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: We examined the feasibility of a full-length gene analysis for the drug resistance-related genes inhA, katG, rpoB, pncA, rpsL, embB, eis, and gyrA using ion semiconductor next-generation sequencing (NGS) and compared the results with those obtained from conventional phenotypic drug susceptibility testing (DST) in multidrug-resistant Mycobacterium tuberculosis (MDR-TB) isolates. METHODS: We extracted genomic DNA from 30 pure MDR-TB isolates with antibiotic susceptibility profiles confirmed by phenotypic DST for isoniazid (INH), rifampin (RIF), ethambutol (EMB), pyrazinamide (PZA), amikacin (AMK), kanamycin (KM), streptomycin (SM), and fluoroquinolones (FQs) including ofloxacin, moxifloxacin, and levofloxacin. Enriched ion spheres were loaded onto Ion PI Chip v3, with 30 samples on a chip per sequencing run, and Ion Torrent sequencing was conducted using the Ion AmpliSeq TB panel (Life Technologies, USA). RESULTS: The genotypic DST results revealed good agreement with the phenotypic DST results for EMB (Kappa 0.8), PZA (0.734), SM (0.769), and FQ (0.783). Agreements for INH, RIF, and AMK+KM were not estimated because all isolates were phenotypically resistant to INH and RIF, and all isolates were phenotypically and genotypically susceptible to AMK+KM. Moreover, 17 novel variants were identified: six (p.Gly169Ser, p.Ala256Thr, p.Ser383Pro, p.Gln439Arg, p.Tyr597Cys, p.Thr625Ala) in katG, one (p.Tyr113Phe) in inhA, five (p.Val170Phe, p.Thr400Ala, p.Met434Val, p.Glu812Gly, p.Phe971Leu) in rpoB, two (p.Tyr319Asp and p.His1002Arg) in embB, and three (p.Cys14Gly, p.Asp63Ala, p.Gly162Ser) in pncA. CONCLUSIONS: Ion semiconductor NGS could detect reported and novel amino acid changes in full coding regions of eight drug resistance-related genes. However, genotypic DST should be complemented and validated by phenotypic DSTs.
Amikacin ; Clinical Coding ; Complement System Proteins ; DNA ; Drug Resistance* ; Ethambutol ; Fluoroquinolones ; Isoniazid ; Kanamycin ; Levofloxacin ; Mycobacterium tuberculosis* ; Mycobacterium* ; Ofloxacin ; Pyrazinamide ; Rifampin ; Semiconductors* ; Streptomycin

Enterococcus spp. are normally present in the gastrointestinal tracts of animals and humans, but can cause opportunistic infections that can be transmitted to other animals or humans with integrated antibiotic resistance. To investigate if this is a potential risk in military working dogs (MWDs), we analyzed antibiotic resistance patterns and genetic relatedness of Enterococcus spp. isolated from fecal samples of MWDs of four different age groups. Isolation rates of Enterococcus spp., Enterococcus (E.) faecalis, and E. faecium, were 87.7% (57/65), 59.6% (34/57), and 56.1% (32/57), respectively, as determined by bacterial culture and multiplex PCR. The isolation rate of E. faecalis gradually decreased with age (puppy, 100%; adolescent, 91.7%; adult, 36.4%; and senior, 14.3%). Rates of resistance to the antibiotics ciprofloxacin, gentamicin, streptomycin, sulfamethoxazole/trimethoprim, imipenem, and kanamycin among Enterococcus spp. increased in adolescents and adults and decreased in senior dogs, with some isolates having three different antibiotic resistance patterns. There were indistinguishable pulsed-field gel electrophoresis patterns among the age groups. The results suggest that Enterococcus is horizontally transferred, regardless of age. As such, periodic surveillance studies should be undertaken to monitor changes in antibiotic resistance, which may necessitate modification of antibiotic regimens to manage antibiotic resistance transmission.
Adolescent ; Adult ; Animals ; Anti-Bacterial Agents ; Ciprofloxacin ; Dogs* ; Drug Resistance, Microbial* ; Electrophoresis, Gel, Pulsed-Field ; Enterococcus faecalis* ; Enterococcus faecium* ; Enterococcus* ; Gastrointestinal Tract ; Gentamicins ; Humans ; Imipenem ; Kanamycin ; Korea* ; Military Personnel* ; Multiplex Polymerase Chain Reaction ; Opportunistic Infections ; Streptomycin

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

Treatment of multidrug-resistant tuberculosis (MDR-TB) is challenging because of the high toxicity of second-line drugs and the longer treatment duration required compared with drug-susceptible TB. The efficacy of treatment for MDR-TB is poorer than that for drug-susceptible TB. The selection of drugs in MDR-TB is based on previous treatment history, drug susceptibility results, and TB drug resistance patterns in the each region. Recent World Health Organization guidelines recommend the use of least 4 second-line drugs (a newer fluoroquinolone, an injectable agent, prothionamide, and cycloserine or para-aminosalicylic acid) in addition to pyrazinamide. The kanamycin is the initial choice of injectable durgs, and newer fluoroquinolones include levofloxacin and moxifloxacin. For MDR-TB, especially cases that are extensively drug-resistant, group 5 drugs such as linezolid, clofazimine, and amoxicillin/clavulanate need to be included. New agents with novel mechanisms of action that can be given for shorter durations (9-12 months) for MDR-TB are under investigation.
Clofazimine ; Cycloserine ; Drug Resistance ; Extensively Drug-Resistant Tuberculosis ; Fluoroquinolones ; Kanamycin ; Levofloxacin ; Linezolid ; Prothionamide ; Pyrazinamide ; Tuberculosis ; Tuberculosis, Multidrug-Resistant* ; World Health Organization

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

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

OBJECTIVETo optimize the process of tomato genetic transformation, screening and seed selection using multiepitope antigenic gene (MAG) and truncated major surface antigen 1 (tSAG1) of Toxoplasma gondii as the target insert genes.

METHODSTomato high-frequency regeneration system was optimized with different choices of media and explants. The genetic transformation procedure was optimized using different tomato cultivars, explants, culture temperatures, media and acetosyringone (AS) supplements. Three concentrations of kanamycin were utilized for resistant selection of the transgenic candidate roots. The selected lines were trained, transplanted to soil and grown in a greenhouse till maturity. Sterile seeding using kanamycin-incorporated medium was conducted for screening transgenic tomato generations.

RESULTS AND CONCLUSIONCotyledons were better than hypocotyls as the regeneration explants. The regeneration rate of cotyledons reached 98% (59/60) using the optimized regeneration medium ZB3. The culture medium and temperature were the key factors for tomato transgenic shoot induction. The number of transgenic buds increased significantly at the appropriate temperature condition (23-/+1 degrees celsius;), and AS of 100 micromol/L in the medium before inoculation also significantly raised transformation rate. The budding rate of Zhongshu No.5 cotyledons was 35% (28/81) using the medium ZB2 under (23-/+1) degrees celsius;. Kanamycin at 80 mg/L was optimal for transgenic plantlet rooting selection with the rooting rate of 48% (31/65). 117 transgenic lines were obtained. Non-transgenic tomato plant growth, especially the root and elongation, was inhibited obviously with kanamycin at 100 mg/L or above, and the roots became purple and lacked lateral roots. The transgenic tomato seeds could be selected effectively with kanamycin at 150 mg/L.

Animals ; Antigens, Protozoan ; genetics ; Drug Resistance ; Kanamycin ; pharmacology ; Lycopersicon esculentum ; drug effects ; genetics ; Plants, Genetically Modified ; drug effects ; genetics ; Protozoan Proteins ; genetics ; Seeds ; drug effects ; genetics ; Toxoplasma ; genetics ; Transformation, Genetic

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