Evaluation of six decontamination procedures for isolation of Mycobacterium avium complex from avian feces

Abstract

Culture is considered the gold standard for definitive diagnosis of mycobacterial infections. However, consensus about the most suitable culture procedure for isolation of nontuberculous mycobacteria is lacking. The study compared the recoveries of mycobacteria after decontamination of spiked and fresh avian feces with 4% sodium hydroxide (NaOH), 12% sulfuric acid (H2SO4), or 1% cetylperidinium chloride (CPC), with and without mixture of three antibiotics, namely vancomycin (VAN, 100 μg/ml), nalidixic acid (NAL, 100 μg/ml), and amphotericin B (AMB, 100 μg/ml). The antibiotic mixture was referred to as VNA. Decontamination procedures were evaluated using two (n = 2) avian fecal samples spiked with 106, 104, and 102 CFU/ml of Mycobacterium avium subsp. avium (ATCC 15769) and fresh avian feces (n = 42). M. avium subsp. avium was detected on the culture media from spiked samples (106 and 104 CFU/ml) decontaminated with NaOH, NaOH-VNA, H2SO4, and H2SO4 -VNA for 2−6 weeks. These bacteria were detected in 2–4 weeks when using CPC and CPC-VNA. M. avium subsp. avium cannot be isolated on culture media from spiked samples (102 CFU/ml) decontaminated with any decontaminating agent. Two mycobacterial isolates, namely, Mycobacterium terrae and M. engbaekii, were isolated from field samples decontaminated with NaOH and CPC-VNA. With regard to the contamination rate, the use of CPC-VNA showed lower contamination rates (5.5% and 19.0%) from spiked and field samples than those of the other methods (NaOH: 22.2% and 59.5%, NaOH-VNA: 16.7% and 21.4%, H2SO4: 11.1% and 40.5%, H2SO4-VNA: 5.5% and 21.4%, and CPC: 66.7% and 50%). In conclusion, the decontamination of fecal samples following a two-step procedure with 1% CPC and VNA can ensure high recovery rate of many mycobacteria with the lowest contamination in cultures.

Non-Tuberculous Mycobacteria: Molecular and Physiological Bases of Virulence and Adaptation to Ecological Niches

“…Both culture-dependent and independent approaches rely on the intrinsic characteristics of the type of matrices tested. The isolation of mycobacteria, namely the slow-growing, is very difficult from microbially rich environments, for which harsh decontamination is recommended [ 317 ]. In addition, the intrinsic characteristics of several species of Mycobacterium require specific micronutrients (e.g., M. haemophilum ), growth enhancers (e.g., M. genavense ), optimal pH, temperature, and oxygen levels, which allied to a particular sensibility to decontamination (e.g., M. ulcerans ) sets obstacles to bacteriological isolation [ 252 , 318 ].…”

Isolation of Mycobacterium avium and other nontuberculous mycobacteria in chickens and captive birds in peninsular Malaysia

“…Previous studies have shown that time for samples processing is important factor affecting primary isolation of mycobacteria [18]. Given that our previous study [24] reported that CPC-VNA can control the contamination rate of L-J cultures better than other methods, we postulate that the contamination rate (27.9%) of L-J cultures in this study is acceptable.…”

“…Although the recovery of mycobacteria on L-J culture was very low, we postulate that this low isolation rate may reflect the real occurrence of mycobacteria in chickens and birds. Furthermore, CPC-VNA decontamination method may has a favorable impact on culture sensitivity by reducing the contamination rate [14,[23][24][25]36] and increasing the number of positive AFB culture. Isolation of environmental mycobacteria like M. avium, M. terrae and M. engbaekii, which do not possess IS901, may not be pathogenic to the birds because no clinical relevance of these species have been reported [38].…”

“…Preparation of CPC, VNA antibiotic cocktail, and L-J culture were described previously [24]. Briefly, CPC (1%) solution was prepared by adding 5 g of CPC (Merck, Germany) and 10 g of NaCl (Merck, Germany) in 500 mL distilled water.…”

“…Additionally, sensitivity of primary culture for mycobacteria can be improved when a gentle chemical agent is used to kill unwanted bacteria present in the specimen [23]. Studies have shown that the Cetylperidinium chloride -Sodium chloride (CPC) method is favorable alternative method to isolate mycobacteria [24,25] as it is less toxic to mycobacteria [24,25], more effective in controlling culture contamination [23,24], supports early growth of mycobacteria [23,26] and does not need neutralization as its inhibitory effects are neutralized on egg based media [27]. Likewise, the incorporation of a VNA antibiotic cocktail composed by vancomycin (VAN), nalidixic acid (NAL) and amphotericin (AMB), in the decontamination of sample has improved the sensitivity of the primary culture (CPC 27.8% and CPC-VAN 66.7%) and reduced the contamination of cultures (CPC 66.7% % and CPC-VAN 19%) [24].…”