Description of a novel selective medium (Abscessus Selective Agar; ASA) for the isolation of Mycobacterium abscessus complex

Abstract

Aims and objectives: Mycobacterium abscessus complex consisting of M. abscessus subsp. massiliense, M. abscessus subsp. bolletii and M. abscessus subsp. abscessus, have now emerged as clinically important NTM pathogens in patients with cystic fibrosis (CF). Culturally, these organisms are difficult to isolate from sputum amongst mixed populations of extremely rapidly growing Gram-negatives, particularly Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Achromobacter spp. Our objective was to develop a highly selective medium, not requiring decontamination of CF sputum but which exploited M. abscessus complex s resistance to several antibiotics. Methods: ASA medium was formulated employing Glucose, 16g/l; Agar, 20g/l; Yeast extract, 30g/l; Peptone, 6.8g/l; Chloramphenicol, 50mg/l; Ceftazidime, 32mg/l; Colistin, 24mg/l; Trimethoprim, 21.33mg/l; Sulfamethoxazole, 106.67mg/l and Novobiocin, 50mg/l. Mycobacterium abscessus complex organisms consisting of M. abscessus subsp. massiliense (n=2), M. abscessus subsp. bolletii (n=2) and M. abscessus subsp. abscessus (n=2) [circa 106 colony forming units (CFU)] were plated onto ASA medium and cultured aerobically for 2 weeks at 30oC. In addition, the following non-NTM organisms were also plated onto ASA medium, as above, Gram-positive: Enterococcus faecalis, Staphylococcus aureus (n=6), Staphylococcus aureus (mucoid), MRSA (Hospital-associated), MRSA (Livestock-associated cc398); Gram-negative: Achromobacter sp. (n=4), Burkholderia gladioli, Burkholderia multivorans, Escherichia coli (n=2), Pseudomonas aeruginosa (n=7), Salmonella enterica subsp. enterica serotype Nottingham, Salmonella enterica subsp. enterica serotype Typhimurium & Stenotrophomonas maltophilia. Following incubation, growth was assessed semiquantitatively, as +++, ++, +, few or no growth. Results: ASA medium supported the prolific growth (+++) of all M. abscessus complex organisms, but inhibited all non NTM organisms from growing (no growth). Conclusions: ASA medium is highly selective, which allowed for the proliferation of all members of the Mycobacterium abscessus complex, but successfully inhibited all other bacterial flora in CF sputum. This medium shows potential in isolating M. abscessus complex organisms from polymicrobial specimens/environments.