Processing Respiratory Specimens with C18-Carboxypropylbetaine: Development of a Sediment Resuspension Buffer That Contains Lytic Enzymes To Reduce the Contamination Rate and Lecithin To Alleviate Toxicity

The C18-carboxypropylbetaine (CB-18) procedure for processing respiratory specimens for the detection of mycobacteria was shown to provide significant increases in sensitivity by smear and culture. However, the procedure also produced increased contamination, a loss in liquid culture sensitivity, and a reduction in smear specificity. Because of these observations, the toxicity of CB-18 and the nature of the contamination were characterized. Preincubation in 1 mM CB-18 impacted viability in a time-dependent fashion, but the magnitude of the loss was species and isolate dependent. Mycobacterium tuberculosis isolates were the most susceptible, losing 20 to 30% of the CFU within 30 min and 30 to 60% after 3 h, whereas Mycobacterium avium and Mycobacterium fortuitum isolates were unaffected by CB-18. In liquid culture, when the concentration of CB-18 exceeded 5 μg/ml, there was an impact on growth characteristics for the most susceptible M. tuberculosis isolate. In contrast, M. fortuitum isolates were able to grow in 100 μg of CB-18 per ml. In liquid culture, the deleterious effects of CB-18 were enhanced in the presence of antibiotics, whereas growth on solid media was not similarly affected. Supplementation of the resuspension buffer with 0.15% lecithin alleviated toxicity. Initial attempts to modify the CB-18 procedure to control contamination incorporated acids or alkalis; however, losses in culture sensitivity occurred. Studies to identify these contaminants led to the development of a sediment resuspension buffer that contained lytic enzymes to combat contamination and lecithin to alleviate toxicity. This formulation included lysozyme, zymolyase, and Cytophaga and Trichoderma extracts and was seen to reduce contamination to acceptable levels (<5%).