7H11 Agar Research Articles

Simplified diagnostic susceptibility testing of mycobacteria against thiacetazone, hydroxylamine, p-nitrobenzoic acid and picric acid

In order to simplify diagnostic susceptibility testing of mycobacteria against thiacetazone, hydroxylamine, p-nitrobenzoic acid and picric acid, we modified the standard procedure by using Middlebrook 7H11 agar in a single quadrant petri dish. This plate can be inoculated and read in parallel with routine susceptibility tests done by the proportion method. We present results of 449 consecutive routine strains tested with our method.

Drug susceptibility testing in tuberculosis: A comparison of the proportion methods using Lowenstein-Jensen, middlebrook 7H10 and 7H11 agar media and a radiometric method

In the present investigation, the conventional drug susceptibility testing procedures using the 1% proportion method on Löwenstein-Jensen (LJ) and Middlebrook 7H10/7H11 agar media were compared with a recent radiometric method. The latter using “Bactec 460-TB” apparatus and a special broth medium (Becton and Dickinson Diagnostic Instrument Systems, Towson, MD, USA) gave drug susceptibility results within a week as compared to 4–5 weeks with the LJ method, and 3–4 weeks using 7H10/7H11 agar media. This report describes the critical concentrations retained for each of the methods and the methodology used. Also, the compatibility of the results obtained with the radiometric method with those obtained with conventional 7H11 agar method was perfect. In this report, we justify the choice of the 7H11 agar method instead of the LJ method as our conventional drug susceptibility procedure, and suggest also that radiometric drug susceptibility determination by Bactec 460-TB apparatus may be the method of choice for routine, rapid drug susceptibility determinations.

New antibiotics, resorcinomycins A and B: antibacterial activity of resorcinomycin A against mycobacteria in vitro.

Resorcinomycin A, N-[(S)-alpha-guanidino-3,5-dihydroxy-4-isopropylphenylacetyl]glyci ne, S-RSM-A, a new antibiotic produced by Streptoverticillium roseoverticillatum, has an antibacterial spectrum directed towards mycobacterial species. It is not active against Gram-negative and Gram-positive bacteria, except mycobacteria and weakly active against mycoplasmas. In vitro activities of the S-, R- and S,R-isomers of RSM-A against atypical mycobacterial strains were compared with those of streptomycin (SM) and kanamycin (KM) by 2-fold agar dilution methods using Middlebrook 7H10 agar medium. Although R-RSM-A and S,R-RSM-A were comparable or inferior to both antibiotics, the antimycobacterial activity of S-RSM-A was superior to that of SM and KM.

Quality control of individual components used in Middlebrook 7H10 medium for mycobacterial susceptibility testing.

The acceptability of different lots of commercial components which constitute our basal medium for susceptibility testing of mycobacteria was evaluated. The basal medium consisted of Middlebrook 7H10 agar supplemented with 10% oleic acid-albumin-dextrose-catalase and 0.5% glycerol. Studies were performed by using three separate microbiologic assays, and results were compared with parallel tests on previously standardized and acceptable lots of media. Components were rejected if comparison with standardized medium showed a major change in growth support or susceptibility status of any reference strain to any antimicrobial agent tested. Of the components tested in such a manner, 7 of 23 (30%) lots of 10% oleic acid-albumin-dextrose-catalase, 2 of 13 (15%) lots of Middlebrook 7H10 agar, and 0 of 5 lots of glycerol were found to be unacceptable. This study demonstrates that individual lots of components of this basal medium may vary significantly in their suitability for susceptibility testing, and failure to detect such variation may dramatically affect susceptibility profiles.

Minimal Inhibitory Concentrations of Isoniazid, Rifampin, Ethambutol, and Streptomycin againstMycobacterium tuberculosisStrains Isolated before Treatment of Patients in Taiwan

Minimal inhibitory concentrations (MICs) of isoniazid (INH), rifampin (RMP), ethambutol (EMB), and streptomycin (SM) for susceptible "wild" M. tuberculosis strains isolated from Taiwanese patients were within the limits previously reported for strains isolated in the United States. The highest agar-determined MICs (in 7H10 and 7H11 agar) corresponded well with the critical concentrations established for these media. The highest MICs found radiometrically in 7H12 broth were significantly lower than the critical concentrations proposed for this medium. On the basis of an evaluation of the highest broth-determined MICs found in this and in the previous study (1), we suggest that the following MICs, when determined radiometrically, should be used as breakpoints to classify the strain as "susceptible": for INH, 0.1 microgram/ml or less; for RMP, 0.5 microgram/ml or less; for EMB, 4.0 micrograms/ml or less; for SM, 2.0 micrograms/ml or less.

Susceptibility of Mycobacterium avium complex to various two-drug combinations of antituberculosis agents.

The drug susceptibility of two untypable strains of Mycobacterium avium complex were studied in 7H10 agar plates containing ethambutol (EMB), isoniazid (INH), rifampicin (RMP), ethionamide (ETH), and streptomycin (SM) alone and in two-drug combinations. The effective dose inhibiting 75% of the mycobacterial population (ED 75) was estimated by a regression analysis on the probit transformed inhibition percentages and plotted on an isobologram for each combination. No major discrepancies were found between strains. Five combinations (RMP plus INH, RMP plus EMB, EMB plus SM, INH plus EMB, and ETH plus INH) showed synergistic effect, whereas five other combinations (ETH plus EMB, ETH plus RMP, ETH plus SM, SM plus RMP, and SM plus INH) showed antagonistic effect. These in vitro results are not in combination with the known results of treatment of the M. avium diseases. We conclude that the effect of drug combinations against M. avium may be strain dependent and that it is important to determine this effect in vitro before setting up a treatment protocol.

Comparative in vitro and in vivo activity of rifabutin and rifampicin against mycobacterium avium complex

In vitro antimicrobial activity of rifabutin and rifampicin against various mycobacteria, including the Mycobacterium avium complex, was evaluated by the agar dilution method, using 7H10 agar medium. The activity of rifabutin based on MIC 50 and MIC 90 was higher than that of rifampicin, against all the acid-fast organisms tested. Microbicidal activity of rifabutin against the M. avium complex phagocytosed in mouse peritoneal or alveolar macrophages was greater than that of rifampicin. Both rifabutin and rifampicin had therapeutic effects against murine infections induced by M. avium complex. Rifabutin was somewhat more effective than rifampicin in mice.

MIC as a quantitative measurement of the susceptibility of Mycobacterium avium strains to seven antituberculosis drugs

MICs of isoniazid, rifampin, ethionamide, streptomycin, amikacin, kanamycin, and capreomycin were determined for Mycobacterium avium complex strains by two methods: broth dilution in 7H12 medium radiometrically and agar dilution on 7H10 agar plates. The broth-determined MICs of all drugs with the exception of isoniazid were two to eight times lower than the agar-determined MICs for most of the tested M. avium strains, which is probably due to the higher absorption and degradation of the drugs in solid media. The MICs, especially those determined in broth, are suggested as quantitative measurements of the degree of susceptibility of M. avium complex strains. For a certain percentage of the M. avium strains the broth-determined MICs were within the limits of MICs found for wild susceptible Mycobacterium tuberculosis strains. These M. avium strains were classified as presumably susceptible. In contrast to M. tuberculosis, the MICs for M. avium strains had a wide range. When the MICs for M. avium strains were only one dilution higher than those for M. tuberculosis, they were tentatively classified as moderately susceptible. The designation moderately resistant or resistant, respectively, is suggested for those M. avium strains for which the MICs were at or above the concentrations achievable in blood. The quantitation of the degree of susceptibility by the MICs and the tentative interpretation of the MICs are suggested for future use in clinical trials as a means of evaluating the patients' responses to chemotherapy compared with the degree of susceptibility of the initial strain isolated before treatment.

Comparison of media used for the primary isolation of Mycobacteriurm bovis by veterinary and medical diagnostic laboratories

Veterinary and medical laboratories engaged in the cultural diagnosis of bovine or human tuberculosis were requested to supply samples of the media that they routinely use for the primary isolation of M. bovis. Fourteen laboratories supplied 7 basic media types; these were Lowenstein-Jensen, Stonebrink's, modified Middlebrook 7H11 agar, tuberculosis bovine blood agar, egg yolk agar, Gerloff's egg and Herrold's egg yolk. Two strains of M. bovis were used to test the media, strain AN5, a glycerol-tolerant laboratory strain and M86/90 a glycerol-sensitive wildtype strain. AN5 grew well on all media with the exception of Herrold's and strain M86/90 did not grow on media containing glycerol and grew poorly on Herrold's medium. It is recommended that Lowenstein-Jensen with pyruvate (but without glycerol), Stonebrink's, modified Middlebrook 7H11 and tuberculosis bovine blood agar should be considered the media of choice for the primary isolation of M. bovis. Egg yolk agar also proved adequate for this purpose in the trial. This medium may be suitable for routine use but to date experience with its use is limited.

Comparison of three methods for recovery of Mycobacterium avium complex from blood specimens

Three methods were used for the recovery of mycobacteria from blood specimens obtained from acquired immunodeficiency syndrome patients: (i) inoculation of 7H11 agar plates with a concentrated specimen, (ii) inoculation of 7H12 BACTEC vials with a concentrated specimen, and (iii) inoculation of 7H13 BACTEC vials with a nonconcentrated specimen. In this study, we examined 255 specimens and obtained positive mycobacterial growth in 47 of them. Among these 47 cultures, 40 were found to be positive by all three methods, and the total recovery rates in relation to these culture-positive specimens were 94% for method 1, 89% for method 2, and 96% for method 3. The advantages and disadvantages of these three methods are discussed.

Rifabutine: minimal inhibitory and bactericidal concentrations for Mycobacterium tuberculosis.

The minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) of rifabutine (ansamycin LM427, spiropiperidyl rifamycin) against rifampin-susceptible and rifampin-resistant strains of Mycobacterium tuberculosis were determined. The MICs for 17 rifampin-susceptible strains were low, falling in a narrow range of 0.06 microgram/ml or less when determined by either the agar-dilution method (in 7H10 or 7H11 agar) or in 7H12 broth (radiometrically or by sampling and colony-forming units [CFU/ml] determination). The MICs of rifabutine for 21 rifampin-resistant strains were much higher, ranging from 0.25 to 16.0 micrograms/ml. The MBCs of rifabutine were 0.125 or 0.25 microgram/ml for rifampin-susceptible strains, and were 4.0 and 32.0 micrograms/ml for two tested rifampin-resistant strains. The MIC/MBC ratio was 1:4 for rifampin-susceptible strains.

Broth microdilution testing of susceptibilities to 30 antimicrobial agents of Mycobacterium avium strains from patients with acquired immune deficiency syndrome.

A total of 31 strains of Mycobacterium avium complex isolated from patients with acquired immune deficiency syndrome were tested for susceptibility to 30 antimicrobial agents by using microdilution trays containing dried antimicrobial agents. MICs were determined over a period of 7 days of growth in a broth medium (7HSF) that is equivalent to 7H11 agar. MICs obtained by this method showed good agreement with MICs determined by the agar dilution method. Strains could be divided into two groups by their antimicrobial susceptibility patterns. All group 1 strains (8 of the 31 strains tested) were at least moderately susceptible to inhibition by a variety of beta-lactam antimicrobial agents, including amoxicillin-clavulanic acid and cefmenoxime. Group 2 strains (23 of 31) were susceptible only to amikacin (22 of 23 strains). All 31 strains were resistant to oxacillin, clindamycin, erythromycin, tetracycline, chloramphenicol, vancomycin, nitrofurantoin, and aztreonam at the highest concentration of antimicrobial agent present in the microdilution trays. The addition of Tween 80 to 7HSF broth increased the susceptibility of M. avium complex to many of the antimicrobial agents tested. Killing of M. avium complex (i.e., less than or equal to 1% survival after 7 days) was found to vary for different strains and antimicrobial agents. Killing of some strains by amoxicillin-clavulanic acid, carbenicillin, azlocillin, cefmenoxime, cefotaxime, amikacin, and ampicillin occurred at concentrations of antimicrobial agent that are achievable in serum. Further studies are needed to determine whether any of these antimicrobial agents has activity against M. avium complex cells that have been ingested by macrophages.

A more accurate method for measurement of tuberculocidal activity of disinfectants.

The current Association of Official Analytical Chemists method for testing tuberculocidal activity of disinfectants has been shown to be inaccurate and to have a high degree of variability. An alternate test method is proposed which is more accurate, more precise, and quantitative. A suspension of Mycobacterium bovis BCG was exposed to a variety of disinfectant chemicals and a kill curve was constructed from quantitative data. Data are presented that show the discrepancy between current claims, determined by the Association of Official Analytical Chemists method, of selected commercially available products and claims generated by the proposed method. The effects of different recovery media were examined. The data indicated that Mycobacteria 7H11 and Middlebrook 7H10 agars were equal in recovery of the different chemically treated cells, with Lowenstein-Jensen agar having approximately the same recovery rate but requiring incubation for up to 3 weeks longer for countability. The kill curves generated for several different chemicals were reproducible, as indicated by the standard deviations of the slopes and intercepts of the linear regression curves.

Determination of minimal inhibitory concentrations of antituberculosis drugs by radiometric and conventional methods.

Minimal inhibitory Concentrations (MIC) of 5 antituberculosis drugs (isoniazid, rifampin, streptomycin, ethionamide, and ethambutol) were determined by the radiometric (BACTEC) broth method and by the agar plate proportion method. Seventeen M. tuberculosis strains, isolated from patients before treatment, were tested. The MIC values of 4 of the 5 drugs (the exception was streptomycin) were 2 to 4 times lower in 7H12 broth than in 7H11 agar. The broth-determined MIC were also at least 2 to 4 times lower than the achievable serum concentrations. The broth-determined MIC are probably much closer to the true MIC values than those determined in agar plates because of the lower degree of absorption and degradation in the liquid medium. The radiometric broth method is a simple and rapid quantitative method for accurate determination of the MIC values of the antituberculosis drugs. The data obtained in this study will be used for further evaluation of the MIC values as complementary or alternative to the conventional qualitative testing against critical concentrations.

Susceptibility testing of Mycobacterium tuberculois to pyrazinamide

One hundred and sixty clinical isolates of Mycobacterium tuberculois were tested for their sensitivity to pyrazinamide (PZA) using the conventional proportion method (Middlebrook 7H10 agar) and the results compared to the pyrazinamidase (PZase) activity of the strains. The correlation between strains susceptible to PZA and the production of PZase was 99%. None of the isolates were exclusively resistant to PZA. At present the rate of initial PZA resistance in Kanton Zurich is 0.6%.

Laboratory diagnosis of mycobacterial infections in patients with acquired immunodeficiency syndrome.

Disseminated mycobacterial infections are commonly seen in acquired immunodeficiency syndrome (AIDS) patients, and laboratory culture is the best method for diagnosing these infections. In addition to conventional agar media, we used BACTEC 12A (Johnston Laboratories, Inc., Towson, Md.) broth medium for culture. More isolates of Mycobacterium avium complex and Mycobacterium tuberculosis were recovered from 12A broth than from Lowenstein-Jensen or Middlebrook 7H11 agar. Also, the average detection time of these mycobacteria was the earliest with 12A broth. Stool examination has been helpful in diagnosing mycobacterial disease in AIDS patients, and in this study both acid-fast stain and culture of fecal material was necessary for efficient detection of mycobacteria. Another sensitive and practical method for detecting mycobacterial infections in patients with AIDS is the Isolator lysis-centrifugation system (Du Pont Co., Wilmington, Del.) which offers the advantage of quantitating the degree of mycobacteremia. Laboratories should be alerted to the possibility of mixed mycobacterial infection in patients with AIDS, and positive cultures should be repeatedly examined to detect coinfection with a slower-growing mycobacterium such as M. tuberculosis as well as M. avium complex.

Use of a modified 7H-11 agar to increase growth rate of Mycobacterium bovis from bovine tissues.

Australian Veterinary JournalVolume 63, Issue 10 p. 348-349 Use of a modified 7H–11 agar to increase growth rate of Mycobacterium bovis from bovine tissues G M VEERMAN, G M VEERMAN Queensland Department of Primary Industries, Animal Research Institute, Yeerongpilly 4105Search for more papers by this authorJ G PIKE, J G PIKE Queensland Department of Primary Industries, Animal Research Institute, Yeerongpilly 4105Search for more papers by this authorJ L FOX, J L FOX Queensland Department of Primary Industries, Animal Research Institute, Yeerongpilly 4105Search for more papers by this author G M VEERMAN, G M VEERMAN Queensland Department of Primary Industries, Animal Research Institute, Yeerongpilly 4105Search for more papers by this authorJ G PIKE, J G PIKE Queensland Department of Primary Industries, Animal Research Institute, Yeerongpilly 4105Search for more papers by this authorJ L FOX, J L FOX Queensland Department of Primary Industries, Animal Research Institute, Yeerongpilly 4105Search for more papers by this author First published: October 1986 https://doi.org/10.1111/j.1751-0813.1986.tb02890.xCitations: 4AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article.Citing Literature Volume63, Issue10October 1986Pages 348-349 RelatedInformation

Nonweekend schedule for BACTEC drug susceptibility testing of Mycobacterium tuberculosis.

Determination of the drug susceptibility of Mycobacterium tuberculosis by conventional methods using an agar-based medium may take 3 weeks or more to complete. On the other hand, results on positive cultures are generally available in 4 to 7 days with the radiometric (BACTEC, Johnston Laboratories, Towson, Md.) procedure. One disadvantage to the latter is the requirement to determine the quantity of 14CO2 in each test vial on a daily basis from the day of inoculation. Growth index readings often must be made over weekends, adding to the work load of clinical laboratories during periods of reduced staff or necessitating compensatory pay or time. Susceptibility tests with streptomycin, isoniazid, ethambutol, and rifampin against 104 M. tuberculosis strains were performed by the submerged disk method, the recommended BACTEC method with daily growth index readings, and the radiometric procedure with readings delayed for 2 days after inoculation. Criteria for interpretation of "delayed" tests were established. Drug concentrations tested included some modifications of those available commercially. Overall agreement for the four drugs by the three methods was greater than 90%. We conclude that under our test conditions a schedule of inoculation of radiometric test vials on Friday with growth index readings commencing on Monday gives susceptibility results that correlate well with the daily BACTEC method and with a conventional 7H10 agar method.

Determination of in vitro susceptibility of Mycobacterium tuberculosis to cephalosporins by radiometric and conventional methods.

Among eight cephalosporins and cephamycins tested in preliminary in vitro screening against Mycobacterium tuberculosis, the most promising for further study was found to be ceforanide, followed by ceftizoxime, cephapirin, and cefotaxime. Moxalactam, cefoxitin, cefamandole, and cephalothin were found to be not active enough against M. tuberculosis to be considered for further in vitro studies. The antibacterial activity of various ceforanide concentrations was investigated by three methods: (i) the dynamics of radiometric readings (growth index) in 7H12 broth; (ii) the number of CFU in the same medium; and (iii) the proportion method on 7H11 agar plates. There was a good correlation among the results obtained with these methods. The MIC for most strains ranged from 6.0 to 25.0 micrograms/ml. The BACTEC radiometric method is a reliable, rapid, and convenient method for preliminary screening and determination of the level of antibacterial activity of drugs not commonly used against M. tuberculosis.

Radiometric studies with gas-liquid and thin-layer chromatography for rapid demonstration of hemin dependence and characterization of Mycobacterium haemophilum.

Eight isolates of Mycobacterium haemophilum were evaluated by radiometric methods to determine whether this test system could support the growth of these organisms as well as demonstrate their growth requirements for iron complexes such as hemin, ferric ammonium citrate, and blood. In addition, gas-liquid and thin-layer chromatography were evaluated to determine whether these procedures could further differentiate M. haemophilum from other mycobacteria. During the initial 24 to 48 h, there was no significant difference between the radiometric test broths containing iron complexes and control broths without iron supplementation. After 48 h, the test growth index readings rapidly increased, and control broth readings leveled off and declined. The mean growth index reading of the test broths after 6 days of incubation was 100 times that of the controls. The mean incubation time with supplemented 7H10 agar was 17 days. The use of radiometric media resulted in the demonstration of hemin dependence by M. haemophilum significantly earlier than with 7H10 agar. Of the three supplements studied, whole blood provided the greatest growth rate, followed by ferric ammonium citrate and hemin. When 12 species of mycobacteria other than M. haemophilum were radiometrically evaluated, no isolate demonstrated an iron complex requirement. Gas-liquid and thin-layer chromatography procedures were able to rapidly differentiate M. haemophilum from the other 12 Mycobacterium species.