiÃticos, cautelas com a dieta enteral e precauÃÃes de contato, para os pacientes com diarreia nosocomial. / Nosocomial diarrhea (ND) is a healthcare - associated infections (HAI) with increasing incidence and severity. It is proposed to determine the incidence of ND, the associated risk factors and the incidence of disease associated to Clostridium difficile (C. difficile). For this, a case - control study, pairing patients by age, sex, length of admission, and clinical diagnosis was conducted 06 / Feb/12 to 05/Fev/13 in the University Hospital of the UFC. Cases: patients with DN and controls: patients without ND. Nosocomial diarrhea is defined as watery stools, three or more times within 24 hours, over 12 hours without further diagnostic procedures or inflammatory causes. ND was detected by active surveillance, visiting the patients of Hematology, Liver and Renal Transplant. DN was defined as loose stools, 3 or more times in 24 hours, with duration longer than 12 hours, without other inflammatory causes or diagnostic procedures. The ELISA TOX A / B II test was used to detect toxin A and/or B and to diagnose C. difficile associated disease. Others HAI were investigated by the notification records of nosocomial infection (NI). The overall rate of Nosocomial infection was 7.17 %. The incidence of DN in the wards of Hematology, Liver and Renal Transplant was 4.80% (44/925) and C. difficile associated with DN was 0.12 % (01/925). Toxins A/B were detected in the case of C. difficile [1/43 (2.32%)] and controls [3/72 (4.17%)]. DN was significantly associated with previous use > 6 antimicrobials per patient, beyond the prior use of ciprofloxacin, metronidazole, polymyxin B and enteral feeding (p ≤ 0.05). Patients with ND remained in hospital longer, had more vomiting, cramps and fever, verifying high statistical significance (p ≤ 0.05). Other identified HAI were mainly urinary infection 54% (15/28), followed by bacterial bloodstream infection 32% (8/28), surgical site infection 11% (3/28) and soft tissue infection 4% (1/28). ND entails risks to the already debilitated patients. The data demonstrate the presence of endemic C. difficile. The updated of the local epidemiology guide control measures NI, such as judicious use of antibiotics, enteral feeding precautions and contact precautions for patients with nosocomial diarrhea.

Master of Science / Department of Plant Pathology / Revathi Govind / Clostridium difficile, a gram-positive, anaerobic bacterium, is a major cause of antibiotic-related diarrhea and pseudomembraneous colitis. In the last decades, C. difficile has emerged as a major threat because of its tendency to cause frequent and severe disease. Because of the severity of the infection and its high rate of recurrence, there is a significant financial burden on healthcare systems. Antibiotic treatments are a primary risk factor for the development of C. difficile infection because they disrupt the normal gut flora in the host, enabling the antibiotic resistant bacterium to colonize the colon. Most of the resistance mechanisms in C. difficile reported to date can be classified as either antibiotic-degrading enzymes or modification of target sites. Another mechanism that can contribute to antibiotic resistance in C. difficile is the extrusion of antimicrobial compounds by efflux pumps. The goal of this project was to provide initial insights into the roles and mechanisms of a putative efflux pump complex. To do this, a number of experiments were designed to provide information about the structures, localization, and functions of this protein complex. It was determined that acidic pH conditions and a small number of antimicrobials, including inorganic compounds, organic compounds, fungicides, and antibiotics, inhibit growth of a C. difficile mutant lacking this pump system. Interestingly, higher NaCl in the medium and alkaline pH seem to promote the growth of a C. difficile mutant lacking this pump or, surprisingly, only inhibit growth of the wild type strain. The experiments performed in this project suggest that this efflux pump might have an essential role in C. difficile physiology, possibly by serving as an efflux pump for toxic metabolites.

Class of 2014 Abstract / Specific Aims: Clostridium difficile associated diarrhea (CDAD) frequently occurs in patients exposed to broad-spectrum antibiotics which can result in a life threatening illness. The role of probiotics in the prevention of CDAD is not well established and many medical centers across the United States are opting to remove probiotics from common CDAD prophylaxis. We aim to evaluate the efficacy of lactobacillus probiotics during the use of broad-spectrum antibiotic therapy in the acute care setting for the prophylaxis of CDAD at Kindred Hospital. Methods: We performed a single center, retrospective data analysis efficacy trial of inpatients receiving beta-lactam, fluoroquinolone or clindamycin antibiotics from the Kindred Hospital database. Two study groups will be compared: patients who received lactobacillus probiotic therapy based on protocol since May 2011 and patients who did not receive probiotic therapy. The presence or absence of CDAD will be used to evaluate probiotic efficacy. Main Results: Of the ### patients screened, ## were assigned to the treatment group and ## were assigned to the non-treatment group, a total of ## patients were analyzed for the primary endpoint. CDAD occurred in ## patients (xx%) receiving probiotic therapy while CDAD occurred in ## patients (xx%) not receiving probiotic therapy (relative risk [RR]: xx.x; p=0.xxx). Conclusion: [Anticipated] We identified no statistically significant evidence that the use of lactobacillus was effective in the prevention of CDAD. Further knowledge of the pathophysiology of CDAD and proper antibiotic use is needed for future studies.

Class of 2014 Abstract / Specific Aims: The incidence of Clostridium difficile (C. Diff) infections in pediatric patients has continually risen, which could be caused by the emergence of a hyper virulent strain, specifically NAP1/B1/027. The objectives of the study were to evaluate the incidence of strain type, compare treatment(s) prescribed, treatment duration, rate of infection recurrence based on strain and severity, rates of re-infection or recurrence, and treatment failures for patients less than 6 months and up to 18 years of age. Methods: A retrospective study of patients admitted to an academic medical center with detection of C. diff toxin was performed. Data analyses included descriptive and inferential statistics to examine demographics, strain type, infection severity, and treatment failure. Main Results: Fourty-five patients with C. Diff toxin detection were included in study analyses and the median age was 6.2 [0.31- 17.9 years]. Oral or intravenous metronidazole was prescribed as initial therapy in 89% of the patients. Strain type was available in 77% of patients, with NAP1/B1/027 detected in 31% of stool samples tested. Within 21 days after initial toxin detection, there was a 13% rate of clinical failure or death, although none directly associated with C. Diff. Within days 22 - 65 after initial toxin detection, there was a 16% rate of recurrence or reinfection. Initial therapy selection, therapy duration, and rate of recurrence or reinfection were not significantly associated with NAP1/B1/027 strain type. Conclusion: Despite variability in severity of infection, the majority of pediatric patients with C. Diff were treated with metronidazole and were infected with a non-B1/NAP1/027 strain.

Class of 2012 Abstract / Specific Aims: To identify the number of Clostridium difficile-associated disease (CDAD) cases in hospitalized oncology patients between 2005 and 2009. To identify the occurrence of mortality, comorbidities, and the amount of hospital charges in this patient population. Predictive variables for mortality and hospital charges were also identified. Methods: Data retrieved from the Nationwide Inpatient Sample were used to retrospectively identify the number of CDAD cases in hospitalized oncology patients, the occurrence of mortality and comorbidities, and the amount of hospital charges incurred between 2005 and 2009. Multivariate logistic regression was used to identify predictors of mortality and a lognormal regression was used to identify predictors of inpatient charges. Main Results: From 2005 to 2009, we identified 210,449 cases of CDAD in hospitalized adult patients with cancer. Total inpatient charges for all CDAD cases over the five-year period exceeded 18 billion dollars. The percentage of cases that resulted in death was 13%. Variables that were found to be predictors of mortality included increased age (OR 1.019, CI 1.018-1.020), chronic pulmonary disease (OR 1.231, CI 1.191-1.272), coagulopathy (OR 2.085, CI 2.011-2.162), liver disease (OR 1.159, CI 1.072-1.253), fluid and electrolyte disorders (OR 1.561, CI 1.518-1.605), renal failure (OR 1.405, CI 1.349-1.462), and weight loss (OR 1.408, CI 1.362-1.456)(all p<.001). Conclusions: This study identified a large number of CDAD cases in hospitalized adult cancer patients. Several factors that appeared to be predictive of mortality and inpatient charges were also identified, which may be useful knowledge for clinicians who need to identify at-risk patients.

Clostridium difficile is a leading cause of nosocomial infection in North America and a considerable challenge to healthcare professionals in hospitals and nursing homes. The Gram-positive bacterium produces two exotoxins, toxin A (TcdA) and toxin B (TcdB), which are the major virulence factors responsible for C. difficile-associated disease (CDAD) and are targets for CDAD therapy. In this work, recombinant single-domain antibody fragments (VHHs) which target the cell receptor binding domains of TcdA or TcdB were isolated from an immune, llama phage display library and characterized. Four VHHs (A4.2, A5.1, A20.1, and A26.8) were potent neutralizers of the cytopathic effects of TcdA in an in vitro assay and the neutralizing potency was enhanced when VHHs were administered in combinations. Epitope mapping experiments revealed that some synergistic combinations consisted of VHHs recognizing overlapping epitopes, an indication that factors other than mere epitope blocking are responsible for the increased neutralization. Binding assays revealed TcdA-specific VHHs neutralized TcdA by binding to sites other than the carbohydrate binding pocket of the toxin. The TcdB-specific VHHs failed to neutralize TcdB, as did a panel of human VL antibodies isolated from a synthetic library. To enhance the stability of the C. difficile TcdA-specific VHHs for oral therapeutic applications, the VHHs were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. The mutant VHHs were found to be well expressed, were non-aggregating monomers, retained low nM affinity for TcdA, and were capable of in vitro TcdA neutralization. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants without compromising inherent VHH trypsin resistance. Collectively, the second disulfide not only increased VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance. These are all desirable characteristics for the design of protein-based oral therapeutics. In conclusion, llama VHHs represent a class of novel, non-antibiotic inhibitors of infectious disease virulence factors such as C. difficile toxins.

Nosocomial infections represent for the health care providers serious economical and also health complications. The aim of this thesis is a comprehensive presentation of nosocomial infections. Second part focuses on a bacteria Clostridium difficile. This thesis analyzes epidemiological situation in a particular medical care facility together with the cost of therapy. The incidence of Clostridium difficile is, despite of the worldwide increasing trend, decreasing in the selected facility. Significant differences exist among individual departments in the amount of samples collected as well as in their rate of positivity for the bacteria presence. Data collected are applied to a German pharmacoeconomic model which compares vancomycin therapy with fidaxomicin therapy. On a group of 100 patients it was proved that more efficient is, despite of its higher price, treatment by fidaxomicin because it leads to a lower percentage of recurrences and morbidity and mortality decrease.