Journal of Microbiology

Volume 55(8); August 2017

Journal Articles

Uliginosibacterium flavum sp. nov. isolated from an artificial lake: Ji Young Kang , Jeesun Chun , Kwang Yeop Jahng; J. Microbiol. 2017;55(8):595-599. Published online July 28, 2017; DOI: https://doi.org/10.1007/s12275-017-7002-6

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Abstract: A Gram-negative, facultative anaerobic, rod-shaped, motile by means of a polar flagellum, greenish-yellow-pigmented bacterial strain (designated strain JJ3220T) was isolated from an artificial lake in South Korea and characterized using a polyphasic approach. The 16S rRNA gene sequence of strain JJ3220T indicated that the isolate belongs to the family Rhodocyclaceae, and that it exhibits 96.4% similarity to Uliginosibacterium paludis KBP-13T. The major cellular fatty acids of the novel strain were C14:0, C16:0, and summed feature 3 (C16:1 ω6c and/or C16:1 ω7c). Strain JJ3220T had flexirubin-type pigments. The DNA G+C content of the strain was 62.8%. The major respiratory quinone and major polar lipid of strain JJ3220T were ubiquinone-8 and phosphatidylethanolamine, respectively. Based on the morphological and physiological properties and biochemical evidence presented, it can be concluded that strain JJ3220T represents a novel species of the genus Uliginosibacterium. The type strain Uliginosibacterium flavum is JJ3220T (=KACC 17644T =JCM 19465T).

Genetic variation and phylogenetic relationships of the ectomycorrhizal Floccularia luteovirens on the Qinghai-Tibet Plateau: Rui Xing , Qing-bo Gao , Fa-qi Zhang , Peng-cheng Fu , Jiu-li Wang , Hui-ying Yan , Shi-long -Chen; J. Microbiol. 2017;55(8):600-606. Published online July 4, 2017; DOI: https://doi.org/10.1007/s12275-017-7101-4

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Abstract: Floccularia luteovirens, as an ectomycorrhizal fungus, is widely distributed in the Qinghai-Tibet Plateau. As an edible fungus, it is famous for its unique flavor. Former studies mainly focus on the chemical composition and genetic structure of this species. However, the phylogenetic relationship between genotypes remains unknown. In this study, the genetic variation and phylogenetic relationship between the genotypes of F. luteovirens in Qinghai-Tibet Plateau was estimated through the analysis on two protein-coding genes (rpb1 and ef-1α) from 398 individuals collected from 24 wild populations. The sample covered the entire range of this species during all the growth seasons from 2011 to 2015. 13 genotypes were detected and moderate genetic diversity was revealed. Based on the results of network analysis, the maximum likelihood (ML), maximum parsimony (MP), and Bayesian inference (BI) analyses, the genotypes H-1, H-4, H-6, H-8, H-10, and H-11 were grouped into one clade. Additionally, a relatively higher genotype diversity (average h value is 0.722) and unique genotypes in the northeast edge of Qinghai- Tibet plateau have been found, combined with the results of mismatch analysis and neutrality tests indicated that Southeast Qinghai-Tibet plateau was a refuge for F. luteovirens during the historical geological or climatic events (uplifting of the Qinghai-Tibet Plateau or Last Glacial Maximum). Furthermore, the present distribution of the species on the Qinghai-Tibet plateau has resulted from the recent population expansion. Our findings provide a foundation for the future study of the evolutionary history and the speciation of this species.

Pseudaminobacter granuli sp. nov., isolated from granules used in a wastewater treatment plant: Young Ki Hahn , Minseok S. Kim , Wan-Taek Im; J. Microbiol. 2017;55(8):607-611. Published online July 28, 2017; DOI: https://doi.org/10.1007/s12275-017-7257-y

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Abstract: A Gram negative, aerobic, non-motile and rod-shaped bacterial strain designated as Gr-2T was isolated from granules used in a wastewater treatment plant in Korea, and its taxonomic position was investigated using a polyphasic approach. Strain Gr-2T grew at 18–37°C (optimum temperature, 30°C) and a pH of 6.0–8.0 (optimum pH, 7.0) on R2A agar medium. Based on 16S rRNA gene phylogeny, the novel strain showed a new branch within the genus Pseudaminobacter of the family Phyllobacteriaceae, and formed clusters with Pseudaminobacter defluvii THI 051T (98.9%) and Pseudaminobacter salicylatoxidans BN12T (98.7%). The G+C content of the genomic DNA was 63.6%. The predominant respiratory quinone was ubiquinone-10 (Q-10) and the major fatty acids were cyclo-C19:0 ω8c, C18:1 ω7c, and iso-C17:0. The overall polar lipid patterns of Gr-2T were similar to those determined for the other Pseudaminobacter species. DNA-DNA relatedness values between strain Gr-2T and its closest phylogenetically neighbors were below 18%. Strain Gr-2T could be differentiated genotypically and phenotypically from the recognized species of the genus Pseudaminobacter. The isolate therefore represents a novel species, for which the name Pseudaminobacter granuli sp. nov. is proposed with the type strain Gr-2T (=KACC 18877T =LMG 29567T).

Imipenem-resistant Gram-negative bacterial isolates carried by persons upon medical examination in Korea: So Yeon Kim , Sang Yop Shin , Ji-Young Rhee , Kwan Soo Ko; J. Microbiol. 2017;55(8):612-618. Published online July 18, 2017; DOI: https://doi.org/10.1007/s12275-017-6555-8

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Abstract: Carbapenem-resistant Gram-negative bacteria (CR-GNB) have emerged and disseminated worldwide, become a great concern worldwide including Korea. The prevalence of fecal carriage of imipenem-resistant Gram-negative bacteria (IRGNB) in persons in Korea was investigated. Stool samples were collected from 300 persons upon medical examination. Samples were screened for IR-GNB by using MacConkey agar with 2 μl/ml imipenem. Species were identified by 16S rRNA gene sequence analysis, and antimicrobial susceptibility was determined by the broth microdilution method. In total, 82 IR-GNB bacterial isolates were obtained from 79 (26.3%) out of 300 healthy persons. Multilocus sequence typing analysis showed very high diversity among IR P. aeruginosa, S. maltophilia, and E. cloacae isolates, and pulsedfield gel electrophoresis revealed five main pulsotypes of IR P. mirabilis. As for the presence of metallo-β-lactamases (MBLs), only one IMP-25-producing S. marcescens isolate was identified. Although only one carbapenemase-producing isolate was identified, the high colonization rates with IRGNB isolates in this study is notable because carriers may be a reservoir for the dissemination of resistant pathogens within the community as well as in health care institutions.

Morphologies and phenotypes in Bacillus subtilis biofilms: Xiaoling Wang , Shuo Meng , Jingshi Han; J. Microbiol. 2017;55(8):619-627. Published online July 4, 2017; DOI: https://doi.org/10.1007/s12275-017-7041-z

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Abstract: In this study, we explored Bacillus subtilis biofilm growth under various conditions such as the use of substrates with different stiffnesses and nutrient levels using a well-developed optical imaging technique to spatially and temporally track biofilm growth. We also developed a quantitative method to characterize B. subtilis biofilm morphologies under various growth conditions. To determine biofilm rim irregularities, we used the dimensionless P2A ratio, defined as P2/4πA, where P is the perimeter and A is the area of the biofilm. To estimate biofilm thickness from transmission images, we developed a calibration procedure based on Beer- Lambert’s law and cross sectioning. Furthermore, to determine the distributions of different B. subtilis cell phenotypes during biofilm growth, we used a triple-fluorescence-labeled B. subtilis strain that expressed motility, matrix production, and sporulation. Based on this work, we are able to tune biofilm growth by changing its growing environment.

NMR-based metabolomics reveals the metabolite profiles of Vibrio parahaemolyticus under ferric iron stimulation: Jun Zhou , Chenyang Lu , Dijun Zhang , Chennv Ma , Xiurong Su; J. Microbiol. 2017;55(8):628-634. Published online July 28, 2017; DOI: https://doi.org/10.1007/s12275-017-6551-z

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Abstract: Vibrio parahaemolyticus is a halophilic bacterium endemic to coastal areas, and its pathogenicity has caused widespread seafood poisoning. In our previous research, the protein expression of V. parahaemolyticus in Fe3+ medium was determined using isobaric tags for relative and absolute quantitation (iTRAQ). Here, nuclear magnetic resonance (NMR) was used to detect changes in the V. parahaemolyticus metabolome. NMR spectra were obtained using methanol-water extracts of intracellular metabolites from V. parahaemolyticus under various culture conditions, and 62 metabolites were identified, including serine, arginine, alanine, ornithine, tryptophan, glutamine, malate, NAD+, NADP+, oxypurinol, xanthosine, dCTP, uracil, thymine, hypoxanthine, and betaine. Among these, 21 metabolites were up-regulated after the stimulation of the cells by ferric iron, and 9 metabolites were down-regulated. These metabolites are involved in amino acid and protein synthesis, energy metabolism, DNA and RNA synthesis and osmolality. Based on these results, we conclude that Fe3+ influences the metabolite profiles of V. parahaemolyticus.

A rapid and simple method for identifying bacterial polar lipid components in wet biomass: Tuan Manh Nguyen , Jaisoo Kim; J. Microbiol. 2017;55(8):635-639. Published online July 4, 2017; DOI: https://doi.org/10.1007/s12275-017-7092-1

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Abstract: There are marked differences between wet and freeze-dried cells with regard to the identification of polar lipid components. The determination of the polar lipid composition of freeze-dried cells is well established. However, several approaches to identifying polar lipid components in wet cells have met with limited success owing to the presence of non-polar compounds in the extracts, resulting in a lipid composition with a narrow scope. In this study, we surveyed the lipid profiles of the wet biomasses of three Gram-positive (Microbacterium lacticum, Rhodococcus koreensis, and Streptomyces longwoodensis) and two Gram-negative (Pseudomonas aeruginosa and Novosphingobium capsulatum) bacteria; the results were comparable in quality to those obtained using a standard freeze-dried approach. Moreover, our improved method ensures simple lipid extraction. Overall, the results of the analysis showed minor lipid profile differences between the two approaches with regard to quantity, and lipid identification was consistent in both methods for all species.

Effect of amikacin on cell wall glycopeptidolipid synthesis in Mycobacterium abscessus: So-Young Lee , Hee-Youn Kim , Byoung-Jun Kim , Hong Kim , Seung-hyeok Seok , Bum-Joon Kim , Yoon-Hoh Kook; J. Microbiol. 2017;55(8):640-647. Published online July 28, 2017; DOI: https://doi.org/10.1007/s12275-017-6503-7

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Abstract: Cultivation of the smooth colony Mycobacterium abscessus at the sub-minimum inhibitory concentration (MIC) of amikacin changed its growth pattern including its colony morphology (smooth to rough) and cell arrangement (dispersed to cord formation). In addition, reduced sliding motility and biofilm formation were observed. The amount of glycogpetidolipid (GPL) and mRNA expression of key genes involved in GPL synthesis were decreased in the amikacin-treated M. abscessus strain. An in vitro infection assay revealed that the amikacin-treated smooth M. abscessus strain induced more pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) than that of the smooth strain in murine macrophage cells. These results suggest that long-term exposure to a low concentration of amikacin causes a physical change in the cell wall which may increase its virulence.

Composition and abundance of microbiota in the pharynx in patients with laryngeal carcinoma and vocal cord polyps: Hongli Gong , Boyan Wang , Yi Shi , Yong Shi , Xiyan Xiao , Pengyu Cao , Lei Tao , Yuezhu Wang , Liang Zhou; J. Microbiol. 2017;55(8):648-654. Published online July 28, 2017; DOI: https://doi.org/10.1007/s12275-017-6636-8

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Abstract: The pharynx is an important site of microbiota colonization, but the bacterial populations at this site have been relatively unexplored by culture-independent approaches. The aim of this study was to characterize the microbiota structure of the pharynx. Pyrosequencing of 16S rRNA gene libraries was used to characterize the pharyngeal microbiota using swab samples from 68 subjects with laryngeal cancer and 28 subjects with vocal cord polyps. Overall, the major phylum was Firmicutes, with Streptococcus as the predominant genus in the pharyngeal communities. Nine core operational taxonomic units detected from Streptococcus, Fusobacterium, Prevotella, Granulicatella, and Veillonella accounted for 21.3% of the total sequences detected. However, there was no difference in bacterial communities in the pharynx from patients with laryngeal cancer and vocal cord polyps. The relative abundance of Firmicutes was inversely correlated with Fusobacteria, Proteobacteria, Actinobacteria, and Bacteroidetes. The correlation was evident at the genus level, and the relative abundance of Streptococcus was inversely associated with Fusobacterium, Leptotrichia, Neisseria, Actinomyces, and Prevotella. This study presented a profile for the overall structure of the microbiota in pharyngeal swab samples. Inverse correlations were found between Streptococcus and other bacterial communities, suggesting that potential antagonism may exist among pharyngeal microbiota.

Development of recombinant Yarrowia lipolytica producing virus-like particles of a fish nervous necrosis virus: Van-Trinh Luu , Hye Yun Moon , Jee Youn Hwang , Bo-Kyu Kang , Hyun Ah Kang; J. Microbiol. 2017;55(8):655-664. Published online July 28, 2017; DOI: https://doi.org/10.1007/s12275-017-7218-5

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Abstract: Nervous necrosis virus (NNV) causes viral encephalopathy and retinopathy, a devastating disease of many species of cultured marine fish worldwide. In this study, we used the dimorphic non-pathogenic yeast Yarrowia lipolytica as a host to express the capsid protein of red-spotted grouper nervous necrosis virus (RGNNV-CP) and evaluated its potential as a platform for vaccine production. An initial attempt was made to express the codon-optimized synthetic genes encoding intact and N-terminal truncated forms of RGNNV-CP under the strong constitutive TEF1 promoter using autonomously replicating sequence (ARS)-based vectors. The full-length recombinant capsid proteins expressed in Y. lipolytica were detected not only as monomers and but also as trimers, which is a basic unit for formation of NNV virus-like particles (VLPs). Oral immunization of mice with whole recombinant Y. lipolytica harboring the ARSbased plasmids was shown to efficiently induce the formation of IgG against RGNNV-CP. To increase the number of integrated copies of the RGNNV-CP expression cassette, a set of 26S ribosomal DNA-based multiple integrative vectors was constructed in combination with a series of defective Ylura3 with truncated promoters as selection markers, resulting in integrants harboring up to eight copies of the RGNNVCP cassette. Sucrose gradient centrifugation and transmission electron microscopy of this high-copy integrant were carried out to confirm the expression of RGNNV-CPs as VLPs. This is the first report on efficient expression of viral capsid proteins as VLPs in Y. lipolytica, demonstrating high potential for the Y. lipolytica expression system as a platform for recombinant vaccine production based on VLPs.

Characterization and phylogenetic analysis of Varicella-zoster virus strains isolated from Korean patients: Min Ho Kim , Jeong Seon Jeon , In Kyo Kim , Ji Seon Park , Hosun Park , Ok Sarah Shin , Chan Hee Lee; J. Microbiol. 2017;55(8):665-672. Published online July 28, 2017; DOI: https://doi.org/10.1007/s12275-017-7171-3

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Abstract: Varicella-zoster virus (VZV) is a causative agent of chickenpox in primary infection and shingles after its reactivation from latency. Complete or almost-complete genomic DNA sequences for various VZV strains have been reported. Recently, clinical VZV strains were isolated from Korean patients whose genome was sequenced using high-throughput sequencing technology. In this study, we analyzed single nucleotide polymorphism (SNP) of VZV strains to genetically characterize Korean clinical isolates. Phylogenetic analyses revealed that three Korean strains, YC01, YC02, and YC03, were linked to clade 2. Comprehensive SNP analysis identified 86 sites specific for the 5 VZV clades. VZV strains isolated from Korea did not form a phylogenetic cluster. Rather, YC02 and YC03 clustered strongly with Chinese strain 84-7 within clade 2, more specifically cluster 2a. Signature sequences for the cluster 2a were identified and found to play an important role in the separation of cluster 2a strains from other clade 2 strains, as shown in substitution studies. Further genetic analysis with additional strains isolated from Japan, China, and other Asian countries would provide a novel insight into the significance of two distinct subclades within clade 2.

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