Magister Pharmaceuticae - MPharm / Antimicrobial resistance is a global concern associated with increased morbidity and mortality. It has been estimated that, by 2050, the continuous escalation of antimicrobial resistance, globally, will result in more deaths per year, compared to cancer and diabetes. The direct and indirect impact of ineffective antibiotics, and therefore, antimicrobial resistance, will be hardest felt by low and middle-income countries, as the financial burden will be too great to manage. Carbapenems are considered the last line of antimicrobials to treat multidrug-resistant bacterial infections. They are the preferred choice to treat infections, presenting with extended-spectrum beta-lactamases (ESBL) producing Enterobacteriacea. Various strains of bacteria that have become resistant, due to the selective pressure, as a result of carbapenem over use, are referred to as Carbapenem-resistant Enterobacteriaceae (CRE). / 2022

Contamination is a prevalent issue with reprocessed levered endoscopes. The number of infections caused by resistant enterobacteriaceae in patients due to contaminated endoscopes increased to the point that the United States Food and Drug Administration released a safety alert to health care facilities that perform endoscopic retrograde cholangiopancreatography (ERCP). The purpose of this descriptive project was to evaluate if levered endoscopes used in ERCP procedures met high level disinfection criteria, were properly processed, and were germ free after reprocessing. The project was supported by 2 theories: the middle range theory of patient advocacy and the germ theory. Data (counts and percentages) were collected from testing 150 endoscopes at each of 4 facilities within an organization regarding the effectiveness of the reprocessing of the levered endoscopes. According to the project findings, there was a 7% average germ-free failure rate across the sites after the initial reprocessing. The cleaning process of the levered endoscopes allowed bacteria to remain on the scopes after the manufacturer-recommended cleaning was completed at the sites. Standardization of the organization's cleaning process and improvement in the national protocols were recommended. The project supported protecting the safety of endoscopy patients by identifying that the cleaning process could be improved to prevent introduction of infectious bacteria through a procedure. The results will be informative for laboratory staff who clean levered endoscopes, physicians who use the scopes in patient procedures, patients who undergo the procedures, and nurses who are tasked with improving patient safety in perioperative environments.

No description available.

Commercial production of catfish is the leading aquaculture industry in the United States. Production in Mississippi, Louisiana, Alabama, and Arkansas representing 96% of the total catfish sales in the USA. In 2017, the USDA-FSIS added testing of generic Escherichia coli and Enterococcus to the sampling of Siluriformes. The incidence of Escherichia coli in fish and seafood has been associated with possible fecal matter contamination. However, this indicator bacteria could also be a part of the natural microflora of catfish ponds where processing plants collect hybrid catfish. Aerobes, Psychrotrophs, Coliforms, Escherichia coli, and Enterobacteriaceae counts among environmental, liquid and fish samples were tested every season and periodically during one day of operation. Generic Escherichia coli counts were greater in late Summer (P ≤ 0.05) for liquid samples (1 log CFU/g) . During the day, Escherichia coli appeared to be more recurrent in the afternoon at the skinning step (P ≤ 0.05).

According to CDC, every year at least 2 million people are affected and 23,000 dies as a result of antibiotic resistance in U.S. It is considered one of the biggest threats to global health. More and more bacterial infections are becoming harder to treat. One such infection is fire blight, one of the most destructive disease of apple and pear trees. It is caused by bacteria Erwinia amylovora and its outbreaks have been known to destroy entire orchards in a single season. The conventional method of treatments includes use of antibiotics like streptomycin and oxytetracycline but the incidences like presence of multi-drug resistant bacteria in the mammals grazing in the fields have raised concerns. Phage therapy is considered one of the few ways available to combat bacterial resistance and prevent fire blight. In this method, a cocktail of highly lytic bacteriophages is prepared and sprayed on the trees at different time intervals. Bacteriophages are an “intelligent” drug. They multiply at the site of the infection until there are no more bacteria and then they are excreted back into the nature. These phenomena make them more efficient than an antibiotic, which kills all kind of bacteria including good bacteria and can be maintained in the environment for long periods of time. These qualities of bacteriophage have resulted in many commercially available phage therapies. The initial part of this research focuses on isolation, characterization and genomic comparison of bacteriophages that infect a plant pathogen E.amylovora of Erwiniaceae family of Enterobacteriales order. In this study, 28 novel bacteriophages were isolated, fully sequenced, characterized and grouped into seven families based on phage homology. To take this further, we characterized a novel jumbo family of bacteriophages that has a small burst size of 4.6-4.9 and are most similar to bacteriophages that infect Pseudomonas and Ralstonia rather than Enterobacteriales bacteria by protein similarity. These bacteriophages are shown to infect Erwinia and Pantoea bacterial strains, but no infection of 9 other bacterial strains tested, was seen, under laboratory conditions. The results of this work provide an insight on special characteristics that makes bacteriophage so unique and adaptable. The final part of this research explores the enormous diversity of bacteriophages. In 2014 Grose and Casjens grouped 337 fully sequenced tailed phages into 56 diverse clusters (32 lytic and 24 temperate). We further expanded our current understanding of these clusters by performing the comprehensive analysis of genomes and proteomes of 1037 tailed bacteriophages, posted on GenBank. The results of this work provide insights into diversity and relatedness of bacteriophages and the data is posted on GenBank.

Bacteriophages (phages) are the smallest biological entity on the planet. They provide vast amounts of valuable knowledge to biologists. Phage genomes are relatively simple compared to the organisms they infect (prokaryotes) and yet continually point to the complexity surrounding molecular- and microbiological mechanisms of life. By studying phages we can learn of the systems of gene expression, protein interaction and DNA organization. Phages are useful not only from an academic perspective, but may also have useful clinical applications. In the face of the rise of antibiotic-resistant bacterial “super pathogens”, scientists and researchers turn to phages as alternative treatments to these types of infections. Phages are capable of infecting and killing even the deadliest of bacterial pathogens, such as carbapenem-resistant Enterobacteriaceae (CRE) or Bacillus anthracis, and may prove increasingly useful in the future for combatting harmful pathogens. This thesis looks at several aspects of phage biology—from the underlying genetics contributing to phage virulence, to the clinical application of phage therapy to treat infections. First, a look at CRE-Klebsiella pneumoniae isolates and phages capable of infecting some strains may reveal a potential therapeutic approach in the future. Additionally, genomic analysis reveals interesting features that may explain aspects of phage virulence and evolutionary history. Then, a collection of genetically diverse phages is used in infection assays on pathogenic strains of Bacillus anthracis to establish the first-reported phages capable of infecting these strains. Finally, the process of preparing phage samples for therapeutic application is explored in-depth to conclude with discussion of clinical application. During the course of these projects over 25 phages were isolated, as many phage genomes were assembled and annotated, resulting in the preparation of two genome announcements and near-completion of two publishable first-author papers (chapters II and III). In addition, participation in a variety of collaborative efforts may lead to a handful of co-author papers and on various topics, including phage biology and application.

This investigation has for its aim the determination, in a measure, of the degree of personal sanitation existent among students at North Texas State Teachers College, Denton, Texas.

The intestinal tract of virtually any metazoan, including mammals, is colonized with a complex microbial community to which we refer as intestinal or gut microbiota. One of the roles of the healthy intestinal microbiota is to protect the host against gut colonization with pathogenic bacteria through a phenomenon known as colonization resistance (CR). Dysbiosis of the intestinal microbiota, usually as a result of an antibiotic treatment, may lead to the disruption of the CR, and subsequent colonization with bacterial pathogens. However, and despite the importance, the role of the microbiota dysbiosis on the gut colonization by many bacterial pathogens, such as multidrug resistant Enterobacteriaceae, has not been elucidated: the members of the microbiota that confer CR and factors that promote colonization remain mostly unknown. The general aim of this thesis has been to improve the understanding of the role of the microbiota dysbiosis in gut colonization by bacterial pathogens. For this purpose, 3 projects have been established. In the first project we tried to elucidate the role of the microbiota dysbiosis on colonization by multidrug resistant Enterobacteriaceae (MRE) in mice. In the second project we investigated the risk factors and members of the microbiota associated with the MRE colonization in hospitalized patients. MRE infections represent a great threat for hospitalized patients. Specifically, acute leukemia patients are often colonized with MRE, probably due to the impaired CR as a result of intensive antibiotic treatments these patients receive. In the third project we studied the role of the microbiota dysbiosis on the development of Epizootic Rabbit Enteropathy (ERE). ERE is a severe gastrointestinal disease with a high percentage of mortality that occurs in young rabbits during first weeks post-weaning. ERE rabbits have been shown to suffer microbiota dysbiosis during the development of the disease. Moreover, the disease could be reproduced by contact between healthy and sick animals and by administration of cecal contents from ERE rabbits to healthy rabbits, suggesting that a pathogenic agent may be involved in the development of this intestinal pathology, although no causative agent has been identified until now. / El tracto intestinal de prácticamente cualquier metazoo, incluidos los mamíferos, está colonizado por una compleja comunidad microbiana a la que nos referimos como microbiota intestinal. Uno de los papeles de la microbiota intestinal es proteger al huésped contra la colonización intestinal con bacterias patógenas a través de un fenómeno conocido como resistencia a la colonización (RC). La disbiosis de la microbiota intestinal, a menudo como resultado de un tratamiento antibiótico, puede conducir a la alteración de la RC y posterior colonización por patógenos bacterianos. Sin embargo, y pese a su importancia, el papel de la disbiosis de la microbiota en la colonización intestinal por muchos patógenos bacterianos, como son las Enterobacterias multirresistentes, no se ha esclarecido: los miembros de la microbiota que confieren RC y los factores que promueven la colonización siguen siendo desconocidos. El objetivo general de esta tesis ha sido mejorar la comprensión del papel de disbiosis de la microbiota en la colonización intestinal por patógenos bacterianos. Para ello se han establecido tres proyectos. En el primer proyecto investigamos el papel de disbiosis de la microbiota intestinal en la colonización por Enterobacterias multiresistentes (MRE) en ratones. En el segundo proyecto investigamos los factores de riesgo y los miembros de la microbiota asociados con la colonización por MRE en pacientes hospitalizados. Las infecciones por MRE representan una gran amenaza para los pacientes hospitalizados. Específicamente, MRE a menudo colonizan los pacientes con leucemia aguda, probablemente debido a que la RC está alterada como resultado de tratamientos antibióticos intensivos recibidos por estos pacientes. En el tercer proyecto investigamos el papel de la disbiosis microbiana en desarollo de Enteropatía Epizoótica de Conejo (ERE). ERE es una enfermedad gastrointestinal severa con un alto porcentaje de mortalidad que ocurre en conejos jóvenes durante las primeras semanas después del destete. Se ha demostrado que los conejos con ERE sufren disbiosis microbiana después del inicio de la enfermedad, aunque no está claro el papel de la disbiosis en el desarollo de la enfermedad. Además, la enfermedad puede ser reproducida por contacto entre animales sanos y enfermos y por la administración del contenido cecal de conejos con ERE a conejos sanos, lo que sugiere que un agente patógeno podría estar implicado en el desarrollo de esta patología intestinal, aunque hasta ahora no se ha logrado identificar ningún agente causal. / El tracte intestinal de pràcticament qualsevol metazoo, inclosos els mamífers, està colonitzat per una complexa comunitat microbiana a la qual ens referim com microbiota intestinal. Un dels papers de la microbiota intestinal és protegir a l'hoste contra la colonització intestinal amb bacteris patògens a través d'un fenomen conegut com a resistència a la colonització (RC). La disbiosis de la microbiota intestinal, frecuentment com a resultat d'un tractament antibiòtic, pot conduir a l'alteració de la RC i posterior colonització per patògens bacterians. No obstant això, i malgrat la seva importància, el paper de la disbiosis de la microbiota en la colonització intestinal per molts patògens bacterians, com són les Enterobacteries multirresistentes, no s'ha esclarit: els membres de la microbiota que confereixen RC i els factors que promouen la colonització segueixen sent desconeguts. L'objectiu general d'aquesta tesi ha estat millorar la comprensió del paper de la disbiosis de la microbiota en la colonització intestinal per patògens bacterians. Per a això s'han establert tres projectes. En el primer projecte vam investigar el paper de la disbiosis de la microbiota intestinal en la colonització per Enterobacteries multiresistentes (MRE) en ratolins. En el segon projecte, investiguem els factors de risc i els membres de la microbiota associats amb la colonització per MRE en pacients hospitalitzats. Les infeccions per MRE representen una gran amenaça per als pacients hospitalitzats. Específicament, MRE sovint colonitza els pacients amb leucèmia aguda, probablement a causa de que la RC està alterada com a resultat de tractaments antibiòtics intensius rebuts per aquests pacients. En el tercer projecte, vam investigar el paper de la disbiosis microbiana en desenvolupament de l'Enteropatía Epizoótica de Conill (ERE). ERE és una malaltia gastrointestinal severa amb un alt percentatge de mortalitat que ocorre en conills joves durant les primeres setmanes després del deslleti. S'ha demostrat que els conills amb ERE sofreixen disbiosis microbiana després de l'inici de la malaltia, encara que no és clar el paper de la disbiosis en el desenvolupament de la malaltia. A més, la malaltia pot ser reproduïda per contacte entre animals sans i malalts i per l'administració del contingut cecal de conills amb ERE a conills sans, la qual cosa suggereix que un agent patogen podria estar implicat en el desenvolupament d'aquesta patologia intestinal, encara que fins ara no s'ha aconseguit identificar cap agent causal. / Djukovic, A. (2017). Role of intestinal dysbiosis on gut colonization by bacterial pathogens [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90415

Após o surgimento e disseminação das β-lactamases de amplo espectro em membros da família Enterobacteriaceae, os antibióticos carbapenêmicos (imipenem, meropenemeertapenem) têm sido considerados a terapia de escolha devido à estabilidade apresentada contra estas enzimas. A desvantagem destes antibióticos é a sua capacidade de induzir resistência aos β-lactâmicos e a outros antibióticos quimicamente não relacionados. O imipenem tem favorecido a indução de cefalosporinases cromossômicas (AmpC) e também tem sido relacionado, in vivo, com a seleção de mecanismos intrínsecos de resistência, contribuindo com o perfil multi -droga resistente (MDR). Esse perfil é freqüentemente associado à diminuição da permeabilidade por alteração na síntese de porinas em conjunto com um aumento da atividade de bombas de efluxo, as quais não permitem o estabelecimento de uma concentração ativa do antibiótico no interior da célula bacteriana. O presente trabalho teve como objetivo avaliar o estabelecimento do perfil MDR em enterobactérias provenientes de isolados clínicos em função da exposição a diferentes concentrações de imipenemin vitro. A seleção do grupo das amostras estudadas foi feito por meio da determinação do perfil de sensibilidade dos isolados, tipagem molecular e ensaio de hidrólise de Imipenem. Nos isolados selecionados para a indução foi realizada numa etapa inicial (etapa basal) a análise de porinas de membrana externa por SDS-PAGE e o estudo de genes codificadores de β-lactamases pela técnica de PCR. O estudo do estabelecimento do perfil MDR foi feito por meio de passagens sucessivas das amostras em meio contendo concentrações sub-inibitórias de imipenem seguido de análise fenotípica (CIM e acúmulo do antibiótico intracelular e SDS-PAGE), e a análise da expressão gênica de genes associados a permeabilidade de membrana (ompC, ompF eAcrA) e genes reguladores(marA e ompR). Após a indução com o imipenem, 77% dos isolados induzidos aumentaram a CIM para os carbapenêmicos, mudando assim o perfil de resistência observado na etapa basal Também foi afetado o perfil de resistência para outros antibióticos não relacionados a β-lactámicos, porém numa percentagem menor. Com relação à alteração da permeabilidade, a perda de porina foi observada apenas para um isolado, no entanto a diminuição na expressão gênica de Omp36 foi significativa desde o começo da indução. A expressão da bomba de efluxoAcrAB foi afetada pela indução com imipenem, aumentando significativamente a expressão de AcrA, enquanto os reguladores estudados, MarA e OmpR tiveram a sua expressão induzida pelo imipenem. Foi possível observar também associação do nível de expressão gênica do regulador MarA com a expressão de AcrA,porém não foi possível observar uma associação estatisticamente significativa deste regulador com o perfil de expressão de OMPs. A indução de OmpR foi associado com um aumento da expressão de RNAm de Omp35, já para Omp36 foi possível observar apenas uma tendência na repressão deste gene. O estudo da resposta destes genes reguladores e determinantes de resistência, em resposta à exposição ao com o imipenem in vitro, permitiu reportar o comportamento molecular da bactéria numa resposta adaptativa no estagio inicial do estabelecimento do fenótipo MDR. A utilização de isolados clínicos com diversos determinantes de resistência permitiu observar a variabilidade nas respostas adaptativas das enterobacterias, o que é fundamental para a compreensão dos mecanismos de adaptação da bactéria e sua contribuição na falha terapêutica. / After emergence and broad dissemination of extended spectrum β-lactamases into the Enterobacteriaceae family, the carbapenemic antibiotics (imipenem, meropenem and ertapenem) have been considered the chosen therapy in the treatment of nosocomial infections by the stability that these antibiotics show to these enzymes. The disadvantage of carbapenems is theirs capacity to induce resistance against β-lactamics and to other chemically unrelated antibiotics. The imipenem has been shown to induce chromosomal cephalosporinases (AmpC) and it was also related, in vivo, with the selection of intrinsic mechanism leading to multi-drug resistance profile (MDR). This profile is usually associated with membrane impermeability due to reduced outer membrane porin synthesis with an incremented activity of efflux pumps, which results in a reduced concentration of antibiotics inside the bacteria. This study aimed to evaluate the establishment of the MDR profile in Enterobacteriaceae from clinical isolates by exposure to different concentrations of imipenem in vitro. The selection of the study group was performed by determination of antibiotic susceptibility profile,molecular typing and hydrolysis assay of imipenem. In the selected isolates submitted to induction, in an initial step (baseline), was performed the outer membrane porin analysis by SDS-PAGE and the gene-specific amplification of B-lactamase enzymes by PCR. The study of the establishment of MDR was performed by progressive passages with subclinical concentrations of imipenem, followed each one by the evaluation of phenotypic profile (MIC, accumulation antibiotic in celland SDS-PAGE) and gene expression analysisof genes related to membrane permeability (ompC, ompF and acrA) and regulatory genes(MarA and ompR). After induction with imipenem, 77 % of the isolates increased the MIC for the carbapenems, changing the resistance profile at the baseline. In a lesser percentage, the resistance profile to other β-lactams-unrelated antibiotics was also affected. Loss of porin was observed only for an isolated, however a significantly decreased Omp36 mRNA expression was observed from the start of induction. The expression of the efflux pump AcrAB ,was also affected by the imipenem induction, significantly increasing the AcrA gene expression, whereas the studied regulatory genes,MarA and OmpR,were induced by the imipenem. It was also possible to observe an association between the expression of the regulator MarA and the expression of AcrA, nevertheless no association was observed between this regulator and OMPs . OmpR induction was associated with an increased Omp35mRNA expression, however only a trend for the repression of Omp36was observed. The study of the response of these regulatory genes and genetic determinants of resistance, in response to the imipenem exposure in vitro, allowed to report the molecular behavior of the bacteria in an adaptive response in the initial stage of the establishment of a MDR phenotype. The use of clinical isolates with diverse resistance determinants allowed observing the variability in adaptive responses in enterobacteria, which is important to understand the adaptive mechanisms of bacteria to this antibiotic, the involvement in the emergence of the MDR profile and its contribution to the treatment failure.

O objetivo deste trabalho foi investigar a presença de bactérias produtoras de β-lactamases adquiridas na microbiota Gram-negativa comensal de humanos e animais domésticos em uma comunidade remota na região da Floresta Amazônica. De março a julho de 2013 foram coletadas amostras de fezes de indivíduos atendidos e funcionários de um centro assistencial em saúde restrito a comunidades indígenas e de swab retal de animais de companhia da comunidade. Nas amostras de humanos foram detectados isolados de Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes, Enterobacter kobei e Morganella morganii, carregando os genes blaTEM-1, blaCTX-M-15, blaCTX-M-14, blaCTX-M-8 e blaGES-5. Nas amostras de animais foram detectados apenas isolados de E. coli carregando os genes blaTEM-1, blaCTX-M-14, blaCTX-M-2 e blaCTX-M-8. Foi observada a relação clonal entre isolados de E. coli de origem humana e de origem animal. Estes resultados demonstram a disseminação de um problema endêmico em áreas urbanas para uma comunidade, em teoria, com baixa exposição a antibacterianos. / The aim of this study was to investigate the presence of acquired β-lactamase in the commensal Gram-negative microbiota of humans and domestic animals of a remote community in the Amazon Forest region. From March to July 2013 stool samples were collected from individuals attended in a health care center restricted to indigenous communities and from the local staff, and rectal swab samples were collected from companion animals in the community. In the human samples were detected Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes, Enterobacter kobei and Morganella morganii isolates harboring blaTEM-1, blaCTX-M-15, blaCTX-M-14, blaCTX-M-8 e blaGES-5 genes. In the animal samples only E. coli strains harboring blaTEM-1, blaCTX-M-14, blaCTX-M-2 and blaCTX-M-8 were detected. The clonal relatedness between E. coli strains from human and animal samples was observed. These results demonstrate the dissemination of an urban endemic problem to a community, in theory, with low antimicrobial exposure.