Growth Of Mycobacterium Avium Complex Research Articles

Omadacycline drug susceptibility testing for non-tuberculous mycobacteria using oxyrase to overcome challenges with drug degradation

BackgroundDrug susceptibility testing (DST) protocol of omadacycline against non-tuberculous mycobacteria has not yet been established. We developed a method to accurately determine MIC omadacycline MIC against Mycobacterium abscessus (Mab), Mycobacterium avium-complex (MAC), and Mycobacterium kansasii (Mkn). MethodsFirst, we identified the oxyrase concentration not affecting Mab, MAC, and Mkn growth followed by omadacycline MIC experiments with and without oxyrase using reference and clinical strains. ResultsOxyrase 0.5 % (v/v) stabilized omadacycline in the culture medium. The median omadacycline MIC was 1 mg/L for Mab and 8 mg/L for Mkn. For MAC, the median omadacycline MIC was 2 mg/L for M. avium, 256 mg/L for M. intracellulare, and 4 mg/L for M. chimaera (p < 0.0001). Wilcoxon matched-pairs signed rank test revealed statistically lower MICs with oxyrase for all MAC subspecies (p < 0.0001), all Mab subspecies (p < 0.0001), and Mkn (p = 0.0002). The decrease in MICs with oxyrase was 17/18 of Mab, 14/19 of Mkn, 8/8 of M. avium, 4/5 M. chimera, but only 11/18 of M. intracellulare (p < 0.013). ConclusionUse of 0.5 % oxyrase could be a potential solution to reliable and reproducible omadacycline MIC of Mab. However, oxyrase demonstrated a variable effect in reducing MICs against MAC and Mkn.

In vitro and intracellular activities of novel thiopeptide derivatives against macrolide-susceptible and macrolide-resistant Mycobacterium avium complex.

Unsatisfactory outcomes following long-term multidrug treatment in patients with Mycobacterium avium complex (MAC) pulmonary disease have urged us to develop novel antibiotics. Thiopeptides, a class of peptide antibiotics derived from natural products, have potential as drug candidates that target bacterial ribosomes, but drug development has been hampered due to their extremely poor solubility. Here, we evaluated three new compounds (AJ-037, AJ-039, and AJ-206) derived from the thiopeptide micrococcin P2 with enhanced aqueous solubility; the derivatives were generated based on structure-activity relationship analysis. We conducted in vitro drug susceptibility and intracellular antimycobacterial activity testing of the three thiopeptide derivatives against various MAC strains, including macrolide-resistant MAC clinical isolates. These compounds showed low MICs against MAC, similar to that of clarithromycin (CLR). In particular, two compounds, AJ-037 and AJ-206, had intracellular antimycobacterial activities, along with synergistic effects with CLR, and inhibited the growth of MAC inside macrophages. Moreover, these two compounds showed in vitro and intracellular anti-MAC activities against macrolide-resistant MAC strains without showing cross-resistance with CLR. Taken together, the results of the current study suggest that AJ-037 and AJ-206 can be promising anti-MAC agents for the treatment of MAC infection, including for macrolide-resistant MAC strains. IMPORTANCE Novel antibiotics for the treatment of MAC infection are urgently needed because the treatment outcomes using the standard regimen for Mycobacterium avium complex (MAC) pulmonary disease remain unsatisfactory. Here, we evaluated three novel thiopeptide derivatives (AJ-037, AJ-039, and AJ-206) derived from the thiopeptide micrococcin P2, and they were confirmed to be effective against macrolide-susceptible and macrolide-resistant MAC. Our thiopeptide derivatives have enhanced aqueous solubility through structural modifications of poorly soluble thiopeptides. The thiopeptide derivatives showed minimal inhibitory concentrations against MAC that were comparable to clarithromycin (CLR). Notably, two compounds, AJ-037 and AJ-206, exhibited intracellular antimycobacterial activities and acted synergistically with CLR to hinder the growth of MAC within macrophages. Additionally, these compounds demonstrated in vitro and intracellular anti-MAC activities against macrolide-resistant MAC strains without showing any cross-resistance with CLR. We believe that AJ-037 and AJ-206 can be promising anti-MAC agents for the treatment of MAC infections, including macrolide-resistant MAC strains.

Coexistence of lung cancer in a patient with non-tuberculous mycobacteria

Nontuberculous mycobacteria (NTM) may mimic lung carcinoma as a solitary mass or miliary nodules, the coexistence of NTM and lung carcinoma is rare. The diagnosis may be delayed due to a high initial index of suspicion for mycobacterium tuberculosis complex rather than malignancy in an endemic country like India. We report a rare case of 47-year-old male who presented with exertional dyspnea, cough with expectoration and fever was initially treated with anti-tuberculous drugs following a mycobacterium avium complex growth in BAL cultures. Despite treatment, patient’s condition worsened, and computed tomography (CT) guided biopsy was done which showed adenocarcinoma. This case report highlights that a lung biopsy is a necessity for accurate diagnosis, and a positive NTM culture doesn't always exclude a concomitant lung carcinoma.

Lady Windermere Syndrome: Historical Perspective and Report of a Rare Case

Abstract Introduction/Objective Lady Windermere syndrome refers to Mycobacterium avium complex (MAC) pulmonary disease with an isolated lingular or middle lobe pattern of involvement. This is thought to be due to habitual suppression of the cough voluntarily. Typical demographics are that of elderly female patient. Although the etiology remains unclear high prevalence of pectus excavatum, scoliosis and mitral valve prolapse has also been noted in these patients. The purpose of this report this to describe a case of this syndrome. Methods/Case Report A 64-year-old female with a history of breast carcinoma status post bilateral mastectomies, and thyroid papillary microcarcinoma, status post thyroidectomy, presented with hemoptysis. Clinically and radiological findings were consistent with pneumonia which improved but did not completely resolve following antibiotic treatment. Computerized tomography (CT) revealed progressive infiltrates and bronchoscopy cultures grew Mycobacterium avium complex (MAC). Repeat antibiotic therapy resulted in only marginal improvement. Repeat CT showed diffuse bronchiectatic changes with scattered nodularity and tree-in-bud opacities, with majority of involvement in the right middle lobe and lingula. Multiple interventions were employed to ameliorate the patient’s condition including a positive expiratory pressure device, gastroesophageal reflux disease (GERD) precautions and combinations of 2 drug therapies without significant improvement in her condition. It was ultimately decided that she may benefit from a right middle lobectomy. Histologic sections from the lobectomy specimen showed necrotizing and non-necrotizing granulomatous inflammation and bronchiolectasis. Rare acid-fast microorganisms were identified on AFB special satin. Lady Windermere syndrome was suggested in the diagnostic comment after expert consultation. Patient’s symptoms improved after surgery and additional samples show no growth of MAC. Results (if a Case Study enter NA) NA Conclusion Although rendering a definite diagnosis is challenging in such cases, the constellation of clinical, radiologic, and histologic finding can be used to raise the possibility of Lady Windermere syndrome.

Synthesis and in vitro analysis of novel gallium tetrakis(4-methoxyphenyl)porphyrin and its long-acting nanoparticle as a potent antimycobacterial agent

Bacterial heme uptake pathways offer a novel target for antimicrobial drug discovery. Recently, gallium (Ga) porphyrin complexes were found to be effective against mycobacterial heme uptake pathways. The goal of the current study is to build on this foundation and develop a new Ga(III) porphyrin and its nanoparticles, formulated by a single emulsion-evaporation technique to inhibit the growth of Mycobacterium avium complex (MAC) with enhanced properties. Gallium 5,10,15,20-tetrakis(4-methoxyphenyl)porphyrin chloride (GaMeOTP) was synthesized from 5,10,15,20-tetrakis(4-methoxyphenyl)porphyrin and GaCl3. GaMeOTP showed enhanced antimicrobial activity against MAC104 and some clinical M. avium isolates. The synthesized Ga(III) porphyrin antimicrobial activity resulted in the overproduction of reactive oxygen species. Our study also demonstrated that F127 nanoparticles encapsulating GaMeOTP exhibited a smaller size than GaTP nanoparticles and a better duration of activity in MAC-infected macrophages compared to the free GaMeOTP. The nanoparticles were trafficked to endosomal compartments within MAC-infected macrophages, likely contributing to the antimicrobial activity of the drug.

Abdominal Malakoplakia Secondary to Disseminated Mycobacterium Avium Complex after Lung Transplant

Introduction Malakoplakia is a rare granulomatous inflammation in response to a bacterial infection. It is incited by incomplete macrophagic degradation of bacteria. The presence of these remnant phagosomes on pathology is considered pathognomonic for malakoplakia and known as Michaelis-Gutmann bodies (MGB). There are few case reports describing malakoplakia after lung transplant and none of which describe disseminated Mycobacterium avium Complex (MAC) induced malakoplakia. Case Report A 60-year-old female, with history of hypogammaglobulinemia and double lung transplant for bronchiectasis, presented with abdominal pain 6 months after transplant. She was diagnosed with acute diverticulitis and treated with IV antibiotics. Subsequently, she was readmitted with perforated diverticulum and a new complex phlegmon by CT imaging. In spite of antibiotics, the phlegmon enlarged. She underwent exploratory laparotomy that revealed a dense abdominal mass. The histopathologic exam of this mass showed MGB, by PAS and Von Kossa stains, diagnostic of malakoplakia. Tissue cultures grew VRE and E. coli, but intra-peritoneal fluid cultures grew MAC. However, tissue PCR and blood cultures were negative. MAC was deemed a contaminant and no treatment was rendered. The malakoplakia was thought to be secondary to VRE and E. Coli infection for which she was treated appropriately, and immunosuppression was lowered. She was readmitted with ongoing pain, and CT of abdomen showed further increase in size of mass. Repeat AFB blood cultures were positive for MAC. She was started on MAC therapy with clinical and radiographic improvement. Summary Since differential of malakoplakia could include malignancy, it is imperative to obtain tissue sampling. In this case, despite early sampling, diagnosis was delayed due to the uncertainty surrounding the isolated growth of MAC with above negative testing. This discordance could be explained by only a 70% sensitivity of PCR with negative AFB smear. Notably, false negatives may occur if organisms are present in low numbers. Patient had positive AFB blood cultures with negative respiratory cultures suggesting an extrapulmonary etiology of disseminated MAC. This in combination with continued growth of the mass despite culture directed antibiotics and improvement following initiation of MAC therapy supports the diagnosis of MAC induced malakoplakia.

Long-term follow-up of post-cardiac surgery Mycobacterium chimaera infections: A 5-center case series

ObjectivesIn multiple countries, endovascular/disseminated Mycobacterium chimaera infections have occurred in post-cardiac surgery patients in association with contaminated, widely-distributed cardiac bypass heater-cooler devices. To contribute to long-term characterization of this recently recognized infection, we describe the clinical course of 28 patients with 3–7 years of follow-up for survivors. MethodsIdentified at five hospitals in the United States 2010–2016, post-cardiac surgery patients in the cohort had growth of Mycobacterium avium complex (MAC)/M. chimaera from a sterile site or surgical wound, or a clinically compatible febrile illness with granulomatous inflammation on biopsy. Case follow-up was conducted in May 2019. ResultsOf 28 patients, infection appeared to be localized to the sternum in four patients. Among 18 with endovascular/disseminated infection who received combination anti-mycobacterial treatment and had sufficient follow-up, 39% appeared to have controlled infection (>12 months), 56% died, and one patient is alive with relapsed bacteremia. While the number of patients is small and interpretation is limited, four (67%) of six patients who had cardiac prosthesis removal/replacement appeared to have controlled infection compared to three (25%) of 12 with retained cardiac prosthesis (p >0.14; Fisher's exact test). ConclusionsGiven poor response to treatment and potential for delayed relapses, post-cardiac surgery M. chimaera infection warrants aggressive treatment and long-term monitoring.

Microbiological Persistence in Patients With Mycobacterium avium Complex Lung Disease: The Predictors and the Impact on Radiographic Progression.

Persistent growth of Mycobacterium avium complex (MAC) in the lungs indicates continuous infection in MAC lung disease (MAC-LD), but its clinical significance has not been investigated. We aimed to evaluate the predictors of persistent culture-positivity for MAC (MAC-PP) and its impact on radiographic deterioration in MAC-LD. Patients with MAC-LD at multiple medical centers from 2011 to 2016 were enrolled retrospectively. Microbiological persistence of MAC-LD was defined as MAC-PP exceeding 1 year, in contrast with the negative-conversion group. The outcome was radiographic progression, namely, increased number of involved lung areas or cavitary formation. Among 126 patients with MAC-LD, 75 (60%) were in the MAC-PP group; these patients had a higher proportion of radiographic progression (54%) than patients in the negative-conversion group (odds ratio [OR], 3.318; 95% confidence interval, 1.146-9.612). Independent predictors of MAC-PP were low body mass index (BMI), radiographic nodular-bronchiectatic (NB) pattern, and increase in the highest grade of acid-fast bacilli smear (AFS). Patients with BMI <21 kg/m2, NB pattern, and positive AFS had an OR of 17.7 for MAC-PP, and those with ≥2 of the factors had a 4.5-fold increased OR for MAC-PP relative to the comparison group. Other than MAC-PP, the highest AFS grade and no anti-MAC treatment were correlated with radiographic progression. Microbiological persistence in patients with MAC-LD is not uncommon and leads to an increased risk of radiographic progression. The predictors of MAC-PP are low BMI, NB pattern, and high AFS grade; if these risk factors are present, anti-MAC treatment should be seriously considered.

Overexpression of RORγt Enhances Pulmonary Inflammation after Infection with Mycobacterium Avium.

Mycobacterium avium complex (MAC) is the most common cause of nontuberculous mycobacterial disease in humans. The role of Th17 immunity in the pathogenesis of intracellular bacteria, such as MAC, is not currently understood. Transcription factor RAR-related orphan receptor gamma t (RORγt) is known as the master regulator for Th17 cell development. Here, we investigated the role of RORγt in host responses against MAC infection. Wild-type (WT) mice and RORγt-overexpressing mice were infected with MAC via intratracheal inoculation. Systemic MAC growth was not different between WT mice and RORγt-overexpressing mice. However, neutrophilic pulmonary inflammation following MAC infection was enhanced in RORγt-overexpressing mice compared with that in WT mice. The cytokine expression shifted toward a Th17 phenotype in the lungs of RORγt-overexpressing mice following MAC infection; the levels of IL-6 and IL-17 were significantly higher in the lung of these mice than in WT mice. In addition to the increase in IL-17 single-positive T cells, T cells producing both IL-17 and interferon-γ were elevated in the lung of RORγt-overexpressing mice following MAC infection. These findings suggest that RORγt overexpression-mediated Th17 bias contributes to local inflammation rather than systemic responses, by regulating neutrophil recruitment into the sites of infection during MAC infection.

Role of Th1/Th17 Balance Regulated by T-bet in a Mouse Model of Mycobacterium avium Complex Disease

Th1 immune responses are thought to be important in protection against intracellular pathogens. T-bet is a critical regulator for Th1 cell differentiation and Th1 cytokine production. The aim of this study was to determine the role of T-bet in host defense against Mycobacterium avium complex (MAC) infection. Wild-type mice, T-bet-deficient mice, and T-bet-overexpressing mice were infected with MAC via intratracheal inoculation. Macrophages and dendritic cells obtained from these mice were incubated with MAC. T-bet-deficient mice were highly susceptible to MAC, compared with wild-type mice and T-bet-overexpressing mice. Neutrophilic pulmonary inflammation was also enhanced in T-bet-deficient mice, but attenuated in T-bet-overexpressing mice, following MAC infection. Cytokine expression shifted toward Th1 in the lung and spleen of T-bet-overexpressing mice, but toward Th17 in T-bet-deficient mice. IFN-γ supplementation to T-bet-deficient mice reduced systemic MAC growth but did not reduce pulmonary inflammation. In contrast, neutralization of IL-17 in T-bet-deficient mice reduced pulmonary inflammation but did not affect mycobacterial growth in any organs tested. T-bet-deficient T cells tended to differentiate toward Th17 cells in vitro following exposure to MAC. Treatment with NO donor suppressed MAC-induced Th17 cell differentiation of T-bet-deficient T cells. This study identified that the fine balance between Th1 and Th17 responses is essential in defining the outcome of MAC disease. T-bet functions as a regulator for Th1/Th17 balance and is a critical determinant for host resistance to MAC infection by controlling cytokine and NO levels.

Perspective of pulmonary MAC infection treatment

Chemotherapy of pulmonary MAC (Mycobacterium avium complex) infection has been almost universally agreed with the multidrugs regimen that contains Clarithromycin (CAM), Rifampin (RFP), Ethambtol (EB), and aminoglycoside in case of advanced stage. One of the reason for the multidrugs regimen which is similar to tuberculous chemotherapy is to inhibit the emersion of resistant MAC strain. The other reasons, enhancement of anti microbial activity and response to polyclonal infection are unique to the MAC chemotherapy. In the current MAC chemotherapy, both CAM and aminoglycoside are main axes because only they can suppress the growth of MAC alone respectively. Efficacy of CAM was revealed through the randomized controlled trials of disseminated MAC infection with HIV and that consequences applied to pulmonary MAC infection treatment. CAM is not effective unless exceed 2 microg/ml blood concentration. RFP decreases CAM blood concentration remarkably, but the regimens contained RFP and CAM are superior clinically to the regimens without RFP. There seemed to be unknown pharmacological mechanisms with RFP. Although the advantage of aminoglycosides is easily achieved high blood concentration, if aminoglycoside dosage is exceed 15 mg/kg, the possibility of auditory disturbance increase. About the duration of MAC chemotherapy, many guidelines recommended that one year continuation after the negative conversion of sputum culture. It is not the evidence but an expert opinion. We often experience recurrences several months later after the all drugs are ceased. The interval days to positive conversion of sputum culture from the day of completion of chemotherapy are randomly distributed with weibull's equation. It suggests that exogenous re-infection may cause the recurrence of pulmonary MAC infection as pointed out by Wallace Jr. Considering these issues, we have the conception of pulmonary MAC infection chemotherapy as follows. 1. Full dose induction chemotherapy (two years). 2. Maintenance chemotherapy (one year). 3. Preventive chemotherapy (one year). These conceptions have to be the problem validated. However, these current chemotherapies are not effective adequately, we need the combination treatment with surgical resection when indicated as a localized focus for example. Generally chemotherapy could not cured the destructed bronchial lesion due to MAC infection as like as local bronchiectasis or cavities. Consequently, the chemotherapy just after the surgical resection of destructed focus is most appropriate period.

Nontuberculous mycobacteriosis; the present status and in the future. Mechanisms of host resistance to Mycobacterium avium complex and Mycobacterium tuberculosis infection

In order to know profiles of the antimicrobial action of some microbicidal effector molecules against Mycobacterium avium complex (MAC) and M. tuberculosis (MTB), profiles of collaborating effects among reactive nitrogen intermediates (RNI), free fatty acids (FFA), and reactive oxygen intermediates (ROI) were studied, RNI and FFA exerted synergistic effects in killing MAC and MTB, while the combination of ROI (H2O2-mediated halogenation system) with FFA conversely caused antagonism. The combination of RNI with ROI displayed additive effects in killing MTB, whereas the same combination showed antagonistic effects against MAC. Murine peritoneal macrophages (M phi s) produced and/or released these three antimicrobial effectors in the order ROI, FFA, and RNI in response to cellular stimulation induced by their contact with MAC or MTB organisms. These findings indicate that the collaborating effect of RNI with FFA is crucial for M phi-mediated intracellular killing of MAC and MTB. Secondly, we examined the modes of bacterial growth of MAC and MTB in murine peritoneal M phi s and A-549 type II alveolar epithelial cell line. The growth rate of these organisms was much larger in A-549 cells than in M phi s. In addition, the growth rate of high-virulence MAC (N-260 strain) was significantly larger than that of low-virulence MAC (N-444 strain), when they were residing in M phi or A-549 cells. Although a high virulence MTB (strain Kurono) also showed much more rapid growth in M phi s than did low-virulence MTB (strain H37Ra), such a phenomenon was not observed for their intracellular growth in A-549 cells. MTB exhibited strong cytotoxic effects against M phi s but not against A-549 cells when resided in these cells. On the other hand, MAC organisms did not cause cytotoxicity even in M phi s. Although MAC and MTB infections caused significant increase in RNI production by M phi s but not by A-549 cells, there was no significant relationship between the degree of M phi RNI production by a given mycobacterial organism and its virulence. These findings indicate some important roles of type II alveolar epithelial cells as a target cell for primary invasion and transient growth of mycobacterial organisms in the host lungs.

Mechanism of bacterial regrowth at the sites of infection in Mycobacterium avium complex-infected mice during treatment with chemotherapeutic agents

Although various antimicrobial drugs show appreciable bactericidal activity in the early phase (2 to 4 weeks after infection) of Mycobacterium avium complex (MAC) infections in mice, no drug, as far as we know, can continue to exert the growth inhibiting activity against the bacteria at the site of infection in the progressed stage. Here, we studied the mechanisms of the bacterial regrowth which usually starts around 2-4 weeks after infection. First, the changes in the level of TNF-alpha, IFN-gamma, IL-6 and IL-10 in the lungs and spleen during the course of MAC infections was examined. Tissue levels of TNF-alpha and IL-10 increased around weeks 2 to 4, then rapidly decreased thereafter, and returned to the normal levels by week 8, while levels of IFN-gamma and IL-6 remained very low throughout the observation period. In this experiment, the bacterial CFUs rapidly decreased during the first 2 weeks of the treatment with a rifamycin derivative, KRM-1648, and thereafter the regrowth of the organisms was observed even in mice treated continuously with KRM-1648, although the rate of bacterial growth in the treated mice was much lower than in untreated control mice. Second, effect of either anti-TGF-beta or anti-IL-10 antibody on intracellular growth of MAC in human monocytes cultured in vitro in the medium with or without addition of TNF-alpha or IFN-gamma were examined. Anti-TGF-beta and anti-IL-10 antibodies potently reduced the bacterial growth in monocytes. Effects of TNF-alpha and IFN-gamma in reducing the bacterial growth was potentiated by the addition of either anti-TGF-beta or anti-IL-10 antibody. Third, anti-IL-10 antibody augmented to some extent the chemotherapeutic efficacy of KRM-1648 against MAC infection, when the drug was given to mice at weeks 2 and 4 after infection. From these results, it is suggested that IL-10 derived from MAC-infected macrophages in response to stimulation with some bacterial components, such as lipoarabinomannan, might downregulate the antimicrobial function of host macrophages against MAC.

Rifampicin-loaded liposomes for the passive targeting to alveolar macrophages: in vitro and in vivo evaluation

Mycobacterium avium complex (MAC), the most frequent cause of opportunistic nontuberculous pulmonary infection, is made up of a group of intracellular pathogens that are able to survive and multiply inside lung alveolar macrophages. As nebulized liposomes are reported to be effective to target antibacterial agents to macrophages, in this work we have prepared and characterized re-dispersible freeze-dried rifampicin (RFP)-loaded vesicles by using soy lecithin (SL) and a commercial, enriched mixture of soy phosphatidylcholine (Phospholipon 90, P90) with or without cholesterol. The obtained results showed that RFP could be loaded stably in SL vesicles only when cholesterol was not present in the film preparation, whereas with P90 vesicles, the highest stability was obtained with formulations prepared with P90/cholesterol 7:1 or 4:1 molar ratios. RFP-liposome aerosols were generated using an efficient high-output continuous-flow nebulizer, driven by a compressor. After the experiments, nebulization efficiency (NE%) and nebulization efficiency of the encapsulated drug (NEED%) were evaluated. The results of our study indicated that nebulization properties and viscosity of formulations prepared with the low-transition-temperature phospholipids, SL and P90, are affected by vesicle composition. However, all formulations showed a good stability during nebulization and they were able to retain more than 65% of the incorporated drug. The effect of liposome encapsulation on lung levels of RFP following aerosol inhalation was determined in rats. The in vitro intracellular activity of RFP-loaded liposomes against MAC residing in macrophage-like J774 cells was also evaluated. Results indicated that liposomes are able to inhibit the growth of MAC in infected macrophages and to reach the lower airways in rats.

Recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) or tumour necrosis factor-alpha (TNF-alpha) activate human alveolar macrophages to inhibit growth of Mycobacterium avium complex.

We investigated the effects of certain macrophage-active cytokines on the phagocytosis and growth inhibition of Mycobacterium avium complex (MAC) by human alveolar macrophages (AM). We also evaluated the effects of pretreatment with each cytokine on the superoxide anion release (O2-) from AM. The cytokines that we used were recombinant GM-CSF, natural type TNF-alpha, recombinant interferon-gamma (IFN-gamma), and recombinant IL-2. We found that phagocytosis by the various cytokine-stimulated AM was similar to that of unstimulated AM. On the other hand, significant growth inhibition of MAC was observed in the macrophages treated with GM-CSF or TNF-alpha, while no growth inhibition of MAC was observed in the macrophages treated with IFN-gamma or IL-2. Pretreatment with all cytokines tested enhanced the O2- release from AM, but there was no correlation between the enhancement of O2- release and the growth inhibition of MAC. Thus, we concluded that GM-CSF or TNF-alpha could activate AM to inhibit growth of MAC, probably not through the enhanced production of reactive oxygen intermediates.

Cloning and Characterization of CYP51 from Mycobacterium avium

Mycobacterium avium complex (MAC) causes chronic lung disease in immunocompetent people and disseminated infection in patients with AIDS. MAC is intrinsically resistant to many conventional antimycobacterial agents, it develops drug resistance rapidly to macrolide antibiotics, and patients with MAC infection experience frequent relapses or the inability to completely eradicate the infection with current treatment. Treatment regimens are prolonged and complicated by drug toxicity or intolerances. We sought to identify biochemical pathways in MAC that can serve as targets for novel antimycobacterial treatment. The cytochrome P450 enzyme, CYP51, catalyzes an essential early step in sterol metabolism, removing a methyl group from lanosterol in animals and fungi, or from obtusifoliol in plants. Azoles inhibit CYP51 function, leading to an accumulation of methylated sterol precursors. This perturbation of normal sterol metabolism compromises cell membrane integrity, resulting in growth inhibition or cell death. We have cloned and characterized a CYP51 from MAC that functions as a lanosterol 14alpha-demethylase. We show the direct interactions of azoles with purified MAC-CYP51 by absorbance and electron paramagnetic resonance spectroscopy, and determine the minimum inhibitory concentrations (MICs) of econazole, ketoconazole, itraconazole, fluconazole, and voriconazole against MAC. Furthermore, we demonstrate that econazole has a MIC of 4 mug/ml and a minimum bacteriocidal concentration of 4 mug/ml, whereas ketoconazole has a MIC of 8 mug/ml and a minimum bacteriocidal concentration of 16 mug/ml. Itraconazole, voriconazole, and fluconazole did not inhibit MAC growth to any significant extent.

Successful HLA-identical bone marrow transplantation in a patient with interferon-γ receptor 2 deficiency and disseminated Mycobacterium avium complex infection

Interferon gamma receptor-2 (IFNγR2) deficiency is characterized by increased susceptibility to nontuberculous mycobacteria and intracellular pathogens. We report successful HLA-identical bone marrow transplantation (BMT) of a patient with this defect. A Qatari male born to consanguineous parents presented with a CMV positive urine culture, while blood and urine mycobacterial cultures were initially negative. Bacille-Calmette-Guerin (BCG) vaccine was not administered due to a history of IFN-γ R2 deficiency in his deceased older sister. The patient was found to have the same defect. Parents initially refused BMT. He received prophylactic azithromycin but developed disseminated Mycobacterium avium complex (MAC) infection requiring an aggressive regimen including amikacin, azithromycin, ethambutol, linezolid, rifampin and moxifloxacin. CMV infection was treated with ganciclovir, foscarnet, and CMV immune globulin. Splenectomy was performed at age 9 months, due to massive hypersplenism with subsequent growth of MAC. CMV DNA was positive until age 11 months, while mycobacterial blood cultures were positive until age 10 months. At age 13 months, he underwent bone marrow transplantation from his HLA-identical father. Pre-transplant conditioning included busulfan, cyclophosphamide, and antithymocyte globulin (ATG). Post-transplant graft vs. host disease prophylaxis included methotrexate and cyclosporine. RFLP analysis revealed 98% donor cells on day +32 post-transplant. Transplant course was complicated by Clostridium difficile infection, fevers, hypertension and thrombocytopenia, but he is stable 94 days post-transplantation. Conclusions Our case is the only living patient who has undergone successful BMT for a defect in the IFNγR2. We conclude that bone marrow transplantation for IFNγR2 deficiency is an effective means of correcting this immunodeficiency.

Efficacies of cyclodextrin-complexed and liposome-encapsulated clarithromycin against Mycobacterium avium complex infection in human macrophages

Cyclodextrins and liposomes have been used in recent years as drug delivery vehicles, improving the bioavailability and therapeutic efficacy of many poorly water-soluble drugs. In this study, we used two approaches to enhance the availability of the poorly water-soluble antibiotic, clarithromycin, by inclusion complex formation and by liposome-encapsulation. We examined the efficacies of these formulations against Mycobacterium avium complex (MAC) in human peripheral blood monocyte-derived macrophages. The water solubility of clarithromycin was enhanced by about 700-fold by complexation with cyclodextrin. The use of a rapid radiometric (BACTEC) method for the detection of MAC growth and susceptibility showed identical MICs against MAC for both the free and complexed drug. The anti-MAC efficacy of the cyclodextrin complex of clarithromycin in macrophages was slightly lower than the free drug, probably due to the high stability of the inclusion complex. At higher drug concentrations, Liposome-encapsulated clarithromycin was slightly more effective against intracellular MAC growth than the free drug.

Effector molecules in expression of the antimicrobial activity of macrophages against Mycobacterium avium complex: roles of reactive nitrogen intermediates, reactive oxygen intermediates, and free fatty acids.

We studied microbicidal activities of reactive nitrogen intermediates (RNI), free fatty acids (FFA), and reactive oxygen intermediates (ROI) against Mycobacterium avium complex (MAC) and the mode of macrophage (mphi) production of these effectors. (1) Intracellular growth of MAC in murine peritoneal mphis was accelerated by scavengers for ROI or RNI and inhibitors of nitric oxide synthase or phospholipase A2, indicating roles of ROI, RNI, and FFA in mphi anti-MAC functions. (2) Acidified NaNO2-derived RNI, FFA (linolenic and arachidonic acids), and the H2O2-mediated halogenation system exhibited a significant anti-MAC bactericidal activity. The combination of RNI with FFA showed a synergistic effect. However, the H2O2-halogenation system in combination with either RNI or FFA showed an antagonism. When Listeria monocytogenes (Lm) was used as a target organism, the combinations of RNI + FFA and RNI + H2O2-halogenation gave a synergistic effect, whereas FFA + H2O2-halogenation showed an antagonism in exerting bactericidal activity. In addition, when ROI generated by the xanthine oxidase-acetaldehyde system was combined with RNI, anti-Lm but not anti-MAC activity was potentiated. (3) ROI production by murine peritoneal mphis was observed immediately after contact with MAC organisms (MAC stimulation) and ceased within 2 h. FFA release was seen 1-24 h after MAC stimulation. RNI production was initiated from 3 h and increased during the first 36 h and continued at least for 4 days. These findings suggest that RNI and FFA rather than ROI are important effectors of anti-MAC functions of mphis, and the collaborating action of RNI with FFA temporarily participates in mphi-mediated killing of MAC in the relatively early phase after MAC stimulation.

Bidirectional effects of IFN-gamma on growth of Mycobacterium avium complex in murine peritoneal macrophages.

The effects of macrophage stimulation with interferon-gamma (IFN-gamma) before or after infection on the intracellular growth of Mycobacterium avium complex (MAC) were investigated. Treatment of murine peritoneal macrophages before infection with IFN-gamma (50 U/ml) for 24 h and 48 h, but not for 72 h, was associated with 41% and 52% significant MAC growth inhibition, respectively. NG-monomethyl-L-arginine (NMA) did not affect the preinfection antimycobacterial activity of IFN-gamma, thus indicating that nitric oxide was not involved in this phenomenon. In contrast, treatment of macrophages with IFN-gamma (50 U/ml) for 24 h and 48 h after infection was ineffective, whereas treatment for 72 h caused some MAC growth promotion. The use of NMA suppressed the IFN-gamma-mediated MAC growth, suggesting that nitric oxide may affect postinfection microbicidal function of macrophages. These results suggest that activation of macrophages with IFN-gamma before or after infection may direct the course of the infection and that nitric oxide may be detrimental more than beneficial for MAC-infected macrophages.