Oral Health in Down Syndrome

Oral health in Down Syndrome (DS) individuals has some peculiar aspects that must be considered in the follow up of these patients. In this chapter, we will focus on the oral and maxillofacial morphological alteration, the most prevalent oral pathologies as well as preventive measures and strategies for pathologies management in this population. Also, future research on oral health of DS will be discussed

Raoultella ornithinolytica: an opportunistic pathogen in the oral cavity of chronic kidney disease patients

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Gut microbiome in hemodialysis patients treated with calcium acetate or treated with sucroferric oxyhydroxide : a pilot study

It has been proved that the gut microbiome is altered in patients with chronic kidney disease. This contributes to chronic inflammation and increases cardiovascular risk and mortality, especially in those undergoing hemodialysis. Phosphate binders may potentially induce changes in their microbiome. This trial aimed to compare the changes in the gut microbiome of hemodialysis patients treated with calcium acetate to those treated with sucroferric oxyhydroxide. Twelve hemodialysis patients were distributed to receive calcium acetate or sucroferric oxyhydroxide for 5 months. Blood samples (for biochemical analysis) and stool samples (for microbiome analysis) were collected at baseline, 4, 12, and 20 weeks after treatment initiation. Fecal DNA was extracted and a 16S rRNA sequencing library was constructed targeting the V3 and V4 hypervariable regions. Regarding clinical variables and laboratory parameters, no statistically significant differences were observed between calcium acetate or sucroferric oxyhydroxide groups. When analyzing stool samples, we found that all patients were different (p = 0.001) among themselves and these differences were kept along the 20 weeks of treatment. The clustering analysis in microbial profiles grouped the samples of the same patient independently of the treatment followed and the stage of the treatment. These results suggest that a 5-month treatment with either calcium acetate or sucroferric oxyhydroxide did not modify baseline diversity or baseline bacterial composition in hemodialysis patients, also about the high-variability profiles of the gut microbiome found among these patients

Agro-industrial wastes as alternative substrates for the production of prebiotic with Zymomonas mobilis

Fructooligosaccharides (FOS) are promising prebiotics in the relevant and increasing market of functional food. However, to achieve a more sustainable process, the industrial production of FOS should use cheap substrates. Zymomonas mobilis (ZM) has great potential for the production of FOS due to the presence of native enzymes (levansucrase) capable of metabolizing sucrose. In addition, ZM can use different carbon sources, such as molasses and sugarcane juice, which make the FOS production process cost-effective. In this study, sugarcane molasses (a potential replacement of sucrose) and corn step liquor (CSL) (a potential replacement of yeast extract (YE)), were used as nutrients for FOS production using ZM in an in vivo bioprocess approach. FOS production process from sucrose was first optimized and 52 g/L of FOS with a yield of 0.16 g/g was obtained. Afterwards, molasses and CSL were used as alternative nutrients. After studying different combinations of CSL and YE, the highest amount of FOS (54 g/L, with a yield of 0.18 g/g) was obtained with 12 g/L of CSL and 8 g/L of YE. In addition, 45 g/L of FOS were produced from molasses containing 200 g/L of sucrose, with a yield of 0.3 g/g. With this approach, it was possible to reduce around 5.5-times the cost associated with the FOS production medium. Moreover, this study proposed a sustainable process for the valorization of agro-industrial wastes contributing to the future Circular (Bio)Economy and the EU Green Deal.Cláudia Amorim, João Rainha, Beatriz B. Cardoso and Daniela Gomes acknowledge their grants (2020.0029.CEECIND, SFRH/BD/138325/2018, SFRH/BD/132324/2017 and SFRH/BD/04433/2020, respectively) from Portuguese Foundation for Science and Technology (FCT). The study received financial support from Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and by LABBELS – Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020.info:eu-repo/semantics/publishedVersio

Characterization of oral enterobacteriaceae prevalence and resistance profile in chronic kidney disease patients undergoing peritoneal dialysis

Chronic Kidney Disease (CKD) is a growing public-health concern worldwide. Patients exhibit compromised immunity and are more prone to infection than other populations. Therefore, oral colonization by clinically relevant members of the Enterobacteriaceae family, major agents of both nosocomial and dialysis-associated infections with frequent prevalence of antibiotic resistances, may constitute a serious risk. Thus, this study aimed to assess the occurrence of clinically relevant enterobacteria and their antibiotic resistance profiles in the oral cavity of CKD patients undergoing peritoneal dialysis (CKD-PD) and compare it to healthy controls. Saliva samples from all the participants were cultured on MacConkey Agar and evaluated regarding the levels of urea, ammonia, and pH. Bacterial isolates were identified and characterized for antibiotic resistance phenotype and genotype. The results showed that CKD-PD patients exhibited significantly higher salivary pH, urea, and ammonia levels than controls, that was accompanied by higher prevalence and diversity of oral enterobacteria. Out of all the species isolated, only the prevalence of Raoultella ornithinolytica varied significantly between groups, colonizing the oral cavity of approximately 30% of CKD-PD patients while absent from controls. Antibiotic resistance phenotyping revealed mostly putative intrinsic resistance phenotypes (to amoxicillin, ticarcillin, and cephalothin), and resistance to sulfamethoxazole (~43% of isolates) and streptomycin (~17%). However, all isolates were resistant to at least one of the antibiotics tested and multidrug resistance isolates were only found in CKD-PD group (31,6%). Mobile genetic elements and resistance genes were detected in isolates of the species Raoultella ornithinolytica, Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, and Enterobacter asburiae, mostly originated from CKD-PD patients. PD-related infection history revealed that Enterobacteriaceae were responsible for ~8% of peritonitis and ~ 16% of exit-site infections episodes in CKD-PD patients, although no association was found to oral enterobacteria colonization at the time of sampling. The results suggest that the CKD-induced alterations of the oral milieu might promote a dysbiosis of the commensal oral microbiome, namely the proliferation of clinically relevant Enterobacteriaceae potentially harboring acquired antibiotic resistance genes. This study highlights the importance of the oral cavity as a reservoir for pathobionts and antibiotic resistances in CKD patients undergoing peritoneal dialysis.info:eu-repo/semantics/publishedVersio

Optimization of fructooligosaccharides production in Zymomonas mobilis

Dissertação de mestrado em BiotecnologiaOs frutooligosacarídeos (FOS) são prebióticos promissores no mercado crescente dos alimentos funcionais. Industrialmente, são produzidos a partir da sacarose por ação das enzimas frutosiltransferase (FTase) ou b-frutofuranosidase (FFase). No entanto, este processo apresenta baixas conversões e gera misturas com baixa pureza devido às elevadas quantidades de glucose libertadas durante a produção dos FOS. A Zymomonas mobilis é uma bactéria que possui enzimas nativas (levanosucrase) capazes de converter a sacarose em FOS. O principal objetivo deste trabalho foi eliminar a atividade da invertase na Z. mobilis e avaliar o uso de resíduos agro-industriais como substratos de baixo custo na produção de FOS. Inicialmente, o melaço e o licor de maceração de milho (CSL) foram avaliados como fontes alternativas de carbono e azoto. O meio composto por 200 g L-1 de sacarose em melaço, 12 g L-1 de CSL e 8 g L-1 de extrato de levedura (CSLM) foi considerado o mais promissor para produzir FOS. Em matraz, foram produzidos 58,15 ± 0,21 g L-1 de FOS (1,211 ± 0,004 g L-1 h-1 ; 0,307 ± 0,003 gFOS gsacarose-1 ) numa mistura que incluí a 1-questose, a 6-questose, a nistose e a neonistose. A realização de ensaios à escala de biorreator em modo descontínuo permitiu aumentar o rendimento do processo em 1,6 vezes (0,482 ± 0.008 gFOS gsacarose-1 ). A deleção do gene da invertase do genoma de Z. mobilis ZM4 foi realizada por recombinação homóloga utilizando um plasmídeo suicida. Após introdução deste plasmídeo na Z. mobilis e a recombinação homóloga, obteve-se a estirpe Z. mobilis SacC-. Com esta estirpe modificada observou se uma redução de 70 % na produção de monossacarídeos e um aumento de 9 vezes na produção de levano comparativamente à estirpe selvagem. A implementação do modo de operação semi-contínuo por pulsos utilizando o meio CSLM permitiu produzir 41,92 ± 2,52 g L-1 de FOS (0,29 ± 0,02 g L-1 h-1 ; 0,25 ± 0,07 gFOS gsacarose-1 ). Rendimentos e produtividades semelhantes foram obtidos à escala de biorreator. De modo a melhorar a produção de FOS, o gene que nativo da levanosucrase (sacB) foi sobre-expresso. A construção deste plasmídeo foi concluída com êxito, porém a sua transformação em Z. mobilis SacC não foi conseguida no decorrer desta dissertação. De acordo com o no nosso conhecimento, este trabalho descreve pela primeira vez a produção de FOS, com uma estirpe geneticamente modificada de Z. mobilis, num processo de uma só etapa utilizando resíduos agro-industriais. Embora estes resultados sejam promissores, será necessária uma otimização adicional para que se possa estabelecer um processo competitivo.Fructooligosaccharides (FOS) are promising prebiotics in the growing market of functional foods. Industrially, these compounds are produced from sucrose by the action of fructosyltransferase (FTase) or b-fructofuranosidase (FFase) enzymes. However, this process has low conversion yields and generates mixtures with low purity due to the high amounts of glucose released during its production. Zymomonas mobilis is a well-known ethanol producing bacterium with native enzymes (levansucrase) able to convert sucrose into FOS. The main goal of this work was to use synthetic biology approaches to eliminate invertase activity in Z. mobilis and further study the potential use of agro-industrial by-products as low cost substrates in FOS production. Firstly, molasses and corn steep liquor (CSL) were evaluated as alternative carbon and nitrogen sources. Medium containing 200 g L-1 of sucrose in molasses, 12 g L-1 of CSL and 8 g L-1 of YE (CSLM) was found to be the most promising medium for FOS production. In shake flask, 58.15 ± 0.21 g L-1 of FOS were synthesised (1.211 ± 0.004 g L-1 h-1 , 0.307 ± 0.003 gFOS gsucrose-1 ), resulting in a diverse FOS profile that included 1-kestose, 6-kestose, nystose and neonystose. When the batch process was studied at bioreactor scale, a 1.6-fold increase in FOS yield was observed (0.482 ± 0.008 gFOS gsucrose-1 ). Invertase deletion from the Z. mobilis ZM4 genome was accomplished by homologous recombination of an engineered suicide plasmid. After introducing this plasmid into Z. mobilis and homologous recombination, a sacC disrupted strain was obtained (Z. mobilis SacC-). Using this strain, a 70 % reduction in monosaccharide production and a 9.0-fold increase in levan formation was observed when compared to the wild-type strain. Implementation of a fed-batch approach with CSLM medium allowed to produce 41.92 ± 2.52 g L-1 of FOS (0.29 ± 0.02 g L-1 h-1 , 0.25 ± 0.07 gFOS gsucrose-1 ). When the process was studied at bioreactor scale, similar yields and productivities were obtained. To further improve FOS production using Z. mobilis strains, we aimed to overexpress its native levansucrase gene (sacB). Plasmid construction was successfully completed, however transformation into Z. mobilis SacC was unsuccessful during this dissertation. To our knowledge, this work describes for the first time the production of FOS using a genetically modified Z. mobilis strain in a one-step approach with agro-waste residues. While these results are promising, still additional optimization is needed to establish a competitive process

The urogenital microbiome in chronic kidney disease patients on peritoneal dialysis

Introduction and objectives: Diabetes, dyslipidemia, older age, gender, urinary tract infections, and recent antibiotic intake have been associated with a decrease in the urobiome richness and other fluctuations in this microbiome. Gut and blood microbiome have been reported to be altered in patients with chronic kidney disease (CKD), and specifically in peritoneal dialysis (PD) patients. Still, there are currently no studies describing the urogenital microbiome in CKD-PD patients. In this study we characterized the urobiome profile in 46 PD patients and analyzed its clinical and inflammatory parameters. Materials and methods: Mid-stream urine, fecal and blood samples were collected from 46 patients undergoing PD at Centro Hospitalar Universitário de São João (CHUSJ) in Porto, Portugal. Exclusion criteria were age under 18 years old, inability to give informed consent, history of infection in the last three months, and antibiotic intake in the last three months. The microbiome communities were analyzed by amplification and sequencing of the V3–V4 region of the bacterial 16S rRNA gene. Correlations with the patients’ clinical data and inflammatory profile were performed. Results: CKD-PD patients presented a unique urobiome profile dominated by Bacillota, Actinomycetota and Pseudomonadota and characterized by a lower Shannon diversity than fecal and blood microbiome. The taxonomic profiles of urogenital samples were organized in multiple subtypes dominated by populations of Lactobacillus, Staphylococcus, Streptococcus, Gardnerella, Prevotella, Escherichia-Shigella, being similar to other non-PD-CKD patients. Gender, sCD14, residual diuresis and history of peritonitis were significantly associated to variations in the urobiome. Although not reaching statistical significance, diabetes and the time on PD also showed association with particular taxonomic groups. Depletion of Gardnerella, Staphylococcus, Corynebacterium, Lactobacillus or Dermabacter populations correlated with CKD-PD patients with history of diabetes, history of peritonitis and altered levels of sCD14. Conclusions: Our results highlight urogenital microbiome as a potential partner and/or marker in the overall health state of CKD-PD patients. Resumen: Introducción y objetivos: Diabetes, dislipemia, edad avanzada, género, infecciones del tracto urinario y toma reciente de antibióticos, entre otras, se han asociado a una disminución en la riqueza del urobioma y a otras fluctuaciones de dicho microbioma.Recientemente, se han descrito alteraciones en losmicrobiomas intestinal y en sangreen pacientes con enfermedad renal crónica (ERC) y, específicamente, en pacientes en diálisis peritoneal (DP).A pesar de ello, aún no existen estudios que describan el microbioma urogenital en pacientes en DP. En el presente trabajo, caracterizamos el urobioma en 46 pacientes en DP. Pacientes y métodos: Se recogieron muestras de orina (micción espontánea), heces y sangre de 46 pacientes en DP del Centro HospitalarUniversitário de São João en Oporto, Portugal. Los criterios de exclusión fueron edad menor a 18 años, incapacidad para entenderel consentimiento informado, e historia de infección y toma de antibióticos en los últimos 3 meses. Las comunidades microbiológicas fueron analizadas por amplificación y secuenciación de las regiones V3-V4 del 16S rRNA bacteriano. Se realizaron correlaciones con los datos clínicos y el perfil inflamatorio de los pacientes. Resultados: Los pacientes en DP presentaron un urobioma único dominado por Bacillota, Actinomycetota yPseudomonadota, y caracterizado por una menor diversidad de Shannon que los microbiomas en sangre e intestinal. Los perfiles taxonómicos de las muestras urogenitales se organizaron en múltiples subtipos dominados por poblaciones de Lactobacillus, Staphylococcus, Streptococcus, Gardnerella, Prevotella, Escherichia-Shigella, siendo similar al descrito para otros pacientes con ERC no en DP.Género, factor sCD14, diuresis residual yantecedentes de peritonitis se asociaron de forma significativa a cambios en el urobioma. Diabetes y tiempo en DP también se asociaron con grupos taxonómicos específicos, aunque no de forma estadísticamente significativa. La deplección de Gardnerella, Staphylococcus, Corynebacterium, Lactobacillus o Dermabacterse correlacionó con antecedentes de diabetes, historia de peritonitis y niveles alterados de sCD14. Conclusiones: Nuestros resultados subrayan la importancia del microbioma urogenital como potencial biomarcadordel estado de salud de nuestros pacientes en DP

Vertical Transmission and Antifungal Susceptibility Profile of Yeast Isolates from the Oral Cavity, Gut, and Breastmilk of Mother–Child Pairs in Early Life

Yeast acquisition begins at birth; however, the contribution of the mother on yeast transmission to the offspring and associated resistance is yet to be clarified. The aim of this study was to explore the vertical transmission of yeasts and their antifungal susceptibility profile in early life. Oral, fecal, and breastmilk samples were collected from 73 mother–child pairs four to twelve weeks after delivery and cultured on Sabouraud dextrose agar with chloramphenicol. The isolates were identified by MALDI-TOF MS. The vertical transmission was studied by microsatellite genotyping. Antifungal susceptibility was determined for fluconazole, voriconazole, miconazole, anidulafungin, and nystatin by broth microdilution assay, following CLSI–M60 guidelines. A total of 129 isolates were identified from 53% mother–child pairs. We verified the vertical transmission of Candida albicans (n = three mother–child pairs) and Candida parapsilosis (n = one mother–child pair) strains, including an antifungal resistant strain transmitted from breastmilk to the gut of a child. Most isolates were susceptible to the tested antifungals, with the exception of four C. albicans isolates and one R. mucilaginosa isolate. The vertical transmission of yeasts happens in early life. This is the first work that demonstrated the role of the mother as a source of transmission of antifungal-resistant yeasts to the child