Middlebrook 7H9 Broth Research Articles

UV Light Inactivation of Mycobacterium avium subsp. paratuberculosis in Milk as Assessed by FASTPlaque TB 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).

Synthesis and <i> in vitro</i> antitubercular activity of 7-substituted fluoroquinolones

Three novel fluoroquinolones with general structure 1-t-butyl-7-[4-substituted (piperazin-1-yl/piperidin-1-yl)]-6-fluoro-4-oxo-1,4-dihydro-quinoline-3-carboxylic acids were synthesized and evaluated for antitubercular activity in vitro against M. tuberculosis H37Rv in Middlebrook 7H9 broth using sparfloxacin, ciprofloxacin and isoniazid as standards. Test compounds were found to be less potent than sparfloxacin; however, one of the compounds was more potent than ciprofloxacin.

Examination of Mycobacterium tuberculosis sigma factor mutants using low-dose aerosol infection of guinea pigs suggests a role for SigC in pathogenesis

Secondary sigma factors in bacteria direct transcription of defence regulons in response to specific stresses. To identify which sigma factors in the human respiratory pathogen Mycobacterium tuberculosis are important for adaptive survival in vivo, defined null mutations were created in individual sigma factor genes. In this study, in vitro growth virulence and guinea pig pathology of M. tuberculosis mutants lacking functional sigma factors (SigC, SigF, or SigM) were compared to the parent strain, H37Rv. None of the mutant strains exhibited a growth deficiency in Middlebrook 7H9 broth, nor were any impaired for intracellular replication in the human monocytic macrophage cell-line THP-1. Following low-dose aerosol infection of guinea pigs, however, differences could be detected. While a SigM mutant resulted in lung and spleen granulomas of comparable composition to those found in H37Rv-infected animals, a SigF mutant was partially attenuated, exhibiting necrotic spleen granulomas and ill-defined lung granulomas. SigC mutants exhibited attenuation in the lung and spleen; notably, necrotic granulomas were absent. These data suggest that while SigF may be important for survival in the lung, SigC is likely a key regulator of pathogenesis and adaptive survival in the lung and spleen. Understanding how SigC mediates survival in the host should prove useful in the development of anti-tuberculosis therapies.

Identification of a Novel Protein with a Role in Lipoarabinomannan Biosynthesis in Mycobacteria

All species of Mycobacteria synthesize distinctive cell walls that are rich in phosphatidylinositol mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). PIM glycolipids, having 2-4 mannose residues, can either be channeled into polar PIM species (with 6 Man residues) or hypermannosylated to form LM and LAM. In this study, we have identified a Mycobacterium smegmatis gene, termed lpqW, that is required for the conversion of PIMs to LAM and is highly conserved in all mycobacteria. A transposon mutant, Myco481, containing an insertion near the 3' end of lpqW exhibited altered colony morphology on complex agar medium. This mutant was unstable and was consistently overgrown by a second mutant, represented by Myco481.1, that had normal growth and colony characteristics. Biochemical analysis and metabolic labeling studies showed that Myco481 synthesized the complete spectrum of apolar and polar PIMs but was unable to make LAM. LAM biosynthesis was restored to near wild type levels in Myco481.1. However, this mutant was unable to synthesize the major polar PIM (AcPIM6) and accumulated a smaller intermediate, AcPIM4. Targeted disruption of the lpqW gene and complementation of the initial Myco481 mutant with the wild type gene confirmed that the phenotype of this mutant was due to loss of LpqW. These studies suggest that LpqW has a role in regulating the flux of early PIM intermediates into polar PIM or LAM biosynthesis. They also suggest that AcPIM4 is the likely branch point intermediate in polar PIM and LAM biosynthesis.

生物発光法による抗らい菌活性測定法の開発

Antimicrobial susceptibility testing of Mycobacterium leprae by non-radioactive bioluminescence assay was developed. Optimization of the assay conditions such as temperature and time for ATP extraction, bacteria dose, preparation of bacteria suspension and pH of culture medium was carried out using M. leprae Thai 53 strain. Samples of bacterial suspension of M. leprae were first treated with filamentous cell treatment reagent at room temperature for 30 minutes and ATP was extracted from the leprosy bacilli by heating at 60 degrees for five minutes. Luciferin luciferase was added to the extract after cooling to room temperature followed by measurement of relative light units (RLU) of each sample using a luminometer. The concentrations of the drugs used for the evaluation of antimicrobial activities of rifampin (RFP), clofazimine (CLF), ofloxacin (OFLX) and clarithromycin (CAM) were 0.125, 0.50, 2.0 and 8.0 microg/ml respectively. Middlebrook 7H9 broth medium was used (pH6.6) as the basal medium and the bacilli were cultivated at 32 C for 0-14 days. ATP was extracted from 0.1 ml of culture suspension and inhibition of the luminescent activity was calculated. The results were compared to that obtained by radio-active CO2 detection system, Buddemeyer method which is commonly used for measuring anti-M. leprae activity. There was a good correlation between the results obtained by ATP method on the tenth day of culture and the results obtained by Buddemeyer method on the seventh day of culture. ATP method may be useful for the determination of drug susceptibility ofM. leprae.

Determination of in vitro synergy when three antimicrobial agents are combined against Mycobacterium tuberculosis

We determined the in vitro antimycobacterial activity of rifampicin, isoniazid and a third agent in combination using a three-dimensional chequerboard in Middlebrook 7H9 broth microdilutions. Of 28 agents screened, ethambutol, streptomycin, clarithromycin, minocycline, ciprofloxacin, levofloxacin, sparfloxacin, gatifloxacin and sitafloxacin were potentially synergistic. A further three-dimensional chequerboard assay quantitatively looked for synergy against ten clinical isolates of Mycobacterium tuberculosis, including seven multidrug-resistant isolates. Sitafloxacin, gatifloxacin and clarithromycin showed significant synergy, with fractional inhibitory concentration indices ranging from 0.41 to 0.79, 0.39 to 0.90 and 0.48 to 0.95, respectively. It is concluded that three-dimensional chequerboard assay can quantitatively determine antimycobacterial synergy, and that fluoroquinolones and antibacterial agents such as clarithromycin are effective against multidrug-resistant isolates of M. tuberculosis when combined with rifampicin and isoniazid.

Investigation of the impact of simulated commercial centrifugation and microfiltration conditions on levels of Mycobacterium avium ssp. paratuberculosis in milk

The potential for physical removal of Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) from milk by centrifugation and microfiltration was investigated by simulating commercial processing conditions in the laboratory by means of a microcentrifuge and syringe filters, respectively. Results indicated that both centrifugation of preheated milk (60°C) at 7000 × g for 10 s, and microfiltration through a filter of pore size 1.2 µm, were capable of removing up to 95–99.9% of M. paratuberculosis cells from spiked whole milk and Middlebrook 7H9 broth suspensions, respectively. Centrifugation and microfiltration may therefore have potential application within the dairy industry as pretreatments to reduce M. paratuberculosis contamination of raw milk.

Compartmentalization of Lipid Biosynthesis in Mycobacteria

The plasma membrane of Mycobacterium sp. is the site of synthesis of several distinct classes of lipids that are either retained in the membrane or exported to the overlying cell envelope. Here, we provide evidence that enzymes involved in the biosynthesis of two major lipid classes, the phosphatidylinositol mannosides (PIMs) and aminophospholipids, are compartmentalized within the plasma membrane. Enzymes involved in the synthesis of early PIM intermediates were localized to a membrane subdomain termed PMf, that was clearly resolved from the cell wall by isopyknic density centrifugation and amplified in rapidly dividing Mycobacterium smegmatis. In contrast, the major pool of apolar PIMs and enzymes involved in polar PIM biosynthesis were localized to a denser fraction that contained both plasma membrane and cell wall markers (PM-CW). Based on the resistance of the PIMs to solvent extraction in live but not lysed cells, we propose that polar PIM biosynthesis occurs in the plasma membrane rather than the cell wall component of the PM-CW. Enzymes involved in phosphatidylethanolamine biosynthesis also displayed a highly polarized distribution between the PMf and PM-CW fractions. The PMf was greatly reduced in non-dividing cells, concomitant with a reduction in the synthesis and steady-state levels of PIMs and amino-phospholipids and the redistribution of PMf marker enzymes to non-PM-CW fractions. The formation of the PMf and recruitment of enzymes to this domain may thus play a role in regulating growth-specific changes in the biosynthesis of membrane and cell wall lipids.

Activity of capuramycin analogues against Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium intracellulare in vitro and in vivo.

The antimycobacterial activities of RS-112997, RS-124922 and RS-118641, three capuramycin analogues that inhibit phospho-N-acetylmuramyl-pentapeptide translocase, were tested against clinical isolates of Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium intracellulare. MICs were determined by the broth microdilution method using a modified Middlebrook 7H9 broth. RS-118641 was the most potent compound overall. The MIC50/90 (mg/L) results for RS-118641 were: M. tuberculosis, 1/2; multidrug-resistant (MDR) M. tuberculosis, 0.5/2; M. avium, 4/8; and M. intracellulare, 0.06/0.5. No statistically significant differences in MIC distributions were observed between non-MDR and MDR M. tuberculosis for any of the capuramycin analogues tested. In order to evaluate the therapeutic efficacy of RS-112997 and RS-124922 in a murine lung model of tuberculosis, both compounds were administered intranasally at 0.1 or 1 mg/mouse/day for 12 days. The mycobacterial load in the lungs was significantly lower in all treatment groups than in the untreated controls. Additional experiments were performed to evaluate the therapeutic efficacy of the three compounds against the M. intracellulare infection in mice. All compounds were administered intranasally at 0.1 mg/mouse/day for 21 days. The mycobacterial load in the lungs was significantly lower in all treatment groups than in the untreated controls. These results suggest that capuramycin analogues exhibit strong antimycobacterial potential and should be considered for further evaluation in the treatment of M. tuberculosis and M. avium-M. intracellulare complex infections in humans.

Comparison of inhibitory effect of rifalazil and rifampicin against Mycobacterium ulcerans infection induced in mice

Buruli ulcer is a human skin disease caused by Mycobacterium ulcerans infection, which is characterized by massive skin ulceration and persistent necrotic change. In recent years Buruli ulcer has rapidly emerged as an increasingly important cause of human morbidity around the world. The disease is endemic at least 32 countries in Africa, Western Pacific, Asia and South America, and it is considered the third most common mycobacterial infection of humans after tuberculosis and leprosy. An effective chemotherapeutic regimen against Buruli ulcer disease has not been established to date. In this study, the inhibitory effect of rifalazil (RLZ) against M. ulcerans was assessed in experimentally infected mice and compared to that of rifampicin (RFP). Five-week-old BALB/c female mice were challenged with 25 microliters (CFU = 4 x 10(4) of M. ulcerans cultured in Middlebrook 7H9 broth in bilateral hind footpads. Mice were administered per os with a suspension of RLZ or RFP at 2.5, 5, or 10 mg/kg once daily 5 times per week starting from one day up to 6 weeks after infection. During the treatment, mice were observed weekly for footpad skin lesions and examined for footpad swelling. In addition, CFU enumeration was done on both hind footpads and spleen at 2, 4, and 6 weeks after initiating treatment. In the infected control mice group, slightly erythematous lesions and moderate swelling of footpads were observed 4 weeks after the infection. Ulcerative lesion was observed 6 weeks after the infection. Mean log10 CFU/footpad (FP) was 5.22 on day 1 after the infection and increased to 5.56, 6.29, and 7.33 at 2, 4, and 6 weeks after treatment was initiated in the treated groups. On the other hand, no visible erythema, swelling or ulcerative lesion in footpads were observed in RLZ-administered groups. Furthermore, log10 CFU/FP decreased to 4.14 after only 2 weeks of initiating treatment in 2.5 mg/kg administered group, i.e. the lowest dose employed group. Log10 CFU/FP decreased to < 2.1 in 6 weeks in the 10 mg/kg administered group, which was close to the detection limit (< 1.7) of the CFU assay. By contrast, inhibitory effect on disease progression and reduction of CFU were observed only in the group of mice given 10 mg/kg among RFP-administered groups: the reduction of CFU was not observed in the early period but 6 weeks after initiating treatment. These results clearly demonstrate that the in vivo anti-M.ulcerans activity of RLZ is much higher than RFP. RLZ activity against M. ulcerans can be expected to control the disease progression in the clinical applications.

Intracellular lipophilic inclusions of mycobacteria in vitro and in sputum.

Although most mycobacterial lipids are thought to be associated with the cell envelope, the authors previously observed substantial deposits of intracellular lipophilic material. A Nile-red-based cytological assay was used to determine factors which affect the presence and natural history of intracellular lipophilic inclusions (ILIs) in Mycobacterium smegmatis. Development of ILIs was associated with stationary-phase cultures in broth and with aged (6 days) colonies on agar. Using variants of Youmans' defined medium, the frequency and size of ILIs was observed to be minimal in carbon-poor medium. ILIs were observed to form within 15 min after provision of fatty acids to the medium and after a period of several days in nitrogen-poor medium. Analysis of the non-polar lipid extracts of ILI-rich and -poor preparations indicated that the triacylglycerols (TAGs) were a major component of the inclusions. The acyl substituents of the TAGs varied according to whether they were formed in Middlebrook 7H9 broth, in low-nitrogen Youmans' broth or rapidly after oleic acid supplementation of Youmans' broth. These studies support a storage function for TAGs in mycobacteria in addition to their previously suggested occurrence as components of the cell envelope. To assess a possible role for ILIs in Mycobacterium tuberculosis, a combined acid-fast (Auramine)/Nile red assay was applied to heavily positive sputum samples from patients with tuberculosis. Strong intracellular Nile red signals were obtained from acid-fast cells, indicating that ILI occur in M. tuberculosis in vivo. This may reflect a distinct physiological state of these cells, which it has not been possible to reproduce in vitro. These findings indicate that the uptake of long-chain fatty acids and TAG biosynthetic and degradative pathways are important aspects of mycobacterial lipid metabolism, meriting further investigation.

Menadione-catalyzed luminol chemiluminescent assay for viability of Mycobacterium bovis.

Stable luminol chemiluminescence was observed 10 min after the addition of menadione to a suspension of Mycobacterium bovis homogenized in Middlebrook 7H9 broth base including OADC enrichment. The chemiluminescence intensity was proportional to the absorbance of the bacterial suspension at 600 nm in a range of 0.005 to 0.15. Luminol chemiluminescence disappeared after 10 min incubation of M. bovis at over 60% of ethanol or 4 days of cultivation of M. bovis in the presence of 40 microg/ml of streptomycin. The bacterium showing the disappearance of chemiluminescence could not grow after being washed, suggesting that the inhibition concentration of the antimicrobials can be estimated on the basis of the disappearance of chemiluminescence. Menadione-catalyzed luminol chemiluminescent assay was rapid and sensitive in comparison to turbidimetry, tetrazolium (WST-8) reduction assay, and the assay using the Mycobacteria growth indicator tube (MGIT).

Application of IS900 PCR for Detection of Mycobacterium avium subsp. paratuberculosis Directly from Raw Milk

A polymerase chain reaction (PCR)-based assay was developed for detection of insertion sequence 900 (IS900) of Mycobacterium avium subsp. paratuberculosis in raw milk. This IS900 PCR assay included DNA extraction and PCR assay using commercially available kits. The DNA extraction and PCR assay were optimized to detect the IS900 sequence directly from raw milk. The IS900 PCR assay was evaluated by inoculating raw bulk milk and Middlebrook's 7H9 broth with 0 to 108 cfu/ml of each of four American Type Culture Collection strains of M. paratuberculosis. Under experimental conditions, both milk culture on Herrold's egg yolk medium slants, and IS900 PCR could detect 10 to 100 cfu/ml of M. paratuberculosis. Detection of M. paratuberculosis by IS900 PCR was consistent (24/24 PCR assays) when about 100 cfu/ml were present, whereas detection was variable (12/24 PCR assays) at concentrations as low as 10 cfu/ml. Based on the findings of the experimental study, IS900 PCR was further evaluated with pooled quarter milk samples from 211 cows from five herds with known history of Johne's disease. Out of 211 animals examined, nine (4%) and 69 (33%) were positive for M. paratuberculosis by milk culture and IS900 PCR from milk, respectively. A total of 20 bulk tank milk sample aliquots (one sample, four aliquots from each herd) were also examined, of which 10 (50%) were positive for M. paratuberculosis by IS900 PCR. By contrast, only one out of 20 (5%) bulk tank milk sample aliquots was positive by culture. The IS900 PCR amplified product of 229-bp obtained on testing of quarter milk and bulk tank milk samples was confirmed to be the IS900 of M. paratuberculosisy by DNAsequence analysis. The results of this study suggest that M. paratuberculosis can be detected directly from quarter milk and bulk tank milk by IS900 PCR.

Differential expression of sigE by Mycobacterium tuberculosis during intracellular growth

The Mycobacterium tuberculosis sigE gene encodes a sigma factor that is a member of the extracytoplasmic function subfamily of sigma factors. Using RT-PCR we demonstrated that sigE is expressed in M. tuberculosis bacilli during growth in human macrophages beginning after 30 min but before 6 h after infection through at least 5 days after infection, but that sigE is not expressed by M. tuberculosis bacteria during growth in Middlebrook 7H9 broth medium. However, sigE expression can be induced by treatment of broth cultures with hydrogen peroxide. Further,sigE is not expressed by M. tuberculosis bacilli during attachment or growth in type II pneumocytes. Using a green fluorescent protein (GFP) reporter gene fused to the sigE promoter, we observed induction of GFP expression following macrophage infection. Western blotting confirmed that sigE protein expression correlated with mRNA expression in induced systems. Analysis of the region of the M. tuberculosis genome encoding sigE suggested it is part of an operon consisting of sigE-orf1-htrA-orf2. The data presented in this report showed that sigE is differentially expressed by M. tuberculosis bacilli in macrophages and might play a role in the pathogenesis of this organism.

Binding of bovine growth hormone to Mycobacterium avium ss paratuberculosis

Mycobacterium avium ss paratuberculosis causes a chronic progressive enteritis in cattle and other ruminants referred to as Johne's disease. It also has been suggested by some as possibly being associated with Crohn's disease in humans. In a previous study we observed that incubation of bovine monocytes with recombinant bovine growth hormone (bGH) altered the ingestion and intracellular growth of M. avium ss paratuberculosis in vitro. This led us to investigate whether bGH also has a direct effect on M. avium ss paratuberculosis. We observed that addition of bGH (5 μg/ml) had a direct inhibitory effect on the growth of M. avium ss paratuberculosis in Middlebrook 7H9 broth. In contrast, the growth of Mycobacterium smegmatis was unaffected, even at a bGH concentration of 50 μg/ml. Using 125I-bGH we observed high affinity binding ( K d=1.32 nM) of bGH to M. avium ss paratuberculosis, with an estimated 204 binding sites per bacillus. To the best of our knowledge, this is the first report of a mammalian hormone binding to this important enteric pathogen.

Laboratory media for the cultivation of tubercle bacillus

A variety of different media for the cultivation of mycobacteria have been described but a few of them are in use today. Those currently used can be characterized by three basic types. The first is egg-based media represented by Ogawa and Löwenstein-Jensen. The second type is agar-based media; the most common one are Middlebrook 7H10 and 7H11. The third type is liquid media such as Middlebrook 7H9. Several weeks of incubation may be required for the isolation of M. tuberculosis on solid media. Substantial improvement in the time to detection and the recovery rate was realized by using broth-based culture system such as the BACTEC 460TB, Septi-Chek AFB, MGIT and BACTEC 9000. In the BACTEC 460TB system, the mycobacteria is detected radiometrically. The processed specimen is added to a modified 7H9 medium (BACTEC 12B) containing 14C-labeled palmitic acid and an antibiotic complex, PANTA. Mycobacterial growth can be ascertained by the liberation of 14CO2 and detected by BACTEC 460TB instrument. The Septi-Chek AFB is a biphasic medium which combines broth and solid media. The liquid medium is a modified Middlebrook 7H9 in a carbon-dioxide-enriched culture bottle. After inoculation of the sample, the bottle is capped with a slide consisting of three solid media; a non-selective Middlebrook 7H11 agar, an egg-based medium, and chocolate agar. A novel system is the MGIT, which is a nonradiometric broth method for the detection of mycobacteria from clinical specimens. The MGIT consists of a modified Middlebrook 7H9 broth and a sensor embedded in silicone on the bottom of a tube. The appearance of orange-colored fluorescence in the sensor when excited indicates the growth of mycobacteria. MB Redox is a modified, serum-supplemented Kirchner medium containing p-indonitrotetrazolium violet (INT) as an indicator of microbial growth. The INT is reduced by the redox system of the mycobacteria to deep violet-colored formazan. This substance is water insoluble and is reduced to the cell surface, by which bacterial clamps can be easily detected by their violet color. At present, the egg-based media are the first choice for the culture of clinical samples. However, there are advantages to each type of medium and not all strains of mycobacteria can be recovered on a single medium. Therefore, it is recommended that one representative of each type of medium be used for primary isolation; one example in Japan may be Ogawa egg medium in combination with Middlebrook 7H11 and MGIT.

Genetic evaluation for rifampicin-susceptibility of Mycobacterium tuberculosis

We evaluated the relationship between rifampicin (RFP)-susceptibility and genetic alterations in rpoB gene of clinically isolated Mycobacterium tuberculosis strains, collected from different geographic locations within Japan. Alterations in rpoB gene were detected by PCR-direct sequencing analysis, and the minimum inhibitory concentrations (MICs) of RFP were determined by broth microdilution method using Middlebrook 7H9 broth. One hundred and sixty six epidemiologically unrelated M. tuberculosis strains were examined. A total of 99 genetic alterations in the 69 bp core region of rpoB gene were detected in 95 out of 166 strains. Among them, 96 out of 166 strains showed RFP-resistant phenotypes with MICs > or = 2 micrograms/ml. Examination of the correlation between the MICs of RFP and amino acid substitutions in 69 bp core region of rpoB gene revealed that 58 out of 59 strains containing amino acid substitution in codon 531 showed highly RFP-resistant phenotypes with MICs > or = 64 micrograms/ml. In contrast, a variable level of RFP-susceptibility was observed among strains containing amino acid substitutions in either codon 516 or 526. On the other hand, the MICs of three strains with a point mutation in either codon 515 or 533 were all < or = 1 microgram/ml. Our results suggest that rpoB gene sequencing is useful for not only the detection of RFP-resistant M. tuberculosis strains, but also the prediction of RFP-susceptibility of the strains.

Isolation of a 33-kDa protein antigen from delipidified Mycobacterium tuberculosis H 37 Rv

A 33-kDa protein (TB33) was isolated from a delipidated cell sonicate (CS) of Mycobacterium tuberculosis H37Rv (grown in Middlebrook 7H9 broth supplemented with glucose) using immobilized metal affinity chromatography (IMAC) on a nickel-nitrilotriacetic acid (Ni-NTA) column. TB33 could not be isolated from the culture filtrate (CF) of M. tuberculosis H37Rv using Ni-NTA. TB33 was recognized by monoclonal antibodies (mAb) known to react with proteins of M. tuberculosis with a molecular mass of 33/34 kDa; namely, mAb F126-5, F67-1 and F126-2. The N-terminal amino acid sequence of TB33 was found to be Xaa-Xaa-Thr-Pro-Ala-Asp-Val-Ser/Cys-Asn-Val-Ala-Ile and thus, shows identity with the N-terminal of antigen 84 of M. tuberculosis except for two mismatches. Antibodies to TB33 could be raised in mice by administering four injections of TB33 (40 micrograms total protein). Sera from tuberculosis patients reacted with TB33, while those from normal healthy individuals did not.

A new genetic assay for rifampicin susceptibility of Mycobacterium tuberculosis.

A new method for testing rifampicin (RFP) susceptibility of Mycobacterium tuberculosis was developed. This method is based on detection of the internal sequence derived from 71-kDa heat shock protein mRNA in tubercle bacilli heat-treated in the presence of RFP. The target sequence was amplified by reverse transcription and PCR, followed by agarose gel electrophoretic analysis. No amplification occurred in one RFP-susceptible strain by exposure to 45 degrees C for 45 min in Middlebrook 7H9 broth containing RFP (10 micrograms/ml) after overnight incubation at 37 degrees C. On the other hand, an amplified 275-bp product was obtained from the RFP-resistant strain MY-129. In a subsequent trial using 65 clinical isolates, this method defined their RFP susceptibility levels as well as the verification of the MICs obtained by the conventional agar dilution method, with the exception of one RFP-susceptible strain. Thus, this method provides a rapid and practical system to determine RFP susceptibility in M. tuberculosis.

Activities of the benzoxazinorifamycin KRM 1648 and ethambutol against Mycobacterium avium complex in vitro and in macrophages.

KRM 1648 is a 4-aminobenzoxazine derivative of rifamycin S with potent in vitro activity against the Mycobacterium avium complex (MAC); the MIC for 90% of 24 MAC isolates from AIDS patients was 0.25 microgram/ml as determined by a radiometric broth macrodilution assay. KRM 1648 was bactericidal for MAC isolates in Middlebrook 7H9 broth, with a reduction in viability of 1 to 4 orders of magnitude over 72 h. In human macrophages, KRM 1648 also was bactericidal, with a reduction of 3 to 4 orders of magnitude in CFU per ml of macrophage lysate at a concentration of 1 microgram/ml; however, the bactericidal activity varied approximately 10-fold among the three MAC serovars tested. In growth medium, ethambutol potentiated the effect of KRM 1648, but this potentiation was modest when tested against MAC in macrophages and also varied between MAC strains. KRM 1648 has potential as an antimycobacterial agent for MAC disease, perhaps in combination with other agents so that the use of lower dosages of KRM 1648 than are needed with other rifamycins may be possible.