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In the present work, we evaluated the effect of gatifloxacin on the evolution of experimental murine infection with Nocardia brasiliensis using linezolid as a control. Gatifloxacin was injected subcutaneously at 100 mg/kg body weight every 8 h for 4 weeks. This compound was equally as efficient as linezolid in reducing the production of lesions.Mycetoma is a multietiological subcutaneous infection caused by true fungi and aerobic actinomycetes observed in many tropical and subtropical countries (8). In Mexico, 98% of the total cases are produced by actinomycetes and about 86% are produced by Nocardia brasiliensis (3). The highest cure rates (70%) have been obtained with the use of sulfamethoxazole-trimethoprim. In our dermatology clinic we have added amikacin to the sulfamethoxazole-trimethoprim combination to treat severe cases of mycetoma or those cases involving subjacent organs, obtaining a cure rate of about 95% in a series of 52 patients (7). However, in some cases, the use of these antimicrobials carries the risk of side effects, or we can have the development of bacterial resistance, making necessary the search for new therapeutic alternatives.Quinolones are a group of antimicrobials with no or poor in vitro activity against Nocardia brasiliensis (2). However, the most recent compounds of this class, such as moxifloxacin, gatifloxacin, and garenoxacin, have been observed to be highly active in vitro against this microorganism (6), exhibiting MICs similar to those presented by linezolid and amikacin, which are active in human infections. Therefore, it is important to confirm the activity of these quinolones in animal infections before using them in human subjects. In the present work, we used a murine animal model to study the activity of one of these quinolones, gatifloxacin, and compared its effectiveness with that of linezolid.For the animal assays, we utilized Nocardia brasiliensis HUJEG-1, which has been utilized in previous studies (2). The MICs of this strain, determined by the broth microdilution method, are 0.25 g/ml for gatifloxacin and 0.12 g/ml for linezolid.For the determination of the plasma levels of gatifloxacin and linezolid, several doses of these compounds were used. Linezolid was used at 10 mg/kg body weight, 25 mg/kg, and 50 mg/kg, and gatifloxacin at 50 mg/kg, 75 mg/kg, and 100 mg/kg. Eight-to 12-week-old female BALB/c mice were injected subcutaneously with the antimicrobials. For each dose tested, 27 mice were utilized; 24 were injected with the selected dose, and 3 mice were not injected to represent time zero. Next, 500-l blood samples were taken from the infraorbital sinus of each mouse, which previously had undergone general anesthesia with ethylic ether. The samples were taken from groups of three mice each at the following time intervals: 0 min, 20 min, 40 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 10 h. After sample collection, the plastic tubes containing the blood were centrifuged and the plasma was separated and frozen at Ϫ70°C. Plasma concentrations were determined by using a prev...

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BackgroundSubculturing has been extensively used to attenuate human pathogens. In this work we studied the effect of continuous subculturing of Nocardia brasiliensis HUJEG-1 on virulence in a murine model.MethodsNocardia brasiliensis HUJEG-1 was subcultured up to 130 times on brain heart infusion over four years. BALB/c mice were inoculated in the right foot pad with the bacteria subcultured 0, 40, 80, 100 and 130 times (T0, T40, T80 T100 and T130). The induction of resistance was tested by using T130 to inoculate a group of mice followed by challenge with T0 12 weeks later. Biopsies were taken from the newly infected foot-pad and immunostained with antibodies against CD4, CD8 and CD14 in order to analyze the in situ immunological changes.ResultsWhen using T40, T80 T100 and T130 as inoculums we observed lesions in 10, 5, 0 and 0 percent of the animals, respectively, at the end of 12 weeks. In contrast, their controls produced mycetoma in 80, 80, 70 and 60% of the inoculated animals. When studying the protection of T130, we observed a partial resistance to the infection. Immunostaining revealed an intense CD4+ lymphocytic and macrophage infiltrate in healing lesions.ConclusionsAfter 130 in vitro passages of N. brasiliensis HUJEG-1 a severe decrease in its virulence was observed. Immunization of BALB/c mice, with these attenuated cells, produced a state of partial resistance to infection with the non-subcultured isolate.

BackgroundMycetoma is a neglected, chronic, and deforming infectious disease caused by fungi and actinomycetes. In Mexico, N. brasiliensis is the predominant etiologic agent. Therapeutic alternatives are necessary because the current drug regimens have several disadvantages. Benzothiazinones (BTZ) are a new class of candidate drugs that inhibit decaprenyl-phosphoribose-epimerase (DprE1), an essential enzyme involved in the cell wall biosynthesis of Corynebacterineae.Methodology/Principal findingsIn this study, the in vitro activity of the next generation BTZ, PBTZ169, was tested against thirty Nocardia brasiliensis isolates. The MIC50 and MIC90 values for PBTZ169 were 0.0075 and 0.03 μg/mL, respectively. Because Nocardia is a potential intracellular bacterium, a THP-1 macrophage monolayer was infected with N. brasiliensis HUJEG-1 and then treated with PBTZ169, resulting in a decrease in the number of colony-forming units (CFUs) at a concentration of 0.25X the in vitro value. The in vivo activity was evaluated after infecting female BALB/c mice in the right hind food-pad. After 6 weeks, treatment was initiated with PBTZ169 and its activity was compared with the first generation compound, BTZ043. Both BTZ compounds were administered at 100 mg/kg twice daily by gavage, and sulfamethoxazole/trimethoprim (SXT), at 100 mg/kg sulfamethoxazole, was used as a positive control. After 22 weeks of therapy, only PBTZ169 and SXT displayed statistically significant activity.ConclusionThese results indicate that DprE1 inhibitors may be useful for treating infections of Nocardia and may therefore be active against other actinomycetoma agents. We must test combinations of these compounds with other antimicrobial agents, such as linezolid, tedizolid or SXT, that have good to excellent in vivo activity, as well as new DprE1 inhibitors that can achieve higher plasma levels.