Herbert Seiler

Fourier-Transform Infrared Microspectroscopy, a Novel and Rapid Tool for Identification of Yeasts

ABSTRACT Fourier-transform infrared (FT-IR) microspectroscopy was used in this study to identify yeasts. Cells were grown to microcolonies of 70 to 250 μm in diameter and transferred from the agar plate by replica stamping to an IR-transparent ZnSe carrier. IR spectra of the replicas on the carrier were recorded using an IR microscope coupled to an IR spectrometer, and identification was performed by comparison to reference spectra. The method was tested by using small model libraries comprising reference spectra of 45 strains from 9 genera and 13 species, recorded with both FT-IR microspectroscopy and FT-IR macrospectroscopy. The results show that identification by FT-IR microspectroscopy is equivalent to that achieved by FT-IR macrospectroscopy but the time-consuming isolation of the organisms prior to identification is not necessary. Therefore, this method also provides a rapid tool to analyze mixed populations. Furthermore, identification of 21 Debaryomyces hansenii and 9 Saccharomyces cerevisiae strains resulted in 92% correct identification at the strain level for S. cerevisiae and 91% for D. hansenii, which demonstrates that the resolution power of FT-IR microspectroscopy may also be used for yeast typing at the strain level.

Identification of coryneform bacteria and related taxa by Fourier-transform infrared (FT-IR) spectroscopy.

An extensive Fourier-transform infrared (FT-IR) spectroscopy database for the identification of bacteria from the two suborders Micrococcineae and Corynebacterineae (Actinomycetales, Actinobacteria) as well as other morphologically similar genera was established. The database consists of averaged IR spectra from 730 reference strains, covering 220 different species out of 46 genera. A total of 192 species are represented by type strains. The identity of 352 reference strains was determined by comparative 16S rDNA sequence analysis and, if necessary, strains were reclassified accordingly. FT-IR frequency ranges, weights and reproducibility levels were optimized for this section of high-G+C gram-positive bacteria. In an internal validation, 98.1% of 208 strains were correctly identified at the species level. A simulated external validation which was carried out using 544 strains from 54 species out of 16 genera resulted in a correct identification of 87.3% at the species level and 95.4% at the genus level. The performance of this identification system is well within the range of those having been reported in the literature for the identification of coryneform bacteria by phenotypical methods. Coryneform and related taxa display a certain degree of overlapping distribution of different taxonomical markers, leading to a limited differentiation capacity of non-genotypical identification methods in general. However, easy handling, rapid identification within 25 h starting from a single colony, a satisfactory differentiation capacity and low cost, render FT-IR technology clearly superior over other routine methods for the identification of coryneform bacteria and related taxa.

Identification of yeasts and coryneform bacteria from the surface microflora of brick cheeses.

Coryneform bacteria and yeasts of 21 brick cheeses from six German dairies, produced by using undefined ripening cultures, were identified. Arthrobacter nicotianae, Brevibacterium linens, Corynebacterium ammoniagenes, Corynebacterium variabilis and Rhodococcus fascians were found in significant numbers. Out of 148 coryneform isolates 36 could not be identified at the species level. With the exception of a large rennet cheese, the coryneform microflora of rennet and acid cured cheeses were similar, but the cheeses had clearly different yeast populations. Debaryomyces hansenii and Galactomyces geotrichum prevailed in rennet cheeses while Kluyveromyces marxianus and Pichia membranaefaciens were the main species found in acid cured cheese. The dominance of Yarrowia lipolytica probably indicates an improper yeast population, resulting in poor cheese quality. Some of the species identified are potential candidates for designing a defined ripening culture for rennet red smear cheese.

Polyphasic identification of wild yeast strains isolated from Greek sourdoughs

A total of forty-five wild yeast strains were isolated from five traditional Greek wheat sourdoughs. Strains were identified using the classical identification technique along with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole cell proteins (SDS-PAGE), Fourier transform-infrared spectroscopy (FT-IR) and the randomly amplified polymorphic DNA-polymerase chain reaction analysis (RAPD-PCR). The latter methods confirmed the classical identification. According to the results obtained, fourteen strains were identified as Saccharomyces cerevisiae strains, twenty-five as Pichia membranaefaciens strains and six as Yarrowia lipolytica.

Identification of Yoghurt-spoiling Yeasts with 18S rRNA-targeted Oligonucleotide Probes

Summary 18S rRNA-targeted oligonucleotide probes were designed for rapid and reliable identification of yeasts involved in spoilage of dairy products. Candida parapsilosis, Candida glabrata, Clavispora lusitaniae, Debaryomyces hansenii, Dekkera bruxellensis, Hanseniaspora uvarum, Pichia anomala, Pichia membranaefaciens, Rhodotorula glutinis, Saccharomyces cerevisiae and Saccharomycopsis capsularis were identified by dot blot hybridization assays using species-specific, digoxygenin-labeled probes which were deduced from comparative analysis of highly variable regions of 18S rRNA. The eucarya-specific probe EUK516 gave intense signals detected by epifluorescense microscopy when hybridized in situ to all yeast species tested. Whole cell hybridization experiments revealed that the 3′-end of the target molecule is a suitable site for fluorescently labeled species-specific nucleic acid probes which detect S. cerevisiae, P. anomala, D. hansenii and D. bruxellensis in situ . Other variable regions of the 18S rRNA tested for species-specific probes apparently were not accessible to in situ hybridization. S. cerevisiae and P. anomala were detected in yoghurt using the fluorescently labeled probes Sce1711 and Pan1710.

Quantification of micro‐organisms in binary mixed populations by Fourier transform infrared (FT‐IR) spectroscopy

H. OBERREUTER, F. MERTENS, H. SEILER and S. SCHERER.2000.Fourier Transform Infrared (FT‐IR) spectroscopy was used for the first time to determine the ratios of different microorganisms in mixtures. Exemplarily, systems composed of two food‐associated yeast species (Saccharomyces cerevisiae/Hanseniaspora uvarum) and two yoghurt lactic acid bacteria (Lactobacillus acidophilus/Streptococcus salivarius ssp. thermophilus) were investigated. Determination of the cell number ratio in the lactic acid bacteria system was possible with a minimal prediction accuracy of ±16 ratio percentage points while the minimum accuracy of prediction in the yeast two‐component system was ± 4% (both at a 95% confidence level). These results show that FT‐IR spectroscopy is potentially a rapid method for the quantification of cell ratios in mixtures of two different microorganisms, provided that the cell ratio does not drop below a certain, system‐specific threshold.

Reliable identification of closely related Issatchenkia and Pichia species using artificial neural network analysis of Fourier-transform infrared spectra

A reliable identification system for closely related species of the genera Issatchenkia and Pichia was established, using artificial neural network‐based Fourier‐transform infrared (FTIR) spectroscopy; 16 common Pichia species and all five known Issatchenkia species were included. A total of 238 strains isolated from a large variety of habitats were used as reference strains to generate an artificial neural network (ANN) identification system. This system consists of 10 single subnets connected to an ANN with four consecutive levels. An internal validation of the system, using unknown spectra of each reference strain, yielded an identification rate of 99.2%. To evaluate the performance of the ANN in routine diagnostics, 1608 spectra of 179 strains unknown to the ANN were used as a test dataset in an external validation. An overall identification rate of 98.6%, including a success rate of 100% for two common species, P. anomala and P. membranifaciens, demonstrates considerable potential of this FTIR‐based artificial neural network for the identification of closely related yeast species. Copyright

Domibacillus robiginosus gen. nov., sp. nov., isolated from a pharmaceutical clean room.

A novel red-pigmented bacterial strain, designated WS 4628(T), was isolated from a pharmaceutical clean room of a vaccine-producing company and was investigated in a taxonomic study using a polyphasic approach. The strain was Gram-stain-positive, strictly aerobic, motile, catalase-positive and produced spherical to slightly ellipsoidal endospores in rods. The genomic DNA G+C content was 44.1 mol%. The major fatty acids were anteiso-C15:0, iso-C15:0 and anteiso-C17:0 and the predominant quinone was MK-6. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, an unidentified phosphoglycolipid and an unidentified phospholipid. meso-diaminopimelic acid (type A1γ) was present in the cell-wall peptidoglycan and the major whole-cell sugars were glucose and ribose. The closest phylogenetic neighbours were identified as Bacillus badius ATCC 14574(T) (95.8% 16S rRNA gene sequence similarity), Bacillus indicus Sd/3(T) (94.8%), Jeotgalibacillus alimentarius YKJ-13(T) (94.8%) and Bacillus cibi JG-30(T) (94.8%). Phylogenetic, physiological, biochemical and morphological differences between strain WS 4628(T) and its closest relatives in the families Bacillaceae and Planococcaceae suggest that this strain represents a novel species in a new genus in the family Bacillaceae for which the name Domibacillus robiginosus gen. nov., sp. nov. is proposed; the type strain of the type species is WS 4628(T) (=DSM 25058(T)=LMG 26645(T)).

Psychroflexus halocasei sp. nov., isolated from a microbial consortium on a cheese

A novel halophilic, Gram-reaction-negative, strictly aerobic, non-motile, rod-shaped and oxidase- and catalase-positive bacterial strain, designated WCC 4520(T), was isolated from a semi-hard, Raclette-type cheese. The colonies were yellow-orange; flexirubin-type pigments were not found. The strain hydrolysed gelatin, hippurate, tyrosine and Tweens 20 and 80. Optimal growth was observed with 6-8% (w/v) NaCl, at pH 7-8 and at 27-30 °C. The genomic DNA G+C content was 33.6 mol%. In phylogenetic analysis based on 16S rRNA gene sequences, strain WCC 4520(T) appeared to be a member of the family Flavobacteriaceae and the closest phylogenetic neighbours were identified as Psychroflexus gondwanensis DSM 5423(T) (94.0% 16S rRNA gene sequence similarity) and Psychroflexus salinarum CCUG 56752(T) (94.0%). The predominant cellular fatty acids were iso-C(15:0), anteiso-C(15:0), iso-C(15:1) G and iso-C(17:0) 3-OH. The only detected quinone was MK-6 and the major polar lipids were phosphatidylethanolamine, an unidentified aminolipid and an unidentified polar lipid. Minor polar lipids and traces of polyamines were also detected. On the basis of the data presented, strain WCC 4520(T) represents a novel species of the genus Psychroflexus, for which the name Psychroflexus halocasei sp. nov. is proposed. The type strain is WCC 4520(T) ( = LMG 25857(T) = CCUG 59705(T)).

Lysinibacillus meyeri sp. nov., isolated from a medical practice.

A gram-positive, oxidase- and catalase-positive, strictly aerobic and motile bacterium, designated WS 4626(T), was isolated from a medical practice. Spherical endospores were formed terminally in swollen rods. The genomic DNA G+C content was 37.1 mol%. Cells contained iso-C15 : 0, anteiso-C15 : 0, iso-C17 : 1ω10c, anteiso-C17 : 0 and iso-C17 : 0 as the predominant cellular fatty acids and MK-7 and MK-6 as the major isoprenoid quinones. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol, the cell-wall peptidoglycan was type A4α, L-Lys-D-Asp and the major cell-wall sugar was xylose. The closest phylogenetic relatives were Lysinibacillus xylanilyticus XDB9(T) (96.7 % 16S rRNA gene sequence similarity) and Lysinibacillus odysseyi 34hs-1(T) (96.5 %). DNA-DNA relatedness between the isolate and L. odysseyi DSM 18869(T) was very low (6 %). On the basis of the data presented, strain WS 4626(T) represents a novel species of the genus Lysinibacillus, for which the name Lysinibacillus meyeri sp. nov. is proposed. The type strain is WS 4626(T) ( = DSM 25057(T) = LMG 26643(T)).