Effects of Age and Region on Fecal Microflora in Elderly Subjects Living in Bama,Guangxi, China

Intestinal microflora analysis was performed on 52 healthy elderly subjects of different ages and in different regions in Bama County, Guangxi, China. The participants were assigned to three groups depending on their age and location: longevous (group M; mean age = 98 years; n = 21); rural younger elderly (group S; mean age = 70 years; n = 18); and urban elderly (group C; mean age = 82 years; n = 13). Ten groups of bacteria were quantified using real-time PCR. Age-related differences were observed in the number of Clostridium coccoides–Eubacterium rectale—there were more in longevous participants. Region affected the numbers of Bacteroides—Prevotella and Clostridium perfringens subgroup, and longevous participants had significantly more of the two bacterial groups than urban elderly participants. Region-related effects were also observed for the relative abundance of E. coli, and rural elderly participants had a lower proportion. Both age and regional effects were observed in the amount of total bacteria, and longevous participants had higher numbers than urban elderly participants. A significantly higher proportion of lactobacilli was observed in rural younger elderly participants than urban elderly participants, but independent age or regional effects did not contribute to this difference. This study suggests that age and region can affect the intestinal microflora of elderly people.     

Studies of Intestinal Microflora VI. Effect of X Irradiation on the Fecal Microflora of the Rat

The effect of sublethal, midlethal, and lethal doses of total-body X irradiation on the fecal microbial population of the rat was studied. No changes were observed in animals receiving sublethal doses of X ray. Midlethal and lethal doses produced an increase in the numbers of fecal coliforms, staphylococci, streptococci, and fungi; these changes were transient in rats that survived, but were more marked and persisted during life in those that died. The possible role of these alterations in intestinal microflora in post-irradiation infection and in death is discussed.     

Fecal Calprotectin in Healthy Children Aged 1-4 Years

Objective

Calprotectin has been well emulated recently in adults as well as in children. The aim of this study was to assess fecal calprotectin concentrations in healthy children aged from 1 to 4 years.

Methods

Volunteers were enlisted from 3 nurseries. A brief questionnaire was used to ensure these children meet the inclusion criteria, and some clinical and sociodemographic factors were collected. Anthro software (version 3.1) was used to calculated Length-for-age Z-scores (LAZ), weight-for-age Z-scores (WAZ), and weight-for-length Z-scores (WLZ) respectively. Fecal calprotectin was detected by a commercially available ELISA.

Results

In total 274 children were recruited, with age ranging from 1 to 4 years old. The median FC concentration was 83.19 μg/g [range 4.58 to 702.50 μg/g, interquartile range (IQR) 14.69–419.45 μg/g] or 1.92 log10 μg/g (range 0.66 log10 to 2.85 log10 μg/g, IQR 1.17 log10-2.62 log10 μg/g). All of the children were divided into three groups, 1–2 years (12–24 months), 2–3 years (24–36 months), 3–4 years (36–48 months), with median FC concentrations 96.14 μg/g (1.98 log10 μg/g), 81.48 μg/g (1.91 log10 μg/g), 65.36 μg/g (1.82 log10 μg/g), respectively. There was similar FC level between boys and girls. FC concentrations showed a downward trend by the growing age groups. A statistic difference was found in FC concentrations among groups 1–2 years, 2–3 years and 3–4 years (P = 0.016). In inter-groups comparison, a significant difference was found between children aged 1–2 years and children aged 3–4 years (P = 0.007). A negative correlation trend was found between age and FC concentration (Spearman''s rho = -0.167, P = 0.005) in all the participants. A simple correlation was performed among WLZ, WAZ, birth weight, or birth length with FC, and there was no correlation being observed.

Conclusion

Children aged from 1 to 4 years old have lower FC concentrations compared with healthy infants (<1years), and higher FC concentrations when comparing with children older than 4 years and adults.     

Fecal Calprotectin Concentrations in Healthy Children Aged 1-18 Months

Objective

Fecal calprotectin (FC) is an established biomarker of gut inflammation. The aim of this study was to evaluate FC concentrations in healthy children between 1 and 18 months of age.

Methods

Healthy children aged 1-18 months were enrolled in this study at the Department of Children''s Health Care in Shanghai, China. Children’s stool samples were collected and analyzed, and FC concentration was determined using a commercially available enzyme-linked immunosorbent assay (ELISA). The children''s weights and lengths were measured. Parents were asked to complete a brief questionnaire regarding several clinical and sociodemographic factors.

Results

The FC concentrations were unevenly distributed; the median FC concentration was 174.3 μg/g (range: 6.0-1097.7 μg/g) or 2.241 log10 μg/g (range: 0.775-3.041 log10 μg/g) for all 288 children. The children were divided into several age groups: 1-3 months, 3-6 months, 6-9 months, 9-12 months and 12-18 months. The median FC concentrations for these age groups were 375.2 μg/g (2.574 log10 μg/g), 217.9 μg/g (2.338 log10 μg/g), 127.7 μg/g (2.106 log10 μg/g), 96.1 μg/g (1.983 log10 μg/g) and 104.2 μg/g (2.016 log10 μg/g), respectively. A significant correlation between age and FC concentration was found (r=-0.490, p<0.001). A simple correlation analysis of weight-for-length Z-scores or weight-for-age Z-scores vs. FC concentrations showed that these variables were negatively correlated (Spearman’s rho=-0.287, p<0.001; Spearman’s rho=-0.243, p<0.001, respectively).

Conclusions

The FC levels of children aged 1-18 months exhibit a downward trend with increasing age and are greater than the normal levels observed in healthy adults. In healthy children aged <6 months, FC levels are high. In children aged 6-18 months, FC concentrations are relatively low but are still higher than those of children aged >4 years.     

Microflora and Invert Sugars in Juice from Healthy Tissue of Stored Sugarbeets

Bacterial populations increased in juice of healthy tissue of sugarbeet roots stored at 5 C. Average counts showed a sixfold increase after 150 days of storage. Invert sugar levels increased over threefold in “American 4 Hybrid A” and remained fairly constant in “Mono-Hy D-2.” The former cultivar also had significantly higher bacterial colony counts than the latter before 90 days of storage. Of 36 isolates identified, 16 were Pseudomonas spp. including P. chlororaphis; 6 Bacillus spp. including B. subtilis; 5 Arthrobacter spp. including A. globiformis; 4 yeasts; 2 Erwinia spp.; 2 Flavobacterium spp. including F. aquatile; and Streptomyces longisporus. Isolates of all genera except S. longisporus were able to hydrolyze sucrose in vitro.     

Analysis of the Fecal Microflora of Human Subjects Consuming a Probiotic Product Containing Lactobacillus rhamnosus DR20

The composition of the fecal microflora of 10 healthy subjects was monitored before (6-month control period), during (6-month test period), and after (3-month posttest period) the administration of a milk product containing Lactobacillus rhamnosus DR20 (daily dose, 1.6 × 10⁹ lactobacilli). Monthly fecal samples were examined by a variety of methods, including bacteriological culture analysis, fluorescent in situ hybridization with group-specific DNA probes, denaturing gradient gel electrophoresis of the V2-V3 region of 16S rRNA genes amplified by PCR, gas-liquid chromatography, and bacterial enzyme activity analysis. The composition of the Lactobacillus population of each subject was analyzed by pulsed-field gel electrophoresis of bacterial DNA digests in order to differentiate between DR20 and other strains present in the samples. Representative isolates of lactobacilli were identified to the species level by sequencing the V2-V3 region of their 16S rRNA genes and comparing the sequences obtained (BLAST search) to sequences in the GenBank database. DR20 was detected in the feces of all of the subjects during the test period, but at different frequencies. The presence of DR20 among the numerically predominant strains was related to the presence or absence of a stable indigenous population of lactobacilli during the control period. Strain DR20 did not persist at levels of >10² cells per g in the feces of most of the subjects after consumption of the product ceased; the only exception was one subject in which this strain was detected for 2 months during the posttest period. We concluded that consumption of the DR20-containing milk product transiently altered the Lactobacillus and enterococcal contents of the feces of the majority of consumers without markedly affecting biochemical or other bacteriological factors.     

Shiga Toxin 2-Encoding Bacteriophages in Human Fecal Samples from Healthy Individuals

Shiga toxin-converting bacteriophages (Stx phages) carry the stx gene and convert nonpathogenic bacterial strains into Shiga toxin-producing bacteria. Previous studies have shown that high densities of free and infectious Stx phages are found in environments polluted with feces and also in food samples. Taken together, these two findings suggest that Stx phages could be excreted through feces, but this has not been tested to date. In this study, we purified Stx phages from 100 fecal samples from 100 healthy individuals showing no enteric symptoms. The phages retrieved from each sample were then quantified by quantitative PCR (qPCR). In total, 62% of the samples carried Stx phages, with an average value of 2.6 × 10⁴ Stx phages/g. This result confirms the excretion of free Stx phages by healthy humans. Moreover, the Stx phages from feces were able to propagate in enrichment cultures of stx-negative Escherichia coli (strains C600 and O157:H7) and in Shigella sonnei, indicating that at least a fraction of the Stx phages present were infective. Plaque blot hybridization revealed lysis by Stx phages from feces. Our results confirm the presence of infectious free Stx phages in feces from healthy persons, possibly explaining the environmental prevalence observed in previous studies. It cannot be ruled out, therefore, that some positive stx results obtained during the molecular diagnosis of Shiga toxin-producing Escherichia coli (STEC)-related diseases using stool samples are due to the presence of Stx phages.     

Relation between Serum Cholesterol and Triglyceride Concentration and Haemoglobin Values in Non-anaemic Healthy Persons

In a study of 2,458 healthy non-anaemic subjects a positive correlation was found between haemoglobin levels and serum cholesterol and triglyceride levels. This may be due to simple changes in plasma volume, which may increase when the haemoglobin concentration decreases. Such an association between plasma lipid levels and haemoglobin values might be of importance in the aetiology of coronary heart disease, as a high haemoglobin value is known to be a “risk factor” in myocardial infarction.     

Fecal Bacterial Microbiota of Healthy Free-Ranging,Healthy Corralled,and Chronic Diarrheic Corralled Rhesus Macaques (Macaca mulatta)

A clinical challenge to nearly every primate facility in North America is chronic idiopathic diarrhea (CID), the pathogenesis of which has yet to be fully elucidated. However, wild macaques appear resistant to CID, a trend that we observed in the free-ranging population of the Caribbean Primate Research Center. The gastrointestinal microbiota has been shown to have a significant role in the pathogenesis of disease and in maintaining normal health and development of the gut. In humans, chronic diarrhea is associated with alteration of the gut microbiota, which has lower bacterial diversity than does the microbiota of healthy humans. The current study was designed to describe and compare the fecal bacterial microbiota of healthy corralled, CID corralled, and healthy, free-ranging macaques. Fresh fecal samples were collected from healthy corralled (HC; n = 30) and CID (n = 27) rhesus macaques and from healthy macaques from our free-ranging colony (HF; n = 43). We excluded macaques that had received antibiotics during the preceding 60 d (90 d for healthy animals). Bacterial DNA was extracted, and the V4 region of the 16S rRNA gene was sequenced and compared with known databases. The relative abundance of Proteobacteria was higher in CID animals than HC animals, but otherwise few differences were found between these 2 groups. HF macaques were differentially enriched with Christensenellaceae and Helicobacter, which are highly associated with a ‘healthy’ gut in humans, as compared to corralled animals, whereas CID animals were enriched with Proteobacteria, which are associated with dysbiosis in other species. These results indicate that environment has a greater influence than health status on the gut microbiota. Furthermore, the current data provided targets for future studies on potential clinical interventions, such as probiotics and fecal transplants.

Chronic idiopathic diarrhea (CID; also called idiopathic chronic diarrhea and chronic enterocolitis) is a clinical challenge that plagues nearly every large primate facility in North America. For example, the Oregon National Primate Center reports that CID comprises nearly 30% of their clinical caseload.²⁰ At the Caribbean Primate Research Center, a review of the medical records database at the Sabana Seca Field Station (SSFS), where animals are housed in large, outdoor corrals, indicates that treatment for diarrhea comprises nearly 50% of the clinical caseload.Information on CID in wild macaques is sparse, and an exact cause for CID in research macaques has not been identified, despite extensive study. Fecal bacterial culture has yielded mixed results, with no specific pathogen consistently isolated from animals with CID. An increased prevalence of Campylobacter, Shigella, and Yersinia species in animals with chronic diarrhea compared with healthy animals has been reported.⁵⁹ However, the overall prevalence in diarrheic animals was around 25% for Campylobacter and well below 25% for Shigella and Yersinia.⁵⁹ Similarly, one study reported that approximately 30% of chronic diarrheic animals had at least one historic bout of diarrhea that was culture positive and 40% culture positive for Campylobacter at the time of necropsy.³⁸ Others have reported that fecal cultures are regularly negative for these and other common gastrointestinal pathogen,^28,38 which is consistent with our experience.The collective, interacting genomes of the symbiotic microorganisms in the gastrointestinal tract are referred to as the gastrointestinal microbiome.³⁴ The microbiome has a significant role in the pathogenesis of disease and contributes to normal health and development of the gut.^19,67 In humans, chronic diarrhea due to Clostridium difficile infection is associated with alteration of the gut microbiota (also known as dysbiosis), which has lower bacterial diversity than does the microbiota of healthy humans. This finding led to the successful use of fecal bacterial transplantation to restore the flora to normal.^17,39 Similarly, our group identified significant differences in the bacterial microbiota and enrichment of Proteobacteria (a phylum associated with dysbiosis) in diarrheic calves and horses as compared with healthy ones.^3,23 We also reported that diarrheic calves had lower relative abundance of genes responsible for metabolism of various nutrients, indicating that nutrient availability can be altered in diarrheic states.²¹ A better understanding of the organisms present in the gut of healthy and diarrheic macaques may offer new insights into the pathogenesis of this condition, and lead to new approaches to prevent and treat CID in NHP.The current study was designed to describe and compare the fecal bacterial microbiota of healthy free-ranging, semiwild rhesus macaques (HF group), healthy macaques living in large, outdoor corrals (HC group), and corralled macaques with CID. The composition of the fecal bacterial microbiota from these 3 groups was compared to determine whether differences in bacterial composition are present among the groups. Identification of such changes may provide feasible starting points for studying the role of the intestinal microbiota in the pathophysiology of CID and possible treatment and preventive measures.     

Molecular Profiling of the Clostridium leptum Subgroup in Human Fecal Microflora by PCR-Denaturing Gradient Gel Electrophoresis and Clone Library Analysis

A group-specific PCR-based denaturing gradient gel electrophoresis (DGGE) method was developed and combined with group-specific clone library analysis to investigate the diversity of the Clostridium leptum subgroup in human feces. PCR products (length, 239 bp) were amplified using C. leptum cluster-specific primers and were well separated by DGGE. The DGGE patterns of fecal amplicons from 11 human individuals revealed host-specific profiles; the patterns for fecal samples collected from a child for 3 years demonstrated the structural succession of the population in the first 2 years and its stability in the third year. A clone library was constructed with 100 clones consisting of 1,143-bp inserts of 16S rRNA gene fragments that were amplified from one adult fecal DNA with one forward universal bacterial primer and one reverse group-specific primer. Eighty-six of the clones produced the 239-bp C. leptum cluster-specific amplicons, and the remaining 14 clones did not produce these amplicons but still phylogenetically belong to the subgroup. Sixty-four percent of the clones were related to Faecalibacterium prausnitzii (similarity, 97 to 99%), 6% were related to Subdoligranulum variabile (similarity, ~99%), 2% were related to butyrate-producing bacterium A2-207 (similarity, 99%), and 28% were not identified at the species level. The identities of most bands in the DGGE profiles for the same adult were determined by comigration analysis with the 86 clones that harbored the 239-bp group-specific fragments. Our results suggest that DGGE combined with clone library analysis is an effective technique for monitoring and analyzing the composition of this important population in the human gut flora.     

Winter Temperature as a Constraint to the Migration of Preindustrial Peoples

The dispersion of preindustrial peoples is strongly related to winter temperature in two surprising respects. Based on a classification of societies by language, we introduce a statistical model for their dispersion and show that (1) the 10°C (50°F) winter temperature isotherm has been an effective constraint to migration and expansion, and (2) the dispersion of language phyla has been remarkably homogeneous in a certain temperature scale, in contrast to the marked heterogeneity in terms of geographical dispersion , [climate, language, migration]     

Normalizing Electrocardiograms of Both Healthy Persons and Cardiovascular Disease Patients for Biometric Authentication

Although electrocardiogram (ECG) fluctuates over time and physical activity, some of its intrinsic measurements serve well as biometric features. Considering its constant availability and difficulty in being faked, the ECG signal is becoming a promising factor for biometric authentication. The majority of the currently available algorithms only work well on healthy participants. A novel normalization and interpolation algorithm is proposed to convert an ECG signal into multiple template cycles, which are comparable between any two ECGs, no matter the sampling rates or health status. The overall accuracies reach 100% and 90.11% for healthy participants and cardiovascular disease (CVD) patients, respectively.     

The Effect of Sampling and Storage on the Fecal Microbiota Composition in Healthy and Diseased Subjects

Large-scale cohort studies are currently being designed to investigate the human microbiome in health and disease. Adequate sampling strategies are required to limit bias due to shifts in microbial communities during sampling and storage. Therefore, we examined the impact of different sampling and storage conditions on the stability of fecal microbial communities in healthy and diseased subjects. Fecal samples from 10 healthy controls, 10 irritable bowel syndrome and 8 inflammatory bowel disease patients were collected on site, aliquoted immediately after defecation and stored at -80°C, -20°C for 1 week, at +4°C or room temperature for 24 hours. Fecal transport swabs (FecalSwab, Copan) were collected and stored for 48-72 hours at room temperature. We used pyrosequencing of the 16S gene to investigate the stability of microbial communities. Alpha diversity did not differ between all storage methods and -80°C, except for the fecal swabs. UPGMA clustering and principal coordinate analysis showed significant clustering by test subject (p<0.001) but not by storage method. Bray-Curtis dissimilarity and (un)weighted UniFrac showed a significant higher distance between fecal swabs and -80°C versus the other methods and -80°C samples (p<0.009). The relative abundance of Ruminococcus and Enterobacteriaceae did not differ between the storage methods versus -80°C, but was higher in fecal swabs (p<0.05). Storage up to 24 hours (at +4°C or room temperature) or freezing at -20°C did not significantly alter the fecal microbial community structure compared to direct freezing of samples from healthy subjects and patients with gastrointestinal disorders.     

Hemodynamics and Function of Resistance Arteries in Healthy Persons and End Stage Renal Disease Patients

Introduction

Cardiovascular disease is the leading cause of death in patients with end stage renal disease (ESRD). The vasodilator mechanisms in small resistance arteries are in earlier studies shown to be reduced in patients with end stage renal disease. We studied whether endothelium dependent vasodilatation were diminished in ESRD patients and the interaction between the macro- and microcirculation.

Methods

Eleven patients with ESRD had prior to renal transplant or insertion of peritoneal dialysis catheter measured pulse wave velocity. During surgery, a subcutaneous fat biopsy was extracted. Resistance arteries were then dissected and mounted on a wire myograph for measurements of dilator response to increasing concentrations of acetylcholine after preconstriction with noradrenaline. Twelve healthy kidney donors served as controls.

Results

Systolic blood pressure was elevated in patients compared to the healthy controls; no difference in the concentration of asymmetric dimethyl arginine was seen. No significant difference in the endothelium dependent vasodilatation between patients and controls was found. Correlation of small artery properties showed an inverse relationship between diastolic blood pressure and nitric oxide dependent vasodilatation in controls. Pulse pressure was positively correlated to the total endothelial vasodilatation in patients. A negative association between S-phosphate and endothelial derived hyperpolarisation-like vasodilatation was seen in resistance arteries from controls.

Conclusion

This study finds similar vasodilator properties in kidney patients and controls. However, correlations of pulse pressure and diastolic blood pressure with resistance artery function indicate compensating measures in the microcirculation during end stage renal disease.