Su Kyoung Shin

Metagenomic Insights into the Bioaerosols in the Indoor and Outdoor Environments of Childcare Facilities

Airborne microorganisms have significant effects on human health, and children are more vulnerable to pathogens and allergens than adults. However, little is known about the microbial communities in the air of childcare facilities. Here, we analyzed the bacterial and fungal communities in 50 air samples collected from five daycare centers and five elementary schools located in Seoul, Korea using culture-independent high-throughput pyrosequencing. The microbial communities contained a wide variety of taxa not previously identified in child daycare centers and schools. Moreover, the dominant species differed from those reported in previous studies using culture-dependent methods. The well-known fungi detected in previous culture-based studies (Alternaria, Aspergillus, Penicillium, and Cladosporium) represented less than 12% of the total sequence reads. The composition of the fungal and bacterial communities in the indoor air differed greatly with regard to the source of the microorganisms. The bacterial community in the indoor air appeared to contain diverse bacteria associated with both humans and the outside environment. In contrast, the fungal community was largely derived from the surrounding outdoor environment and not from human activity. The profile of the microorganisms in bioaerosols identified in this study provides the fundamental knowledge needed to develop public health policies regarding the monitoring and management of indoor air quality.

Cochleicola gelatinilyticus gen. nov., sp. nov., Isolated from a Marine Gastropod, Reichia luteostoma.

A yellow, rod-shaped, non-motile, gram-negative, and strictly aerobic bacterial strain, designated LPB0005(T), was isolated from a marine gastropod, Reichia luteostoma. Here the genome sequence was determined, which comprised 3,395,737 bp with 2,962 protein-coding genes. The DNA G+C content was 36.3 mol%. The 16S rRNA gene sequence analysis indicated that the isolate represents a novel genus and species in the family Flavobacteriaceae, with relatively low sequence similarities to other closely related genera. The isolate showed chemotaxonomic properties within the range reported for the family Flavobacteriaceae, but possesses many physiological and biochemical characteristics that distinguished it from species in the closely related genera Ulvibacter, Jejudonia, and Aureitalea. Based on phylogenetic, phenotypic, and genomic analyses, strain LPB0005(T) represents a novel genus and species, for which the name Cochleicola gelatinilyticus gen. nov., sp. nov. is proposed. The type strain is LPB0005(T) (= KACC 18693(T) = JCM 31218(T)).

Reclassification of Serpens flexibilis Hespell 1977 as Pseudomonas flexibilis comb. nov., with Pseudomonas tuomuerensis Xin et al. 2009 as a later heterotypic synonym

Serpens flexibilis was proposed in 1977 and approved in 1980 without the 16S rRNA gene sequence information. The sequence of S. flexibilis became available in 2010, after the publication of Pseudomonas tuomuerensis in 2009. Our preliminary phylogenetic analyses indicated that these two strains share high sequence similarity and therefore showed strong potential to be united into a single species. To clarify the taxonomic status of the two species, a polyphasic taxonomy study was conducted including whole genome sequencing. The value of average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between the genome sequences of S. flexibilis ATCC 29606(T) and P. tuomuerensis JCM 14085(T) were 98.1% and 89.0%, respectively. The phenotypic and chemotaxonomic properties including enzymatic activities, substrate utilization profiles, and fatty acids, supported that the two taxa have no pronounced difference and should thus constitute a single species. Therefore, we propose to transfer Serpens flexibilis Hespell 1977 to the genus Pseudomonas as Pseudomonas flexibilis comb. nov. (type strain=ATCC 29606(T)), with Pseudomonas tuomuerensis Xin et al. 2009 as a later heterotypic synonym of Pseudomonas flexibilis.

Polaribacter vadi sp. nov., isolated from a marine gastropod

A Gram-reaction-negative, non-motile, yellow-pigmented, NaCl-requiring bacterial strain, designated LPB0003T, was isolated from a marine gastropod, the granulated dogwhelk (Thais luteostoma). The complete genome sequence of strain LPB0003T (GenBank accession number CP017477), sized 3.81 Mb, had 3296 protein-coding genes. 16S rRNA gene sequence analysis indicated that strain LPB0003T belongs to the genus Polaribacter within the family Flavobacteriaceae with sequence similarities of 95.3-97.8 % to other species of the genus Polaribacter. The average nucleotide identities with closely related species were not higher than 80 %. The isoprenoid quinone (MK-6) and DNA G+C content (29.6 mol%) of strain LPB0003T fell within the expected ranges for the genus Polaribacter. The major polar lipids of the isolate were phosphatidylethanolamine, two unidentified aminolipids, an unidentified phospholipid, an unidentified glycolipid, and two unidentified lipids. The predominant cellular fatty acids were iso-C15 : 0, iso-C15  : 0 3-OH, C16 : 1ω7c and/or C16 : 1ω6c (summed feature 3), and C15 : 1ω6c. Based on phylogenetic, genomic and phenotypic data presented in this study, strain LPB0003T should be classified as a novel species of the genus Polaribacter, for which the name Polaribacter vadi sp. nov. is proposed. The type strain is LPB0003T (=KACC 18704T=JCM 31217T).

Real-time selective monitoring of allergenic Aspergillus molds using pentameric antibody-immobilized single-walled carbon nanotube-field effect transistors

Airborne fungus, including Aspergillus species, is one of the major causes of human asthma. Conventional immunoassay or DNA-sequencing techniques, although widely used, are usually labor-intensive, time-consuming and expensive. In this paper, we demonstrate a sensor for the rapid detection of Aspergillus niger, a well-known allergenic fungal species, using single-walled carbon nanotube (SWNT) field effect transistors (FETs) functionalized with pentameric antibodies that specifically bind to Aspergillus species. This strategy resulted in the real time and highly sensitive and selective detection of Aspergillus due to an electrostatic gating effect from the Aspergillus fungus. This mechanism is in contrast to a previously reported Aspergillus sensor, which was based on mobility modulation from Aspergillus adsorption. Also, our sensor shows a much wider detection range from 0.5 pg mL−1 to 10 μg mL−1 with a lower detection limit of 0.3 pg mL−1. The resulting SWNT-FET was able to selectively detect Aspergillus molds in the presence of more concentrated amounts of other mold species such as Alternaria alternata, Cladosporium cladosporioides, and Penicillium chrysogenum. We expect that our results can be used in real-time monitoring of the indoor air quality of a variety of public facilities for the elderly and children, who are more vulnerable to environmental biohazards.

Fully Automated Field-Deployable Bioaerosol Monitoring System Using Carbon Nanotube-Based Biosensors

Much progress has been made in the field of automated monitoring systems of airborne pathogens. However, they still lack the robustness and stability necessary for field deployment. Here, we demonstrate a bioaerosol automonitoring instrument (BAMI) specifically designed for the in situ capturing and continuous monitoring of airborne fungal particles. This was possible by developing highly sensitive and selective fungi sensors based on two-channel carbon nanotube field-effect transistors (CNT-FETs), followed by integration with a bioaerosol sampler, a Peltier cooler for receptor lifetime enhancement, and a pumping assembly for fluidic control. These four main components collectively cooperated with each other to enable the real-time monitoring of fungi. The two-channel CNT-FETs can detect two different fungal species simultaneously. The Peltier cooler effectively lowers the working temperature of the sensor device, resulting in extended sensor lifetime and receptor stability. The system performance was verified in both laboratory conditions and real residential areas. The system response was in accordance with reported fungal species distribution in the environment. Our system is versatile enough that it can be easily modified for the monitoring of other airborne pathogens. We expect that our system will expedite the development of hand-held and portable systems for airborne bioaerosol monitoring.

Flavobacterium gilvum sp. nov., isolated from stream water

A cream-colored, non-gliding, aerobic Flavobacterium strain, designated EM1308T, was isolated from stream water. Gene sequence analysis of the 16S rRNA indicated that this isolate is closely related to Flavobacterium glycines NBRC 105008T (97.3 %) and Flavobacterium piscis CCUG 60099T (97.2 %). To evaluate the genomic relatedness of the isolate with its neighbors, the whole genome sequences of strain EM1308T and type strains of F. glycines and F. piscis were determined. The average nucleotide identities revealed that strain EM1308T is independent from other Flavobacterium species. The properties of major cellular fatty acids, polar lipids, menaquinone, and DNA G+C content of the isolate were within the general range for the genus Flavobacterium, but many biochemical and physiological characteristics distinguished the isolate from previously known species. Thus, strain EM1308T represents a novel species of the genus Flavobacterium, for which the name Flavobacterium gilvum sp. nov. is proposed. The type strain is EM1308T (= KACC 18113T = JCM 30144T).

Flavobacterium crassostreae sp. nov., isolated from Pacific oyster.

A yellow, rod-shaped, Gram-reaction-negative bacterial strain, designated LPB0076T, was isolated from a Pacific oyster. Results of 16S rRNA gene sequence analyses indicated that the strain represented a member of the genus Flavobacterium. It had the highest sequence similarity to the type strains of Flavobacterium frigidarium (97.6 %) and Flavobacterium omnivorum (97.0 %), and its similarities with all other species of the genus Flavobacterium were below 97.0 %. Its genome size (3.02 Mb), DNA G+C content (36.0 mol%), predominant cellular fatty acids (anteiso-C15 : 0, iso-C15 : 0 and C16 : 1ω7c and/or C17 : 1ω6c), and major polar lipid (phosphatidylethanolamine) were similar to those described previously for members of the genus Flavobacterium. In contrast, a number of phenotypic characteristics, including the inability to grow microaerophilically, absence of flexirubin-type pigments and gliding motility and differences in enzymatic reactions, clearly distinguished LPB0076T from other species of the genus Flavobacterium. The polyphasic data presented in this study indicate that this isolate should be classified as representing a novel species of the genus Flavobacterium. The name Flavobacterium crassostreae sp. nov. is therefore proposed for the isolate, with the type strain being LPB0076T (=KACC 18706T=JCM 31219T).

Wolbachia sequence typing in butterflies using pyrosequencing

Wolbachia is an obligate symbiotic bacteria that is ubiquitous in arthropods, with 25-70% of insect species estimated to be infected. Wolbachia species can interact with their insect hosts in a mutualistic or parasitic manner. Sequence types (ST) of Wolbachia are determined by multilocus sequence typing (MLST) of housekeeping genes. However, there are some limitations to MLST with respect to the generation of clone libraries and the Sanger sequencing method when a host is infected with multiple STs of Wolbachia. To assess the feasibility of massive parallel sequencing, also known as next-generation sequencing, we used pyrosequencing for sequence typing of Wolbachia in butterflies. We collected three species of butterflies (Eurema hecabe, Eurema laeta, and Tongeia fischeri) common to Korea and screened them for Wolbachia STs. We found that T. fischeri was infected with a single ST of Wolbachia, ST41. In contrast, E. hecabe and E. laeta were each infected with two STs of Wolbachia, ST41 and ST40. Our results clearly demonstrate that pyrosequencing-based MLST has a higher sensitivity than cloning and Sanger sequencing methods for the detection of minor alleles. Considering the high prevalence of infection with multiple Wolbachia STs, next-generation sequencing with improved analysis would assist with scaling up approaches to Wolbachia MLST.

Gramella salexigens sp. nov., isolated from seawater

A Gram-stain-negative, rod-shaped, aerobic bacterial strain, designated LPB0144T was isolated from the seawater of South Sea, Korea. The 16S rRNA gene sequence was found to share the highest sequence similarity to Gramella sediminilitoris GHTF-27T (97.7 %) and the strain branched within the radiation of the genus Gramella in phylogenetic trees. Thus, the taxonomic position of the novel isolate was investigated using a polyphasic approach and complete genome sequencing. Strain LPB0144T has a circular chromosome of 2.98 Mb with DNA G+C content of 38.2 mol%. The genome includes 2604 protein-coding genes and three copies of rRNA operons. The detected respiratory quinone (MK-6) and the major polar lipid (phosphatidylethanolamine) resemble the chemotaxonomic profile of other Gramella species. The major cellular fatty acid profile (iso-C15 : 0, iso-C16 : 0, iso-C17 : 0 3-OH and anteiso-C15 : 0) is also within the range of Gramella, but detailed composition and amounts were found to be different from those of closely related neighbours. Many biochemical and physiological characteristics also distinguished the isolate from other species within the genus Gramella. On the basis of polyphasic taxonomic data obtained here, we propose strain LPB0144T as a novel Gramella species, for which the name Gramella salexigens sp. nov. is proposed. The type strain is LPB0144T (=KACC 18894T=JCM 31560T).