• Journal of Microbiology and Biotechnology
• /
• 제16권2호
• /
• pp.256-263
• /
• 2006

In the model legume Medicago truncatula, two mutants, sickle and sunn, exhibit morphologically and genetically distinct hypernodulation phenotypes. However, efforts to isolate the single recessive and single semidominant genes for sickle and sunn, respectively, by map-based cloning have so far been unsuccessful, partly due to the absence of clones that enable walks from linked marker positions. To help resolve these difficulties, a new bacterial artificial chromosome (BAC) library was constructed using BamHI-digested genomic fragments. A total of 23,808 clones were collected from ligation mixtures prepared with double-size-selected high-molecular-weight DNA. The average insert size was 116 kb based on an analysis of 88 randomly selected clones using NotI digestion and pulsed-field gel electrophoresis. About 18.5% of the library clones lacked inserts. The frequency of the BAC clones carrying chloroplast or mitochondrial DNA was 0.98% and 0.03%, respectively. The library represented approximately 4.9 haploid M. truncatula genomes. Hybridization of the BAC clone filters with a $C_{0}t-l$ DNA probe revealed that approximately 37% of the clones likely carried repetitive sequence-enriched DNA. An ordered array of pooled BAC DNA was screened by polymerase chain reactions using 13 sequence-characterized molecular markers that belonged to the eight linkage groups. Except for two markers, one to five positive BAC clones were obtained per marker. Accordingly, the sickle- and sunn-linked BAC clones identified herein will be useful for the isolation of these biotechnologically important genes. The new library will also provide clones that fill the gaps between preexisting BAC contigs, facilitating the physical mapping and genome sequencing of M. truncatula.

• 한국동물위생학회지
• /
• 제34권1호
• /
• pp.37-43
• /
• 2011

This study was conducted to investigate the prevalence and genotypes of extended-spectrum ${\beta}$-lactamase (ESBL) in 377 Escherichia coli isolated from healthy and sick animals. Two isolates (0.5%), each of which were isolated from diseased swine and chicken, respectively, were confirmed as ESBL producing isolates by double disk synergy test, and showed a multidrug resistant phenotype. Minimum inhibitory concentration of cefotaxime for the two ESBL producing isolates were 3~4 times higher than those of ceftazidime, respectively. By PCR and sequencing, one isolate from swine have both $bla_{CTX-15}$ and $bla_{TEM-1}$, and one isolate from chicken have $bla_{CTX-15}$ and $bla_{TEM-116}$. Also, these genes were transferred to E. coli J53 by conjugation. These two isolates showed unrelated pulsed-field gel electrophoresis. To our knowledge, this is the first time that $bla_{TEM-116}$ gene was identified in E. coli isolated from animals in Korea. These results suggest more prudent use of third- generation cephalosporins, and surveillance and monitoring for ESBL producing E. coli in both animals and their environments should be necessary.

• 대한수의학회지
• /
• 제60권3호
• /
• pp.173-177
• /
• 2020

Edible offal is easily contaminated by Escherichia coli (E. coli) and fluoroquinolone (FQ)-resistant E. coli is considered a serious public health problem, thus, this study investigated the genetic characteristics of FQ-resistant E. coli from edible offal. A total of 22 FQ-resistant E. coli isolates were tested. A double mutation in each gyrA and parC led the highest MIC. Four (18.2%) isolates carried plasmid-mediated quinolone resistance genes. The fimH, eaeA, escV, astA, and iucC genes were confirmed. Seventeen isolates (77.3%) were positive for plasmid replicons. The isolates showed high genetic heterogeneity based on pulsed-field gel electrophoresis patterns.

• Journal of Microbiology
• /
• 제45권5호
• /
• pp.466-472
• /
• 2007

A total of 98 vancomycin-resistant Enterococcus faecium (VREF) isolates (58 isolates from patients and 40 isolates from poultry) were compared based on their antimicrobial susceptibility, Tn1546 element organization, and pulsed-field gel electrophoresis (PFGE) patterns. This comparison aided in determining the relationships between the groups of isolates. All the VREF isolates harbored the vanA gene; however, 29 (29.6%) of the isolates exhibited the VanB phenotype-vanA genotype. Furthermore, the VREF isolates from humans and poultry exhibited distinct antimicrobial resistance patterns. The PCR mapping of the Tn1546 elements exhibited 12 different transposon types (A to L). The VREF isolates of poultry were classified into types A to D, whereas the human isolates were classified into types E to L. A PFGE analysis demonstrated a high degree of clonal heterogeneity in both groups of isolates; however, the distinct VREF clones appeared in each group of isolates. The deletion of the vanX-vanY genes or insertion of IS1216V in the intergenic region from the vanX-vanY genes is directly associated with the incongruence of the VanB phenotype-vanA genotype in human VREF isolates. These data suggest that the VREF isolates exhibit distinct phenotypic and genotypic traits according to their origins, which suggests that no evidence exists to substantiate the clonal spread or transfer of vancomycin resistance determinants between humans and poultry.

• Journal of Microbiology and Biotechnology
• /
• 제18권11호
• /
• pp.1853-1857
• /
• 2008

Salmonella contamination in chicken meat was studied with 100 chicken meat samples purchased from 55 shops located in various regions. A total of 21 isolates of Salmonella enterica were isolated from 21 chicken meat samples from four shops located at open markets, whereas there were none from supermarkets with well-equipped cold systems. Among these, 18 isolates were identified as Salmonella enterica serotype Haardt (S. Haardt) and three isolates were S. enterica serotype Muenchen. When the minimal inhibitory concentrations of the S. Haardt isolates were assayed with the agar dilution method to determine susceptibility to ampicillin, chloramphenicol, sulfisoxazole, tetracycline, and nalidixic acid, all 18 isolates were resistant to tetracycline and nalidixic acid and nine of these were resistant to ampicillin. These isolates showed reduced susceptibility to eight fluoroquinolones including ciprofloxacin, enrofloxacin, levofloxacin, gatifloxacin, gemifloxacin, moxifloxacin, norfloxacin, and ofloxacin. When quinolone resistance determining regions of gyrA and gyrB were sequenced, every isolate had the same missense mutation Ser83$\rightarrow$Tyr (TCC$\rightarrow$+TAC) in gyrA, whereas no mutation was found in gyrB. Pulsed-field gel electrophoresis with XbaI revealed a close relationship among these isolates, suggesting a contamination of raw chicken meat with clonal spread of nalidixic acid-resistant and quinolone-reduced susceptibility S. Haardt in chickens. Results in this study show the importance of a well-equipped cold system and the prudent use of fluoroquinolone in chickens to prevent the occurrence of quinolone-resistant isolates.

• 한국동물위생학회지
• /
• 제34권3호
• /
• pp.217-226
• /
• 2011

Salmonella spp. is of increasing public health concern as causative pathogens of food poisoning. The aim of this study was to investigate the serotypes and antimicrobial resistance pattern of Salmonella spp. isolated from duck farms in Daegu-Gyeongbuk province. Also, S. Enteritidis and S. Typhimurium isolates were further examined for plasmid analysis, phage typing and pulsed-field gel electrophoresis (PFGE). A total of 34 Salmonella spp. (16.4%) were isolated from duck farms and ten serotypes were identified. The predominant serotypes were S. Typhimurium (23.5%) S. Fyris (17.6%) and S. Haardt (11.8%), S. Agona and S. Enteritidis (respectively 8.8%). Of 34 Salmonella isolates, 15 (44.1%) isolates were resistant to at least one antimicrobial agent and multiple resistance (resistance to more than 4 drugs) was observed in 9 strains (26.5%). The high resistance was found to streptomycin (32.4%), tetracycline (29.4%), ampicillin, kanamycin and nalidixic acid (respectively, 26.5%), all Salmonella isolates were susceptible to cefoxitin, cefotaxime, gentamicin, amikacin and ciprofloxacin. All S. Enteritidis and S. Typhimurium isolates were found to contain only one plasmid (ca. 54 or 55kb, respectively). Among the S. Enteritidis isolates, two phage types were found, PT32a and PT1c, respectively, one isolates did not react with any of the phages used. Whereas, all S. Typhimurium isolates were RDNC (reacts but does not conform). PFGE showed to be a useful typing method better than plasmid analysis and phage typing for discrimination of isolates especially, S. Typhimurium isolates. Our results indicated that the serotypes of Salmonella isolates are widely distributed in duck farms, further epidemiological studies should be carried out.

• 약학회지
• /
• 제57권2호
• /
• pp.132-138
• /
• 2013

Acinetobacter baumannii is gram-negative bacilli that can be widely found in environments. Recently, A. baumannii emerged as a serious nosocomial infection. A total of 92 A. baumannii were isolated from hospitalized patients in Seoul, Korea, between December 2010 and April 2011. Antimicrobial susceptibility testing was investigated using CLSI agar dilution methods. Tigecycline non-susceptible A. baumannii isolates were investigated by repetitive extragenic palindromic sequence-based PCR (rep-PCR). Pulsed-field gel electrophoresis was performed to determine the epidemiological relationships. All clinical isolates showed high-level resistance to the most commonly used antibiotics: Ciprofloxacin (87.0%), Ampicillin/sulbactam (82.6%), Cefotaxime (81.5%), Ceftazidime (80.4%). Moreover, 50.0% of these isolates were non-susceptible to tigecycline. When evaluated by RAPD analysis, generated distinct band ranging in size from 1kb to 8k band varying from 4 to 10 bands. Stricter surveillance and more rapid detection are essential to prevent the spread of multi drug resistant A. baumannii.

• Journal of Microbiology and Biotechnology
• /
• 제16권10호
• /
• pp.1518-1522
• /
• 2006

In the present study, it was observed how the phage-host system that is naturally reproduced in activated sludge is affected by the host inoculation. The system of Microlunatus phosphovorus and its phages was selected as the phage-host system native to an activated sludge system operated for 19 days under sequencing anaerobic-aerobic conditions with glutamate as the main carbon source. The phage-host system related to M. phosphovorus was monitored by plaque assay for the phages and by fluorescent in situ hybridization (FISH) for the bacterial host. In addition, the whole phage structure was also monitored by pulsed-field gel electrophoresis (PFGE). During the first 9 days, the phage-host system was more or less steady at approx. 9% (FISH/ DAPI) for M. phosphovorus and approx. 10,000 PFU/ml for its lytic phages. Microlunatus phosphovorus JCM9379 was inoculated into the activated sludge on day 10. Right after the inoculation, M. phosphovorus was approx. 24% (FISH/DAPI) whereas its lytic phages dropped down to approx. 500 PFU/ ml. After the host inoculation (within 9 days), however, the phage-host system eventually reverted to its original level in each population. On the other hand, the whole phage structure was not significantly changed by M. phosphovorus inoculation but stable throughout the process operation. Only the minor change that four phage groups gradually became abundant after the host inoculation was observed.

• Journal of Microbiology and Biotechnology
• /
• 제24권3호
• /
• pp.421-426
• /
• 2014

To characterize the extended-spectrum beta-lactamases (ESBLs) in diarrheagenic Escherichia coli from Korea in 2008-2011, we screened seven enterotoxigenic E. coli (ETEC) and one enteroaggregative E. coli (EAEC) that produce ESBLs from a nationwide survey. All eight isolates produced CTX-M-type ESBLs, including CTX-M-12 (n = 4), CTX-M-14 (n = 2), and CTX-M-15 (n = 2). PCR-based replicon typing indicated that the $bla_{CTX-M-12}$ genes of four ETEC isolates were carried on a conjugative IncF plasmid, whereas the $bla_{CTX-M-14}$ of one EAEC was located on an IncK plasmid. This is the first report of the occurrence of $bla_{CTX-M}$ genes in clinical isolates of EAEC in Korea. The ESBL-producing isolates were shown to be different based on pulsed-field gel electrophoresis and multilocus sequence typing, whereas the four isolates with CTX-M-12 were clonally related. These observations raise an alarm for the spread of plasmid-mediated resistance to ESBL among diarrheagenic E. coli.

• Journal of Microbiology and Biotechnology
• /
• 제23권1호
• /
• pp.85-87
• /
• 2013

An outbreak of Staphylococcus aureus infections occurred in a university with an enrollment of 80 students in the city of Daejon, Republic of Korea. All nine S. aureus isolates from patients (n = 7), staff members (n = 1), and the fried chicken served as the lunch (n = 1) harbored the enterotoxin A gene and showed an identical antibioticresistant profile, PFGE banding pattern (STAS16.001), and sequence type, ST 6. These results suggested that the outbreak was associated with eating the fried chicken that had been handled by an infected staff member. This case report demonstrated a practical approach to identifying the source and transmission of an infection.