• Restorative Dentistry and Endodontics
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• v.36 no.6
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• pp.498-505
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• 2011

Objectives: The purpose of this in vivo study was to investigate the microbial diversity in symptomatic and asymptomatic canals with primary endodontic infections by using GS FLX Titanium pyrosequencing. Materials and Methods: Sequencing was performed on 6 teeth (symptomatic, n = 3; asymptomatic, n = 3) with primary endodontic infections. Amplicons from hypervariable region of the small-subunit ribosomal RNA gene were generated by polymerized chain reaction (PCR), and sequenced by means of the GS FLX Titanium pyrosequencing. Results: On average, 10,639 and 45,455 16S rRNA sequences for asymptomatic and symptomatic teeth were obtained, respectively. Based on Ribosomal Database Project Classifier analysis, pyrosequencing identified the 141 bacterial genera in 13 phyla. The vast majority of sequences belonged to one of the seven phyla: Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, Proteobacteria, Spirochetes, and Synergistetes. In genus level, Pyramidobacter, Streptococcus, and Leptotrichia constituted about 50% of microbial profile in asymptomatic teeth, whereas Neisseria, Propionibacterium, and Tessaracoccus were frequently found in symptomatic teeth (69%). Grouping the sequences in operational taxonomic units (3%) yielded 450 and 1,997 species level phylotypes in asymptomatic and symptomatic teeth, respectively. The total bacteria counts were significantly higher in symptomatic teeth than that of asymptomatic teeth (p < 0.05). Conclusions: GS FLX Titanium pyrosequencing could reveal a previously unidentified high bacterial diversity in primary endodontic infections.

• Journal of Microbiology and Biotechnology
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• v.15 no.1
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• pp.14-21
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• 2005

By using PCR with nirS gene primers, three nirSharboring denitrifying bacteria (strain N6, strain N23, and strain R13) were newly isolated from activated sludge of a weak municipal wastewater treatment plant. Small-subunit rRNA gene-based analysis indicated that strain N6, strain N23, and strain R13 were closely related to Arthrobacter sp.,Staphylococcus sp., and Bacillus sp., respectively. In an attempt to identify their roles in biological nitrate and nitrite removal from sewage, we investigated their specific denitrification rates (SDNRs) for $NO_-^3$ - and $NO_-^2$ - in various cultures. All purecultures of each isolated nirS-harboring bacterial strain could remove $NO_-^3$ - and $NO_-^2$ - simultaneously in high efficiency, and the carbon requirements for $NO_-^3$ - removal of strain N6 and strain R13 were effectively low at 3.1 and 4.1 g COD/g $NO_3N$, respectively. In the case of mix-cultures of the strains (N6+N23, N6+R13, N23+R13, and N6+N23+R13), their SDNRs for $NO_-^3$ - were also effective, and their carbon requirements for $NO_-^3$ - removal were also effective at 3.0- 3.8 g COD/g NO3N. However, all tested mix-cultures accumulated $NO_-^2$ - in their culture media. On the other hand, the continuous culture of activated sludge mixed with strain N6 showed no significant increase of $NO_-^3$ - removal in comparison with strain N6's pure culture. These results suggest that nitrate and nitrite removal in biological wastewater treatment might be dependent on complicated bacterial interactions, including several effective denitrifying bacteria isolated in this study, rather than the specific bacterial types present and the number of bacterial types in activated sludge.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.6
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• pp.880-884
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• 2001

Rumen microbiology research has undergone several evolutionary steps: the isolation and nutritional characterization of readily cultivated microbes; followed by the cloning and sequence analysis of individual genes relevant to key digestive processes; through to the use of small subunit ribosomal RNA (SSU rRNA) sequences for a cultivation-independent examination of microbial diversity. Our knowledge of rumen microbiology has expanded as a result, but the translation of this information into productive alterations of ruminal function has been rather limited. For instance, the cloning and characterization of cellulase genes in Escherichia coli has yielded some valuable information about this complex enzyme system in ruminal bacteria. SSU rRNA analyses have also confirmed that a considerable amount of the microbial diversity in the rumen is not represented in existing culture collections. However, we still have little idea of whether the key, and potentially rate-limiting, gene products and (or) microbial interactions have been identified. Technologies allowing high throughput nucleotide and protein sequence analysis have led to the emergence of two new fields of investigation, genomics and proteomics. Both disciplines can be further subdivided into functional and comparative lines of investigation. The massive accumulation of microbial DNA and protein sequence data, including complete genome sequences, is revolutionizing the way we examine microbial physiology and diversity. We describe here some examples of our use of genomics- and proteomics-based methods, to analyze the cellulase system of Ruminococcus flavefaciens FD-1 and explore the genome of Ruminococcus albus 8. At Illinois, we are using bacterial artificial chromosome (BAC) vectors to create libraries containing large (>75 kbases), contiguous segments of DNA from R. flavefaciens FD-1. Considering that every bacterium is not a candidate for whole genome sequencing, BAC libraries offer an attractive, alternative method to perform physical and functional analyses of a bacterium's genome. Our first plan is to use these BAC clones to determine whether or not cellulases and accessory genes in R. flavefaciens exist in clusters of orthologous genes (COGs). Proteomics is also being used to complement the BAC library/DNA sequencing approach. Proteins differentially expressed in response to carbon source are being identified by 2-D SDS-PAGE, followed by in-gel-digests and peptide mass mapping by MALDI-TOF Mass Spectrometry, as well as peptide sequencing by Edman degradation. At Ohio State, we have used a combination of functional proteomics, mutational analysis and differential display RT-PCR to obtain evidence suggesting that in addition to a cellulosome-like mechanism, R. albus 8 possesses other mechanisms for adhesion to plant surfaces. Genome walking on either side of these differentially expressed transcripts has also resulted in two interesting observations: i) a relatively large number of genes with no matches in the current databases and; ii) the identification of genes with a high level of sequence identity to those identified, until now, in the archaebacteria. Genomics and proteomics will also accelerate our understanding of microbial interactions, and allow a greater degree of in situ analyses in the future. The challenge is to utilize genomics and proteomics to improve our fundamental understanding of microbial physiology, diversity and ecology, and overcome constraints to ruminal function.

• Mycobiology
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• v.51 no.3
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• pp.115-121
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• 2023

The fungal strain KNUF-22-18B, belonging to Cucurbitariaceae, was discovered from a stink bug (Hygia lativentris) during the investigation of insect microbiota in Chungnam Province, South Korea. The colonies of the strain KNUF-22-18B were wooly floccose, white to brown in the center on oatmeal agar (OA), and the colonies were buff, margin even, and colorless, reverse white to yellowish toward the center on malt extract agar (MEA). The strain KNUF-22-18B produced pycnidia after 60 days of culturing on potato dextrose agar, but pycnidia were not observed on OA. On the contrary, N. keratinophila CBS 121759^T abundantly formed superficial pycnidia on OA and MEA after a few days. The strain KNUF-22-18B produced chlamydospores subglobose to globose, mainly in the chain, with a small diameter of 4.4-8.8 ㎛. At the same time, N. keratinophila CBS 121759^T displayed a globose terminal with a diameter of 8-10 ㎛. A multilocus phylogeny using the internal transcribed spacer regions, 28S rDNA large subunit, b-tubulin, and RNA polymerase II large subunit genes further validated the uniqueness of the strain. The detailed description and illustration of the proposed species as Neocucurbitaria chlamydospora sp. nov. from Korea was strongly supported by molecular phylogeny.

• Parasites, Hosts and Diseases
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• v.51 no.5
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• pp.557-562
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• 2013

In contrast to the gradual reduction in the number of locally transmitted malaria cases in China, the number of imported malaria cases has been increasing since 2008. Here, we report a case of a 39-year-old Chinese man who acquired Plasmodium ovale wallikeri infection while staying in Ghana, West Africa for 6 months in 2012. Microscopic examinations of Giemsa-stained thin and thick blood smears indicated Plasmodium vivax infection. However, the results of rapid diagnostic tests, which were conducted 3 times, were not in agreement with P. vivax. To further check the diagnosis, standard PCR analysis of the small-subunit rRNA gene was conducted, based on which a phylogeny tree was constructed. The results of gene sequencing indicated that this malaria is a variant of P. ovale (P. ovale wallikeri). The infection in this patient was not a new infection, but a relapse of the infection from the one that he had contracted in West Africa.

• Korean Journal of Environmental Biology
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• v.38 no.2
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• pp.216-221
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• 2020

A eukaryotic marine microalga was isolated from Jungmun Saekdal Beach, Jeju Island, Korea and an integrated approach, including molecular phylogeny and morphology, was used to determine its taxonomical status. Molecular phylogenetic evidence inferred from the small subunit (SSU) 18S rRNA sequence and internal transcribed spacer (ITS) secondary structure analysis clearly showed that the isolate belonged to the recently described species, Jaagichlorella roystonensis. Distinctive morphological keys of the species were also observed by light microscopy and scanning/transmission electron microscopy(S/TEM). In this study, a Korean marine J. roystonensis species was described for the first time and was subsequently added to the national culture collections in Korea.

• Parasites, Hosts and Diseases
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• v.52 no.3
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• pp.299-304
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• 2014

This study aimed to identify the assemblages (or subassemblages) of Giardia duodenalis by using normal or nested PCR based on 4 genetic loci: glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi), ${\beta}$-giardin (bg), and small subunit ribosomal DNA (18S rRNA) genes. For this work, a total of 216 dogs' fecal samples were collected in Guangdong, China. The phylogenetic trees were constructed with MEGA5.2 by using the neighbor-joining method. Results showed that 9.7% (21/216) samples were found to be positive; moreover, 10 samples were single infection (7 isolates assemblage A, 2 isolates assemblage C, and 1 isolate assemblage D) and 11 samples were mixed infections where assemblage A was predominant, which was potentially zoonotic. These findings showed that most of the dogs in Guangdong were infected or mixed-infected with assemblage A, and multi-locus sequence typing could be the best selection for the genotype analysis of dog-derived Giardia isolates.

• Parasites, Hosts and Diseases
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• v.54 no.4
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• pp.455-460
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• 2016

Blastocystis is a common zoonotic enteric protozoan that has been classified into 17 distinct subtypes (STs). A cross-sectional study was conducted to determine the prevalence and subtype distributions of Blastocystis in villagers living along the Chao Phraya River, Ayutthaya Province, Thailand, and to assess the risk of zoonotic infection. In total, 220 stool samples were collected, and DNA was extracted. PCR and sequencing were performed with primers targeting the small-subunit ribosomal RNA (SSU rRNA) genes. Blastocystis was present in 5.9% (13/220) of samples, and ST3 (5.0%; 11/220) was the predominant subtype, followed by ST2 (0.45%; 1/220) and ST6 (0.45%; 1/220). Phylogenetic trees were constructed with the maximum-likelihood method based on the Hasegawa-Kishino-Yano + G + I model, neighbor-joining, and maximum parsimony methods. The percentage of bootstrapped trees in which the associated taxa clustered together was relatively high. All the sequences of the Blastocystis-positive samples (KU051524-KU051536) were closely related to those from animals (pig, cattle, and chicken), indicating a zoonotic risk. Therefore, the villagers require proper health education, especially regarding the prevention of parasitic infection, to improve their personal hygiene and community health. Further studies are required to investigate the Blastocystis STs in the animals living in these villages.

• Parasites, Hosts and Diseases
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• v.53 no.1
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• pp.119-124
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• 2015

The objective of this study was to genetically characterize isolates of Giardia duodenalis and to determine if zoonotic potential of G. duodenalis could be found in stray cats from urban and suburban environments in Guangzhou, China. Among 102 fresh fecal samples of stray cats, 30 samples were collected in Baiyun district (urban) and 72 in Conghua district (suburban). G. duodenalis specimens were examined using light microscopy, then the positive specimens were subjected to PCR amplification and subsequent sequencing at 4 loci such as glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi), ${\beta}$-giardin (bg), and small subunit ribosomal RNA (18S rRNA) genes. The phylogenetic trees were constructed using obtained sequences by MEGA5.2 software. Results show that 9.8% (10/102) feline fecal samples were found to be positive by microscopy, 10% (3/30) in Baiyun district and 9.7% (7/72) in Conghua district. Among the 10 positive samples, 9 were single infection (8 isolates, assemblage A; 1 isolate, assemblage F) and 1 sample was mixed infection with assemblages A and C. Based on tpi, gdh, and bg genes, all sequences of assemblage A showed complete homology with AI except for 1 isolate (CHC83). These findings not only confirmed the occurrence of G. duodenalis in stray cats, but also showed that zoonotic assemblage A was found for the first time in stray cats living in urban and suburban environments in China.

• Parasites, Hosts and Diseases
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• v.53 no.1
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• pp.13-19
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• 2015

Blastocystis sp. is a common zoonotic intestinal protozoa which has been classified into 17 subtypes (STs). A cross-sectional study was conducted to determine the prevalence and subtype distribution of Blastocystis in villagers living on the Thai-Myanmar border, where the risk of parasitic infection is high. A total of 207 stool samples were collected and DNA was extracted. PCR and sequencing using primers targeting small-subunit ribosomal RNA (SSU rRNA) gene were performed. The prevalence of Blastocystis infection was 37.2% (77/207). ST3 (19.8%; 41/207) was the predominant subtype, followed by ST1 (11.6%; 24/207), ST2 (5.3%; 11/207), and ST4 (0.5%; 1/207). A phylogenetic tree was reconstructed using the maximum likelihood (ML) method based on the Hasegawa-Kishino-Yano + G + I model. The percentage of bootstrapped trees in which the associated taxa clustered together was relatively high. Some sequences of Blastocystis positive samples (TK18, 39, 46, 71, and 90) were closely related to animals (pig and cattle) indicating zoonotic risks. Therefore, proper health education in parasitic prevention for the villagers should be promoted to improve their personal hygiene. Further longitudinal studies are required to monitor the prevalence of parasitic infections after providing health education and to investigate Blastocystis ST in animals living in these villages.