Evaluation of Various Culture Media for Detection of Rapidly Growing Mycobacteria from Patients with Cystic Fibrosis

Abstract

cIsolation of nontuberculous mycobacteria (NTM) from the sputum of patients with cystic fibrosis (CF) is challenging due to overgrowth by rapidly growing species that colonize the lungs of patients with CF. Extended incubation on Burkholderia cepacia selective agar (BCSA) has been recommended as an expedient culture method for the isolation of rapidly growing NTM in this setting. The aim of this study was to assess five selective media designed for the isolation of Burkholderia cepacia complex, along with two media designed for the isolation of mycobacteria (rapidly growing mycobacteria [RGM] medium and Middlebrook 7H11 agar), for their abilities to isolate NTM. All seven media were challenged with 147 isolates of rapidly growing mycobacteria and 185 isolates belonging to other species. RGM medium was then compared with the most selective brand of BCSA for the isolation of NTM from 224 sputum samples from patients with CF. Different agars designed for the isolation of B. cepacia complex varied considerably in their inhibition of other bacteria and fungi. RGM medium supported the growth of all isolates of mycobacteria and was more selective than any other medium. NTM were recovered from 17 of 224 sputum samples using RGM medium, compared with only 7 samples using the most selective brand of BCSA (P ‫؍‬ 0.023). RGM medium offers a superior option, compared to other selective agars, for the isolation of rapidly growing mycobacteria from the sputum of patients with CF. Furthermore, the convenience of using RGM medium enables routine screening for rapidly growing NTM in all submitted sputum samples from patients with CF. Burkholderia cepacia complex (BCC) and nontuberculous mycobacteria (NTM) are both recognized as potentially important pathogens when isolated from the lungs of patients with cystic fibrosis (CF). The use of a selective culture medium is recommended for the isolation of BCC (1), and several brands of such media are commercially available. Rapidly growing mycobacteria (RGM) represent a subset of NTM that generate colonies on solid culture media within 7 days of incubation (2). The predominant species of NTM within the CF population in Europe is Mycobacterium abscessus complex (MABSC) (3-5), and there is convincing evidence that the prevalence of infection by MABSC in the CF population is increasing (3,(5)(6)(7). This rapidly growing species comprises three subspecies, i.e., Mycobacterium abscessus subsp. abscessus, Mycobacterium abscessus subsp. bolletii, and Mycobacterium abscessus subsp. massiliense.Culture of mycobacteria from sputum samples has traditionally relied on methods that were designed to accommodate slowly growing mycobacteria, particularly Mycobacterium tuberculosis, which is comparatively rare among cystic fibrosis patients. Such methods are laborious and expensive, as they involve chemical decontamination of samples and subsequent culture on both liquid and solid media (8, 9). Furthermore, contamination of cultures by faster growing microorganisms may mean that cultures have to be aban...

Performance of RGM Medium for Isolation of Nontuberculous Mycobacteria from Respiratory Specimens from Non-Cystic Fibrosis Patients

“…However, some Gram-negative bacteria, such as B. cepacia complex and Achromobacter sp., could break through and grow on RGM medium. This observation was consistent with the results from previous studies (12,13,15). In our routine mycobacterial culture, we also use additional medium (Mitchison 7H11 selective agar) to enhance growth of mycobacteria by inhibiting other nonmycobacterial organisms.…”

“…The respiratory specimens comprised 160 sputa/tracheal aspirates and 43 bronchoalveolar lavage (BAL) fluids which were submitted for routine AFC without additional specimens requested for the purposes of this study. The RGM medium was prepared and kindly provided by the Microbiology Department, Freeman Hospital, Newcastle upon Tyne, UK, as previously described (12,13). RGM plates may be obtained from this source until they are commercially available.…”

“…Recently, a new selective medium (RGM medium), which is composed of Middlebrook 7H9-based agar supplemented with oleic acid-albumin-dextrose-catalase (OADC) and four antimicrobial agents (12,13), has been shown to have high performance for the recovery of rapidly growing mycobacteria from respiratory specimens from CF patients (12)(13)(14)(15). RGM medium was superior to Burkholderia cepacia complex selective medium and showed performance equivalent to or higher than that of the MGIT system.…”

“…RGM medium was superior to Burkholderia cepacia complex selective medium and showed performance equivalent to or higher than that of the MGIT system. An added advantage was the ability of RGM medium to inhibit growth of other nonmycobacterial organisms in CF patients' respiratory specimens without requirement for a decontamination step (12)(13)(14)(15). The use of an extended incubation period of 28 days allows RGM medium to be used for the isolation of all NTM, rather than simply rapidly growing species, but there are only limited data on the recovery of slowly growing species (15).…”

Comparison of Mycobacterial Growth Indicator Tube with Culture on RGM Selective Agar for Detection of Mycobacteria in Sputum Samples from Patients with Cystic Fibrosis

“…RGM medium was prepared from its basic ingredients at the Microbiology Department, Freeman Hospital, Newcastle upon Tyne, United Kingdom, as previously described (13) and transferred by courier to Bedford Hospital where the culture of the sputum samples was performed. As the medium has been shown to have a shelf life of 12 weeks at 4°C (C. L. Preece and J. D. Perry, unpublished data), two batches of medium were used for the 5-month evaluation.…”