Jane Tang

Identification of Mycobacteria by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry

ABSTRACT Classical methods for identification of Mycobacterium species rely on morphology and biochemical profiles. Speciation of a Mycobacterium isolate using these standard methods is a lengthy process based on subjective data interpretation. In this study, Mycobacterium species were characterized by utilizing matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). This technology is designed to provide a characteristic mass spectral fingerprint based on desorbed ions from the cell surface. Thirty-seven strains were analyzed; these represented thirteen species and five subspecies that included the Mycobacterium tuberculosis complex and the M. avium-M. intracellulare complex, as well as rapid- and slow-growing mycobacteria. All 37 strains were analyzed in triplicate, and a database was generated. This method produced species-specific patterns for all but 1 of the 37 isolates and provided reliable differentiation at the strain level. The data suggest that whole-cell MALDI-TOF MS has potential as a rapid and reproducible method for the identification and characterization of Mycobacterium species.

A polymerase chain reaction based method for detecting Mycoplasma/Acholeplasma contaminants in cell culture

A detection system that utilizes a primer mixture in a nested polymerase chain reaction for detecting Mycoplasma contaminants in cell cultures is described. Primers were designed to amplify the spacer regions between the 16S and 23S ribosomal RNA genes of Mycoplasma and Acholeplasma. This detection system was able to detect 20-180 colony forming units per milliliter of sample. Eight commonly encountered Mycoplasma and Acholeplasma contaminants, which include Mycoplasma (M.) arginini, M. fermentans, M. hominis, M. hyorhinis, M. orale, M. pirum, M. salivarium, and Acholeplasma laidlawii, were consistently amplified. Mycoplasma contaminants generated a single DNA band of 236-365 base pairs (bp), whereas A. laidlawii produced a characteristic two-band pattern of 426 and 219 bp amplicons. Species identification could be achieved by size determination and restriction enzyme digestion. Minor cross-reactions were noted with a few closely related gram positive bacteria and DNA from rat cell lines. A Mycoplasma Detection Kit for detecting Mycoplasma contaminants in cell cultures has been developed based on this approach.

Captured Diversity in a Culture Collection: Case Study of the Geographic and Habitat Distributions of Environmental Isolates Held at the American Type Culture Collection

The use of molecular techniques to assess prokaryotic diversity independent of the need for enrichment culture has profoundly changed how we view and study microbial diversity. As molecular data have accumulated over the past 15 years, these data are now resulting in a healthy debate about how much we really know about prokaryotic diversity in a wide range of environments (8, 21–23, 32, 44, 55). A number of recent articles have discussed these questions, and opinions range from the position that prokaryotic diversity is essentially infinite, with the existence of millions of potential species, (6, 10), to arguments that microbial diversity is reasonably finite (17, 26). In light of this debate concerning the results of theoretical considerations and molecule-based surveys, it is interesting to take stock of the holdings of the environmental prokaryotes available at the American Type Culture Collection (ATCC) to determine how these holdings reflect overall trends in microbial diversity in different habitats. The principle mission of bioresource centers, such as the ATCC, is to serve as living stock collections that acquire diverse biological materials for redistribution to researchers throughout the world. The ATCC has about two-thirds of the type strains of prokaryotic species in the world. In many cases, multiple strains of a given species are acquired. As these materials are accessioned, data relevant to their provenance and physiology are collected. These data are an underutilized asset of bioresource centers. For example, the data on the source locations of isolates can provide information about the relative sampling efforts for different geographic regions. Concordantly, data concerning the environmental habitats of isolates can provide important information about the diversity of genera that are associated with certain habitat types. No articles were found in a literature search that specifically analyzed culture collections as a metric for assessing our knowledge of larger patterns of microbial diversity. In this article we present an analysis of the holdings of environmental prokaryotes at the ATCC with regard to the geographical and environmental habitats from which they were isolated. The term environmental is broadly defined to include all organisms that are not pathogenic to humans or animals or that are not otherwise human associated (see below). Selected habitats for which holdings at the ATCC are the most abundant were compared to recently published findings of workers who used cultivation-independent methods to assess microbial diversity. This provided perspective on how well the “captured diversity” in a culture collection represents the diversity which may exist in nature.

Proteocatella sphenisci gen. nov., sp. nov., a psychrotolerant, spore-forming anaerobe isolated from penguin guano

A novel, obligately anaerobic, psychrotolerant bacterium, designated strain PPP2T, was isolated from guano of the Magellanic penguin (Spheniscus magellanicus) in Chilean Patagonia. Cells were Gram-stain-positive, spore-forming, straight rods (0.7-0.8x3.0-5.0 microm) that were motile by means of peritrichous flagella. Growth was observed at pH 6.7-9.7 (optimum pH 8.3) and 2-37 degrees C (optimum 29 degrees C). Growth was observed between 0 and 4% (w/v) NaCl with optimum growth at 0.5% (w/v). Strain PPP2T was a catalase-negative chemo-organoheterotroph that was capable of fermentative metabolism. Peptone, bacto-tryptone, Casamino acids, oxalate, starch, chitin and yeast extract were utilized as substrates. The major metabolic products were acetate, butyrate and ethanol. Strain PPP2T was resistant to ampicillin, but sensitive to tetracycline, chloramphenicol, rifampicin, kanamycin, vancomycin and gentamicin. The DNA G+C content of strain PPP2T was 39.5 mol%. Phylogenetic analysis revealed that strain PPP2T was related most closely to Clostridium sticklandii SR (approximately 90% 16S rRNA gene sequence similarity). On the basis of phylogenetic analysis and phenotypic characteristics, strain PPP2T is considered to represent a novel species of a new genus, for which the name Proteocatella sphenisci gen. nov., sp. nov. is proposed. The type strain of Proteocatella sphenisci is PPP2T (=ATCC BAA-755T=JCM 12175T=CIP 108034T).