Microbial Material Research Articles

Non-cultural methods of human microflora evaluation for the benefit of crew medical control in confined habitat

Current control of human microflora is a great problem not only for the space medicine but also for practical health care. Due to many reasons its realization by classical bacteriological method is difficult in practical application or cannot be done. To evaluate non-cultural methods of microbial control of crews in a confined habitat we evaluated two different methods. The first method is based on digital treatment of microbial visual images, appearing after gram staining of microbial material from natural sample. This way the rate between gram-positive and gram-negative microbe could be gained as well as differentiation of rods and cocci could be attained, which is necessary for primary evaluation of human microbial cenosis in remote confined habitats. The other non-culture method of human microflora evaluation is gas chromatomass spectrometry (gcms) analysis of swabs gathered from different body sites. Gc-ms testing of swabs allows one to validate quantitative and special microflora based on specific lipid markers analysis.

Extracellular biological organic matters in microbial fuel cell using sewage sludge as fuel

The microbial fuel cell (MFC) was applied to produce electricity using sewage sludge as a fuel. The extracellular biological organic matter (EBOM) of sludge, before and after MFC operation, was extracted using ammonium hydroxide whose hydrophobicity was characterized with XAD resin fractionation technique. Following MFC operation, the hydrophilic fraction (HPI) of EBOM was enriched (48.0%–64.5%), the hydrophobic acid (HPO-A) fraction was reduced (32.0%–14.5%), and the dissolved organic carbon (DOC) was removed by 36.8% and the sludge aromaticity decreased by 65.7%. The fluorescence tests indicated that the MFC removed the aromatic proteins-like and soluble microbial byproduct-like materials in sludge. Fourier-transform infrared (FT-IR) results showed that the aliphatic (C–H related) components, hydrocarbon and carbohydrate were easily hydrolyzed and biodegraded in the studied MFC.

Comparative community genomics in the Dead Sea: an increasingly extreme environment

Owing to the extreme salinity ( approximately 10 times saltier than the oceans), near toxic magnesium levels (approximately 2.0 M Mg(2+)), the dominance of divalent cations, acidic pH (6.0) and high-absorbed radiation flux rates, the Dead Sea represents a unique and harsh ecosystem. Measures of microbial presence (microscopy, pigments and lipids) indicate that during rare bloom events after exceptionally rainy seasons, the microbial communities can reach high densities. However, most of the time, when the Dead Sea level is declining and halite is precipitating from the water column, it is difficult to reliably measure the presence of microorganisms and their activities. Although a number of halophilic Archaea have been previously isolated from the Dead Sea, polar lipid analyses of biomass collected during Dead Sea blooms suggested that these isolates were not the major components of the microbial community of these blooms. In this study, in an effort to characterize the perennial microbial community of the Dead Sea and compare it with bloom assemblages, we performed metagenomic analyses of concentrated biomass from hundreds of liters of brine and of microbial material from the last massive Dead Sea bloom. The difference between the two conditions was reflected in community composition and diversity, in which the bloom was different and less diverse from the residual brine population. The distributional patterns of microbial genes suggested Dead Sea community trends in mono- and divalent cation metabolisms as well as in transposable elements. This may indicate possible mechanisms and pathways enabling these microbes to survive in such a harsh environment.

A common basis for facilitated legitimate exchange of biological materials proposed by the European Culture Collections' Organisation

Being charged with the task of accessioning and supplying of living microbiological material, microbial culture collections are institutions that play a central role between the interests of a variety of user communities. On the one side are the providers of living microbiological material, such as individual scientists, institutions and countries of origin and on the other side are the various kinds of recipients/users of cultures of microorganisms from academia and industry. Thus, providing access to high quality biological material and scientific services while at the same time observing donor countries' rights, intellectual property rights, biosafety and biosecurity aspects poses demanding challenges. E.g. donor countries rights relate to Article 15 of the Convention on Biological Diversity: "Contracting parties …. recognize the sovereign rights of states over their natural resources …. shall facilitate access to resources … and not impose restrictions that run counter to the aims of the Convention. Access to natural resources shall be by mutually agreed terms and subject to prior informed consent ..." The use of a proposed standard contract by culture collections is discussed as a way of contractually safeguarding the existing research commons, while observing the new rights established in the Convention on Biological Diversity as well as other existing and new legislation impacting on the accessibility of living microbial material.

A common basis for facilitated legitimate exchange of biological materials proposed by the European Culture Collections' Organisation

Being charged with the task of accessioning and supplying of living microbiological material, microbial culture collections are institutions that play a central role between the interests of a variety of user communities. On the one side are the providers of living microbiological material, such as individual scientists, institutions and countries of origin and on the other side are the various kinds of recipients/users of cultures of microorganisms from academia and industry. Thus, providing access to high quality biological material and scientific services while at the same time observing donor countries' rights, intellectual property rights, biosafety and biosecurity aspects poses demanding challenges. E.g. donor countries rights relate to Article 15 of the Convention on Biological Diversity: Contracting parties …. recognize the sovereign rights of states over their natural resources …. shall facilitate access to resources … and not impose restrictions that run counter to the aims of the Convention. Access to natural resources shall be by mutually agreed terms and subject to prior informed consent ... The use of a proposed standard contract by culture collections is discussed as a way of contractually safeguarding the existing research commons, while observing the new rights established in the Convention on Biological Diversity as well as other existing and new legislation impacting on the accessibility of living microbial material.

In this issue: Biotechnology Journal 12/2009

AbstractGenome‐scale in silico modelingMilne et al., Biotechnol. J. 2009, 4, 1653–1670Driven by advancements in high‐throughput biological technologies and the growing number of sequenced genomes, the construction of in silico models at the genome scale has provided powerful tools to investigate a vast array of biological systems and applications. Nathan Price and colleagues review comprehensively the use of such models in industrial and medical biotechnology, including biofuel generation, food production, and drug development. As such, genome‐scale models can provide a basis for rational genome‐scale engineering and synthetic biology. Genome‐scale in silico models promise to extend their application and analysis scope to become a transformative tool in biotechnology.From metagenomics to metaproteomicsTuffin et al., Biotechnol. J. 2009, 4, 1671–1683Metagenomics emerged in the late 1990s as a tool for accessing and studying the collective microbial genetic material in the environment and has been widely predicted to reach new dimensions of the protein sequence space. A decade on, researchers from South Africa see that while several novel enzyme activities and protein structures have been identified the greatest advancement has been made in the isolation of novel protein sequences, some of which have no close relatives, form deeply branched lineages and even represent novel families. However, there is much room for improvement in the methods employed that need to be addressed in order to access novel biocatalytic activities.Recombinant secondary metabolitesSchäfer et al., Biotechnol. J. 2009, 4, 1684–1703Plants produce a high diversity of natural products or secondary metabolites which have interesting biological properties and quite a number are of medicinal importance. Their functions range from the protection against herbivores and/or microbial pathogens to defend against abiotic stress, e.g. UV‐B exposure. Because the production of valuable natural products, such as the anticancer drugs paclitaxel, vinblastine or camptothecin in plants is a costly process, biotechnological alternatives to produce these alkaloids more economically become more and more important. This review provides an overview of the state of art to produce alkaloids in recombinant microorganisms, such as bacteria or yeast. In a longterm perspective, it will probably be possible to generate gene cassettes for complete pathways, which could then be used for the production of valuable natural products in bioreactors or for metabolic engineering of crop plants.

Metagenomic gene discovery: How far have we moved into novel sequence space?

Metagenomics emerged in the late 1990s as a tool for accessing and studying the collective microbial genetic material in the environment. The advent of the technology generated great excitement, as it has provided new opportunities and technologies for studying the wealth of microbial genetic diversity in the environment. Metagenomics has been widely predicted to access new dimensions of protein sequence space. A decade on, we review how far we have actually moved into new sequence space (and other aspects of protein space) using metagenomic tools. While several novel enzyme activities and protein structures have been identified through metagenomic strategies, the greatest advancement has been made in the isolation of novel protein sequences, some of which have no close relatives, form deeply branched lineages and even represent novel families. This is particularly true for glycosyl hydrolases and lipase/esterases, despite the fact that these activities are frequently screened for in metagenomic studies. However, there is much room for improvement in the methods employed and they will need to be addressed so that access to novel biocatalytic activities can be widened.

Effect of Electrode Shape on Power and Internal Resistance in Benthic Microbial Fuel Cell Material on Marine Sediment

As the electrode structure has a great effect on the performance of the benthic microbial fuel cell (BMFC), several graphite electrodes with different shapes (column, plane disk and tubular shape for example) are designed in this paper. The maximum power density (Pm) of BMFC-c and BMFC-d are 20.2 mW•m-2 and 14.9 mW•m-2 respectively, and the internal resistances are 333 Ω and 598 Ω respectively. Three cells are composed of three different sizes of graphite tubes, and their internal diameter of these electrodes are 2.5 cm (called it as BMFC-I for short); 1.0 cm (BMFC-II) and 0 cm (column shape for comparison, BMFC-III) respectively. Test results show that the Pm of BMFC-I, BMFC-II and BMFC-III are 13, 11 and 16 mW•m-2 respectively, and their internal resistances are 435 Ω, 488 Ω and 419 Ω respectively. Results show that the column structure electrode has a lower internal resistance and a higher power density than the disk and tubular structure electrode.

Characterizing the release of different composition of dissolved organic matter in soil under acid rain leaching using three-dimensional excitation–emission matrix spectroscopy

Although excitation–emission matrix spectroscopy (EEMS) has been widely used to characterize dissolved organic matter (DOM), there has no report that EEMS has been used to study the effects of acid rain on DOM and its composition in soil. In this work, we employed three-dimensional EEMS to characterize the compositions of DOM leached by simulated acid rain from red soil. The red soil was subjected to leaching of simulated acid rain of different acidity, and the leached DOM presented five main peaks in its EEMS: peak-A, related to humic acid-like (HA-like) material, at Ex/Em of 310–330/395–420 nm; peak-B, related to UV fulvic acid-like (FA-like) material, at Ex/Em of 230–280/400–435 nm; peak-C and peak-D, both related to microbial byproduct-like material, at Ex/Em of 250–280/335–355 nm and 260–280/290–320 nm, respectively; and peak-E, related to simple aromatic proteins, at Ex/Em of 210–240/290–340 nm. EEMS analysis results indicated that most DOM could be lost from red soil in the early phase of acid rain leaching. In addition to the effects of the pH of acid rain, the loss of DOM also depended on the properties of its compositions and the solubility of their complexes with aluminum. HA-like and microbial byproduct-like materials could be more easily released from red soil by acid rain at both higher pH (4.5 and 5.6) and lower pH (2.5 and 3) than that at middle pH (3.5). On the contrary, FA-like material lost in a similar manner under the action of different acid rains with pH ranging from 2.5 to 5.6.

Effects of reclamation and regeneration processes on organic matter from estuarine soils and sediments

Three adjacent areas in the Urdaibai Estuary (NW Spain) comprising a reclaimed salt marsh, a natural salt marsh and a regenerated salt marsh were sampled in order to study edaphic processes and changes in organic matter (OM) composition related to reclamation and regeneration of soils and sediments in estuarine environments. For this, a general analytical description of the soil and sediment profiles was carried out and the OM was characterized using solid state 13C nuclear magnetic resonance (NMR) spectroscopy and analytical pyrolysis–gas chromatography/mass spectrometry (Py–GC/MS). The reclaimed salt marsh displayed very different characteristics from the natural salt marsh throughout the whole profile, indicating an important degree of change in soil quality and, particularly, in OM composition caused by the reclamation. At this reclaimed site, characterized by oxic conditions and a lack of tidal influence, transformation of the former salt marsh to grassland resulted in input of OM richer in carbohydrates and with a lower contribution of aromatic compounds than the OM in the salt marsh soils. On the other hand, the edaphic characteristics of the regenerated marsh were similar to those of the natural marsh and the vegetation cover was the same. However, the natural marsh accumulated twice the amount of organic carbon at the surface as the regenerated marsh. Furthermore, the natural marsh contained more aromatic material, as detected with NMR and the Py–GC/MS results indicated a greater presence of microbial material. These differences indicate that the regenerated marsh has not completely recovered its natural characteristics, despite the long period (more than 40y) since the beginning of the regeneration process. We propose the composition of soil OM as a suitable long term indicator of reclamation and regeneration processes in the salt marsh environments.

Asian sand dust aggravates allergic rhinitis in guinea pigs induced by Japanese cedar pollen

Asian sand dust (ASD) contains microbial materials, sulfate (SO42−), and nitrate (NO3−), and is derived from air pollutants in East China. ASD reportedly causes adverse respiratory health effects; a case in point is aggravated allergen-associated experimental lung eosinophilia. Guinea pigs were administered normal saline (control), ASD (0.3 mg/animal), ASD (0.6 mg/animal), Japanese cedar pollen (JCP) (0.2 mg/kg body weight), JCP + ASD (0.3 mg/animal), or JCP + ASD (0.6 mg/animal), into their nasal cavities at seven weekly intervals. The number of sneezes, amount of nasal secretions, and nasal obstructing response were measured as indices of nasal responses. Total immunoglobulin E (IgE) antibodies in serum and the number of eosinophils, histamine, and arachidonic acid metabolites in nasal cavity lavage fluids (NCLF) were also measured. ASD enhanced the JCP-associated nasal obstructing response, but not the number of sneezes or amount of nasal secretions. ASD enhanced JCP-associated cysteinyl leukotrienes (C4, D4, E4) and histamine production in NCLF. ASD augmented the number of eosinophils in NCLF and total IgE in serum induced by JCP. ASD enhanced eosinophil recruitment in the nasal mucosa, and goblet cell proliferation in the nasal epithelium induced by JCP. These results suggest that ASD enhances the nasal allergic reaction induced by repeated JCP administration in guinea pigs.

Denture disinfection by microwave irradiation: A randomized clinical study

Objective This study evaluated the clinical effectiveness of two exposure times of microwave irradiation on the disinfection of complete dentures. Methods Biofilm samples were collected from dentures of 30 patients, who were randomly divided into two experimental groups of 15 subjects each: Group 1—patients had their maxillary denture microwaved for 3 min (650 W); Group 2—patients had their maxillary denture microwaved for 2 min (650 W). Denture biofilm samples were taken with swabs, before (left side surfaces) and after (right side surfaces) microwave irradiation. All microbial material was plated on selective media for Candida spp., Staphylococcus spp., mutans streptococci and a non-selective media. After incubation (48 h/37 °C), the number of colony-forming units (cfu/mL) was counted. Microorganisms which grew on selective media were identified using biochemical methods. The data were statistically analyzed by Kruskal–Wallis test, followed by Dunn's post-test ( α = 0.05). Results Microwave irradiation for 3 min (Group 1) resulted in sterilization of all dentures evaluated. After microwave irradiation for 2 min (Group 2), a significant decrease in Candida spp. ( P = 0.0062), Staphylococcus spp. ( P = 0.0178), mutans streptococci ( P = 0.0047) and non-identified species ( P < 0.0001) was achieved in comparison with the cfu/mL obtained before irradiation. The colonies grown after 2 min of microwave irradiation were identified as Candida albicans, non-aureus Staphylococci and Streptococcus mutans. Conclusion Microwave irradiation for 3 min may be a potential treatment to prevent cross-contamination.

Promoting human health through forests: overview and major challenges

This review aims to contribute to the ongoing discussion about human health, global change, and biodiversity by concentrating on the relationships between forests and human health. This review gives a short overview of the most important health benefits that forests provide to humans, and the risks that forests may pose to human health. Furthermore, it discusses the future challenges for the research on the links between forests and human health, and for delivering health through forests in practice. Forests provide enormous possibilities to improve human health conditions. The results of a vast amount of research show that forest visits promote both physical and mental health by reducing stress. Forests represent rich natural pharmacies by virtue of being enormous sources of plant and microbial material with known or potential medicinal or nutritional value. Forest food offers a safety net for the most vulnerable population groups in developing countries, and healthy forest ecosystems may also help in regulation of infectious diseases. Utilizing forests effectively in health promotion could reduce public health care budgets and create new sources of income. Main challenges to delivering health through forests are due to ecosystem and biodiversity degradation, deforestation, and climate change. In addition, major implementation of research results into practice is still lacking. Inadequate implementation is partly caused by insufficient evidence base and partly due to the lack of policy-makers' and practitioners' awareness of the potential of forests for improving human health. This calls for strong cooperation among researchers, policy-makers, and practitioners as well as between different sectors, especially between health and environmental professionals.

A Novel Fold in the TraI Relaxase–Helicase C-Terminal Domain Is Essential for Conjugative DNA Transfer

TraI relaxase-helicase is the central catalytic component of the multiprotein relaxosome complex responsible for conjugative DNA transfer (CDT) between bacterial cells. CDT is a primary mechanism for the lateral propagation of microbial genetic material, including the spread of antibiotic resistance genes. The 2.4-A resolution crystal structure of the C-terminal domain of the multifunctional Escherichia coli F (fertility) plasmid TraI protein is presented, and specific structural regions essential for CDT are identified. The crystal structure reveals a novel fold composed of a 28-residue N-terminal alpha-domain connected by a proline-rich loop to a compact alpha/beta-domain. Both the globular nature of the alpha/beta-domain and the presence as well as rigidity of the proline-rich loop are required for DNA transfer and single-stranded DNA binding. Taken together, these data establish the specific structural features of this noncatalytic domain that are essential to DNA conjugation.

Humic substances in estuarine soils colonized by Spartina maritima

This study characterizes humic substances (HS) from two soils colonized by Spartina maritima at different physiographical positions in estuarine environments on the north-western coast of the Iberian Peninsula: the Villaviciosa site, a stand close to the main tidal channel, and the Ortigueira site, located in the low salt marsh. Humic and fulvic acids were extracted from the soils and characterized qualitatively by the following spectroscopic techniques: Fourier transform infrared spectroscopy, fluorescence spectroscopy and solid-state 13C nuclear magnetic resonance. The characterized HS showed a predominance of low humified compounds with a high proportion of aliphatic components and a low degree of aromaticity. The HS composition differed substantially between sites. In the Villaviciosa soil, the large amount of nonpolar aliphatic components and the very low degree of aromaticity may indicate a significant contribution of marine organic matter and/or microbial material to the HS. However, in the HS from the Ortigueira soil, the higher proportion of polysaccharides together with the presence of lignin-derived compounds may indicate greater inputs of vascular plant material. The δ 13C isotopic composition of the bulk soils highlights the large input of Spartina maritima debris to the Ortigueira site, whereas in the Villaviciosa site, organic contributions from this C4 vascular plant were not so evident. The results indicate that in these soils colonized by Spartina maritima, physiographical position has an important effect on the composition of soil HS and, therefore, must be considered in the study of organic matter characteristics in such estuarine environments.

The Role of Calcium Ions in the Photocatalytic Oxidation of Humic Acid at Neutral pH

Humic acids (HAs) are natural organic matter derived from the decomposition of plant, algal, and microbial materials. They belong to the group of the most predominant type of natural organic matter present in ground and surface waters. HAs affect the mobility and bioavailability of aquatic contaminants. However, if they are left unremoved from the water before water treatment processes, they can form carcinogenic disinfection by-products, such as trihalomethanes, haloacetic acids, and other halogenated disinfection by-products, that can pose a threat to human beings. An advanced oxidation process using UV light and a commercially available titanium dioxide was used to oxidize HA at a pH that is similar to that of natural water. The effect of adding calcium ions to the adsorption and the photocatalytic oxidation of HAs was studied. The effect of varying the TiO(2) load was also investigated. The experiment was done using a photochemical batch reactor equipped with a mercury lamp emitting light with wavelengths of 310-580 nm. The absorbances by the samples were determined at wavelengths of 254 nm and 436 nm, which represent the aromatic-compound content of and the color of the solution, respectively. Results indicated calcium ions have an effect on both the adsorption and the photocatalytic oxidation of HA at a pH within 8.0 +/- 0.5. Calcium ions facilitated adsorption of HA onto the surface of TiO(2) and resulted to faster photocatalytic oxidation. The data were plotted with respect to the normalized absorbances and irradiation time.

FBI, Allied Scientists Describe Analytic Side of Anthrax Investigation

A “body of powerful evidence” identifies both “the origin and the perpetrator of the 2001 Bacillus anthracis mailings,” says Vahid Majidi of the Federal Bureau of Investigation (FBI) in Washington, D.C. That series of incidents in 2001 led to five deaths and 17 additional cases of pneumonia— and a massive investigation that, after various twists and turns led FBI officials to name as its prime and final suspect, Bruce E. Ivins, a microbiologist with the U.S. Army Medical Research Institute for Infectious Diseases, Fort Detrick, Md. (USAMRIID), who committed suicide last July. Several weeks later, Majidi, who heads the FBI Weapons of Mass Destruction Directorate, several colleagues from FBI, and several microbiologists and other scientists who helped in analyzing anthrax-related evidence met with reporters in August to summarize scientific findings that link those spores to Ivins, albeit without referring to him directly during their discussions. FBI officials also explicitly declined to discuss other evidence linking Ivins to the 2001 anthrax crimes, restricting discussions to microbial forensics and other more traditional procedures used for analyzing microbial and related materials. Some of those procedures helped in determining that the spores contained “no intentional additives,” despite many reports that they had been chemically altered to be “more dispersible,” Majidi says. Eventual microscopic analysis and X-ray imaging indicate that silicon oxide (silica) associated with the spore samples is on the “inside of the spore coat” and also is found “in vegetative cells,” adds Joseph Michael from Sandia National Laboratories in Albuquerque, N.M., who served as one of many outside analysts for FBI during the investigation.

Effect of immunostimulants on the haemolymph haemagglutinins of tiger shrimpPenaeus monodon

The levels of haemagglutinins in Penaeus monodon, following administration of immunostimulants, β-glucans and/or vibrio bacterin either orally or by immersion, were studied. The freshly drawn haemolymph was incubated with microbial materials like β-glucans/vibrio bacterin and serum obtained after coagulation was administered to naïve animals. The immunostimulant treatments either via immersion, feeding or injection were found to cause an increase (P<0.006) in haemagglutination activity (HA) of the haemolymph against mouse erythrocytes. Injection of saline or heterologous haemolymph caused an increase in the HA, but injection of haemolymph serum obtained by clotting haemolymph in the presence of vibrio bacterin or glucan did not bring about an increase in HA. There was no change in the haemolymph protein profile of the groups receiving immunostimulants through immersion or feed. Two protein bands (27 and 30 kDa), which were present in the uninjected group, were found to be overexpressed in the haemolymph-injected groups. Three bands of 17, 21 and 23 kDa, which were absent in control or saline-injected groups, were present in all the haemolymph serum-injected groups. The study indicates that modulation of HA may partly account for the immunomodulatory activity of immunostimulants like β-glucan or vibrio bacterins.

The use of DNA from non-human sources

Biological material is found ubiquitously during a forensic investigation. Currently the focus of an examination is primarily on human biological material to provide a link between items and to identify the source of the human material. Much associative evidence may exist in the form of botanical, microbial or non-human animal material that is either overlooked or not examined. Further there are crimes that are specifically against animals, such as the trade in endangered species. This paper covers many of the uses of DNA in animal and wildlife crimes. The scope of the paper does not cover the wide area of botanical and microbial examinations that are possible but will focus on the use of DNA to identify a species or to link a sample to a particular organism.

The Effects of Microbial Materials Adhered to Asian Sand Dust on Allergic Lung Inflammation

Asian sand dust (ASD) containing microbiological materials, sulfate (SO(4)(2)), and nitrate (NO(3)(-) ) derived from air pollutants in East China, reportedly cause adverse respiratory health effects. ASD aggravates ovalbumin (OVA)-associated experimental lung eosinophilia. In this study, the toxic materials adsorbed onto ASD were excluded by heat treatment at 360 degrees C for 30 min. The effects of nonheated ASD or heated ASD (H-ASD) toward the allergic lung inflammation were compared in murine lungs. ICR mice were administered intratracheally with normal saline (control), H-ASD, ASD, OVA, OVA + H-ASD, and OVA + ASD, four times at 2-week intervals. ASD only increased neutrophils in bronchoalveolar lavage fluids (BALFs) along with pro-inflammatory mediators, such as keratinocyte chemoattractant (KC). H-ASD and ASD enhanced eosinophil recruitment induced by OVA in the alveoli and in the submucosa of the airway, which has a goblet cell proliferation in the bronchial epithelium. The two ASDs synergistically increased interleukin-5 (IL-5), monocyte chemotactic protein-3 (MCP-3), and eotaxin, which were associated with OVA, in BALF. The enhancing effects were much greater in ASD than in H-ASD. The two ASDs induced the adjuvant effects to specific IgE and IgG1 production by OVA. In the in vitro study using RAW264.7 cells, ASD increased the expression of Toll-like receptor 2 (TLR 2) mRNA but not TLR4 mRNA. H-ASD caused no expression of either TLR mRNA. These results suggest that the aggravated lung eosinophilia by ASD may be due to activation of Th2-associated immune response via the activation of TLR2 by microbial components adhered to ASD.