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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 (R 2 > 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 (R 2 ) 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

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., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

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., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020. Published by Elsevier B.V.)

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 (MS 2 ) 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.

An antibiotic-producing actinobacterium, designated isolate B375 T , 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 B375 T was most closely related to Williamsia serinedens DSM 45037 T and Williamsia spongiae DSM 46676 T 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 C 16:0 (23.3%), C 18:0 10-methyl (23.2%) and C 18:1 ω9c (21.6%) as the major cellular fatty acids. The strain B375 T inhibited growing of Staphylococcus aureus and Colletotrichum gloeosporioides strains and was considered a producer of antimicrobial compounds. Based on the data obtained, the isolate B375 T (= CBMAI 1090 T  = DSM 46677 T ) 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.

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.

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., FEV 1 , 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/m 3 , 118 EU/m 3 , and 3.6 mg/m 3 , 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., (© 2017 Wiley Periodicals, Inc.)

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.

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., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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.

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.

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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)).

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)-β-D-GlcpNAc-(1→4)-β-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.

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.

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.

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Strain 210417(T), which forms highly branched substrate and aerial mycelia, is a Gram-positive, aerobic and non-motile actinomycete isolated from mangrove rhizosphere soil. 16S rRNA gene sequence analysis showed that the strain should be classified in the genus Nonomuraea, being most closely related to the type strains of Nonomuraea coxensis (99.6 %) and Nonomuraea bangladeshensis (99.3 %). Chemotaxonomic properties [madurose as the major sugar in the cell wall; meso-diaminopimelic acid and N-acetylmuramic acid in the peptidoglycan; MK-9(H(4)) as the major menaquinone; iso-C(16 : 0) (24.1 %) as major fatty acid; and phospholipid pattern type IV] are consistent with the assignment of strain 210417(T) to the genus Nonomuraea. Strain 210417(T) could be differentiated from the closely related species N. coxensis and N. bangladeshensis by morphological, physiological, biochemical and chemotaxonomic properties, 16S rRNA gene sequence analysis and DNA-DNA hybridization results. It is therefore proposed that strain 210417(T) represents a novel species of the genus Nonomuraea, for which the name Nonomuraea wenchangensis sp. nov. is given; the type strain is 210417(T) ( = CGMCC 4.5598(T)  = DSM 45477(T)).

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)).

By the method of intermittent leaching aerobic incubation, this paper studied the mineralization of three kinds of microbes-derived amino sugar (glucosamine, muramic acid, and galactosamine) in black soil of Northeast China, and the responses to glucose addition and glucose plus nitrogen amendment. The mineralization of the amino sugars was compound-specific. During incubation period, the content of muramic acid decreased by 25.4%, while that of glucosamine decreased by 7.1%, suggesting that bacteria-derived muramic acid was more inclined to be mineralized, compared with fungi-originated glucosamine. However, the mineralized amount of glucosamine (68.4 mg x kg(-1)) was greater than that of muramic acid (15.4 mg x kg(-1)). Both glucose addition and glucose plus nitrogen amendment improved the contents of glucosamine and muramic acid significantly, but the effect varied. The mineralization of galactosamine was much slower, and less affected by exogenous substances addition, indicating that galactosamine was more stable in test soil.

In agricultural and other environments, inhalation of airborne microorganisms is linked to respiratory disease development. Bacterial endotoxins, peptidoglycans, and fungi are potential causative agents, but relative microbial characterization and inflammatory comparisons amongst agricultural dusts are not well described. The aim of this study was to determine the distribution of microbial endotoxin, 3-hydroxy fatty acids (3-OHFA), muramic acid, and ergosterol and evaluate inflammatory responses in human monocytes and bronchial epithelial cells with various dust samples. Settled surface dust was obtained from five environments: swine facility, dairy barn, grain elevator, domestic home (no pets), and domestic home with dog. Endotoxin concentration was determined by recombinant factor C (rFC). 3-OHFA, muramic acid, and ergosterol were measured using gas chromatography-mass spectrometry. Dust-induced inflammatory cytokine secretion in human monocytes and bronchial epithelial cells was evaluated. Endotoxin-independent dust-induced inflammatory responses were evaluated. Endotoxin and 3-OHFA levels were highest in agricultural dusts. Muramic acid, endotoxin, 3-OHFA, and ergosterol were detected in dusts samples. Muramic acid was highest in animal farming dusts. Ergosterol was most significant in grain elevator dust. Agricultural dusts induced monocyte tumor necrosis factor (TNF) alpha, interleukin (IL)-6, IL-8, and epithelial cell IL-6 and IL-8 secretion. Monocyte and epithelial IL-6 and IL-8 secretion was not dependent on endotoxin. House dust(s) induced monocyte TNFalpha, IL-6, and IL-8 secretion. Swine facility dust generally produced elevated responses compared to other dusts. Agricultural dusts are complex with significant microbial component contribution. Large animal farming dust(s)-induced inflammation is not entirely dependent on endotoxin. Addition of muramic acid to endotoxin in large animal farming environment monitoring is warranted.

The muramic acid (MurA) assay is a powerful tool for the detection and quantification of bacteria with no need to enrich samples by culturing. However, the analysis of MurA in mixed biological and environmental matrices is potentially more complex than analysis in isolated bacterial cells. In this study, we employed one commonly used procedure for extraction of MurA from environmental samples and found that the presence of streptomycin interfered with the determination of MurA by creating chemical species that coeluted with the aldononitrile derivative of MurA prepared in this method. On a molar basis, streptomycin yields a signal that is approximately 0.67 times that of MurA. Mass spectrometry analysis confirmed that the interference from hydrolyzed streptomycin is not actually by MurA, but rather is likely to be N-methyl glucosamine. Because streptomycin is widely applied for selective growth of eukaryotes both in situ and in vitro, our findings may have implications for the significance of results from MurA assays. We conclude that MurA remains an effectual bacterial biomarker due to its unique bacterial origin, but care must be applied in interpreting results from the assay when performed in the presence of streptomycin.

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.

Two Gram-negative, non-motile, coccoid or rod-shaped, chemoheterotrophic bacteria designated strains YM21-132(T) and YM27-005(T) were isolated from marine animals, and were subjected to a polyphasic taxonomic examination. Phylogenetic analyses based on 16S rRNA gene sequences indicated that the two isolates belong to the genus Rubritalea of the phylum 'Verrucomicrobia' (subdivision 1). The novel isolates shared approximately 97-98 % sequence similarity with each other and showed 93-97 % similarity with Rubritalea species of the family Verrucomicrobiaceae. The level of DNA-DNA relatedness between strains YM21-132(T) and YM27-005(T) was less than 70 %, which is accepted as the phylogenetic definition of a species. Both strains produced reddish carotenoid pigments and squalene. The cell wall peptidoglycan of both strains contained muramic acid and meso-diaminopimelic acid. The G+C contents of the genomic DNA were 48.0 mol% (strain YM21-132(T)) and 50.3 mol% (strain YM27-005(T)). The presence of MK-8 and MK-9 as the major isoprenoid quinones, and iso-C(14 : 0), iso-C(16 : 0) and C(16 : 1)omega7c as the major cellular fatty acids supported the identification of the two novel strains as members of the genus Rubritalea. On the basis of polyphasic taxonomic studies, it was concluded that these strains should be classified as representing two novel, separate species in the genus Rubritalea within the phylum 'Verrucomicrobia', for which the names Rubritalea spongiae sp. nov. (type strain YM21-132(T)=MBIC08281(T)=KCTC 12906(T)) and Rubritalea tangerina sp. nov. (type strain YM27-005(T)=MBIC08282(T)=KCTC 12907(T)) are proposed.

A polyphasic taxonomic study was performed on strain YM26-026(T), which was isolated from acid-treated sediment in Kamaishi, Japan. The bacterial cells were pale-pink-pigmented, Gram-negative, obligately aerobic, non-spore-forming, spherical and non-motile. Phylogenetic analysis based on 16S rRNA gene sequences showed that the novel isolate was a member of the phylum 'Verrucomicrobia' and shared approximately 84-87 % sequence similarity with strains of the class Opitutae that have been cultivated to date. Strain YM26-026(T) produced pale-pink pigments of carotenoid. beta-Lactam antibiotic susceptibility tests and amino acid analysis of cell-wall hydrolysates indicated that the novel isolate did not contain muramic acid or diaminopimelic acid in the cell wall, suggesting that the strain lacks peptidoglycan. The G+C content of the DNA of strain YM26-026(T) was 54.0 mol%. Menaquinone-7 was the major quinone and C(14 : 0) and C(18 : 1)omega9c were the major fatty acids. On the basis of polyphasic taxonomic studies, it was concluded that strain YM26-026(T) represents a new genus of the family Puniceicoccaceae within the phylum 'Verrucomicrobia', for which the name Cerasicoccus arenae gen. nov., sp. nov. is proposed. The type strain is YM26-026(T) (=MBIC08280(T)=KCTC 12870(T)).

Five Gram-negative, white-pigmented, spherical, chemoheterotrophic bacteria were isolated from seawater from Japan and the Republic of Palau by use of an in situ cultivation technique. Phylogenetic analyses based on 16S rRNA gene sequences indicated that the five novel isolates, 02PA-Ca-133(T), YM14-201(T), H-MN57(T), H-MN48 and MN1-156, were closely affiliated to members of subdivision 4 within the phylum 'Verrucomicrobia'. The novel isolates shared 96-100 % sequence similarity with each other and showed less than 90 % similarity with the cultivated strains of subdivision 4. DNA-DNA relatedness values between strains 02PA-Ca-133(T), YM14-201(T) and H-MN57(T) were less than 70 %; the value commonly accepted as the threshold for the phylogenetic definition of a species. Antibiotic susceptibility tests and amino acid analysis of cell-wall hydrolysates indicated that the novel isolates did not contain muramic acid or diaminopimelic acid in their cell walls, suggesting that these strains lack peptidoglycan. The DNA G+C contents of the five strains were 51-57 mol%. The major menaquinone was MK-7 and C(16 : 0), C(16 : 1)omega 7c and anteiso-C(15 : 0) were the major fatty acids. On the basis of polyphasic taxonomic evidence, it is concluded that these strains should be classified as representing a new genus and three novel species in subdivision 4 of the phylum 'Verrucomicrobia', for which the names Pelagicoccus mobilis gen. nov., sp. nov. [type strain 02PA-Ca-133(T) (=MBIC08004(T)=IAM 15422(T)=KCTC 13126(T))], Pelagicoccus albus sp. nov. [type strain YM14-201(T) (=MBIC08272(T)=IAM 15421(T)=KCTC 13124(T))] and Pelagicoccus litoralis sp. nov. [type strain H-MN57(T) (=MBIC08273(T)=IAM 15423(T)=KCTC 13125(T))] are proposed.

An obligately aerobic, Gram-negative, non-spore-forming, non-motile, spherical bacterium, designated strain 04OKA010-24(T), was isolated from seawater surrounding the hard coral Galaxea fascicularis L., collected at Majanohama, Akajima, Japan, and was subjected to a polyphasic taxonomic study. Phylogenetic analyses based on the 16S rRNA gene sequence indicated that the new strain represented a member of the phylum 'Verrucomicrobia' and shared 84-95 % sequence similarity with cultivated strains of 'Verrucomicrobia' subdivision 4. Amino acid analysis of the cell-wall hydrolysate indicated the absence of muramic acid and diaminopimelic acid, which suggested that the strain did not contain peptidoglycan in the cell wall. The G+C content of the DNA was 53.9 mol%. MK-7 was the major menaquinone and C(14 : 0), C(18 : 1)omega9c and C(18 : 0) were the major fatty acids. On the basis of these data, it was concluded that strain 04OKA010-24(T) represents a novel species in a new genus in subdivision 4 of the phylum 'Verrucomicrobia', for which the name Coraliomargarita akajimensis gen. nov., sp. nov. is proposed. The type strain of Coraliomargarita akajimensis is 04OKA010-24(T) (=MBIC06463(T)=IAM 15411(T)=KCTC 12865(T)).

A soil isolate, strain CC-AB07T, was characterized using a polyphasic approach. This organism had chemotaxonomic and morphological properties consistent with its classification in the genus Gordonia. 16S rRNA gene sequence analysis showed that the novel strain formed a monophyletic branch at the periphery of the evolutionary radiation occupied by the genus Gordonia, its closest neighbours being the type strains of Gordonia alkanivorans, Gordonia amicalis, Gordonia bronchialis, Gordonia desulfuricans, Gordonia polyisoprenivorans and Gordonia rhizosphera. The novel isolate was distinguished from all of these type strains using a range of phenotypic properties and by gyrB gene sequence analysis. It was evident from the genotypic and phenotypic data that strain CC-AB07T should be classified as representing a novel species in the genus Gordonia, for which the name Gordonia soli sp. nov. is proposed. The type strain is CC-AB07T (=BCRC 16810T=DSM 44995T).

Two actinomycete strains, TT2-10(T) and TT2-3, which produced long spore chains (more than 10 spores per chain), were isolated from peat swamp forest soil in Pattaloong Province, Thailand. Their taxonomic positions were determined using a polyphasic approach. The chemotaxonomic characteristics of these strains coincided with those of the family Micromonosporaceae, i.e. cell-wall chemotype II, muramic acid of the N-glycolyl type, whole-cell sugar pattern D and type II phospholipids. Analysis of the 16S rRNA gene sequences also indicated that these strains constitute a distinct lineage within the family Micromonosporaceae, sharing 91.3-93.8 % sequence similarity with members of this family. On the basis of their phenotypic and genotypic characteristics and their phylogenetic position, these strains represent a novel genus and species, for which the name Actinocatenispora thailandica gen. nov., sp. nov. is proposed. The type strain of Actinocatenispora thailandica is strain TT2-10(T) (=JCM 12343(T)=PCU 235(T)=DSM 44816(T)).

Simple, selective yet sensitive methods to quantify low-abundance bacterial biomarkers derived from complex samples are required in clinical, biological, and environmental applications. In this report, a new strategy to integrate sample pretreatment with chemical analysis is investigated using on-line preconcentration with chemical derivatization by CE and UV detection. Single-step enantioselective analysis of muramic acid (MA) and diaminopimelic acid (DAP) was achieved by CE via sample enrichment by dynamic pH junction with ortho-phthalaldehyde/N-acetyl-L-cysteine labeling directly in-capillary. The optimized method resulted in up to a 100-fold enhancement in concentration sensitivity compared to conventional off-line derivatization procedures. The method was also applied toward the detection of micromolar levels of MA and DAP excreted in the extracellular medium of Escherichia coli bacterial cell cultures. On-line preconcentration with chemical derivatization by CE represents a unique approach for conducting rapid, sensitive, and high-throughput analyses of other classes of amino acid and amino sugar metabolites with reduced sample handling, where the capillary functions simultaneously as a concentrator, microreactor, and chiral selector.

Geobacillus tepidamans GS5-97(T) is a novel Gram-positive, moderately thermophilic bacterial species that is covered by a glycosylated surface layer (S-layer) protein. The isolated and purified S-layer glycoprotein SgtA was ultrastructurally and chemically investigated and showed several novel properties. By SDS-PAGE, SgtA was separated into four distinct bands in an apparent molecular mass range of 106-166 kDa. The three high molecular mass bands gave a positive periodic acid-Schiff staining reaction, whereas the 106-kDa band was nonglycosylated. Glycosylation of SgtA was investigated by means of chemical analyses, 600-MHz nuclear magnetic resonance spectroscopy, and electrospray ionization quadrupole time-of-fight mass spectrometry. Glycopeptides obtained after Pronase digestion revealed the glycan structure [-->2)-alpha-L-Rhap-(1-->3)-alpha-D-Fucp-(1-->](n=approximately 20), with D-fucopyranose having never been identified before as a constituent of S-layer glycans. The rhamnose residue at the nonreducing end of the terminal repeating unit of the glycan chain was di-substituted. For the first time, (R)-N-acetylmuramic acid, the key component of prokaryotic peptidoglycan, was found in an alpha-linkage to carbon 3 of the terminal rhamnose residue, serving as capping motif of an S-layer glycan. In addition, that rhamnose was substituted at position 2 with a beta-N-acetylglucosamine residue. The S-layer glycan chains were bound via the trisaccharide core -->2)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1--> to carbon 3 of beta-D-galactose, which was attached in O-glycosidic linkage to serine and threonine residues of SgtA of G. tepidamans GS5-97(T).

In previous articles [Anal. Biochem. 284 (2000) 201; J. Lipid Res. 43 (2002) 794], we reported that the GC/MS identification and quantification of nearly all constituents of glycolipids could be obtained on the same sample in a single GC/MS analysis as heptafluorobutyrate derivatives of the products liberated using acid-catalyzed methanolysis. The same type of data could be obtained on glycoproteins and proteoglycans [Biochemistry 42 (2003) 8342]. These experiments were performed on material from higher organisms, and there was no evidence that bacteria-specific constituents could also be identified and quantified. The current article reports that the GC/MS analysis of compounds liberated by acid-catalyzed methanolysis as heptafluorobutyrate derivatives allows the simultaneous qualitative and quantitative determinations of pentoses, deoxyhexoses, hexoses, hexosamines, uronic acids, Kdo, Mur, heptose, Kdn, and neuraminic acid as well as of most fatty acids (including hydroxylated fatty acids). This approach provides a way of obtaining fingerprints of bacterial constituents and quantification of the overall effect of gene inactivation or of culture conditions.

The chemical dynamics of marine dissolved organic nitrogen (DON), a reservoir featuring surface accumulations even in areas where nitrogen limits productivity, have yet to be resolved. We exploited differences in the acid lability of amide bonds within high-molecular-weight (HMW) DON to show that vertical DON profiles result in part from the presence of two chemically distinct pools of amide. Half of HMWDON in surface waters is present as N-acetyl amino polysaccharides. In contrast, nearly all deep-sea HMWDON, and therefore, most HMWDON, is present in amides that resist both chemical hydrolysis and biological degradation.

We used gas chromatography--tandem mass spectrometry to analyze microbial components in 85 samples of airborne dust from schools in Jordan, Sweden, and Poland. To collect the samples, we allowed dust to settle on plexiglass plates hanging in the breathing zone in school buildings during both summer and winter. In each of the three countries, we conducted such sampling in two schools: one in an urban environment and the other in rural surroundings. The microbial marker profiles differed significantly between the schools and seasons. For example, samples from Jordan contained remarkably low levels of ergosterol (marker of fungal biomass) and high levels of 3-hydroxy acids (markers of lipopolysaccharide) of 10, 12, and 14 carbon chain lengths relative to such acids of 16 and 18 carbons in comparison with samples from Sweden and Poland. This dissimilarity in 3-hydroxy fatty acid distribution indicates significant differences in the populations of Gram-negative bacteria. We also noted that muramic acid (marker of bacterial biomass) exhibited the smallest variation between schools and seasons. In summary, our results demonstrate that exposure to microorganisms in indoor air in school buildings may differ markedly between countries, between seasons, and between urban and rural environments.

Exposure to organic dust originating from biofuels can cause adverse health effects. In the present study we have assessed the dustiness in terms of microbial components and particles of various biofuels by using a rotating drum as a dust generator. Microbial components from straw, wood chips, wood pellets and wood briquettes were quantified by several methods. Excellent correlations (r >/= 0.85, P < 0.0001) were found: between lipopolysaccharide (LPS) (as determined by 3-hydroxy fatty acid analysis) and endotoxin (as determined by a Limulus test), cultivable bacteria, total number of bacteria and muramic acid; between endotoxin and cultivable bacteria, total number of bacteria and muramic acid; between total number of bacteria and muramic acid; between cultivable fungi and total number of fungi. Straw was dustier than the other biofuels in terms of actinomycetes, bacteria, muramic acid, endotoxin, LPS, particle mass and number of particles. One of the wood chips studied and the straws had comparatively high dustiness in terms of fungi, while both wood pellets and wood briquettes had comparatively low dustiness in terms of all microbial components. An initially high particle generation rate of straw and wood chips decreased over time whereas the particle generation rate of wood briquettes and wood pellets increased during a 5 min rotation period. Particles of non-microbial origin may be the determining factor for the health risk in handling briquettes and pellets. Straw dust contained significantly more microorganisms per particle than did wood chip dust, probably because bacteria were most abundant in straw dust. The concentrations of endotoxin and fungi were high in wood and straw dust; dust from one of the straws contained 3610 EU/mg and dust from one of the chips contained 7.3 x 10(6) fungal spores/mg. An exposure to 3 mg of straw or wood chips dust/m(3) (the Swedish and Danish OEL of unspecific inhalable dust) could cause exposures to endotoxin and fungi higher than levels were health symptoms are seen to develop. The very different levels of dustiness in terms of particles and microbial components of different biofuels shows that dustiness is an important health-relevant factor to consider when choosing among biofuels and when designing worksites for handling of biofuels.

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.

Muramic acid (MA) is a unique amino sugar that is a constituent of the peptidoglycan (PG) present in prokaryotic cell walls. MA can serve as a marker for quantifying bacterial load, e.g. in indoor environments, by using gas chromatography-tandem mass spectrometry (GC-MS-MS). We demonstrated recently that the methyl ester O-methyl acetate (MMA) derivative can be used to detect MA in house dust by ion-trap GC-MS-MS. However, since the MMA derivative is not formed from free MA quantification was not optimal. Here we report 1) significant improvements in sample preparation of the MMA derivative and 2) an evaluation of the performance of derivative, using for comparison the alditol acetate derivative, the gold standard in quantitative trace analysis of MA in complex matrices. The MMA derivative was analysed using an MS instrument with internal ionization and the alditol acetate derivative was analysed using an instrument with external ionization. 13C-labelled cyanobacteria, containing MA in their PG, were used as the internal standard. A linear relationship was found between the two methods in studies on 27 parallel samples of airborne dust from school classes collected on filters. Although the analytical sensitivity of the MMA derivatives was somewhat slightly lower than of the alditol acetate derivative, this may be due to differences in yield of derivative, sample clean-up efficiency, or different performance of the GC columns or MS instruments. However preparation of the MMA derivative is quick and compatible with preparation of methyl esters of 3-hydroxy fatty acids (used as markers of Gram negative endotoxin) allowing the levels of both markers to be determined in the same dust sample. In conclusion, the MMA procedure can be used to determine MA in environmental samples with good reproducibility provided the concentration of the 13C-labelled MA internal standard in the cyanobacteria is first determined with an alternative method.

Airborne dust samples from damp (n = 9) and control (n = 9) residences were analyzed for microorganisms (molds and bacteria), bacterial markers (3-hydroxy fatty acids and muramic acid), and adsorbed volatile organic compounds (VOCs). The number of mold species was greater in the damp residences than in the controls (23 vs.18) and nine mold species were found only in damp residences. The levels of 3-hydroxy fatty acids and muramic acid correlated better in damp residences than in controls, indicating that damp conditions affect the bacterial flora of airborne dust. Identifications made by culture and microscopy of the major molds found, i.e. Aspergillus, Cladosporium, and Penicillum, coincided with the identification of VOCs known to be produced by these species. A number of additional VOCs irritating to the skin, eyes, or respiratory tract were also found. The results from this pilot study illustrate the diversity of microorganisms and VOCs present in the indoor environment and suggest that analysis of airborne dust may help to assess human exposure to microorganisms and chemical compounds.

Background: Bacterial cell wall (CW) arthritis develops in susceptible strains of rats after a single intraperitoneal injection of the CW from certain bacterial species, both pathogenic and non-pathogenic. For the development of chronic bacterial CW arthritis, the structure of the bacterial peptidoglycan (PG) has been found to be decisive., Objective: To define the role of PG subtypes in the pathogenesis of chronic bacterial CW arthritis., Method: Arthritis was induced with CWs of Lactobacillus plantarum, L casei B, L casei C, and L fermentum. Gas chromatography-mass spectrometry was used to measure the presence of CW derived muramic acid in the liver and to determine PG subtypes. CWs were also tested for their resistance to lysozyme in vitro., Results: These results and those published previously indicate that PGs of CWs which induce chronic arthritis, no matter whether they were derived from strains of Streptococcus, Bifidobacterium, Collinsella, or Lactobacillus, all have lysine as the third amino acid of the PG stem peptide, representing PG subtypes A3alpha and A4alpha. Those strains which induce only transient acute arthritis or no arthritis at all do not have lysine in this position, resulting in different PG subtypes., Conclusions: In vivo degradation of only those PGs with the subtypes A3alpha and A4alpha leads to the occurrence of large CW fragments, which persist in tissue and have good proinflammatory ability. CWs with other PG subtypes, even if they are lysozyme resistant, do not cause chronic arthritis, because the released fragments are not phlogistic. It is emphasised that a variety of microbial components not causing inflammation have been found in animal and human synovial tissue.

The mre genes of Escherichia coli and Bacillus subtilis are cell shape determination genes. Mutants affected in mre function are spheres instead of the normal rods. Although the mre determinants are not required for viability in E. coli, the mreB determinant is an essential gene in B. subtilis. Conflicting results have been reported as to whether the two membrane-associated proteins MreC and MreD are essential proteins. Furthermore, although the MreB protein has been studied in some detail, the roles of the MreC and MreD proteins in cell shape determination are unknown. We constructed a strain of B. subtilis in which expression of the mreC determinant is dependent upon the addition of isopropyl-beta-D-thiogalactopyranoside to the culture medium. Utilizing this conditional strain, it was shown that mreC is an essential gene in B. subtilis. Furthermore, it was shown that cells lacking sufficient quantities of MreC undergo morphological changes, namely, swelling and twisting of the cells, which is followed by cell lysis. Electron microscopy was utilized to demonstrate that a polymeric material accumulated at one side of the division septum of the cells and that the presence of this material correlated with the bending of the cell. The best explanation for the results is that the MreC protein is involved in the control of septal versus long-axis peptidoglycan synthesis, that cells lacking MreC perform aberrant septal peptidoglycan synthesis, and that lysis results from a deficiency in long-axis peptidoglycan synthesis.

Objective: To study the presence of bacterial components in the synovial tissue (ST) of patients with advanced rheumatoid arthritis (RA)., Methods: ST was collected during joint surgery from 41 RA patients. Tissue from 39 patients with osteoarthritis (OA), 4 patients with undifferentiated inflammatory arthritis (UA), and 3 cases of accidental deaths served as controls. The pan-bacterial polymerase chain reaction (PCR) with primers for the 23S ribosomal RNA (rRNA) and 16S rRNA genes was used to detect bacterial DNA. In addition, synovial fluid (SF) samples from patients with chlamydial reactive arthritis (ReA) were also examined by the same method. The positive controls, bacterial DNA or ST spiked with different living bacteria, were analyzed alongside clinical samples. Most of the ST samples were also analyzed by gas chromatography-mass spectrometry (GC-MS) for determining the presence of bacteria-derived muramic acid. Strict precautions were followed in the clinics and the laboratory to prevent contamination., Results: In GC-MS analysis, muramic acid was observed in the ST from 4 of 35 RA patients and from 2 of 14 OA patients, but not in ST from 2 patients with UA and 3 cadavers. Bacterial DNA was not detected by either one of the PCR primers used in ST from 42 patients with RA and 39 patients with OA. However, 5 of 15 SF samples from ReA patients were PCR positive. The sensitivity of GC-MS to detect muramic acid was 2 pg/injected amount (227 pg muramic acid/mg ST), and that of the pan-bacterial PCR was 2-20 bacteria colony forming units/reaction., Conclusion: These results indicate that a bacterial component, muramic acid, is detectable by GC-MS in ST from a few patients with advanced RA or OA. However, no bacterial DNA was detectable by PCR.

An integrated procedure is presented whereby gas chromatography-ion trap mass spectrometry is used to determine chemical markers of gram-negative bacterial lipopolysaccharide (3-hydroxy fatty acids with 10 to 18 carbon atoms), gram-positive bacteria (branched-chain fatty acids with 15 and 17 carbon atoms), bacterial peptidoglycan (muramic acid), and fungal biomass (ergosterol) in samples of settled house dust. A hydrolysate of (13)C-labeled cyanobacterial cells is used as an internal standard for the first three markers. These analyses require two dust samples, one for 3-OH fatty acids, branched-chain fatty acids, and muramic acid and another for ergosterol. The method may be used to characterize microbial communities in environmental samples.

An automated derivatization instrument has been developed for the preparation of alditol acetates from bacterial hydrolysates for analysis by gas chromatography-mass spectrometry (GC-MS). The current report demonstrates the utility of the automated instrument for the more demanding task of trace analysis of muramic acid (Mur) in airborne dust using gas chromatography-tandem mass spectrometry (GC-MS(2)). Conditions for efficient derivatization of Mur, vital for trace analysis, are rigorous including lactam and imido group formation under anhydrous conditions. Furthermore, as the detection limit is lowered, possible contamination or carry-over of samples becomes an increasingly greater consideration and must not occur. The instrument meets these criteria and was successfully used for assaying the levels of Mur in laboratory air, which were found to be much lower than in the previous studies of heavily occupied schools and agricultural environments. The potential for GC-MS(3) in further lowering the detection limit was also demonstrated.

Muramic acid (Mur) is present in the cell wall of Eubacteria and serves as a chemical marker for the trace detection of bacteria and bacterial cell wall debris in complex matrices. There have been numerous studies using a variety of derivatives of Mur, particularly in combination with gas chromatography-tandem mass spectrometry (GC-MS-MS) where the detection limit has been steadily lowered. A stable, halogenated derivative, the pentafluorobenzyl oxime (PFBO) acetate of Mur, has been developed by others and successfully used for GC with electron-capture detection. The current report is the first use of this derivative for GC-MS-MS analysis of Mur, or indeed any other carbohydrate, using negative ion chemical ionization (NICI) with GC-MS-MS. Mur was readily detected in settled surface dust (166 ng/mg), as well as dust collected from indoor air (1.4-5.9 ng/mg). Analyses of Mur as a PFBO acetate by GC-NICI-MS-MS or as alditol acetates by electron impact GC-electron impact ionization MS-MS serve as complementary approaches for trace detection in complex matrices.

It has been hypothesized that bacterial debris may accumulate in tissues of the reticuloendothelial system (RES) serving as an inflammatory stimulus for human disease. In support of this hypothesis, muramic acid (Mur), a component of bacterial peptidoglycan (PG), has previously been reported to be present in culture-negative human spleen. High-performance liquid chromatography (HPLC) was employed in these analyses, and a peak was detected at the retention time of Mur. However, HPLC is best used as a screening technique, and it is vital that these tentative observations be reexamined by the state-of-the-art approach (gas chromatography-tandem mass spectrometry [GC-MS(2)]). Indeed, in the present work using GC-MS(2), Mur was not detected in six out of seven human spleens previously examined by HPLC. However, Mur was categorically detected at minute concentrations, 50 ppb, in one spleen. In conclusion, since Mur is not generally found in culture-negative human spleen, in future studies, these tissues can serve as negative controls. The study of Mur levels in inflammation (e.g., reactive arthritis) could prove important in testing the hypothesis that bacterial debris persisting in tissues could serve as a depot inciting diseases of unknown etiology.

Two almost-identical strains of Eubacterium aerofaciens isolated from the normal human gut flora were used. The cell wall (CW) of one strain with a peptidoglycan (PG) type A4alpha induces chronic arthritis in the rat after a single intraperitoneal injection, whereas CW of the other with PG type A4beta induces only a transient acute arthritis. The CW of the arthritogenic E. aerofaciens was a twofold-more-potent stimulator of the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and monocyte chemoattractant protein 1 (MCP-1) than the nonarthritogenic CW. After degradation with mutanolysin, the capacity of the arthritogenic PG to stimulate production of TNF-alpha and MCP-1 was significantly increased, whereas that of the nonarthritogenic PG was significantly decreased. In other words, after enzyme degradation the arthritogenic PG had a four- to fivefold-stronger stimulatory capacity than that of the enzyme-treated nonarthritogenic PG. These findings indicate that the arthritogenicity of CW or a PG is not dependent on the enzyme resistance alone but also on how the PG fragments released by enzyme degradation stimulate the production of proinflammatory cytokines.

The use of chemical marker analysis for characterising microbial communities in organic dust samples is exemplified in a comparative study of dusts collected in a home and a swine confinement building, respectively. The chemical markers studied included 3-hydroxy fatty acids (markers of endotoxin), ergosterol (marker of fungal biomass), and muramic acid (marker of peptidoglycan/bacterial biomass). Samples were hydrolysed and subjected to various chemical manipulations for rendering the markers suitable for gas chromatography-tandem mass spectrometry analysis. Considerable differences between the dust samples were revealed. Swine dust contained 46 ng/mg of ergosterol (house dust 2.1 ng/mg), 0.096 nmol/mg of endotoxin (house dust 0.020 nmol/mg), and 483 ng/mg of muramic acid (house dust 366 ng/mg). The 3-hydroxy fatty acid and muramic acid results demonstrated a much higher proportion of Gram-negative bacteria to Gram-positives in swine dust than in house dust, and ergosterol results demonstrated a much higher proportion of fungi. The different distribution of 3-hydroxy fatty acids in the 2 samples illustrated differences in their flora of Gram-negative bacteria. The described method allows accurate determination of markers even when present down to trace levels in chemically complex matrices and should be useful in evaluating the role of microorganisms in the development of occupational lung disease, e.g. in agricultural environments.

Muramic acid serves as a marker for the presence of bacterial cell wall debris in mammalian tissues. There have been a number of controversial and sometimes conflicting results on assessing the levels of muramic acid in health and disease. The present report is the first to use the state-of-the art technique, gas chromatography-tandem mass spectrometry, to identify and quantify the levels of muramic acid in tissues. Muramic acid was not found in normal rat brain or spleen. However, when tissues were spiked with muramic acid, it was readily identified. The detection limit was <1 ng of muramic acid/100 mg (wet weight) of tissue. The levels of muramic acid reported in diseased human spleen and spleen of arthritic rats, previously injected with bacterial cell walls, were 100- to 1,000-fold higher. In the present study, muramic acid was also readily detected in the cerebrospinal fluid of patients with pneumococcal meningitis (6.8 to 3,900 ng of muramic acid/ml of cerebrospinal fluid). In summary, there can be an enormous difference in the levels of muramic acid found in different mammalian tissues and body fluids in health and disease. This report could have great impact in future studies assessing the role of bacterial cell wall remnants in the pathogenesis of certain human inflammatory diseases.