• Journal of Microbiology and Biotechnology
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• v.23 no.3
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• pp.430-435
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• 2013

Multidrug resistance, especially multidrug efflux mechanisms that extrude structurally unrelated cytotoxic compounds from the cell by multidrug transporters, is a serious problem and one of the main reasons for the failure of therapeutic treatment of infections by pathogenic microorganisms as well as of cancer cells. Streptococcus mutans is considered one of the primary causative agents of dental caries and periodontal disease, which comprise the most common oral diseases. A fragment of chromosomal DNA from S. mutans KCTC3065 was cloned using Escherichia coli KAM32 as host cells lacking major multidrug efflux pumps. Although E. coli KAM32 cells were very sensitive to many antimicrobial agents, the transformed cells harboring a recombinant plasmid became resistant to several structurally unrelated antimicrobial agents such as tetracycline, kanamycin, rhodamin 6G, ampicillin, acriflavine, ethidium bromide, and tetraphenylphosphonium chloride. This suggested that the cloned DNA fragment carries a gene encoding a multidrug efflux pump. Among 49 of the multidrug-resistant transformants, we report the functional gene cloning and characterization of the function of one multidrug efflux pump, namely MdeA from S. mutans, which was expressed in E. coli KAM32. Judging from the structural and biochemical properties, we concluded that MdeA is the first cloned and characterized multidrug efflux pump using the proton motive force as the energy for efflux drugs.

• Microbiology and Biotechnology Letters
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• v.31 no.3
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• pp.230-234
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• 2003

The Bacillus subtilis YvaE protein, the small multidrug resistance (SMR) family (TC #2.A. 7.1), is shown to catalyze efflux of multiple cationic drugs including many disinfectants, when it was cloned and expressed in Escherichia coli. When the yvaD gene was coexpressed with yvaE gene, the yvaD protein, encoded within a single operon with the yvaE gene, is shown to counteract the action ofYvaE. By ethidium efflux analysis, the cells harvoring a vector with yvaE gene showed a rapid ethidium efflux, compared with the control cells. These results clearly suggest that YvaE mediates drug export from the cell cytoplasm.

• Biomedical Science Letters
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• v.27 no.2
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• pp.59-68
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• 2021

Multidrug-resistant strain of Acinetobacter baumannii (MDRAB) is an emerging pathogen in health care facilities, preventing MDRAB is a public health concern. We conducted this experiment on a clinical isolate of A. baumannii with two main goals: the role of the efflux pump system in the stress provision of carbapenem and the response to the transcription level of the efflux pump gene. A total of 34 strains of A. baumannii was isolated from the Yangsan Hospital of Pusan National University. First, when we compared and observed the expression of the efflux pump gene and antibacterial resistance to carbapenem, a strong correlation was observed between carbapenem resistance and overexpression of adeB (P=0.0056). Second, a correlation between the efflux pump and concentration gradient and tolerance to carbapenem stress at the AdeABC efflux pump genes transcription level was confirmed. Our results revealed that the expression of the AdeABC efflux pump is an important resistance determinant in obtaining antibiotic resistance of the carbapenem group in A. baumannii.

• Journal of Microbiology and Biotechnology
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• v.30 no.7
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• pp.982-995
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• 2020

A putative multidrug efflux gene, yddA, was cloned from the Escherichia coli K-12 strain. A drug-sensitive strain of E. coli missing the main multidrug efflux pump AcrB was constructed as a host and the yddA gene was knocked out in wild-type (WT) and drug-sensitive E. coliΔacrB to study the yddA function. Sensitivity to different substrates of WT E.coli, E. coliΔyddA, E. coliΔacrB and E. coliΔacrBΔyddA strains was compared with minimal inhibitory concentration (MIC) assays and fluorescence tests. MIC assay and fluorescence test results showed that YddA protein was a multidrug efflux pump that exported multiple substrates. Three inhibitors, ortho-vanadate, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and reserpine, were used in fluorescence tests. Ortho-vanadate and reserpine significantly inhibited the efflux and increased accumulation of ethidium bromide and norfloxacin, while CCCP had no significant effect on YddA-regulated efflux. The results indicated that YddA relies on energy released from ATP hydrolysis to transfer the substrates and YddA is an ABC-type multidrug exporter. Functional study of unknown ATP-binding cassette (ABC) superfamily transporters in the model organism E. coli is conducive to discovering new multidrug resistance-reversal targets and providing references for studying other ABC proteins of unknown function.

• Journal of Microbiology and Biotechnology
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• v.26 no.12
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• pp.2043-2050
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• 2016

Exploring novel antibiotics is necessary for multidrug-resistant pathogenic bacteria. Because the probiotics in soybean food have antimicrobial activities, we investigated their effects on multidrug-resistant Acinetobacter baumannii. Nineteen multidrug-resistant A. baumannii strains were clinically isolated as an experimental group and 11 multidrug-sensitive strains as controls. The growth rates of all bacteria were determined by using the analysis for xCELLigence Real-Time Cell. The combination of antibiotics showed synergistic effects on the strains in the control group but no effect on the strains in the experimental group. Efflux pump gene adeS was absent in all the strains from the control group, whereas it exists in all the strains from the experimental group. Furthermore, all the strains lost multidrug resistance when an adeS inhibitor was used. One strain of probiotics isolated from soybean food showed high antimicrobial activity for multidrug-resistant A. baumannii. The isolated strain belongs to Bacillus subtilis according to 16S RNA analysis. Furthermore, E. coli showed multidrug resistance when it was transformed with the adeS gene from A. baumannii whereas the resistant bacteria could be inhibited completely by isolated Bacillus subtilis. Thus, probiotics from soybean food provide potential antibiotics against multidrug-resistant pathogenic bacteria.

• Korean Journal of Microbiology
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• v.49 no.2
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• pp.126-130
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• 2013

The major aim of this study was to investigate the distribution of genes that encode multidrug efflux pumps in Enterococci spp. isolates from bovine milk samples and antibiotic resistance patterns of these strains. Of the 245 isolates, 44.1% showed ampicillin resistance, 79.2% showed erythromycin resistance, 76.3% showed tetracycline resistance and 36.3% showed chloramphenicol resistance. In case of vancomycin and ciprofloxacin, all of the isolates were susceptible to these antibiotics. Of the 245 enterococcal isolates, 82.1% have MFS type eme(A) gene, 72.7% have ABC type efr(A) gene, 77.1% have ABC type efr(B) gene, and 71.8% have ABC type lsa gene. In case of Enterococcus faecalis, the original strain for these genes, 92.5% have eme(A), 87.4% have efr(A), 88.4% have efr(B), and 88.4% have lsa. Interestingly, in case of different species of Enterococci, eme(A) was also detected in four strains of E. faecium, seven strains of E. avium, four strains of E. durans and two strains of E. raffinosus. efr(A) was also detected in two strains of E. faecium and two strains of E. durans and efr(B) was also detected in four strains of E. faecium, five strains of E. avium and four strains of E. durans. This means the possibility of co-transfer of resistance genes between Enterococci species in natural environment. These results are the first report describing the presence of same multidrug efflux pumps in different species of Enterococci in Korea.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.16
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• pp.6849-6853
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• 2014

Multidrug resistance (MDR) is a major impediment to successful chemotherapy of gastric cancer. Our aim was to establish an epirubicin-resistant cell subline (AGS/EPI) and to elucidate the mechanisms involved in acquired EPI resistance. The AGS/EPI cell subline developed by exposing parental AGS cells to stepwise increasing concentrations of EPI demonstrated 2.52-fold resistance relative to the AGS cell line, and mRNA expression of the ATP-dependent drug-efflux pump P-glycoprotein (Pgp), more recently known as ABCB1 protein, was similarly upregulated. An AGS/EPI cell subline could thus be effectively established, and MDR mechanism of these cells was shown to be related to the overexpression of mRNA of the ABCB1 gene.

• Journal of Food Hygiene and Safety
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• v.32 no.2
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• pp.135-140
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• 2017

The aim of this study was to investigate the distribution of genes that encode multi-drug efflux pumps and virulence factors in Enterococcus faecalis isolated from retail meat and antibiotic resistance patterns of these strains. Of the 277 retail meat samples, 93 Enterococcus faecalis were isolated. The strains exhibited resistance to ampicillin (35.5%), chloramphenicol (6.4%), ciprofloxacin (4.3%), erythromycin (18.3%), levofloxacin (0%), quinupristin-dalfopristin (76.3%), tetracycline (45.2%), teicoplanin (0%) and vancomycin (0%). The strains were positive for MFS type eme(A) (100%), ABC type efr(A) (100%), ABC type efr(B) (98.9%) and ABC type lsa (91.4%) efflux pump gene. The strains were positive for gelE (68.8%), ace (90.3%), asa1 (47.3%), efaA (91.4%) and esp (12.9%) virulence gene. This research will help to assess the hazards associated with the occurrence of drug resistance among enterococci from retail meat. Therefore, it is necessary to monitor enterococcus spp. isolated from retail meat continuously.

• Biomolecules & Therapeutics
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• v.19 no.2
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• pp.174-180
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• 2011

Experiments were carried out to determine the role of Raf-1 kinase in the development of drug resistance to paclitaxel in v-H-ras transformed NIH 3T3 fibroblasts (Ras-NIH 3T3). We established a multidrug-resistant cell line (Ras-NIH 3T3/Mdr) from Ras-NIH 3T3 cells by stepwise increases in paclitaxel. Drug sensitivity assays indicated that the $IC_{50}$ value for drug-resistant Ras-NIH 3T3/Mdr cells was more than 1 ${\mu}M$ paclitaxel, 10- or more-fold higher than for the parental Ras-NIH 3T3 cells. Western blot and RT-PCR analysis showed that the drug efflux pump a P-glycoprotein were highly expressed in Ras-NIH 3T3/Mdr cells, while not being detectable in Ras-NIH 3T3 cells. Additionally, verapamil, which appears to inhibit drug efflux by acting as a substrate for P-glycoprotein, completely reversed resistance to paclitaxel in Ras-NIH 3T3/Mdr cell line, indicating that resistance to paclitaxel is associated with overexpression of the multidrug resistance gene. Interestingly, Ras-NIH 3T3/Mdr cells have higher basal Raf-1 activity compared to Ras-NIH 3T3 cells. Unexpectedly, however, the colocalization of Raf-1 and its negative regulator Spry2 was less observed in cytoplasm of Ras-NIH 3T3/Mdr cells due to translocation of Spry2 around the nucleus in the perinuclear zone, implying that Raf-1 may be released from negative feedback inhibition by interacting with Spry2. We also showed that shRNA-mediated knockdown of Raf-1 caused a moderate increase in cell susceptibility to paclitaxel. Thus, the results presented here suggest that a Raf-1-dependent pathway plays an important role in the development of acquired drug-resistance.

• Korean Journal of Microbiology
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• v.52 no.4
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• pp.428-436
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• 2016

The emergence of antibiotic-resistant bacteria has been increased and become a public health concern worldwide. Many bacterial infections can be sequentially treated with different types of antibiotics. Thus, this study was designed to evaluate the changes in survival, antibiotic susceptibility, mutant frequency, ${\beta}$-lactamase activity, biofilm formation, and gene expression in Klebsiella pneumoniae after exposure to sequential antibiotic treatments of ciprofloxacin and meropenem. Treatments include control (CON; no addition), 1/2 MIC ciprofloxacin addition (1/2CIP), 2 MIC ciprofloxacin addition (2CIP), initial 1/2 MIC ciprofloxacin addition followed by 1/2 MIC meropenem (8 h-incubation) and 2 MIC ciprofloxacin (16 h-incubation) (1/2CIP-1/2MER-2CIP), initial 1/2 MIC ciprofloxacin addition followed by 1/2 MIC meropenem (8 h-incubation) and 2 MIC meropenem (16 h-incubation) (1/2CIP-1/2MER-2MER), and initial 1/2 MIC ciprofloxacin addition followed by 2 MIC ciprofloxacin(8 h-incubation) and 2 MIC meropenem(16 h-incubation) (1/2CIP-2CIP-2MER). No growth of K. pneumoniae was observed for the 2CIP throughout the incubation period. The numbers of planktonic cells varied with the treatments (7~10 log CFU/ml), while those of biofilm cells were not significantly different among treatments after 24-h incubation, showing approximately 7 log CFU/ml. Among the sequential treatments, the least mutant frequency was observed at the 1/2CIP-1/2MER-2CIP (14%). Compared to the CON, 1/2CIP-2CIP-2MER decreased the sensitivity of K. pneumoniae to piperacillin, cefotaxime, and nalidixic acid. The highest ${\beta}$-lactamase activity was 22 nmol/min/ml for 1/2CIP-1/2MER-2CIP, while the least ${\beta}$-lactamase activity was 6 nmol/min/ml for 1/2CIP-2CIP-2MER. The relative expression levels of multidrug efflux pump-related genes (acrA, acrB, and ramA) were increased more than 2-fold in K. pneumoniae exposed to 1/2CIP-1/2MER-2MER and 1/2CIP-2CIP-2MER. The results suggest that the sequential antibiotic treatments could change the antibiotic resistance profiles in K. pneumoniae.