María Isabel Queipo-Ortuño

Gut microbiota in children with type 1 diabetes differs from that in healthy children: a case-control study

BackgroundA recent study using a rat model found significant differences at the time of diabetes onset in the bacterial communities responsible for type 1 diabetes modulation. We hypothesized that type 1 diabetes in humans could also be linked to a specific gut microbiota. Our aim was to quantify and evaluate the difference in the composition of gut microbiota between children with type 1 diabetes and healthy children and to determine the possible relationship of the gut microbiota of children with type 1 diabetes with the glycemic level.MethodsA case-control study was carried out with 16 children with type 1 diabetes and 16 healthy children. The fecal bacteria composition was investigated by polymerase chain reaction-denaturing gradient gel electrophoresis and real-time quantitative polymerase chain reaction.ResultsThe mean similarity index was 47.39% for the healthy children and 37.56% for the children with diabetes, whereas the intergroup similarity index was 26.69%. In the children with diabetes, the bacterial number of Actinobacteria and Firmicutes, and the Firmicutes to Bacteroidetes ratio were all significantly decreased, with the quantity of Bacteroidetes significantly increased with respect to healthy children. At the genus level, we found a significant increase in the number of Clostridium, Bacteroides and Veillonella and a significant decrease in the number of Lactobacillus, Bifidobacterium, Blautia coccoides/Eubacterium rectale group and Prevotella in the children with diabetes. We also found that the number of Bifidobacterium and Lactobacillus, and the Firmicutes to Bacteroidetes ratio correlated negatively and significantly with the plasma glucose level while the quantity of Clostridium correlated positively and significantly with the plasma glucose level in the diabetes group.ConclusionsThis is the first study showing that type 1 diabetes is associated with compositional changes in gut microbiota. The significant differences in the number of Bifidobacterium, Lactobacillus and Clostridium and in the Firmicutes to Bacteroidetes ratio observed between the two groups could be related to the glycemic level in the group with diabetes. Moreover, the quantity of bacteria essential to maintain gut integrity was significantly lower in the children with diabetes than the healthy children. These findings could be useful for developing strategies to control the development of type 1 diabetes by modifying the gut microbiota.

Influence of red wine polyphenols and ethanol on the gut microbiota ecology and biochemical biomarkers

BACKGROUND Few studies have investigated the effect of dietary polyphenols on the complex human gut microbiota, and they focused mainly on single polyphenol molecules and select bacterial populations. OBJECTIVE The objective was to evaluate the effect of a moderate intake of red wine polyphenols on select gut microbial groups implicated in host health benefits. DESIGN Ten healthy male volunteers underwent a randomized, crossover, controlled intervention study. After a washout period, all of the subjects received red wine, the equivalent amount of de-alcoholized red wine, or gin for 20 d each. Total fecal DNA was submitted to polymerase chain reaction(PCR)-denaturing gradient gel electrophoresis and real-time quantitative PCR to monitor and quantify changes in fecal microbiota. Several biochemical markers were measured. RESULTS The dominant bacterial composition did not remain constant over the different intake periods. Compared with baseline, the daily consumption of red wine polyphenol for 4 wk significantly increased the number of Enterococcus, Prevotella, Bacteroides, Bifidobacterium, Bacteroides uniformis, Eggerthella lenta, and Blautia coccoides-Eubacterium rectale groups (P < 0.05). In parallel, systolic and diastolic blood pressures and triglyceride, total cholesterol, HDL cholesterol, and C-reactive protein concentrations decreased significantly (P < 0.05). Moreover, changes in cholesterol and C-reactive protein concentrations were linked to changes in the bifidobacteria number. CONCLUSION This study showed that red wine consumption can significantly modulate the growth of select gut microbiota in humans, which suggests possible prebiotic benefits associated with the inclusion of red wine polyphenols in the diet. This trial was registered at controlled-trials.com as ISRCTN88720134.

Gut Microbiota Composition in Male Rat Models under Different Nutritional Status and Physical Activity and Its Association with Serum Leptin and Ghrelin Levels

Background Several evidences indicate that gut microbiota is involved in the control of host energy metabolism. Objective To evaluate the differences in the composition of gut microbiota in rat models under different nutritional status and physical activity and to identify their associations with serum leptin and ghrelin levels. Methods In a case control study, forty male rats were randomly assigned to one of these four experimental groups: ABA group with food restriction and free access to exercise; control ABA group with food restriction and no access to exercise; exercise group with free access to exercise and feed ad libitum and ad libitum group without access to exercise and feed ad libitum. The fecal bacteria composition was investigated by PCR-denaturing gradient gel electrophoresis and real-time qPCR. Results In restricted eaters, we have found a significant increase in the number of Proteobacteria, Bacteroides, Clostridium, Enterococcus, Prevotella and M. smithii and a significant decrease in the quantities of Actinobacteria, Firmicutes, Bacteroidetes, B. coccoides-E. rectale group, Lactobacillus and Bifidobacterium with respect to unrestricted eaters. Moreover, a significant increase in the number of Lactobacillus, Bifidobacterium and B. coccoides–E. rectale group was observed in exercise group with respect to the rest of groups. We also found a significant positive correlation between the quantity of Bifidobacterium and Lactobacillus and serum leptin levels, and a significant and negative correlation among the number of Clostridium, Bacteroides and Prevotella and serum leptin levels in all experimental groups. Furthermore, serum ghrelin levels were negatively correlated with the quantity of Bifidobacterium, Lactobacillus and B. coccoides–Eubacterium rectale group and positively correlated with the number of Bacteroides and Prevotella. Conclusions Nutritional status and physical activity alter gut microbiota composition affecting the diversity and similarity. This study highlights the associations between gut microbiota and appetite-regulating hormones that may be important in terms of satiety and host metabolism.

Impact of the gut microbiota on the development of obesity and type 2 diabetes mellitus

Obesity and its associated disorders are a major public health concern. Although obesity has been mainly related with perturbations of the balance between food intake and energy expenditure, other factors must nevertheless be considered. Recent insight suggests that an altered composition and diversity of gut microbiota could play an important role in the development of metabolic disorders. This review discusses research aimed at understanding the role of gut microbiota in the pathogenesis of obesity and type 2 diabetes mellitus (TDM2). The establishment of gut microbiota is dependent on the type of birth. With effect from this point, gut microbiota remain quite stable, although changes take place between birth and adulthood due to external influences, such as diet, disease and environment. Understand these changes is important to predict diseases and develop therapies. A new theory suggests that gut microbiota contribute to the regulation of energy homeostasis, provoking the development of an impairment in energy homeostasis and causing metabolic diseases, such as insulin resistance or TDM2. The metabolic endotoxemia, modifications in the secretion of incretins and butyrate production might explain the influence of the microbiota in these diseases.

Diagnostic Yield of a PCR Assay in Focal Complications of Brucellosis

ABSTRACT In order to evaluate the diagnostic yield of a PCR assay for patients with focal complications of brucellosis, we studied by PCR and by conventional microbiological techniques 34 nonblood samples from 32 patients with different focal forms of brucellosis. The samples from patients with brucellosis were paired to an equal number of control samples from the same locations of patients whose illnesses had different etiologies. Thirty-three of the 34 nonblood samples (97%) from the brucellosis patients were positive by PCR, whereasBrucella spp. were isolated from only 29.4% of the conventional cultures. For 11.4% of the patients, the confirmatory serological tests were either negative or showed titers below the diagnostic range. Two patients (6.2%) from the control group, both with tuberculous vertebral osteomyelitis, had a positive PCR result. The brucella PCR of blood from these two patients was also positive, and the two strains of Mycobacterium tuberculosisisolated were analyzed by the brucella PCR, with no evidence of amplification. These results show that the PCR assay is far more sensitive than conventional cultures, and this, coupled with its speed and reduction in risk to laboratory workers, makes this technique a very useful tool for the diagnosis of focal complications of brucellosis.

Chronic hepatosplenic abscesses in brucellosis. Clinico-therapeutic features and molecular diagnostic approach ☆

In order to analyze the clinical and therapeutic features of chronic hepatosplenic abscesses, and to define the diagnostic yield of new molecular techniques, we describe seven cases, four hepatic and three splenic, of this uncommon complication of Brucellosis. Onset of symptoms in all cases was insidious and the diagnostic delay considerable. Abdominal CT scan showed large, poorly defined lesions, with heterogeneous attenuation and thick central calcifications surrounded by hypointense areas. Histologically, all cases presented granulomas with central necrosis, a polymorphic infiltrate, few giant cells and peripheral fibrosis. The diagnostic yield with conventional microbiologic techniques was poor, whereas a Brucella PCR-assay of a tissue or pus sample was positive in all six cases in which it was performed. Conservative therapy with antibiotics, either alone or combined with percutaneous drainage, failed in all cases, so that in this type of lesion, the treatment of choice should be medical-surgical, in order to guarantee excision of the central calcium nucleus responsible for the persistence of the infection.

Preparation of Bacterial DNA Template by Boiling and Effect of Immunoglobulin G as an Inhibitor in Real-Time PCR for Serum Samples from Patients with Brucellosis

ABSTRACT Real-time PCR is a widely used tool for the diagnosis of many infectious diseases. However, little information exists about the influences of the different factors involved in PCR on the amplification efficiency. The aim of this study was to analyze the effect of boiling as the DNA preparation method on the efficiency of the amplification process of real-time PCR for the diagnosis of human brucellosis with serum samples. Serum samples from 10 brucellosis patients were analyzed by a SYBR green I LightCycler-based real-time PCR and by using boiling to obtain the DNA. DNA prepared by boiling lysis of the bacteria isolated from serum did not prevent the presence of inhibitors, such as immunoglobulin G (IgG), which were extracted with the template DNA. To identify and confirm the presence of IgG, serum was precipitated to separate and concentrate the IgG and was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The use of serum volumes above 0.6 ml completely inhibited the amplification process. The inhibitory effect of IgG in serum samples was not concentration dependent, and it could be eliminated by diluting the samples 1/10 and 1/20 in water. Despite the lack of the complete elimination of the IgG from the template DNA, boiling does not require any special equipment and it provides a rapid, reproducible, and cost-effective method for the preparation of DNA from serum samples for the diagnosis of brucellosis.

Endotoxin increase after fat overload is related to postprandial hypertriglyceridemia in morbidly obese patients

The low-grade inflammation observed in obesity has been associated with a high-fat diet, though this relation is not fully understood. Bacterial endotoxin, produced by gut microbiota, may be the linking factor. However, this has not been confirmed in obese patients. To study the relationship between a high-fat diet and bacterial endotoxin, we analyzed postprandial endotoxemia in morbidly obese patients after a fat overload. The endotoxin levels were determined in serum and the chylomicron fraction at baseline and 3 h after a fat overload in 40 morbidly obese patients and their levels related with the degree of insulin resistance and postprandial hypertriglyceridemia. The morbidly obese patients with the highest postprandial hypertriglyceridemia showed a significant increase in lipopolysaccharide (LPS) levels in serum and the chylomicron fraction after the fat overload. Postprandial chylomicron LPS levels correlated positively with the difference between postprandial triglycerides and baseline triglycerides. There were no significant correlations between C-reactive protein (CRP) and LPS levels. The main variables contributing to serum LPS levels after fat overload were baseline and postprandial triglyceride levels but not glucose or insulin resistance. Additionally, superoxide dismutase activity decreased significantly after the fat overload. Postprandial LPS increase after a fat overload is related to postprandial hypertriglyceridemia but not to degree of insulin resistance in morbidly obese patients.

Clinical Findings, Therapeutic Approach, and Outcome of Brucellar Vertebral Osteomyelitis

BACKGROUND Osteoarticular complications are the most common focal complications of brucellosis. Although vertebral osteomyelitis is the most frequent location in adults >30 years of age, little information is available about this serious complication of brucellosis, and great confusion surrounds its prognosis and the most appropriate treatment. METHODS We undertook a descriptive, retrospective, observational study of 96 patients who received a diagnosis of brucella vertebral osteomyelitis from September 1982 through December 2005 at a tertiary care hospital. All of the patients were treated for 3 months, after which they were followed up monthly for the first 3 months and then at 2-month intervals for the subsequent 6 months. RESULTS The incidence of vertebral osteomyelitis was 10.4%. The mean diagnostic delay was 12.7 weeks. Inflammatory spinal pain (occurring in 94.8% of patients) and fever (91.7%) were the most relevant clinical characteristics. Eight patients (8.3%) had motor weakness or paralysis. Paravertebral masses, epidural masses, and psoas abscesses were detected in 45.8%, 27.1%, and 10.4% of patients, respectively. Sixty-three patients (65.6%) received medication only, and 33 (34.4%) required surgical therapy in addition to medication. Twenty percent of patients experienced therapeutic failure. Attributable mortality was 2.1%, and severe functional sequelae were apparent in 6.2% of the patients. No significant differences were seen between patients who were treated with doxycycline-streptomycin and those treated with doxycycline-rifampicin. CONCLUSIONS Vertebral osteomyelitis is a serious complication of brucellosis. It generates a high rate of therapeutic failure and functional sequelae. In the absence of more-powerful controlled studies, the duration of treatment of brucellar vertebral osteomyelitis should be 3 months.

Development and Evaluation of a PCR-Enzyme-Linked Immunosorbent Assay for Diagnosis of Human Brucellosis

ABSTRACT In order to overcome some of the limitations of conventional microbiological techniques in the diagnosis of human brucellosis, a simple PCR-enzyme-linked immunosorbent assay (PCR-ELISA) was developed. After amplification of a 223-bp sequence of a gene that codes for the synthesis of an immunogenetic membrane protein specific for the Brucella genus (BCSP31), the digoxigenin-labeled amplified product was hybridized with a biotinylated capture probe which was complementary to the inner part of the amplicon. The hybrid was captured on streptavidin-coated microtiter plates and detected by using an antidigoxigenin Fab-peroxidase conjugate. The detection limit of the PCR-ELISA in a background of 3.5 μg of human genomic DNA was 10 fg (two bacterial cells). The PCR-ELISA showed an analytical sensitivity higher than that of ethidium bromide staining and equal to that obtained by conventional PCR followed by dot blot hybridization. In 59 peripheral blood samples from 57 consecutive patients with active brucellosis and 113 control samples, the PCR-ELISA was found to be 94.9% sensitive and 96.5% specific, whereas the sensitivity of the blood culture was only 70.1%. Since the assay can be performed in 1 day, is very reproducible, is easily standardized, and avoids the risk of infection in laboratory workers, this PCR-ELISA seems to be a practical and reliable tool for the diagnosis of human brucellosis.