Muramic Acid Concentrations Research Articles

Dietary muramidase increases ileal amino acid digestibility of wheat and corn-based broiler diets without affecting endogenous amino acid losses

The objective of these studies was to evaluate the impact of dietary muramidase (MUR) on endogenous amino acids (AA) losses and digestibility of nutrients in wheat and corn-based broiler diets. In experiment 1, the effect of dietary MUR on the flow of endogenous AA (EAA) at the jejunum and terminal ileum of broilers were assessed using either the nitrogen (N) free diet method (NFD) or the highly digestible protein diet method (HDP; 100 g casein/kg diet). Sialic acid and muramic acid concentrations were measured in the jejunal content. In experiment 2, a 2x2x2 factorial arrangement of treatments with 2 base grains (wheat or corn), with low or high metabolizable energy (ME) levels, and without or with MUR supplementation was implemented. All diets contained phytase, xylanase, and cellulase. Apparent ileal digestibility (AID) of dry matter (DM), protein (CP), amino acids (AA), crude fat, and energy, as well as the apparent total tract metabolizability (ATTM) of DM, CP, and gross energy (GE) were determined. The standardized ileal digestibility (SID) of AA was obtained by correcting AID values for basal ileal EAA obtained from chicks fed with NFD or HDP in experiment 1, jejunal EAA flow of all AA was higher (P < 0.001) compared to the ileum, but this effect was method dependent. Jejunal, but not ileal, EAA flow measured with HDP was higher compared to NFD, as well as sialic acid (P < 0.001) and muramic acid (P < 0.004) concentrations. Muramidase inclusion had no effect on basal EAA flow, independently of the segment and the method used. In experiment 2, dietary MUR supplementation increased the AID of CP (P < 0.05), all AA, and tended (P=0.07) to increase the AID of GE, independently of the cereal type used. However, ATTM of DM and GE, but not CP, increased with MUR inclusion compared with the control treatments, especially in wheat and low ME diets (P < 0.05). In conclusion, MUR supplementation improved AID of CP and AA without affecting EAA losses and increases energy utilization.

Alder expansion increases soil microbial necromass carbon in a permafrost peatland of Northeast China

Northern peatlands have undergone widespread expansion of nitrogen (N)-fixing woody plants such as alder species. However, how alder expansion alters soil microbial necromass carbon (C) is unclear in these ecosystems. We compared the differences in soil stoichiometric ratios among C, nitrogen (N), and phosphorus (P), microbial biomass C (MBC) and N (MBN), basal respiration rate, net N mineralization rate, glucosamine, muramic acid, and galactosamine at 0–10 cm, 10–20 cm, and 20–40 cm layers between Alnus sibirica patches (alder patches) and open peatlands in a permafrost peatland in the Heilongjiang Province of China, and assessed the impact of alder expansion on soil bacterial, fungal, and total necromass C in this peatland. Compared with open peatlands, alder patches have lower C:N ratio at each soil layer but have higher C:P ratio at 10–20 cm layer. Across soil layers, alder patches had higher soil MBC, MBN, basal respiration rate, and net N mineralization rate, but lower soil MBC:MBN ratio than open peatlands. Moreover, alder expansion enhanced the concentrations of glucosamine and muramic acid at 0–10 cm layer, but only increased muramic acid concentration at 20–40 cm layer. Accordingly, soil bacterial, fungal, and total necromass C were increased by alder expansion at 0–10 cm layer, whereas only soil bacterial necromass C was enhanced at 20–40 cm layer. In addition, bacterial necromass C correlated negatively with both soil C:N and MBC:MBN ratios. These results suggest that alder expansion promotes microbial necromass C accumulation in surface soils, and highlight that altered C:N stoichiometry drives alder expansion-induced shifts in soil bacterial necromass C in northern peatlands.

Divergent Accumulation of Microbial Residues and Amino Sugars in Loess Soil after Six Years of Different Inorganic Nitrogen Enrichment Scenarios

Amino sugars are key microbial biomarkers for determining the contribution of microbial residues in soil organic matter (SOM). However, it remains largely unclear as to what extent inorganic nitrogen (N) fertilization can lead to the significant degradation of SOM in alkaline agricultural soils. A six-year field experiment was conducted from 2013 to 2018 to evaluate the effects of chronic N enrichment on microbial residues, amino sugars, and soil biochemical properties under four nitrogen (urea, 46% N) fertilization scenarios: 0 (no-N, control), 75 (low-N), 225 (medium-N), and 375 (high-N) kg N ha−1. The results showed that chronic N enrichment stimulated microbial residues and amino sugar accumulation over time. The medium-N treatment increased the concentration of muramic acid (15.77%), glucosamine (13.55%), galactosamine (18.84%), bacterial residues (16.88%), fungal residues (11.31%), and total microbial residues (12.57%) compared to the control in 2018; however, these concentrations were comparable to the high-N treatment concentrations. The ratio of glucosamine to galactosamine and of glucosamine to muramic acid decreased over time due to a larger increase in bacterial residues as compared to fungal residues. Microbial biomass, soil organic carbon, and aboveground plant biomass positively correlated with microbial residues and amino sugar components. Chronic N enrichment improved the soil biochemical properties and aboveground plant biomass, which stimulated microbial residues and amino sugar accumulation over time.

Earthworms negate the adverse effect of arbuscular mycorrhizae on living bacterial biomass and bacterial necromass accumulation in a subtropical soil

Earthworms and arbuscular mycorrhizal fungi (AMF) have substantial individual effects on the supply and absorption of soil nutrients in terrestrial ecosystems. How their interactions regulate the soil living microbial community and microbial necromass accumulation remains unknown. We hypothesized that the effect of their interaction on the microbial community and necromass accumulation may relate to their controls on status of soil N and P. We conducted a greenhouse experiment manipulating the AMF Rhizophagus intraradices and the earthworm Pontoscolex corethrurus in microcosms planted with a fern Dicranopteris dichotoma. Phospholipid fatty acids and amino sugars were used to indicate the living microbial community and necromass accumulation, respectively. The AMF consistently reduced the biomass of bacterial groups and the concentration of total amino sugars, glucosamine and muramic acid, in the absence of earthworms, but had no effect in their presence. The earthworms increased the concentration of soil total dissolved N, water-soluble N and water-soluble P, and the activity of β–1,4–N-acetylglucosaminidase and phosphatase, and the amount of P in total plant biomass. Redundancy analysis showed that the bacterial biomass was positively correlated with most soil labile N indices and negatively correlated with the amount of N in total plant biomass. Pearson correlation revealed that the bacterial nutritional stress indicator Sat/Mono was negatively correlated with soil NH4+ concentration and positively correlated with the amount of N in total plant biomass. The results suggested that earthworms might negate the suppression effect of AMF on bacterial abundance and necromass accumulation by alleviating AMF-induced N deficiency.

Personal exposure of dairy workers to dust, endotoxin, muramic acid, ergosterol, and ammonia on large-scale dairies in the high plains Western United States

ABSTRACTDairy workers experience a high degree of bioaerosol exposure, composed of an array of biological and chemical constituents, which have been tied to adverse health effects. A better understanding of the variation in the magnitude and composition of exposures by task is needed to inform worker protection strategies. To characterize the levels and types of exposures, 115 dairy workers grouped into three task categories on nine farms in the high plains Western United States underwent personal monitoring for inhalable dust, endotoxin, 3-hydroxy fatty acids (3-OHFA), muramic acid, ergosterol, and ammonia through one work shift. Eighty-nine percent of dairy workers were exposed to endotoxin at concentrations exceeding the recommended exposure guidelines (adjusted for a long work shift). The proportion of workers with exposures exceeding recommended guidelines was lower for inhalable dust (12%), and ammonia (1%). Ergosterol exposures were only measurable on 28% of samples, primarily among medical workers and feed handlers. Milking parlor workers were exposed to significantly higher inhalable dust, endotoxin, 3-OHFA, ammonia, and muramic acid concentrations compared to workers performing other tasks. Development of large modern dairies has successfully made progress in reducing worker exposures and lung disease prevalence. However, exposure to endotoxin, dust, and ammonia continues to present a significant risk to worker health on North American dairies, especially for workers in milking parlors. This study was among the first to concurrently evaluate occupational exposure to assayable endotoxin (lipid A), 3-hydroxy fatty acids or 3-OHFA (a chemical measure of cell bound and noncell-bound endotoxins), muramic acid, ergosterol, and ammonia among workers on Western U.S. dairies. There remains a need for cost-effective, culturally acceptable intervention strategies integrated in OHS Risk Management and production systems to further optimize worker health and farm productivity.

Inflammatory potential in relation to the microbial content of settled dust samples collected from moisture-damaged and reference schools: results of HITEA study

Aiming to identify factors causing the adverse health effects associated with moisture-damaged indoor environments, we analyzed immunotoxicological potential of settled dust from moisture-damaged and reference schools in relation to their microbiological composition. Mouse RAW264.7 macrophages were exposed to settled dust samples (n=25) collected from moisture-damaged and reference schools in Spain, the Netherlands, and Finland. After exposure, we analyzed production of inflammatory markers [nitric oxide (NO), tumor necrosis factor-α (TNF-)α, interleukin (IL)-6, and macrophage inflammatory protein (MIP)2] as well as mitochondrial activity, viability, apoptosis, and cell cycle arrest. Furthermore, particle counts, concentration of selected microbial groups as well as chemical markers such as ergosterol, 3-hydroxy fatty acids, muramic acid, endotoxins, and glucans were measured as markers of exposure. Dust from moisture-damaged schools in Spain and the Netherlands induced stronger immunotoxicological responses compared to samples from reference schools; the responses to Finnish samples were generally lower with no difference between the schools. In multivariate analysis, IL-6 and apoptosis responses were most strongly associated with moisture status of the school. The measured responses correlated with several microbial markers and numbers of particles, but the most important predictor of the immunotoxicological potential of settled dust was muramic acid concentration, a marker of Gram-positive bacteria.

The use of multivariate curve resolution methods to improve the analysis of muramic acid as bacterial marker using gas chromatography–mass spectrometry: An alternative method to gas chromatography–tandem mass spectrometry

In analysis of muramic acid (MA) as bacterial marker, two dominant disturbing factors lead the researchers to use gas chromatography-tandem mass spectrometry (GC-MS/MS) technique instead of gas chromatography-mass spectrometry (GC-MS). These factors are the trace concentration of MA and fundamental disturbance of base line mass channels in GC-MS technique. This study aimed to utilize multivariate curve resolution (MCR) methods combined with GC-MS to improve the analysis of MA. First, the background and noise in GC-MS analysis were corrected and reduced using MCR methods. In addition, the MA overlapped peaks were resolved to its pure chromatographic and mass spectral profiles. Then the two-way response of each component was reconstructed by the outer product of the pure chromatographic and mass spectral profiles. The overall volume integration (OVI) method was used for quantitative determination. The MA peak area was decreased dramatically after the background correction and noise reduction. The findings severely ratify the appropriateness of using MCR techniques combined with GC-MS analysis as a simple, fast and inexpensive method for the analysis of MA in complex mixtures. The proposed method may be considered as an alternative method to GC-MS/MS for thorough analysis of the bacterial marker.

A Mid‐Infrared Spectroscopy Method to Determine the Glucosamine, Galactosamine, and Muramic Acid Concentrations in Soil Hydrolysates

The current method for determining amino sugars in soils involves a derivatization process which is time‐consuming and involves working with and disposing of hazardous chemicals. This study aims to evaluate the potential of mid‐infrared (MIR) spectroscopy to predict amino sugar concentrations in soil. Two separate sets of the same 60 soil samples were hydrolyzed and purified, and then one set of samples was transformed into amino sugar derivatives for gas chromatography (GC) determination and the other set of samples was used for MIR spectra collection without further processing. The GC‐measured concentrations of amino sugars were calibrated, using test‐set data set (n = 20) and leave‐one‐out data set, against the MIR spectral data with partial least squares (PLS) regression. Based on the values of coefficient of determination (R2) and residual prediction deviation (RPD), the calibration models for amino sugars were good for both data sets, with R2 values ranging from 0.82 to 0.98 and RPD values from 2.38 to 7.76. For test‐set validation, predictions were excellent for total amino sugars (R2 = 0.96, RPD = 5.14), glucosamine (R2 = 0.97, RPD = 6.27), galactosamine (R2 = 0.94, RPD = 4.16), and good for muramic acid (R2 = 0.84, RPD = 2.52). For leave‐one‐out cross‐validation, predictions were excellent for glucosamine (R2 = 0.91, RPD = 3.28), good for total amino sugars (R2 = 0.86, RPD = 2.70) and galactosamine (R2 = 0.81, RPD = 2.29), but relatively poor for muramic acid (R2 = 0.61, RPD = 1.61). We concluded that the MIR spectroscopy has high potential to estimate the concentrations of amino sugars in soil with significant savings in time, cost, and chemicals.

A longitudinal study of sick building syndrome among pupils in relation to microbial components in dust in schools in China

There are few longitudinal studies on sick building syndrome (SBS), which include ocular, nasal, throat, and dermal symptoms, headache, and fatigue. We studied the associations between selected microbial components, fungal DNA, furry pet allergens, and incidence and remission of SBS symptoms in schools in Taiyuan, China. The study was based on a two-year prospective analysis in pupils (N=1143) in a random sample of schools in China. Settled dust in the classrooms was collected by vacuum cleaning and analyzed for lipopolysaccharide (LPS), muramic acid (MuA), and ergosterol (Erg). Airborne dust was collected in Petri dishes and analyzed for cat and dog allergens and fungal DNA. The relationship between the concentration of allergens and microbial compounds and new onset of SBS was analyzed by multi-level logistic regression. The prevalence of mucosal and general symptoms was 33% and 28%, respectively, at baseline, and increased during follow-up. At baseline, 27% reported at least one symptom that improved when away from school (school-related symptoms). New onset of mucosal symptoms was negatively associated with concentration of MuA, total LPS, and shorter lengths of 3-hydroxy fatty acids from LPS, C14, C16, and C18. Onset of general symptoms was negatively associated with C18 LPS. Onset of school-related symptoms was negatively associated with C16 LPS, but positively associated with total fungal DNA. In general, bacterial compounds (LPS and MuA) seem to protect against the development of mucosal and general symptoms, but fungal exposure measured as fungal DNA could increase the incidence of school-related symptoms.

Soil microorganisms and the growth of Lupinus albus on a high metal soil in the presence of EDTA

A pot experiment was designed with the objective of determining whether the presence of ethylenediaminetetraacetic acid (EDTA) and the resulting mobilization of heavy metals have any affect on: (i) soil microorganisms, (ii) growth of L. albus, and (iii) microbial colonization of roots. There was no effect of the different treatments on the contents of soil microbial biomass C and microbial biomass N. Increasing addition of EDTA to soil led to proportionate increases in extractable C and N, being roughly equivalent to the added amount. Increasing EDTA addition to soil led also to a proportionate increase in mobile heavy metals. Plant height, total amount of shoot and root C were not affected by EDTA addition. Fungal ergosterol in the lupine roots showed a 5- to 8-fold increase in the 0.1 and 0.2% EDTA treatments in comparison with the control. In contrast, EDTA addition did not affect fungal glucosamine or bacterial muramic acid concentrations in the lupine roots. Increasing EDTA addition to soil led also to a proportionate increase in the metal concentrations in the lupine shoots. The concentrations of heavy metals in the lupine shoots and in the NH4NO3 soil extracts were all highly significantly correlated.

Litter decomposition in fertilizer treatments of vegetable crops under irrigated subtropical conditions

In the coastal Batinah plain of Oman, a litterbag experiment was carried out in an irrigated field, investigating the effects of organic fertilization and mineral fertilization on the cultivation of carrots and cauliflower. Two straw varieties and two green-harvested crops were used, simulating the properties of green manures. The loss of C in the litterbags declined in the order maize (−94%) > alfalfa (−89%) > wheat (−80%) > canola (−69%). For all these materials, the concentration of muramic acid, as an indicator of bacterial C, as well as galactosamine was generally increased in comparison with the initial values. In contrast, fungal glucosamine and consequently also the ratio of fungal C/bacterial C declined for canola and wheat straw. The loss of N, P, and S was generally smaller than that of C and showed strong substrate-specific patterns. Fertilization and crop cultivation had no effect on C losses. Organic fertilization resulted in significant increases in S, Mg, and Al in the litterbags in comparison with mineral fertilization. Cultivation of carrots led to significantly lower ash, N, P, Ca, K, Na, and Al concentrations than cultivation of cauliflower. Organic fertilization and carrot cultivation both led to stronger fungal colonization of the litter retained in the litterbags in comparison with mineral fertilization and cauliflower cultivation, respectively. More information is required on the interactions between initial plant surface colonizing microorganisms and soil-derived colonizers.

Determination of bacterial load in house dust using qPCR, chemical markers and culture

In this study, we developed two novel qPCR-assays for the detection of bacteria in house dust; one that determines the total bacterial amount and another that detects Gram-positive and Gram-negative bacteria separately. The methods were tested in silico and in vitro with microbial strains and vacuum cleaner dust samples, and validated in relation to culture and chemical marker analysis. We also compared the results of these three types of methods (qPCR, culture and chemical marker analysis) in 211 house dust samples from farming and non-farming environments. Microbial concentrations determined by the new qPCR assays (median 7.2 x 10(5) cell equivalents mg(-1)) were about two orders of magnitude higher than concentrations obtained by culture (median 6.7 x 10(3) cfu mg(-1)). The median concentration of muramic acid was 25.67 ng mg(-1) and that of 3-hydroxy fatty acids, expressed as LPS(10-16) was 26.14 pg mg(-1). Correlations between qPCR and chemical markers were moderate, while correlations between culture and qPCR and chemical markers were low to moderate. All the methods used in this study showed that the microbial concentrations are statistically significantly higher (p < 0.001, Mann-Whitney) in farming than non-farming environments.As a conclusion, all tested methods can be used for determining the bacterial load in dust samples, but none of the methods was superior to the others. The results obtained with these methods represent different aspects of bacterial exposure and therefore the results are not expected to be identical with each other.

Microbial colonisation of roots as a function of plant species

Fifteen plants species were grown in the greenhouse on the same soil and sampled at flowering to obtain rhizosphere soil and root material. In both fractions, the data on fungal and bacterial tissue obtained by amino sugar analysis were compared with the total microbial biomass based on fumigation-extraction and ergosterol data. The available literature on glucosamine concentrations in fungi and on muramic acid concentrations in bacteria was reviewed to prove the possibility of generating conversion values for general use in root material. All microbial properties analysed revealed strong species-specific differences in microbial colonisation of plant roots. The root material contained considerable amounts of microbial biomass C and biomass N, reaching mean levels of 10.9 and 1.4 mg g −1 dry weight, respectively. However, the majority of CHCl 3 labile C and N, i.e. 89 and 55% was root derived. The average amount of ergosterol was 13 μg g −1 dry weight and varied between 0.0 for Phacelia roots and 45.5 μg g −1 dry weight for Vicia roots. The ergosterol content in root material of mycorrhizal and non-mycorrhizal plant species did not differ significantly. Fungal glucosamine was converted to fungal C by multiplication by 9 giving a range of 7.1–25.9 mg g −1 dry weight in the root material. Fungal C and ergosterol were significantly correlated. Bacterial C was calculated by multiplying muramic acid by 45 giving a range from 1.7 to 21.6 mg g −1 dry weight in the root material. In the root material of the 15 plant species, the ratio of fungal C-to-bacterial C ranged from 1.0 in mycorrhizal Trifolium roots to 9.5 in non-mycorrhizal Lupinus roots and it was on average 3.1. These figures mean that the microbial tissue in the root material consists on average of 76% fungal C and 24% bacterial C. The differences in microbial colonisation of the roots were reflected by differences in microbial indices found in the rhizosphere soil, most strongly for microbial biomass C and ergosterol, but to some extent also for glucosamine and muramic acid.

A novel GC/MS technique to assess 15N and 13C incorporation into soil amino sugars

Amino sugars have been used as biomarker to indicate microorganism contribution to soil organic matter turnover and sequestration. However, there is no direct gas chromatograph mass spectrometry (GC/MS) approach to assess microbial synthesis of amino sugars in soil. We developed a novel method which combines laboratory incubation of substrate containing 15N or 13C and a GC/MS technique to trace 15N or 13C isotope changes in three amino sugars, glucosamine, galactosamine, and muramic acid. Sample preparation followed the procedure of Zhang and Amelung (1996) [Zhang, X., Amelung, W., 1996. Gas chromatographic determination of muramic acid, glucosamine, galactosamine, and mannosamine in soils. Soil Biology and Biochemistry 28, 1201–1206.]. The GC/MS determination was conducted using a full scan mode with both electronic ionization (EI) and chemical ionization (CI) sources. The CI source was suitable for all of the three amino sugars, while the EI source was not applicable to muramic acid due to its low sensitivity in the determination as well as low concentration of muramic acid in soil. The enrichment of 15N or 13C in amino sugars during incubation was estimated by calculating the atom percentage excess (APE). 15N incorporation was evaluated according to fragment ( F) abundance ratio of mass F+1 to F, whilst 13C incorporation was estimated according to the ratio of mass F+ n to F ( n is skeleton carbon number in the fragment). This novel method was assessed by using two soil samples (a Kandiudult and a Udoll) incubated with either 15N-amonium or U- 13C-glucose. The results indicate that the GC/MS determination is reproducible, thus this technique is useful in detecting the microbial synthesis of amino sugars in soil, and especially it should be possible when looking at the position or how much labeled carbon and nitrogen atoms have been incorporated.

Microbial exposure of rural school children, as assessed by levels of N-acetyl-muramic acid in mattress dust, and its association with respiratory health

Background Endotoxin exposure has been shown to be associated with a decreased prevalence of atopic sensitization and symptoms. Yet endotoxin represents only a part of the indoor microbial exposure. Muramic acid, a constituent of peptidoglycan, is present in gram-negative and gram-positive bacteria in the environment and may therefore serve as an additional marker of microbial exposure. Objective To study the factors determining the level of indoor exposure to muramic acid/peptidoglycan, as well as its potential association with respiratory health. Methods In 553 farm and nonfarm school children from Austria, Switzerland, and Germany, mattress dust muramic acid concentrations were determined, and health was assessed by using IgE measurements and questionnaire information. Results The muramic acid concentration was found to be significantly higher in dust from farm children's mattresses than in dust from nonfarm children's mattresses (157 vs 131 ng/mg). Children with higher mattress dust muramic acid concentrations had a significantly lower prevalence of wheezing (odds ratio of highest vs lowest tertile of muramic acid concentration, 0.3; 95% CI, 0.1-0.9), regardless of farming status and endotoxin exposure. The association for asthma was similar, and no association was found with atopic sensitization. Conclusion Next to endotoxin, muramic acid provides us with an independent marker of microbial exposure. Unlike endotoxin, muramic acid was inversely associated with wheezing rather than with atopic sensitization.

Amino sugars and muramic acid—biomarkers for soil microbial community structure analysis

Characterizing functional and phylogenetic microbial community structure in soil is important for understanding the fate of microbially-derived compounds during the decomposition and turn-over of soil organic matter. This study was conducted to test whether amino sugars and muramic acid are suitable biomarkers to trace bacterial, fungal, and actinomycetal residues in soil. For this aim, we investigated the pattern, amounts, and dynamics of three amino sugars (glucosamine, mannosamine and galactosamine) and muramic acid in the total microbial biomass and selectively cultivated bacteria, fungi, and actinomycetes of five different soils amended with and without glucose. Our results revealed that total amino sugar and muramic acid concentrations in microbial biomass, extracted from soil after chloroform fumigation varied between 1 and 27 mg kg −1 soil. In all soils investigated, glucose addition resulted in a 50–360% increase of these values. In reference to soil microbial biomass-C, the total amino sugar- and muramic acid-C concentrations ranged from 1–71 g C kg −1 biomass-C. After an initial lag phase, the cultivated microbes revealed similar amino sugar concentrations of about 35, 27 and 17 g glucosamine-C kg −1 TOC in bacteria, fungi, and actinomycetes, respectively. Mannosamine and galactosamine concentrations were lower than those for glucosamine. Mannosamine was not found in actinomycete cultures. The highest muramic acid concentrations were found in bacteria, but small amounts were also found in actinomycete cultures. The concentrations of the three amino sugars studied and muramic acid differed significantly between bacteria and the other phylogenetic microbial groups under investigation (fungi and actinomycetes). Comparison between the amino sugar and muramic acid concentrations in soil microbial biomass, extracted after chloroform fumigation, and total concentrations in the soil showed that living microbial biomass contributed negligible amounts to total amino sugar contents in the soil, being at least two orders of magnitude greater in the soils than in the soil inherent microbial biomass. Thus, amino sugars are significantly stabilized in soil.

Detection of bacterial components in synovial tissue from patients with inflammatory arthritis by using PCR with pan bacterial 23S rRNA and 16S rRNA primers, and gas chromatography-mass spectrometry

Using PCR for 16S rRNA, the presence of bacterial DNA in synovial tissue (ST) from a variety of inflammatory arthritides has been reported. To confirm this, we have applied the PCR with pan bacterial 23S rRNA and 16S rRNA primers, which both methods have been used successfully for bacterial identification in various clinical samples. ST were collected at joint surgery from 81 patients: 42 rheumatoid arthritis (RA), 31 osteoarthritis (OA), 8 other inflammatory arthritides. Extremely strict precautions were followed in the clinics and laboratory to prevent contamination. Bacterial DNA could not been detected by PCR with pan 23S rRNA and 16S rRNA in any of the samples. The positive controls, including bacterial DNA and human DNA, were run with each sample, and were always positive. Further, using the same method, 5/15 (33%) synovial fluid samples from patients with Chlamydia reactive arthritis were PCR positive. The PCR sensitivity was 2-20 CFU/reaction determined by mixing the living bacteria with ST and using exactly the same experimental procedure as with the patient samples. Gas chromatography-mass spectrometry has been applied to detect muramic acid (bacterial cell wall specific chemical component) in ST. Preliminary results suggest that low concentration of muramic acid can be detected in the ST from some patients with inflammatory arthritis. Our results show that bacterial DNA in ST from RA and OA could not been detected by PCR for 23S rRNA and 16S rRNA. Instead, muramic acid could be detected by gas chromatography-mass spectrometry. These observations indicate that the presence of bacterial DNA in ST might not be as prevalent as previously suggested. Nevertheless, the bacterial components may exist in ST.

Total and viable airborne bacterial load in two different agricultural environments using gas chromatography-tandem mass spectrometry and culture: a prototype study.

Airborne exposure to bacterial components found in agricultural environments can lead to pulmonary inflammation. Total (viable and nonviable) bacterial load was monitored in a stable and a dairy by a new approach, gas chromatography-tandem mass spectrometry measurement of muramic acid, a component of gram positive and gram negative bacterial peptidoglycan. Also used to assess the gram negative bacterial load were 3-hydroxy fatty acids, markers of bacterial lipopolysaccharide. Culture, an established procedure for assessing the viable bacterial portion of airborne dust, served as a basis for comparison. The muramic acid and 3-hydroxy fatty acid concentrations (total C12:0, C14:0, and C16:0) showed a correlation with an R2 of 0.81. Dust and muramic acid levels also correlated. However, although relative muramic acid levels were lower in the stable than the dairy, colony forming units (CFU) were considerably higher in the stable. The total bacterial load (estimated from muramic acid values) for both the stable and dairy was also higher than would have been predicted from culture. These results suggest that nonculture based approaches and culture provide complementary but independent measurements of airborne biopollution.

Desert system microbial biomass determined by phospholipid phosphate and muramic acid measurement

The effect of soil moisture on the biomass of the natural microbial communities in the Negev Desert was studied during 1991–1993 using measurements of phospholipid phosphate and muramic acid. The immediate response of the microbial communities to varying amounts of nitrogen added as a single pulse was also studied. Two different weather conditions were observed during the study period: (1) the winter of 1992, which was very cold and snowy, with intermittent rainfall occurring at the end of winter and beginning of spring; (2) the winter of 1993 which was warmer, without snow, but with periodical rainfall occurring through early spring. Soil samples collected from the 0–10 cm and 10–20 cm depths during 1991–1992 showed significant changes in soil moisture and phospholipid phosphate and muramic acid concentrations following precipitation events. The greatest concentration of phospholipid phosphate was observed in December 1991. The concentrations of phospholipid phosphate at the two sample depths were 4–50 times greater than those found during other months. The concentration of muramic acid (31·19 mg g−1) was greatest in March at 0–10 cm depth, as compared with the greatest concentration (46·37 mg g−1) at 10–20 cm depth, which occurred in January. These muramic acid concentrations were 2–3 times greater than those found during other months. In 1992–93, soil samples were collected from 0–10 cm depth areas amended with three different concentrations of nitrogen (25, 50 or 100 kg NH4NO3 ha−1) and from unamended soil. Fluxes of the microbial communities (phospholipid phosphate and muramic acid) were correlated with the nitrogen treatments and diurnal fluctuations in soil moisture. The greatest concentrations of phospholipid phosphate and muramic acid were found in soil treated with 50 or 100 kg NH4NO3 ha−1. Our results demonstrated that phospholipid phosphate and muramic acid concentrations were greatest and more stable after nitrogen addition than in control soils lacking nitrogen amendments. This paper demonstrates that the soil microbiota in the Negev Desert ecosystem are dependent on the moisture and nitrogen content of the soil and are influenced by seasonal variations in weather conditions, as well as by individual precipitation levels.

Prolonged simulated acid rain treatment in the subarctic: Effect on the soil respiration rate and microbial biomass

Humus chemistry and respiration rate, ATP, ergosterol, and muramic acid concentration as measures of chemical properties, microbial activity, biomass, and indicators of fungal and bacterial biomass were studied in a long-term acid rain experiment in the far north of Finnish Lapland. The treatments used in this study were dry control, irrigated control (spring water, pH 6), and two levels of simulated acid rain (pH 4 and pH 3). Originally (1985–1988), simulated acid rain was prepared by adding both H2SO4 and HNO3 (1.9:1 by weight). In 1989 the treatments were modified as follows. In subarea 1 the treatments continued unchanged (H2SO4+HNO3 in rain to pH 4 and pH 3), but in subarea 2 only H2SO4 was applied. The plots were sampled in 1992. The acid application affected humus chemistry by lowering the pH, cation exchange capacity, and base saturation (due to a decrease in Ca and Mg) in the treatment with H2SO4+HNO3 to pH 4 (total proton load over 8 years 2.92 kmol ha-1), whereas the microbial variables were not affected at this proton load, and only the respiration rate decreased by 20% in the strongest simulated acid rain treatment (total proton load 14.9 kmol ha-1). The different ratios of H2SO4+HNO3 in subareas 1 and 2 did not affect the results.