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Isolation of nontuberculous mycobacteria from soil using Middlebrook 7H10 agar with increased malachite green concentration.

ABSTRACT: Environmental exposure is considered to be responsible for nontuberculous mycobacterial infections in humans. To facilitate the isolation of mycobacteria from soil, Middlebrook 7H10 agar was optimized as an enhanced selective medium by increasing the concentration of malachite green. A series of modified Middlebrook 7H10 agar media with malachite green concentrations ranging from 2.5 to 2500 mg/L was evaluated using 20 soil samples decontaminated with 3% sodium dodecyl sulfate plus 2% NaOH for 30 min. Among these modified Middlebrook 7H10 media, the medium with malachite green at a concentration of 250 mg/L, i.e., at the same concentration as in Löwenstein-Jensen medium, was the most effective in terms of the number of plates with mycobacterial growth. This medium was further evaluated with 116 soil samples. The results showed that 87.1% (101/116) of the samples produced mycobacterial growth, and 15 samples (12.9%) produced no mycobacterial growth. Of the plates inoculated with the soil samples, each in duplicate, 5.2% (12/232) showed late contamination. In total, 19 mycobacterial species were isolated, including seven (36.8%) rapidly growing mycobacteria and 12 (63.2%) slowly growing mycobacteria. Our results demonstrate that the modified Middlebrook 7H10 agar with 250 mg/L malachite green is useful for the primary isolation of nontuberculous mycobacteria from soil.

SUBMITTER: Hu Y

PROVIDER: S-EPMC5364124 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Isolation of nontuberculous mycobacteria from soil using Middlebrook 7H10 agar with increased malachite green concentration.

Hu Yuli Y Yu Xinglong X Zhao Dun D Li Runcheng R Liu Yang Y Ge Meng M Hu Huican H

AMB Express 20170323 1

Environmental exposure is considered to be responsible for nontuberculous mycobacterial infections in humans. To facilitate the isolation of mycobacteria from soil, Middlebrook 7H10 agar was optimized as an enhanced selective medium by increasing the concentration of malachite green. A series of modified Middlebrook 7H10 agar media with malachite green concentrations ranging from 2.5 to 2500 mg/L was evaluated using 20 soil samples decontaminated with 3% sodium dodecyl sulfate plus 2% NaOH for 3 ...[more]

PMID: 28337735

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Project description:The environment is the likely source of most nontuberculous mycobacteria (NTM) involved in human infections, especially pulmonary, skin, and soft tissue infections. In order to measure the prevalence of NTM in different aquatic ecosystems, we tried to standardize the culture methods used for surface water testing since many procedures have been described previously. Cultivation of mycobacteria requires long-term incubation in rich media and inactivation of rapidly growing microorganisms whose growth impedes observation of mycobacterial colonies. Consequently, the two criteria used for evaluation of the methods examined were (i) the rate of inhibition of nontarget microorganisms and (ii) the efficiency of recovery of mycobacteria. We compared the competitive growth of Mycobacterium chelonae and M. avium with nontarget microorganisms on rich Middlebrook 7H11-mycobactin medium after treatment by several chemical decontamination methods that included acids, bases, detergent, or cetylpyridinium chloride (CPC) with and without an antibiotic cocktail, either PANTA (40 U/ml polymyxin, 4 microg/ml amphotericin B, 16 microg/ml nalidixic acid, 4 microg/ml trimethoprim, and 4 microg/ml azlocillin) or PANTAV (PANTA plus 10 microg/ml vancomycin). Our results showed that treatment for 30 min with CPC (final concentration, 0.05%) of water concentrated by centrifugation, followed by culture on a rich medium supplemented with PANTA, significantly decreased the growth of nontarget microorganisms (the concentrations were 6.2 +/- 0.4 log(10) CFU/liter on Middlebrook 7H11j medium and 4.2 +/- 0.2 log(10) CFU/liter on Middlebrook 7H11j medium containing PANTA [P < 0.001]), while the effect of this procedure on NTM was not as great (the concentrations of M. chelonae on the two media were 7.0 +/- 0.0 log(10) CFU/liter and 6.9 +/- 0.0 log(10) CFU/liter, respectively, and the concentrations of M. avium were 9.1 +/- 0.0 log(10) CFU/liter and 8.9 +/- 0.0 log(10) CFU/liter, respectively). We propose that this standardized culture procedure could be used for detection of NTM in aquatic samples.

Project description:Nontuberculous mycobacterial (NTM) species are assuming public health importance in pulmonary diseases; they are increasingly being isolated, and importantly, most NTMs do not respond to routine tuberculosis (TB) drugs. This study aimed to identify NTMs isolated from pulmonary TB cases and also determine their susceptibility to streptomycin (STR), isoniazid (INH), and rifampicin (RIF).A total of 1755 mycobacterial isolates, obtained between August 2012 and July 2014, from 2036 smear-positive pulmonary cases were identified using polymerase chain reaction amplification of IS6110, and hsp65 gene sequencing analysis. Drug susceptibility testing (DST) was then performed for the identified NTMs against STR, INH, and RIF using microplate Alamar blue assay. The results were analyzed against patients' biodata for statistical associations.Of the 1755 analyzed isolates, we identified 43 (2.5%) NTMs, which included 18 (41.9%) Mycobacterium intracellulare, 13 (30.2%) Mycobacterium avium subs. paratuberculosis, 5 (11.3%) Mycobacterium abscessus, 3 (7.0%) each of Mycobacterium mucogenicum and Mycobacterium colombiense, and 1 (2.3%) Mycobacterium simiae. Patients infected with NTMs (52.0%) were more likely to be human immunodeficiency virus-positive (P = 0.001, odds ratio = 6.6, 95% confidence interval = 2.7-16.2) than those infected with M. tuberculosis complex (5.8%). All the 43 (100%) NTMs were resistant to INH, whereas 32 (74%) and 19 (44%) were resistant to RIF and STR, respectively. Furthermore, 16 (37.2%) NTMs were resistant to all three drugs, 20 were resistant to INH and RIF, and 3 were resistant to STR and INH. All the M. abscessus isolates were resistant to all the three drugs, whereas all the M. avium isolates were resistant to INH and RIF, but only three were resistant to STR. Among the M. intracellulare isolates, 8, 18, and 15 isolates were resistant to STR, INH, and RIF, respectively.The observed high-resistance level to INH and RIF supports the need for rapid species identification and DST of nonresponding TB cases before retreatment.

Dataset Information

Isolation of nontuberculous mycobacteria from soil using Middlebrook 7H10 agar with increased malachite green concentration.

Publications

Isolation of nontuberculous mycobacteria from soil using Middlebrook 7H10 agar with increased malachite green concentration.

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