PROCESSING RESPIRATORY SPECIMENS WITH C-18-CARBOXYPROPYLBETAINE - DEVELOPMENT OF A SEDIMENT RESUSPENSION BUFFER THAT CONTAINS LYTIC ENZYMESTO REDUCE THE CONTAMINATION RATE AND LECITHIN TO ALLEVIATE TOXICITY

The C-18-carboxypropylbetaine (CB-18) procedure for processing respira tory specimens for the detection of mycobacteria was shown to provide significant increases in sensitivity by smear and culture. However, th e procedure also produced increased contamination, a loss in liquid cu lture sensitivity, and a reduction in smear specificity. Because of th ese observations, the toxicity of CB-18 and the nature of the contamin ation were characterized. Preincubation in 1 mM CB-18 impacted viabili ty in a time-dependent fashion, but the magnitude of the loss was spec ies and isolate dependent. Mycobacterium tuberculosis isolates were th e most susceptible, losing 20 to 30% of the CFU within 30 min and 30 t o 60% after 3 h, whereas Mycobacterium avium and Mycobacterium fortuit um isolates were unaffected by CB-18. In liquid culture, when the conc entration of CB-18 exceeded 5 mu g/ml, there was an impact on growth c haracteristics for the most susceptible M. tuberculosis isolate. In co ntrast, M. fortuitum isolates were able to grow in 100 mu g of CB-18 p er mi. In liquid culture, the deleterious effects of CB-18 were enhanc ed in the presence of antibiotics, whereas growth on solid media was n ot 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 t hese contaminants led to the development of a sediment resuspension bu ffer 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 contami nation to acceptable levels (<5%).