• Genomics & Informatics
• /
• v.11 no.4
• /
• pp.272-276
• /
• 2013

Sequence analysis of the 16S rRNA gene has been widely used for the classification of microorganisms. However, we have been unable to clearly identify five Flavobacterium species isolated from a freshwater by using the gene as a single marker, because the evolutionary history is incomplete and the pace of DNA substitutions is relatively rapid in the bacteria. In this study, we tried to classify Flavobacterium species through multilocus sequence analysis (MLSA), which is a practical and reliable technique for the identification or classification of bacteria. The five Flavobacterium species isolated from freshwater and 37 other strains were classified based on six housekeeping genes: gyrB, dnaK, tuf, murG, atpA, and glyA. The genes were amplified by PCR and subjected to DNA sequencing. Based on the combined DNA sequence (4,412 bp) of the six housekeeping genes, we analyzed the phylogenetic relationship among the Flavobacterium species. The results indicated that MLSA, based on the six housekeeping genes, is a trustworthy method for the identification of closely related Flavobacterium species.

• Microbiology and Biotechnology Letters
• /
• v.48 no.3
• /
• pp.276-286
• /
• 2020

Probiotics are live microorganisms that, when administered in adequate amounts, confer health benefits to the host. This study was conducted for the isolation of potential lactic acid bacteria (LAB) with probiotic properties from goat milk and yogurt. Several tests were conducted in vitro using the standard procedures for evaluating the inhibitory spectra of LAB against pathogenic bacteria; tolerance to NaCl, bile salt, and phenol; hemolytic, milk coagulation, and bile salt hydrolase activities; gastrointestinal transit tolerance; adhesion properties; and antibiotic susceptibility. Among 40 LAB strains screened according to culture characteristics, five isolates exhibited antagonistic properties. Three were identified as Pediococcus acidilactici, and two were identified as Enterococcus faecium, exploiting 16S rRNA gene sequencing. All the isolates succeeded in the gastrointestinal transit tolerance assay and successively colonized mucosal epithelial cells. Based on the results of these in vitro assays, both P. acidilactici and E. faecium can be considered as potential probiotic candidates.

• Journal of Microbiology and Biotechnology
• /
• v.20 no.5
• /
• pp.862-870
• /
• 2010

Cyperus rotundus L. is a perennial herb that was found to be dominating an area in northeast Brazil previously contaminated with petroleum. In order to increase our knowledge of microorganism-plant interactions in phytoremediation, the bacterial community present in the rhizosphere and roots of C. rotundus was evaluated by culture-dependent and molecular approaches. PCR-DGGE analysis based on the 16S rRNA gene showed that the bacterial community in bulk soil, rhizosphere, and root samples had a high degree of similarity. A complex population of alkane-utilizing bacteria and a variable nitrogen-fixing population were observed via PCR-DGGE analysis of alkB and nifH genes, respectively. In addition, two clone libraries were generated from alkB fragments obtained by PCR of bulk and rhizosphere soil DNA samples. Statistical analyses of these libraries showed that the compositions of their respective populations were different in terms of alkB gene sequences. Using culturedependent techniques, 209 bacterial strains were isolated from the rhizosphere and rhizoplane/roots of C. rotundus. Dot-blot analysis showed that 17 strains contained both alkB and nifH gene sequences. Partial 16S rRNA gene sequencing revealed that these strains are affiliated with the genera Bosea, Cupriavidus, Enterobacter, Gordonia, Mycoplana, Pandoraea, Pseudomonas, Rhizobium, and Rhodococcus. These isolates can be considered to have great potential for the phytoremediation of soil with C. rotundus in this tropical soil area.

• Food Science of Animal Resources
• /
• v.27 no.4
• /
• pp.509-516
• /
• 2007

Starters of lactic acid bacteria(LAB) were isolated from the commercial yoghurt products and the four isolates have been studied on their identification and some physiological characteristics. For the purpose of identification, microscopic examination, API test, and 16s rRNA gene sequencing were conducted. Isolate A from a yogurt product of local dairy company A was shown to be Gram-positive rod-shaped bacterium. All strains isolated were turned out to be as Lactobacillus paracasei by using a API 50 CHL kit. In contrast, isolate A was identified as a strain of Lactobacillus helveticus based on the 16S rRNA sequencing data, and L. casei ssp. casei for both B and D and L. paracasei for C. All the isolates survived the simulated gastric juice, pH 2.0 within 3 hours and sharply decreased in viability so that no viable cell was observed after 4.5 hours incubation. In addition, the four isolated strains were almost identical in antibiotic susceptibility to six different kinds of antibiotics including erythromycin ($15\;{\mu}g$), ampicillin ($10\;{\mu}g$), gentamycin ($10\;{\mu}g$), neomycin ($30\;{\mu}g$), but rather resistant to colistin ($10\;{\mu}g$) and streptomycin ($10\;{\mu}g$). It was noteworthy that four isolates were confirmed to produce antibacterial substance against foodborne pathogens of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli 0157:H7 as test organisms based on the inhibitory zones on an MRS soft agar medium. At presence, the inhibitory factor is unknown so that further studies are required to ascertain the active factor responsible for the inhibitory activities.

• Journal of Microbiology and Biotechnology
• /
• v.24 no.11
• /
• pp.1464-1472
• /
• 2014

The microbial community structures of two continuous stirred tank reactors digesting turkey manure with pine wood shavings as well as chicken and swine manure were investigated. The reactor fed with chicken/swine wastes displayed the highest organic acids concentration (up to 15.2 g/l) and ammonia concentration (up to 3.7 g/l ammonium nitrogen) and generated a higher biogas yield (up to $366ml/g_{VS}$) compared with the reactor supplied with turkey wastes (1.5-1.8 g/l of organic acids and 1.6-1.7 g/l of ammonium levels; biogas yield was up to $195ml/g_{VS}$). The microbial community diversity was assessed using both sequencing and profiling terminal restriction fragment length polymorphisms of 16S rRNA genes. Additionally, methanogens were analyzed using methyl coenzyme M reductase alpha subunit (mcrA) genes. The bacterial community was dominated by members of unclassified Clostridiales with the prevalence of specific clostridial phylotypes in each reactor, indicating the effect of the substrate type on the community structure. Of the methanogenic archaea, methanogens of the genus Methanosarcina were found in high proportions in both reactors with specific methanosarcinas in each reactor, whereas the strict hydrogenotrophic methanogens of Methanoculleus sp. were found at significant levels only in the reactor fed with chicken/swine manure (based on the analyses of 16S rRNA gene). This suggests that among methanogenic archaea, Methanosarcina species which have different metabolic capabilities, including aceticlastic and hydrogenotrophic methanogenesis, were mainly involved in anaerobic digestion of turkey wastes.

• Journal of Dairy Science and Biotechnology
• /
• v.24 no.1
• /
• pp.19-28
• /
• 2006

This study was conducted to isolate antilisterial strains of the Bifidobacterium isolates from the infant feces. The bifidobacteria were isolated anaerobically on BL agar and screened for their inhibitory activity on the MRS-cysteine medium against three foodborne pathogens: Listeria monocytogenes, Bacillus cereus, and Staphylococcus aureus. Among the 52 bifidobacterial isolates, 5 strains(A24, Bl, B6, B10, and Bl2) were finally selected based on their stronger antilisterial activity against Listeria monocytogenes than other isolates tested. Morphologically, all the isolates were typically shown Y-and V-shaped under electron microscopic examination. Each isolate was primarily subjected to identification by a polymerase chain reaction(PCR) using a genus-specific primer designed for targeting the 16S rRNA gene sequence, and confirmed the primary identification data using an API-kit(Biomeriuex, France), commercially available product for identification based on biochemical and physiological traits. Of the isolates with antilisterial activity, strain A24 was finally confirmed as the Bifidobacterium longum A24.

• Journal of Microbiology and Biotechnology
• /
• v.28 no.4
• /
• pp.551-560
• /
• 2018

Bellflower root (Platycodon grandiflorum), which belongs to the Campanulaceae family, is a perennial grass that grows naturally in Korea, northeastern China, and Japan. Bellflower is widely consumed as both food and medicine owing to its high nutritional value and potential therapeutic effects. Since foodborne disease outbreaks often come from vegetables, understanding the public health risk of microorganisms on fresh vegetables is pivotal to predict and prevent foodborne disease outbreaks. We investigated the microbial communities on the bellflower root (n = 10). 16S rRNA gene amplicon sequencing targeting the V6-V9 regions of 16S rRNA genes was conducted via the 454-Titanium platform. The sequence quality was checked and phylogenetic assessments were performed using the RDP classifier implemented in QIIME with a bootstrap cutoff of 80%. Principal coordinate analysis was performed using the weighted Fast UniFrac distance. The average number of sequence reads generated per sample was 67,192 sequences. At the phylum level, bacterial communities from the bellflower root were composed primarily of Proteobacteria, Firmicutes, and Actinobacteria in March and September samples. Genera Serratia, Pseudomonas, and Pantoea comprised more than 54% of the total bellflower root bacteria. Principal coordinate analysis plots demonstrated that the microbial community of bellflower root in March samples was different from those in September samples. Potential pathogenic genera, such as Pantoea, were detected in bellflower root samples. Even though further studies will be required to determine if these species are associated with foodborne illness, our results indicate that the 16S rRNA gene-based sequencing approach can be used to detect pathogenic bacteria on fresh vegetables.

• Food Science of Animal Resources
• /
• v.39 no.4
• /
• pp.585-600
• /
• 2019

Gouda cheese, one of most popular cheeses in the Korea, has been produced from only pasteurized milk in Korean dairy farms. Recently, it has become legally possible to produce ripened cheese manufactured with raw milk in Korea. In the present study, we investigated the physico-chemical and microbiological characteristics of Gouda cheese manufactured with raw (R-GC) or pasteurized milk (P-GC) during manufacturing and ripening. Particularly, this study characterized the bacterial community structure of two cheese types, which are produced without pasteurization during ripening based on next generation sequencing of 16S rRNA gene amplicons. During ripening, protein and fat content increased slightly, whereas moisture content decreased in both P-GC and R-GC. At the 6 wk of ripening, R-GC became softer and smoother and hence, the values of hardness and gumminess, chewiness in R-GC was lower than that of P-GC. Metagenomic analysis revealed that the bacterial genera used a starter cultures, namely Lactococcus and Leuconostoc were predominant in both P-GC and R-GC. Moreover, in R-GC, the proportion of coliform bacteria such as Escherichia, Leclercia, Raoultella, and Pseudomonas were detected initially but not during ripening. Taken together, our finding indicates the potential of manufacturing with Gouda cheese from raw milk and the benefits of next generation sequencing for microbial community composition during cheese ripening.

• Journal of Microbiology and Biotechnology
• /
• v.12 no.2
• /
• pp.312-317
• /
• 2002

Indigenous lactobacilli were isolated from vaginas of Korean women for possible use in ecological treatment of bacterial vaginosis. Vaginal swab samples were obtained from a gynecological clinic and streaked on Rogosa SL agar plates to select the most predominant lactobacilli in each sample. The preliminary identification of the isolates as lactobacilli was based on microscopic observation of Gram-positive rod-shaped cell morphology. The initial characterization was performed on 108 isolates in terms of their cell surface hydrophobicity (CSH), antimicrobial activity, and hydrogen peroxide (H₂O₂) production capability, and 10 isolates were then selected for further molecular identification. For a rapid procedure to identify lactobacilli, polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses of the l6S rRNA genes were applied. The 10 selected lactobacilli and 9 different reference strains of Lactobacillus spp. were characterized by PCR-RFLP where the amplified l6S rDNA was digested with 7 different restriction endonucleases prior to analysis. DNA sequencing of the 16S rRNA gene of one particular isolate, KLB 46, that had been identified as L. crispatus by the PCR-RFLP analysis, further confirmed its identity as L. crispatus.

• Microbiology and Biotechnology Letters
• /
• v.51 no.4
• /
• pp.474-483
• /
• 2023

Members of the genus Streptomyces produce more than 70% of antibiotics. The rise in antibiotic resistance globally enhanced the search for novel species with the ability to produce new bioactive compounds. This study was initiated to investigate different regions in Jordan for previously uncultured and rare Streptomyces species capable of producing novel antimicrobial compounds especially active against bacteria resistant to antibiotics. A total of 191 Streptomyces strains were isolated from 26 soil samples collected from different geographic regions in Jordan. Isolates were characterized based on colony and cellular morphology as well as using 16S rRNA gene sequencing. These isolates were screened for their ability to produce antibiotics by the perpendicular-cross streak method, and then tested by well diffusion method against tested pathogens. Fifty-four isolates showed potential to produce antimicrobial products especially active against resistant bacteria, 20.1% of the isolates showed inhibitory effect against Staphylococcus aureus, 16.9% against clinical MSSA strains, and 18.0% against MRSA: whereas only 4.2% against Esherichia coli, 3.2% against Klebsiella pneumonia, 2.7% against Pseudomonas aeruginosa, and 10.0% against clinical Candida albicans. Three isolates were selected for further identification due to their antibacterial activity against S. aureus, MRSA, and MSSA. These isolates were identified as follows; Streptomyces aburaviensis DSa3, Streptomyces alboniger SAb7 and Streptomyces misionensis ZAb2, based on cultural, biochemical characteristics and molecular analysis of the 16S rRNA.