Mirja Salkinoja-Salonen

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.

Quantitative Analysis of Cereulide, the Emetic Toxin of Bacillus cereus, Produced under Various Conditions

ABSTRACT This paper describes a quantitative and sensitive chemical assay for cereulide, the heat-stable emetic toxin produced by Bacillus cereus. The methods previously available for measuring cereulide are bioassays that give a toxicity titer, but not an accurate concentration. The dose of cereulide causing illness in humans is therefore not known, and thus safety limits for cereulide cannot be indicated. We developed a quantitative and sensitive chemical assay for cereulide based on high-performance liquid chromatography (HPLC) connected to ion trap mass spectrometry. This chemical assay and a bioassay based on boar sperm motility inhibition were calibrated with purified cereulide and with valinomycin, a structurally similar cyclic depsipeptide. The boar spermatozoan motility assay and chemical assay gave uniform results over a wide range of cereulide concentrations, ranging from 0.02 to 230 μg ml−1. The detection limit for cereulide and valinomycin by HPLC-mass spectrometry was 10 pg per injection. The combined chemical and biological assays were used to define conditions and concentrations of cereulide formation by B. cereus strains F4810/72, NC7401, and F5881. Cereulide production commenced at the end of logarithmic growth, but was independent of sporulation. Production of cereulide was enhanced by incubation with shaking compared to static conditions. The three emetic B. cereus strains accumulated 80 to 166 μg of cereulide g−1 (wet weight) when grown on solid medium. Strain NC7401 accumulated up to 25 μg of cereulide ml−1 in liquid medium at room temperature (21 ± 1°C) in 1 to 3 days, during the stationary growth phase when cell density was 2 × 108 to 6 × 108 CFU ml−1. Cereulide production at temperatures at and below 8°C or at 40°C was minimal.

Variation of Microcystin Content of Cyanobacterial Blooms and Isolated Strains in Lake Grand-Lieu (France)

A bstractCyanobacterial blooms were sampled at five locations in Lake Grand-Lieu on seven different occasions during May–October 1994. Strains of Microcystis aeruginosa and Anabaena circinalis were isolated from the samples. Microcystins were detected in freeze-dried field samples and the isolated strains by HPLC. The toxins were present in the blooms sampled between June and October. The microcystin content in the blooms varied with site and time, from undetectable concentrations to 0.23 mg g−1. The highest concentrations of microcystin were found in blooms sampled in September. Microcystin-LR and microcystins with retention times close to the retention time of [Dha7]microcystin-RR (probably varieties of microcystin-RR) were found in the field samples. Sixteen of the 98 isolated M. aeruginosa strains and 2 of the 24 A. circinalis strains produced microcystins. The total amount of microcystins varied from undetectable concentrations to 5.06 mg g−1 in the M. aeruginosa isolates, and from undetectable concentrations to 1.86 mg g−1 in the A. circinalis strains. Microcystin-LR was the main toxin found in strains of M. aeruginosa, but was not present in strains of A. circinalis. Both microcystin-producing strains and strains that did not produce microcystin coexisted in the bloom samples.

Characterization of Nodularia strains, cyanobacteria from brackish waters, by genotypic and phenotypic methods

An investigation was undertaken of the genetic diversity of Nodularia strains from the Baltic Sea and from Australian waters, together with the proposed type strain of Nodularia spumigena. The Nodularia strains were characterized by using a polyphasic approach, including RFLP of PCR-amplified 16S rRNA genes, 16S rRNA gene sequencing, Southern blotting of total DNA, repetitive extragenic palindromic- and enterobacterial repetitive intergenic consensus-PCR, ribotyping and phenotypic tests. With genotypic methods, the Nodularia strains were grouped into two clusters. The genetic groupings were supported by one phenotypic property: the ability to produce nodularin. In contrast, the cell sizes of the strains were not different in the two genetic clusters. 16S rRNA gene sequences indicated that all the Nodularia strains were closely related, despite their different origins. According to this study, two genotypes of Nodularia exist in the Baltic Sea. On the basis of the taxonomic definitions of Komarek et al. (Algol Stud 68, 1-25, 1993), the non-toxic type without gas vesicles fits the description of Nodularia sphaerocarpa, whereas the toxic type with gas vesicles resembles the species N. spumigena and Nodularia baltica.

Degradation of polychlorinated phenols by Rhodococcus chlorophenolicus

SummaryAn actinomycete, Rhodococcus chlorophenolicus, isolated from a pentachlorophenol-degrading mixed bacterial culture is a polychlorophenol degrader. It was shown to oxidize pentachlorophenol into carbon dioxide and to metabolize also 2,3,4,5-,2,3,4,6-, and 2,3,5,6-tetrachlorophenol, 2,3,4-, 2,3,5-, 2,3,6-, 2,4,6-, and 2,4,5-trichlorophenol, 2,5-, and 2,6-dichlorophenol and tetrachloro-p-hydroquinone in an inducible manner. Pentachlorophenol set on the synthesis of enzymes required for the metabolism of all these chlorophenols and of tetrachloro-p-hydroquinone. 2,4,5-, and 2,4,6-trichlorophenol and 2,5-, and 2,6-dichlorophenol were degraded by R. chlorophenolicus cells only if these had previous contact to pentachlorophenol. Other chlorophenols mentioned were able to set on the synthesis of enzymes for their own degradation. 2,3,4,5-, and 2,3,4,6-tetrachlorophenol, and 2,3,5-, 2,4,5-, and 3,4,5-trichlorophenol were more toxic to R. chlorophenolicus than the other chlorophenols, but nevertheless 2,3,4,5-, and 2,3,4,6-tetrachlorophenol and 2,3,5-trichlorophenol were readily degraded by the bacteria.

Dust‐borne bacteria in animal sheds, schools and children's day care centres

A total of 316 bacterial strains, including psychrophiles, mesophiles and thermophiles, were isolated and identified from indoor dusts in schools, childrens day care centres and animal sheds. Several species which had not previously been reported from indoor environments were found: Sphingomonas, Brevibacterium, Nocardiopsis, Deinococcus and Rhodococcus/Gordona. A new psychrophilic actinomycete genus was also found in animal sheds, representing a new undescribed peptidoglycan type and an unusual whole‐cell fatty acid composition. The indoor dusts of animal sheds contained mainly the Gram‐negative genera Pseudomonas, Pantoea, Flavobacterium and Xanthomonas early in the indoor feeding season, but changed to a composition dominated by Bacillus, Micrococcus and mesophilic and thermophilic actinomycetes towards the end of the season. The dust contained, and air‐borne bacterial flora in schools and day care centres were dominated by, Gram‐positive bacilli and actinomycetes, notably Bacillus cereus, Brevibacillus brevis, B. licheniformis, B. subtilis and species of Arthrobacter, Corynebacterium, Rhodococcus/Gordona, Nocardiopsis sp., Deinococcus, Staphylococcus and Micrococcus. Indoor air and dust contained Klebsiella oxytoca, Acinetobacter calcoaceticus, Ac. lwoffi, Bacillus cereus and Nocardiopsis dassonvillei with the status of hazard group II. Indoor dusts of animal sheds contained eight different 3‐hydroxy fatty acids, the 2‐hydroxy fatty acid 14: 0 and two 10‐methyl fatty acids, whereas in dusts from schools and day care centres, these were below the detection level (< 3·5 ng mg−1). The 3‐ and 2‐hydroxy fatty acids could be assigned to one or more of the dust‐contained cultivable strains, but 10‐methyl C16: 0 was not present in any of the strains isolated. The dusts from schools and childrens day care centres contained 0·2–0·3 ng of endotoxin mg−1 and 0·5–1·4 ng of β‐d‐ glucan mg−1, whereas the dusts from animal sheds contained more 0·3–41 ng mg−1 and 8–35 ng mg−1, respectively.

Bioreclamation of chlorophenol-contaminated soil by composting

SummaryMicrobiological decontamination of technical chlorophenol-containing soil by composting was studied. In two 50 m3 windrows the concentration of chlorophenols went down from 212 mg kg-1 to 30 mg kg-1 in 4 summer months and after the second summer of composting it was only 15 mg kg-1. All chlorophenol congeners present in the technical chlorophenol were degraded, but the main dimeric impurities, polychlorinated phenoxyphenols were recalcitrant. The contaminated soil was found to contain chlorophenol-degrading microbes, 5x106 cfu g-1 of dry windrow soil. Laboratory experiments with samples from the windrow compost showed that chlorophenols were truly degraded and that chlorophenol loss by evaporation was less than 1.5% under the circumstances studied. Laboratory experiments also showed that degradation of chlorophenols (120 mg kg-1) was accelerated when sterilized contaminated soil was inoculated with Rhodococcus chlorophenolicus (mineralizer of several chlorophenols) or naturally occurring microbes of the field composts. Biomethylation of chlorophenols in the composts was insignificant compared to biodegradation.

Description of Four Pentachlorophenol-Degrading Bacterial Strains as Sphingomonas chlorophenolica sp. nov.

Summary The chemotaxonomy, physiology and ultrastructure were studied of four pentachlorophenol (PCP) degrading strains known as Arthrobacter sp. ATCC 33790, Flavobacterium sp. ATCC 39723, Pseudomonas sp. RA2 and Pseudomonas sp. SR3. All of them were Gram-negative rods, and electron microscopy revealed an outer membrane in each of the four strains. The whole cell fatty acid compositions of the strains were similar: from 56 to 62% of octadecenoic and from 7 to 15% each of 2-hydroxymyristic, cis-9-hexadecenoic and hexadecanoic acids. All strains contained sphingolipids and no Iipopolysaccharide. The main respiratory quinone was ubiquinone 10, and the G+C contents of the DNA ranged from 63 to 67 mol%. On the basis of chemotaxonomic data, biochemical and physiological properties, and whole cell protein profiles, the four pentachlorophenol-degrading strains Arthrobacter sp. ATCC 33790, Flavobacterium sp. ATCC 39723, Pseudomonas sp. RA2 and Pseudomonas sp. SR3 were reclassified in the genus Sphingomonas as a new species S. chlorophenolica sp. nov.

Description of Chlorophenol-Degrading Pseudomonas sp. Strains KF1T, KF3, and NKF1 as a New Species of the Genus Sphingomonas, Sphingomonas subarctica sp. nov.

Gram-negative polychlorophenol-degrading bacterial strains KF1T (T = type strain), KF3, and NKF1, which were described previously as Pseudomonas saccharophila strains, were studied by chemotaxonomic, genetic, and physiological methods and by electron microscopy and compared with selected xenobiotic compound-degrading bacteria. These strains contained sphingolipids with d-18:0, d-20:1, and d-21:1 as the main dihydrosphingosines, ubiquinone 10 as the main respiratory quinone, and spermidine as the major polyamine, and the DNA G + C content was 66 mol%. The cellular fatty acids included about 60% octadecenoic acid, 9% 2-hydroxymyristic acid, 14% cis-9-hexadecenoic acid, and 10% hexadecanoic acid. These strains exhibited less than 97% 16S ribosomal DNA sequence similarity to all of the other taxa studied. In the DNA-DNA reassociation studies the highest levels of reassociation between these strains and previously described species were less than 40%. Thin sections of cells of strains KF1T, KF3, and NKF1 were examined by electron microscopy, and the results showed that the cells had peculiar concentrically arranged layered membranous blebs that extruded from the outer membrane, especially at the cell division points. On the basis of the results of this study, polychlorophenol-degrading strains KF1T, KF3, and NKF1 are considered members of a new species of the genus Sphingomonas, Sphingomonas subarctica. The polycyclic aromatic hydrocarbon-degrading organism Sphingomonas paucimobilis EPA 505 was closely related to Sphingomonas chlorophenolica as determined by chemotaxonomic, phylogenetic, and physiological criteria. The xenobiotic compound degraders Alcaligenes sp. strain A175 and Pseudomonas sp. strain BN6 were identified as members of species of the genus Sphingomonas.

Predominance of Gram-positive bacteria in house dust in the low-allergy risk Russian Karelia

Simple living conditions and farming environment have been associated with reduced risk for allergic diseases such as atopy and asthma but the factors responsible for this effect remain unresolved. We examined the bacterial composition of house dusts obtained from Finnish and Russian Karelia, two adjacent areas with high and low occurrence of atopic diseases respectively. Two dust mixes, both composed of 10 randomly selected dust samples from 349 Finnish and 417 Russian Karelian households were studied for bacterial biomarkers (DNA, Limulus-active endotoxin, 3-OH fatty acids, muramic acid) and for 16S rRNA gene sequences. Overall, the DNA cloning revealed more taxons (94 different genera) of dustborne bacteria than seen in any previous study on residential environments. Majority (67%) of the bacterial DNA clones in house dust from the low-allergy Russian Kareliarepresented Gram-positive bacteria (Firmicutes and Actinobacteria), predominantly Staphylococcaceae and Corynebacteriaceae. Russian Karelian dust showed up to 20-fold higher contents of muramic acid (marker of Gram-positive bacteria) and a sevenfold higher number of clones of animal-associated species, whereas in Finnish Karelian dust Gram-negatives (mainly Proteobacteria) predominated. Clones of plant-associated bacterial species and of chloroplast, indicating plant biomass, were more numerous in Finnish than in Russian Karelian dust. In conclusion, this study revealed major disparities between Finnish and Russian house dusts. The higher bacterial content and the predominance of Gram-positive bacteria in Russian dust may have implications for occurrence of atopy.