Nymphs of the Oriental cockroach (Blatta orientalis) as passive vectors of causal agents of avian tuberculosis and paratuberculosis

The potential transmission of the causal agent of paratuberculosis Mycobacterium avium ssp. paratuberculosis and avian tuberculosis Mycobacterium avium ssp. avium (Actinomycetales: Mycobacteriaceae) by nymphs of the Oriental cockroach Blatta orientalis L. (Blattodea: Blattidae) was investigated by oral infection with mycobacterial suspensions and examination of their droppings and bodies. Both the subspecies of M. avium were isolated from droppings at 3 days post-infection and M. a. avium was found in homogenized bodies at 10 days post-infection. The identity of M. a. avium and M. a. paratuberculosis isolates was demonstrated by Restriction Fragment Length Polymorphism (RFLP) analysis. The M. a. avium isolate used as the inoculum and the isolates from the bodies and droppings of the nymphs were shown to be virulent in chickens. The results show that orally infected nymphs of B. orientalis can harbour and shed viable and virulent mycobacteria. This hazard should be considered in the implementation of control measures against mycobacterial infections of animals and humans, which should include destruction of all developmental stages of cockroaches and prevention of their access to materials that can be contaminated by mycobacteria.     

UV light inactivation of Mycobacterium avium subsp. paratuberculosis in milk as assessed by FASTPlaqueTB phage assay and culture

UV light inactivation of Mycobacterium avium subsp. paratuberculosis in Middlebrook 7H9 broth and whole and semiskim milk was investigated using a laboratory-scale UV machine that incorporated static mixers within UV-penetrable pipes. UV treatment proved to be less effective in killing M. avium subsp. paratuberculosis suspended in milk (0.5- to 1.0-log(10) reduction per 1,000 mJ/ml) than that suspended in Middlebrook 7H9 broth (2.5- to 3.3-log(10) reduction per 1,000 mJ/ml). The FASTPlaqueTB phage assay provided more rapid enumeration of surviving M. avium subsp. paratuberculosis (within 24 h) than culture on Herrold's egg yolk medium (6 to 8 weeks). Despite the fact that plaque counts were consistently 1 to 2 log(10) lower than colony counts throughout the study, UV inactivation rates for M. avium subsp. paratuberculosis derived using the phage assay and culture results were not significantly different (P = 0.077).     

Various stages in the life cycle of syrphid flies (Eristalis tenax; diptera: Syrphidae) as potential mechanical vectors of pathogens causing mycobacterial infections in pig herds

We defined the role of the syrphid fly Eristalis tenax in the survival and transmission of mycobacteria in pigs. The conditionally pathogenic mycobacterial (CPM) species Mycobacterium chelonae was isolated from 10 % of liquid dung samples, and both M. chelonae and another CPM species M. fortuitum were isolated from 7 (78 %) of the examined E. tenax larvae collected from the same location. Mycobacteriosis of the lymph nodes of pigs from 3 infected farms was caused by M. avium subsp. avium, M. avium subsp. hominissuis, and M. fortuitum. M. avium subsp. avium and M. avium subsp. hominissuis of identical genotype and serotypes and M. fortuitum were isolated from 7 (1.9 %) larvae, 2 (7.4 %) puparia, and one (1.6 %) imago. The count of colony forming units isolated from larval skin covering (pouch) was higher (p < or = 0.01) than that isolated from the internal organs of larvae. These results showed the potential for E. tenax larvae to spread mycobacteria throughout pig herds and the surrounding environment.     

流式细胞术检测胞内重组耻垢分枝杆菌的活力研究

目的建立一种快速检测胞内分枝杆菌活力的方法。方法将一定量培养至对数生长期的含pMV-eis的重组耻垢分枝杆菌感染U937巨噬细胞,以含空质粒的耻垢分枝杆菌为对照,吞噬作用2 h后洗去胞外细菌,再分别培养4、12、24和48 h后收集细胞并裂解之。获得的胞内细菌用FDA荧光染料染色后用流式细胞仪检测死亡率,并与平板菌落计数法进行比较。结果流式细胞仪检测出感染12 h后重组耻垢分枝杆菌胞内死亡率较对照组均有显著下降（P〈0.05）,流式细胞仪检测法与平板菌落计数法相比差异无统计学意义（P〉0.05）。结论流式细胞术与传统的平板计数法相比具有快速、敏感、方便的特点,可用于分枝杆菌活菌快速检测。     

Inhibition of cytokines expression in human microglia infected by virulent and non-virulent mycobacteria

The pathogenesis of tuberculosis (TBC) meningitis is still unknown. As shown by previous studies, human microglia can be the target of mycobacteria, but no data are available about their cellular response to infection. Consequently, we studied the expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) and IL-10 in human microglia pure cultures infected with the two variants of Mycobacterium avium (domed-opaque (SmD) and transparent (SmT)) and with Mycobacterium tuberculosis. Results showed that microglia was productively infected by mycobacteria which could grow inside the cells. Mycobacteria internalization was more rapid for M. avium, but M. tuberculosis infection turned out to be more efficient due to the incorporation of densely packed bacteria. TNF-alpha expression was not affected by M. avium, whereas an increase followed by a decrease was observed in M. tuberculosis. Both IL-1 and IL-10 cytokine expression was rapidly inhibited by infection with the more virulent bacteria, whereas the non-pathogenic one had almost no effect. Also, the expression of the co-stimulatory molecule CD137, a member of tumor necrosis factor receptor family, was affected by infection with virulent mycobacteria. Our results show that microglia response to mycobacterial infection is modulated in correlation with virulence, mainly toward inhibition of inflammatory response. This observation might be one of the mechanisms by which non-pathogenic mycobacteria are quickly eliminated, explaining one of the bases of virulence.     

Characterization of the intracellular survival of Mycobacterium avium ssp. paratuberculosis: phagosomal pH and fusogenicity in J774 macrophages compared with other mycobacteria

The phagosomes containing viable pathogenic mycobacteria, such as Mycobacterium ( M .) tuberculosis and Mycobacterium avium ssp. avium ( M. avium ), are known to be limited in their ability to both acidify and fuse with late (but not early) endocytic organelles. Here, we analysed the pH and fusogenicity of phagosomes containing M. avium ssp. paratuberculosis ( M. ptb ), the causative agent of paratuberculosis in ruminants. Using the murine J774 macrophage cell line, we compared viable and heat-killed M. ptb and, in addition, viable or dead M. avium , as well as two non-pathogenic mycobacteria, Mycobacterium smegmatis and Mycobacterium gordonae . Electron microscopic analysis revealed that M. ptb persisted intracellularly in phagosomes for up to 15 days. The phagosomes containing live M. ptb and M. avium were significantly reduced in their ability to acquire some markers for the endocytic pathway, such as internalized calcein, BSA–gold or the membrane protein Lamp 2. However, they were almost completely accessible to 70 kDa fluorescein isothiocyanate (FITC)–dextran and Lamp 1. Overall, the phagosomes containing dead pathogenic mycobacteria behaved similarly to the ones containing live non-pathogenic mycobacteria in all experiments. Using FITC–dextran in a novel fluorescence-activated cell sorting (FACS)-based method, we could also show that the bulk of endocytic compartments, including phagosomes, were only very mildly acidified to ≈ pH 6.3 over at least 72 h in J774 cells infected with live M. ptb and M. avium . In contrast, J774 cells treated with heat-killed M. ptb or BSA-coated latex beads showed substantial acidification of the phagosome/endocytic compartments to a pH value of ≈ 5.2. After infection with M. smegmatis and M. gordonae , acidification was initially (1–5 h after infection) inhibited, but increased after longer infection to levels similar to those with dead mycobacteria.     

Role of lipids in killing mycobacteria by macrophages: evidence for NF-κB-dependent and -independent killing induced by different lipids

We have shown that several lipids can modulate the macrophage innate immune response against mycobacteria and enhance their killing. Since NF-κB is required for mycobacterial killing, we tested the ability of lipids to activate NF-κB in uninfected macrophages and those infected with mycobacteria. In uninfected cells, sphingomyelin (SM), phosphatidylinositol-4-phosphate (PIP) and arachidonic acid (AA) enhanced NF-κB activation and the cell surface expression of CD69, a macrophage activation marker regulated by NF-κB. Sphingosine (Sph), sphingosine-1-phosphate (S1P), diacylglycerol (DAG), eicosapentanoic acid (EPA) and phosphatidyl choline (PC) failed to activate either NF-κB or CD69. Ceramide (Cer) activated CD69 expression without activating NF-κB. In Mycobacterium smegmatis- infected cells, NF-κB was transiently activated in a manner that was enhanced by SM, PIP and AA. In contrast Mycobacterium avium mostly repressed NF-κB activation and only SM and AA could induce its partial activation. While lipids that activate NF-κB in uninfected cells tend to kill mycobacteria in macrophages Sph and S1P failed to activate NF-κB under most conditions but nevertheless enhanced killing of M. smegmatis , M. avium and M. tuberculosis H37Rv. Our results argue that both NF-κB-dependent and -independent mechanisms are involved in macrophage killing of mycobacteria and that both mechanisms can be enhanced by selected lipids.     

Factors Affecting the Determination of Respiratory Activity on the Basis of Cyanoditolyl Tetrazolium Chloride Reduction with Membrane Filtration

Deficiencies in traditional bacterial enumeration techniques which rely on colony formation have led to the use of total direct counting methods, such as the acridine orange direct count technique for the enumeration of planktonic bacteria. As total direct counts provide no information on the viability or activity of the organisms, demonstration of respiratory activity with the fluorochrome cyanoditolyl tetrazolium chloride (CTC) has been employed. We have modified this technique by performing filtration prior to CTC incubation. Cells captured on a polycarbonate membrane were incubated on absorbent pads saturated with medium containing CTC. Following counterstaining with DAPI (4(prm1),6-diamidino-2-phenylindole) total and respiring cells were enumerated by epifluorescence microscopy. Factors affecting CTC reduction by Klebsiella pneumoniae, Salmonella typhimurium, and Escherichia coli K-12 were investigated. With K. pneumoniae, nutrient additions to the CTC medium did not increase the number of respiring cells detected. CTC reduction by all three organisms decreased in response to an increase of the pH of the CTC medium above pH 6.5. Increasing phosphate concentrations contributed to this inhibitory effect. CTC-membrane filter counts of K. pneumoniae, S. typhimurium, and E. coli K-12 and of bacteria in well water corresponded closely with plate counts (r = 0.987). The results show that careful attention should be given to the composition of CTC-containing media which are used to enumerate respiring bacteria. With an appropriate medium, reliable enumeration of respiring bacteria can be achieved within a few hours.     

Differential release of tumor necrosis factor-α from murine peritoneal macrophages stimulated with virulent and avirulent species of mycobacteria

Abstract The ability of Mycobacterium tuberculosis H37Rv and H37Ra, M. bovis BCG and M. smegmatis to induce the secretion of tumor necrosis factor-α (TNF-α) by cultured murine peritoneal macrophages is inversely related to their virulence. The avirulent species of mycobacteria which were unable to persist in macrophages were capable of inducing significant levels of TNF-α compared to that formed in cultures infected with the virulent M. tuberculosis H37Rv. This difference was also associated with an inherent toxicity by live H37Rv for macrophage cultures. Heat-killed H37Rv was non-toxic and induced significant levels of TNF-α; in contrast, live and heat-killed suspensions of avirulent mycobacteria had an equivalent ability to trigger TNF-α secretion. The TNF-α response was dose-dependent, related directly to the percentage of infected cells, and peaked 6–12 h post-infection. An early and vigorous TNF-α response appears to be a marker of macrophage resistance, while the downregulation of this response seems associated with macrophage toxicity and unrestricted mycobacterial growth.     

Fluorescent acid-fast microscopy for measuring phagocytosis of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum by Tetrahymena pyriformis and their intracellular growth

Fluorescent acid-fast microscopy (FAM) was used to enumerate intracellular Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum in the ciliated phagocytic protozoan Tetrahymena pyriformis. There was a linear relationship between FAM and colony counts of M. avium cells both from cultures and within protozoa. The Ziehl-Neelsen acid-fast stain could not be used to enumerate intracellular mycobacteria because uninfected protozoa contained acid-fast, bacterium-like particles. Starved, 7-day-old cultures of T. pyriformis transferred into fresh medium readily phagocytized M. avium, M. intracellulare, and M. scrofulaceum. Phagocytosis was rapid and reached a maximum in 30 min. M. avium, M. intracellulare, and M. scrofulaceum grew within T. pyriformis, increasing by factors of 4- to 40-fold after 5 days at 30 degrees C. Intracellular M. avium numbers remained constant over a 25-day period of growth (by transfer) of T. pyriformis. Intracellular M. avium cells also survived protozoan encystment and germination. The growth and viability of T. pyriformis were not affected by mycobacterial infection. The results suggest that free-living phagocytic protozoa may be natural hosts and reservoirs for M. avium, M. intracellulare, and M. scrofulaceum.     

Haemopoiesis in mice genetically lacking granulocyte-macrophage colony stimulating factor during chronic infection with Mycobacterium avium

In order to test the role of granulocyte-macrophage colony stimulating factor (GM-CSF) in haemopoiesis during chronic infection, mice with a targeted disruption of the gene for GM-CSF were infected intraperitoneally with the facultative intracellular pathogen, Mycobacterium avium. The bacteria spread to lungs, liver and spleen and persisted for more than 10 weeks at levels between 105 and 106 CFU. Bacterial numbers did not differ significantly between infected GM-CSF-/- and wild-type mice, making this an excellent model in which to study the effects of GM-CSF deficiency on haemopoietic cells without complications of interpretation relating to differences in bacterial load. Haemopoietic colony forming cells (CFC) in the bone marrow of GM-CSF-/- mice before infection were not different from wild-type. However, whereas CFC in wild-type mice increased 1.5-fold with infection, GM-CSF-/- mice were unable to increase their CFC and numbers were significantly lower than in infected wild-type mice. Cells attracted to the peritoneal cavity of the GM-CSF-/- mice following i.p. injection of bacteria were notably lacking in the large, granular macrophages of activated appearance, which were a feature in wild-type mice. Nitric oxide production by peritoneal cells from GM-CSF-/- mice was deficient. Thus, GM-CSF is not critical for haemopoiesis during chronic infection, but in its absence the mice are unable to increase their output of haemopoietic cells and there are deficiencies in macrophage activation.     

Establishment of a sepsis model following implantation of<Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>-infected fibrin clot into the peritoneal cavity of mice

Successful establishment of sepsis by entrapping a dose of 150 colony forming units of Klebsiella pneumoniae in a fibrin clot following implantation into the peritoneal cavity of mice is reported. The dose in the fibrin clot gave 50% mortality in mice, spread over a period of one week. All the infected mice showed positive blood culture up to 6 d post-infection; histopathology revealed inflammatory changes in both liver and spleen. Introduction of K. pneumoniae into experimental mice without entrapment in fibrin clot caused no mortality and blood culture remained positive only up to 2 d; histopathology of liver and spleen throughout the period of study showed relatively mild inflammatory changes, which almost cleared during 14 d post-infection. The use of the fibrin-clot model may thus be considered to be useful in studying both the initial and the persisting stage of infection in the peritoneum, whence a slow release of bacteria into the blood takes place which finally leads to sepsis and septicemia.     

Increased intracellular growth of Mycobacterium avium in HIV-1 exposed monocyte-derived dendritic cells

Dendritic cells (DC) are the most potent antigen-presenting cells, and form a link between the innate and adaptive immune system. They sample the periphery of the body for antigens and present them to T cells to elicit a proper immune response. It has been shown that dendritic cells phagocytose mycobacteria, but there have been conflicting reports as to whether the bacteria are capable of intracellular replication in DCs. Mycobacterium avium is a facultative intracellular bacterium, part of the Mycobacterium avium complex (MAC) of mycobacteria and are commonly seen as opportunistic pathogens in patients infected by Human immunodeficiency virus type 1 (HIV-1). To clarify the issue of whether DCs are capable of controlling the intracellular growth of M. avium and whether this control is lost upon HIV-1 exposure, we investigated the intracellular replication of M. avium in monocyte-derived dendritic cells and compared it to bacterial growth in dendritic cultures exposed to HIV-1 for 24 h. Our results show that exposure of DCs to HIV-1 promotes or facilitates the intracellular growth of M. avium.     

Elemental analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and Mycobacterium smegmatis-containing phagosomes indicates pathogen-induced microenvironments within the host cell's endosomal system

Mycobacterium avium and Mycobacterium tuberculosis are human pathogens that infect and replicate within macrophages. Both organisms live in phagosomes that fail to fuse with lysosomes and have adapted their lifestyle to accommodate the changing environment within the endosomal system. Among the many environmental factors that could influence expression of bacterial genes are the concentrations of single elements within the phagosomes. We used a novel hard x-ray microprobe with suboptical spatial resolution to analyze characteristic x-ray fluorescence of 10 single elements inside phagosomes of macrophages infected with M. tuberculosis and M. avium or with avirulent M. smegmatis. The iron concentration decreased over time in phagosomes of macrophages infected with Mycobacterium smegmatis but increased in those infected with pathogenic mycobacteria. Autoradiography of infected macrophages incubated with (59)Fe-loaded transferrin demonstrated that the bacteria could acquire iron delivered via the endocytic route, confirming the results obtained in the x-ray microscopy. In addition, the concentrations of chlorine, calcium, potassium, manganese, copper, and zinc were shown to differ between the vacuole of pathogenic mycobacteria and M. smegmatis. Differences in the concentration of several elements between M. avium and M. tuberculosis vacuoles were also observed. Activation of macrophages with recombinant IFN-gamma or TNF-alpha before infection altered the concentrations of elements in the phagosome, which was not observed in cells activated following infection. Siderophore knockout M. tuberculosis vacuoles exhibited retarded acquisition of iron compared with phagosomes with wild-type M. tuberculosis. This is a unique approach to define the environmental conditions within the pathogen-containing compartment.     

omp25c基因对马耳他布鲁菌疫苗株M5的毒力及免疫保护性的影响

目的：初步评价马耳他布鲁菌M5疫苗株omp25c基因对其毒力及免疫保护性的影响。方法：利用同源重组的方法,用卡那霉素抗性基因替换M5的omp25c（BMEI1829）基因,得到缺失突变株M5Δomp25c;分别用M5Δomp25c和M5免疫小鼠,在免疫后不同时间点处死小鼠,通过脾脏细菌计数分析缺失突变株在小鼠体内的毒力,通过检测IgG和IFN-γ的水平分析缺失突变株在小鼠体内诱导的体液和细胞免疫应答能力,通过攻毒实验评价突变株的免疫保护效果。结果：与M5株相比,M5Δomp25c在小鼠脾脏内的存活时间较短,在第4周时未能检出;M5Δomp25c免疫小鼠诱导产生的IgG水平在第4周达到最高,第6周开始下降;M5Δomp25c免疫小鼠诱导分泌的IFN-γ水平在第4周达到最高为790pg/mL,第6周时浓度降至530pg/mL,整体趋势显著低于阳性对照组;接种了M5Δomp25c的小鼠用布鲁菌强毒株16M攻毒后,免疫保护效果也下降。结论：缺失omp25c的突变株毒力减弱,诱导的体液和细胞免疫水平及免疫保护效果下降,说明omp25c基因是马耳他布鲁菌M5疫苗株的毒力相关基因,对疫苗株M5的免疫应答和免疫保护效果有一定的影响。     

Maximum growth rate of Mycobacterium avium in continuous culture or chronically infected BALB/c mice

Mycobacterium avium is a human pathogen which may cause either chronic or disseminated disease and the organism exhibits a slow rate of growth. This study provides information on the growth rate of the organism in chronically infected mice and its maximal growth rate in vitro. M. avium was grown in continuous culture, limited for nitrogen with 0.5 mM ammonium chloride and dilution rates that ranged from 0.054 to 0.153 h-1. The steady-state concentration of ammonia nitrogen and M. avium cells for each dilution rate were determined. The bacterial saturation constant for growth-limiting ammonia was 0.29 mM (4 micrograms nitrogen/ml) and, from this, the maximal growth rate for M. avium was estimated to be 0.206 h-1 or a doubling time of 3.4 h. BALB/c mice were infected intravenously with 3 x 10(6) colony-forming units and a chronic infection resulted, typical of virulent M. avium strains. During a period of 3 months, the number of mycobacteria remained constant in the lungs, but increased 30-fold and 8,900-fold, respectively, in the spleen and mesenteric lymph nodes. The latter increase appeared to be due to proliferation in situ. The generation time of M. avium in the mesenteric lymph nodes was estimated to be 7 days.     

Illegitimate recombination: An efficient method for random mutagenesis in Mycobacterium avium subsp. hominissuis

ABSTRACT: BACKGROUND: The genus Mycobacterium (M.) comprises highly pathogenic bacteria such as M. tuberculosis as well as environmental opportunistic bacteria called non-tuberculous mycobacteria (NTM). While the incidence of tuberculosis is declining in the developed world, infection rates by NTM are increasing. NTM are ubiquitous and have been isolated from soil, natural water sources, tap water, biofilms, aerosols, dust and sawdust. Lung infections as well as lymphadenitis are most often caused by M. avium subsp. hominissuis (MAH), which is considered to be among the clinically most important NTM. Only few virulence genes from M. avium have been defined among other things due to difficulties in generating M. avium mutants. More efforts in developing new methods for mutagenesis of M. avium and identification of virulence-associated genes are therefore needed. RESULTS: We developed a random mutagenesis method based on illegitimate recombination and integration of a Hygromycin-resistance marker. Screening for mutations possibly affecting virulence was performed by monitoring of pH resistance, colony morphology, cytokine induction in infected macrophages and intracellular persistence. Out of 50 randomly chosen Hygromycin-resistant colonies, four revealed to be affected in virulence-related traits. The mutated genes were MAV_4334 (nitroreductase family protein), MAV_5106 (phosphoenolpyruvate carboxykinase), MAV_1778 (GTP-binding protein LepA) and MAV_3128 (lysyl-tRNA synthetase LysS). CONCLUSIONS: We established a random mutagenesis method for MAH that can be easily carried out and combined it with a set of phenotypic screening methods for the identification of virulence-associated mutants. By this method, four new MAH genes were identified that may be involved in virulence.     

Blocking the receptor for IL-10 improves antimycobacterial chemotherapy and vaccination

Novel approaches are required for the prevention and therapy of mycobacterial infections since the only vaccine in use, bacillus Calmette-Guérin, is poorly effective and chemotherapy is long and often ineffective in sterilizing the infection. We used a mouse model of Mycobacterium avium infection to address the usefulness of a mAb able to block IL-10R both in treatment of primary infections and in conventional multidrug therapy and subunit vaccination. Treatment of infected mice with this mAb during the entire period of experimental infection had little impact on the course of M. avium infection, with a slight improvement in the resistance of infected mice observed in the liver and spleen at day 30 of infection, which was associated with increased macrophage activation and priming of CD4(+) T cells for IFN-gamma production. Administration of this mAb later in infection had no effect on its course, but improved the effectiveness of chemotherapy when the latter was started in a chronic phase of infection. Also, the anti-IL-10R mAb acted as an adjuvant in the induction of protective immunity upon vaccination with a mycobacterial subunit preparation.     

In vivo efficiency of targeted norfloxacin against persistent, isoniazid-insensitive, Mycobacterium bovis BCG present in the physiologically hypoxic mouse liver

Persistence of Mycobacterium tuberculosis is a hypoxia-inducible state in which the bacteria are phenotypically insensitive to currently available antituberculous drugs. In humans, persistent M. tuberculosis is found in granulomatous lesions, either inside macrophages or in necrotic tissue, where the partial oxygen pressure (pO(2)) is very low. Persistent bacteria can remain silent for decades before overt tuberculosis develops. Due to insensitivity to classical drugs, M. tuberculosis persistence prevents rapid and definitive clearance of bacteria. Consequently, therapeutic molecules are required that are both active against persistent bacilli and able to reach their intramacrophagic location. In contrast to its native form, norfloxacin is active in vivo against Mycobacterium bovis BCG present in the lungs when temporarily linked to a macromolecular carrier targeted to macrophages. To study the efficiency of this macromolecular prodrug targeted to persistent mycobacteria confined inside macrophages, we established a short-term in vivo model based on the physiological pO(2) differences between lungs, spleen and liver. Whereas lungs and spleen are well oxygenated, the liver has a low pO(2) due to its portal irrigation. Therefore, studying mycobacteria in the liver yields information about in vivo persistent bacilli exposed to low pO(2). To our knowledge, no similar short-term in vivo model has been published to date. Using this model, we demonstrated the insensitivity to isoniazid of M. bovis BCG present in hypoxic sites, and showed that norfloxacin given as a mannosylated macrophage-targeted prodrug was able to kill these isoniazid-insensitive mycobacteria. This demonstrates that intracellular persistent mycobacteria are amenable to antibiotic treatment.