Early Life Intestinal Microbiota in Health and in Atopic Eczema

Decrease in microbial contacts in affluent societies is considered to lie behind the rise in allergic and other chronic inflammatory diseases during the last decades. Indeed, deviations in the intestinal microbiota composition and diversity have been associated with several diseases, such as atopic eczema. However, there is no consensus yet on what would constitute a beneficial or harmful microbiota. The aim of this thesis was to study the microbiota development in healthy infants and to characterize intestinal microbiota signatures associated with disease status and severity in infants with atopic eczema. The methodological aim was to compare and optimize methods for DNA extraction from fecal samples to be used in high-throughput microbiota analyses. It was confirmed that the most critical step in successful microbial DNA extraction from fecal samples is the mechanical cell lysis procedure. Based on this finding, an efficient semi-automated extraction process was developed that can be scaled for use in high-throughput platforms such as phylogenetic microarray used in this series of studies. By analyzing a longitudinal motherchild cohort for 3 years it was observed that the microbiota development is a gradual process, where some bacterial groups reach the degree of adult-type pattern earlier than others. During the breast-feeding period, the microbiota appeared to be relatively simple, while major diversification was found to start during the weaning process. By the age of 3 years, the child’s microbiota composition started to resemble that of an adult, but the bacterial diversity has still not reached the full diversity, indicating that the microbiota maturation extends beyond this age. In addition, at three years of age, the child’s microbiota was more similar to mother’s microbiota than to microbiota of nonrelated women.In infants with atopic eczema, a high total microbiota diversity and abundance of butyrate-producing bacteria was found to correlate with mild symptoms at 6 months. At 18 months, infants with mild eczema had significantly higher microbiota diversity and aberrant microbiota composition when compared to healthy controls at the same age. In conclusion, the comprehensive phylogenetic microarray analysis of early life microbiota shows the synergetic effect of vertical transmission and shared environment on the intestinal microbiota development. By the age of three years, the compositional development of intestinal microbiota is close to adult level, but the microbiota diversification continues beyond this age. In addition, specific microbiota signatures are associated with the existence and severity of atopic eczema and intestinal microbiota seems to have a role in alleviating the symptoms of this disease.

Immunoglobulin production is impaired in protein-deprived mice and can be restored by dietary protein supplementation

Most contacts with food protein and microbiota antigens occur at the level of the gut mucosa. In animal models where this natural stimulation is absent, such as germ-free and antigen-free mice, the gut-associated lymphoid tissue (GALT) and systemic immunological activities are underdeveloped. We have shown that food proteins play a critical role in the full development of the immune system. C57BL/6 mice weaned to a diet in which intact proteins are replaced by equivalent amounts of amino acids (Aa diet) have a poorly developed GALT as well as low levels of serum immunoglobulins (total Ig, IgG, and IgA, but not IgM). In the present study, we evaluated whether the introduction of a protein-containing diet in 10 adult Aa-fed C57BL/6 mice could restore their immunoglobulin levels and whether this recovery was dependent on the amount of dietary protein. After the introduction of a casein-containing diet, Aa-fed mice presented a fast recovery (after 7 days) of secretory IgA (from 0.33 to 0.75 mg/mL, while in casein-fed mice this value was 0.81 mg/mL) and serum immunoglobulin levels (from 5.39 to 10.25 mg/mL of total Ig). Five percent dietary casein was enough to promote the restoration of secretory IgA and serum immunoglobulin levels to a normal range after 30 days feeding casein diet (as in casein-fed mice - 15% by weight of diet). These data suggest that the defect detected in the immunoglobulin levels was a reversible result of the absence of food proteins as an antigenic stimulus. They also indicate that the deleterious consequences of malnutrition at an early age for some immune functions may be restored by therapeutic intervention later in life.

Comparison of the effects of lactated Ringer solution with and without hydroxyethyl starch fluid resuscitation on gut edema during severe splanchnic ischemia

The type of fluid used during resuscitation may have an important impact on tissue edema. We evaluated the impact of two different regimens of fluid resuscitation on hemodynamics and on lung and intestinal edema during splanchnic hypoperfusion in rabbits. The study included 16 female New Zealand rabbits (2.9 to 3.3 kg body weight, aged 8 to 12 months) with splanchnic ischemia induced by ligation of the superior mesenteric artery. The animals were randomized into two experimental groups: group I (N = 9) received 12 mL·kg-1·h-1 lactated Ringer solution and 20 mL/kg 6% hydroxyethyl starch solution; group II (N = 7) received 36 mL·kg-1·h-1 lactated Ringer solution and 20 mL/kg 0.9% saline. A segment from the ileum was isolated to be perfused. A tonometric catheter was placed in a second gut segment. Superior mesenteric artery (Q SMA) and aortic (Qaorta) flows were measured using ultrasonic flow probes. After 4 h of fluid resuscitation, tissue specimens were immediately removed for estimations of gut and lung edema. There were no differences in global and regional perfusion variables, lung wet-to-dry weight ratios and oxygenation indices between groups. Gut wet-to-dry weight ratio was significantly lower in the crystalloid/colloid-treated group (4.9 ± 1.5) than in the crystalloid-treated group (7.3 ± 2.4) (P < 0.05). In this model of intestinal ischemia, fluid resuscitation with crystalloids caused more gut edema than a combination of crystalloids and colloids.

Gut permeability to lactulose and mannitol differs in treated Crohn's disease and celiac disease patients and healthy subjects

The gut barrier monitors and protects the gastrointestinal tract from challenges such as microorganisms, toxins and proteins that could act as antigens. There is evidence that gut barrier dysfunction may act as a primary disease mechanism in intestinal disorders. The aim of the present study was to evaluate the barrier function towards sugars after the appropriate treatment of celiac disease and Crohn's disease patients and compare the results with those obtained with healthy subjects. Fifteen healthy volunteers, 22 celiac disease patients after 1 year of a gluten-free diet, and 31 Crohn's disease patients in remission were submitted to an intestinal permeability test with 6.0 g lactulose and 3.0 g mannitol. Six-hour urinary lactulose excretion in Crohn's disease patients was significantly higher than in both celiac disease patients (0.42 vs 0.15%) and healthy controls (0.42 vs 0.07%). Urinary lactulose excretion was significantly higher in celiac disease patients than in healthy controls (0.15 vs 0.07%). Urinary mannitol excretion in Crohn's disease patients was the same as healthy controls (21 vs 21%) and these values were significantly higher than in celiac disease patients (10.9%). The lactulose/mannitol ratio was significantly higher in Crohn's disease patients in comparison to celiac disease patients (0.021 vs 0.013) and healthy controls (0.021 vs 0.003) and this ratio was also significantly higher in celiac disease patients compared to healthy controls (0.013 vs 0.003). In spite of treatment, differences in sugar permeability were observed in both disease groups. These differences in the behavior of the sugar probes probably reflect different mechanisms for the alterations of intestinal permeability.

Variação da microbiota natural e de pseudomonas aeruginosa em água mineral não carbonatada embalada em diferentes materiais durante o armazenamento a 30°c ± 1°c

Garrafas de PVC, polipropileno e vidro adequadamente limpas e sanificadas foram enxaguadas e enchidas com água mineral de uma fonte localizada no município de Lindóia-SP, e a seguir armazenadas a 30°C ± 1°C. Foram enumerados os microrganismos heterotróficos totais nos meios Ágar Padrão para Contagem e Ágar R2A imediatamente após engarrafamento. A variação da contaminação foi seguida através de exames periódicos. A variação da população de Pseudomonas aeruginosa foi estudada inoculando garrafas contendo água mineral com uma suspensão de P. aeruginosa ATCC 10145 de maneira a se obter uma contaminação de aproximadamente 10² UFC/ml. A população da bactéria foi avaliada periodicamente durante o tempo de armazenamento usando o meio Ágar Pseudomonas P. Houve aumento da população dos microrganismos heterotróficos totais nos primeiros 30 dias de armazenamento para depois diminuir de maneira irregular e ficar aproximadamente constante até completar 6 meses de observação. As contagens de P. aeruginosa aumentaram sensivelmente nas 2 primeiras semanas de estocagem diminuindo ligeiramente a seguir até atingir níveis próximos ao inicial. Não foram constatadas diferenças entre os 3 tipos de materiais de embalagem comparados.

A microbiota da carcaça e da carne ovina tratada com ácido acético, embalada a vácuo e maturada por 48 dias

Foi avaliada a carga de bactérias mesófilas e coliformes totais e fecais na superfície da carcaça ovina após 12 horas do abate. Posteriormente, a carne foi cortada em fatias de aproximadamente 3cm de espessura, imersas por um minuto em ácido acético 1% ou em água, embaladas individualmente a vácuo e armazenadas a 1°C. Nos dias 3,13, 23, 33 e 48 de maturação as carnes foram analisadas para bactérias mesófilas e psicrotróficas, mofos e leveduras, coliformes totais e fecais, salmonela e clostrídios sulfito-redutores. As contagens de bactérias mesófilas e psicrotróficas foram menores (p<0,05) nas carnes tratadas com ácido acético que nas não tratadas nos dias 13 e 23 e nos dias 3 e 13 de maturação, respectivamente. A contagem de mofos e leveduras foi menor (p<0,05), nas carnes tratadas em relação às não tratadas, apenas nos dias 13 e 23 de maturação. Após 3 dias de maturação as carnes tratadas mostraram menores (p<0,05) contagens de coliformes fecais e totais que as não tratadas. As carnes apresentaram presença de salmonela com 3, 13 e 23 dias de maturação mais não com 33 e 48. Não foi detectada presença de clostrídios sulfito-redutores durante todo o período de maturação. Pode-se concluir que o tratamento com ácido e as condições de maturação utilizadas neste experimento mantêm baixas contagens de microrganismos mesófilos, psicrotróficos, mofos e leveduras e coliformes por 13 dias. Durante este período, porém, inibição do crescimento de salmonelas somente foi detectada após 33 dias de maturação da carne ovina.

Microbiota contaminante em repolho minimamente processado

A microbiota contaminante de repolho minimamente processado foi avaliada durante as etapas de sanitização e estocagem sob atmosfera modificada passiva em embalagens com diferentes taxas de permeabilidade a O2 e CO2 e a 1ºC, 5ºC e 12ºC. A sanitização do repolho por 10min., à temperatura ambiente, em soluções sanitizantes de hipoclorito de sódio a 200mgL-1, de composto orgânico clorado a 200mgL-1 ou ácido acético a 1% reduziu em, no máximo, 1,8log10 UFCg-1 a população de microrganismos aeróbios mesófilos. A concentração de CO2 no interior das embalagens variou significativamente (P<0,05) ao longo de 15 dias de estocagem do repolho minimamente processado mantido tanto a 1ºC como a 5ºC. Nessas mesmas condições, não foi constatada variação na população de aeróbios e anaeróbios mesófilos e de psicrotróficos. Em avaliação subjetiva, o repolho minimamente processado apresentou-se em condições adequadas de consumo aos 20 dias de estocagem, a 1ºC e 5ºC, nas embalagens de filmes de alta permeabilidade ao O2. Quando acondicionado em bandejas plásticas transparentes, seladas com filme de PVC termoencolhível, o produto apresentou, no vigésimo dia, a 5ºC, características sensoriais indesejáveis. O repolho minimamente processado mantido a 12ºC apresentou sinais evidentes de deterioração após cinco dias, manifestados pelo aparecimento de manchas escuras, limosidade e odor desagradável, e teve um aumento de até 3log10 UFCg-1 na população inicial de aeróbios e anaeróbios mesófilos e de psicrotróficos.

Influência de frações da parede celular de levedura (Saccharomyces cerevisiae) sobre os índices séricos de glicose e lipídios, microbiota intestinal e produção de ácidos graxos voláteis (AGV) de cadeias curtas de ratos em crescimento

Os índices séricos de glicose e lipídios, a microbiota intestinal e a produção de ácidos graxos voláteis de cadeias curtas (AGV) foram determinados em ratos Wistar submetidos às dietas: padrão (AIN-P), padrão modificada (AIN-M) e às dietas contendo frações de parede celular de levedura: glicana insolúvel (GI), manana (M) e glicana mais manana (G+M), como única fonte de fibra alimentar. O fracionamento da parede celular (PC) foi realizado por processos físicos e químicos de extração, centrifugação e secagem em "spray dryer". Os índices séricos foram dosados através de "kits" comerciais. A microbiota e a produção de AGV foram determinadas nos conteúdos intestinais, incluindo cólon, ceco e reto. Considerando os níveis de colesterol no tempo (T0) e no tempo 28 (T28), as dietas AIN-P, AIN-M e M apresentaram efeito hipocolesterolêmico, tendo em vista que a composição das dietas eram de natureza hipercolesterolêmica. Em relação à glicose sérica, no tempo (T0) observou-se uma elevação geral da glicemia, sugerindo um efeito hiperglicêmico das dietas estudadas. A dieta G+M foi a que apresentou valores significantemente mais elevados de lipídios séricos no tempo T14, e os níveis mais baixos foram observados na dieta M e na dieta GI no T14 e nas dietas AIN-M e AIN-P. A dieta AIN-P foi a que apresentou valor significantemente mais elevado de triacilgliceróis nos tempos T14 e T28. Os níveis mais baixos nos tempos T14 foram constatados para as dietas G+M e GI e no tempo T28 para as dietas AIN-M e M. De um modo geral, não houve modificações significativas na microbiota intestinal dos animais em nenhuma das dietas. Dentre os AGV, o ácido acético foi o predominante, seguido do propiônico e do butírico, em todas as dietas estudadas.

Microbiota of sausages obtained by spontaneous fermentation produced in the South of Brazil

The study of the ecology of fermented sausage is fundamental to understand the physical and chemical changes that happen during fermentation and maturation. The aim of the present study was to determine the microbiological characteristics of sausages produced by spontaneous fermentation. Fifty samples of sausages produced in the South of Brazil by different small manufacturers were analyzed for the following microbiota: aerobic mesophilic bacteria; Micrococcaceae; mold and yeast; lactic acid bacteria; total and fecal coliforms; coagulase-positive Staphylococcus, and Salmonella. In most samples (72%), the count of lactic bacteria was higher than 6 log10 cfu.g-1, and the samples with the highest counts were above 8 log10 cfu.g-1. The counts of Micrococcaceae in most samples were between 5 log10 and 7 log10 cfu.g-1. With respect to the presence of molds and yeasts, there was a significant variation among the samples with counts ranging from 2 log10 cfu.g-1 and 6 log10 cfu.g-1. From the data obtained, it was possible to conclude that 24% of the analyzed samples did not comply with the current law in Brazil since the levels of fecal coliforms or coagulase-positive Staphylococcus exceeded the maximum limit allowed.

Frog meat microbiota (Lithobates catesbeianus) used in infant food

Captive breeding of bullfrog (Lithobates catesbeianus) is of great economic potential, mainly for its thighs and leather. The nutritional quality of frog meat includes properly balanced amino acids with a protein profile of high biological value, low fat and low cholesterol, and high digestibility due to its short chain molecule structure. It is recommended by doctors and nutritionists, especially for protein restricted children or malnourished children. Aiming to aggregate value to the segment and offer a product with nutritional properties that meet the need of children aged six months and above, a meat product based on the composition of frog meat was developed experimentally. To ensure raw material quality after bleaching and deboning, the microbiota present in the frog meat was determined. The analyses were performed according to Brazilian laws. It was observed that the resident and transient microbiota met the standards set by regulations. The results found were: mesophyll 4.5 x 10(4) CFU/g; Staphylococcus coagulase positive 2.0 x 10² CFU/g; negative for Salmonella sp. and Aeromonas spp. The findings indicate that the raw material showed satisfactory sanitation even in terms of family industry.

Use of starter cultures isolated from native microbiota of artisanal sausage in the production of Italian sausage

The most promising microorganisms for use as starter cultures are those isolated from the native microbiota of traditional fermented products. The aim of this study was to evaluate the use of lactic acid bacteria selected from the native microbiota of sausages produced by spontaneous fermentation as starter cultures for the production of sausage. Strains of Lactobacillus plantarum 503 and 341 have the potential for use as starter cultures in the manufacture of Italian sausage type. The population of lactic acid bacteria in sausages was >8 log CFU.g-1 during fermentation, which caused the pH to decrease to <4.5. This decrease in pH and the water activity of < 0.90 of sausages ensures the safety and preservation of this product. Sausages produced with these lactic cultures fulfill the requirements for microbiological quality and composition of Italian sausage type. Our results suggest the possibility of using these starter cultures for the production of sausages with peculiar characteristics that contribute to the identity of the product.

Extensive characterization of Campylobacter jejuni chicken isolates to uncover genes involved in the ability to compete for gut colonization

Background: Campylobacter jejuni is responsible for human foodborne enteritis. This bacterium is a remarkable colonizer of the chicken gut, with some strains outcompeting others for colonization. To better understand this phenomenon, the objective of this study was to extensively characterize the phenotypic performance of C. jejuni chicken strains and associate their gut colonizing ability with specific genes. Results: C. jejuni isolates (n = 45) previously analyzed for the presence of chicken colonization associated genes were further characterized for phenotypic properties influencing colonization: autoagglutination and chemotaxis as well as adhesion to and invasion of primary chicken caecal cells. This allowed strains to be ranked according to their in vitro performance. After their in vitro capacity to outcompete was demonstrated in vivo, strains were then typed by comparative genomic fingerprinting (CGF). In vitro phenotypical properties displayed a linear variability among the tested strains. Strains possessing higher scores for phenotypical properties were able to outcompete others during chicken colonization trials. When the gene content of strains was compared, some were associated with different phenotypical scores and thus with different outcompeting capacities. Use of CGF profiles showed an extensive genetic variability among the studied strains and suggested that the outcompeting capacity is not predictable by CGF profile. Conclusion: This study revealed a wide array of phenotypes present in C. jejuni strains, even though they were all recovered from chicken caecum. Each strain was classified according to its in vitro competitive potential and its capacity to compete for chicken gut colonization was associated with specific genes. This study also exposed the disparity existing between genetic typing and phenotypical behavior of C. jejuni strains.

Diversity and antimicrobial activity of Lactic Acid Bacteria from the gut of marine fish Rastrelliger kanagurta against fish, shrimp and human pathogens

Emergence of antibiotic resistance among aquaculture pathogens has made it necessary to look into environment friendly, effective and sustainable methods such as probiotic and immunostimulants among others.. In the present study, LAB were isolated from the gut of fish species namely Rastrelliger kanagurta and analyzed for their antibacterial activity against various fish, shrimp and human pathogens. Different LAB species such as Lactobacillus plantarum, L. bulgaricus, L. brevis and L. viridiscens were encountered in the gut of R. kanagurta. Several strains showed good activity against fish, shrimp and human pathogens. LAB from the gut of such marine species may be developed as possible probiont for environment friendly health management of fresh water, estuarine and marine species currently exploited in aquaculture

Gut Associated Lactic Acid Bacteria Isolated from the Estuarine Fish Mugil cephalus: Molecular Diversity and Antibacterial Activities against Pathogens

In the present study we address the issue on gut associated lactic acid bacteria (LAB) isolated from the intestine of estuarine fish Mugil cephalus using de Man Rogossa and Sharpe (MRS) agar. LAB isolates were identified biochemically and screened for their ability to inhibit in vitro growth of various fish, shrimp and human pathogens. Most of the LAB isolates displayed an improved antagonism against fish pathogens compared to shrimp and human pathogens. Selected representative strains displaying high antibacterial activity were identified using 16S rRNA gene sequence analysis. Of the selected strains Lactobacillus brevis was the most predominant. Four other species of Lactobacillus, Enterobacter hormaechei and Enterobacter ludwigii were also identified. It was also observed that even among same species, considerable diversity with respect to substrate utilization persisted. Considering the euryhaline nature of grey mullet (Mugil cephalus), the LAB isolated from the gut possessed good tolerance to varying salt concentrations. This finding merits further investigation to evaluate whether the isolated LAB could be used as probiotics in various fresh and sea water aquaculture