Muramic Acid Content Research Articles

Dietary muramidase improved growth performance, feed efficiency, breast meat yield, and welfare of turkeys from hatch to market

Muramidase is an enzyme that hydrolyzes peptidoglycans of bacterial cell walls and improves performance of broilers in a dose-dependent manner. An experiment was conducted to evaluate muramidase supplementation, at a high or step-down dose, in turkeys from hatch to market. Male, B.U.T. 6 turkey poults were placed in 24 floor pens at 32 birds per pen. Poults were fed 1 of 3 diets from d 1 to 126 of age. There were 8 replicate pens per treatment. The treatments were a control (CTL) diet, the CTL plus muramidase at 45,000 LSU(F)/kg from phase 1 to 6 (BAL45), and the CTL plus muramidase at 45,000 LSU(F)/kg from phase 1 to 3 and decreased to 25,000 LSU(F)/kg from phase 4 to 6 (BAL45-25). Data were analyzed using SAS. The model included treatment and block and means were separated by Fisher LSD test. Birds fed BAL45 were heavier (P < 0.05) and had a greater (P < 0.05) average daily gain compared with birds fed the CTL from hatch to d 126 of age. Birds fed BAL45-25 had a final BW and average daily gain intermediate to or equivalent to birds fed BAL45 at the same phases. Feed conversion ratio was improved (P < 0.05) in birds fed BAL45 compared with birds fed the CTL and intermediate in birds fed BAL45-25. Breast meat yield was greater (P < 0.05) in turkeys fed muramidase, regardless of dose, compared with birds fed the CTL. There was no effect of treatment on muramic acid content in the jejunum digesta or litter scores. The frequency of pododermatitis score 1 was greater (P < 0.05) and score 2 was lower (P < 0.05) in birds fed muramidase, regardless of dose, compared with birds fed the CTL diet. In conclusion, muramidase supplementation improved performance, breast meat yield, feed efficiency and some markers of welfare, proportional to the dose in the diets.

A muramidase from Acremonium alcalophilum hydrolyse peptidoglycan found in the gastrointestinal tract of broiler chickens.

This study evaluates peptidoglycan hydrolysis by a microbial muramidase from the fungus Acremonium alcalophilum in vitro and in the gastrointestinal tract of broiler chickens. Peptidoglycan used for in vitro studies was derived from 5 gram-positive chicken gut isolate type strains. In vitro peptidoglycan hydrolysis was studied by three approaches: (a) helium ion microscopy to identify visual phenotypes of hydrolysis, (b) reducing end assay to quantify solubilization of peptidoglycan fragments, and (c) mass spectroscopy to estimate relative abundances of soluble substrates and reaction products. Visual effects of peptidoglycan hydrolysis could be observed by helium ion microscopy and the increase in abundance of soluble peptidoglycan due to hydrolysis was quantified by a reducing end assay. Mass spectroscopy confirmed the release of hydrolysis products and identified muropeptides from the five different peptidoglycan sources. Peptidoglycan hydrolysis in chicken crop, jejunum, and caecum samples was measured by quantifying the total and soluble muramic acid content. A significant increase in the proportion of the soluble muramic acid was observed in all three segments upon inclusion of the microbial muramidase in the diet.

Effects of nitrogen and phosphorus addition on soil microbial community and amino sugar in a temperate forest on Changbai Mountain, Northeast China

We carried out an experiment including nitrogen addition (N, 50 kg N·hm-2·a-1), phosphorus addition (P, 25 kg P·hm-2·a-1) and both nitrogen and phosphorus addition (NP, 50 kg N·hm-2·a-1+25 kg P·hm-2·a-1) in a natural Korean pine broad-leaved mixed forest on Changbai Mountain to examine the effects of single and combined N and P additions on soil microbial community composition and amino sugar. The results showed that N and P addition significantly reduced total microbial biomass by 19.5% and 24.6% in the organic layer of soil, while P addition significantly reduced the biomass of bacteria and fungi by 23.8% and 19.3%, respectively. In the mineral layer, N, P and NP addition significantly increased total microbial biomass by 94.8%, 230.9%, and 115.0% respectively, while the biomass of bacteria and fungi were significantly increased under all the treatments. The fungi to bacteria ratio (F/B) was significantly increased in the organic layer by N addition, while was decreased in the mineral layer soil by NP addition. The Gram-positive bacteria to Gram-negative bacteria ratio showed positive response to N, P and NP addition. Soil amino sugars responded differently to different treatments. N, P and NP addition significantly decreased glucosamine content by 41.3%, 48.8% and 36.4% in the organic layer, while N and NP addition increased muramic acid content by 43.0% and 71.1%, respectively. The contents of glucosamine and muramic acid in the mineral layer did not change significantly in response to N addition but increased significantly in response to both P addition and NP addition. The glucosamine to muramic acid ratio in the organic layer significantly decreased under fertilization treatments, indicating that N and P addition increased the relative contribution of bacteria to soil organic carbon accumulation. The changes in soil amino sugar contents were closely related to the change in microbial community composition after N and P addition, both of which were affected by changes in soil chemical properties.

Silver nanoparticles: Antimicrobial activity, cytotoxicity, and synergism with N-acetyl cysteine.

The fast progression of nanotechnology has led to novel therapeutic interventions. Antimicrobial activities of silver nanoparticles (Ag NPs) were tested against standard ATCC strains of Staphylococcus aureus (ATCC 9144), Escherichia coli (O157:H7), Pseudomonas aeruginosa (ATCC 27853), and Candida albicans (ATCC 90028) in addition to 60 clinical isolates collected from cancer patients. Antimicrobial activity was tested by disk diffusion method and MIC values for Ag NPs alone and in combination with N-acetylcysteine (NAC) against tested pathogens were determined by broth microdilution method. Ag NPs showed a robust antimicrobial activity against all tested pathogens and NAC substantially enhanced the antimicrobial activity of Ag NPs against all tested pathogens. Synergism between Ag NPs and NAC has been confirmed by checkerboard assay. The effect of Ag NPs on tested pathogens was further scrutinized by Transmission Electron Microscope (TEM) which showed disruption of cell wall in both bacteria and fungi. Ag NPs abrogated the activity of respiratory chain dehydrogenase of all tested pathogens and released muramic acid content from S. aureus in culture. The cytotoxic effect of Ag NPs alone and in combination with NAC was examined using human HepG2 cells and this revealed no cytotoxicity at MIC values of Ag NPs and interestingly, NAC reduced the cytotoxic effect of Ag NPs at concentrations higher than their MIC values. Taken together, Ag NPs have robust antimicrobial activity and NAC substantially enhances their antimicrobial activities against MDR pathogens which would provide a novel safe, effective, and inexpensive therapeutic approach to control the prevalence of MDR pathogens.

Response of soil microorganisms after converting a saline desert to arable land in central Asia

Uncultivated saline lands are reserve resources for arable land without environmental degradation. A field experiment was conducted in a continuously cultivated farm station in Xinjiang province, northwest China, including a saline-desert land. The objective was to investigate the effects of different cotton cultivation periods from 1 to 16 years on soil chemical and soil biological properties. Cultivation led to a decrease in soil electrical conductivity and reduced soil pH from 8.9 in uncultivated land to 7.9. Soil organic C showed a small and variable increase after cultivation. After year 1 of cultivation, the contents of muramic acid, galactosamine and glucosamine were all roughly 30% lower, similarly to those of PLFA in comparison with the uncultivated soil. Further cultivation led to a continuous non-linear 40% increase until year 16 in comparison with the uncultivated soil, which mainly occurred within five years of cultivation. The ratio of fungal C to bacterial C, based on fungal glucosamine and bacterial muramic acid, varied around 0.7 in the uncultivated soil and around 1.4 from the second year on after cultivation. Microbial biomass C showed a 100% increase to contents around 300μgg−1 soil from year 4 to 16 in comparison with the initial values. The microbial biomass C to SOC ratio ranged from 2 to 6% without clear cultivation effects. The ergosterol to microbial biomass C ratio increased from values around 0.04% during the first 3 years to values around 0.2% from year 4 on. Also, the Gram-negative to Gram-positive bacterial PLFA ratio increased with increasing cultivation time, whereas the microbial biomass C to total PLFA ratio varied in a relatively small range around 8.9μgnmol−1.

Amino sugars reflect microbial residues as affected by clay mineral composition of artificial soils

Microbial residues are important contributors to soil organic matter (SOM). However, the effect of mineral composition on microbial activity and thereby SOM development is not well understood. Here, we used artificial soils composed of four different mixtures containing illite, montmorillonite, ferrihydrite and charcoal to study the effect of mineral composition on SOM development. In addition, silt- and sand-sized quartz were used to provide texture, sterilised manure was added as OM source and the mixtures were inoculated with a microbial community extracted from a natural soil. The mixtures were incubated in the dark for 3–18months and amino sugar and muramic acid content of the bulk soil and<20μm fraction were determined. Amino sugar content was generally low and changed only slightly in the<20μm fraction with incubation time, which shows that microbial residues in the form of amino sugars did not preferentially accumulate with time. The montmorillonite-containing mixture had a higher galactosamine and glucosamine content than the others, pointing to greater fungal residue formation. The effect was most pronounced for the bulk soil, indicating that fungal residues accumulated on coarse particulate OM and macroaggregates rather than in the fine fraction. The results illustrate that amino sugars can be a biomarker for studying the effect of mineral composition on microbial residues and SOM formation. However, it is important to consider the entire soil structure in order to gain understanding of the interaction between microorganisms, minerals and SOM.

Predominance of Gram‐positive bacteria in house dust in the low‐allergy risk Russian Karelia

Simple living conditions and farming environment have been associated with reduced risk for allergic diseases such as atopy and asthma but the factors responsible for this effect remain unresolved. We examined the bacterial composition of house dusts obtained from Finnish and Russian Karelia, two adjacent areas with high and low occurrence of atopic diseases respectively. Two dust mixes, both composed of 10 randomly selected dust samples from 349 Finnish and 417 Russian Karelian households were studied for bacterial biomarkers (DNA, Limulus-active endotoxin, 3-OH fatty acids, muramic acid) and for 16S rRNA gene sequences. Overall, the DNA cloning revealed more taxons (94 different genera) of dustborne bacteria than seen in any previous study on residential environments. Majority (67%) of the bacterial DNA clones in house dust from the low-allergy Russian Kareliarepresented Gram-positive bacteria (Firmicutes and Actinobacteria), predominantly Staphylococcaceae and Corynebacteriaceae. Russian Karelian dust showed up to 20-fold higher contents of muramic acid (marker of Gram-positive bacteria) and a sevenfold higher number of clones of animal-associated species, whereas in Finnish Karelian dust Gram-negatives (mainly Proteobacteria) predominated. Clones of plant-associated bacterial species and of chloroplast, indicating plant biomass, were more numerous in Finnish than in Russian Karelian dust. In conclusion, this study revealed major disparities between Finnish and Russian house dusts. The higher bacterial content and the predominance of Gram-positive bacteria in Russian dust may have implications for occurrence of atopy.

Peptidoglycan O Acetylation and Autolysin Profile ofEnterococcus faecalisin the Viable but Nonculturable State

The O acetylation of peptidoglycan occurs specifically at the C-6 hydroxyl group of muramoyl residues. Using a combination of high-performance liquid chromatography-based organic acid analysis and carbohydrate analysis by high-pH anion-exchange chromatography, we determined that strains of Entercoccus durans, E. faecalis, E. faecium, and E. hirae produce O-acetylated peptidoglycan. The levels of O acetylation ranged from 19% to 72% relative to the muramic acid content, and they were found to vary with the growth phase of the culture. Increases of 10 to 40% in O acetylation were observed with cultures entering the stationary phase. Cells of E. faecalis in the viable but nonculturable (VBNC) state had the highest levels of peptidoglycan O acetylation. The presence of this modification to peptidoglycan was shown to inhibit the action of hen egg white lysozyme in a concentration-dependent manner. Zymography using sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels containing either O-acetylated or chemically de-O-acetylated peptidoglycan was used to monitor the production of specific autolysins in E. faecalis. Differences in the expression of specific autolysins were observed with the age of the culture, and VBNC E. faecalis produced the highest levels of these enzymes. This technique also permitted classification of the enterococcal autolysins into enzymes that preferentially hydrolyze either O-acetylated or non-O-acetylated peptidoglycan and enzymes that show no apparent preference for either substrate type.

Effects of microbial loading and sporulation temperature on atmospheric plasma inactivation of Bacillus subtilis spores

Current inactivation studies of Bacillus subtilis spores using atmospheric-pressure glow discharges (APGD) do not consider two important factors, namely microbial loading at the surface of a substrate and sporulation temperature. Yet these are known to affect significantly microbial resistance to heat and hydrogen peroxide. This letter investigates effects of microbial loading and sporulation temperature on spore resistance to APGD. It is shown that microbial loading can lead to a stacking structure as a protective shield against APGD treatment and that high sporulation temperature increases spore resistance by altering core water content and cross-linked muramic acid content of B. subtilis spores.

Thermobaculum terrenum gen. nov., sp. nov.: a non-phototrophic gram-positive thermophile representing an environmental clone group related to the Chloroflexi (green non-sulfur bacteria) and Thermomicrobia.

A novel bacterium was cultivated from an extreme thermal soil in Yellowstone National Park, Wyoming, USA, that at the time of sampling had a pH of 3.9 and a temperature range of 65-92 degrees C. This organism was found to be an obligate aerobic, non-spore-forming rod, and formed pink-colored colonies. Phylogenetic analysis of the 16S rRNA gene sequence placed this organism in a clade composed entirely of environmental clones most closely related to the phyla Chloroflexi and Thermomicrobia. This bacterium stained gram-positive, contained a novel fatty-acid profile, had cell wall muramic acid content similar to that of Bacillus subtilis (significantly greater than Escherichia coli), and failed to display a lipopolysaccharide profile in SDS-polyacrylamide gels that would be indicative of a gram-negative cell wall structure. Ultrastructure examinations with transmission electron microscopy showed a thick cell wall (approximately 34 nm wide) external to a cytoplasmic membrane. The organism was not motile under the culture conditions used, and electron microscopic examination showed no evidence of flagella. Genomic G+C content was 56.4 mol%, and growth was optimal at 67 degrees C and at a pH of 7.0. This organism was able to grow heterotrophically on various carbon compounds, would use only oxygen as an electron acceptor, and its growth was not affected by light. A new species of a novel genus is proposed, with YNP1(T) (T=type strain) being Thermobaculum terrenum gen. nov., sp. nov. (16S rDNA gene GenBank accession AF391972). This bacterium has been deposited in the American Type Culture Collection (ATCC BAA-798) and the University of Oregon Culture Collection of Microorganisms from Extreme Environments (CCMEE 7001).

The effect of lime on microbial activity in spruce (Picea abies L.) forests

The use of lime increased heat output and decreased the C:N ratio (global indicators of biological activity) in the organic horizons of a spruce forest (Picea abies L.) soil. These changes were still present after 18 years. During the same period, the muramic acid content increased slightly, while the concentration of both ergosterol and glucosamine decreases. The ratios of ergosterol or glucosamine to muramic acid decreased significantly after 3 years in the plots that had been irrigated and limed, and after 8 years in the limed non-irrigated plots.

Effect of a soluble bacterial carbohydrate fraction on the viscosity of intestinal contents in healthy subjects and patients with Crohn's disease

Abstract. Supernatants of faecal suspensions from patients with Crohn's disease (CD) showed much lower viscosity than those from healthy subjects. Material responsible for the viscosity could be precipitated with ethanol. Gel filtration indicated that the viscosity was not due to the glycoprotein fraction but to a fraction with higher molecular weight and relatively high contents of muramic acid suggesting a bacterial origin. The concentration and viscosity of this fraction are less in faeces from patients with CD than in that of healthy subjects.

Effect of a soluble bacterial carbohydrate fraction on the viscosity of intestinal contents in healthy subjects and patients with Crohn's disease

Supernatants of faecal suspensions from patients with Crohn's disease (CD) showed much lower viscosity than those from healthy subjects. Material responsible for the viscosity could be precipitated with ethanol. Gel filtration indicated that the viscosity was not due to the glycoprotein fraction but to a fraction with higher molecular weight and relatively high contents of muramic acid suggesting a bacterial origin. The concentration and viscosity of this fraction are less in faeces from patients with CD than in that of healthy subjects.

Cell walls from Actinopolyspora halophila, an extremely halophilic actinomycete

Cell wall components were prepared from Actinopolyspora halophila (strain wt), an extremely halophilic actinomycete requiring a minimum 12% NaCl concentration for growth, and from an erythromycin-resistant strain of A. halophila (strain ER) that required only 6% NaCl for growth. Both cell wall preparations contained glutamic acid, alanine, and diaminopimelic acid in a 1:2:1 molar ratio. On the basis of muramic acid content, peptidoglycans from the wt and ER strains contained 255 and 245 disaccharide units per mg dry weight respectively. In addition, both cell wall preparations contained from 10 to 20% more glucosamine than muramic acid, and equimolar amounts of d-galactose and d-arabinose. Analysis of cell walls before and after digestion with Myxobacter AL-1 protease indicated that nearly all glycan disaccharide units were peptide-substituted and that peptide cross-bridging was facilitated by direct peptide linkages between Ne-diaminopimelic acid and C-terminal alanine. While the peptidoglycan of A. halophila wt was 50% peptide cross-linked, that from A. halophila ER was approximately 67% peptide cross-linked. Chemical modifications involving substitution of non-N-acetylated hexosamines of the cell walls greatly enhanced their sensitivity to lysozyme. Although differences in peptidoglycan structure between the two strains of A. halophila were observed, these probably do not account for the reduced salt requirement for growth of the erythromycin-resistant strain.

Muramic Acid Measurements for Bacterial Investigations in Marine Environments by High-Pressure Liquid Chromatography

Muramic acid, a constituent of procaryotic cell walls, was assayed by high-pressure liquid chromatography in samples from several marine environments (water column, surface microlayer, and sediment) and a bacterial culture. It is used as a microbial biomass indicator. The method gave a good separation of muramic acid from interfering compounds with satisfactory reproducibility. A pseudomonad culture had a muramic acid content of 4.7 x 10 to 5.3 x 10 mug per cell during growth. In natural water samples, highly significant relationships were found between muramic acid concentrations and bacterial numbers for populations of 10 to 10 cells per liter. The muramic acid content in natural marine water decreased from 5.3 x 10 to 1.6 x 10 mug per cell with increasing depth. In coastal sediments exposed to sewage pollution, concentrations of muramic acid, ATP, organic carbon, and total amino acids displayed a parallel decrease with increasing distance from the sewage outlet. Advantages of muramic acid measurement by high-pressure liquid chromatography are its high sensitivity and reduction of preparation steps, allowing a short time analysis.

Gas chromatographic-mass spectrometric determination of muramic acid content and pyrolysis profiles for a group of gram-positive and gram-negative bacteria.

Two gas chromatographic-mass spectrometric methods, one based on chemical derivatisation and the other on analytical pyrolysis, were employed for the characterisation of a diverse group of bacteria. The muramic acid content of some Gram-positive and Gram-negative bacteria (including legionellae) was determined by aldononitrile acetate derivativisation and capillary GC-MS analysis. Muramic acid levels were generally higher in Gram-positive than in Gram-negative bacteria. When applied to the same set of bacteria, pyrolysis GC-MS and non-linear mapping were also able to distinguish Gram-negative from Gram-positive bacteria. The potential of GC-MS techniques for the direct chemical characterisation of bacteria is discussed.

Biomass of suspended bacteria over coral reefs

The biomass of bacteria suspended in water flowing over coral reefs at Lizard Island and Yonge Reef (Northern Great Barrier Reef) was estimated by measurement of muramic acid. Values ranged from 20 mg C m-3 in the open water up to about 60 mg C m-3 over the reef flat. Direct counts of total numbers of free bacteria were made for comparison. Values of around 2.0x109 cells μg-1 muramic acid showed that there was a good agreement between direct counts and muramic acid content of free bacteria in the open water. In samples containing suspended particulate matter, ratios of direct counts to muramic acid concentration were lower, because bacteria on particles could not be counted. Thus, these ratios were used to indicate the proportions of bacteria attached to particles. Changes in the biomass and numbers of bacteria were determined in water masses identified either by a drogue or fluorescein, as they moved across the reefs. In the zone on the outside of the reef, the number of free bacteria decreased compared to open sea water, but total biomass increased, showing that particulate matter containing bacteria was thrown up into suspension. About 50% of bacteria were attached to particles. Water flowing over the reef flats contained much particulate material with bacteria attached. Bacteria constituted between about 5 and 20% of particulate organic carbon.

Improved method using muramic acid to estimate biomass of bacteria in sediments.

A method, which depends on the measurement of muramic acid content to estimate bacterial biomass, has been improved in sensitivity by two orders of magnitude. It is now applicable to any aquatic sediment, whereas previously it was mainly useful in the analysis of gut contents of deposit-feeding animals. Reduced NAD, a product of the oxidation of d-lactate derived from muramic acid, is assayed using bacterial luciferase. The amount of muramic acid in a number of terrestrial and marine bacteria was measured, and found to be lower than that obtained with the previous, less specific, assay procedure. The muramic acid content of a blue-green alga has been measured, thus allowing blue-green algae to be taken into account when estimating bacterial biomass. Experimental evidence is presented which shows that muramic acid in cell wall fragments of bacteria is rapidly degraded by microorganisms in a marine sediment.

Galactomuramic Acid: CHEMICAL SYNTHESIS, PROPERTIES, ASSAY, AND SURVEY IN SEVERAL BACTERIAL SPECIES

Abstract Synthetic d-galacto- and d-glucomuramic acids were prepared from the condensation of l-α-chloropropionic acid with benzyl-2-acetamido-4,6-O-benzylidene-2-deoxy-α-d-galacto- or glucopyranoside. The configuration of the d(-) lactate moiety (i.e. 3-O-(d-1-carboxyethyl)-) obtained by alkaline β elimination, was proven by use of specific d(-)- and l(+)-lactic acid dehydrogenases. Standard methods commonly used for the detection of muramic acid were found to be unsuitable for distinction between the two products. Thus, although the Barker and Summerson, Morgan-Elson, Park-Johnson, and Rondle-Morgan tests were shown to detect both gluco and galacto epimers of muramic acid (color yields differed slightly), such colorimetric assays could not differentiate between the analogues. Ionophoresis did not distinguish the forms of muramic acid, and thin layer chromatography was found to be suitable for distinction only under carefully controlled conditions. Cation exchange chromatography, according to Gardell, was used successfully in the separation of the glucomuramic and galactomuramic acids, but it was found that glucosamine co-elutes with galactomuramic acid. The most rapid and precise detection method was that in which a commercial amino acid analyzer was used. Detection of nanomole quantities of material was possible. The muramic acid content of 16 bacterial species was determined. Only the glucomuramic acid analogue was found.