Multidrug-resistant tuberculosis. 2. Mechanisms of drug-resistance in Mycobacterium tuberculosis--genetic mechanisms of drug-resistance

Multidrug-resistant Mycobacterium tuberculosis infection is now world wide health problem. However, according to the recent advances of molecular biological technics, some of the genetic mechanisms of drug-resistance of M. tuberculosis has been uncovered. Generally, drug-resistance of M. tuberculosis was caused by point mutations in chromosomal gene. In isoniazid (INH) resistant M. tuberculosis, mutations and genetic deletions in catalase-peroxidase gene (katG), inhA gene, or alkyl hydroperoxide reductase gene were reported. We also found that about 15% of INH-resistant M. tuberculosis isolates lacked katG gene, and these isolates showed highly resistance to INH with MIC > or = 64 micrograms/ml. On the other hand, mutations and other genetic alterations in RNA polymerase beta subunit gene (rpoB) were the major mechanisms of resistance to rifampicin (RFP) with high frequencies of 90% or more. Our evaluation of the relationship between RFP susceptibility and genetic alteration in rpoB gene also showed that 95% of RFP-resistant M. tuberculosis isolates involved genetic alterations in 69 bp core region of rpoB gene. Moreover, these genetic alterations in rpoB gene were suspected as the resistant mechanism to other rifamycin antituberculosis drugs, such as rifabutin and KRM-1648. In addition, it was reported that point mutations in 16S rRNA gene (rrs) and ribosomal protein S12 gene (rpsL) induced M. tuberculosis as streptomycin (SM) resistant phenotype. We analyzed genetic alternations in rpsL gene of clinically isolates of M. tuberculosis, about 60% of SM resistant isolates were shown point mutation in this gene ant they were all high SM-resistant with MIC > or = 256 micrograms/ml. Furthermore, nicotinamidase (pncA) gene, DNA gyrase A subunit (gyrA) gene, and embB gene were reported as the responsible gene to pyrazinamide-, quinolone- and ethambutol-resistance, respectively. Although all mechanisms of drug-resistance were still unclear, these informations are very useful and helpful for development of rapid diagnosis system of drug-resistant M. tuberculosis.     

The practical application on the numbers of control in 1:R matched case-control study

OBJECTIVE: To investigate the molecular mechanism of INH-resistance and the relationship between INH-resistance and KatG mutations. METHODS: Use the primers designed from the 5' end of the KatG gene to amplify 282bp fragments from INH-susceptible and -resistant M. tuberculosis isolates. Single strand conformation polymorphism (SSCP) for their PCR products were analysed. RESULTS: No katG gene sequence deletion was observed, mutations were detected in 17 of 30 INH-resistant strains and no gene perturbations were shown in 16 INH-susceptible isolates. CONCLUSION: The results suggested that mutation in katG gene was one of the most important INH-resistant mechanisms in M. tuberculosis.     

Growth of virulent and avirulent Mycobacterium tuberculosis strains in human macrophages

Mycobacterium tuberculosis H37Rv causes progressive disease in animals, whereas the H37Ra strain does not. The relevance of this difference in virulence to human infection is uncertain because these strains have been shown to have similar growth rates in human macrophages. To evaluate the intracellular growth of M. tuberculosis strains in macrophages under conditions similar to those encountered in vivo, we infected human monocyte-derived macrophages with H37Ra, H37Rv, or one of four isolates from tuberculosis patients at a low bacillus-to-macrophage ratio. H37Rv and the patient isolates grew significantly faster than H37Ra, based on the numbers of CFU and acid-fast bacilli. These findings did not result from extracellular mycobacterial growth, differential macrophage viability, or bacillary clumping. In contrast to other published results, these findings indicate that the virulence characteristics of M. tuberculosis strains in animal models are relevant to human tuberculosis infection.     

Characteristics and outcome of anti-glomerular basement membrane disease: a single-center experience

The relative virulence and avirulence of Mycobacterium tuberculosis strains H37Rv and H37Ra were previously defined using animal infection models. To investigate host species' specificity of mycobacterial virulence, growth of the 2 M. tuberculosis strains in human monocyte-derived macrophages in vitro was studied. Mycobacterial growth was evaluated by acid-fast staining, electron microscopy, and colony-forming units (cfu) assay. As expected, the 2 strains demonstrated significantly different growth rates in mouse macrophages in vitro (53 h for H37Rv, 370 h for H37Ra). In marked contrast, in human macrophages the average division times of the strains were nearly equal (80 h for H37Rv and 76 h for H37Ra by cfu measurement, and 96 h for H37Rv and 104 h for H37Ra by acid-fast staining). These findings indicate that observations of mycobacterial virulence in murine systems may not necessarily translate to the human system, in which different mechanisms to control mycobacterial growth may be expressed.     

Mechanisms involved in the intrinsic isoniazid resistance of Mycobacterium avium

Isoniazid (INH), which acts by inhibiting mycolic acid biosynthesis, is very potent against the tuberculous mycobacteria. It is about 100-fold less effective against Mycobacterium avium. This difference has often been attributed to a decreased permeability of the cell wall. We measured the rate of conversion of radiolabelled INH to 4-pyridylmethanol by whole cells and cell-free extracts and estimated the permeability barrier imposed by the cell wall to INH influx in Mycobacterium tuberculosis and M. avium. There was no significant difference in the relative permeability to INH between these two species. However, the total conversion rate in M. tuberculosis was found to be four times greater. Examination of in vitro-generated mutants revealed that the major resistance mechanism for both species is loss of the catalase-peroxidase KatG. Analysis of lipid and protein biosynthetic profiles demonstrated that the molecular target of activated INH was identical for both species. M. avium, however, formed colonies at INH concentrations inhibitory for mycolic acid biosynthesis. These mycolate-deficient M. avium exhibited altered colony morphologies, modified cell wall ultrastructure and were 10-fold more sensitive to treatment with hydrophobic antibiotics, such as rifampin. These findings may significantly impact the design of new therapeutic regimens for the treatment of infections with atypical mycobacteria.     

What significance should we attribute to the detection of MLL fusion transcripts?

Isoniazid (INH) activation in vitro is associated with reduction of the mycobacterial ferric KatG catalase-peroxidase by hydrazine and reaction with O2 to form an oxyferrous enzyme complex. Since this complex could also form directly via reaction of ferric KatG with superoxide, intracellular activation might be responsive to superoxide concentration. When Mycobacterium smegmatis carrying the M. bovis katG gene was treated with nontoxic levels of plumbagin, a generator of superoxide, the bacteriostatic activity of INH increased unless a plasmid-borne superoxide dismutase gene was also present. Thus, endogenous superoxide probably contributes to intracellular activation of INH.     

Oxidative stress response and characterization of the oxyR-ahpC and furA-katG loci in Mycobacterium marinum

Oxidative stress response in pathogenic mycobacteria is believed to be of significance for host-pathogen interactions at various stages of infection. It also plays a role in determining the intrinsic susceptibility to isoniazid in mycobacterial species. In this work, we characterized the oxyR-ahpC and furA-katG loci in the nontuberculous pathogen Mycobacterium marinum. In contrast to Mycobacterium smegmatis and like Mycobacterium tuberculosis and Mycobacterium leprae, M. marinum was shown to possess a closely linked and divergently oriented equivalents of the regulator of peroxide stress response oxyR and its subordinate gene ahpC, encoding a homolog of alkyl hydroperoxide reductase. Purified mycobacterial OxyR was found to bind to the oxyR-ahpC promoter region from M. marinum and additional mycobacterial species. Mobility shift DNA binding analyses using OxyR binding sites from several mycobacteria and a panel of in vitro-generated mutants validated the proposed consensus mycobacterial recognition sequence. M. marinum AhpC levels detected by immunoblotting, were increased upon treatment with H2O2, in keeping with the presence of a functional OxyR and its binding site within the promoter region of ahpC. In contrast, OxyR did not bind to the sequences upstream of the katG structural gene, and katG expression did not follow the pattern seen with ahpC. Instead, a new open reading frame encoding a homolog of the ferric uptake regulator Fur was identified immediately upstream of katG in M. marinum. The furA-katG linkage and arrangement are ubiquitous in mycobacteria, suggesting the presence of additional regulators of oxidative stress response and potentially explaining the observed differences in ahpC and katG expression. Collectively, these findings broaden our understanding of oxidative stress response in mycobacteria. They also suggest that M. marinum will be useful as a model system for studying the role of oxidative stress response in mycobacterial physiology, intracellular survival, and other host-pathogen interactions associated with mycobacterial diseases.     

Identification and characterization of a new organic hydroperoxide resistance (ohr) gene with a novel pattern of oxidative stress regulation from Xanthomonas campestris pv. phaseoli

We have isolated a new organic hydroperoxide resistance (ohr) gene from Xanthomonas campestris pv. phaseoli. This was done by complementation of an Escherichia coli alkyl hydroperoxide reductase mutant with an organic hydroperoxide-hypersensitive phenotype. ohr encodes a 14.5-kDa protein. Its amino acid sequence shows high homology with several proteins of unknown function. An ohr mutant was subsequently constructed, and it showed increased sensitivity to both growth-inhibitory and killing concentrations of organic hydroperoxides but not to either H2O2 or superoxide generators. No alterations in sensitivity to other oxidants or stresses were observed in the mutant. ohr had interesting expression patterns in response to low concentrations of oxidants. It was highly induced by organic hydroperoxides, weakly induced by H2O2, and not induced at all by a superoxide generator. The novel regulation pattern of ohr suggests the existence of a second organic hydroperoxide-inducible system that differs from the global peroxide regulator system, OxyR. Expression of ohr in various bacteria tested conferred increased resistance to tert-butyl hydroperoxide killing, but this was not so for wild-type Xanthomonas strains. The organic hydroperoxide hypersensitivity of ohr mutants could be fully complemented by expression of ohr or a combination of ahpC and ahpF and could be partially complemented by expression ahpC alone. The data suggested that Ohr was a new type of organic hydroperoxide detoxification protein.     

Chemokine production by a human alveolar epithelial cell line in response to Mycobacterium tuberculosis

To investigate the role of chemokines during the initial local response to Mycobacterium tuberculosis in the human lung, we studied chemokine production by the human alveolar epithelial cell line A549 after infection with M. tuberculosis. M. tuberculosis-infected A549 cells produced mRNAs and protein for monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) but not mRNAs for macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES. Chemokine production in response to M. tuberculosis was not dependent on production of tumor necrosis factor alpha, IL-1beta, or IL-6. Two virulent clinical M. tuberculosis isolates, the virulent laboratory strain H37Rv, and the avirulent strain H37Ra elicited production of comparable concentrations of MCP-1 and IL-8, whereas killed M. tuberculosis and three Mycobacterium avium strains did not. The three virulent M. tuberculosis strains grew more rapidly than the avirulent M. tuberculosis strain in the alveolar epithelial cell line, and the three M. avium strains did not grow intracellularly. These findings suggest that intracellular growth is necessary for mycobacteria to elicit production of MCP-1 and IL-8 by alveolar epithelial cells but that virulence and the rate of intracellular growth do not correlate with chemokine production. Alveolar epithelial cells may contribute to the local inflammatory response in human tuberculosis by producing chemokines which attract monocytes, lymphocytes, and polymorphonuclear cells.     

In vitro and in vivo studies on the role of dimethylsulfoxide (DMSO) in resensibilization of bacterial strains resistant to antibiotics and chemotherapeutic agents

Resensibilization in vitro to seven antibiotics under the influence of DMSO was studied in 624 resistant strains of five species of bacteria (E. coli, S. typhi, S. pyogenes, S. viridans, S. aureus), 61 strains of tubercle bacilli resistant to isonicotinic acid hydrazide (INH) and 19 strains of tubercle bacilli resistant to rifampicin (RMP). DMSO in concentrations of 0.1-10.0% caused reversion of sensitivity in strains of E. coli, S. pyogenes and S. viridans. Reversion in vivo of sensitivity to INH of tubercle bacilli was studied in experimental tuberculosis of guinea pigs. Tubercle bacilli previously resistant to INH recovered complete sensitivity to the drug, enabling animals infected with the INH-resistant strain of bacilli to be treated with INH.     

Inhibition of pulmonary granuloma formation in mice by treatment with Mycobacterial protoplasm and immuno-suppressants and its relation to protection against aerosol infection with virulent Mycobacterium tuberculosis

Because the pulmonary granuloma formation by mycobacterial cell walls (CW) is likely mediated by cellular immunity, the effect of specific antigens and various immunosuppressants on pulmonary granuloma formation and protection against virulent m. tuberculosis, strain H37Rv was investigated in mice immunized with oil-treated CW from BCG or M. Tuberculosis, strain Aoyama B. Results of desensitization experiments with specific antigens showed that multiple intravenous injections of BCG protoplasm inhibited the footpad delayed hypersensitivity, macrophage migration inhibitory activity of lung cells and pulmonary granuloma formation as well as the enhancement of resistance against aerosol challenge with M, tuberculosis, strain H37Rv. However, treatment with nonspecific immunosuppressants like con A, silica or carrageenan induced only abrogation of footpad delayed hypersensitivity but not prevention of pulmonary granuloma formation or resistance against aerosol challenge. These results suggest a close relationship between pulmonary granuloma formation and protection against H37Rv in mice immunized with oil-treated mycobacterial CW.     

NADH dehydrogenase defects confer isoniazid resistance and conditional lethality in Mycobacterium smegmatis

Isoniazid (INH) is a highly effective drug used in the treatment and prophylaxis of Mycobacterium tuberculosis infections. Resistance to INH in clinical isolates has been correlated with mutations in the inhA, katG, and ahpC genes. In this report, we describe a new mechanism for INH resistance in Mycobacterium smegmatis. Mutations that reduce NADH dehydrogenase activity (Ndh; type II) cause multiple phenotypes, including (i) coresistance to INH and a related drug, ethionamide; (ii) thermosensitive lethality; and (iii) auxotrophy. These phenotypes are corrected by expression of one of two enzymes: NADH dehydrogenase and the NADH-dependent malate dehydrogenase of the M. tuberculosis complex. The genetic data presented here indicate that defects in NADH oxidation cause all of the mutant traits and that an increase in the NADH/NAD+ ratio confers INH resistance.     

Fluctuating currents in puncture therapy

Antibodies to Mycobacterium tuberculosis antigens H37Rv and reverse strains previously isolated from patients with sarcoidosis with granular isolates were determined in 50 patients with sarcoidosis (including 16 patients isolating granular types) and 56 patients with tuberculosis, by using ELISA and immunoblotting. Serum antibodies from patients with sarcoidosis were ascertained to more commonly react in ELISA with the antigen (ultrasound disintegrant (USDs) obtained from reverse mycobacteria isolated (initially) from patients with sarcoidosis (AGS) than with the USD of the M. tuberculosis H37Rv (AGT) and, on the contrary, serum antibodies from patients with tuberculosis more frequently reacted with the M. tuberculosis H37Rv. The spectrum of serum antibodies from patients with sarcoidosis greatly differed at immunoblotting with AGS and AGT. There was most commonly a reaction with the antigenic determinants 79, 27, 30, and 50 kDa to AGS and that of the determinants 17, 35, 32 kDa to AGT.     

Haemolytic activity of Mycobacterium spp

Haemolytic activity of clinical isolates of Mycobacterium bacilli (98) was determined by the method of King et al., 1993. During 3-h incubation, all M. tuberculosis (MTB) isolates (28) and one out of 38 M. avium-intracellulare (MAI) strains, produced a strong contact-dependent haemolysin (CDH). Six MAI strains expressed a weak CDH. One MAI isolate produced a strong and five other MAI strains a weak contact-independent haemolysin (CIH). Two M. bovis BCG strains and 7 M. vaccae strains did not demonstrate haemolytic activity. The persistence of chosen Mycobacterium strains differing by haemolytic activity, in the spleens of infected C57BL/6 mice was examined. Mycobacteria producing a strong CDH (MTB H37Rv, MTB 101/92, MAI 83/93) or CIH (MAI 475/93) survived in the spleens of nonimmunized or M. bovis BCG-immunized mice for longer time than MAI strains expressing weak haemolytic activity or M. bovis BCG vaccine strain.     

Relationship between lipid composition and antibiotic-resistance to isoniazid, streptomycin, p-aminosalicylic acid, ethambutol, rifampicin in mycobacteria

The content of neutral lipids, peptido-glycolipids and phospholipids, separated by solvent extractions and TLC, was investigated and compared in various strains of Mycobacterium tuberculosis and in Mycobacterium avium. The examined strains could be divided into two groups: (i) those possessing a stable INH-resistance with a chromosomal location for the resistance genes, in which the content of total lipids and the distribution of lipid subfractions were modified with respect to the control strain H37Rv; (ii) those strains, such as the mutants resistant to streptomycin (Sm), ethambutol (EM), p-aminosalicylic acid (PAS) and rifampicin (R/AMP), uncorrelated drugs with different genetic location of the antibiotic-resistance, in which a disturbance of the lipid metabolism does not appeared to be significant. TLC and chemical analysis of INH-mutants suggested that the decrease of the cell lipids was associated with the peptidoglyco-phospholipid fraction, while the neutral lipids were characterized by a most elevated amount of both menaquinone-9 and free fatty acids, and by comparable levels of both glycerides and total fatty acids with respect to the sensitive strain. In the pattern of fatty acid shorter than C26 of all the fractions studied, no significant differences were observed between the H37Rv strain and the other resistant strains, including the M. avium. In the course of the lipid analysis no unusual phospholipids in addition to CL, PE and phosphatidylinositol-oligo-mannoside (PIM) were detected. Reasoning in a speculative way, the dramatic fall of the complex lipid elaboration in the INH-resistant mycobacteria, may underline a role for these substances in the envelope of the mycobacterial cell that defends itself from the selecting attack of the antibiotic molecule and that keeps this phenotypic expression of INH-resistance in the mutants.     

Evidence for differential binding of isoniazid by Mycobacterium tuberculosis KatG and the isoniazid-resistant mutant KatG(S315T)

Isoniazid is a mainstay of antibiotic therapy for the treatment of tuberculosis, but its molecular mechanism of action is unclear. Previous investigators have hypothesized that isoniazid is a prodrug that requires in vivo activation by KatG, the catalase-peroxidase of Mycobacterium tuberculosis, and that resistance to isoniazid strongly correlates with deletions or point mutations in KatG. One such mutation, KatG(S315T), is found in approximately 50% of clinical isolates exhibiting isoniazid resistance. In this work, 1H nuclear magnetic resonance T1 relaxation measurements indicate that KatG and KatG(S315T) each bind isoniazid at a position approximately 12 A from the active site heme iron. Electron paramagnetic resonance spectroscopy revealed heterogeneous populations of high-spin ferric heme in both wild-type KatG and KatG(S315T) with the ratios of each species differing between the two enzymes. Small changes in the proportions of these high-spin species upon addition of isoniazid support the finding that isoniazid binds near the heme periphery of both enzymes. Titration of wild-type KatG with isoniazid resulted in the appearance of a "type I" substrate-induced difference spectrum analogous to those seen upon substrate binding to the cytochromes P450. The difference spectrum may result from an isoniazid-induced change in a portion of the KatG heme iron from 6- to 5-coordinate. Titration of KatG(S315T) with isoniazid failed to produce a measurable difference spectrum indicating an altered active site configuration. These results suggest that KatG(S315T) confers resistance to isoniazid through subtle changes in the isoniazid binding site.     

Mycobacterium tuberculosis invades and replicates within type II alveolar cells

Although Mycobacterium tuberculosis is assumed to infect primarily alveolar macrophages after being aspirated into the lung in aerosol form, it is plausible to hypothesize that M. tuberculosis can come in contact with alveolar epithelial cells upon arrival into the alveolar space. Therefore, as a first step toward investigation of the interaction between M. tuberculosis and alveolar epithelial cells, we examined the ability of M. tuberculosis to bind to and invade alveolar epithelial cells in vitro. The H37Rv and H37Ra strains of M. tuberculosis were cultured to mid-log phase and used in both adherence and invasion assays. The A549 human type II alveolar cell line was cultured to confluence in RPMI 1640 supplemented with 5% fetal bovine serum, L-glutamine, and nonessential amino acids. H37Rv was more efficient in entering A549 cells than H37Ra, Mycobacterium avium, and Escherichia coli Hb101, and nonpiliated strain (4.7% +/- 1.0% of the initial inoculum in 2 h compared with 3.1% +/- 0.8%, 2.1% +/- 0.9%, and 0.03% +/- 0.0%, respectively). The invasion was more efficient at 37 degrees C than 30 degrees C (4.7% +/- 1.0% compared with 2.3% +/- 0.8%). H37Rv and H37Ra were both capable of multiplying intracellularly at a similar ration over 4 days. Binding was inhibited up to 55.7% by anti-CD51 antibody (antivitronectin receptor), up to 55% with anti-CD29 antibody (beta(1) integrin), and 79% with both antibodies used together. Update of M. tuberculosis H37Rv was microtubule and microfilament dependent. It was inhibited by 6l.4% in the presence of 10 micron colchicine and by 72.3% in the presence of 3 micron cytochalasin D, suggesting two separate pathways for uptake. Our results show that M. tuberculosis is capable of invading type II alveolar epithelial cells and raise the possibility that invasion of alveolar epithelial cells is associated with the pathogenesis of lung infection.     

Inhibition of a Mycobacterium tuberculosis beta-ketoacyl ACP synthase by isoniazid

Although isoniazid (isonicotinic acid hydrazide, INH) is widely used for the treatment of tuberculosis, its molecular target has remained elusive. In response to INH treatment, saturated hexacosanoic acid (C26:0) accumulated on a 12-kilodalton acyl carrier protein (AcpM) that normally carried mycolic acid precursors as long as C50. A protein species purified from INH-treated Mycobacterium tuberculosis was shown to consist of a covalent complex of INH, AcpM, and a beta-ketoacyl acyl carrier protein synthase, KasA. Amino acid-altering mutations in the KasA protein were identified in INH-resistant patient isolates that lacked other mutations associated with resistance to this drug.     

The effect of oxygenated mycolic acid composition on cell wall function and macrophage growth in Mycobacterium tuberculosis

There are three major structural classes of mycolic acids in the cell envelope of Mycobacterium tuberculosis (MTB): alpha-, methoxy- and ketomycolate. The two oxygen-containing classes are biosynthetically related through a common alpha-methyl hydroxymycolate intermediate. BCG strains that fail to produce methoxymycolate and instead produce only keto- and alpha-mycolic acids show apparent defects in the O-methyltransferase MMAS-3. Overproduction of MMAS-3 from MTB resulted in a complete replacement of ketomycolate by methoxymycolate in both BCG and MTB. In vitro growth of these recombinant strains lacking ketomycolate was impaired at reduced temperatures but appeared to be normal at 37 degrees C. Glucose uptake was significantly decreased in such strains, but uptake of chenodeoxycholate and glycine was unaffected. Although sensitivity to INH remained unchanged, these cells were found to be hypersensitive to ampicillin and rifampicin. Infectivity of BCG and H37Rv wild type or MMAS-3 overproducers in THP-1 cells was somewhat affected, but the ability of the strains lacking ketomycolate to grow within this macrophage-like cell line was severely compromised. In vivo labelling of mycolic acids during growth of H37Rv within THP-1 cells revealed a substantial increase in ketomycolate and alphamycolate synthesized by intracellularly grown mycobacteria. These results establish a critical role for mycolate composition in proper cell wall function during the growth of MTB in vivo.     

Relationship between streptomycin susceptibility and rpsL mutations of Mycobacterium tuberculosis strains

The relationship between streptomycin (SM) susceptibility and rpsL mutations of Mycobacterium tuberculosis strains was studied. Of 18 clinically isolated SM-resistant M.tuberculosis strains, mutation was suspected in 9 strains (50%) with SM MICs of > or = 256 micrograms/ml by PCR-single strand conformation polymorphism targeting rpsL gene. On the other hand, using PCR-direct sequence method, amino acid substitution caused by single nucleotide point mutation in rpsL gene was demonstrated in 11 out of 18 strains (61%). The same amino acid substitution at codon 43 (Lys-->Arg) was observed in all 11 strains with SM MICs of > or = 256 micrograms/ml. In addition, PCR products obtained from these 11 strains could not be cut by a restriction enzyme, Mbo II, while H37Rv strain and the other 32 strains with SM MICs of < 256 micrograms/ml were cut into 2 fragments. In conclusion, our results suggest that highly SM-resistant M.tuberculosis strains with MICs of > or = 256 micrograms/ml could be rapidly and easily detected by the restriction enzymatic method.