BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) is an increasing public health problem and poses a serious threat to global TB control. Fluoroquinolone (FQ) and aminoglycoside (AG) are essential anti-TB drugs for MDR-TB treatment. REBA MTB-FQ(R) and REBA MTB-KM(R) (M&D, Wonju, Korea) were evaluated for rapid detection of FQ and kanamycin (KM) resistance in MDR-TB clinical isolates. METHODS: M. tuberculosis (n=67) were isolated and cultured from the sputum samples of MDR-TB patients for extracting DNA of the bacilli. Mutations in genes, gyrA and rrs, that have been known to be associated with resistance to FQ and KM were analyzed using both REBA MTB-FQ(R) and REBA MTB-KM(R), respectively. The isolates were also utilized for a conventional phenotypic drug susceptibility test (DST) as the gold standard of FQ and KM resistance. The molecular and phenotypic DST results were compared. RESULTS: Sensitivity and specificity of REBA MTB-FQ(R) were 77 and 100%, respectively. Positive predictive value and negative predictive value of the assay were 100 and 95%, respectively, for FQ resistance. Sensitivity, specificity, positive predictive value and negative predictive value of REBA MTB-KM(R) for detecting KM resistance were 66%, 94%, 70%, and 95%, respectively. CONCLUSION: REBA MTB-FQ(R) and REBA MTB-KM(R) evaluated in this study showed excellent specificities as 100 and 94%, respectively. However, sensitivities of the assays were low. It is essential to increase sensitivity of the rapid drug resistance assays for appropriate MDR-TB treatment, suggesting further investigation to detect new or other mutation sites of the associated genes in M. tuberculosis is required.
Chimera ; DNA ; Drug Resistance ; Drug Resistance, Microbial ; Fluoroquinolones ; Humans ; Kanamycin ; Kanamycin Resistance ; Mycobacterium ; Mycobacterium tuberculosis ; Public Health ; Sputum ; Tuberculosis ; Tuberculosis, Multidrug-Resistant

Ninety nine strains of Escherichia coli isolated from clinical urine specimens in Taegu area were tested for the antimicrobial susceptibility to 20 drugs and studies for molecular and genetic characterization of R-plasmid. All strains were susceptible to amikacin (Ak), moxalactam(Mx), norfloxacin(Nf), ciprofloxacin(Cf) and ofloxacin(Of). One-6.1% of the strains were resistant to tobramycin(To). nalidixic acid(Na), enoxacin(Ex), pefloxacin(Pf) and rifampin(Rf), 17.2-31.3% to gentamicin(Gm), cephalothin(Ct) and cephamandole(Cfm), and 59.6-84.4% to kanamycin (Km), streptomycin(Sm), a.mpicillin(Ap), chloramphenicol(Cm), tetracycline(Tc), sulfisomidine (Su) and trimethoprime(Tp). MIC90 of Ak, Mx, Ex, Nf, Cf, Of and Pf were below the aritimicrobial concentration tested. In multiple drug resistance patterns, resistance to 7 drugs (CmTcSmSuAp TpKm) were most frequently encountered. Except Na and Rf in 66.4 % of resistant strains, most or drug resistance were co-transferred to recipient E.coli RG488 or RG176, indicating that multiple drug resistance was R-plasmid mediated phenomenon. Plasmid profiles for molecular characterization of R-plasmids from B. coli strains were studied through the methods of alkaline SDS lysis and agarose gel electrophoresis. R-plasmids were 40.9-122.3 mega dalton in molecular size. Pst I restriction enzyme digestion patterns of R-plasmid DNAs were examined. R-plasmids with different molecular weights and phenotype markers showed different restriction patterns. pDE9l58 and pDE 9055, which have same molecular weight and phenotype marker except Cfm, showed identical restriction pattern.
Amikacin ; Daegu ; Digestion ; DNA ; Drug Resistance ; Drug Resistance, Microbial* ; Drug Resistance, Multiple ; Electrophoresis, Agar Gel ; Escherichia coli* ; Escherichia* ; Kanamycin ; Molecular Weight ; Phenotype ; Plasmids ; Sulfisomidine

OBJECTIVETo study MIC value of 7 boron derivatives namely [Boric acid (H(3)BO(3)), Anhydrous Borax (Na(2)B(4)O(7)), Sodium Borate (NaBO(2)), Diammonium Tetraborate (NH(4))(2)B(4)O(7), Sodium Perborate (NaBO(3)), Boron Trioxide (B(2)O(3)), Potassium Tetraborate (K(2)B(4)O(7))] on E. coli and P. aeruginosa and their effects on survival of bacteria in lake water and resistance against kanamycin antibiotic.

METHODSMIC values of Boron derivatives and antibiotic were studied by broth microdilution method. The effect of boron derivatives on survival of bacteria in lake water were also determined with plate count.

RESULTSSodium perborate was determined as the most effective substance among the studied substances. Effectiveness increased as temperature increased. E. coli was more affected from P. aeruginosa in 8 mg/mL sodium perborate concentration in lake water. Moreover, it was determined that MIC value of kanamycin antibiotic decreased 200 times by especially treating P. aeruginosa with sodium perborate in lake water. However, it can be stated that this change in resistance did not arise from microorganisms.

CONCLUSIONSodium perborate solution can be used supportedly in kanamycin antibiotic applications for P. aeruginosa. Future studies are necessary to explore the relation between sodium perborate and kanamycin which is effective on P. aeruginosa in lake water.

Borates ; pharmacology ; Escherichia coli ; drug effects ; Kanamycin Resistance ; Lakes ; microbiology ; Microbial Sensitivity Tests ; Pseudomonas aeruginosa ; drug effects

Neomycin-resistance gene is widely used as a selectable marker in eukaryotic expression vector. It codes neomycin phosphotransferase II (NPT II) which confers resistance to various aminoglycoside antibiotic such as G418 and kanamycine. In this work, by site-directed mutagenesis the neo gene mutant was obtained. The expression vector pmDNA using the neo gene mutant as selectable marker has been constructed. After inserting interest luciferase gene, the expression plasmid pmDNAluc + was stably transfected CHO-K1 cells. As a result, the expression positive ratio reaches to approximate 95% and the ratio of high expression colonies is apparently higher than the controls.
Amino Acid Sequence ; Base Sequence ; Drug Resistance ; genetics ; Genetic Markers ; Genetic Vectors ; Kanamycin Kinase ; genetics ; Molecular Sequence Data ; Mutation

Despite global efforts to control tuberculosis (TB), multidrug-resistant TB (MDR-TB) is still a serious problem worldwide. The diagnosis of MDR-TB is based on mycobacterial culture followed by drug susceptibility testing, with results available in weeks to months. This requirement calls for rapid direct tests, especially genotypic tests, in which specimens are amplified directly for the detection of MDR-TB. The treatment of MDR-TB is challenging because of the high toxicity of second-line drugs and the longer treatment duration required compared to drug-susceptible TB. The selection of drugs in MDR-TB is based on the treatment history, drug susceptibility results, and TB drug resistance patterns in each region. Recent World Health Organization guidelines recommend the use of at least four second-line drugs (i.e., a newer fluoroquinolone, an injectable agent, prothionamide, and cycloserine or para-aminosalicylic acid) in addition to pyrazinamide. Kanamycin is the initial choice of an injectable drug, and newer fluoroquinolones include levofloxacin and moxifloxacin. For extensively drug-resistant TB, group 5 drugs such as linezolid and clofazimine need to be included. New drugs such as delamanid and bedaquiline have recently been approved for treating MDR-TB and other agents with novel mechanisms of action that can be given for shorter durations (6-12 months) for MDR-TB are under investigation.
Clofazimine ; Cycloserine ; Diagnosis* ; Drug Resistance ; Fluoroquinolones ; Kanamycin ; Levofloxacin ; Prothionamide ; Pyrazinamide ; Tuberculosis ; Tuberculosis, Multidrug-Resistant* ; World Health Organization

One hundred and thirty trimethoprim-resistant R plasmids derived from of Escherichia coli isolated from clinical specimens and feces of healthy collegians were examined for incompatibility, EcoRI endonuclease restriction fragment pattern, and Southern hybridization with DHFR I, II, III, V, and VII probe. 1. Most trimethoprim-resistant R plasmids were resistant to ampicillin, tetracycline, chloramphenicol, gentamicin, and kanamycin, and showed multiple drug resistance and various antimicrobial resistance patterns. 2. Trimethoprim-resistant R plasmids ranged from 90 to 50 kilobase and 42.3% of R plasmids tested were classified to incompatibilty group Inc FI, Inc FII or Inc FIV, 3. Among 48 random selected R plasmids from various origin, 14 R plasmids (including 9 of 14 Inc FII plasmids and 3 of 14 Inc FI plasmids) hybridized with DHFR VII oligonucleotide probe but others did not respond to any of DHFR probes used. 4. Most R plasmids showed various EcoRI endonuclease fragments and different reaction sites by Southern hybridization. Six plasmids showed identical or nearly identical molecular weight, EcoRI endonuclease fragment patterns and different sites of Southern hybridization. But 2 Inc FII plasmids derived from urine and feces showed identical pattern. These findings, if confirmed by further studies, suggest that normal flora E. coli can act as reservoir of resistant genes and, consequently, as a factor in the dissemination of these genes among enteric pathogens and need to be examined further.
Ampicillin ; Chloramphenicol ; Deoxyribonuclease EcoRI ; Drug Resistance, Multiple ; Escherichia coli* ; Escherichia* ; Feces ; Gentamicins ; Immunodeficiency Virus, Feline ; Kanamycin ; Molecular Biology* ; Molecular Weight ; Plasmids ; R Factors ; Tetracycline ; Trimethoprim Resistance* ; Trimethoprim*

Antitubercular Agents/therapeutic use* ; China/epidemiology* ; Drug Resistance, Multiple, Bacterial ; Extensively Drug-Resistant Tuberculosis ; Humans ; Kanamycin Resistance ; Microbial Sensitivity Tests ; Mycobacterium tuberculosis ; Ofloxacin/pharmacology* ; Tuberculosis, Multidrug-Resistant/epidemiology*

BACKGROUND: Many genes encoding aminoglycoside modifying enzymes (AMEs) on transposon or plasmid were transferred from one strain to another strain and inserted into a staphylococcal chromosomal cassette mec (SCCmec). There are very diverse subtypes in SCCmec type to the insertion of resistant genes. Therefore, we researched the resistance rates of antibiotics and distribution of AME genes according to SCCmec type in MRSA strains. MATERIALS AND METHODS: We isolated 640 Staphylococcus aureus from non-tertiary hospitals in 2004, detected mecA, aac(6')-aph(2"), aph(3')-IIIa, and ant(4')-Ia using the multiplex PCR method, tested antibacterial susceptibility disk diffusion and minimal inhibitory concentration, and determined SCCmec type. RESULTS: Of 640 S. aureus isolates, MRSA rate was 39.7% and all MRSA isolates carried mecA gene. Among 214 MRSA selected, aminoglycoside-resistant rates were 98.1% in kanamycin and tobramycin, 68.7% in gentamicin, 30.8% in amikacin, and 2.8% in netilmicin. The detection rates for aac(6')-aph(2"), aph(3')-IIIa, and ant(4')-Ia were 77.1%, 13.1%, and 53.3%, respectively. Also, SCCmec type was 50.9% in SCCmec type II, 16.4% in type III, and 32.7% in type IV. The genes encoding AMEs were distributed aac(6')-aph(2") (49.5%) and aac(6')-aph(2")/ant(4')-Ia (36.7%) in SCCmec type II, aph(3')-IIIa/aac(6')-aph(2") (60%) and aac(6')-aph(2") (31.4%) in type III, and aac(6')-aph(2")/ant(4')-Ia (41.4%) and ant(4')-Ia (50%) in type IV. CONCLUSION: 39.7% of S. aureus isolated from non-tertiary hospitals was resistant to methicillin. More than 90% of MRSA isolates were detected aac(6')-aph(2") in SCCmec type II and III, and ant(4')-Ia in type IV. With these results, the genes encoding AMEs may be closed related to SCCmec type.
Adenosine ; Amikacin ; Amphotericin B ; Anti-Bacterial Agents ; Diffusion ; Gentamicins ; Kanamycin ; Kanamycin Kinase ; Methicillin ; Methicillin Resistance ; Methicillin-Resistant Staphylococcus aureus ; Multiplex Polymerase Chain Reaction ; Netilmicin ; Plasmids ; Sprains and Strains ; Staphylococcus aureus ; Tobramycin

BACKGROUND: Many genes encoding aminoglycoside modifying enzymes (AMEs) on transposon or plasmid were transferred from one strain to another strain and inserted into a staphylococcal chromosomal cassette mec (SCCmec). There are very diverse subtypes in SCCmec type to the insertion of resistant genes. Therefore, we researched the resistance rates of antibiotics and distribution of AME genes according to SCCmec type in MRSA strains. MATERIALS AND METHODS: We isolated 640 Staphylococcus aureus from non-tertiary hospitals in 2004, detected mecA, aac(6')-aph(2"), aph(3')-IIIa, and ant(4')-Ia using the multiplex PCR method, tested antibacterial susceptibility disk diffusion and minimal inhibitory concentration, and determined SCCmec type. RESULTS: Of 640 S. aureus isolates, MRSA rate was 39.7% and all MRSA isolates carried mecA gene. Among 214 MRSA selected, aminoglycoside-resistant rates were 98.1% in kanamycin and tobramycin, 68.7% in gentamicin, 30.8% in amikacin, and 2.8% in netilmicin. The detection rates for aac(6')-aph(2"), aph(3')-IIIa, and ant(4')-Ia were 77.1%, 13.1%, and 53.3%, respectively. Also, SCCmec type was 50.9% in SCCmec type II, 16.4% in type III, and 32.7% in type IV. The genes encoding AMEs were distributed aac(6')-aph(2") (49.5%) and aac(6')-aph(2")/ant(4')-Ia (36.7%) in SCCmec type II, aph(3')-IIIa/aac(6')-aph(2") (60%) and aac(6')-aph(2") (31.4%) in type III, and aac(6')-aph(2")/ant(4')-Ia (41.4%) and ant(4')-Ia (50%) in type IV. CONCLUSION: 39.7% of S. aureus isolated from non-tertiary hospitals was resistant to methicillin. More than 90% of MRSA isolates were detected aac(6')-aph(2") in SCCmec type II and III, and ant(4')-Ia in type IV. With these results, the genes encoding AMEs may be closed related to SCCmec type.
Adenosine ; Amikacin ; Amphotericin B ; Anti-Bacterial Agents ; Diffusion ; Gentamicins ; Kanamycin ; Kanamycin Kinase ; Methicillin ; Methicillin Resistance ; Methicillin-Resistant Staphylococcus aureus ; Multiplex Polymerase Chain Reaction ; Netilmicin ; Plasmids ; Sprains and Strains ; Staphylococcus aureus ; Tobramycin

The frequency of antibiotic resistance among Salmonella enterica serovar Typhimurium has increased due to the transfer of multiple resistance factors. We detected the 13 antibiotic resistance genes by multiplex-PCR and compared with the results of phage typing and antibiotic disk diffusion for 49 S. typhimurium isolated from food-poisoning outbreaks in Seoul from 1999 to 2002. Resistance genes for tetracycline, streptomycin, ampicillin, sulfonamide, amino-glycoside-modifying enzyme, chloramphenicol, kanamycin, and trimethoprim were detected in 67.3%, 57.1%, 26.5%, 8.1%, 8.1%, 5%, 2.0%, and 0% of isolates, respectively. Overall 28 isolates (57.1%) possessed two or more antibiotic resistance genes. Class 1 integron carrying multidrug resistace genes, ant(3")-IaB, blaPSE, qacE delta1/sul, and tet G were amplified especially in only DT104 isolates. Among the related resistance genes for same antibiotics, strA and strB for streptomycin resistance were simultaneously detected but tetA and tetB for tetracycline were sporadically detected. DT 104 isolates contained only aadA2 and tetG.
Ampicillin ; Anti-Bacterial Agents ; Bacteriophage Typing ; Chloramphenicol ; Diffusion ; Disease Outbreaks ; Drug Resistance, Microbial ; Humans ; Integrons ; Kanamycin ; R Factors ; Salmonella enterica* ; Salmonella* ; Seoul* ; Streptomycin ; Tetracycline ; Trimethoprim