• Journal of Yeungnam Medical Science
• /
• v.12 no.2
• /
• pp.366-385
• /
• 1995

Epidemiological studies are important in both the prevention and treatment of mycobacterial infections. This study was initiated to establish the pulsed-field gel electrophoresis (PFGE) method, which are not yet extensively studied. The most apprpriate restriction endonucleases included DraI, AsnI, and XbaI. The optimal PFGE condition was different according to the enzymes used. Two stage PFGE was performed, in case of DraI first stage was performed with 10 seconds of initial pulse and 15 seconds of final pulse, while the second stage was performed with 60 seconds of initial pulse and 70 seconds of final pulse. The electrophoresis time for DraI-PFGE was 14 hours for each stage. Electrophoresis was performed for 22 hours, in case of XbaI, with 3 seconds of initial pulse and 12 seconds of final pulse. Electrophoresis was performed for 22 hours, in case of AsnI, with 5 seconds of initial pulse and 25 seconds of final pulse. In all cases the voltage of the electrophoresis was maintained constantly at 200 voltage. Standard mycobacterial strains, which included Mycobacterium bovis BCG, M. tuberculosis, and M. fortuitum, could not be differentiated by PFGE analysis. PFGE analysis was performed to differentiate 9 clinically isolated M. fortuitum strains using AsnI. All M. fortuitum strains showed different genotypes except 2 strains. Cluster analysis divided M. fortuitum strains into 2 large groups. PFGE analysis was performed to further differentiate M. fortuitum isolates using XbaI. The undifferentiated 2 M. fortuitum strains showed different PFGE patterns with Xba I. Cluster analysis of the XbaI-PFGE patterns showed more complex grouping than AsnI-PFGE patterns, which showed that XbaI-PFGE analysis was better than AsnI-PFGE in M. fortuitum genotyping. The top dissimilarity values of AsnI-PFGE and XbaI-PFGE were 0.74 and 0.75, respectively. This value was higher than that of arbitrarily primed polymerase chain reaction (AP-PCR) analysis and lower than that of restriction fragment length polymorphism (RFLP) analysis. This suggested that PFGE can be used as a supportive or alternative genotyping method to RFLP analysis.

• Korean Journal of Veterinary Research
• /
• v.33 no.1
• /
• pp.81-85
• /
• 1993

Gel electrophoresis has proven to be one of the most useful of DNA separation and purification. The new technique of pulsed field gel electrophoresis (PFGE) is high resolution separation of large size DNA moleculs. Conventional continuous gel electrophresis can not be separation of large DNA fragments(20~50 k base). Field inversion gel electrophoresis(FIGE) is very useful for large DNA molecules. We have found that a pulse ratio ; 2 : 1, time ; 24hrs., volts ; $10^{volts}/_{cm}$, start ; 0.45sec, end ; 1sec, is most effectively resolves DNA fragment in the 6~50k base.

• Journal of Life Science
• /
• v.16 no.4
• /
• pp.664-671
• /
• 2006

We obtained 424 Salmonella enterica serovar Typhi isolates from sporadic cases of infection in Busan during 1996 to 2005. We investigated the trend of antimicrobial resistance and molecular typing by pulsed-field gel electrophoresis (PFGE). Of the total 424 isolates, 6 strains (1.4%) were multidrug-resistant (MDR) S. enterica serovar Typhi isolates, 2 strains (0.5%) were resistant to only nalidixic acid, and the remaining 416 strains (98.1%) were fully susceptible to the 18 antimicrobial agent. PFGE of XbaI-digested chromosomal DNA was performed on 50 sporadic S. enterica serovar Typhi isolates with the objective of investigating the extent of genetic diversity of these isolates in our region. We could find that these isolates were much more heterogeneous and at least 32 different PFGE patterns were generated according by dice coefficient, between 0.69 and 1.0. Restriction fragment patterns consisted of 13 to 18 fragments ranged in size from 20 to 630 kb. The results confirmed that PFGE would be an useful tool for investigating surveillance of sporadic or outbreak case and assessing clonality for S. enterica serovar Typhi in Busan area. Our finding will be valuable in developing rational strategies to control this pathogen and setting the basis of an effective PulseNet system in Korea.

• Journal of Microbiology and Biotechnology
• /
• v.16 no.10
• /
• pp.1650-1655
• /
• 2006

Bacteria belonging to the genus Paenibacillus are facultatively anaerobic endospore formers and are attracting growing ecological and agricultural interest, yet their genome information is very limited. The present study surveyed the genomic features of P. polymyxa ATCC $842^T$ using pulse-field gel electrophoresis of restriction fragments and sample genome sequencing of 1,747 reads (approximately 17.5% coverage of the genome). Putative functions were assigned to more than 60% of the sequences. Functional classification of the sequences showed a similar pattern to that of B. subtilis. Sequence analysis suggests nitrogen fixation and antibiotic production by P. polymyxa ATCC $842^T$, which may explain its plant growth-promoting effects.

• International Journal of Oral Biology
• /
• v.35 no.1
• /
• pp.7-12
• /
• 2010

The aims of this study were to investigate the nosocomial infection route of methicillin-resistant Staphylococcus aureus (MRSA) and explore preventative methods for this pathogen that involve blocking its dispersion. We cultured MRSA from nasal cavity swabs collected between June and July 2008 that we obtained from eight dental healthcare providers, 32 nurses and the sputum specimens of two patients from our hospital. In addition, we used VITEK 2 equipment to measure drug sensitivity, and we further performed biochemical testing and pulse-field gel electrophoresis (PFGE) to isolate MRSA colonies. The incidence of these bacteria on the nasal swabs was 25.0% from dental clinic healthcare providers, 13.6% from the internal medicine ward nurses and 30.0% from intensive care unit nurses. Moreover, MRSA was detectable in sputum specimens of ward patients. The antimicrobial agents resistance and partial PFGE types of MRSA showed a similar pattern. We suggest from these analyses that nasal cavity infection by MRSA could occur by cross contamination between healthcare providers and patients which underscores the importance of stringent MRSA management practices.

• International Journal of Oral Biology
• /
• v.35 no.4
• /
• pp.185-190
• /
• 2010

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most prevalent pathogens in hospitals. To investigate cross contamination by this bacterium in both dental and medical settings, the pathogens that cause acute pyogenic infection and one of the major microbes responsible for nosocomial infection were isolated from health care providers, nurses and patients. We used VITEK II to measure drug sensitivity, and we further performed biochemical testing, coagulase serotype testing and pulse-field gel electrophoresis (PFGE) for isolated MRSA colonies. The isolation rate of Staphylococcus aureus from nasal swabs was 75.0% from dental health care providers and 18.8% from the medical health care providers. A total of 10 MRSA strains were isolated from 40 health care providers and 2 patients and the prevalent coagulase serotype from patients and health care providers was VII. The antimicrobial drug resistance and partial PFGE types of the isolated MRSA strains showed a similar pattern. These results suggest that MRSA may be one of the principal causes of nosocomial infection in dental and medical hospitals.

• Journal of Microbiology
• /
• v.40 no.1
• /
• pp.63-69
• /
• 2002

A total of twenty-five norfloxacin resistant Escherichia coli were isolated from Joongrang-chun stream, a branch of the Han River in Seoul, Korea from May to July in 2000 and their norfloxacin resistance mechanism was characterized for target site mutation, permeability, and efflux pump. Fourteen iso- lates contained the same three mutations, Ser83→Leu and Asp87→Asn in GyrA and Ser90→ lle in ParC. Six isolates had Ser83→Leu and Asp87→Tyr in GyrA and Ser87→lle in ParC while one isolate had Ser83→Leu and Va1103→Ala in GyrA and Ser80→lle in ParC. Two isolates had mutation(s) in GyrA without any mutation in ParC. Two isolates had Ser80→Arg in ParC instead of the commonly found Ser80→lle. Every norfloxacin resistant isolate had an efflux system but the correlation between the efflux activity and MIC was not observed. The amount of OmpF for norfloxacin permeability decreased in resistant isolates compared to the susceptible strains. When amplified polymorphic DNA (RAPD) and pulse field gel electrophoresis (PFGE) were performed, these isolates showed no similarity to each other or clinical isolates.

• Korean Journal of Microbiology
• /
• v.44 no.4
• /
• pp.305-310
• /
• 2008

Staphylococcus aureus is one of the most significant pathogens and a causative agents of nosocomial infections. The emergence of methicillin resistant S. aureus (MRSA), in particular, has become a major clinical and epidemiological problems worldwide. In this study, we analyzed the toxin genes and investigated molecular epidemiological characteristics of S. aureus isolated from stools of diarrheal patients at the hospitals in Incheon. Of the 609 strains from 2,281 specimens, 173 strains retained enterotoxin; 68 isolates (39.30%), 100 isolates (57.80%) were classified to A and C type, respectively. In the antibiotic susceptibility, all of enterotoxin positive isolates were resistant to oxacillin. Eighty eight strains (50.86%) of 173 MRSA isolate possessed tsst gene, but eta and eth genes were not detected at all. In the detection of MRSA associated genes by PCR method, mecA genes were detected in 167 strains (96.53%). From the result of PFGE analysis, we classified tsst-positive MRSA to 10 types and 24 subtypes. Type A, H and F were the major strains comprised of 57.95% (51 strains), 10.22% (9 strains) and 9.09% (8 strains) respectively.

• Journal of Microbiology and Biotechnology
• /
• v.30 no.12
• /
• pp.1862-1869
• /
• 2020

The spread of plasmid-mediated colistin resistance has posed a serious threat to public health owing to its effects on the emergence of pandrug-resistant bacteria. In this study, we investigated the prevalence and characteristics of mcr-1-positive Escherichia coli isolated from retail meat samples in Korea. In total, 1,205 E. coli strains were isolated from 3,234 retail meat samples in Korea. All E. coli strains were subjected to antimicrobial susceptibility testing and were examined for the presence of mcr-1 gene. All mcr-1-positive E. coli (n = 10, 0.8%) from retail meat were subjected to pulse-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS). The transferability of mcr-1 gene was determined by conjugation assays. The mcr-1-positive strains exhibited diverse clonal types. Our mcr-1 genes were located in plasmids belonged to the IncI2 (n = 1) and IncX4 (n = 8) types, which were reported to be prevalent in Asia and worldwide, respectively. Most mcr-1 genes from mcr-1-positive strains (9/10) were transferable to the recipient strain and the transfer frequencies ranged from 2.4 × 10-3 to 9.8 × 10-6. Our data suggest that the specific types of plasmid may play an important role in spreading plasmid-mediated colistin resistance in Korea. Furthermore, our findings suggest that the retail meat may be an important tool for disseminating plasmid-mediated colistin resistance.

• Korean Journal of Microbiology
• /
• v.42 no.1
• /
• pp.12-18
• /
• 2006

Thirty five Salmonella enterica serovar Typhimurium strains were isolated from diarrheic patients and pigs in Gyeongbuk area from 2003 to 2004. All 35 strains (17 strains from diarrheic patients and 18 from pigs) were resistant to more than one drug and most of strains isolated from pigs were resistant to ampicillin, ohloram-phenicol, streptomycin, sulfamethoxazole-trimethoprime, tetracyclin and nalidixic acid. Each isolate was also screened or the presence of class I, II and III integron gene cassettes. Among 35 strains,3 out of 17 strains isolated from diarrheic patients, carried dhfrX-orfF-aadA2 integron gene cassette and among 18 strains isolated from diseased pigs, 11 strains carried dhfrX-orfF-aadA2 integron gene cassette and 1 strain carried aadA2 integron only. But any class II and class II integron gene cassette were not detected in 35 strains. Thirty five strains were divided by five pulsotypes. Thirty one strains out of thirty five were pulsotype A. Among the remaining 4 strains, one each strain belonged to pulsotype B, C, D and pulsotype E. This data of pulsotypes showed that the widespread of pulsotype A, Salmonella enterica serovar Typhimurium in human and pigs in Gyeongbuk area may have been caused by the dissemination of a few epidemic strains in this area. Thirteen strains contain dhfrX-orfF-aadA2 integron gene cassette showed pulsotype A and one strain contains dhfrX-orfF-aadA2 integron gene cassette showed pulsotype B. One strain contains aadA2 integron showed pulsotype E. But fifteen strains do not contain any integron showed pulsotype A.