• Biomolecules & Therapeutics
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• v.1 no.2
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• pp.177-182
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• 1993

From 84 clinical isolates of Staphylococcus species, ten strains showing inducible resistance to MLS antibiotics were selected by disk agar diffusion method. Colony hybridization was executed using two MLS inducible resistance genes, ermA and ermC, previously identified from S. aureus as probes. S. hemolyticus 401 and S. epidermidis 542 whose genes were not homologous to those probes were finally selected. It was determined that the resistance genes of S. hemolyticus 401 and S. epidermidis 542 were not homologous to ermA, ermC and ermAM by Southern hybridization. S. epidermidis 542 had a plasmid DNA. To know if the plasmid may have genes related to inducible resistance, it was attempted to transform B. subtilis BR151 and S. aureus RN4220 with the plasmid prepared from S. epidermidis 542. It was shown that the gene related to inducible resistance to MLS antibiotics did not exist in this plasmid. These results indicate that two clinical isolates of S. hemolyticus 401 and S. epidermidis 542 had novel genes which were not homologous to MLS resistance genes identified previously. It was assumed that these genes may exist in chromosomal DNA.

• Korean Journal of Microbiology
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• v.49 no.2
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• pp.105-111
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• 2013

The Erm family of adenine-$N^6$ methyltransferases (MTases) is responsible for the development of resistance to macrolide-lincosamide-streptogramin B antibiotics through the methylation of 23S ribosomal RNA. Recently, it has been proposed that well conserved amino acids in ErnC' located in concave cleft between N-terminal 'catalytic' domain and C-terminal 'RNA-binding' domain interacts with substrate RNA. We carried out the site-directed mutagenesis and studied the function of the ErmSF R237 mutant in vitro and in vivo. R237 amino acid residue is located in the concave cleft between two domains. Furthermore this residue is very highly conserved in almost all the Erm family. Purified mutant protein exhibited only 51% enzyme activity compared to wild-type. Escherichia coli with R237A mutant protein compared to the wild-type protein expressing E. coli did not show any difference in its MIC (minimal inhibitory concentration) suggesting that even with lowered enzyme activity, mutant protein was able to efficiently methylate 23S rRNA to confer the resistance on E. coli expressing this protein. But this observation strongly suggests that R237 of ErmSF probably interacts with substrate RNA affecting enzyme activity significantly.

• YAKHAK HOEJI
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• v.38 no.3
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• pp.293-299
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• 1994

Forty nine clinical isolates of S. aureus showing resistance to erythromycin(EM) were selected from 83 strains isolated recently in Korea. Fourteen strains of S. aureus showing inducible resistance to MLS antibiotics were selected by disc agar diffusion method. Colony hydridization was executed using two MLS inducible resistance genes, ermA and ermC, identified previously from S. aureus as probes. S. aureus 375 and S. aureus 507 whose genes were not homologous to those probes were finally selected. It was confirmed that the resistance genes of S. aureus 375 and S. aureus 507 had no homology with those probes in southern hybridization test using ermA, ermC and ermAM as probes. It was determined that S. aureus 375 had a plasmid whose size was about 35 kb. To know if the plasmid may have the genes related to inducible resistance to MLS antibiotics, it was attempted to transform Bacillus subtillis BR151 and S. aureus RN4220 with the plasmid isolated from S. aureus 375. It was shown that the gene related to inducible resistance to MLS antibiotics did not exist in this plasmid. These results indicate that two clinical isolates of S. aureus showing inducible resistance to MLS antibiotics have novel genes that have no homology with MLS resistance genes identified so far. It is assumed that these genes may exist in chromosomal DNA.

• Korean Journal of Microbiology
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• v.28 no.1
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• pp.27-34
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• 1990

Two macrolide-lincosamide-streptogramin B (MLS) antibiotic resistance genes, one expressed inducibly and the other expressed constitutively were recognized from a single Staphylococcus aureus DH1 strain. The inducible MLS resistance gene was isolated and cloned from the R-plasmid pDE1(7.4kb) and the constitutive gene was from chromosomal DNA. Base sequence of the inducible MLS resistance gene (1.2kb) was determined and found as same that of pE194. The restriction map of the cloned constitutive MLS resistance gene was compared with that of the inducible gene. Two genes have same restriction map except leader region. In the constitutive gene there is no leader region which is doing major role in inducible expression.

• Food Science of Animal Resources
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• v.37 no.5
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• pp.670-681
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• 2017

The aim of this study was to isolate enterococci in Sucuk, a traditional Turkish dry-fermented sausage and to analyze isolates for their biodiversity, antibiotic resistance patterns and the presence of some antibiotic resistance genes. A total of 60 enterococci strains were isolated from 20 sucuk samples manufactured without using a starter culture and they were identified as E. faecium (73.3%), E. faecalis (11.7%), E. hirae (8.3%), E. durans (3.3%), E. mundtii (1.7%) and E. thailandicus (1.7%). Most of the strains were found resistant to rifampin (51.67%) followed by ciprofloxacin (38.33%), nitrofurantoin (33.33%) and erythromycin (21.67%). All strains were found susceptible to ampicillin. Only E. faecium FYE4 and FYE60 strains displayed susceptibility to all antibiotics. Other strains showed different resistance patterns to antibiotics. E. faecalis was found more resistant to antibiotics than other species. Most of the strains (61.7%) displayed resistance from between two and eight antibiotics. The ermB, ermC, gyrA, tetM, tetL and vanA genes were detected in some strains. A lack of correlation between genotypic and phenotypic analysis for some strains was detected. The results of this study indicated that Sucuk manufactured without using a starter culture is a reservoir of multiple antibiotic resistant enterococci. Consequently, Sucuk is a potential reservoir for the transmission of antibiotic resistance genes from animals to humans.

• Korean Journal of Veterinary Service
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• v.45 no.3
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• pp.171-180
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• 2022

The aim of this study was to investigate the prevalence of antimicrobial resistance and virulence factors in staphylococci isolated from canine otitis externa. A total 295 causative microorganisms were isolated. The most common isolated species were Staphylococcus (S) pseudintermedius (94 isolates) followed by Pseudomonas aeruginosa (60 isolates), S. schleiferi (25 isolates), Escherichia coli (23 isolates) and Proteus mirabilis (20 isolates). Staphylococci isolates were showed high resistance to penicillin (78.6%), erythromycin (55.9%), tetracycline (52.4%), clindamycin (51.7%) and ciprofloxacin (42.8%). Of the 145 staphylococci isolates, 49 (33.8%) methicillin-resistant staphylococci (MRS) were observed, distributed among S. pseudintermedius (n=34), S. schleiferi (n=6), S. epidermis (n=4), S. hominis (n=2), S. aureus, S. caprae and S. saprophyticus (n=1, respectively). Forty-three (87.8%) of 49 MRS and 10 (10.4%) of 96 methicillin-susceptibility staphylococci harbored mecA gene. About 80% of MRS were multidrug-resistant with resistance to at least one antibiotic in three or more antibiotic classes. Resistance genes blaZ (93/114, 81.5%), ermB (35/81, 43.2%), ermC (3/81, 3.7%), aacA-aphD (50/54, 92.5%), tetM (69/76, 90.7%) and tetK (6/76, 7.8%) were detected among resistant isolates. Virulence factors genes lukF and lukS were found in 100%(145/145) and 43.4%(63/145), respectively. Genes encoding ermA, eta, etb and tsst were not detected. To the best of our knowledge, this is the first study which investigated for the presence of genes encoding antimicrobial resistance and staphylococcal toxins in staphylococci isolated from canine otitis externa. A continuous monitoring and surveillance program to prevent antimicrobial resistance in companion animals is demanded.

• Korean Journal of Microbiology
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• v.46 no.2
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• pp.148-151
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• 2010

The aim of this study was to investigate the erythromycin resistance patterns of Enterococci sp. present in cow milk. A total 110 erythromycin resistant Enterococci were isolated from milk samples; E. faecalis (n=101), E. avium (n=7), and E. durans (n=2). The minimum inhibitory concentration of erythromycin against 110 Enterococci were determined. The 66.3% of Entercocci (n=73) showed high level resistance (${\geq}64$ mg/ml). Among 110 isolates, 86.3% (n=95) showed $cMLS_B$ phenotype and 13.6% (n=15) showed $iMLS_B$ The aim of this study was to investigate the erythromycin resistance patterns of Enterococci sp. present in cow milk. A total 110 erythromycin resistant Enterococci were isolated from milk samples; E. faecalis (n=101), E. avium (n=7), and E. durans (n=2). The minimum inhibitory concentration of erythromycin against 110 Enterococci were determined. The 66.3% of Entercocci (n=73) showed high level resistance (${\geq}64$ mg/ml). Among 110 isolates, 86.3% (n=95) showed $cMLS_B$ phenotype and 13.6% (n=15) showed $iMLS_B$ phenotype. All of isolates have erm(B) determinant, 75.45% (n=83) have mef(A) an efflux system determinant. The majority of Enetrcococci isolated from raw milk samples in northern area of Gyeonggi-Do showed high level of resistance to erythromycin.

• KSBB Journal
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• v.20 no.3
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• pp.220-227
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• 2005

Doxorubicin is an anthracycline-family polyketide compound with a very potent anti-cancer activity, typically produced by Streptomyces peucetius. To understand the potential target biosynthetic genes critical for the doxorubicin everproduction, a doxorubicin-specific DNA microarray chip was fabricated and applied to reveal the growth-phase-dependent expression profiles of biosynthetic genes from two doxorubicin-overproducing strains along with the wild-type strain. Two doxorubicin-overproducing 5. peucetius strains were generated via over-expression of a dnrl (a doxorubicin-specific positive regulatory gene) and a doxA (a gene involved in the conversion from daunorubicin to doxorubicin) using a streptomycetes high expression vector containing a strong ermE promoter. Each doxorubicin-overproducing strain was quantitatively compared with the wild-type doxorubicin producer based on the growth-phase-dependent doxorubicin productivity as well as doxorubicin biosynthetic gene expression profiles. The doxorubicin-specific DNA microarray chip data revealed the early-and-steady expressions of the doxorubicin-specific regulatory gene (dnrl), the doxorubicin resistance genes (drrA, drrB, drrC), and the doxorubicin deoxysugar biosynthetic gene (dnmL) are critical for the doxorubicin overproduction in S. peucetius. These results provide that the relationship between the growth-phase-dependent doxorubicin productivity and the doxorubicin biosynthetic gene expression profiles should lead us a rational design of molecular genetic strain improvement strategy.

• Korean Journal of Microbiology
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• v.51 no.3
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• pp.256-262
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• 2015

In order to study the effect of the receptor protein (SeaR), which is isolated from Saccharopolyspora erythraea, we introduced the SeaR gene to Streptomyces virginiae as host strains. An effective transformation procedure for S. virginiae was established based on transconjugation by Escherichia coli ET12567/pUZ8002 with a ${\varphi}C31$-derived integration vector, pSET152, which contained int, oriT, attP, and $ermEp^{\ast}$ (erythromycin promotor). Therefore, the pEV615 was introduced into S. virginiae by conjugation and integrated at the attB locus in the chromosome of the recipients by the ${\varphi}C31$ integrase (int) function. Transformants of S. virginiae containing the SeaR gene were confirmed by PCR and transcriptional expression of the SeaR gene in the transformants was analyzed by RT-PCR, respectively. And, we examined the production time of virginiamycins in the culture media of both the transformants and the wild type. The production time of virginiamycins in the wild type and transformants was the same. When 100 ng/ml of synthetic $VB-C_6$ was added to the state of 6 or 8 hour cultivation of wild type and transformants, respectively, the virginiamycins production was induced, meaning that the virginiamycins production in the wild type was detected 2 h early than transformants. From these results, SeaR expression was also affected to virginiamycins production in transformants derived from S. virginiae. In this study, we showed that the SeaR protein worked as a repressor in transformants.

• Journal of Microbiology and Biotechnology
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• v.33 no.2
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• pp.211-218
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• 2023

A cryptic plasmid (pTH32) was characterized from Tetragenococcus halophilus 32, an isolate from jeotgal, Korean traditional fermented seafood. pTH32 is 3,198 bp in size with G+C content of 35.84%, and contains 4 open reading frames (ORFs). orf1 and orf2 are 456 bp and 273 bp in size, respectively, and their translation products showed 65.16% and 69.35% similarities with RepB family plasmid replication initiators, respectively, suggesting the rolling-circle replication (RCR) mode of pTH32. orf3 and orf4 encodes putative hypothetical protein of 186 and 76 amino acids, respectively. A novel Tetragenococcus-Escherichia coli shuttle vector, pMJ32E (7.3 kb, Em^r), was constructed by ligation of pTH32 with pBluescript II KS(+) and an erythromycin resistance gene (ErmC). pMJ32E successfully replicated in Enterococcus faecalis 29212 and T. halophilus 31 but not in other LAB species. A pepA gene, encoding aminopeptidase A (PepA) from T. halophilus CY54, was successfully expressed in T. halophilus 31 using pMJ32E. The transformant (TF) showed higher PepA activity (49.8 U/mg protein) than T. halophilus 31 cell (control). When T. halophilus 31 TF was subculturd in MRS broth without antibiotic at 48 h intervals, 53.8% of cells retained pMJ32E after 96 h, and only 2.4% of cells retained pMJ32E after 14 days, supporting the RCR mode of pTH32. pMJ32E could be useful for the genetic engineering of Tetragenococcus and Enterococcus species.