Influence of Dietary Zinc Oxide and Copper Sulfate on the Gastrointestinal Ecosystem in Newly Weaned Piglets

Dietary doses of 2,500 ppm ZnO-Zn reduced bacterial activity (ATP accumulation) in digesta from the gastrointestinal tracts of newly weaned piglets compared to that in animals receiving 100 ppm ZnO-Zn. The amounts of lactic acid bacteria (MRS counts) and lactobacilli (Rogosa counts) were reduced, whereas coliforms (MacConkey counts) and enterococci (Slanetz counts, red colonies) were more numerous in animals receiving the high ZnO dose. Based on 16S rRNA gene sequencing, the colonies on MRS were dominated by three phylotypes, tentatively identified as Lactobacillus amylovorus (OTU171), Lactobacillus reuteri (OTU173), and Streptococcus alactolyticus (OTU180). The colonies on Rogosa plates were dominated by the two Lactobacillus phylotypes only. Terminal restriction fragment length polymorphism analysis supported the observations of three phylotypes of lactic acid bacteria dominating in piglets receiving the low ZnO dose and of coliforms and enterococci dominating in piglets receiving the high ZnO dose. Dietary doses of 175 ppm CuSO₄-Cu also reduced MRS and Rogosa counts of stomach contents, but for these animals, the numbers of coliforms were reduced in the cecum and the colon. The influence of ZnO on the gastrointestinal microbiota resembles the working mechanism suggested for some growth-promoting antibiotics, namely, the suppression of gram-positive commensals rather than potentially pathogenic gram-negative organisms. Reduced fermentation of digestible nutrients in the proximal part of the gastrointestinal tract may render more energy available for the host animal and contribute to the growth-promoting effect of high dietary ZnO doses. Dietary CuSO₄ inhibited the coliforms and thus potential pathogens as well, but overall the observed effect of CuSO₄ was limited compared to that of ZnO.     

Taxonomic Identification of Commensal Bacteria Associated with the Mucosa and Digesta throughout the Gastrointestinal Tracts of Preweaned Calves

Bacterial colonization in the gastrointestinal tracts (GIT) of preweaned calves is very important, since it can influence early development and postweaning performance and health. This study investigated the composition of the bacteria along the GIT (rumen, jejunum, ileum, cecum, and colon) of preweaned bull calves (3 weeks old) using pyrosequencing to understand the segregation of bacteria between the mucosal surface and digesta. Phylogenetic analysis revealed that a total of 83 genera belonging to 13 phyla were distributed throughout the GIT of preweaned calves, with the Firmicutes, Bacteroidetes, and Proteobacteria predominating. Quantitative PCR (qPCR) analysis of selected abundant bacterial genera (Prevotella, Bacteroides, Lactobacillus, and Faecalibacterium) revealed that their prevalence was significantly different among the GIT regions and between mucosa- and digesta-associated communities. Rumens contained the most diverse bacterial population, consisting of 47 genera, including 16 rumen-specific genera, followed by the large intestine and then the small intestine. Bacterial species richness was higher at the mucosal surface than in the local digesta, with the exception of the rumen. The majority of bacteria found on the rumen epithelial surface and within the small intestine could not be identified due to a lack of known genus-level information. Thus, future studies will be required to fully characterize the microbiome during the development of the rumens and the mucosal immune systems of newborn calves. This is the first study to analyze in depth the bacterial composition of the GIT microbiome in preweaned calves, which extends previous findings regarding early rumen colonization and bacterial segregation between mucosa- and digesta-associated microbial communities.     

Dietary Leucine Supplementation Improves the Mucin Production in the Jejunal Mucosa of the Weaned Pigs Challenged by Porcine Rotavirus

The present study was mainly conducted to determine whether dietary leucine supplementation could attenuate the decrease of the mucin production in the jejunal mucosa of weaned pigs infected by porcine rotavirus (PRV). A total of 24 crossbred barrows weaned at 21 d of age were assigned randomly to 1 of 2 diets supplemented with 1.00% L-leucine or 0.68% L-alanine (isonitrogenous control) for 17 d. On day 11, all pigs were orally infused PRV or the sterile essential medium. During the first 10 d of trial, dietary leucine supplementation could improve the feed efficiency (P = 0.09). The ADG and feed efficiency were impaired by PRV infusion (P<0.05). PRV infusion also increased mean cumulative score of diarrhea, serum rotavirus antibody concentration and crypt depth of the jejunal mucosa (P<0.05), and decreased villus height: crypt depth (P = 0.07), goblet cell numbers (P<0.05), mucin 1 and 2 concentrations (P<0.05) and phosphorylated mTOR level (P<0.05) of the jejunal mucosa in weaned pigs. Dietary leucine supplementation could attenuate the effects of PRV infusion on feed efficiency (P = 0.09) and mean cumulative score of diarrhea (P = 0.09), and improve the effects of PRV infusion on villus height: crypt depth (P = 0.06), goblet cell numbers (P<0.05), mucin 1 (P = 0.08) and 2 (P = 0.07) concentrations and phosphorylated mTOR level (P = 0.08) of the jejunal mucosa in weaned pigs. These results suggest that dietary 1% leucine supplementation alleviated the decrease of mucin production and goblet cell numbers in the jejunal mucosa of weaned pigs challenged by PRV possibly via activation of the mTOR signaling.     

Influence of the Biliary System on Biliary Bacteria Revealed by Bacterial Communities of the Human Biliary and Upper Digestive Tracts

Biliary bacteria have been implicated in gallstone pathogenesis, though a clear understanding of their composition and source is lacking. Moreover, the effects of the biliary environment, which is known to be generally hostile to most bacteria, on biliary bacteria are unclear. Here, we investigated the bacterial communities of the biliary tract, duodenum, stomach, and oral cavity from six gallstone patients by using 16S rRNA amplicon sequencing. We found that all observed biliary bacteria were detectable in the upper digestive tract. The biliary microbiota had a comparatively higher similarity with the duodenal microbiota, versus those of the other regions, but with a reduced diversity. Although the majority of identified bacteria were greatly diminished in bile samples, three Enterobacteriaceae genera (Escherichia, Klebsiella, and an unclassified genus) and Pyramidobacter were abundant in bile. Predictive functional analysis indicated enhanced abilities of environmental information processing and cell motility of biliary bacteria. Our study provides evidence for the potential source of biliary bacteria, and illustrates the influence of the biliary system on biliary bacterial communities.     

Prevalence and Abundance of Uncultivated Megasphaera-Like Bacteria in the Human Vaginal Environment

Cultivation-independent analysis of 16S rRNA gene sequences in vaginal samples revealed two previously unrecognized, uncultivated Megasphaera-like phylotypes. Phylogenetic analysis and environmental distribution suggest that these Megasphaera types may be unique to the vaginal environment. Quantitative PCR suggests that both phylotypes are present in higher concentrations in women with bacterial vaginosis.     

Types and Distribution of Anaerobic Bacteria in the Large Intestine of Pigs

An examination was made of various sites along the length of the swine large intestine, using strictly anaerobic culture methods. Sites were separated by differential washing into fractions described as lumenal content, lumenal surface layer, and intestinal wall tissue. Direct microscopic clump counts averaged 13.3 × 10¹⁰ organisms per g (dry weight) of material in the lumenal content, 14.0 × 10¹⁰ in the surface layer, and 5.1 × 10¹⁰ in the intestinal wall tissue. Both direct microscopic counts and viable culture counts were higher from the lumenal content and surface layer than from the intestinal tissue at all sites sampled in the intestine. Cultural counts averaged 56.2% of the direct microscopic counts in lumenal content and surface layer and 20.2% in intestinal tissue. Over 90% of the bacteria isolated were gram positive and consisted mainly of gram-positive cocci, lactobacilli, eubacteria, and clostridia. Of 192 isolates recovered, only 124 could be assigned to recognized species.     

A Mutation in the N Domain of Escherichia coli Lon Stabilizes Dodecamers and Selectively Alters Degradation of Model Substrates

Escherichia coli Lon, an ATP-dependent AAA⁺ protease, recognizes and degrades many different substrates, including the RcsA and SulA regulatory proteins. More than a decade ago, the E240K mutation in the N domain of Lon was shown to prevent degradation of RcsA but not SulA in vivo. Here, we characterize the biochemical properties of the E240K mutant in vitro and present evidence that the effects of this mutation are complex. For example, Lon^E240K exists almost exclusively as a dodecamer, whereas wild-type Lon equilibrates between hexamers and dodecamers. Moreover, Lon^E240K displays degradation defects in vitro that do not correlate in any simple fashion with degron identity, substrate stability, or dodecamer formation. The Lon sequence segment near residue 240 is known to undergo nucleotide-dependent conformational changes, and our results suggest that this region may be important for coupling substrate binding with allosteric activation of Lon protease and ATPase activity.     

Biotic Interactions and Sunlight Affect Persistence of Fecal Indicator Bacteria and Microbial Source Tracking Genetic Markers in the Upper Mississippi River

The sanitary quality of recreational waters that may be impacted by sewage is assessed by enumerating fecal indicator bacteria (FIB) (Escherichia coli and enterococci); these organisms are found in the gastrointestinal tracts of humans and many other animals, and hence their presence provides no information about the pollution source. Microbial source tracking (MST) methods can discriminate between different pollution sources, providing critical information to water quality managers, but relatively little is known about factors influencing the decay of FIB and MST genetic markers following release into aquatic environments. An in situ mesocosm was deployed at a temperate recreational beach in the Mississippi River to evaluate the effects of ambient sunlight and biotic interactions (predation, competition, and viral lysis) on the decay of culture-based FIB, as well as molecularly based FIB (Entero1a and GenBac3) and human-associated MST genetic markers (HF183 and HumM2) measured by quantitative real-time PCR (qPCR). In general, culturable FIB decayed the fastest, while molecularly based FIB and human-associated genetic markers decayed more slowly. There was a strong correlation between the decay of molecularly based FIB and that of human-associated genetic markers (r², 0.96 to 0.98; P < 0.0001) but not between culturable FIB and any qPCR measurement. Overall, exposure to ambient sunlight may be an important factor in the early-stage decay dynamics but generally was not after continued exposure (i.e., after 120 h), when biotic interactions tended to be the only/major influential determinant of persistence.     

Source of Nitrous Oxide Emissions during the Cow Manure Composting Process as Revealed by Isotopomer Analysis of and amoA Abundance in Betaproteobacterial Ammonia-Oxidizing Bacteria

A molecular analysis of betaproteobacterial ammonia oxidizers and a N₂O isotopomer analysis were conducted to study the sources of N₂O emissions during the cow manure composting process. Much NO₂⁻-N and NO₃⁻-N and the Nitrosomonas europaea-like amoA gene were detected at the surface, especially at the top of the composting pile, suggesting that these ammonia-oxidizing bacteria (AOB) significantly contribute to the nitrification which occurs at the surface layer of compost piles. However, the ¹⁵N site preference within the asymmetric N₂O molecule (SP = δ¹⁵N^α − δ¹⁵N^β, where ¹⁵N^α and ¹⁵N^β represent the ¹⁵N/¹⁴N ratios at the center and end sites of the nitrogen atoms, respectively) indicated that the source of N₂O emissions just after the compost was turned originated mainly from the denitrification process. Based on these results, the reduction of accumulated NO₂⁻-N or NO₃⁻-N after turning was identified as the main source of N₂O emissions. The site preference and bulk δ¹⁵N results also indicate that the rate of N₂O reduction was relatively low, and an increased value for the site preference indicates that the nitrification which occurred mainly in the surface layer of the pile partially contributed to N₂O emissions between the turnings.The very sensitive greenhouse gas nitrous oxide (N₂O) has a 296 times higher impact than CO₂ (39) and is also responsible for ozone depletion (10). Agricultural activities such as the use of nitrate fertilizers, livestock production, and manure management, including composting, are known to be important sources of N₂O emissions (18). To devise a strategy to mitigate N₂O emissions, it is essential to understand its sources in detail. However, the sources of N₂O emissions during the composting process are still largely unclear.In the composting process, a part of NH₄⁺-N is known to be processed through nitrification-denitrification and emitted as N₂ and N₂O. Nitrous oxide is known to be generated through both the nitrification and denitrification processes as intermediate products or by-products. Nitrous oxide emission is a very complex process because denitrifying bacteria are phylogenetically diverse (60), and nitrifiers are also known to utilize the denitrification process even under aerobic conditions (42). It is thus very difficult to estimate the relative contributions of nitrification and denitrification in actual N₂O emissions from the environment. Until now, there has been insufficient knowledge about the relative contributions of these processes to N₂O emissions during the animal manure composting process. Measurement of the actual contributions of N₂O emissions from compost piles in the field is therefore critical to establishing a strategy of mitigating N₂O emissions.Recently, a high-precision analytical technique for determining intramolecular ¹⁵N site preference in asymmetric molecules of N₂O was developed (47). Since N₂O has two N atoms within the molecule (central and outer N), distribution of a stable isotope, ¹⁵N, results in the distribution of three isotopomers, such as ¹⁵N¹⁵NO, ¹⁵N¹⁴NO, and ¹⁴N¹⁵NO. By using this newly developed innovative technique, the latter two types of molecules, which exist abundantly in the environment, can be individually measured. The difference in δ¹⁵N between δ¹⁵N^α and δ¹⁵N^β is the so-called site preference (SP = δ¹⁵N^α − δ¹⁵N^β, where ¹⁵N^α and ¹⁵N^β represent the ¹⁵N/¹⁴N ratios at the center and end sites of the nitrogen atoms, respectively). The site preference enabled us to identify the source and sinks of N₂O in the environment (48, 49, 50, 56). Using this technique, Sutka et al. (44) found that the site preference for N₂O from hydroxylamine oxidation (∼33‰) and nitrite reduction (∼0‰) differs in a pure culture study and noted that this difference can be used to distinguish the relative contributions of nitrification and denitrification sources to N₂O emissions. There have still been only several reported studies which applied this measurement technique to field N₂O samples (48, 53) or referred to the relative contributions of nitrification and denitrification. To our knowledge, the present study is the first to apply this isotopomer analysis technique to the determination of N₂O sources in the composting process. We specifically used this technique to understand the actual contributions of nitrification and denitrification to N₂O emissions during the cow manure composting process.Ammonia oxidation, the conversion of ammonium to nitrite via hydroxylamine, is an initial step of the nitrification-denitrification process and is critical to the nitrogen cycle in the terrestrial environment (4, 24). In the nitrification process, N₂O is generated as a by-product when ammonia oxidizers convert hydroxylamine to nitrite (35). Since NO₂⁻-N and NO₃⁻-N accumulate in the latter stages of the composting process (29, 30), it is obvious that nitrifiers are active in compost piles. Therefore, it is important to clarify the role and significance of ammonia oxidizers in N₂O emissions during the composting process. However, since the pure culture isolation method is so difficult and time-consuming, little is known about these ammonia oxidizers. A molecular approach based on PCR has been recently developed and has to date been used to target the ammonia monooxygenase gene (amoA) or 16S rRNA gene of betaproteobacterial ammonia oxidizers in soil, wetlands, and marine sediments (2, 3, 6, 7, 13, 32, 52). Using these techniques, substantial information about uncultured ammonia-oxidizing bacteria (AOB) that are partially or wholly responsible for nitrification in the environment will become available. Since the microbial community drastically changes through the composting process (19, 29), and a high accumulation of nitrite or nitrate will occur, especially in the latter half of the process (30), we continuously sampled and analyzed the diversity and abundance of AOB throughout the process. Our objectives in this study were to elucidate the sources of N₂O emissions during the cow manure composting process by combining the isotopomer analysis and molecular analysis of betaproteobacterial AOB.     

Abundance and Diversity of Ammonia-Oxidizing Bacteria and Archaea in Cold Springs on the Qinghai-Tibet Plateau

The cold springs underlain by gas hydrates on the Qinghai-Tibet Plateau (QTP) are similar to deep-sea cold seeps with respect to methane biogeochemistry. Previous studies have shown that ammonia oxidizing bacteria (AOB) and archaea (AOA) are actively present and play important roles in the carbon/nitrogen cycles in cold seeps. Studying AOA and AOB communities in the QTP cold springs will be of great importance to our understanding of carbon and nitrogen cycling dynamics related to the underlying gas hydrates on the QTP. Thus, the abundance and diversity of AOB and AOA in sediments of four cold springs underlain by gas hydrates on the QTP were determined by using quantitative polymerase chain reaction and amoA gene (encoding ammonia monooxygenase involved in ammonia oxidation) phylogenetic analysis. The results showed that the AOB and AOA amoA gene abundances were at 10³–10⁴ copies per gram of the sediments in the investigated cold springs. The AOB population consisted of Nitrosospira and Nitrosomonas in contrast with the mere presence of Nitrosospira in marine cold seeps. The AOB diversity was higher in cold springs than in cold seeps. The AOA population was mainly composed of Nitrososphaera, in contrast with the dominance of Nitrosopumilus in cold seeps. The terrestrial origin and high level of dissolved oxygen of the cold springs may be the main factors accounting for the observed differences in AOB and AOA populations between the QTP cold springs and marine cold seeps.     

Bacterial Responses to Different Dietary Cereal Types and Xylanase Supplementation in the Intestine of Broiler Chicken

Several studies were carried out to investigate the influence of dietary cereals differing in soluble non starch polysaccharides (NSP) content and a xylanase preparation on selected bacterial parameters in the small intestine of broiler chicken. Compared to a maize diet colony forming units (CFU) of mucosa associated bacteria were higher in a wheat/rye diet, most notably for enterobacteria and enterococci. Xylanase supplementation to the wheat/rye diet generally led to lower CFU, especially in the first week of life. However, xylanase supplementation also displayed higher in vitro growth potentials for enterobacteria and enterococci. Bacterial growth of luminal samples in minimal media supplemented with selected NSP showed that the wheat/rye diet enhanced bacterial capacities to utilize NSP only in ileal samples. The xylanase application generally shifted respective maximum growth to the proximal part of the small intestine. The presence of soluble NSP from wheat or rye in the diet per se did not enhance bacterial NSP hydrolyzing enzyme activities in the small intestine, but xylanase supplementation resulted in higher 1,3-1,4- g - glucanase activity. Compared to a maize diet the activity of bacterial bile salt hydrolases in samples of the small intestine was not increased due to inclusion of wheat/rye or triticale to the diet. However, xylanase supplementation led to a reduction with a corresponding increase of lipase activity. It was concluded that dietary cereals producing high intestinal viscosities lead to increased overall bacterial activity in the small intestine. The supplementation of a xylanase to cereal based diets producing high intestinal viscosity, changes composition and metabolic potential of bacterial populations and may specifically influence fat absorption in young animals.     

Role of Productivity and Protozoan Abundance for the Occurrence of Predation-resistant Bacteria in Aquatic Systems

Top-down control of lower trophic levels, e.g., bacteria, has been suggested to increase along aquatic productivity gradients. The response by the bacterial community may be to become more predation resistant in highly productive environments. To test this hypothesis, samples were taken from 20 aquatic systems along a productivity gradient (dissolved organic carbon from 7 to 71 mg/L), during late summer. The results showed that the biomass of bacteria, phytoplankton, and ciliates increased along the gradient (r2 = 0.532, 0.426, and 0.758, P < 0.01, respectively). However, the organisms did not increase equally, and the ratio of protozoan to bacterial biomass showed a 100-fold increase along the gradient. Ciliates dominated the protozoan biomass in the more nutrient-rich waters. The edibility of colony-forming bacteria was tested using a ciliate predator, Tetrahymena pyriformis. Bacterial edibility was found to decrease with increases in nutrient richness and ciliate biomass in the aquatic systems (r2 = 0.358, P < 0.01; r2 = 0.242, P < 0.05, respectively). Quantile regression analysis indicated that the selection pressures on edible bacteria were increasing along the productivity gradient. Thus, inedible forms of bacteria were selected for in the transition from oligotrophic to eutrophic conditions. Isolated bacteria were distributed among the alpha-, beta-, and gamma- Proteobacteria and the Actinobacteria and Firmicutes taxa. We conclude that bacterial predation resistance increases in nutrient-rich waters with high protozoan predation.     

上消化道出血在ICU的诊治近况

近年来,ICU在急重症上消化道出血救治中的作用越来越受到重视。上消化道出血是重症监护病房(ICU)内常见的危重病症 之一,与多器官功能不全密切相关,是多种危重病引起的常见并发症,病情严重者甚至危及生命。引起上消化道出血的病因很多, 其中非特异性粘膜异常、非甾体类抗炎药和抗血小板药物的不合理使用逐渐引起临床的重视。本文对国内外临床诊断和治疗上 消化道出血的近况进行了回顾分析,进一步阐述上消化道出血与多器官功能不全之间的联系,为多学科联合诊断提供参考。     

Differential Expression of the Middle East Respiratory Syndrome Coronavirus Receptor in the Upper Respiratory Tracts of Humans and Dromedary Camels

Middle East respiratory syndrome coronavirus (MERS-CoV) is not efficiently transmitted between humans, but it is highly prevalent in dromedary camels. Here we report that the MERS-CoV receptor—dipeptidyl peptidase 4 (DPP4)—is expressed in the upper respiratory tract epithelium of camels but not in that of humans. Lack of DPP4 expression may be the primary cause of limited MERS-CoV replication in the human upper respiratory tract and hence restrict transmission.     

Influence of Season and Plant Species on the Abundance and Diversity of Sulfate Reducing Bacteria and Ammonia Oxidizing Bacteria in Constructed Wetland Microcosms

Constructed wetlands offer an effective means for treatment of wastewater from a variety of sources. An understanding of the microbial ecology controlling nitrogen, carbon and sulfur cycles in constructed wetlands has been identified as the greatest gap for optimizing performance of these promising treatment systems. It is suspected that operational factors such as plant types and hydraulic operation influence the subsurface wetland environment, especially redox, and that the observed variation in effluent quality is due to shifts in the microbial populations and/or their activity. This study investigated the biofilm associated sulfate reducing bacteria and ammonia oxidizing bacteria (using the dsrB and amoA genes, respectively) by examining a variety of surfaces within a model wetland (gravel, thick roots, fine roots, effluent), and the changes in activity (gene abundance) of these functional groups as influenced by plant species and season. Molecular techniques were used including quantitative PCR and denaturing gradient gel electrophoresis (DGGE), both with and without propidium monoazide (PMA) treatment. PMA treatment is a method for excluding from further analysis those cells with compromised membranes. Rigorous statistical analysis showed an interaction between the abundance of these two functional groups with the type of plant and season (p?<?0.05). The richness of the sulfate reducing bacterial community, as indicated by DGGE profiles, increased in planted vs. unplanted microcosms. For ammonia oxidizing bacteria, season had the greatest impact on gene abundance and diversity (higher in summer than in winter). Overall, the primary influence of plant presence is believed to be related to root oxygen loss and its effect on rhizosphere redox.