The Effect of Diet and Exercise on Intestinal Integrity and Microbial Diversity in Mice

Background

The gut microbiota is now known to play an important role contributing to inflammatory-based chronic diseases. This study examined intestinal integrity/inflammation and the gut microbial communities in sedentary and exercising mice presented with a normal or high-fat diet.

Methods

Thirty-six, 6-week old C57BL/6NTac male mice were fed a normal or high-fat diet for 12-weeks and randomly assigned to exercise or sedentary groups. After 12 weeks animals were sacrificed and duodenum/ileum tissues were fixed for immunohistochemistry for occludin, E-cadherin, and cyclooxygenase-2 (COX-2). The bacterial communities were assayed in fecal samples using terminal restriction fragment length polymorphism (TRFLP) analysis and pyrosequencing of 16S rRNA gene amplicons.

Results

Lean sedentary (LS) mice presented normal histologic villi while obese sedentary (OS) mice had similar villi height with more than twice the width of the LS animals. Both lean (LX) and obese exercise (OX) mice duodenum and ileum were histologically normal. COX-2 expression was the greatest in the OS group, followed by LS, LX and OX. The TRFLP and pyrosequencing indicated that members of the Clostridiales order were predominant in all diet groups. Specific phylotypes were observed with exercise, including Faecalibacterium prausnitzi, Clostridium spp., and Allobaculum spp.

Conclusion

These data suggest that exercise has a strong influence on gut integrity and host microbiome which points to the necessity for more mechanistic studies of the interactions between specific bacteria in the gut and its host.     

Isolation and characterization of three chitinases from Trichoderma harzianum.

Three proteins which display chitinase activity were purified from the supernatants of Trichoderma harzianum CECT 2413 grown in minimal medium supplemented with chitin as the sole carbon source. Purification was carried out after protein precipitation with ammonium sulphate, adsorption to colloidal chitin and digestion, and, finally, chromatofocusing. By this procedure, two chitinases of 42 kDa (CHIT42) and 37 kDa (CHIT37) were purified to homogeneity, as judged by SDS/PAGE and gel filtration, whereas a third, of 33 kDa (CHIT33), was highly purified. The isoelectric points for CHIT42, CHIT37 and CHIT33 were 6.2, 4.6 and 7.8, respectively. The three enzymes displayed endochitinase activities and showed different kinetic properties. CHIT33 was able to hydrolyze chitin oligomers of a polymerization degree higher than n = 4, its Km for colloidal chitin being 0.3 mg/ml. CHIT42 and CHIT37 were able to hydrolyze chitin oligomers with a minimal polymerization degree of n = 3, their Km values for colloidal chitin being 1.0 mg/ml and 0.5 mg/ml respectively. With regard to their lytic activity with purified cell walls of the phytopathogenic fungus Botrytis cinerea, a hydrolytic action was observed only when CHIT42 was present. Antibodies against CHIT42 and CHIT37 specifically recognized the proteins and did not display cross-reaction, suggesting that each protein is encoded by a different gene.     

Intermediate states between P* and Pf in bacterial reaction centers,as detected by the fluorescence kinetics

The fluorescence lifetimes of the reaction centers isolated from the wild-type Rhodopseudomonas sphaeroides purple bacterium and those from the R26 mutant strain, lacking the carotenoid, were measured at low redox potential. In addition to the prompt fluorescence occurring directly from P* and the long delayed emission related to the radical pair state Pf, two other components are present. We suggest that they may come from intermediate states between P* and Pf, or reflect the stabilization of Pf itself.     

Experimentally broken faecal sacs affect nest bacterial environment,development and survival of spotless starling nestlings

Nestlings of most avian species produce faecal sacs, which facilitate the removal of nestlings’ excrements by parents, thereby reducing proliferation of potentially pathogenic microorganisms and/or detectability by predators and parasites. The nest microbial environment that birds experience during early life might also affect their development and thus, faecal sacs facilitating parental removal may be a strategy to decrease bacterial contamination of nests that could harm developing nestlings. Here, we tested this hypothesis by experimentally broken faecal sacs and spreading them in nests of spotless starlings Sturnus unicolor, thereby avoiding their removal by adults. In accordance with the hypothesis, experimental nests harboured higher bacterial density than control nests. Nestlings in experimental nests were of smaller size (tarsus length) and experienced lower probability of survival (predation) than those in control nests. Moreover, nestlings in experimental nests tended to suffer more from ectoparasites than those in control nests. We discuss the possible pivotal role of bacteria producing chemical volatiles that ectoparasites and predators might use to find avian nests, and that could explain our experimental results in starlings.     

Presence of Pathogens and Indicator Microbes at a Non-Point Source Subtropical Recreational Marine Beach

Swimming in ocean water, including ocean water at beaches not impacted by known point sources of pollution, is an increasing health concern. This study was an initial evaluation of the presence of indicator microbes and pathogens and the association among the indicator microbes, pathogens, and environmental conditions at a subtropical, recreational marine beach in south Florida impacted by non-point sources of pollution. Twelve water and eight sand samples were collected during four sampling events at high or low tide under elevated or reduced solar insolation conditions. The analyses performed included analyses of fecal indicator bacteria (FIB) (fecal coliforms, Escherichia coli, enterococci, and Clostridium perfringens), human-associated microbial source tracking (MST) markers (human polyomaviruses [HPyVs] and Enterococcus faecium esp gene), and pathogens (Vibrio vulnificus, Staphylococcus aureus, enterovirus, norovirus, hepatitis A virus, Cryptosporidium spp., and Giardia spp.). The enterococcus concentrations in water and sand determined by quantitative PCR were greater than the concentrations determined by membrane filtration measurement. The FIB concentrations in water were below the recreational water quality standards for three of the four sampling events, when pathogens and MST markers were also generally undetectable. The FIB levels exceeded regulatory guidelines during one event, and this was accompanied by detection of HPyVs and pathogens, including detection of the autochthonous bacterium V. vulnificus in sand and water, detection of the allochthonous protozoans Giardia spp. in water, and detection of Cryptosporidium spp. in sand samples. The elevated microbial levels were detected at high tide and under low-solar-insolation conditions. Additional sampling should be conducted to further explore the relationships between tidal and solar insolation conditions and between indicator microbes and pathogens in subtropical recreational marine waters impacted by non-point source pollution.Global estimates indicate that each year more than 120 million cases of gastrointestinal disease and 50 million cases of severe respiratory diseases are caused by swimming and bathing in wastewater-polluted coastal waters (42). Swimming-related illness is attributed predominantly to exposure to microbial pathogens, which enter the water through point sources, such as sewage outfalls. Water quality at beaches may also be impacted by non-point sources, such as storm water runoff, sand resuspension, animal fecal inputs, and human bather shedding (8, 12, 22, 47, 59).The concentration of indicator microorganisms in a body of recreational water is used to estimate the health risk to bathers. These microbes serve as surrogates for microbial pathogens. Studies show that the U.S. Environmental Protection Agency (EPA)-recommended indicator microbe for marine beaches, enterococci, shows a significant correlation with illness in marine beaches impacted by point source pollution (38, 54). However, a similar correlation has not been identified at beaches impacted by non-point source pollution or subtropical marine beaches (17, 29, 38, 54).The failure to consistently demonstrate an association between enterococci and illness at non-point source beaches calls into question the ability of indicator microbes to predict the presence of pathogens. Studies conducted on the west coast of the United States have shown that indicators are often not correlated with measured pathogens at non-point source beaches (31, 32, 33, 37). Additional studies conducted in a subtropical environment, such as that of South Florida, where this study was conducted, have repeatedly shown the limited accuracy of indicator microbe standards for determining the presence of pathogens (27, 35). This lack of correlation is understandable since an indicator microbe, such as enterococci, may come from relatively low-risk sources of fecal pollution and therefore may not be related to human or other high-risk sources of fecal pollution and pathogens (9). It has also been shown in both subtropical and temperate climates that indicator bacteria can multiply in the environment, resulting in a false impression of increased microbial pollution and pathogen presence (4, 7, 19, 24, 41, 45, 57, 58). Environmental factors, such as tide, rain, and solar insolation, can also have significant and varying effects on the levels of indicator and pathogenic microbes (21, 24, 33).The lack of correlation between pathogens and indicator microbes at non-point source beaches can result in two problematic scenarios. If indicator microbes are absent and pathogens are present (false-negative scenario), regulatory monitoring may fail to identify the potential adverse health effects on bathers due to the pathogens. This problem is likely to occur since indicator bacteria are less resistant to environmental stresses and disinfection at wastewater treatment plants than certain pathogens (6, 15). However, if indicator microbes are present and pathogens are absent (false-positive scenario), there can be unnecessary economic losses due to recreational beach advisories and/or closures. A 4-month closure of Huntington Beach in 1999 due to microbial standard violations resulted in the loss of millions of dollars in tourism income to the business community and almost 2 million dollars in beach closure investigation fees (55; for a review, see reference 27).Given these two possible scenarios, the relationship between indicator microbes and pathogens under different environmental conditions at non-point source beaches representing different geographic and climatic settings should be assessed further. Investigation of this relationship would require a large sample size in order to establish possible significant associations between the various factors and targets. The objective of this study was to conduct a preliminary evaluation of the presence of indicator microbes and pathogens and the possible association between indicator microbes, pathogen measurements, and environmental conditions at a subtropical recreational marine beach in South Florida. Because of cost limitations when multiple targets, including pathogens, are screened, this study was not intended to establish a conclusive relationship between the various factors and targets but was intended to provide insight into both the presence of organisms and possible associations which should be investigated further. Such information would be useful for understanding the potential health risks to bathers from non-point sources of microbes and would also contribute to determining the appropriateness of using indicator microbes to monitor the water quality at non-point source beaches. Although previous studies have assessed the presence of either viral, protozoan, or bacterial pathogens along with indicator microbes in point or non-point source recreational beach waters (27, 33, 37, 47), to our knowledge, this is the first study to assess the presence of all three classes of pathogens (viral, protozoan, and bacterial) as well as indicator microbes at a non-point source recreational beach. This study is also the first study to sample for all these microbes in both water and sand at a non-point source recreational beach. Through analysis of the various microbes under different targeted environmental conditions, this study also included a preliminary evaluation of the sources of microbial contaminants and pathogens and the effectiveness of various analytical methods for microbe detection. The latter part of the study included a comparison of three different methods for enterococcus enumeration, as well as an innovative method for simultaneously concentrating protozoans and viruses from water samples.     

Detection of 2,4,6-Trinitrotoluene-Utilizing Anaerobic Bacteria by 15N and 13C Incorporation

2,4,6-Trinitrotoluene (¹⁵N or ¹³C labeled) was added to Norfolk Harbor sediments to test whether anaerobic bacteria use TNT for growth. Stable-isotope probing (SIP)-terminal restriction fragment length polymorphism (TRFLP) detected peaks in the [¹⁵N]TNT cultures (60, 163, and 168 bp). The 60-bp peak was also present in the [¹³C]TNT cultures and was related to Lysobacter taiwanensis.It has been estimated that there are over 1 million cubic yards of material contaminated with 2,4,6-trinitrotoluene (TNT) in the United States at concentrations as high as 600,000 to 700,00 mg/kg of material (9). Marine and estuarine sediments have also been impacted through the manufacturing, use, and/or disposal of TNT. Microbial biodegradation of these pollutants in situ is preferable due to the large volume of contaminated soils/sediments. However, it is unclear whether in situ bacteria can utilize TNT as a nitrogen or carbon source. Under aerobic conditions, TNT appears to be largely unavailable to bacteria but can be used by a variety of fungi as a carbon and nitrogen source (7). Under anaerobic conditions, only a few bacterial strains (Clostridium and Desulfovibrio strains and Pseudomonas sp. strain JLR11) have been reported to utilize TNT as a sole nitrogen source (6, 7). It is widely believed that nitroaromatic compounds cannot serve as growth substrates under anaerobic conditions in situ (11), and coamendment strategies are suggested for stimulating TNT transformation to 2,4,6-triaminotoluene (TAT) (1, 7, 18). Given these difficulties, there is no direct evidence that TNT can be biodegraded in situ and there is little proof that anaerobic bacteria can utilize TNT as a sole carbon or nitrogen source in organic-rich sediments. This study tested whether bacteria in Norfolk Harbor sediment are able to incorporate nitrogen (N) or carbon (C) from TNT into biomass under sulfidogenic conditions using stable-isotope probing (SIP). The findings indicate that bacteria assimilate ¹⁵N and ¹³C from TNT into their genomes during anaerobic incubations (2 to 35 days). Interestingly, one small-subunit (SSU) gene, related to Lysobacter taiwanensis, was observed in both the ¹⁵N and the ¹³C incubations.     

Probing the Role of the Internal Disulfide Bond in Regulating Conformational Dynamics in Neuroglobin

In this report, we demonstrate that the internal disulfide bridge in human neuroglobin modulates structural changes associated with ligand photo-dissociation from the heme active site. This is evident from time-resolved photothermal studies of CO photo-dissociation, which reveal a 13.4± 0.9 mL mol⁻¹ volume expansion upon ligand photo-release from human neuroglobin, whereas the CO dissociation from rat neuroglobin leads to a significantly smaller volume change (ΔV = 4.6 ± 0.3 mL mol⁻¹). Reduction of the internal disulfide bond in human neuroglobin leads to conformational changes (reflected by ΔV) nearly identical to those observed for rat Ngb. Our data favor the hypothesis that the disulfide bond between Cys⁴⁶ and Cys⁵⁵ modulates the functioning of human neuroglobin.