Development of analytical methods for the determination of 3-hydroxy fatty acids and muramic acid as bacterial markers in airborne particles and settled dust.

Inhalation of airborne bacteria in indoor environments is known to be associated with respiratory diseases. Analytical methods for the determination of 3-hydroxy fatty acids (3-OHFAs) and muramic acid (MA) as chemical markers of gram-negative and gram-positive bacteria, respectively, were developed for airborne particle and dust samples in this study. 3-OHFAs as markers of endotoxin were released and esterified during the hydrolysis process under methanolic acid conditions, and their hydrolysates, i.e., 3-OHFA methyl esters, were cleaned up by solid-phase extraction using silica sorbent that provided more effective separation from interferents than polymeric sorbent through elution pattern. The SPE eluent was analyzed by GC-MS/MS measurement after the trimethylsilylation reaction. The recovery of the method ranged from 82.1 % to 103.2 %, with a limit of detection ranging from 0.5 to 1.1 ng/filter and good linearity (R2 > 0.991). For the analysis of MA, muramic acid methyl ester (MAME), a product formed during methanolic hydrolysis, was selected as a specific marker of peptidoglycan. It was the first proposed compound identified and confirmed with MS and MS/MS spectra using high-resolution measurement. In particular, the measurement of MAME providing 12.5 times greater sensitivity than MA with the application of the LC-MS/MS method is one of the notable findings of this study. The recovery by simple liquid extraction was 99.4 % following the removal of the hydrophobic matrix and neutralization with solvent reconstruction. The method displayed a LOD of 0.7 ng/filter and linearity (R2) of 0.997 through a simple pretreatment process. Both developed methods were applied and evaluated by determining 3-OHFAs and MA in airborne particles collected from multipurpose facilities and settled dust in the laboratory and office.

Peptidoglycan Deacetylases in Bacterial Cell Wall Remodeling and Pathogenesis.

The bacterial cell wall peptidoglycan (PG) is a dynamic structure that is constantly synthesized, re-modeled and degraded during bacterial division and growth. Postsynthetic modifications modulate the action of endogenous autolysis during PG lysis and remodeling for growth and sporulation, but also they are a mechanism used by pathogenic bacteria to evade the host innate immune system. Modifications of the glycan backbone are limited to the C-2 amine and C-6 hydroxyl moieties of either GlcNAc or MurNAc residues. This paper reviews the functional roles and properties of peptidoglycan de-Nacetylases (distinct PG GlcNAc and MurNAc deacetylases) and recent progress through genetic studies and biochemical characterization to elucidate their mechanism of action, 3D structures, substrate specificities and biological functions. Since they are virulence factors in pathogenic bacteria, peptidoglycan deacetylases are potential targets for the design of novel antimicrobial agents.

Quantitation of amino sugar stereoisomer and muramic acid biomarkers by hydrophilic interaction liquid chromatography-mass spectrometry.

A rapid separation and quantitation of the stereoisomer amino sugars glucosamine, galactosamine, and mannosamine, along with muramic acid, is needed. These compounds, when their quantities are accurate, can be used to understand the origin and fate of natural organic matter (NOM) in the environment. These target molecules are biomarkers of fungi and bacteria and allow the deconvolution of microbial transformations and degradation of NOM in a wide variety of environmental matrices. Analytical methods applied to this suite of biomarkers are needed to understand carbon and nitrogen biogeochemistry with a changing global climate. Traditional separations of these analytes by gas chromatography require sample derivatization, as does reverse phase liquid chromatography. In contrast, ion chromatography can separate the analytes directly, but requires a separate analytical method to quantify muramic acid. In this work we present a direct analysis of all these molecules using hydrophilic liquid interaction chromatography. Solvent composition, buffer strength, pH, flow rate, and column temperature were optimized. The method can separate these four compounds and the biopolymeric precursor molecule N-acetylglucosamine in a single run in under 8 min with equivalent resolution to the best previously reported separations that did not require derivatization prior to analysis. Detection of the analytes was performed by both tandem and time-of-flight mass spectrometry. The method was assessed for its quantitative capabilities through i) peak area assignment, ii) check standards with ratios of the target analytes likely to be present in real samples, iii) an injection internal standard, and iv) quantitative analysis of real soil hydrolysates by external calibration and standard addition approaches. Across their expected analytical ranges the response for each analyte was highly linear with good accuracy (<25%) and precision (<15%) over three orders of magnitude. Detection limits of 20 µg L-1 were found for galactosamine and 5 µg L-1 for the remainder of the analytes, comparable to the majority of other methods reported in the literature. Overall, this new approach can directly and rapidly quantify amino sugars recovered in environmental hydrolysates.

Peptidoglycan-type analysis of the N-acetylmuramic acid auxotrophic oral pathogen Tannerella forsythia and reclassification of the peptidoglycan-type of Porphyromonas gingivalis.

BACKGROUND: Tannerella forsythia is a Gram-negative oral pathogen. Together with Porphyromonas gingivalis and Treponema denticola it constitutes the "red complex" of bacteria, which is crucially associated with periodontitis, an inflammatory disease of the tooth supporting tissues that poses a health burden worldwide. Due to the absence of common peptidoglycan biosynthesis genes, the unique bacterial cell wall sugar N-acetylmuramic acid (MurNAc) is an essential growth factor of T. forsythia to build up its peptidoglycan cell wall. Peptidoglycan is typically composed of a glycan backbone of alternating N-acetylglucosamine (GlcNAc) and MurNAc residues that terminates with anhydroMurNAc (anhMurNAc), and short peptides via which the sugar backbones are cross-linked to build up a bag-shaped network. RESULTS: We investigated T. forsythia's peptidoglycan structure, which is an essential step towards anti-infective strategies against this pathogen. A new sensitive radioassay was developed which verified the presence of MurNAc and anhMurNAc in the cell wall of the bacterium. Upon digest of isolated peptidoglycan with endo-N-acetylmuramidase, exo-N-acetylglucosaminidase and muramyl-L-alanine amidase, respectively, peptidoglycan fragments were obtained. HPLC and mass spectrometry (MS) analyses revealed the presence of GlcNAc-MurNAc-peptides and the cross-linked dimer with retention-times and masses, respectively, equalling those of control digests of Escherichia coli and P. gingivalis peptidoglycan. Data were confirmed by tandem mass spectrometry (MS2) analysis, revealing the GlcNAc-MurNAc-tetra-tetra-MurNAc-GlcNAc dimer to contain the sequence of the amino acids alanine, glutamic acid, diaminopimelic acid (DAP) and alanine, as well as a direct cross-link between DAP on the third and alanine on the fourth position of the two opposite stem peptides. The stereochemistry of DAP was determined by reversed-phase HPLC after dabsylation of hydrolysed peptidoglycan to be of the meso-type. CONCLUSION: T. forsythia peptidoglycan is of the A1γ-type like that of E. coli. Additionally, the classification of P. gingivalis peptidoglycan as A3γ needs to be revised to A1γ, due to the presence of meso-DAP instead of LL-DAP, as reported previously.

Williamsia aurantiacus sp. nov. a novel actinobacterium producer of antimicrobial compounds isolated from the marine sponge.

An antibiotic-producing actinobacterium, designated isolate B375T, was isolated from marine sponge Glodia corticostylifera collected from Praia Guaecá, São Paulo, Brazil (23°49S; 45°25W), and its taxonomic position established using data from a polyphasic study. The organism showed a combination of morphological, physiological, biochemical and chemotaxonomic characteristics consistent with its classification in the genus Williamsia. Comparative 16S rRNA gene sequence analysis indicated that the strain B375T was most closely related to Williamsia serinedens DSM 45037T and Williamsia spongiae DSM 46676T and having 99.43% and 98.65% similarities, respectively, but was distinguished from these strains by a low level of DNA-DNA relatedness (53.2-63.2%) and discriminatory phenotypic properties. Chemotaxonomic investigations revealed the presence of cell-wall chemotype IV and N-glycolated muramic acid residues present in the wall cells. The cells contained C16:0 (23.3%), C18:0 10-methyl (23.2%) and C18:1 ω9c (21.6%) as the major cellular fatty acids. The strain B375T inhibited growing of Staphylococcus aureus and Colletotrichum gloeosporioides strains and was considered a producer of antimicrobial compounds. Based on the data obtained, the isolate B375T (= CBMAI 1090T = DSM 46677T) should, therefore, be classified as the type strain of a novel species of the genus Williamsia, for which the name Williamsia aurantiacus sp. nov. is proposed.

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

Dairy 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.

Pulmonary function and airway inflammation among dairy parlor workers after exposure to inhalable aerosols.

BACKGROUND: Inhalation exposure to organic dust causes lung inflammation among agricultural workers. Due to changes in production and work organization, task-based inhalation exposure data, including novel lung inflammation biomarkers, will inform exposure recommendations for dairy farm workers. METHODS: Linear regression was used to estimate the associations of airborne exposure to dust concentration, endotoxin, and muramic acid with pulmonary outcomes (i.e., FEV1 , exhaled nitric oxide). Logistic regression was used to estimate associations with self-reported pulmonary symptoms. RESULTS: Mean exposure concentration to inhalable dust, endotoxin, and muramic acid were 0.55 mg/m3 , 118 EU/m3 , and 3.6 mg/m3 , respectively. We found cross-shift differences for exhaled nitric oxide (P = 0.005) and self-reported pulmonary symptoms (P = 0.008) but no association of exposure with respiratory outcomes. CONCLUSIONS: Inhalation exposures during parlor tasks, which were lower than previously reported and were not associated with cross-shift measures of pulmonary health among dairy workers. Modern milking parlor designs may be contributing to lower inhalation exposure. Am. J. Ind. Med. 60:255-263, 2017. © 2017 Wiley Periodicals, Inc.

Muramic and dipicolinic acids in atmospheric particulate matter as biomarkers of bacteria and bacterial spores.

Airborne bacteria are components of the atmospheric aerosol particles and can be responsible of allergic disease, regardless of their viability. In this paper, we report a method for the determination of total (viable and nonviable) bacterial content in airborne particles, using muramic and dipicolinic acids as biomarkers of bacteria and bacterial spores, respectively. The analytical procedure was optimized with bacteria and spores of Bacillus subtilis. After extraction and purification, the two biomarkers were analyzed by HPLC-ESI-MS/MS and their percentage was evaluated to be used as conversion factor. The present method for the determination of the total bacterial content was then applied to environmental samples, after a proper collection in an urban site. Thanks to the use of a low pressure impactor, capable of fractionating particles into the range of 0.03-10 µm, it was also possible to study the bacterial content in ultrafine, fine, and coarse particulate matter. The results from this study showed that muramic and dipicolinic acids can be determined together in one chromatographic run in reversed phase ion pair chromatography. Bacteria were more abundant than bacterial spores in the urban atmosphere, both showing a higher concentration in the coarse fraction of particles, although bacteria and bacterial spore amounts per unit mass of ultrafine particles were higher than in fine and coarse particles.

Endotoxin, ergosterol, muramic acid and fungal DNA in dust from schools in Johor Bahru, Malaysia--Associations with rhinitis and sick building syndrome (SBS) in junior high school students.

This paper studied associations between ocular symptoms, rhinitis, throat and dermal symptoms, headache and fatigue in students by ethnicity and in relation to exposure to chemical microbial markers and fungal DNA in vacuumed dust in schools in Malaysia. A total of 462 students from 8 randomly selected secondary schools in Johor Bahru, Malaysia, participated (96% response rate). Dust was vacuumed from 32 classrooms and analysed for levels of five types of endotoxin as 3-hydroxy fatty acids (C10, C12, C14, C16 and C18 3-OH), muramic acid, ergosterol and five sequences of fungal DNA. Multiple logistic regression was applied. Totally 11.9% reported weekly ocular symptoms, 18.8% rhinitis, 15.6% throat and 11.1% dermal symptoms, 20.6% headache and 22.1% tiredness. Totally 21.1% reported pollen or furry pet allergy (atopy) and 22.0% parental asthma or allergy. Chinese students had less headache than Malay and Indian had less rhinitis and less tiredness than Malay. Parental asthma/allergy was a risk factor for ocular (odds ratio=3.79) and rhinitis symptoms (OR=3.48). Atopy was a risk factor for throat symptoms (OR=2.66), headache (OR=2.13) and tiredness (OR=2.02). There were positive associations between amount of fine dust in the dust samples and ocular symptoms (p<0.001) and rhinitis (p=0.006). There were positive associations between C14 3-OH and rhinitis (p<0.001) and between C18 3-OH and dermal symptoms (p=0.007). There were negative (protective) associations between levels of total endotoxin (LPS) (p=0.004) and levels of ergosterol (p=0.03) and rhinitis and between C12 3-OH and throat symptoms (p=0.004). In conclusion, the amount of fine dust in the classroom was associated with rhinitis and other SBS symptoms and improved cleaning of the schools is important. Endotoxin in the school dust seems to be mainly protective for rhinitis and throat symptoms but different types of endotoxin could have different effects. The ethnic differences in symptoms among the students deserve further attention.

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.

Predictors of microbial agents in dust and respiratory health in the Ecrhs.

BACKGROUND: Dampness and mould exposure have been repeatedly associated with respiratory health. However, less is known about the specific agents provoking or arresting health effects in adult populations. We aimed to assess predictors of microbial agents in mattress dust throughout Europe and to investigate associations between microbial exposures, home characteristics and respiratory health. METHODS: Seven different fungal and bacterial parameters were assessed in mattress dust from 956 adult ECRHS II participants in addition to interview based home characteristics. Associations between microbial parameters and the asthma score and lung function were examined using mixed negative binomial regression and linear mixed models, respectively. RESULTS: Indoor dampness and pet keeping were significant predictors for higher microbial agent concentrations in mattress dust. Current mould and condensation in the bedroom were significantly associated with lung function decline and current mould at home was positively associated with the asthma score. Higher concentrations of muramic acid were associated with higher mean ratios of the asthma score (aMR 1.37, 95%CI 1.17-1.61). There was no evidence for any association between fungal and bacterial components and lung function. CONCLUSION: Indoor dampness was associated with microbial levels in mattress dust which in turn was positively associated with asthma symptoms.

Asthma and the hygiene hypothesis. Does cleanliness matter?

RATIONALE: The early hygiene hypothesis explained the development of allergies by a lack of infections; nowadays, the aspect of excessive cleanliness in affluent populations seems to have replaced this concept. Yet, no investigation has shown that home or personal cleanliness relate to allergic diseases. OBJECTIVES: To relate personal and home cleanliness to risk of asthma and allergies. METHODS: Comprehensive questionnaire information on home or personal cleanliness and allergic health conditions at school age was collected in 399 participants of the urban Perinatale Asthma Umwelt Langzeit Allergie Studie (PAULA) birth cohort. Bacterial markers were assessed in floor and mattress dust and were related to cleanliness and allergic diseases. MEASUREMENTS AND MAIN RESULTS: Personal cleanliness was inversely related to bacterial compounds on floors and mattresses, whereas home cleanliness effectively reduced dust amount but not microbial markers. Exposure to muramic acid related to a lower prevalence of school-age asthma (adjusted odds ratio, 0.59 [95% confidence interval, 0.39; 0.90]). Mattress endotoxin in the first year of life was inversely associated with atopic sensitization (0.73 [0.56-0.96]) and asthma at school age (0.72 [0.55-0.95]). Despite the associations of dust parameters both with cleanliness and allergic health conditions, the development of allergies was not related to home and personal cleanliness. CONCLUSIONS: Bacterial exposure in house dust determined childhood asthma and allergies. Personal cleanliness, such as washing hands, and home cleanliness were objectively reflected by dust parameters in homes. However, neither personal nor home cleanliness was associated with a risk for asthma and allergies. Other microbial components in house dust not affected by personal hygiene are likely to play a role.

Determinants, reproducibility, and seasonal variation of bacterial cell wall components and viable counts in house dust.

The objectives of this study were (i) to assess the determinants that affect concentrations of the bacterial cell wall components 3-hydroxy fatty acids (3-OH FAs) and muramic acid and of total viable bacteria and actinomycetes in house dust; and (ii) to examine the seasonal variation and reproducibility of these bacterial cell wall components in house dust. A number of lifestyle and environmental factors, mostly not consistent for different bacterial measures but commonly including the type of dwelling and farming (number of livestock), explained up to 37% of the variation of the bacterial concentrations in 212 homes in Eastern Finland. The reproducibility of 3-OH FAs and muramic acid measurements in house dust were studied in five urban homes and were found to be generally high (ICC 74-84%). Temporal variation observed in repeated sampling of the same home throughout a year was more pronounced for 3-OH FAs determinations (ICC 22%) than for muramic acid (ICC 55-66%). We conclude that determinants vary largely for different types of bacterial measurements in house dust; the measured parameters represent different aspects of the bacterial content indoors. More than one sample is needed to describe bacterial concentrations in house dust in the home environment due to large temporal variation.

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.

Luteolibacter yonseiensis sp. nov., isolated from activated sludge using algal metabolites.

A Gram-negative, rod-shaped, aerobic bacterial strain, designated EBTL01(T), was isolated from activated sludge by using metabolites of microalgae Ankistrodesmus gracilis SAG278-2. Phylogenetic analyses based on 16S rRNA gene sequence showed that strain EBTL01(T) belongs to the family Verrucomicrobiaceae, class Verrucomicrobiae, and is related most closely to Luteolibacter pohnpeiensis A4T-83(T) (95.5 % sequence similarity) and Luteolibacter algae A5J-41-2(T) (95.2 %). The G+C content of the genomic DNA of strain EBTL01(T) was 56.3 mol% and the menaquinone MK-9 was detected as the predominant quinone. Major fatty acid components were iso-C14 : 0, C16 : 1ω7c and C16 : 0. The amino acids of the cell-wall peptidoglycan contained muramic acid and meso-diaminopimelic acid. These profile results supported the affiliation of strain EBTL01(T) to the genus Luteolibacter. On the other hand, based on chemotaxonomic properties and phenotypic characteristics, strain EBTL01(T) could be clearly differentiated from its phylogenetic neighbours. Therefore, strain EBTL01(T) represents a novel species of the genus Luteolibacter, for which the name Luteolibacter yonseiensis sp. nov. is proposed. The type strain is EBTL01(T) ( = KCTC 23678(T) = JCM 18052(T)).

Structure of a peptidoglycan-related polysaccharide from Providencia alcalifaciens O45.

A polysaccharide was isolated from the opportunistic human pathogen Providencia alcalifaciens O45:H26 by extraction with aqueous phenol and studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including two-dimensional ROESY and H-detected (1)H,(13)C HSQC experiments. The polysaccharide contains N-acetylglucosamine and N-acetylmuramic acid (D-GlcpNAc3Rlac) amidated with L-alanine and has the following structure: →4)-ß-D-GlcpNAc-(1→4)-ß-D-GlcpNAc3(Rlac-L-Ala)-(1→. The polysaccharide possesses a remarkable structural similarity to the bacterial cell wall peptidoglycan. It is not unique to the strain studied but is common to strains of at least four P. alcalifaciens O-serogroups (O3, O24, O38, and O45). No evidence was obtained that the polysaccharide is associated with the LPS, and hence it might represent a bacterial capsule component.

GC-based detection of aldononitrile acetate derivatized glucosamine and muramic acid for microbial residue determination in soil.

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. Current strategies for microbial analysis include both traditional laboratory culture-dependent techniques and those based on direct extraction and determination of certain biomarkers. Few among the diversity of microbial species inhabiting soil can be cultured, so culture-dependent methods introduce significant biases, a limitation absent in biomarker analysis. The glucosamine, mannosamine, galactosamine and muramic acid have been well served as measures of both the living and dead microbial mass, of these the glucosamine (most abundant) and muramic acid (uniquely from bacterial cell) are most important constituents in the soil systems. However, the lack of knowledge on the analysis restricts the wide popularization among scientific peers. Among all existing analytical methods, derivatization to aldononitrile acetates followed by GC-based analysis has emerged as a good option with respect to optimally balancing precision, sensitivity, simplicity, good chromatographic separation, and stability upon sample storage. Here, we present a detailed protocol for a reliable and relatively simple analysis of glucosamine and muramic acid from soil after their conversion to aldononitrile acetates. The protocol mainly comprises four steps: acid digestion, sample purification, derivatization and GC determination. The step-by-step procedure is modified according to former publications. In addition, we present a strategy to structurally validate the molecular ion of the derivative and its ion fragments formed upon electron ionization. We applied GC-EI-MS-SIM, LC-ESI-TOF-MS and isotopically labeled reagents to determine the molecular weight of aldononitrile acetate derivatized glucosamine and muramic acid; we used the mass shift of isotope-labeled derivatives in the ion spectrum to investigate ion fragments of each derivatives. In addition to the theoretical elucidation, the validation of molecular ion of the derivative and its ion fragments will be useful to researchers using δ(13)C or ion fragments of these biomarkers in biogeochemical studies.

Endospore abundance, microbial growth and necromass turnover in deep sub-seafloor sediment.

Two decades of scientific ocean drilling have demonstrated widespread microbial life in deep sub-seafloor sediment, and surprisingly high microbial-cell numbers. Despite the ubiquity of life in the deep biosphere, the large community sizes and the low energy fluxes in this vast buried ecosystem are not yet understood. It is not known whether organisms of the deep biosphere are specifically adapted to extremely low energy fluxes or whether most of the observed cells are in a dormant, spore-like state. Here we apply a new approach--the D:L-amino-acid model--to quantify the distributions and turnover times of living microbial biomass, endospores and microbial necromass, as well as to determine their role in the sub-seafloor carbon budget. The approach combines sensitive analyses of unique bacterial markers (muramic acid and D-amino acids) and the bacterial endospore marker, dipicolinic acid, with racemization dynamics of stereo-isomeric amino acids. Endospores are as abundant as vegetative cells and microbial activity is extremely low, leading to microbial biomass turnover times of hundreds to thousands of years. We infer from model calculations that biomass production is sustained by organic carbon deposited from the surface photosynthetic world millions of years ago and that microbial necromass is recycled over timescales of hundreds of thousands of years.

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.

Yuhushiella deserti gen. nov., sp. nov., a new member of the suborder Pseudonocardineae.

A thermotolerant, Gram-stain-positive, aerobic, sporangium-forming actinomycete, strain RA45(T), was isolated from a desert region in Xinjiang Uigur Autonomous Region, north-western China. Comparative analysis of the 16S rRNA gene sequence and phenotypic characterization revealed that strain RA45(T) belonged phylogenetically to the family Pseudonocardiaceae of the suborder Pseudonocardineae. Strain RA45(T) showed more than 5  % 16S rRNA gene sequence divergence from recognized species of genera in the family Pseudonocardiaceae, forming a distinct lineage within the evolutionary radiation occupied by the genera Amycolatopsis, Prauserella, Thermocrispum, Saccharomonospora, Saccharopolyspora and Sciscionella, but distinct from each of them. The affiliation to the family was supported by the presence of suborder- and family-specific 16S rRNA signature nucleotides, a DNA G+C content of 69.9 mol%, the presence of meso-diaminopimelic acid, ribose, arabinose, glucose and galactose, which are characteristic components of cell-wall chemotype IV of actinomycetes, the presence of menaquinone MK-9(H4) as the major respiratory lipoquinone, a lack of mycolic acids and the presence of an N-acetylated type of muramic acid. However, strain RA45(T) differed from known genera of the family in its polar lipid composition: the major phospholipids were phosphatidylethanolamine, phosphatidylinositol mannosides, phosphatidylmethylethanolamine, diphosphatidylglycerol, phospholipids of unknown structure and phospholipids of unknown structure containing glucosamine (phospholipid type IV). Based on its morphological, chemotaxonomic and phylogenetic characteristics, strain RA45(T) is considered to represent a novel species of a new genus in the family Pseudonocardiaceae, for which the name Yuhushiella deserti gen. nov., sp. nov. is proposed. The type strain of Yuhushiella deserti is RA45(T) (=CGMCC 4.5579(T) =JCM 16584(T)).