Paleomicrobiology: Revealing Fecal Microbiomes of Ancient Indigenous Cultures

Coprolites are fossilized feces that can be used to provide information on the composition of the intestinal microbiota and, as we show, possibly on diet. We analyzed human coprolites from the Huecoid and Saladoid cultures from a settlement on Vieques Island, Puerto Rico. While more is known about the Saladoid culture, it is believed that both societies co-existed on this island approximately from 5 to 1170 AD. By extracting DNA from the coprolites, followed by metagenomic characterization, we show that both cultures can be distinguished from each other on the basis of their bacterial and fungal gut microbiomes. In addition, we show that parasite loads were heavy and also culturally distinct. Huecoid coprolites were characterized by maize and Basidiomycetes sequences, suggesting that these were important components of their diet. Saladoid coprolite samples harbored sequences associated with fish parasites, suggesting that raw fish was a substantial component of their diet. The present study shows that ancient DNA is not entirely degraded in humid, tropical environments, and that dietary and/or host genetic differences in ancient populations may be reflected in the composition of their gut microbiome. This further supports the hypothesis that the two ancient cultures studied were distinct, and that they retained distinct technological/cultural differences during an extended period of close proximity and peaceful co-existence. The two populations seemed to form the later-day Taínos, the Amerindians present at the point of Columbian contact. Importantly, our data suggest that paleomicrobiomics can be a powerful tool to assess cultural differences between ancient populations.     

Effects of Orange Juice Formulation on Prebiotic Functionality Using an In Vitro Colonic Model System

A three-stage continuous fermentative colonic model system was used to monitor in vitro the effect of different orange juice formulations on prebiotic activity. Three different juices with and without Bimuno, a GOS mixture containing galactooligosaccharides (B-GOS) were assessed in terms of their ability to induce a bifidogenic microbiota. The recipe development was based on incorporating 2.75g B-GOS into a 250 ml serving of juice (65°Brix of concentrate juice). Alongside the production of B-GOS juice, a control juice – orange juice without any additional Bimuno and a positive control juice, containing all the components of Bimuno (glucose, galactose and lactose) in the same relative proportions with the exception of B-GOS were developed. Ion Exchange Chromotography analysis was used to test the maintenance of bimuno components after the production process. Data showed that sterilisation had no significant effect on concentration of B-GOS and simple sugars. The three juice formulations were digested under conditions resembling the gastric and small intestinal environments. Main bacterial groups of the faecal microbiota were evaluated throughout the colonic model study using 16S rRNA-based fluorescence in situ hybridization (FISH). Potential effects of supplementation of the juices on microbial metabolism were studied measuring short chain fatty acids (SCFAs) using gas chromatography. Furthermore, B-GOS juices showed positive modulations of the microbiota composition and metabolic activity. In particular, numbers of faecal bifidobacteria and lactobacilli were significantly higher when B-GOS juice was fermented compared to controls. Furthermore, fermentation of B-GOS juice resulted in an increase in Roseburia subcluster and concomitantly increased butyrate production, which is of potential benefit to the host. In conclusion, this study has shown B-GOS within orange juice can have a beneficial effect on the fecal microbiota.     

Comparison of Sewage and Animal Fecal Microbiomes by Using Oligotyping Reveals Potential Human Fecal Indicators in Multiple Taxonomic Groups

Most DNA-based microbial source tracking (MST) approaches target host-associated organisms within the order Bacteroidales, but the gut microbiota of humans and other animals contain organisms from an array of other taxonomic groups that might provide indicators of fecal pollution sources. To discern between human and nonhuman fecal sources, we compared the V6 regions of the 16S rRNA genes detected in fecal samples from six animal hosts to those found in sewage (as a proxy for humans). We focused on 10 abundant genera and used oligotyping, which can detect subtle differences between rRNA gene sequences from ecologically distinct organisms. Our analysis showed clear patterns of differential oligotype distributions between sewage and animal samples. Over 100 oligotypes of human origin occurred preferentially in sewage samples, and 99 human oligotypes were sewage specific. Sequences represented by the sewage-specific oligotypes can be used individually for development of PCR-based assays or together with the oligotypes preferentially associated with sewage to implement a signature-based approach. Analysis of sewage from Spain and Brazil showed that the sewage-specific oligotypes identified in U.S. sewage have the potential to be used as global alternative indicators of human fecal pollution. Environmental samples with evidence of prior human fecal contamination had consistent ratios of sewage signature oligotypes that corresponded to the trends observed for sewage. Our methodology represents a promising approach to identifying new bacterial taxa for MST applications and further highlights the potential of the family Lachnospiraceae to provide human-specific markers. In addition to source tracking applications, the patterns of the fine-scale population structure within fecal taxa suggest a fundamental relationship between bacteria and their hosts.     

Nondigestible Oligosaccharides Enhance Bacterial Colonization Resistance against Clostridium difficile In Vitro

Clostridium difficile is the principal etiologic agent of pseudomembranous colitis and is a major cause of nosocomial antibiotic-associated diarrhea. A limited degree of success in controlling C. difficile infection has been achieved by using probiotics; however, prebiotics can also be used to change bacterial community structure and metabolism in the large gut, although the effects of these carbohydrates on suppression of clostridial pathogens have not been well characterized. The aims of this study were to investigate the bifidogenicity of three nondigestible oligosaccharide (NDO) preparations in normal and antibiotic-treated fecal microbiotas in vitro and their abilities to increase barrier resistance against colonization by C. difficile by using cultural and molecular techniques. Fecal cultures from three healthy volunteers were challenged with a toxigenic strain of C. difficile, and molecular probes were used to monitor growth of the pathogen, together with growth of bifidobacterial and bacteroides populations, over a time course. Evidence of colonization resistance was assessed by determining viable bacterial counts, short-chain fatty acid formation, and cytotoxic activity. Chemostat studies were then performed to determine whether there was a direct correlation between bifidobacteria and C. difficile suppression. NDO were shown to stimulate bifidobacterial growth, and there were concomitant reductions in C. difficile populations. However, in the presence of clindamycin, activity against bifidobacteria was augmented in the presence of NDO, resulting in a further loss of colonization resistance. In the absence of clindamycin, NDO enhanced colonization resistance against C. difficile, although this could not be attributed to bifidobacterium-induced inhibitory phenomena.     

Fermentation of Fructooligosaccharides and Inulin by Bifidobacteria: a Comparative Study of Pure and Fecal Cultures

The utilization of fructooligosaccharides (FOS) and inulin by 55 Bifidobacterium strains was investigated. Whereas FOS were fermented by most strains, only eight grew when inulin was used as the carbon source. Residual carbohydrates were analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection after batch fermentation. A strain-dependent capability to degrade fructans of different lengths was observed. During batch fermentation on inulin, the short fructans disappeared first, and then the longer ones were gradually consumed. However, growth occurred through a single uninterrupted exponential phase without exhibiting polyauxic behavior in relation to the chain length. Cellular β-fructofuranosidases were found in all of the 21 Bifidobacterium strains tested. Four strains were tested for extracellular hydrolytic activity against fructans, and only the two strains which ferment inulin showed this activity. Batch cultures inoculated with human fecal slurries confirmed the bifidogenic effect of both FOS and inulin and indicated that other intestinal microbial groups also grow on these carbon sources. We observed that bifidobacteria grew by cross-feeding on mono- and oligosaccharides produced by primary inulin intestinal degraders, as evidenced by the high hydrolytic activity of fecal supernatants. FOS and inulin greatly affected the production of short-chain fatty acids in fecal cultures; butyrate was the major fermentation product on inulin, whereas mostly acetate and lactate were produced on FOS.     

Amodiaquine-induced Agranulocytosis: Toxic Effect of Amodiaquine in Bone Marrow Cultures In Vitro

A case of agranulocytosis is reported in which amodiaquine, to which the patient had been exposed, was found to be toxic to the patient''s bone marrow cells when these were cultured in an agar colony system in vitro. This technique should be investigated in other patients with agranulocytosis as a possible means of detecting toxic agents.     

In Vitro Fermentation of Breast Milk Oligosaccharides by Bifidobacterium infantis and Lactobacillus gasseri

It has been proposed that human milk oligosaccharides (HMO) function as a prebiotic for bifidobacteria, yet this activity has not been adequately investigated. In this study, Bifidobacterium infantis was shown to ferment purified HMO as a sole carbon source, while another gut commensal, Lactobacillus gasseri, did not ferment HMO. Our results support the hypothesis that HMO selectively amplify bacterial populations in the infant intestine.     

In Vitro Cultivation of Borrelia duttonii on Cultures of SflEp Cells

Borrelia duttonii strain 406 K, a causative agent of relapsing fever, could not be cultivated in vitro in currently available media for borreliae. We have developed an in vitro cultivation system by using SflEp cell cultures. The average increases of the number of borreliae, when inoculated with 1.0 × 10⁵ organisms per ml from infected mice, were 23-fold and 150-fold in the primary culture and the 3rd subculture, respectively. Even a single borrelia could propagate in this cultivation system. This system will be useful for immunological and physiological studies on uncultivable Borrelia strains.     

In Vitro Growth of Mycobacterium lepraemurium under the Influence of Macrophage Cultures

Elongation and limited multiplication of Mycobacterium lepraemurium was observed extracellularly when the bacilli spotted on a coverslip were placed face to face with cultures of mouse peritoneal macrophages adhering to the inside of a test tube held at an angle of 15°. There was no doubt that certain growth-promoting but unstable factors were released from the macrophages.     

Comparative In Vitro Study of Six Carbamazepine Products

The purpose of present study was to evaluate commercial preparations of carbamazepine tablets with respect to drug release through a defined sequence of experiments using Minitab software. The compliance of products with respect to United States Pharmacopeia (USP) dissolution test and comparison of the products with respect to drug release in different dissolution conditions is reported in the present paper. The different dissolution conditions studied include dissolution medium (1% SLS in purified water, 0.1 N HCl), volume (900 and 1,000 ml), rpm (50 rpm, 75 rpm). Studies indicated that all six products complied with USP dissolution criteria. However, the extent of influence of dissolution conditions on drug release was varied among the products. Distinct dissolution profiles were observed and there was no correlation with disintegration time in certain products. The in vitro dissolution experimentation helped in identifying the discriminatory dissolution conditions and also the formulations that were unaffected with change of dissolution variables. In summary, commercial preparations of carbamazepine vary widely in their dissolution behavior in multi dissolution run experimentation. Identifying this behavior of the products was essential as an in vitro tool for screening a good and a bad formulation.     

In Vitro Evaluation of Frozen-Thawed Stallion Semen: A Review

The article reviews methods used for in vitro evaluation of sperm, with particular emphasis on frozen-thawed stallion sperm. The techniques, limitations of the methods and correlations with fertility results are discussed. Very few studies have tried to find correlation between fertility of frozen stallion semen and laboratory tests. It is difficult and expensive to inseminate an adequate number of mares to achieve statistically significant differences. Significant, but low correlations have been demonstrated between the foaling rate and subjective motility of sperm incubated for 2 h and 4 h at 37°C and hypoosmotic swelling test after 0 and 3 h of incubation. Significant correlations have been reported between the pregnancy rate and viability of propidium iodide-stained sperm assessed by flow cytometry as well as for glass wool and Sephadex filtration tests. No correlations have been detected between fertility and motility immediately after thawing. In spite of that, motility estimation by light microscope is the most commonly used method to evaluate frozen-thawed stallion sperm. Computer assisted automatic sperm analyzers have replaced light microscopy in research projects, but so far nobody has been able to demonstrate a correlation between fertility of frozen stallion semen and any of the motility parameters obtained by these instruments.     

Comparative Evaluation of Fecal Fat Excretion Induced by Orlistat and Chitosan

Objective: To compare the effects of chitosan and orlistat on fecal fat excretion. Research Methods and Procedure: A randomized, open‐label, two‐period sequential design study was used. A total of 12 healthy adult volunteers within 20% of their ideal body weight entered a 7‐day run‐in diet period before being randomized to orlistat (120 mg) or chitosan (890 mg) three times daily for 7 days. Subjects then crossed over treatment regimens for an additional 7‐day period. Subjects followed a standardized diet (2500 kcal/d, 30% as fat) for the entire 21‐day study. Feces were collected on days 4 to 7 of the run‐in period (baseline) and during the two treatment periods. Mean daily fecal fat excretion was measured at baseline and during each treatment regimen. Results: Mean baseline fecal fat excretion for all subjects was 1.36 ± 0.45 g/d. During orlistat treatment, mean fecal fat excretion significantly increased from baseline (+16.13 ± 7.27 g/d; p < 0.001). No significant effect was observed with chitosan (+0.27 ± 1.02 g/d; p = 0.379). Fecal fat excretion was significantly greater with orlistat than with chitosan (p < 0.001; 95% confidence intervals: 11.73; 20.00 g/d). Discussion: This study provides additional evidence of the inhibitory effect of orlistat on dietary fat absorption. Chitosan, however, has no effect on fecal fat excretion.     

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors ^1,2 and providing growth promoting adhesion molecules ³ and extracellular matrix molecules ⁴. In addition they myelinate the demyelinated axons at the site of injury ⁵.However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes ^6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules ^8-13. In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer ^14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) ^16,17, FGF/Heparin ¹⁸, Eph/Ephrins¹⁹.This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.Download video file.^{(64M, mov)}     

Enhancing the Prebiotic Relevance of a Set of Covalently Self-Assembling, Autorecombining RNAs Through In Vitro Selection

An in vitro form of the self-splicing group-I intron interrupting the Azoarcus tRNA^Ile was shortened by ~10% with the removal of helix P6a. This deletion reduced the reverse-splicing activity of the ribozyme about 10-fold. Through in vitro selection, this activity was restored in several low-error mutants. A number of mutations were found that improved reverse-splicing activity through both increased k _obs and better folding. The deletion mutant could be fragmented into as many as three discrete pieces, which, when incubated together, were capable of covalent self-assembly through energy-neutral transesterification reactions, a process called autorecombination. A subset of the mutations identified through in vitro selection for reverse-splicing were exaptations in that they were also shown to augment the autorecombination reactions, leading to higher yields of covalently self-assembled products, making this the smallest such system yet discovered.     

In Vitro Kinetic Analysis of Fermentation of Prebiotic Inulin-Type Fructans by Bifidobacterium Species Reveals Four Different Phenotypes

Kinetic analyses of bacterial growth, carbohydrate consumption, and metabolite production of 18 Bifidobacterium strains grown on fructose, oligofructose, or inulin were performed. A principal component analysis of the data sets, expanded with the results of a genetic screen concerning the presence of a β-fructofuranosidase gene previously encountered in Bifidobacterium animalis subsp. lactis DSM 10140^T, revealed the existence of four clusters among the bifidobacteria tested. Strains belonging to a first cluster could not degrade oligofructose or inulin. Strains in a second cluster could degrade oligofructose, displaying a preferential breakdown mechanism, but did not grow on inulin. Fructose consumption was faster than oligofructose degradation. A third cluster was composed of strains that degraded all oligofructose fractions simultaneously and could partially break down inulin. Oligofructose degradation was substantially faster than fructose consumption. A fourth, smaller cluster consisted of strains that shared high fructose consumption and oligofructose degradation rates and were able to perform partial breakdown of inulin. For all strains, a metabolic shift toward more acetate, formate, and ethanol production, at the expense of lactate production, was observed during growth on less readily fermentable energy sources. No correlation between breakdown patterns and the presence of the β-fructofuranosidase gene could be detected. These variations indicate niche-specific adaptation of bifidobacteria and could have in vivo implications on the strain specificity of the stimulatory effect of inulin-type fructans on bifidobacteria.     

Characterisation of Simple In Vitro Cultures of Striga hermonthica Suitable for Metabolic Studies

Abstract: The aim of this study is to develop simplified models for standardised screenings of xenobiotics, especially targeted against mannitol production, to control the harmful parasite, S. hermonthica. Chlorophyllous protoplasts and calli were produced from the young leaves of the parasite. Best yield from protoplast isolation was obtained when leaf segments were incubated at 30 °C, in the light, under shaking in an enzyme cocktail containing 2 % cellulase Onozuka R10, 0.1 % Pectolyase and sorbitol 1 M as the osmoticum. Oxygen exchange measurements, as well as labelling experiments with ¹⁴C-bicarbonate, emphasised a significant decrease in photosynthetic capacity of protoplasts, mannitol remaining, however, a major primary product of photosynthesis. Initiation of cell cultures was unsuccessful and instability of protoplasts prevents their standardised utilisation for herbicide screening. In contrast, globular calli produced first on MS medium containing 0.5 mg L^-1 NAA, 2.5 mg L^-1 BAP and 2 % sucrose were stable for two years, after monthly subculturing on fresh medium. Sucrose substitution by mannose in the medium did not change kinetic growth and stability. Potential autotrophy was lost for calli by increasing exogenous sugar level. Biochemical analyses and labelling experiments with ¹⁴C-bicarbonate or ¹⁴C-sucrose or -mannose showed that carbon partitioning is modified in calli, in comparison with young leaves or protoplasts, sucrose or mannose accumulation being favoured in sucrose- or mannose-fed calli, respectively. However, carbon flow towards mannitol was more marked in calli growing on high mannose medium. Stability and preservation of an active mannitol biosynthetic pathway allows planning of xenobiotic assays with calli as a simplified model for Striga hermonthica.     

Effects of Mitochondrial Inhibitors on Intraerythrocytic Plasmodium falciparum in In Vitro Cultures1

ABSTRACT. Malarial parasites infecting mammalian hosts are considered to be homolactate fermentors at their asexual intraerythrocytic developmental stage; however, existing ultrastructural and biochemical evidence suggest that their acristate mitochondria could be involved in energy metabolism. In the present study, inhibitors of mitochondrial function including compounds which act on NADH and succinate dehydrogenases, electron transport and mitochondrial ATPase, as well as uncouplers, were found to inhibit the growth and propagation of the human parasite Plasmodium falciparum in in vitro cultures at concentrations that specifically affect mitochondrial functions. Direct measurement of parasite protein and nucleic acid synthesis in synchronized cultures showed that throughout the parasite life cycle both processes were inhibited, the latter process being more sensitive. These results strongly suggest that intraerythrocytic malarial parasites require mitochondrial energy production.