• Title/Summary/Keyword: rep-PCR

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Survey in consumers and distribution stages bacteriological analysis for fresh raw beef in Gwangju area, Korea (광주지역 소 생고기 선호도 및 유통단계별 세균학적 분석)

  • Na, Ho-Myung;Bae, Seong-Yeol;Koh, Ba-Ra-Da;Jang, Mi-Sun;Sung, Chang-Min;Kim, Ji-Yeon;Park, Heon-Gyu;Mun, Yong-Un;Kim, Yong-Hwan
    • Korean Journal of Veterinary Service
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    • v.35 no.4
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    • pp.313-319
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    • 2012
  • Consumer's preference and microbial inspections on fresh raw beef were carried out to understand the actual market status in Gwanju, Korea. Over 15 questions on questionnaire by 1,111 randomly selected respondents between April and May in 2011, results showed 65.5% positive on eating fresh raw beef, 63.8% negative on good hygiene condition of fresh raw beef, and 72.5% positive on the secure of the hygiene-safety for priority program, respectively. For microbial inspections, a total of 302 samples were collected from fresh raw beef purchased from slaughterhouse (n=122), transport (n=69) and consumer (n=81) stage, from lettuce (n=30) at consumer stage. The aerobic plate count (APC), E. coli count and food borne bacteria such as Salmonella spp., Listeria monocytogenes, Staphylococcus(S.) aureus and E. coli O157:H7 were tested in the samples. As results, the level of count on APC of fresh raw beef ranged $6{\times}10^1{\sim}1.8{\times}10^5CFU/g$ from slaughterhouse, $2{\times}10^2{\sim}8.3{\times}10^5CFU/g$ from transport stage and $1{\times}10^2{\sim}4{\times}10^5CFU/g$ from consumer stage. The level of count on E. coli of fresh raw beef ranged $1{\sim}9{\times}10^1CFU/g$ from slaughterhouse, $1{\sim}7{\times}10CFU/g$ from transport stage and $1{\sim}5.5{\times}10CFU/g$ from consumer stage. In total, 26 S. aureus were isolated, 10 (14.5%) from fresh raw beef at transport stage, 12 (14.8%) from fresh raw beef and 4 (13.3%) from lettuce at consumer stage. Enterotoxin of S. aureus was not detected among 26 isolates. All S. aureus isolates were typed using a DiversiLab$^{TM}$ rep-PCR system for genetic similarity test, showing over 95% of genetic relationship amon isolates.

Two Pathogenic Groups in Acidovorax valerianellae Causing Bacterial Black Spot on the Various Crop Plants (다양한 작물에서 세균검은점무늬병을 일으키는 Acidovorax valerianellae의 병원성이 다른 2그룹)

  • Kim, Hye-Seong;Kim, Young-Tak;Park, Kyoung-Soo;Lee, Ji-Hye;Lee, Hyok-In;Cha, Jae-Soon
    • Research in Plant Disease
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    • v.23 no.4
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    • pp.314-321
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    • 2017
  • Acidovorax valerianellae had been reported a causal agent of bacterial black spot disease on corn salad in France, 2003 and on watermelon in Korea 2011. In this study, difference in host specificity between 2 groups, corn salad strains and watermelon strains, of Acidovorax valerianellae was recognized and compared. In the pathogenicity test, all 5 watermelon strains showed pathogenicity on the 6 Cucurbitaceae plants but not on corn salad, whereas 4 corn salad strains showed pathogenicity only on the corn salad. Utilization of Biolog substrates was different between watermelon strains and corn salad strains on 4 substrates, Malonic Acid, ${\alpha}-Hydroxybutyric$ Acid, ${\alpha}-Keto$ Butyric Acid, and Glycyl-L Glutamic Acid. The phylogenetic tree built with the 16S rDNA sequences showed that all of A. valerianellae stains was grouped into 1 clade separating from the other species of Acidovorax genus. Within A. valerianellae clade, watermelon strains and corn salad strains were separated into 2 sub-groups. REP-PCR analysis also separated the two groups. Host specificity, substrate utilization, and some genetic characteristics suggested that there are two pathogenic groups, watermelon group and corn salad group in A. valerianellae.

Analysis of Foodborne Pathogens in Brassica campestris var. narinosa microgreen from Harvesting and Processing Steps (어린잎채소의 생산 및 가공 공정 중 식중독 미생물 분석)

  • Oh, Tae Young;Baek, Seung-Youb;Choi, Jeong Hee;Jeong, Moon Cheol;Koo, Ok Kyung;Kim, Seung Min;Kim, Hyun Jung
    • Journal of Applied Biological Chemistry
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    • v.59 no.1
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    • pp.63-68
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    • 2016
  • This study was performed to assess the microbiological quality of Brassica campestris var. narinosa microgreen from harvesting and processing steps. The samples were analyzed for total viable cell counts (TVC), coliforms, Enterobacteriaceae, Escherichia coli, Salmonella spp., Listeria monocytogenes, Vibrio parahaemolyticus, Bacillus cereus, and Staphylococcus aureus. The total viable counts of microgreen (whole leaves) and environment samples from harvesting steps were higher than 6.8 log CFU/g and the contamination level of coliforms in the samples were 3.2 log CFU/g and 3.5 log CFU/g of microgreen and soil, respectively. In case of microgreen samples collected from processing steps, the contamination level of TVC and coliforms were higher in raw materials than samples obtained from later stages of processing, i.e. washing, drain, and final products. The contamination levels of B. cereus in raw materials and environments decreased approximately 1.4 log CFU/g in final products. S. aureus was detected in soil samples but Salmonella spp., Listeria monocytogenes, Vibrio parahaemolyticus and pathogenic E. coli was not detected. In order to identify the sources of contamination for microgreen, the genetic similarity of B. cereus isolates obtained from harvesting and processing steps were compared using the repetitive-sequence-based polymerase chain reaction method. B. cereus isolates obtained from harvesting environments and microgreen were clustered with a similarity greater than 95%. In case of B. cereus isolates obtained from microgreen and environmental samples at processing steps showed low genetic similarity.

Isolation and Characterization of Soil Bacteria Degrading a Fungicide Defenoconazole (살균제 디페노코나졸 분해 세균 분리 및 특성 분석)

  • Ahn, Jae-Hyung;Ro, Yu-Mi;Lee, Gwan-Hyeong;Park, InCheol;Kim, Wan-Gyu;Han, Byeong-Hak;You, Jaehong
    • The Korean Journal of Pesticide Science
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    • v.20 no.4
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    • pp.349-354
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    • 2016
  • Triazole fungicides occupy an important portion in the global fungicide market and are relatively persistent in soil compared to the other fungicides, suggesting possible adverse effects of the fungicides on human health and environment. In this study, we tried to isolate microorganisms from orchard soils, which can decompose the triazole fungicides, tebuconazole, fluquinconazole, and difenoconazole. Only difenoconazole was completely degraded in the enrichment culture, from which several difenoconazole-degrading bacteria were isolated. They showed the same rep-PCR pattern thus only one strain, C8-2, was further studied. The strain was identified as Sphingomonas sp. C8-2 based on its 16S rRNA gene sequence and decomposed 100 mg/L of difenoconazole in a minimum medium to an unknown metabolite with a molecular weight of 296 within 24 hours. The inhibition effect of the metabolite against representative soil microorganisms significantly decreased compared to that of difenoconazole thus the bacterial strain is expected to be used for the detoxification of difenoconazole in soil and crop.

Molecular Characterization and Antimicrobial Susceptibility of Biofilm-forming Acinetobacter baumannii Clinical Isolates from Daejeon, Korea (대전지역에서 분리된 생물막 형성 Acinetobacter baumannii 임상분리주의 분자유전학적 특성과 항균제 감수성양상)

  • Sung, Ji Youn
    • Korean Journal of Clinical Laboratory Science
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    • v.50 no.2
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    • pp.100-109
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    • 2018
  • The emergence and dissemination of multidrug-resistant (MDR) Acinetobacter baumannii isolates have been reported worldwide, with most of these possessing the ability to form biofilms. Biofilm formation is an important virulence factor associated with the resistance to disinfection and desiccation. This study examined the genetic basis of antimicrobial resistance mechanisms of biofilm-forming A. baumannii clinical isolates. Imaging and quantification of biofilms were performed by a crystal violet assay and 46 biofilm-forming A. baumannii isolates were selected. Subsequently, 16 isolates belonging to different clones were identified using REP-PCR, and detection of the antimicrobial determinants in the isolates was carried out. The 16 isolates included 9 non-MDR and 7 MDR isolates. The mean biomass $OD_{560}$ values of the non-MDR (0.96) and MDR (1.05) isolates differed but this difference was not significant. In this study, most biofilm-forming MDR A. baumannii isolates contained various antimicrobial resistance determinants ($bla_{OXA-23}$, armA, and mutations of gyrA and parC). On the other hand, most biofilm-forming non-MDR A. baumannii isolates did not contain antimicrobial resistance determinants. These results suggest that there is little correlation between the biofilm-forming ability and antimicrobial susceptibility in A. baumannii isolates. In addition, the emergence of MDR A. baumannii clinical isolates is generally caused by mutations of the genes associated with antimicrobial resistance and/or the acquisition of various antimicrobial resistance determinants.

Genetic and Phenotypic Diversity of Parathion-Degrading Bacteria Isolated from Rice Paddy Soils

  • Choi, Min-Kyeong;Kim, Kyung-Duk;Ahn, Kyong-Mok;Shin, Dong-Hyun;Hwang, Jae-Hong;Seong, Chi-Nam;Ka, Jong-Ok
    • Journal of Microbiology and Biotechnology
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    • v.19 no.12
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    • pp.1679-1687
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    • 2009
  • Three parathion-degrading bacteria and eight pairs of bacteria showing syntrophic metabolism of parathion were isolated from rice field soils, and their genetic and phenotypic characteristics were investigated. The three isolates and eight syntrophic pairs were able to utilize parathion as a sole source of carbon and energy, producing p-nitrophenol as the intermediate metabolite during the complete degradation of parathion. Analysis of the 16S rRNA gene sequence indicated that the isolates were related to members of the genera Burkholderia, Arthrobacter, Pseudomonas, Variovorax, and Ensifer. The chromosomal DNA patterns of the isolates obtained by polymerasechain-reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences were distinct from one another. Ten of the isolates had plasmids. All of the isolates and syntrophic pairs were able to degrade parathion-related compounds such as EPN, p-nitrophenol, fenitrothion, and methyl parathion. When analyzed with PCR amplification and dot-blotting hybridization using various primers targeted for the organophosphorus pesticide hydrolase genes of previously reported isolates, most of the isolates did not show positive signals, suggesting that their parathion hydrolase genes had no significant sequence homology with those of the previously reported organosphophate pesticide-degrading isolates.

Genetic and Phenotypic Diversity of Fenitrothion-Degrading Bacteria Isolated from Soils

  • Kim, Kyung-Duk;Ahn, Jae-Hyung;Kim, Tae-Sung;Park, Seong-Chan;Seong, Chi-Nam;Song, Hong-Gyu;Ka, Jong-Ok
    • Journal of Microbiology and Biotechnology
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    • v.19 no.2
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    • pp.113-120
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    • 2009
  • Twenty-seven fenitrothion-degrading bacteria were isolated from different soils, and their genetic and phenotypic characteristics were investigated. Analysis of the 16S rDNA sequence showed that the isolates were related to members of the genera Burkholderia, Pseudomonas, Sphingomonas, Cupriavidus, Corynebacterium, and Arthrobacter. Among the 27 isolates, 12 different chromosomal DNA fingerprinting patterns were obtained by polymerase chain reaction(PCR) amplification of repetitive extra genic palindromic(REP) sequences. The isolates were able to utilize fenitrothion as a sole source of carbon and energy, producing 3-methyl-4-nitrophenol as the intermediate metabolite during the complete degradation of fenitrothion. Twenty-two of 27 isolates were able to degrade parathion, methyl-parathion, and p-nitrophenol but only strain BS2 could degrade EPN(O-ethyl-O-p-nitrophenyl phenylphosphorothioate) as a sole source of carbon and energy for growth. Eighteen of the 27 isolates had plasmids. When analyzed with PCR amplification and dot-blotting hybridization using various specific primers targeted to the organophosphorus pesticide hydrolase genes of the previously reported isolates, none of the isolates showed positive signals, suggesting that the corresponding genes of our isolates had no significant sequence homology with those of the previously isolated organophosphate pesticide-degrading bacteria.

Genetic and Phenotypic Diversity of Carbofuran-Degrading Bacteria Isolated from Agricultural Soils

  • Shin, Dong-Hyeon;Kim, Dong-Uk;Seong, Chi-Nam;Song, Hong-Gyu;Ka, Jong-Ok
    • Journal of Microbiology and Biotechnology
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    • v.22 no.4
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    • pp.448-456
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    • 2012
  • Thirty-seven carbofuran-degrading bacteria were isolated from agricultural soils, and their genetic and phenotypic characteristics were investigated. The isolates were able to utilize carbofuran as a sole source of carbon and energy. Analysis of the 16S rRNA gene sequence indicated that the isolates were related to members of the genera Rhodococcus, Sphingomonas, and Sphingobium, including new types of carbofuran-degrading bacteria, Bosea and Microbacterium. Among the 37 isolates, 15 different chromosomal DNA patterns were obtained by polymerase chain reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences. Five of the 15 representative isolates were able to degrade carbofuran phenol, fenoxycarb, and carbaryl, in addition to carbofuran. Ten of the 15 representative isolates had 1 to 8 plasmids. Among the 10 plasmid-containing isolates, plasmid-cured strains were obtained from 5 strains. The cured strains could not degrade carbofuran and other pesticides anymore, suggesting that the carbofuran degradative genes were on the plasmid DNAs in these strains. When analyzed with PCR amplification and dot-blot hybridization using the primers targeting for the previously reported carbofuran hydrolase gene (mcd), all of the isolates did not show any positive signals, suggesting that their carbofuran hydrolase genes had no significant sequence homology with the mcd gene.

Analysis of Plasmid pJP4 Horizontal Transfer and Its Impact on Bacterial Community Structure in Natural Soil

  • KIM TAE SUNG;KIM MI SOON;JUNG MEE KUM;JOE MIN JEONG;AHN JAE HYUNG;OH KYOUNG HEE;LEE MIN HYO;KIM MIN KYUN;KA JONG OK
    • Journal of Microbiology and Biotechnology
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    • v.15 no.2
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    • pp.376-383
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    • 2005
  • Alcaligenes sp. JMP228 carrying 2,4­dichlorophenoxyacetic acid (2,4-D) degradative plasmid pJP4 was inoculated into natural soil, and transfer of the plasmid pJP4 to indigenous soil bacteria was investigated with and without 2,4-D amendment. Plasmid pJP4 transfer was enhanced in the soils treated with 2,4-D, compared to the soils not amended with 2,4-D. Several different transconjugants were isolated from the soils treated with 2,4-D, while no indigenous transconjugants were obtained from the unamended soils. Inoculation of the soils with both the donor Alcaligenes sp. JMP228/pJP4 and a recipient Burkholderia cepacia DBO 1 produced less diverse transconjugants than the soils inoculated with the donor alone. Repetitive extragenic palindromic-polymerase chain reaction (REP-PCR) analysis of the transconjugants exhibited seven distinct genomic DNA fingerprints. Analysis of 16S rDNA sequences indicated that the transconjugants were related to members of the genera Burkholderia and Pandoraea. Denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA genes revealed that inoculation of the donor caused clear changes in the bacterial community structure of the 2,4-D­amended soils. The new 16S rRNA gene bands in the DGGE profile corresponded with the 16S rRNA genes of 2,4-D­degrading transconjugants isolated from the soil. The results indicate that introduction of the 2,4-D degradative plasmid as Alcaligenes sp. JMP228/pJP4 has a substantial impact on the bacterial community structure in the 2,4-D-amended soil.

Genetic Analysis and Serological Detection of Novel O-Antigen Gene Clusters of Plesiomonas shigelloides

  • Wang, Xiaochen;Xi, Daoyi;Li, Yuehua;Yan, Junxiang;Zhang, Jingyun;Guo, Xi;Cao, Boyang
    • Journal of Microbiology and Biotechnology
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    • v.31 no.4
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    • pp.520-528
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    • 2021
  • Plesiomonas shigelloides, a member of the family Vibrionaceae, is a gram-negative, rod-shaped, facultative anaerobic bacterium with flagella. P. shigelloides has been isolated from such sources as freshwater, surface water, and many wild and domestic animals. P. shigelloides contains 102 O-antigens and 51 H-antigens. The diversity of O-antigen gene clusters is relatively poorly understood. In addition to O1 and O17 reported by other laboratories, and the 12 O serogroups (O2, O10, O12, O23, O25, O26, O32, O33, O34, O66, O75, and O76) reported previously by us, in the present study, nine new P. shigelloides serogroups (O8, O17, O18, O37, O38, O39, O44, O45, and O61) were sequenced and annotated. The genes for the O-antigens of these nine groups are clustered together in the chromosome between rep and aqpZ. Only O38 possesses the wzm and wzt genes for the synthesis and translocation of O-antigens via the ATP-binding cassette (ABC) transporter pathway; the other eight use the Wzx/Wzy pathway. Phylogenetic analysis using wzx and wzy showed that both genes are diversified. Among the nine new P. shigelloides serogroups, eight use wzx/wzy genes as targets. In addition, we developed an O-antigen-specific PCR assay to detect these nine distinct serogroups with no cross reactions among them.