Rapid identification of Mycobacterium avium clinical isolates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

BACKGROUND: Rapid and accurate discrimination of Mycobacterium avium from other mycobacteria is essential for appropriate therapeutic management and timely intervention for infection control. However, routine clinical identification methods for M. avium are both time consuming and labor intensive. In the present study, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to identify specific cellular protein pattern for rapid identification of M. avium isolates. METHODS: A total of 40 clinically relevant Mycobacterium strains comprising 13 distinct species were enrolled for the MALDI-TOF MS identification. A 10-minute extraction-free examination procedure was set up to obtain mass spectral fingerprints from whole bacterial cells. RESULTS: The characteristic mass spectral peak patterns in the m/z (mass/charge ratio) range of 5-20 kDa can be obtained within 10 minutes. The species-specific mass spectra for M. avium is identified and can be differentiated from as Mycobacterium strains. This technique shortens and simplifies the identification procedure of MALDI-TOF MS and may further extend the mycobacterial MALDI-TOF MS database. CONCLUSION: Simplicity and rapidity of identification procedures make MALDI-TOF MS an attractive platform in routine identification of mycobacteria. MALDI-TOF MS is applicable for rapid discrimination of M. avium from other Mycobacterium species, and shows its potential for clinical application.

Identification of legionella species by use of an oligonucleotide array.

The genus Legionella contains a diverse group of motile, asaccharolytic, nutritionally fastidious gram-negative rods. Legionella pneumophila is the most important human pathogen, followed by L. micdadei, L. longbeachae, L. dumoffii, and other rare species. Accurate identification of Legionella spp. other than L. pneumophila is difficult because of biochemical inertness and phenotypic identity of different species. The feasibility of using an oligonucleotide array for identification of 18 species of Legionella was evaluated in this study. The method consisted of PCR amplification of the macrophage infectivity potentiator mip gene, followed by hybridization of the digoxigenin-labeled PCR products to a panel of 30 oligonucleotide probes (16- to 24-mers) immobilized on a nylon membrane. A collection of 144 target strains (strains we aimed to identify) and 50 nontarget strains (44 species) were analyzed by the array. Both test sensitivity (144/144 strains) and specificity (50/50 strains) of the array were 100%. The whole procedure for identification of Legionella species by the array can be finished within a working day, starting from isolated colonies. It was concluded that species identification of clinically relevant Legionella spp. by the array method is very reliable and can be used as an accurate alternative to conventional or other molecular methods for identification of Legionella spp.

Pulsed-field gel electrophoresis analysis of Vibrio vulnificus strains isolated from Taiwan and the United States.

Vibrio vulnificus is a marine bacterium that causes human wound infections and septicemia with a high mortality rate. V. vulnificus strains from different clinical and environmental sources or geographic regions have been successfully characterized by ribotyping and several other methods. Pulsed-field gel electrophoresis (PFGE) is a highly discriminative method, but previous studies suggested that it was not suitable for examining the correlation of V. vulnificus strains from different origins. We employed PFGE to determine its efficacy for characterizing V. vulnificus strains from different geographic regions, characterizing a total of 153 strains from clinical and environmental origins from the United States and Taiwan after SfiI or NotI digestion. V. vulnificus strains showed a high intraspecific diversity by PFGE after SfiI or NotI digestion, and about 12% of the strains could not be typed by the use of either of these enzymes. For PFGE with SfiI digestion, most of the clinical and environmental strains from the United States were grouped into cluster A, while the strains from Taiwan were grouped into other clusters. Clinical strains from the United States showed a higher level of genetic homogeneity than clinical strains from Taiwan, and environmental strains from both regions showed a similarly high level of heterogeneity. PFGE with NotI digestion was useful for studying the correlation of clinical strains from the United States and Taiwan, but it was not suitable for analyzing environmental strains. The results showed that PFGE with SfiI digestion may be used to characterize V. vulnificus strains from distant geographic regions, with NotI being a recommended alternative enzyme.