Hans-Jürgen Busse

Notes on the characterization of prokaryote strains for taxonomic purposes.

Taxonomy relies on three key elements: characterization, classification and nomenclature. All three elements are dynamic fields, but each step depends on the one which precedes it. Thus, the nomenclature of a group of organisms depends on the way they are classified, and the classification (among other elements) depends on the information gathered as a result of characterization. While nomenclature is governed by the Bacteriological Code, the classification and characterization of prokaryotes is an area that is not formally regulated and one in which numerous changes have taken place in the last 50 years. The purpose of the present article is to outline the key elements in the way that prokaryotes are characterized, with a view to providing an overview of some of the pitfalls commonly encountered in taxonomic papers.

Proposed minimal standards for describing new taxa of aerobic, endospore-forming bacteria

Minimal standards for describing new taxa within the aerobic endospore-forming bacteria are proposed, following Recommendation 30b of the Bacteriological Code (1990 Revision). These minimal standards are recommended as guidelines to assist authors in the preparation of descriptions for novel taxa. They encourage broad polyphasic characterization and the construction of descriptions that are practically useful in routine diagnostic laboratories. The proposals have been endorsed by the Subcommittee on the Taxonomy of the Genus Bacillus and Related Organisms of the International Committee on Systematics of Prokaryotes.

Discrimination of members of the family Pasteurellaceae based on polyamine patterns

In a study of the classification of members of the family Pasteurellaceae, the polyamine patterns of 101 strains were analyzed. These strains included the type strains of species belonging to the genera Actinobacillus, Haemophilus, and Pasteurella and additional strains of selected species, as well as numerous unnamed strains. Members of the genus Actinobacillus sensu stricto were characterized by the presence of 1,3-diaminopropane as the predominant compound. In the majority of the species of the genus Haemophilus sensu stricto 1,3-diaminopropane was also the major compound in the polyamine pattern. In contrast, Haemophilus intermedius subsp. gazogenes and Haemophilus parainfluenzae were characterized by high levels of 1,3-diaminopropane, cadaverine, and putrescine. These results confirmed the findings of Dewhirst et al. (F. E. Dewhirst, B. J. Paster, I. Olsen, and G. J. Fraser, Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 279:35–44, 1993), who demonstrated that H. parainfluenzae is phylogenetically only distantly related to the type species of the genus Haemophilus, Haemophilus influenzae. The phylogenetic diversity of the genus Pasteurella sensu stricto determined by Dewhirst et al. was also reflected to some extent by different polyamine patterns. The common characteristics found in Pasteurella multocida, Pasteurella canis, Pasteurella dagmatis, Pasteurella stomatis, and Pasteurella sp. strain B were high levels of putrescine and spermidine and the presence of the unusual triamine sym-norspermidine. Pasteurella avium, Pasteurella gallinarum, and Pasteurella volantium contained high concentrations of 1,3-diaminopropane and spermidine. Pasteurella langaa contained only high concentrations of 1,3-diaminopropane, and Pasteurella anatis was characterized by the presence of 1,3-diaminopropane as the predominant compound and high levels of putrescine and spermidine. Our data demonstrate that polyamine patterns are useful for discrimination within the family Pasteurellaceae.

Brucella microti sp. nov., isolated from the common vole Microtus arvalis

Two Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains CCM 4915(T) and CCM 4916), isolated from clinical specimens of the common vole Microtus arvalis during an epizootic in the Czech Republic in 2001, were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA (rrs) and recA gene sequence similarities, both isolates were allocated to the genus Brucella. Affiliation to Brucella was confirmed by DNA-DNA hybridization studies. Both strains reacted equally with Brucella M-monospecific antiserum and were lysed by the bacteriophages Tb, Wb, F1 and F25. Biochemical profiling revealed a high degree of enzyme activity and metabolic capabilities not observed in other Brucella species. The omp2a and omp2b genes of isolates CCM 4915(T) and CCM 4916 were indistinguishable. Whereas omp2a was identical to omp2a of brucellae from certain pinniped marine mammals, omp2b clustered with omp2b of terrestrial brucellae. Analysis of the bp26 gene downstream region identified strains CCM 4915(T) and CCM 4916 as Brucella of terrestrial origin. Both strains harboured five to six copies of the insertion element IS711, displaying a unique banding pattern as determined by Southern blotting. In comparative multilocus VNTR (variable-number tandem-repeat) analysis (MLVA) with 296 different genotypes, the two isolates grouped together, but formed a separate cluster within the genus Brucella. Multilocus sequence typing (MLST) analysis using nine different loci also placed the two isolates separately from other brucellae. In the IS711-based AMOS PCR, a 1900 bp fragment was generated with the Brucella ovis-specific primers, revealing that the insertion element had integrated between a putative membrane protein and cboL, encoding a methyltransferase, an integration site not observed in other brucellae. Isolates CCM 4915(T) and CCM 4916 could be clearly distinguished from all known Brucella species and their biovars by means of both their phenotypic and molecular properties, and therefore represent a novel species within the genus Brucella, for which the name Brucella microti sp. nov. with the type strain CCM 4915(T) (=BCCN 07-01(T)=CAPM 6434(T)) is proposed.

Classification and identification of bacteria: current approaches to an old problem. Overview of methods used in bacterial systematics

Most of the bacterial species are still unknown. Consequently, our knowledge about bacterial ecology is poor and expectations about specialized species with novel enzymatic functions or new products are high. Thus, bacterial identification is a growing field of interest within microbiology. In this review, suitability of developments for identification based on miniaturized biochemical and physiological investigations of bacteria are evaluated. Special emphasis is given to chemotaxonomic methods such as analysis of quinone system, fatty acid profiles, polar lipid patterns, polyamine patterns, whole cell sugars, peptidoglycan diaminoacids, as well as analytical fingerprinting methods and cellular protein patterning. 16S rDNA sequencing introduced to investigate the phylogenetic relationships of bacteria, nucleic acids hybridization techniques and G + C content determination are discussed as well as restriction fragment length polymorphism (RFLP), macrorestriction analysis and random amplified polymorphic DNA (RAPD). The importance of the different approaches in classification and identification of bacteria according to phylogenetic relationships are demonstrated on selected examples.

Chryseobacterium hispalense sp. nov., a plant-growth-promoting bacterium isolated from a rainwater pond in an olive plant nursery, and emended descriptions of Chryseobacterium defluvii, Chryseobacterium indologenes, Chryseobacterium wanjuense and Chryseobacterium gregarium

A novel non-motile, Gram-staining-negative, yellow-pigmented bacterium, designated AG13(T), isolated from a rain water pond at a plant nursery in Spain and characterized as a plant-growth-promoting bacterium, was investigated to determine its taxonomic status. The isolate grew best over a temperature range of 15-40 °C, at pH 5.0-8.0 and with 0-4 % (w/v) NaCl. Chemotaxonomic and molecular characteristics of the isolate matched those described for members of the genus Chryseobacterium. The DNA G+C content of the novel strain was 37.2 mol%. The strain had a polyamine pattern with sym-homospermidine as the major compound and produced flexirubin-type pigments. MK-6 was the dominant menaquinone and the major cellular fatty acids were iso-C15 : 0, C17 : 1ω9c and iso-C17 : 0 3-OH. The main polar lipids were phosphatidylethanolamine, aminolipids and several unidentified lipids. The 16S rRNA gene showed 92.0-97.2 % sequence similarity with those of the members of the genus Chryseobacterium. Based on chemotaxonomic and phenotypic traits, and DNA-DNA hybridizations with the type strains of the most closely related species, the isolate is proposed to represent a novel species, Chryseobacterium hispalense, type strain AG13(T) ( = DSM 25574(T) = CCUG 63019(T)). Emended descriptions of the species Chryseobacterium defluvii, Chryseobacterium indologenes, Chryseobacterium wanjuense and Chryseobacterium gregarium are also provided.

Chemotaxonomic characterisation of Sphingomonas.

Based on published results and investigations done for this study, chemotaxonomic characteristics of all validly described species of the genus Sphingomonas, as well as unnamed strains of this genus and related genera such as Rhizomonas and Blastomonas, are presented. All representatives of this group, here designated as sphingomonads, contain ubiquinone (Q-10). The two different polyamine patterns characterized either by the predominant polyamine sym-homospermidine or spermidine separate the sphingomonads into two major groups. Complex polar lipid profiles were found in sphingomonads in addition to the characteristic compound sphingoglycolipid. Identical profiles were found only in a few phylogenetically highly related species. Common to all sphingomonads is a fatty acid composition with 2-hydroxy fatty acids (14:0 2OH in all currently recognized species) and the lack of 3-hydroxy acids, which distinguishes them from taxa outside this group. Qualitative and quantitative differences in the fatty acid compositions, in several cases, were also suitable for identification at the species level. Thus, the differences in the chemotaxonomic characteristics demonstrate that the analyses of these low molecular weight cell compounds are suitable for classification of sphingomonads.

Environmental monitoring in four European museums

In a European multidisciplinary research project concerning environmental diagnostics, museums have been selected, having different climate and pollution conditions, i.e.: Correr Museum, Venice (Italy); Kunsthistorisches Museum, Vienna (Austria); Royal Museum of Fine Arts, Antwerp (Belgium); Sainsbury Centre for Visual Arts, Norwich (UK). Some field tests investigated the microclimate, the gaseous and particulate air pollution and the biological contamination to suggest mitigative techniques that may reduce the potential for damage in the long run. Potential risk factors are generated by imbalance in temperature and humidity, generated by heating, air conditioning or ventilating system (HVAC), or the building structures, exchange of outside air, or large visitor numbers. HVAC may also enhance indoor gaseous pollution. Plants and carpets represent potential niches for bacterial colonisation. Pollutants and particles have been recognised having partly external and partly internal origin. Tourism has a direct negative impact, i.e. transport of external particles, release of heat, vapour and CO2, as well as generation of turbulence, which increases the deposition rate of particulate matter. However, the main problem is that the microclimate has been planned for the well being of visitors during only the visiting time, disregarding the needs of conservation that requires a constant climate by day and by night. In some of these cases, better environmental niches have been obtained with the help of showcases. In other cases, showcases worsened the situation, especially when incandescent lamps were put inside

Halococcus salifodinae sp. nov., an Archaeal Isolate from an Austrian Salt Mine

A novel extremely halophilic archaeon (archaebacterium) was isolated from rock salt obtained from an Austrian salt mine. The deposition of the salt is thought to have occurred during the Permian period (225 × 106 to 280 × 106 years ago). This organism grew over a pH range of 6.8 to 9.5. Electron microscopy revealed cocci in tetrads or larger clusters. The partial 16S rRNA sequences, polar lipid composition, and menaquinone content suggested that this organism was related to members of the genus Halococcus, while the whole-cell protein patterns, the presence of several unknown lipids, and the presence of pink pigmentation indicated that it was different from previously described coccoid halophiles. We propose that this isolate should be recognized as a new species and should be named Halococcus salifodinae. The type strain is Blp (= ATCC 51437 = DSM 8989). A chemotaxonomically similar microorganism was isolated from a British salt mine.

Rapid identification of Staphylococcus epidermidis

During the collection of airborne bacteria in a museum in England some bacterial strains were isolated which due to their fatty acid profiles were clearly identified as members of the genus Staphylococcus. As fatty acid compositions of coagulase-negative staphylococci are very similar, differing only in quantities but not in qualities, further identification at the species level without a fatty acid database was not achieved. Investigation of the isolates using the Staph ID 32 API system resulted in an identification of the isolates as Staphylococcus epidermidis (probabilities of 79.7-95.5%). For further genotypic characterization of these isolates, some Staphylococcus epidermidis strains from different sources and the type strains of Staphylococcus aureus, Staphylococcus capitis, Staphylococcus epidermidis, Staphylococcus gallinarum, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus warneri and Staphylococcus xylosus were subjected to repetitive-sequence PCR, including enterobacterial repetitive intergenic consensus (ERIC) PCR, BOX-PCR and repetitive extragenic palindromic unit sequence (REP) PCR. ERIC- and BOX-PCR yielded a species-specific banding pattern for all Staphylococcus epidermidis strains. Furthermore, all staphylococcal reference strains investigated exhibited distinct banding patterns, clearly distinguishable from that of Staphylococcus epidermidis. No species-specific banding patterns could be observed after REP-PCR. As species identification of coagulase-negative staphylococci by fatty acid analyses and biochemical tests is known to be difficult ERIC- and BOX-PCR seem to be excellent tools for the identification of Staphylococcus epidermidis isolates.