Comparison of Culture Methods for Isolation of Nontuberculous Mycobacteria from Surface Waters

Abstract

ABSTRACT 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 μg/ml amphotericin B, 16 μg/ml nalidixic acid, 4 μg/ml trimethoprim, and 4 μg/ml azlocillin) or PANTAV (PANTA plus 10 μg/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.