WO2008134298A1 - In vitro model of latent mycobacterial infection - Google Patents
In vitro model of latent mycobacterial infection Download PDFInfo
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- WO2008134298A1 WO2008134298A1 PCT/US2008/061110 US2008061110W WO2008134298A1 WO 2008134298 A1 WO2008134298 A1 WO 2008134298A1 US 2008061110 W US2008061110 W US 2008061110W WO 2008134298 A1 WO2008134298 A1 WO 2008134298A1
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- Prior art keywords
- mycobacterium
- mtb
- macrophages
- cells
- low
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Classifications
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/36—Adaptation or attenuation of cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Definitions
- the present invention relates to the field of infectious diseases and, more particularly, to a pure culture of Mycobacterium which exhibits latency, including resistance to rifampicin and storage of increased lipids, and to a method for generating such a culture.
- Tuberculosis remains the leading cause of preventable deaths in the world with 100 million new infections and two million deaths each year.
- TB is caused by Mycobacterium tuberculosis (hereinafter also referred to by the abbreviation "Mtb”), an acid-fast bacillus that is transmitted primarily via the respiratory route.
- Mtb Mycobacterium tuberculosis
- the aerosol containing the pathogen is released from people with active TB when they cough or sneeze.
- Mtb multiplies within the vacuoles in the macrophage, avoids fusion with the acidic lysosomes and eludes the host defenses.
- the pathogen goes into a non- replicating, drug-resistant, latent state.
- the protective response by the immune system at the site of infection results in the formation of a granuloma that contains the infection and prevents its spread.
- Live bacilli have reportedly been isolated from granulomas or tubercles in the lungs of persons with clinically inactive tuberculosis, regarded as the latent form of TB, indicating that the organism can persist in granulomatous lesions for decades. It is estimated that one-third of the world population has latent TB. These individuals are asymptomatic latent carriers who exhibit no signs of disease.
- latent Mtb persists in a non-replicating state.
- Antibiotics used to treat bacterial infection are usually active against growing bacteria but not against the dormant pathogen. Correlation between antibiotic activity and bacterial growth state in streptomycin-dependent Mtb was shown almost 30 years ago. The antibiotic-resistance of non-growing bacteria is due to changes in bacterial metabolism or physiological state and is described as phenotypic resistance.
- the phenotypic resistance has been classified into three types based on the physiological state of bacteria as stationary phenotyopic resistance, persister phenotypic resistance and phenotypic resistance in dormant bacteria.
- Mtb displays dormancy-related phenotypic resistance which is demonstrated by the Cornell mouse model.
- the phenotypic resistance is exemplified by resistance to the antibiotic Rifampicin (Rif) and is regarded as one of the hallmarks of latent TB. The mechanism of phenotypic resistance in dormant Mtb is not clearly understood.
- Mtb in the host uses fatty acids as the major source of energy. It is well known that glyoxylate cycle is used by organisms that live on fatty acids. In recent years the important role of isocitrate lyase, a key enzyme uniquely used in the glyoxylate cycle, was shown to be required for the persistence of Mtb in the host demonstrating the central role played by fatty acid catabolism in persistence. However, the source of fatty acids used by the pathogen remains unclear.
- TG triacylglycerol
- TG is an important storage form of lipid that accumulates in species belonging to the actinomycetes family, particularly Mtb. Intracellular TG inclusion bodies were detected in mycobacteria isolated from organ lesions and Mycobacterium bovis BCG was reported to preferentially use TG within macrophages indicating that TG is probably used as an energy source by Mtb during the course of the disease. We have shown that TG accumulates when Mtb is subjected to hypoxia or nitric oxide treatment that led to a dormancy-like state in culture. We identified fifteen members of a novel class of diacylglycerol acyltransferase genes which we designated as tgs (triacylglycerol synthase).
- tgs triacylglycerol synthase
- WE waxy esters
- Mtb also stores WE but the genes involved in the synthesis of WE and the growth conditions that cause its accumulation have not been identified.
- the basic mechanisms used for biosynthesis of WE were first elucidated in our laboratory several decades ago and the enzymatic strategy described more recently.
- Rv3391 and Rv1543 encode acyl-CoA reductases involved in WE synthesis in Mtb.
- Rv3391 has been reported to be upregulated under nutrient stress conditions.
- WE accumulates under stress conditions that lead to a dormancy-like state and the accumulated WE is utilized upon starvation. This utilization was reduced in NpY mutant, indicating the involvement of NpY in WE hydrolysis.
- Mtb can produce and use both major energy storage forms, TG and WE, and both forms are likely to be used for successfully going through dormancy.
- WE may also be a component of the cell wall lipids that control permeability.
- the present invention advantageously provides a method of inducing latency in Mycobacterium, the method comprising growing a pure culture of Mycobacterium exposed to multiple stress conditions, the stress conditions including at least a low nutrient culture medium without glycerol, a low pH, a relatively high level of carbon dioxide and a relatively low gas phase oxygen level.
- a latent culture of Mycobacterium growing in vitro is particularly useful in evaluating the effectiveness of antimicrobial compounds against this form of the organism, which is prevalent throughout the world in infected but asymptomatic persons.
- the present invention discloses an in vitro model of latent mycobacterial infection which is useful in testing antimicrobials for activity against the infection in its latent stage.
- the method of the invention includes growing the Mtb in a low nutrient medium comprising approximately 10% Dubos medium, preferably at a pH of approximately 5 and in an atmosphere relatively high in level of carbon dioxide, at approximately 10%. Additionally, the atmosphere includes a relatively low oxygen level of approximately 5%.
- the Mycobacterium is a strain of Mycobacterium tuberculosis.
- Another embodiment of the present invention includes a method of inducing a pure culture of Mycobacterium to become rifampicin resistant and to store an increased lipid content, two hallmarks of latency, the method comprising growing the culture simultaneously exposed to multiple stress conditions, the stress conditions including at least a low nutrient culture medium without glycerol, a low pH, a relatively high level of carbon dioxide and a relatively low gas phase oxygen level.
- the present invention also includes a pure culture, and even a single isolated cell of resistant Mycobacterium generated according to the method disclosed.
- the invention includes an in vitro model of latent tuberculosis, the model comprising an isolated culture of THP1 derived macrophages containing ingested Mycobacterium tuberculosis bacteria and incubated under hypoxic conditions for a time sufficient for the bacteria to accumulate increased lipids therein. More broadly, the invention also provides an in vitro model of latent mycobacterial infection, the model comprising an isolated culture of THP1 derived macrophages containing ingested Mycobacterium spp. cells and incubated under hypoxic conditions for a time sufficient for the bacteria to accumulate increased lipids therein. More broadly still, the invention teaches an in vitro model of mycobacterial infection, the model comprising an isolated culture of THP1 derived macrophages containing ingested Mycobacterium spp. cells.
- the teachings also comprise a method of making a model of latent tuberculosis, the method including inducing cultured THP1 cells to differentiate into macrophages; infecting the macrophages with Mycobacterium tuberculosis bacteria; and incubating the infected macrophages under hypoxia, particularly wherein incubating is for a time sufficient for the bacteria to accumulate increased lipids therein, a hallmark of latency.
- This method is, in general, should also be applicable to other Mycobacterium species as well.
- FIG. 1 is a demonstration of the accumulation of storage lipids in Mtb cells treated for the indicated periods under the multiple stress conditions, according to an embodiment of the present invention. TLC was performed as described; the plates were charred and quantitation was done by densitometry;
- FIG. 2 shows increasing lipid storage bodies in Mtb cells with increasing periods of multiple stress; non-acid fast staining cells (green) and lipid storage body staining (red) increased with time under multiple stresses; cells were stained with Auramine-0 and Nile Red and examined by confocal laser scanning microscopy (Leica TCS SP5) with Z-stacking to get the depth of the scan field; scanned samples were analyzed by LAS AF software for image projection;
- FIG. 3 shows an increase in the percentage of lipid-stained cells and decrease in percentage of acidfast stained cells in Mtb culture when subjected to multiple stresses in vitro;
- FIG. 4 depicts TG accumulation by tgs ⁇ (Rv3130c) and restoration of TG accumulation by complementation under 18 days of multiple stress; equal amounts of lipid were subjected to TLC as in fig 1 ; C- ⁇ f ⁇ s1 , is a complemented mutant;
- FIG. 5 shows real-time PCR measurements of transcript levels of tgs and stress responsive genes in Mtb H37Rv under in vitro multiple stress for 9 days; comparative C 1 method ( ⁇ C T ) was used to quantify and values obtained with starting aerated cells were used to calculate the fold induction;
- FIG. 6 shows expression profiles of genes encoding proteins involved in the glyoxylate cycle during the multiple stress treatment; FIG.
- FIG. 7 depicts a decrease in buoyant density of Mtb cells subjected to multiple stresses; Mtb cells subjected to the multiple stresses were placed on the preformed gradient and centrifuged at 40Og for 20 min; the center tube is a 3 day cell sample mixed with density marker beads; Percoll® gradients were self-formed by centrifugation from a starting solution with a density of 1.0925 gm/ ml; the densities of selected bead layers (p, in gm/ml) are given on the right and the positions of one ml fractions collected for analyses are at the left; numbers below the tubes indicate the number of days under multiple stress; FIG.
- FIG. 8 is a bar graph showing that Alamar Blue assay reveals development of Rif resistance by multiple-stressed Mtb cultures; Mtb cultures subjected to multiple stresses were assayed by the specially adapted Alamar Blue method described in text for resistance to Rif and INH; fluorescence readings above 0 h controls are depicted; FIG. 9 shows real time PCR measurement of transcripts levels of a subset of selected dormancy metabolism and stress responsive genes in Mtb H37Rv under in vitro multiple stresses for 9 and 18 days; a relative quantitation method (ddCt) was used with the 7500 Fast real time system; samples of starter cultures were used as calibrator to calculate the fold induction;
- ddCt relative quantitation method
- FIG. 10 are photomicrographs where Oil Red-O staining reveals lipid droplet accumulation in TDM incubated for 3-days in 1 % O 2 , 5 % CO 2 (a) compared to 0-day control (b);
- FIG. 11 indicates the increase in lipid bodies in TDM infected with Mtb and subjected to hypoxia for 0 and 3-days;
- FIG. 12 shows Mtb within TDM stained with Auramine-O and Nile Red showing spherical fluorescent lipid bodies and lack of acid fast staining
- FIG. 13 shows that TG accumulated by TDM under hypoxia is utilized by Mtb; in A, lipids from uninfected (U) and infected (I) TDM, incubated in 20 % O 2 or 1 % O 2 for 7 days after infection, were resolved on TLC and visualized under UV light after spraying with 2',7'-dichlorofluorescein; in B, lipids of Mtb recovered from TDM incubated in 20% O 2 (i) or 1 % O 2 (ii); solvent was hexane-ether-formic acid (90:10:1 , v/v/v) TG, triacylglycerol, FA, fatty acids;
- FIG. 14 depicts the fatty acid composition of TG from Mtb recovered after TDM infection; after infection with Mtb, TDM were incubated under 1 % O 2 for 7 days; TG from Mtb isolated from TDM was purified by preparative TLC. Fatty acid methyl esters were prepared from Mtb TG and analyzed using a Varian CP-TAP CB column attached to a Varian CP-3900 gas chromatograph under a temperature control program; FIG. 15 depicts transcriptional profiling of genes in Mtb H37Rv from infected TDM under hypoxia;
- FIG. 16 shows that Mtb inside [ 14 C]acetate-labeled lipid-loaded macrophages mobilizes host lipids and accumulates TG enriched in saturated fatty acids;
- A AgNO 3 -impregnated silica-TLC purified from [ 14 C]acetate-labeled lipids of infected macrophages (lane 1 ) and from Mtb recovered from such macrophages (lane 2); solvent system is 1 % methanol in chloroform;
- B is shown reversed-phase TLC analysis of fatty acids methyl esters of TG from infected macrophages (lane 1 ) and from Mtb recovered from infected macrophages (lane 2); the solvent system is acetonitrile:methanol:water:acetic acid (30:70:5:1 , by volume);
- C AgNO 3 -impregnated silica-TLC of fatty acids methyl esters of TG from infected macrophages (lane 1 ) and from M
- TDM infected with Mtb and incubated under hypoxia appear to fuse together;
- TDM infected with Mtb at an MOI of 0.1 and incubated for 7 days under 1 % O 2 were stained with carbolfuschin followed by hematoxylin and eosin (A) or carbolfuschin followed by methylene blue (B); arrows show Mtb; and
- FIG. 1 shows TDM infected with Mtb and incubated under hypoxia appear to fuse together;
- TDM infected with Mtb at an MOI of 0.1 and incubated for 7 days under 1 % O 2 were stained with carbolfuschin followed by hematoxylin and eosin (A) or carbolfuschin followed by methylene blue (B); arrows show Mtb; and FIG.
- TDM-18 shows Mtb inside TDM that accumulate neutral lipids lose acid-fastness; intact TDM harboring Mtb were fixed with 4 % paraformaldehyde overnight and stained with the fluorescent mycolic acid staining dye Auramine-0 (A) followed by the neutral lipid stain Nile Red (B); arrows indicate Mtb that stained strongly for Nile Red but weakly for Auramine-O.
- Nile red staining revealed storage lipid accumulation under the multiple stress conditions. It is well known that Mtb cultures contain a heterogeneous population of cells under different physiological states. As the culture was subjected to multiple stress factors we observed decrease in acid fast staining cells with increasing lipid body staining cells from a barely detectable level to a significant percentage of the total cells by 18 days (FIGS. 2, 3).
- Drug resistance was tested by treatment with 5 ⁇ g/ml Rif for 5 days followed by serial dilution and plating. By 9 days about 10% of the cells were found to be Rif resistant whereas the starting culture contained about 0.03% Rif-resistant cells. Rif-resistance increased up to 18 days, sometimes reaching up to 25% at 5 ⁇ g/ml Rif.
- the f ⁇ s1 (Rv3130c) disruption resulted in the loss of Rif-resistance which was restored in the complemented mutant (Table 1). Hypoxic conditions, that were previously found to cause accumulation of storage lipids, did not cause the cells to develop detectable Rif-resistance at 5 ⁇ g/ml.
- f ⁇ s1 (Rv3130c) disruption resulted in loss of TG accumulation under multiple stress.
- the complemented mutant showed a level of TG accumulation comparable to the wild type (FIG.4).
- MB strain s Days Resistance to Antibiotics (%) INH (0.8 uq/ml) Rif (5.0 uq/ml) WT-
- Table 1 Development of Rif-resistance in wild type H37Rv but not in Rv3130c mutant upon application of multiple stress; complementation restores Rif-resistance. Aliquots were either untreated or treated with Rif (5 ⁇ g/ml) or INH (0.8 ⁇ g/ml). ND, Not determined; d, day.
- the TIGR Pathogen Functional Genomics Resource Center provided the Mtb genome microarray for this study.
- genes that encode enzymes involved in glyoxylate cycle such as isocitrate lyase (aceA) and citrate synthase (gltA1 ) showed significant increase in expression for all time points examined (FIG.6). From these data we infer that the metabolic regulation of cells adapting to the multiple stresses was similar to that observed in persistent bacilli adapting to the phagosomal environment of a macrophage.
- Mtb showed shutdown of both ATP/NAD energy regeneration systems. While gene expression for anaerobic respiration was continuously increased at the later time points, the aerobic respiration was significantly repressed at all the time points.
- the tuberculous granuloma which is thought to be a hypoxic environment, consists of a core of Mb-infected macrophages surrounded by lipid-loaded macrophages, mononuclear phagocytes and lymphocytes enclosed by a fibrous cuff.
- the differentiation of macrophages into lipid-loaded macrophages in tuberculous granulomas is a well-documented observation and the secretion of cytokines by the infected lipid-loaded macrophages probably helps to maintain the granuloma. Histological studies revealed the presence of lipid-loaded macrophages in the granulomas of immunocompetent and HIV-1 infected patients with TB.
- Lipid-loaded macrophages contain abundant cytosolic stores of TG and cholesterol esters.
- hypoxia 1% 02
- M. bovis BCG infection induced the conversion of macrophages into lipid-loaded cells but the nonpathogenic Mycobacterium smegmatis failed to induce lipid body formation.
- lipid bodies within Mto-infected macrophages may have important roles in pathogenesis and possibly in latency.
- Human THP- 1 monocytic cell line-derived macrophages are known to be converted into lipid-loaded macrophages.
- Lipid bodies have been found in Mtb obtained from patients with active disease. However, the origin of these lipids remains unknown.
- the pathogen inside the lipid-loaded macrophages might utilize fatty acids derived from the lipid bodies in the host cells to store lipids within Mtb for later use. Such a possibility was raised by the recent finding that adipocytes might be a home for dormant Mtb in humans.
- Mtb inside adipocytes were found to accumulate lipid bodies while becoming dormant, as indicated by their resistance to killing by drugs.
- the lipid bodies found in the pathogen from patients probably originate from the lipid bodies in the macrophages.
- Our results indicate that Mtb within lipid-loaded macrophages can use the host's TG to accumulate TG within the pathogen and this Mtb becomes Rif resistant meeting our criteria for dormancy.
- THP-1 derived macrophage THP-1 derived macrophage
- THP-1 cells differentiated into macrophages by treatment with 10OnM PMA for 3 days, were incubated for 3 days in 1 % 02 and 5% CO2.
- Oil Red-O staining revealed lipid droplet accumulation in such macrophages (FIG. 2).
- MOI multiplicity of infection
- lipid bodies accumulated in the host cells (FIG.3).
- Mtb cells within the macrophages showed Nile Red stained lipid bodies (FIG. 4).
- Most Mtb cells showed loss of acid fast staining and thus stained only red; a few showed some acid fast and lipid staining (yellow).
- Lipids from the host-lipid bodies were extracted from the supernatant and the lipids from the pelleted
- host or Mtb TG was purified by preparative TLC and the methyl esters generated by BF3/methanol transesterification, were analyzed by capillary GC.
- the amount of fatty acids from the TG isolated from Mtb, recovered for TDM, is more than enough for such GC analysis (FIG. 6) pathogens were extracted.
- TLC analysis of the lipids revealed that TG in the host cells was markedly increased by incubation under hypoxia for 7 days and the levels of TG were lower in infected TDM under hypoxia (FIG. 5A).
- Lipids extracted from Mtb recovered from infected TDM were also analyzed by TLC. We detected increased TG level in Mtb cells recovered from TDM incubated under hypoxic conditions (FIG. 5B).
- the fatty acid composition of the TG from the pathogen was not identical to that of the host TG.
- C16:0, C18:0 and C18:1 fatty acids were the dominant components in both the pathogen and the host.
- Longer chain saturated fatty acids (C24, C26 and C28) that were present in the pathogen TG were absent in the host TG.
- the TG that accumulated in the pathogen probably consisted of fatty acids from the host and some fatty acids generated within the pathogen.
- All primers and Taqman probes were designed using VisualOMP ⁇ software from DNA software, lnc (Ann Arbor, Ml).
- the Taqman probes have a fluorescein reporter dye (FAM) at 5'-end and a Black Hole Quencher (BHQ) at 3'-end.
- FAM fluorescein reporter dye
- BHQ Black Hole Quencher
- Each multiplex and real-time PCR primer was checked for specificity and efficiency. Differences in Mtb specific gene transcripts were quantified by real-time PCR on generated multiplex-PCR products with nested Taqman primers and probes. The overall reliability and sensitivity of the two-step RT-PCR method to quantify gene expression profiling has been discussed in detail elsewhere.
- TDM were infected with Mtb at an MOI of 0.1.
- Mtb cells inside TDM were exposed to antibiotic for 2 days prior to lysis of TDM and recovery of the bacilli.
- the recovered Mtb cells were diluted and plated on agar plates without antibiotic and incubated for 4 weeks after which cfus 5 were enumerated. Antibiotic resistance is expressed as percentage of control without antibiotic. As indicated in Table 2, Mtb recovered from TDM incuba ted
- Table 2 Increase in resistance of Mtb inside lipid-loaded macrophages to Rif and INH. Mtb within TDM incubated for 7 days under 20% O 2 or 1 % O 2 was exposed to antibiotic for 2 days. Mtb cells were then recovered by lysis of TDM and plated on agar plates for cfu determination.
- TDM lipids by incubating the cells with [ 14 C]acetic acid or [ 14 C]oleic acid, under 1 % O 2 for 2 days. These cells were washed three times with sterile phosphate-buffered saline (PBS) to remove unincorporated radiolabel.
- PBS sterile phosphate-buffered saline
- TLC Thin-layer chromatographic
- the fatty acid composition of the Mtb and host TG was analyzed by resolving the intact TG and fatty acid methyl esters derived from TG on reversed-phase silica-TLC and argentation-TLC. Analysis of intact TG from [ 14 C]acetate in TDM was composed of saturated and unsaturated fatty acids. However, the TG of Mtb was predominantly composed of saturated fatty acids as indicated by greater mobility on AgNO 3 -impregnated TLC (FIG. 8 A).
- the TG stored within the pathogen probably includes the fatty acids from the host lipids and fatty acids generated by modification and/or catabolism and resynthesis.
- the biochemical processes involved can be deduced only after further characterization of the TG that accumulates in the host and in the pathogen.
- infected TDM after 7 days under hypoxia were fixed with 4% paraformaldehyde and stained for Mtb with carbolfuschin followed by methylene blue or hematoxylin and eosin to stain the host cell.
- Mtb inside TDM were also stained with the mycolic acid-specific fluorescent dye Auramine-0 followed by Nile Red which stains neutral lipids.
- the Mb-infected TDM which were incubated under hypoxia for 7 days appeared to be fusing together. It is probable that these TDM are in the process of forming multinucleate giant cells (asterisk in FIG.
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CA2685746C (en) * | 2007-04-30 | 2017-02-28 | University Of Central Florida Research Foundation, Inc. | In vitro model of latent mycobacterial infection |
CN102469717B (en) * | 2010-11-08 | 2014-09-17 | 昌硕科技(上海)有限公司 | Clamping module and portable electronic device using same |
KR20170011116A (en) * | 2015-07-21 | 2017-02-02 | 엘지전자 주식회사 | Laundry Treating Apparatus |
CN105125300A (en) * | 2015-09-30 | 2015-12-09 | 崔秋义 | Composite orthodontic arch wire |
RU2702609C1 (en) * | 2018-08-06 | 2019-10-08 | Федеральное государственное бюджетное учреждение "Новосибирский научно-исследовательский институт туберкулеза" Министерства здравоохранения Российской Федерации (ФГБУ "ННИИТ" Минздрава России) | Method for simulating tuberculosis infection in vitro |
US20220033777A1 (en) * | 2018-09-17 | 2022-02-03 | The Lundquist Institute for Biomedical Innovation at Harbor-UCLA Medical Center | Target-primed macrophages and therapeutic uses thereof |
RU2707941C1 (en) * | 2018-11-14 | 2019-12-02 | Федеральное государственное бюджетное учреждение "Новосибирский научно-исследовательский институт туберкулеза" Министерства здравоохранения Российской Федерации (ФГБУ "ННИИТ" Минздрава России) | Method for modeling the dormant state of mycobacterium tuberculosis in vitro |
CN112921107A (en) * | 2020-12-31 | 2021-06-08 | 深圳市慢性病防治中心(深圳市皮肤病防治研究所、深圳市肺部疾病防治研究所) | Heterogeneous drug resistance detection method, kit and application of mycobacterium tuberculosis |
CN113355439A (en) * | 2021-06-07 | 2021-09-07 | 深圳市普瑞康生物技术有限公司 | Novel combined detection method for latent infection and morbidity of mycobacterium tuberculosis |
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Non-Patent Citations (6)
Title |
---|
AUWERX J.: "The human leukemia cell line, THP-1: a multifacetted model for the study of monocyte-macrophage differentiation", EXPERIENTIA, vol. 47, no. 1, 15 January 1991 (1991-01-15), pages 22 - 31, XP000218599 * |
BETTS J.C. ET AL.: "Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistance by gene and protein expression profiling", MOL. MICROBIOL., vol. 43, no. 3, February 2002 (2002-02-01), pages 717 - 731, XP002223692 * |
CHAUHAN: "Effect of Carbon Dioxide on the primary isolation of Mycobacteria", INDIAN JOUR. TUBERCULOSIS, vol. 38, 1991, pages 81 - 85, XP008123502 * |
MUTTUCUMARU ET AL.: "Gene expression profile of Mycobacterium tuberculosis in anon-replicating state", TUBERCULOSIS, vol. 84, May 2004 (2004-05-01), pages 239 - 246, XP008123058 * |
See also references of EP2152853A4 * |
SIRAKOVA T.D. ET AL: "Identification of a diacylglycerol acyltransferase gene involved in accumulation of triacylglycerol in Mycobacterium tuberculosis under stress", MICROBIOLOGY, vol. 152, no. 9, September 2006 (2006-09-01), pages 2717 - 2725, XP008123057 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107941980A (en) * | 2017-11-26 | 2018-04-20 | 浙江省水产技术推广总站 | The remaining ultra performance liquid chromatography tandem mass spectrum rapid assay methods of rifampin in aquatic products |
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EP2152853A4 (en) | 2010-06-16 |
CA2685746A1 (en) | 2008-11-03 |
AU2008245901A1 (en) | 2008-11-06 |
US20130040829A1 (en) | 2013-02-14 |
AU2008245901B2 (en) | 2014-10-16 |
US8211446B2 (en) | 2012-07-03 |
US8012492B2 (en) | 2011-09-06 |
US20090023596A1 (en) | 2009-01-22 |
US8241644B2 (en) | 2012-08-14 |
US20110033915A1 (en) | 2011-02-10 |
EP2152853A1 (en) | 2010-02-17 |
US8551499B2 (en) | 2013-10-08 |
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