Reiner M. Kroppenstedt

A numerical study of the fatty acid patterns of 263 reference strains belonging to the genera Arthrobacter, Aureobacterium, Brevibacterium, Cellulomonas, Clavibacter, Corynebacterium, Curtobacterium, Erysipelothrix, Microbacterium, and Rhodococcus was undertaken based on cultural and chemical standardized techniques. Clustering was by the unweighted pair group method using the correlation coefficient. Two cluster groups could be defined at the 62% level, one containing strains characterized by saturated and monounsaturated fatty acids and the second group characterized by iso- and anteiso-branched fatty acids. Within the first cluster group, a clear separation of strains assigned to the genera Rhodococcus, Erysipelothrix, and Corynebacterium could be achieved. Furthermore, strains of the species Corynebacterium glutamicum, Corynebacterium ammoniagenes, Corynebacterium diphtheriae, 'Corynebacterium ulcerans,' and Erysipelothrix rhusiopathiae could be found in distinct clusters, based on quantitative differences in fatty acid patterns. Within the second cluster group, a high degree of similarity between the genera Aureobacterium, Cellulomonas, Clavibacter, Curtobacterium, and Microbacterium found in phylogenetically based studies could be shown also by fatty acid patterns. Several strains of the plant pathogenic coryneform bacteria assigned to the genus Clavibacter and Curtobacterium flaccumfaciens were found within one cluster, indicating a high similarity between these genera. Strains of the genus Arthrobacter were grouped into three adjacent clusters and could not be differentiated by fatty acid patterns. The results of the study are essentially in line with a previously published numerical survey and with other chemotaxonomic and genetic data. Thus, quantitative fatty acid patterns are recommended for identification of several coryneform bacterial genera. In some cases an identification at the species level is possible.Key words: fatty acid analysis, coryneform bacteria, differentiation, numerical analysis.

Abstract Bacterial menaquinones were separated isocratically on a reverse phase Li Chrosorb RP18 5 μm and a silver loaded ion exchanger. On octyldecylsilica support the separation of the menaquinones depends on their lipophilic character, on the silver column mainly on the number of double bonds in the isoprenyl chain. Comparing the runs of both columns the menaquinones were easily differentiated.

The possibility that the strains included within the Mycobacterium avium complex (MAC), but not belonging either to M. avium or to Mycobacterium intracellulare, may be members of undescribed taxa, has already been questioned by several taxonomists. A very homogeneous cluster of 12 strains characterized by identical nucleotide sequences both in the 16S rDNA and in the 16S-23S internal transcribed spacer was investigated. Similar strains, previously reported in the literature, had been assigned either to the species M. intracellulare on the basis of the 16S rDNA similarity or to the group of MAC intermediates. However, several phenotypical and epidemiological characteristics seem to distinguish these strains from all other MAC organisms. The unique mycolic acid pattern obtained by HPLC is striking as it is characterized by two clusters of peaks, instead of the three presented by all other MAC organisms. All of the strains have been isolated from humans and all but one came from the respiratory tract of elderly people. The clinical significance of these strains, ascertained for seven patients, seems to suggest an unusually high virulence. The characteristics of all the strains reported in the literature, genotypically identical to the ones described here, seem to confirm our data, without reports of isolations from animals or the environment or, among humans, from AIDS patients. Therefore, an elevation of the MAC variant was proposed and characterized here, with the name Mycobacterium chimaera sp. nov.; this increases the number of species included in the M. avium complex. The type strain is FI-01069T (=CIP 107892T=DSM 44623T).

Results of deoxyribonucleic acid (DNA)-ribosomal ribonucleic acid and DNA-DNA hybridizations, together with a phenotypic and chemotaxonomic analysis, revealed that nitrogen-fixing bacteria isolated from roots and stems of sugarcane belong to a new species in the genus Acetobacter, for which the name Acetobacter diazotrophicus sp. nov. is proposed. Strain LMG 7603 (= Döbereiner PA1 5 = ATCC 49037) is the type strain.

A novel genus-specific PCR for mycobacteria with simple identification to the species level by restriction fragment length polymorphism (RFLP) was established using the 16S-23S ribosomal RNA gene (rDNA) spacer as a target. Panspecificity of primers was demonstrated on the genus level by testing 811 bacterial strains (122 species in 37 genera from 286 reference strains and 525 clinical isolates). All mycobacterial isolates (678 strains among 48 defined species and 5 indeterminate taxons) were amplified by the new primers. Among nonmycobacterial isolates, only Gordonia terrae was amplified. The RFLP scheme devised involves estimation of variable PCR product sizes together with HaeIII and CfoI restriction analysis. It yielded 58 HaeIII patterns, of which 49 (84%) were unique on the species level. Hence, HaeIII digestion together with CfoI results was sufficient for correct identification of 39 of 54 mycobacterial taxons and one of three or four of seven RFLP genotypes found in Mycobacterium intracellulare and Mycobacterium kansasii, respectively. Following a clearly laid out diagnostic algorithm, the remaining unidentified organisms fell into five clusters of closely related species (i.e., the Mycobacterium avium complex or Mycobacterium chelonae-Mycobacterium abscessus) that were successfully separated using additional enzymes (TaqI, MspI, DdeI, or AvaII). Thus, next to slowly growing mycobacteria, all rapidly growing species studied, including M. abscessus, M. chelonae, Mycobacterium farcinogenes, Mycobacterium fortuitum, Mycobacterium peregrinum, and Mycobacterium senegalense (with a very high 16S rDNA sequence similarity) were correctly identified. A high intraspecies sequence stability and the good discriminative power of patterns indicate that this method is very suitable for rapid and cost-effective identification of a wide variety of mycobacterial species without the need for sequencing. Phylogenetically, spacer sequence data stand in good agreement with 16S rDNA sequencing results, as was shown by including strains with unsettled taxonomy. Since this approach recognized significant subspecific genotypes while identification of a broad spectrum of mycobacteria rested on identification of one specific RFLP pattern within a species, this method can be used by both reference (or research) and routine laboratories.