Introdução: Enterococcus faecium resistente à vancomicina (EFRV) surgiu como um importante patógeno multirresistente e de etiologia potencialmente letal nas infecções associadas aos cuidados de saúde em todo o mundo. Objetivo: O objetivo deste estudo de coorte retrospectivo foi avaliar os fatores associados à mortalidade em pacientes com bacteremia causadas por EFRV em um grande hospital de referência terciária no sul do Brasil. Métodos: Foram avaliados todos os casos documentados de bacteremia identificados entre maio de 2010 e julho de 2012. Regressão de Cox foi realizada para determinar se as características relacionadas ao hospedeiro ou o tratamento antimicrobiano estavam associadas com a mortalidade por qualquer causa em 30 dias. No total, 35 pacientes documentados com bacteremia por EFRV foram identificados durante o período de estudo. Resultados: A mediana do escore APACHE-II da população do estudo foi 26 (IQR 10). A mortalidade global em 30 dias foi de 65,7%%. Todos isolados de EFRV eram sensíveis à linezolida, daptomicina e quinopristina - dalfopristina. A linezolida foi o único agente antimicrobiano com atividade in vitro contra EFRV que foi administrada à coorte. Após a análise multivariada, o tratamento com linezolida (HR 0,08, 95% CI, 0,02-0,27) e a presença de insuficiência renal aguda no início da bacteremia (HR 4,01, 95% CI, 1,62-9,94) foram associadas de forma independente com o desfecho principal. Conclusão: Apresentação com insuficiência renal aguda e ausência de tratamento com um antibiótico eficaz representa um risco de mortalidade em pacientes com bacteremia por EFRV. / Background: Vancomycin-resistant Enterococcus faecium (VREF) has emerged as a relevant multidrug-resistant pathogen and potentially lethal etiology of health care-associated infections worldwide. Objective: The objective of this retrospective cohort study was to assess factors associated with mortality in patients with VREF bacteremia in a major tertiary referral hospital in southern Brazil. Methods: All documented cases of bacteremia identified between May 2010 and July 2012 were evaluated. Cox regression was performed to determine whether the characteristics related to the host or antimicrobial treatment were associated with the all-cause 30-day mortality. In total, 35 patients with documented VREF bacteremia were identified during the study period. Results: The median APACHE-II score of the study population was 26 (IQR 10). The overall 30-day mortality was 65.7%. All VREF isolates were sensitive to linezolid, daptomycin and quinopristin-dalfopristin. Linezolid was the only antimicrobial agent with in vitro activity against VREF that was administered to the cohort. After multivariate analysis, linezolid treatment (HR, 0.08; 95%CI, 0.02 – 0.27) and presence of acute kidney injury at the onset of bacteremia (HR, 4.01; 95%CI, 1.62 – 9.94) were independently associated with the main outcome. Conclusion: Presentation with acute kidney injury and lack of treatment with an effective antibiotic poses risk for mortality in patients with VREF bacteremia.

Pathogens in low-moisture foods are an emerging food safety concern due to increased survival and thermotolerance in matrices with low water activity. However, limited data is publicly available for the thermotolerance of Listeria monocytogenes, Salmonella spp., and Enterococcus faecium NRRL B-2354 (a Salmonella surrogate). The aims of this study were to identify differences in thermal inactivation rates between these organisms in three different low-moisture foods. Three model low-moisture foods (peanut butter, powder infant formula, and wheat flour) were inoculated with either E. faecium, a Salmonella spp. cocktail, or a L. monocytogenes cocktail using a dry inoculation method for a total of 9 treatments. Samples were heat treated in a hot water bath at predetermined temperatures, and bacterial survival was detected via direct plating on tryptic soy agar with 0.6% yeast extract. In peanut butter and most of the powder infant formula treatments, Salmonella spp. had significantly higher D-values than L. monocytogenes using comparable temperatures (p < 0.05). However, D-values between Salmonella spp. and L. monocytogenes were comparable in wheat flour and one of the treatment temperatures in powder infant formula (p > 0.05). For all but one of the treatments at the same temperature, E. faecium had significantly higher D-values than L. monocytogenes and Salmonella spp. in each food matrix (p < 0.05). The observed matrix effect on thermotolerance for each of the bacteria was reported in descending order as powder infant formula > peanut butter > wheat flour in the majority of the comparable D-values. While Salmonella continues to be the pathogen of concern in low-moisture foods due to survival and outbreaks, these results indicate L. monocytogenes can exhibit similar thermotolerances in relevant model low-moisture foods matrices.

Vegetative, non-sporeforming foodborne pathogens show notable survival and uncanny thermotolerance in low water activity (aw) foods. Controlled studies on Listeria monocytogenes, Salmonella spp., and Enterococcus faecium NRRL B-2354 (a Salmonella surrogate) in a variety of food matrices support thermal process validation studies required to achieve global food safety objectives. In this study, we determined and compared thermal inactivation rates using independent six-strain cocktails of pathogens in three plant-based butters. Direct determinations of decimal reduction times (D-values) for L. monocytogenes, Salmonella, and E. faecium, in corresponding butters were inoculated using peanut oil, almond oil, or sunflower oil. Thermal Death Time (TDT) studies for the organisms were conducted in triplicate. Uniform bagged plant- based butter samples of Salmonella spp. or L. monocytogenes, or E. faecium alone were sandwiched in copper plates immobilized with recessed magnets. Samples underwent rapid heat treatments via water immersion under isothermal conditions ranging from 70°C to 85°C. Bacterial destruction in peanut butter (46% fat, 0.20 aw @ 25°C), almond butter, (50% fat, 0.32 aw @ 25°C), or sunflower butter (56% fat, 0.15 aw @ 25°C) was determined by direct plating. The TDT studies showed Salmonella spp. had consistently higher D-values than L. monocytogenes in all treatments, but pair-wise comparisons found no statistical difference when assessing the thermotolerance of the two pathogens in the individual plant-based butters tested (p > 0.005). These data support Salmonella as the primary pathogen of concern in low water activity foods and show the heat resistance of L. monocytogenes can approximate destruction kinetics observed for Salmonella spp. in low aw matrices. E. faecium exhibited the highest thermotolerance. This further supports the utility of this surrogate for Salmonella spp. and L. monocytogenes in high fat, low-moisture foods similar to the plant-based butters tested. Thermotolerance differences between a dry talc vs. peanut oil-based inoculation procedures in peanut butter were also evaluated. Surprisingly, the oil-based inoculations resulted in lower D- values (p > 0.01) for Salmonella spp. and the surrogate when compared to the dry inoculum.

Limited data are currently available characterizing the thermal resistance of foodborne pathogens in semolina flour and intermediate pasta matrices representative of commercial conditions during mixing, extrusion, and drying. These data are essential to pasta producers seeking to be compliant with federal regulations since Salmonella spp. and Escherichia coli demonstrate survival in wheat flour and dried pasta products. This study investigated the heat resistance of Salmonella, pathogenic E. coli, and E. faecium NRRL B-2354 in raw semolina flour and partially dried pasta intermediates via thermal death time (TDT) studies. This study also assessed the appropriateness of E. faecium NRRL B-2354 as a surrogate in semolina flour and pasta matrices. Inoculated pasta matrices equilibrated to target water activities of 0.85, 0.88, and 0.91 (measured at 25°C) underwent isothermal inactivation treatments at 65°C, 70°C, and 75°C. Serial dilution and direct plating methods allowed for estimation of bacterial survival at each treatment. In representative pasta matrices, the D-values for each microorganism increased as water activity decreased from 0.91 to 0.85. Surprisingly, Salmonella and E. coli did not exhibit significantly different thermal resistance in pasta. The greatest heat resistance was seen in semolina flour (aw 0.45). E. faecium was significantly more thermal resistant than both pathogens in all treatments when the temperature was ≤ 70°C. The results show that E. faecium strain NRRL B-2354 is not an ideal proxy for Salmonella and E. coli in semolina and pasta matrices. Analysis of the TDT data also found that a long-goods pasta drying process can achieve ≥7-log reductions of Salmonella and E. coli when following Good Manufacturing Practices.

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No. of bitstreams: 1 zordao_op_me_arafc.pdf: 1351578 bytes, checksum: bc36b449347fea51a7c4bc4e2a6d703b (MD5) Previous issue date: 2016-07-08 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Objetivos: A utilização de microrganismos probióticos na redução do risco de doenças inflamatórias intestinais (DIIs), incluindo a colite ulcerativa (CU), tem se mostrado promissora, sendo que o efeito benéfico depende da cepa utilizada e da fase da patologia. O objetivo do presente estudo foi averiguar o efeito de um produto probiótico à base de soja, fermentado com Enterococcus faecium CRL 183 e Lactobacillus helveticus 416 e com adição de Bifidobacterium longum ATCC 15707, na fase aguda da colite quimicamente induzida em camundongos. Métodos: O protocolo experimental teve duração total de 14 dias e os animais foram distribuídos aleatoriamente em quatro grupos (n=10): Grupo C - Animais sadios que não receberam os produtos em estudo; Grupo CL - Animais com colite quimicamente induzida e que não receberam os produtos em estudo; Grupo CLF - Animais com colite quimicamente induzida e que receberam o produto fermentado probiótico; Grupo CLP - Animais com colite quimicamente induzida e que receberam o produto não fermentado (placebo). A indução da colite foi feita pela administração de dextran sulfato de sódio (DSS) a 3% dissolvido na água fornecida diariamente aos animais, por um período de sete dias, e os produtos foram administrados ao longo dos 14 dias de estudo. Durante o período experimental foram monitorados os seguintes parâmetros: peso corpóreo dos animais, ingestão hídrica e de ração, consistência e presença de sangue oculto ou aparente nas fezes e composição da microbiota fecal. Ao final do protocolo, os animais foram eutanasiados e o intestino grosso foi removido para realização das análises macroscópica e histológica. Resultados: De acordo com os resultados do índice de atividade da doença (IAD), os animais que consumiram o produto probiótico (CLF) apresentaram redução dos sintomas associados à colite durante o período de indução em comparação aos animais do grupo CL. As análises histológicas evidenciaram que o cólon dos camundongos dos grupos CL e CLP apresentou áreas com infiltrados de células, além de alterações nas criptas, representadas por degeneração ou desaparecimento de tais estruturas. O colón dos animais pertencentes ao grupo CLF exibiu infiltrado de células inflamatórias, sem alteração nas criptas. Os resultados da análise da composição da microbiota fecal mostraram que a ingestão do produto fermentado probiótico resultou no maior aumento na população de Lactobacillus spp. (1,5 log10 UFC/g) e menor aumento de enterobactérias (1,34 log10 UFC/g) ao longo do protocolo. Conclusão: Os resultados obtidos no presente estudo indicam que a ingestão regular do produto fermentado probiótico avaliado pode auxiliar na redução dos sintomas e na preservação da integridade do cólon durante a fase aguda da colite induzida em camundongos / Objectives: The use of probiotic microorganisms in the reduction of inflammatory bowel diseases (IBD) risks, including ulcerative colitis (UC), has been shown promising, and the beneficial effect depends on the strain used and the stage of disease. The objective of this study was to investigate the effect of a probiotic soy product fermented with Enterococcus faecium CRL 183 and Lactobacillus helveticus 416 with addition of Bifidobacterium longum ATCC 15707 in the acute phase of colitis chemically induced in mice. Methods: The experimental protocol had total duration of 14 days and the animals were randomized into four groups (n = 10): Group C - healthy animals that have not received the products under study; CL Group - Animals with chemically induced colitis and who have not received the products under study; CLF Group - Animals with chemically induced colitis and receiving the probiotic fermented product; Group CLP - animals with chemically induced colitis and receiving the unfermented product (placebo). The induction of colitis was made by the administration of dextran sodium sulfate (DSS) 3% dissolved in the drinking water for a period of seven days, and the products were administered over the 14 days of the study. During the trial period the following parameters were monitored: body weight, water and food intake, consistency and presence of hidden or apparent blood in stool and composition of the fecal microbiota. At the end of the protocol, the animals were euthanized and the large intestine was removed to perform the macroscopic and histological analysis. Results: According to the results of disease activity index (DAI), the animals that consumed the probiotic product (CLF) showed a reduction of symptoms associated with colitis during the induction period, compared to the CL group. Histological analysis showed that the colon of mice of CL and CLP groups showed areas with cell infiltrates, as well as changes in the crypts, represented by degeneration or disappearance of such structures. The colon of animals belonging to the CLF group exhibited infiltration of inflammatory cells, without changing in the crypts. The results of the analysis of fecal microbiota composition revealed that the ingestion of the probiotic fermented product resulted in the greatest increase in the population of Lactobacillus spp. (1.5 log10 CFU / g) and less increase of enterobacteria (1.34 log10 CFU / g) over the protocol. Conclusion: The results obtained in this study indicate that regular intake of probiotic fermented product evaluated can help in the reduction of the symptoms and preservation of colon health during the acute colitis induced in mice.

[ES] La obesidad es uno de los principales problemas de salud pública debido a su elevada prevalencia y a las comorbilidades asociadas, lo que se traduce en una reducción considerable de la calidad y esperanza de vida, además de un enorme gasto económico. Su origen es multifactorial y el desarrollo de terapias efectivas para combatirla resulta complejo. La microbiota intestinal ejerce un papel relevante en el mantenimiento del balance energético y salud metabólica. Por ello, las estrategias basadas en la modificación de la microbiota intestinal son consideradas potenciales alternativas para el manejo clínico de la obesidad. No obstante, se requiere un mayor conocimiento sobre cuáles son las especies bacterianas clave en el mantenimiento de la homeostasis energética del hospedador y su modo de acción. El objetivo general de la tesis ha sido identificar nuevas estrategias basadas en la manipulación de la composición y funciones de la microbiota intestinal eficaces contra la obesidad, así como sus mecanismos de interacción con el hospedador. El capítulo primero de la tesis se centra en estudiar los mecanismos de acción por los que Bacteroides uniformis CECT 7771 ejerce efectos protectores frente al desarrollo de la obesidad, tal y como nuestro grupo ha descrito en trabajos previos. A través de un estudio en ratones con obesidad inducida por la dieta hemos demostrado que esta cepa reduce la disfunción metabólica a través de la modulación de la microbiota intestinal y las alteraciones inmunológicas en el intestino y el tejido adiposo asociados a la obesidad. Todos los efectos sobre el eje intestino-tejido adiposo parecen estar mediados por la activación de TLR5. Además, hemos desarrollado una estrategia similar a un simbiótico con el fin de incrementar la eficacia de esta bacteria administrándola a dosis menores. La formulación simbiótica se diseñó en base a la preferencia de B. uniformis CECT 7771 por el salvado de trigo (WBE) como fuente de carbono en cultivos in vitro. La administración conjunta de la bacteria y WBE mostró beneficios inmuno-metabólicos al reducir el aumento de peso corporal y la adiposidad, al tiempo que mejoraron las rutas del metabolismo energético moduladas por insulina y la homeostasis inmunológica intestinal. Además, reforzó la primera línea de defensa inmunológica al aumentar los niveles de butirato y restaurar los niveles de IEL inducidos y las ILC3. En el segundo capítulo hemos llevado a cabo dos estudios preclínicos que describen los efectos de dos nuevas bacterias autóctonas del tracto gastrointestinal humano como potenciales probióticos para el tratamiento de la obesidad identificadas por el grupo. El primer estudio, la administración de Holdemanella biformis CECT 9752 a ratones con obesidad inducida por la dieta redujo los niveles de glucosa en ayuno y mejoró la tolerancia oral a la glucosa de forma independiente a la insulina. A nivel del contenido luminal del intestino grueso, la bacteria incrementó los niveles de ácidos grasos insaturados, potenciales secretagogos lipídicos de GLP-1. A nivel del intestino delgado, incrementó la sensibilidad a GLP-1 de las neuronas vagales aferentes, mecanismo implicado en la producción endógena de glucosa. A nivel hepático, la suplementación con la bacteria redujo la gluconeogénesis y mejoró la sensibilidad a insulina. En el segundo estudio hemos evaluado los efectos inmuno-metabólicos de Phascolarctobacterium faecium DSM 32890, consumidora de succinato y productora de propionato en un modelo animal de obesidad. La administración redujo el aumento de peso corporal y la ingesta de alimentos y mejoró la tolerancia oral a la glucosa. Estos beneficios se asociaron a un aumento sostenido de la hormona intestinal anorexigénica PYY en plasma y una prevención de la hipersecreción de GIP inducida por la dieta rica en grasa. Además, la bacteria normalizó la inmunidad intestinal alterada en la obesidad, y mejoró / [CA] L'obesitat és un dels principals problemas de salut pública a causa de la seua elevada prevalença i a les comorbilitats associades, la qual cosa es tradueix en una reducció considerable de la qualitat i de l'esperança de vida, a més d'una enorme despesa econòmica. El seu origen es multifactorial i el desenvolupament de teràpies efectives per combatre-les resulta complex. La microbiota intestinal exerceix un paper rellevant en el manteniment del balanç energètic i salut metabòlica. Per això, les estrategias basades en la modificació de la microbiota intestinal son considerades hui en dia potencials alternatives per al maneig clínic de l'obesitat. No obstant això, es requereix d'un major coneixement sobre quines son les espècies bacterianes claus al manteniment de l'homeòstasi energètica de l'hoste i la seua manera d'acció. L'objectiu general de la tesi ha sigut identificar noves estratègies basades en la manipulació de la composició i funcions de la microbiota intestinal eficaces contra l'obesitat, així com els seus mecanismes d'interacció amb l'hoste. El capítol primer de la tesi es centra en estudiar els mecanismes d'acció pels quals Bacteroides uniformis CECT 7771 exerceix efectes protectors enfront del desenvolupament de l'obesitat, tal com el nostre grup ha descrit previament. A través d'un estudi en ratolins amb obesitat induïda per la dieta hem demostrat que aquesta soca redueix la disfunció metabòlica a través de la modulació de la microbiota intestinal i les alteracions immunològiques en l'intestí i al teixit adipos epididimal associats a l'obesitat. Tots els efectes sobre l'eix intestí-teixit adipós semblen estar mediades per l'activació de TLR5. A més, hem desenvolupat una estrategia similar a un simbiòtic amb la finalitat d'incrementar l'eficàcia d'aquest bacteri administrant-la a dosis menors. La fomulació simbiòtica es va dissenyar basant-se en la preferència de B. uniformis CECT 7771 pel segó del blat (WBE) com a font de carboni en cultius in vitro. L'administració conjunta de la bacteria i WBE va mostrar beneficis immuno-metabòlics al reduir l'augment de pes corporal i l'adipositat, al mateix temps que van millorar les rutes del metabolisme energètic modulades per insulina i la homeòstasi immunològica intestinal. A més, va reforçar la primera línia de defensa immunològica augmentant els nivells de butirat i restaurant els nivells de IEL induïts i de ILC3. Al segon capítol de la tesi hem dut a terme dos estudis preclínics que descriuen els efectes de dos nous bacteris autòctones del tracte gastrointestinal humà com a potencials probiòtics per al tractament de l'obesitat identificades amb posterioritat pel grup. El primer estudi, l'administració de Holdemanella biformis CECT 9752 a un model animal d'obesitat va reduir els nivells de glucosa en dejú i va millorar la tolerància oral a la glucosa de manera independent a la insulina. A nivell del contingut luminal de l'intestí gros, el bacteri va incrementar els nivells d'àcids grassos insaturats, potencials secretagogos lipídics de GLP-1. A nivell de l'intestí prim, va incrementar la sensibilitat a GLP-1 de les neurones vagals aferents, mecanisme implicat en la producció endògena de glucosa. A nivell hepàtic, la suplementació amb el bacteri va reduir la gluconeogènesi i va millorar la sensibilitat a insulina. En el segon estudi hem avaluat els efectes immuno-metabòlics de Phascolarctobacterium faecium DSM 32890, consumidora de succinat i productora de propionat a un model animal d'obesitat. L'administració va reduir l'augment de pes corporal i l'ingesta d'aliments i va millorar la tolerància oral a la glucosa. Aquests beneficis es van associar a un augment sostingut de l'hormona intestinal anorexigénica PYY en plasma i la prevenció de la hipersecreció de GIP induïda per la dieta rica en greix. A més, el bacteri va normalitzar la immunitat intestinal alterada en l'obesitat i va millorar / [EN] Obesity is one of the main public health problems due to its high prevalence and associated comorbidities, which results in a considerable reduction of the health-related quality of life and life expectancy, as well as in an overwhelming cost to global health economies. It has a multifactorial origin and the development of effective therapies is complex and has become one of the main challenges for society. The gut microbiota plays an important role in the maintenance of energy balance and metabolic health. Therefore, strategies based on gut microbiota modification to beneficially modulate energy metabolism are nowadays considered potential alternatives for the clinical management of obesity. However, the effective implementation of these strategies requires the identification of key bacterial species for the maintenance of host energy homeostasis as well as the better understanding of which mechanisms are behind of their effects. The general objective of this doctoral thesis has been to identify new and effective strategies against obesity based on the manipulation of the gut microbiota composition and function, as well as their mechanisms of interaction with the host. The first chapter of the thesis focuses on studying the mechanisms of action by which Bacteroides uniformis CECT 7771 induces protective effects against the onset of obesity, based on previous studies of our group. An intervention conducted in diet-induced obese mice, we have demonstrated that this strain reduces metabolic dysfunction through the modulation of the intestinal microbiota and immune players obesity associated in both intestine and epididymal white adipose tissue. All the effects on the gut-adipose tissue axis appear to be mediated by TLR5 activation. Moreover, we have developed a symbiotic strategy with the aim of increasing the B. uniformis anti-obesity efficacy at lower doses. The symbiotic formulation was designed based on the preference of B. uniformis CECT 7771 for wheat bran (WBE) as a carbon source in in vitro cultures. The co-administration of the bacteria and WBE showed immuno-metabolic benefits reducing body weight gain and adiposity, along with improving insulin-modulated energy metabolism pathways and intestinal immune homeostasis. In addition, It strengthened the first line of immune defence by increasing butyrate levels and restoring levels of induced IEL and ILC3. In the second chapter of the thesis we have carried out two pre-clinical studies describing the effects of two new autochthonous bacteria of the human gastrointestinal tract as potential probiotics for the treatment of obesity identified later by the group. The first study, the administration of Holdemanella biformis CECT 9752 in an animal model obesity reduced fasting glucose levels and improved oral glucose tolerance in an insulin-independent manner. In the luminal content of the large intestine, the bacteria increased the abundance of unsaturated fatty acids, potential GLP-1 secretagogues. In the small intestine, it could directly increase the GLP-1 sensitivity of vagal afferent neurons, a mechanism involved in hepatic endogenous glucose production. At the hepatic level, the supplementation with the bacteria reduced gluconeogenesis and improved insulin sensitivity. In the second study we have evaluated the immuno-metabolic effects of Phascolarctobacterium faecium DSM 32890 succinate consumer and propionate producer in an animal model of diet-induced obesity. The bacteria reduced body weight gain and food intake and improved oral glucose tolerance. These benefits were associated with a sustained increase in plasma of the anorexigenic gut hormone PYY and a prevention of high-fat diet-induced GIP hypersecretion. In addition, the bacteria normalized the impaired intestinal immunity in obesity and improved intestinal barrier integrity. / This study received funding from the European Union Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 797297 (M.R-P) and from the European Union 7th Framework program under the grant agreement no 613979 (MyNewGut) and grant AGL2017-88801-P from the Spanish Ministry of Economy and Competitiveness (MCIU, Spain). The Santiago Grisolía scholarship (GRISOLIAP/2014/110) to E.F from Generalitat Valenciana and the FPI scholarship (BES-2015-073930) of I López-Almela and the PTA contract (PTA2013-8836-I) of I. Campillo from MCIU are fully acknowledged. / López Almela, I. (2021). Descifrando las funciones de la microbiota intestinal en la obesidad [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/174897

No description available.

Enterococcus faecium è un componente fondamentale del microbiota di diversi alimenti fermentati quali formaggi e salumi e viene spesso isolato in alto numero in alimenti pronti al consumo. É inoltre largamente utilizzato come probiotico sia per l’uomo che per gli animali. Allo stesso tempo, però, questa specie batterica rappresenta una delle cause principali di infezioni nosocomiali quali endocarditi ed infezioni al tratto urinario. Studi recenti hanno dimostato che la specie E. faecium è costituita da due sub-popolazioni principali: la prima è denominate hospital associated (HA) clade “A” ed include la maggior parte dei ceppi responsabili di infezioni umane; la seconda è chiamata community associated (CA) clade “B”, e contiene principalmente ceppi commensali dell’uomo. Analisi più approfondite hanno rivelato un ulteriore suddivisione all’interno del clade A, nel sub-clade A1 (che raggruppa la maggioranza dei ceppi clinici) e nel sub-clade A2, associato agli animali e più sporadicamente ad infezioni umane. Nel 2012, EFSA ha redatto una linea guida per la valutazione della sicurezza di E. faecium usato come probiotico per gli animali, concludendo che i cepi appartenenti all’hospital-associated clade non devono essere utilizzati in nutrizione animale. Comunque, la distinzione tra le due sub-popolazioni è stata fatta utilizzando dati ottenuti prevalentemente da isolati umani e animali e solo un numero limitato di ceppi isolati dagli alimenti è stato considerato. Obiettivo di questa tesi di dottorato è stato quello di valutare la sicurezza di E. faecium negli alimenti fermentati, considerando ceppi isolati da formaggi artigianali e prodotti carnei e utilizzando sia tecniche di genomica che analisi fisiologiche. Nessuno dei ceppi alimentari studiati è risultato parte del clade A1, ma un ceppo isolato da un salame stagionato pronto al consumo ha rivelato diversi tratti tipici dei ceppi A1, tra cui particolari IS, transposase e geni di resistenza agli antibiotici. Questi risultati, così come altri dati, sottolineano la necessità di approfondire le conoscenze circa il ruolo dei ceppi di E. faecium isolati da alimenti come fattore di rischio per la salute umana. / Enterococcus faecium is commonly found in high numbers in ready to eat foods, being a member of the bacterial communities of a variety of fermented foods, including cheese and sausages, and is widely used as human and animal probiotic. However, this bacterial species is a leading cause of nosocomial infection, mainly endocarditis and urinary tract infections. Recent studies have demonstrated that E. faecium species consists of two very distinct clades: the hospital associated (HA) clade “A”, which includes most of the strains responsible for human infections, and the community associated (CA) clade “B”, that contains primarily human commensal isolates. Deeper analysis revealed a further split within clade A into sub-clade A1 (which groups the vast majority of clinical isolates), and sub-clade A2, associated with animals and sporadic human infections. In 2012, the European Food Safety Authority has issued a guideline for the safety assessment of E. faecium used as animal probiotics, concluding the strains belonging to the hospital-associated clade should not be used in animal nutrition. However, the differentiation of the two clades has been performed using data mainly deriving from human and animal isolates, and only a limited number of strains from the food chain were considered. Aim of this doctoral thesis was to assess the safety of E. faecium in fermented food, considering strains isolated from artisanal cheese and meat products, and using both whole genome-based techniques and physiological studies. None of the food isolates studied in this work belong to the epidemic clade A1, however a strain isolated from a ready to eat salami revealed several A1-specific traits, such as specific IS, transposases and antibiotic resistance genes. These results, as well as other data, underline the emergency of deeper understanding the role of E. faecium isolated from fermented foods as risk factor for human health.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2005. / Härtill 5 uppsatser.

[ES] Las bacterias resistentes a múltiples antibióticos, como el Enterococo resistente a vancomicina (ERV), son un problema creciente en los pacientes hospitalizados, por lo que se necesita estrategias alternativas para combatir estos patógenos. Las infecciones causadas por ERV suelen comenzar con la colonización del tracto intestinal, un paso crucial que se afectado por la presencia de la microbiota. Sin embargo, los antibióticos alteran la microbiota y esto promueve la colonización de ERV. Una vez que el patógeno ha colonizado el intestino, alcanza niveles muy altos pudiendo diseminar a otros órganos y pacientes. A pesar de su importancia, se sabe muy poco sobre los genes que codifica para colonizar el intestino y sobre el mecanismo por el cual la microbiota suprime su colonización intestinal, siendo los dos objetivos principales. En primer lugar hemos utilizado una metodología previamente descrita (Zhang et al., 2017, BMC Genomics), basada en la generación de una librería de mutantes por transposición junto a secuenciación masiva, con el fin de identificar los genes codificados por ERV necesarios para la colonización del intestino en ratones. Además, hemos realizado análisis metatranscriptómicos para identificar aquellos genes más expresados. El análisis ha identificado genes cuya interrupción reduce significativamente la colonización intestinal en el intestino grueso. Los genes que más afectaron a la colonización codifican proteínas relacionadas con la absorción o el transporte de diversos nutrientes como los carbohidratos (subunidad EIIAB del transportador PTS de manosa, el regulador transcripcional de la familia LacI, ácido N-acetilmurámico 6-fosfato eterasa) o iones (proteína transportadora dependiente de ATP (ABC) y proteínas del grupo [Fe-S]). El papel de estos genes en la colonización se ha confirmado mediante experimentos de mutagénesis directa y de competición con la cepa salvaje. Además, estos genes afectan a la colonización intestinal con diferentes antibióticos (clindamicina y vancomicina). Para identificar el mecanismo molecular por el cual cada gen afecta a la colonización, hemos realizado experimentos in vitro y ex vivo además del análisis transcriptómico. Los experimentos in vitro confirman que las proteínas del grupo [Fe-S] están involucradas en el transporte iones de hierro, principalmente Fe3+. Por otra parte, los genes de la subunidad EIIAB del transportador de manosa y del ácido N-acetilmurámico 6-fosfato eterasa son necesarios para la utilización de la manosa y el ácido N-acetilmurámico, respectivamente, azúcares que suelen estar presentes en el intestino. También confirmamos que el regulador transcripcional de la familia LacI es un represor que afecta a proteínas transportadoras ABC, probablemente implicadas en la absorción de carbohidratos. Además, algunos de estos genes están codificados principalmente por cepas clínicas de E. faecium y en menor medida por cepas comensales. En segundo lugar, estudiamos los mecanismos de protección de un consorcio de cinco bacterias comensales, que anteriormente se había demostrado que disminuían la colonización intestinal por ERV en ratones. Mediante transcriptómica, metabolómica y los ensayos in vivo observamos que el consorcio bacteriano inhibe el crecimiento de ERV mediante la reducción de nutrientes, concretamente fructosa. Por último, el análisis ARN-Seq in vivo de cada aislado en combinación con los ensayos ex vivo e in vivo demostraron que una sola bacteria (Olsenella sp.) proporciona protección. En conjunto, los resultados obtenidos han identificado la función de genes específicos requeridos por ERV para colonizar el intestino. Además, hemos identificado un mecanismo mediante el cual la microbiota confiere protección. Estos resultados podrían conducir a nuevos enfoques terapéuticos para prevenir las infecciones causadas por este patógeno multiresistente a los antibióticos. / [CA] Els bacteris resistents a múltiples antibiòtics, com el Enterococo resistent a vancomicina (ERV), són un problema creixent en els pacients hospitalitzats, que són resistents a la majoria d'antibiòtics disponibles per la qual cosa es necessita estratègies alternatives per a combatre aquests patògens. Les infeccions causades per ERV solen començar amb la colonització del tracte intestinal, un pas crucial que es veu afectat per la presència de la microbiota. No obstant això, els antibiòtics alteren la microbiota i això promou la colonització de ERV. Una vegada que el patogen ha colonitzat l'intestí, aconsegueix nivells molt alts podent disseminar a altres òrgans i pacients. Malgrat la seua importància, se sap molt poc sobre els gens que codifica ERV per a colonitzar l'intestí i sobre el mecanisme pel qual la microbiota suprimeix la seua colonització intestinal. En primer lloc hem utilitzat una metodologia prèviament descrita (Zhang et al., 2017, BMC Genomics), basada en la generació d'una llibreria de mutants per transposició junt amb seqüenciació massiva, amb la finalitat d'identificar els gens codificats per ERV necessaris per a la colonització de l'intestí en ratolins. A més a més, hem realitzat anàlisi metatranscriptòmics per a identificar aquells gens més expressats. L'anàlisi ha identificat gens quina interrupció redueix significativament la colonització intestinal en l'intestí gros. Els gens que més van afectar la colonització codifiquen proteïnes relacionades amb l'absorció o el transport de diversos nutrients com els carbohidrats (subunitat EIIAB del transportador PTS de manosa, el regulador transcripcional de la família LacI, àcid N-acetilmuràmic 6-fosfat eterasa) o ions (proteïna transportadora dependent d'ATP (ABC) i proteïnes del grup [Fe-S]). El paper d'aquests gens en la colonització s'ha confirmat mitjançant experiments de mutagènesis directa i de competició amb el cep salvatge. A més, aquests gens afecten la colonització intestinal amb diferents antibiòtics (clindamicina i vancomicina). Per a identificar el mecanisme molecular pel qual cada gen afecta a la colonització, hem realitzat experiments in vitro i ex viu a més de l'anàlisi transcriptòmic. Els experiments in vitro confirmen que les proteïnes del grup [Fe-S] estan involucrades en el transport d'ions de ferro, principalment Fe3+. D'altra banda, els gens de la subunitat EIIAB del transportador PTS de manosa i de l'àcid N-acetilmuràmic 6-fosfat eterasa són necessaris per a la utilització de la manosa i l'àcid N-acetilmuràmic, respectivament, sucres que solen estar presents en l'intestí. També confirmem que el regulador transcripcional de la família LacI és un repressor que afecta proteïnes transportadores ABC, probablement implicades en l'absorció de carbohidrats. A més a més, alguns d'aquests gens estan codificats principalment per ceps clínics de E. faecium i en menor mesura per ceps comensals. En segon lloc, estudiem els mecanismes de protecció d'un consorci de cinc bacteris comensals, que adès s'havia demostrat que disminuïen la colonització intestinal per ERV en ratolins. Amb l'ús de transcriptòmica, metabolòmica i els assajos in vivo observem que el consorci bacterià inhibeix el creixement de ERV mitjançant la reducció de nutrients, concretament fructosa. Finalment, l'anàlisi ARN-Seq in vivo de cada aïllat en combinació amb els assajos ex viu i in vivo van demostrar que un sol bacteri (Olsenella sp.) proporciona protecció. En conjunt, els resultats obtinguts han identificat la funció de gens específics requerits per ERV per a colonitzar l'intestí. A més, hem identificat un mecanisme mitjançant el qual la microbiota confereix protecció. Aquests resultats podrien conduir a nous enfocaments terapèutics per a previndre les infeccions causades per aquest patogen multiresistent als antibiòtics. / [EN] Multidrug-resistant bacteria, such as vancomycin-resistant-Enterococcus (VRE), are an increasing problem in hospitalized patients. Some VRE strains can be resistant to most available antibiotics, thus, alternative strategies to antibiotics are urgently needed to combat these challenging pathogens. Infections caused by VRE frequently start by colonization of the intestinal tract, a crucial step that is impaired by the presence of the intestinal microbiota. Administration of antibiotics disrupts the microbiota, which promotes VRE intestinal colonization. Once VRE has colonized the gut, it reaches very high levels, which promotes its dissemination to other organs and its transfer to other patients. Despite the relevance of VRE gut colonization, very little is known about the genes encoded by this pathogen to colonize the gut and about the mechanisms by which the microbiota suppresses VRE gut colonization. In this thesis, we have utilized a previously described methodology (Zhang et al., 2017, BMC Genomics), based on the generation of a transposon mutant library coupled with high-throughput sequencing, in order to identify VRE encoded genes required for colonization of the mouse intestinal tract. In addition, we have performed metatranscriptomic analysis in mice to identify VRE genes specifically expressed in the gut. Our analysis has identified genes whose disruption significantly reduces VRE gut colonization in the large intestine. The genes that most affected VRE gut colonization encoded for proteins related to the uptake or transport of diverse nutrients such as carbohydrates (PTS mannose transporter subunit EIIAB, LacI family DNA-binding transcriptional regulator, N-acetylmuramic acid 6-phosphate etherase) or ions (phosphate ABC transporter ATP-binding protein and proteins from [Fe-S] cluster). The role of these genes in gut colonization has been confirmed through targeted mutagenesis and competition experiments against a wild type strain. Moreover, these genes affect gut colonization under different antibiotic treatments (clindamycin and vancomycin). To elucidate the mechanism by which each gene influences gut colonization, we have performed in vitro and ex vivo experiments besides transcriptomic analysis. In vitro experiments confirm that proteins from [Fe-S] cluster are involved in the transport of different forms of iron ions, mostly Fe3+. On the other hand, the PTS mannose transporter subunit EIIAB and N-acetylmuramic acid 6-phosphate etherase genes are required for the utilization of mannose and N-acetyl-muramic acid, respectively, sugars that are usually present in the intestinal environment. We have also confirmed that LacI family DNA-binding transcriptional regulator is a repressor that affects the expression of genes encoding for an ABC transporter probably involved in the uptake of carbohydrates. Furthermore, we have confirmed that some of these genes are encoded mainly by E. faecium clinical strains but not or to a lower extent by commensal strains. Secondly, we studied the mechanisms of protection of a consortium of five commensals bacteria, previously shown to restrict VRE gut colonization in mice. Functional transcriptomics in combination with targeted metabolomics and in vivo assays performed in this thesis indicated that the bacterial consortium inhibits VRE growth through nutrient depletion, specifically by reducing the levels of fructose. Finally, in vivo RNA-Seq analysis of each bacterial isolate of the consortium in combination with ex vivo and in vivo assays demonstrated that a single bacterium (Olsenella sp.) could recapitulate the protective effect. Altogether, the results obtained have identified the function of specific genes required by VRE to colonize the gut. In addition, we have identified a specific mechanism by which the microbiota confers protection against VRE colonization. These results could lead to novel therapeutic approaches to prevent infections caused by this pathogen. / Flor Duro, A. (2021). Characterization of Genes and Functions Required by Multidrug-resistant Enterococci to Colonize the Intestine [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/166494 / TESIS