The in vitro activities of DA-7157, a novel oxazolidinone, against clinical isolates of Nocardia brasiliensis and Mycobacterium tuberculosis were determined. Equal MIC 50 s and MIC 90 s (0.25 and 0.5 g/ml, respectively) were found for susceptible and multidrug-resistant isolates of M. tuberculosis. The N. brasiliensis isolates showed an MIC 90 of 1 g/ml and an MIC 50 of 1 g/ml. The DA-7157 prodrug, DA-7218, exhibited similar MICs for M. tuberculosis but fivefold-higher MICs for N. brasiliensis.Oxazolidinones are a new, unique class of synthetic antibacterial agents effective against many gram-positive bacteria, including aerobic pathogenic actinomycetes of the genera Mycobacterium, Nocardia, and Actinomadura (3,7,8,9). Linezolid, the first oxazolidinone on the market, has been proven to be active in animal models (2), as well as in clinical trials with patients infected with Nocardia spp. (5) or Mycobacterium tuberculosis (1). Oxazolidinones selectively inhibit bacterial protein synthesis by binding to bacterial 23S rRNA of the 50S subunit and preventing the formation of a functional 70S initiation complex (6). The antibacterial activity of oxazolidinones depends on the affinity for the site of action on the ribosome; therefore, by modifying their chemical structure, new compounds with improved antimicrobial activity can be obtained.Recently, a new oxazolidinone, DA-7157, was shown to be active in vitro against several gram-positive species, with the observation of a level of activity superior to that of linezolid (K. Lee, J. H. Yum, D. Yong, Y. Chong, S. H. Choi, and J. K. Rhee, Abstr. 45th Intersci. Conf. Antimicrob. Agents Chemother., abstr. F-1419Chemother., abstr. F- , 2005. The DA-7157 compound is produced by the metabolism of a highly hydrophilic prodrug, DA-7218 (Fig. 1) . The stock solution of DA-7218 was prepared in distilled water. In order to compare our results, we also performed susceptibility assays with linezolid, a drug previously tested in vitro against M. tuberculosis with good results. The latter was obtained from Pharmacia and Upjohn (Kalamazoo, Mich.), and the stock was prepared in 7H9GC broth. All working solutions were diluted in 7H9GC broth to a 2ϫ concentration prior to their addition to the microplates. Dimethyl sulfoxide controls were also run to determine its possible toxicity on the microorganisms.A total of 95 M. tuberculosis isolates were tested, including 8 isolates resistant to isoniazid, 1 isolate resistant to rifampin, and 25 clinical isolates resistant to both isoniazid and rifampin. The susceptibility assays were performed by the proportion method. For controls, we ran M. tuberculosis H37Rv and H37Ra. In order to determine the susceptibilities to linezolid, DA-7157, and DA-7218, the broth microdilution method with Alamar Blue was utilized (8). In brief, mycobacterial suspensions were prepared in 0.04% (vol/vol) Tween 80-0.2% bovine serum albumin so their turbidities equaled a McFarland turbidity standard of 1. Suspensions were further diluted 1:25 in 7H9GC broth. The rest of...
Although tuberculosis is still a public health problem in Mexico, there is little information about the genetic characteristics of the isolates. In the present study, we analyzed by spoligotyping 180 Mycobacterium tuberculosis clinical isolates from the urban area of Monterrey, Mexico, including drug-susceptible and drug-resistant isolates. The spoligotype patterns were compared with those in the international SITVIT2 spoligotyping database. Four isolates presented spoligotype patterns not found in the database (orphan types); the rest were distributed among 44 spoligo international types (SITs). SIT53 (clade T1) and SIT119 (clade X1) were predominant and included 43 (23.8%) and 28 (15.5%) of the isolates, respectively. In order to determine if there was a dominant spoligotype in the group of multidrug-resistant isolates, 37 of them were analyzed by IS6110-based restriction fragment length polymorphism assays, and scarce clustering of strains with more than five bands was observed. Fourteen isolates of this multidrug-resistant group presented four bands or less and were distributed in four SITs: SIT53 (n ؍ 8), SIT92 (n ؍ 3), SIT70 (n ؍ 2), and SIT3038 (n ؍ 1). When the molecular detection of mutations in the katG and rpoB genes were analyzed in these isolates with low copy numbers of IS6110, only two isolates shared the same IS6110, spoligotyping, and mutations patterns. When the distribution of the spoligotypes was analyzed by age cohort, SIT119 was predominantly found in patients 0 to 20 years old, especially in males, accounting for up to 40% of the isolates. In contrast, SIT53 was more prevalent in older females. This analysis demonstrates the variability of M. tuberculosis isolates in Monterrey and the partial dominance of SIT53 and SIT119 in that area of Mexico.
DA-7867 and DA-7157 are oxazolidinones active against pathogenic aerobic actinomycetes including Nocardia spp. and Mycobacterium tuberculosis. However, the activity of these drugs against nontuberculous mycobacterium (NTM) species is not known. In this work, we compared the susceptibilities of 122 clinical isolates and 29 reference species of both rapidly growing and slowly growing mycobacteria to linezolid, DA-7867, and DA-7157 by the broth microdilution method. The MICs for 50 and 90% of the strains tested (MIC 50 s and MIC 90 s, respectively) of DA-7867 and DA-7157 were lower than those of linezolid. In all of the cases, a MIC 90 of <8 g/ml was observed for all of the species tested in both groups of NTM. For M. kansasii and M. marinum isolates, the MIC 90 s of both DA-7867 and DA-7157 were less than 0.5 g/ml. These results demonstrate the potential of these compounds to treat NTM infections.Nontuberculous mycobacteria (NTM) are associated with a number of clinical diseases, including lymphadenitis, skin abscesses, disseminated infections, and pulmonary infections, in immunocompetent and immunocompromised patients (4). The most commonly isolated species include the Mycobacterium avium complex, M. kansasii, M. marinum, M. chelonae, M. abscessus, and the M. fortuitum group. Because of the increasing numbers of immunocompromised patients, e.g., organ transplant recipients, patients receiving corticosteroid therapy, and patients with human immunodeficiency virus, together with the availability of better technology, the number of species identified is expanding (13). Therapy of these infections is complicated because they are resistant to conventional antitubercular antimicrobials and only a few agents are active.Oxazolidinones are recently developed antimicrobials that inhibit protein synthesis at a site not targeted by other antimicrobials (11). Resistance by mutations or the presence of a resistance determinant has been rarely reported. The first of these compounds to be approved by the Food and Drug Administration, linezolid, is active in vitro against many grampositive actinomycetes, including Nocardia, Actinomadura, and M. tuberculosis (14,15,16), and has been given with successful results to patients infected with Nocardia spp. or M. tuberculosis (6, 7). In the present work, we undertook a comparative study of the in vitro susceptibilities of rapidly growing mycobacteria (RGM) and slowly growing NTM to the two new oxazolidinones DA-7867 and DA-7157 versus the already approved drug linezolid. MATERIALS AND METHODS Microorganisms.A total of 151 NTM isolates, corresponding to 15 species, were analyzed. This number included 122 clinical isolates and 29 reference isolates that were obtained from the culture collection of the University of Texas Health Center at Tyler Mycobacteria/Nocardia Laboratory. Isolates were identified to the species level with a commercial DNA-RNA probe (M. avium complex, M. kansasii) or by PCR restriction analysis of a 441-bp sequence (Telenti fragment) of the hsp65 gene as previously de...
Several techniques have been used to quantify the cytotoxicity produced by Mycobacterium tuberculosis bacilli on cell monolayers; however, they are semi-quantitative or time consuming. Herein, a method based on crystal violet (CV) uptake by THP-1 cell monolayers is described. This colorimetric method quantifies the cytotoxic effect as a function of the number of remaining cells after the infection with M. tuberculosis. Since this micro-organism is not stained by the dye, it does not produce a background that affects absorbance readings. As determined by CV assay (CVA), M. tuberculosis strain H37Rv destroyed 10.5 % of THP-1 cell monolayers at 24 h and 50.52 % at 72 h, while M. tuberculosis strains lacking the complete phospholipase C locus produced a reduced cytotoxic effect. The damage estimated by microscopy corresponded to the effect quantified by CVA. The results show that the use of CVA is a rapid, sensitive and reliable quantitative assay to measure the cytotoxicity of different M. tuberculosis strains. INTRODUCTIONMycobacterium tuberculosis is a bacterial pathogen that produces a detrimental effect on mammalian cell cultures. This effect can be due to the induction of cell apoptosis or necrosis leading to cell death. The phenotype is reported as a cytotoxic or cytopathic effect (Castro-Garza et al., 2002;Danelishvili et al., 2003;Dobos et al., 2000;McDonough & Kress, 1995) and it can be roughly quantified by analysing the altered cell morphology (cell rounding and loss of monolayer integrity) (Daniel et al., 2004;Fischer et al., 1996), setting up a scale to estimate the percentage of rounded or detached cells (Read et al., 1974) or counting the amount of degraded cells by electron microscopy (McDonough & Kress, 1995). A more precise quantitative assay, such as measuring lactate dehydrogenase (LDH) release by using a colorimetric kit, has also been reported (Danelishvili et al., 2003;Dobos et al., 2000). However, most of the above experimental procedures are only semiquantitative or are time-consuming.There are other methods to quantify cytotoxicity; however, they are not practical for use with M. tuberculosis infection systems: exclusion or inclusion of vital dyes requires direct handling of samples, the release of radiolabelled substances increases the biosafety level, and the reduction of coloured compounds such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 4-[3-4(iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) and other tetrazolium salts by bacteria (Franzblau et al., 1998;Gomez-Flores et al., 1995) as well as macrophages (Ferrari et al., 1990) in in vitro infection models would produce a high background and the results obtained would not be accurate.Crystal violet (CV) is a triphenylmethane dye also known as gentian violet. The most commonly used application for this dye is as the primary stain in the Gram-staining procedure. Gillies et al. (1986) A unique characteristic of M. tuberculosis bacilli is their outer lipid bilayer, which is the t...
The in vitro activities of DA-7867, a novel oxazolidinone, and garenoxacin (BMS-284756) were compared to those of linezolid in 67 susceptible and drug-resistant clinical isolates of Mycobacterium tuberculosis. DA-7867 was the most active drug with an MIC 90 of 0.125 g/ml, compared to the MIC 90 s of 4 g/ml of garenoxacin and 2 g/ml of linezolid.Tuberculosis is still a very common infectious disease worldwide, particularly in developing countries, with about 8 million new cases and 3 million deaths every year (7). The infection can be treated properly if an early diagnosis is made and the appropriate therapy is applied. However, in places where drugs are not easily available or delivered, multidrug-resistant strains of Mycobacterium tuberculosis arise and, in some countries, are the predominant strains, with a frequency as high as 14% (5). To date, the therapeutic choices for this disease are scarce, and new drugs are urgently needed. In the present study, we report the susceptibility of M. tuberculosis clinical isolates to garenoxacin (BMS-284756), a newly developed quinolone, and DA-7867, a novel oxazolidinone.Garenoxacin was kindly donated by Bristol-Myers Squibb; DA-7867 was obtained from Dong-A Pharmaceutical Company, Ltd., Yongin, Korea. Stock solutions of garenoxacin and DA-7867 were prepared in 100% dimethyl sulfoxide and 7H9GC (4.7 g of Middlebrook 7H9 broth base [Difco, Detroit, Mich.], 20 ml of 10% [vol/vol] glycerol, 1 g of Bacto Casitone [Difco], 880 ml of distilled water, 100 ml of oleic acid-albumindextrose-catalase [Becton Dickinson, Maryland]) (6) broth, respectively. In order to compare our results, we also ran susceptibility assays for linezolid, a drug previously tested in vitro against M. tuberculosis with good results. The latter was obtained from Pharmacia and Upjohn (Kalamazoo, Mich.), and the stock was prepared in 7H9GC broth. All working solutions were diluted in 7H9GC broth to a 2ϫ concentration prior to their addition to the microplates.Sixty-seven clinical isolates of M. tuberculosis obtained from the Laboratorio Estatal de los Servicios de Salud de Nuevo León were subcultured on Lowenstein-Jensen medium. Twenty-one of the isolates were determined to be resistant to isoniazid and rifampin by the proportion method. In order to determine the susceptibility to garenoxacin, DA-7867, and linezolid, the broth microdilution method with Alamar Blue was utilized (6). In brief, mycobacterial suspensions were prepared in 0.04% (vol/vol) Tween 80-0.2% bovine serum albumin so that their turbidities matched that of the McFarland no. 1 turbidity standard. Suspensions were further diluted 1:25 in 7H9GC broth.Two hundred microliters of sterile deionized water was added to all outer-perimeter wells of sterile 96-well plates (Falcon 3072; Becton Dickinson, Lincoln Park, N.J.) to minimize evaporation of the medium in the test wells during incubation. One hundred microliters of 2ϫ drug solutions were added to the wells in rows B to G. Final drug concentration ranges were as follows: for linezolid and ga...
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