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Evaluation of a new culture medium for isolation of nontuberculous mycobacteria from environmental water samples.

ABSTRACT: Nontuberculous mycobacteria (NTM) are waterborne pathogens commonly found in building water systems where they are a primary concern to vulnerable patient populations and can cause severe disease. The recovery of NTM from environmental samples can be a laborious undertaking and current pre-treatment methods and selective media lack sensitivity. We explored the use of the highly selective Rapidly Growing Mycobacteria (RGM) medium for culturing NTM from environmental water samples compared to existing methods. In total, 223 environmental water samples, including potable and non-potable water, were cultured for NTM using three culture media. In addition to direct culture on RGM medium, each sample was cultured on Middlebrook 7H10 medium and Mitchison 7H11 medium after pre-treatment with 0.2M KCl-HCl. Additionally, 33 distinct species of NTM were inoculated onto RGM medium and 7H10 medium in parallel to directly compare their growth. The use of RGM medium alone without pre-treatment provided a sensitivity (91%) comparable to that offered by culture on both 7H10 and 7H11 with acid pretreatment (combined sensitivity; 86%) with significantly less overgrowth and interference from other organisms on RGM medium. The average concentration of NTM observed on RGM medium alone was comparable to or greater than the NTM concentration on either medium alone or combined. Thirty-three species were examined in parallel and all tested strains of 27 of these species successfully grew on RGM medium, including 19 of 21 from the CDC's healthcare-associated infections species list. RGM medium was successful at recovering environmental NTM without a pre-treatment, greatly reducing labor and materials required to process samples. Simplification of culture processing for environmental NTM will allow for a better assessment of their presence in building water systems and the potential for reduced exposure of susceptible populations.

SUBMITTER: Alexander KJ

PROVIDER: S-EPMC7928522 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Evaluation of a new culture medium for isolation of nontuberculous mycobacteria from environmental water samples.

Alexander Kimberly J KJ Furlong Jennifer L JL Baron Julianne L JL Rihs John D JD Stephenson Dominic D Perry John D JD Stout Janet E JE

PloS one 20210303 3

Nontuberculous mycobacteria (NTM) are waterborne pathogens commonly found in building water systems where they are a primary concern to vulnerable patient populations and can cause severe disease. The recovery of NTM from environmental samples can be a laborious undertaking and current pre-treatment methods and selective media lack sensitivity. We explored the use of the highly selective Rapidly Growing Mycobacteria (RGM) medium for culturing NTM from environmental water samples compared to exis ...[more]

PMID: 33657154

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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:Purpose Nontuberculous mycobacteria (NTM) are ubiquitous bacteria that are naturally resistant to disinfectants and antibiotics and can colonize systems for supplying drinking water. Therefore, this study aimed to evaluate the prevalence of NTM in the drinking water of six hospitals in Tehran, Iran. Methods Totally, 198 water samples were collected. Each water sample was filtered via a membrane filter with a pore size of 0.45 µm and then decontaminated by 0.005% cetylpyridinium chloride. The membrane filters were incubated on two Lowenstein-Jensen media at 25 °C and 37 °C for 8 weeks. The positive cultures were identified with phenotypic tests, and then NTM species were detected according to the hsp65, rpoB, and 16S rDNA genes. Drug susceptibility testing (DST) was also carried out. Results Overall, 76 (40.4%) of the isolates were slowly growing mycobacteria (SGM) and 112 (59.6%) of the ones were rapidly growing mycobacteria (RGM). The most common NTM were Mycobacterium aurum, M. gordonae, M. phocaicum, M. mucogenicum, M. kansasii, M. simiae, M. gadium, M. lentiflavum, M. fortuitum, and M. porcinum. Among these 188 samples, NTM ranged from 1 to > 300 colony-forming unit (CFU) /500 mL, with a median of 182 CFU/500 mL. In the infectious department of all hospitals, the amount of CFU was higher than in other parts of the hospitals. The DST findings in this study indicated the diversity of resistance to different drugs. Among RGM, M. mucogenicum was the most susceptible isolate; however, M. fortuitum showed a different resistance pattern. Also, among SGM isolates, M. kansasii and M. simiae, the diversity of DST indicated. Conclusions The current study showed NTM strains could be an important component of hospital water supplies and a possible source of nosocomial infections according to the CFU reported in this study. The obtained findings also help clarify the dynamics of NTM variety and distribution in the water systems of hospitals in the research area. Supplementary Information The online version contains supplementary material available at 10.1186/s12866-022-02674-z.

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Evaluation of a new culture medium for isolation of nontuberculous mycobacteria from environmental water samples.

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Evaluation of a new culture medium for isolation of nontuberculous mycobacteria from environmental water samples.

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