• Microbiology and Biotechnology Letters
• /
• v.47 no.3
• /
• pp.401-411
• /
• 2019

All Idiomarina species are isolated from saline environments; microorganisms in such extreme habitats develop metabolic adaptations and can produce compounds such as proteases with an industrial potential. ARDRA and 16S rRNA gene sequencing are established methods for performing phylogenetic analysis and taxonomic identification. However, 16S-23S ITS is more variable than the 16S rRNA gene within a genus, and is therefore, used as a marker to achieve a more precise identification. In this study, ten protease producing Idiomarina strains isolated from the Peruvian salterns were characterized using biochemical and molecular methods to determine their bacterial diversity and industrial potential. In addition, comparison between the length and nucleotide sequences of a 16S-23S ITS region allowed us to assess the inter and intraspecies variability. Based on the 16S rRNA gene, two species of Idiomarina were identified (I. zobellii and I. fontislapidosi). However, biochemical tests revealed that there were differences between the strains of the same species. Moreover, it was found that the ITS contains two tRNA genes, $tRNA^{Ile(GAT)}$ and $tRNA^{Ala(TGC)}$, which are separated by an ISR of a variable size between strains of I. zobellii. In one strain of I. zobellii (PM21), we found nonconserved nucleotides that were previously not reported in the $tRNA^{Ala}$ gene sequences of Idiomarina spp. Thus, based on the biochemical and molecular characteristics, we can conclude that protease producing Idiomarina strains have industrial potential; only two I. zobellii strains (PM48 and PM72) exhibited the same properties. The differences between the other strains could be explained by the presence of subspecies.

• Korean Journal of Environmental Biology
• /
• v.24 no.4
• /
• pp.307-313
• /
• 2006

Seasonal variation of marine bacterial community was analyzed in the surface sea water collected from one of the stations locating at Tongyeoung coastal area, Korea. The results obtained by the culture method through identification with the VITEK Microbe ID system after pure culture in the selective medium were compared with those obtained by the DGGE based 16S rRNA PCR method. The composition of the marine bacterial community in the sea water samples harvested in September, 2004, November, 2004, January, 2005, May, 2005 and August, 2005 determined by the culture method showed 5, 5, 4, 6, and 10 strains respectively. Pseudomonas fluorescens and Acinetobacter lwoffii were detected in all seasons. The other strains were identified to be Pseudomonas stutzeri, Sphingomonas paucimobilis, Burkholderia mallei and Chryseobacterium indologenes. In contrast, the 16S rRNA PCR-DGGE method detected 10, 11, 6, 9 and 13 populations respectively in the same sea water samples and the strains were identified to be Acinetobacter lwoffii, Burkholderia mallei, Pseudomonas fluoresence, Actinobacillus ureae, Burkholderia sp., Pseudomonas stutzeri, Roseobacter sp., Vibrio parahaemolyticue, Sphingomonas paucimobilis and Rugeria algocolus. This results indicated that the DGGE based 16S rRNA PCR method was more efficient than the culture method for the grasp of the characteristics of the marine bacterial community.

• Journal of Microbiology and Biotechnology
• /
• v.20 no.8
• /
• pp.1210-1214
• /
• 2010

Many bacteria are involved in the fermentation of doenjang, and Bacillus species are known to perform significant roles. Although SDS-PAGE has been frequently used to classify and identify bacteria in various samples, the microbial diversity in doenjang has not yet been investigated. This study aims to determine the identity and distribution of dominant Bacillus species in doenjang using SDS-PAGE profiles of whole-cell proteins and 16S rRNA gene sequencing. Reference Bacillus strains yielded differential SDS-PAGE banding patterns that could be considered to be highly specific fingerprints. Grouping of bacterial strains isolated from doenjang samples by whole-cell protein patterns was confirmed by analysis of their 16S rRNA gene sequences. B. subtilis was found to be the most dominant strain in most of the samples, whereas B. licheniformis and B. amyloliquefaciens were less frequently found but were also detected in several samples. The results obtained in this study show that a combined identification method using SDS-PAGE profiles of whole-cell proteins and subsequent 16S rRNA gene sequence analysis could successfully identify Bacillus species isolated from doenjang.

• Ocean Science Journal
• /
• v.41 no.2
• /
• pp.113-119
• /
• 2006

Seventeen lactic acid bacterial strains (LAB) were isolated using MRS agar medium from Jeotgal, a Korean fermented food, purchased at the Jukdo market of Pohang. To identify the strains isolated, they were tested by examining their cell morphologies, gram-staining, catalase activity, arginine hydrolase activity, D-L lactate form and carbohydrate fermentation. According to the phenotypic characteristics, three strains were tentatively identified as Lactobacillus spp., ten were Enterococcus spp. (or Streptococcus spp., or Pediococcus spp.) and the rest were Leuconostoc spp. (or Weissella spp.). Five strains among 17 were chosen by preliminary bacteriocin activity test. Four bacterial strains which inhibited both indicator microorganisms were identified by 16S rRNA sequencing. The results are as follows; Leuconostoc mesenteroides (HK 4), Leuconostoc mesenteroides (HK 5), Leuconostoc mesenteroides(HK 11), Streptococcus salivarius(HK 8). In order to check LAB which are showing a high survival rate in gut, we investigated three strains inhibiting both indicator microorganisms in artificial gastric acid and bile juice -all except HK8. The three strains mentioned above grew in extreme low acid conditions.

• International Journal of Oral Biology
• /
• v.41 no.4
• /
• pp.199-208
• /
• 2016

The aim of this study was to identify the non-Aggregatibacter actinomycetemcomitans bacteria grown on the tryptic soy-serum-bacitracin-vancomycin (TSBV) medium, an A. actinomycetemcomitans selective medium. A total of 82 unidentified bacterial isolates from the oral cavities of a Korean population were kindly provide by the Korean Collection for Oral Microbiology. All the clinical isolates were grown on TSBV medium and bacterial DNA purified from each isolate was subjected to PCR with universal primers specific for bacterial 16S rRNA genes (16S rDNAs) sequence. The each bacterial 16S rDNA was amplified by PCR and the nucleotide sequences of it was determined by the dideoxynucleotide chain termination method. They were identified by 16S rDNA sequence comparison method at the specie-level. The data showed that Neisseria spp. (42 strains), Fusobacterium spp. (10 strains), Capnocytophaga spp. (8 strains), Propionibacterium acnes (5 strains), Aggregatibacter aprophilus (4 strains), Campylobacter spp. (5 strains), Veillonella dispar (3 strains), Streptococcus sp. (1 strain), Haemophilus parainfluenzae (1 strain), Leptotrichia wadei (1 strain), Morococcus sp./Neisseria sp. (1 strain), and Staphylococcus sp. (1 strain) were identified. These results could be used to develop a new A. actinomycetemcomitans-selective medium which is more effective than the TSBV medium in future studies.

• Biomedical Science Letters
• /
• v.28 no.4
• /
• pp.317-321
• /
• 2022

Due to COVID-19 pandemic, wearing face masks is obligatory to prevent respiratory virus transmissions in the community. However, there are few studies of the desirable number of wearing a face mask, and how to store them for reuse. Therefore, in this study, a survey was conducted among 208 healthy adults, and 27 kf-94 masks worn for 1, 2, and 3 days were collected. To estimate the risk of bacterial contamination, we analyzed the extent of bacterial contamination of the BHI medium and 16S rRNA gene sequencing. With an increase in the number of days of using the mask, the degree of bacterial contamination of the used mask gradually increased. As a result of 16S rRNA PCR performed for strain identification, Staphylococcus, known as a pathogenic bacterium, was identified the most. In conclusion, we found that wearing a cotton KF mask provides an optimal environment for microbes, which are related to the skin and respiratory system, to thrive. Therefore, it is also important to reduce the risk of bacterial infection of the face mask with appropriate sterilization methods.

• Korean Journal of Microbiology
• /
• v.55 no.1
• /
• pp.1-8
• /
• 2019

Due to the major role played by several species of Streptococcus in the etiology of periodontitis, it is important to assess the pattern of Streptococcus pathogenic pathways within the infected subgingival pockets using a bacterial specific 16S rRNA fragment. From the total of 50 patients with periodontitis included in the study, only 23 Streptococcal isolates were considered for further analyses, in which their 16S rRNA fragments were amplified and sequenced. Then, a comprehensive phylogenetic tree was constructed and in silico prediction was performed for the observed Streptococcal species. The phylogenetic analysis of the subgingival Streptococcal species revealed a high discrimination power of the 16S rRNA fragment to accurately identify three groups of Streptococcus on the species level, including S. salivarius (14 isolates), S. anginosus (5 isolates), and S. gordonii (4 isolates). The employment of state-of-art in silico tools indicated that each Streptococcal species group was characterized with particular transcription factors that bound exclusively with a different 16S rRNA-based secondary structure. In conclusion, the observed data of the present study provided in-depth insights into the mechanism of each Streptococcal species in its pathogenesis, which differ in each observed group, according to the differences in the 16S rRNA secondary structure it takes, and the consequent binding with its corresponding transcription factors. This study paves the way for further interventions of the in silico prediction, with the main conventional in vitro microbiota identification to present an interesting insight in terms of the gene expression pattern and the signaling pathway that each pathogenic species follows in the infected subgingival site.

• Journal of Periodontal and Implant Science
• /
• v.28 no.4
• /
• pp.691-703
• /
• 1998

The 16S rRNA analyzing method is a bacterial identification method that is useful in identifying bacteria which is difficult to do by other means. The following 7 types of bacteria which are Treponema, A. actinomycetemcomitans, P. gingivalis, Fusobacterium, B. forsythus, P. intermedia, P. micros were evaluated in order to study their distribution among patients with adult periodontitis. The 16S rRNA analyzing method was used to compare bacterial distribution among 3 groups. Subgingival plaque acquired from the affected sites(pocket depth ${\geq}6mm$) of 29 patients with adult periodontitis were grouped as the experimental group while plaque from the non-affected sites(pocket depth ${\leq}3mm$) were grouped as control 2 and finally plaque acquired from students with healthy periodontal tissues were grouped as control 1. The results are as follows ; 1. The distribution of Treponema was 12.5% for control 1, 21.4% for control 2 and 75.4% for the experimental group. For A. actinomycetemcomitans the distribution was 0.5%, 19.0%, 44.4% in respect to the order of groups mentioned above. P.gingivalis showed 10.5%, 43.1%, 94.0% distribution, Fusobacterium 33.0%, 48.3%, 81.0% distribution, B. forsythus 9.5%, 17.2%, 65.9% distribution, P. intermedia 1.0%, 12.1%, 26.3% distribution and finally P. micros 5.0%, 19.0%, 48.7% respectively. In all 7 types of bacteria, the experimental group showed higher bacterial distribution compared to the other two groups with statistically significant difference. 2. In the case of Treponema, A. actinomycetemcomitans, gingivalis,Fusobacterium, B. forsythus, P. intermedia, P. micros showed significant difference between control 1 and 2. These results suggest that the 16S rRNA analyzing method which was applied on Koreans for the first time could be utilized and useful in finding potential pathogens of periodontal disease.

• Biomedical Science Letters
• /
• v.9 no.3
• /
• pp.139-144
• /
• 2003

Since water borne infection causes acute diseases and results in spread of diseases by secondary infection, the prevention is very important. Therefore, it is necessary to have a method that is rapid and effective to monitor pathogenic bacteria in drinking water. In this study, we employed a systematic method, Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis, to develop an effective monitoring system for possible bacterial contaminants in drinking water. For this purpose, PCR primers were derived from 992 bp region of the 16s rRNA gene that is highly conserved through the different species of prokaryotes. To test whether the PCR primers designed are indeed useful for detecting all the possible microbial contaminants in the water, the primers were used to amplify 16s rRNA regions of different microbial water-borne pathogens such as E. coli, Salmonella, Yersinia, Listeria, and Staphylococcus. As expected, all of tested microorganisms amplified expected size of PCR products indicating designed PCR primers for 16s rRNA indeed can be useful to amplify all different microbial water-borne pathogens in the water. Furthermore, to test whether these 16s rRNA based PCR primers can detect bacterial populations present in the water, water samples taken from diverse sources, such as river, tap, and sewage, were used for amplification. PCR products were for then subjected for cloning into a T-vector to generate a library containing 16s rRNA sequences from various bacteria. With cloned PCR products, RFLP analysis was done using PCR products digested with restriction enzyme such as Hae III to obtain species-specific RFLP profiles. After PCR-RFLP, the bacterial clones which showed the same RFLP profiles were regarded as the same ones, and the clones which showed distinctive RFLP profiles were subsequently subjected for sequence analysis for species identification. By this PCR-RFLP analysis, we were able to reveal diverse populations of bacteria living in water. In brief, in unsterilized natural river water, over 60 different species of bacteria were found. On the other hand, no PCR products were detected in drinking tap-water. The results from this study clearly indicate that the PCR-RFLP-sequence analysis can be a useful method for monitoring diverse, perhaps pathogenic bacteria contaminated in water in a rapid fashion.

• Journal of Microbiology and Biotechnology
• /
• v.28 no.7
• /
• pp.1112-1121
• /
• 2018

Jeotgal is a Korean traditional fermented seafood with a high concentration of salt. In this study, we isolated lactic acid bacteria (LAB) from galchi (Trichiurus lepturus, hairtail) and myeolchi (Engraulis japonicas, anchovy) jeotgal on MRS agar and MRS agar containing 5% NaCl (MRS agar+5% NaCl), and identified them by using 16S rRNA gene sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as culture-dependent methods. We also performed polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) as a culture-independent method to identify bacterial communities. Five samples of galchi-jeotgal and seven samples of myeolchi-jeotgal were collected from different regions in Korea. A total of 327 and 395 colonies were isolated from the galchi- and myeolchi-jeotgal samples, respectively. 16S rRNA gene sequencing and MALDI-TOF MS revealed that the genus Pediococcus was predominant on MRS agar, and Tetragenococcus halophilus on MRS agar+5% NaCl. PCR-DGGE revealed that T. halophilus, Tetragenococcus muriaticus, and Lactobacillus sakei were predominant in both types of jeotgal. T. halophilus was detected in all samples. Even though the same species were identified by both culture-dependent and -independent methods, many species identified by the culture-dependent methods were not in the bacterial list identified by the culture-independent methods. The distribution of bacteria in galchi-jeotgal was more diverse than in myeolchi-jeotgal. The diverse LAB in galchi- and myeolchi-jeotgals can be further studied as candidates for starter cultures to produce fermented foods.