Identification of mycobacteria through conventional microbiological methods is cumbersome and time-consuming. Recently we have developed a novel bacterial identification method to accurately and rapidly identify different mycobacteria directly from water and clinical isolates. The method utilizes the PCR to amplify a portion of the small subunit rRNA from mycobacteria. The 5' PCR primer has a fluorescent label to allow detection of the amplified product. The PCR product is digested with restriction endonucleases, and an automated DNA sequencer is employed to determine the size of the labeled restriction fragments. Since the PCR product is labeled only at the 5' end, the analysis identifies only the restriction fragment proximal to the 5' end. Each mycobacterial species has a unique 5' restriction fragment length for each specific endonuclease. However, frequently the 5' restriction fragments from different species have similar or identical lengths for a given endonuclease. A set of judiciously chosen restriction enzymes produces a unique set of fragments for each species, providing us with an identification signature. Using this method, we produced a library of 5' restriction fragment sizes corresponding to different clinically important mycobacteria. We have characterized mycobacterial isolates which had been previously identified by biochemical test and/or nucleic acid probes. An analysis of these data demonstrates that this protocol is effective in identifying 13 different mycobacterial species accurately. This protocol has the potential of rapidly (less than 36 h) identifying mycobacterial species directly from clinical specimens. In addition, this protocol is accurate, sensitive, and capable of identifying multiple organisms in a single sample.
We examined potable water in Los Angeles, California, as a possible source of infection in AIDS and non-AIDS patients. Nontuberculous mycobacteria were recovered from 12 (92%) of 13 reservoirs, 45 (82%) of 55 homes, 31 (100%) of 31 commercial buildings, and 15 (100%) of 15 hospitals. Large-restriction-fragment (LRF) pattern analyses were done with AseI. The LRF patterns of Mycobacterium avium isolates recovered from potable water in three homes, two commercial buildings, one reservoir, and eight hospitals had varying degrees of relatedness to 19 clinical isolates recovered from 17 patients. The high number of M. avium isolates recovered from hospital water and their close relationship with clinical isolates suggests the potential threat of nosocomial spread. This study supports the possibility that potable water is a source for the acquisition ofM. avium infections.
Mycobacterium avium is a cause of disseminated disease in AIDS patients. A need for a better understanding of possible sources and routes of transmission of this organism has arisen. This study utilized a PCR typing method designed to amplify DNA segments located between the insertion sequences IS1245 and IS1311 to compare levels of relatedness of M. avium isolates found in patients and foods. Twenty-five of 121 food samples yielded 29 mycobacterial isolates, of which 12 wereM. avium. Twelve food and 103 clinical M. aviumisolates were tested. A clinical isolate was found to be identical to a food isolate, and close relationships were found between two patient isolates and two food isolates. Relatedness between food isolates and patient isolates suggests the possibility that food is a potential source of M. avium infection. This study demonstrates a rapid, inexpensive method for typing M. avium, possibly replacing pulsed-field gel electrophoresis.
A variety of foods collected from local supermarkets and produce stands were examined as possible sources of nontuberculous mycobacterial exposure. Food samples were combined with sterile ultrapure water and manually shaken. To remove large particles, the suspensions were filtered through a sterile strainer, centrifuged, and the supernatants were discarded. The food pellets were stored at -75 degrees C. The pellets were treated with either oxalic acid or sodium hydroxide-sodium citrate solutions to reduce contamination by nonmycobacterial organisms. Decontaminated pellets were cultured on both Middlebrook 7H10C agar and Middlebrook 7H10C agar with supplemental malachite green. Plates were observed for growth at 2 and 8 weeks. Isolates demonstrating acid-fastness were identified to species using polymerase chain reaction and restriction enzyme analysis. Nontuberculous mycobacteria (NTM) were recovered from 25 of 121 foods. Six different species of NTM were isolated, the most predominant being Mycobacterium avium.
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