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, children's 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 children's day care centres contained 0.2-0.3 ng of endotoxin mg-1 and 0.5-1.4 ng of beta-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.     

Identification of thermophilic and marine bacilli from shallow thermal vents by restriction analysis of their amplified 16S rDNA

AIMS: In order to identify 73 thermophilic isolates from shallow, marine thermal vents of Eolian Islands, we compared their restriction patterns of amplified 16S rDNA with those of nine well described Bacillus species and eight Eolian Bacillus strains. METHODS AND RESULTS: This study allowed to assign 57 (78%) isolates to different Bacillus species. Nineteen field strains were recognised as representatives of four described species, namely B. thermodenitrificans, "B. caldolyticus", B. vulcani and B. stearothermophilus. The profiles of 38 isolates matched instead, those of seven Eolian strains (B. thermodenitrificans strain A2, B. licheniformis strain B3-15, and five novel species, represented by Bacillus strain 1bw, Bacillus strain 4-1, Bacillus strain 5-2, Bacillus strain 10-1, Bacillus strain 1as). Among the 16 unidentified isolates, seven restriction patterns were recognised. CONCLUSIONS: This study showed that restriction analysis of amplified 16S rDNA is useful for a rapid and reliable identification of strains belonging to described species as well as for recognition of new species. SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed a high taxonomic diversity among the thermophilic bacilli isolated from Eolian Islands and a distinct distribution of the species within the Eolian hydrothermal vent system.     

Numerical analysis and DNA base compositions of some thermophilic Bacillus species.

Morphological, physiological, and biochemical characteristics and the DNA base compositions of 133 thermophilic Bacillus strains were determined. A total of 54 of these strains were received as identified species (mainly Bacillus stearothermophilus, Bacillus coagulans, Bacillus brevis, and Bacillus licheniformis) from international culture collections, and 79 newly isolated strains, which were isolated mainly from sugar diffusion juices of Italian plants, were also examined. Numerical taxonomy techniques (simple matching coefficient and unweight pair grouping using the mathematical average) and DNA G + C values showed that the strains aggregated into nine clusters. Both B. licheniformis and B. brevis were well separated from the other organisms. B. stearothermophilus and B. coagulans were confirmed as separate clusters and exhibited greater heterogeneity than previously shown. The B. stearothermophilus strains clustered into four groups, three of which have been recognized previously by other authors; the members of the fourth group had distinctive characteristics, including considerable biochemical inertness, an inability to grow at temperatures greater than 60 degrees C, and a high G + C content. Within the B. coagulans cluster the strains with characteristics very similar to those of the new species Bacillus smithii clustered together. However, the remaining strains were still clearly separated into two groups; one of these groups was considered B. coagulans sensu stricto, and the other was distinguished by morphological and biochemical criteria, such as spores which do not swell the sporangia, utilization of citrate, a higher proteolytic activity, and acidification of some carbohydrates. Our results were confirmed by comparing them with distinctive characteristics of recently described thermophilic Bacillus species.     

Sequence diversity of the Bacillus thuringiensis and B. cereus sensu lato flagellin (H antigen) protein: comparison with H serotype diversity

We set out to analyze the sequence diversity of the Bacillus thuringiensis flagellin (H antigen [Hag]) protein and compare it with H serotype diversity. Some other Bacillus cereus sensu lato species and strains were added for comparison. The internal sequences of the flagellin (hag) alleles from 80 Bacillus thuringiensis strains and 16 strains from the B. cereus sensu lato group were amplified and cloned, and their nucleotide sequences were determined and translated into amino acids. The flagellin allele nucleotide sequences for 10 additional strains were retrieved from GenBank for a total of 106 Bacillus species and strains used in this study. These included 82 B. thuringiensis strains from 67 H serotypes, 5 B. cereus strains, 3 Bacillus anthracis strains, 3 Bacillus mycoides strains, 11 Bacillus weihenstephanensis strains, 1 Bacillus halodurans strain, and 1 Bacillus subtilis strain. The first 111 and the last 66 amino acids were conserved. They were referred to as the C1 and C2 regions, respectively. The central region, however, was highly variable and is referred to as the V region. Two bootstrapped neighbor-joining trees were generated: a first one from the alignment of the translated amino acid sequences of the amplified internal sequences of the hag alleles and a second one from the alignment of the V region amino acid sequences, respectively. Of the eight clusters revealed in the tree inferred from the entire C1-V-C2 region amino acid sequences, seven were present in corresponding clusters in the tree inferred from the V region amino acid sequences. With regard to B. thuringiensis, in most cases, different serovars had different flagellin amino acid sequences, as might have been expected. Surprisingly, however, some different B. thuringiensis serovars shared identical flagellin amino acid sequences. Likewise, serovars from the same H serotypes were most often found clustered together, with exceptions. Indeed, some serovars from the same H serotype carried flagellins with sufficiently different amino acid sequences as to be located on distant clusters. Species-wise, B. halodurans, B. subtilis, and B. anthracis formed specific branches, whereas the other four species, all in the B. cereus sensu lato group, B. mycoides, B. weihenstephanensis, B. cereus, and B. thuringiensis, did not form four specific clusters as might have been expected. Rather, strains from any of these four species were placed side by side with strains from the other species. In the B. cereus sensu lato group, B. anthracis excepted, the distribution of strains was not species specific.     

Molecular characterization of Korean Bacillus anthracis isolates by amplified fragment length polymorphism analysis and multilocus variable-number tandem repeat analysis

We analyzed the genetic relationships and molecular characteristics of 34 Bacillus anthracis isolates from soil and clinical samples in various regions of Korea and 17 related Bacillus species, using the amplified fragment length polymorphism (AFLP) and multilocus variable-number tandem repeat (MLVA) approaches. Triplicate AFLP profiles of these strains showed high reproducibility and identified 376 polymorphisms. AFLP phylogenetic analysis of B. anthracis isolates showed a high level of similarity, 0.93, and this monomorphic fragment profile proved to be useful to differentiate B. anthracis strains from other Bacillus species. The B. cereus group was separated from other Bacillus species at a level of similarity of 0.68. Among them, some B. cereus strains showed genetic interspersion with B. thuringiensis strains. The evolutionary pattern of nucleotide differences among B. anthracis strains with the eight MLVA markers showed nine MLVA types. Three MLVA types, M1 to M3, were pathogenic B. anthracis isolates and were assigned as new genotypes belonging to the A4 and B3 clusters, compared with 89 genotypes deduced from previous data. This indicates that differences in cluster prevalence and distribution may be influenced more by MLVA markers on two plasmids loci and human activity. Consequently, we suggest that the novel MLVA type may represent significant evidence for historic adaptation to environmental conditions of the Asian continent, particularly Korea. Therefore, MLVA techniques may be available for molecular monitoring on anthrax-release-related bioterrorism and further study is required for the continuous epidemiological study of variable anthrax collections.     

The hemolytic enterotoxin HBL is broadly distributed among species of the Bacillus cereus group

The prevalence of the hemolytic enterotoxin complex HBL was determined in all species of the Bacillus cereus group with the exception of Bacillus anthracis. hblA, encoding the binding subunit B, was detected by PCR and Southern analysis and was confirmed by partial sequencing of 18 strains. The sequences formed two clusters, one including B. cereus and Bacillus thuringiensis strains and the other one consisting of Bacillus mycoides, Bacillus pseudomycoides, and Bacillus weihenstephanensis strains. From eight B. thuringiensis strains, the enterotoxin gene hblA could be amplified. Seven of them also expressed the complete HBL complex as determined with specific antibodies against the L(1), L(2), and B components. Eleven of 16 B. mycoides strains, all 3 B. pseudomyoides strains, 9 of 15 B. weihenstephanensis strains, and 10 of 23 B. cereus strains carried hblA. While HBL was not expressed in the B. pseudomycoides strains, the molecular assays were in accordance with the immunological assays for the majority of the remaining strains. In summary, the hemolytic enterotoxin HBL seems to be broadly distributed among strains of the B. cereus group and relates neither to a certain species nor to a specific environment. The consequences of this finding for food safety considerations need to be evaluated.     

Putative virulence factor expression by clinical and food isolates of Bacillus spp. after growth in reconstituted infant milk formulae

Forty-seven strains representing 14 different Bacillus species isolated from clinical and food samples were grown in reconstituted infant milk formulae (IMF) and subsequently assessed for adherence to, invasion of, and cytotoxicity toward HEp-2 and Caco-2 cells. Cell-free supernatant fluids from 38 strains (81%) were shown to be cytotoxic, 43 strains (91%) adhered to the test cell lines, and 23 strains (49%) demonstrated various levels of invasion. Of the 21 Bacillus cereus strains examined, 5 (24%) were invasive. A larger percentage of clinically derived Bacillus species (20%) than of similar species tested from the food environment were invasive. Increased invasion occurred after growth of selected Bacillus species in reconstituted IMF containing glucose. While PCR primer studies revealed that many different Bacillus species contained DNA sequences encoding the hemolysin BL (HBL) enterotoxin complex and B. cereus enterotoxin T, not all of these isolates expressed these diarrheagenic genes after growth in reconstituted IMF. Of the 47 Bacillus isolates examined, 3 isolates of B. cereus and 1 isolate of B. subtilis produced the HBL enterotoxin after 18 h of growth in brain heart infusion broth. However, eight isolates belonging to the species B. cereus, B. licheniformis, B. circulans, and B. megaterium were found to produce this enterotoxin after growth in reconstituted IMF when assessed with the B. cereus enterotoxin (diarrheal type) reversed passive latex agglutination (RPLA) kit. It is concluded that several Bacillus species occurring occasionally in clinical specimens and food samples are of potential medical significance due to the expression of putative virulence factors.     

Distribution of Bacillus megaterium QM B1551 plasmids among other B. megaterium strains and Bacillus species

Bacillus megaterium QM B1551 contains seven plasmids. Two are small rolling circle plasmids and five are theta-replicating plasmids with cross-hybridizing replicons that define a new family of very homologous yet compatible theta replicons. Previous sequencing of several of the plasmids has shown genes with high similarity to those on the genomes and plasmids of other Gram-positive bacteria. To test the possible distribution of these plasmids, nine other B. megaterium strains and 20 other Bacillus or related species were tested for the presence of similar replicons, and specific flanking DNA by both hybridization and PCR. The theta replicons were widespread among the B. megaterium strains, and two had one or more of the rolling circle plasmids, but none of the plasmid replicon regions were observed in the other Bacillus or related species. It appears from the data that even though some plasmids carry genes suggesting horizontal transfer, their replicons seem to be unique to B. megaterium, or rarely present in related species.     

环保微生物菌剂常用5种芽胞杆菌的PCR鉴定

枯草芽胞杆菌（Bacillus subtilis）、地表芽胞杆菌（Bacillus licheniformis）、巨大芽胞杆菌（Bacillus megate．rium）、短小芽胞杆菌（Bacillus pumilus）、环状芽胞杆菌（Bacillu scirculans）是5种环保微生物菌剂中常用的芽胞杆菌,我国每年有大量此类菌剂进口。针对这些菌的传统理化检测方法费时费力,检测部门急需建立一种快速、准确的检测方法。利用GenBank上已有gyrB、rpoB、gdh基因序列分别设计和筛选上述菌种的特异性引物,经优化建立PCR检测方法。结果显示,使用所设计的特异引物,每种茼均扩增得到特异单1条带,能准确鉴定上述菌种并且与其他对照芽胞杆菌及非芽胞杆菌无交叉反应,送检样品的PCR检测结果与常规方法检测结果一致。建立的特异PCR方法具有良好的重复性和准确性,可快速鉴定上述菌种,具有很好的推广应用前景。     

Genotyping and toxigenic potential of Bacillus subtilis and Bacillus pumilus strains occurring in industrial and artisanal cured sausages

Artisanal and industrial sausages were analyzed for their aerobic, heat-resistant microflora to assess whether new emerging pathogens could be present among Bacillus strains naturally contaminating cured meat products. Sixty-four isolates were characterized by randomly amplified polymorphic DNA (RAPD)-PCR and fluorescent amplified fragment length polymorphism (fAFLP). The biotypes, identified by partial 16S rRNA gene sequence analysis, belonged to Bacillus subtilis, Bacillus pumilus, and Bacillus amyloliquefaciens species. Both RAPD-PCR and fAFLP analyses demonstrated that a high genetic heterogeneity is present in the B. subtilis group even in strains harvested from the same source, making it possible to isolate 56 different biotypes. Moreover, fAFLP analysis made it possible to distinguish B. subtilis from B. pumilus strains. The strains were characterized for their toxigenic potential by molecular, physiological, and immunological techniques. Specific PCR analyses revealed the absence of DNA sequences related to HBL, BcET, NHE, and entFM Bacillus cereus enterotoxins and the enzymes sphingomyelinase Sph and phospholipase PI-PLC in all strains; also, the immunological analyses showed that Bacillus strains did not react with NHE- and HBL-specific antibodies. However, some isolates were found to be positive for hemolytic and lecithinase activity. The absence of toxigenic potential in Bacillus strains from the sausages analyzed indicates that these products can be considered safe under the processing conditions they were produced; however, great care should be taken when the ripening time is shortened, particularly in the case of traditional sausages, which could contain high amounts of Bacillus strains and possibly some B. cereus cells.     

神农架自然保护区大九湖芽孢杆菌资源调查

对神农架自然保护区大九湖 2 1份土样进行芽孢杆菌的分离 ,共分离得到 1 4 0株纯培养 ,鉴定为 1 1个种 ,处优势地位的四个种分别为腊样芽孢杆菌占 31 .3% ,短芽孢杆菌占 2 5% ,环状芽孢杆菌占 1 2 .5% ,蕈状芽孢杆菌占 1 1 .6%。还分析了嗜中温芽孢杆菌在该地区的分布及其与环境的关系。     

Difference between the spore sizes of Bacillus anthracis and other Bacillus species

AIMS: To determine the size distribution of the spores of Bacillus anthracis, and compare its size with other Bacillus species grown and sporulated under similar conditions. METHODS AND RESULTS: Spores from several Bacillus species, including seven strains of B. anthracis and six close neighbours, were prepared and studied using identical media, protocols and instruments. Here, we report the spore length and diameter distributions, as determined by transmission electron microscopy (TEM). We calculated the aspect ratio and volume of each spore. All the studied strains of B. anthracis had similar diameter (mean range between 0.81 +/- 0.08 microm and 0.86 +/- 0.08 microm). The mean lengths of the spores from different B. anthracis strains fell into two significantly different groups: one with mean spore lengths 1.26 +/- 0.13 microm or shorter, and another group of strains with mean spore lengths between 1.49 and 1.67 microm. The strains of B. anthracis that were significantly shorter also sporulated with higher yield at relatively lower temperature. The grouping of B. anthracis strains by size and sporulation temperature did not correlate with their respective virulence. CONCLUSIONS: The spores of Bacillus subtilis and Bacillus atrophaeus (previously named Bacillus globigii), two commonly used simulants of B. anthracis, were considerably smaller in length, diameter and volume than all the B. anthracis spores studied. Although rarely used as simulants, the spores of Bacillus cereus and Bacillus thuringiensis had dimensions similar to those of B. anthracis. SIGNIFICANCE AND IMPACT OF THE STUDY: Spores of nonvirulent Bacillus species are often used as simulants in the development and testing of countermeasures for biodefence against B. anthracis. The data presented here should help in the selection of simulants that better resemble the properties of B. anthracis, and thus, more accurately represent the performance of collectors, detectors and other countermeasures against this threat agent.     

青海可可西里嗜碱芽胞杆菌资源调查

【目的】了解可可西里嗜碱芽胞杆菌资源多样性及产酶多样性,为芽胞杆菌功能资源挖掘和菌剂开发提供基础。【方法】采用Horikoshi I培养基,通过可培养法分离青海可可西里土壤中的嗜碱芽胞杆菌,利用16S r RNA基因序列初步鉴定分离获得的芽胞杆菌。采用透明圈法分析分离菌株的产蛋白酶、纤维素酶及木聚糖酶活性。【结果】从青海可可西里土壤中共分离获得66株嗜碱芽胞杆菌,根据16S r RNA基因序列相似性分析,发现它们隶属于6个属22个种,分别为芽胞杆菌属(Bacillus)、纤细芽胞杆菌属(Gracilibacillus)、喜盐芽胞杆菌属(Halobacillus)、咸海鲜芽胞杆菌属(Jeotgalibacillus)、类芽胞杆菌属(Paenibacillus)和嗜冷芽胞杆菌属(Psychrobacillus),其中以芽胞杆菌属(Bacillus)为优势属。2株嗜碱芽胞杆菌与它们最近匹配模式菌株的16S r RNA基因序列相似性为97.00%和98.65%,为潜在新种。三种酶活检测结果表明产酶菌株约占总分离菌株的95.00%,其中55株具有产蛋白酶活性,27株具有产纤维素酶活性,8株能够产木聚糖酶。【结论】青海可可西里蕴藏着较丰富的嗜碱芽胞杆菌资源及丰富的产酶资源,为后续嗜碱芽胞杆菌的挖掘提供理论基础。     

Effects of temperature on the growth of psychrophilic bacteria from glaciers.

Growth of five strains of psychrophilic bacteria (four Arthrobacter and one Pseudomonas) isolated from glacial deposits was studied at different temperatures. Three strains were facultative psychrophiles, having an optimum temperature for growth at about 25-28 degrees C and a maximum at about 32-34 degrees C. The two Arthrobacter glacialis strains were found to be obligate psychrophiles with an optimum at 13-15 degrees C and a maximum at 18 degrees C. Arrhenius plots showed that A. glacialis could compete with the facultative psychrophilic bacteria only at 0 degrees C, that is, the temperature of its natural environment. The psychrophilic Arthrobacter species studied here are more resistant to thermal stress than are marine psychrophilic bacteria. For Arthrobacter, in contrast to Pseudomonas, temperatures above the optimum induced formation of filaments and abnormal cells. The culture turbidity increased 10 to 30 times, whereas viable count tended to decrease. The thermal block seems to prevent cell wall synthesis and septation, but at a different step for each species.     

Genetic diversity and involvement in bread spoilage of Bacillus strains isolated from flour and ropy bread

AIMS: To study Bacillus contamination of wheat flour and ropy bread, to analyse genetic diversity of isolated strains and to evaluate the ability of these strains to produce ropy bread. METHODS AND RESULTS: Classical and molecular methods [16S rDNA sequencing and random amplified polymorphic DNA (RAPD)-PCR] were used to identify and type-isolated strains. The predominant species isolated were Bacillus subtilis and B. licheniformis. RAPD analysis demonstrated that the same sample may harbor different strains. Ten of 15 strains of B. subtilis and four of six strains of B. licheniformis were able to cause rope spoilage of the laboratory-baked bread. CONCLUSION: RAPD typing can be useful in the tracking of Bacillus strains during bakery processing and in the understanding of the role of different Bacillus strains in the rope spoilage of bread. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate the variability of Bacillus strains isolated from flour and responsible for rope spoilage of bread.     

Characterization and pathogenic evaluation of Bacillus thuringiensis and Bacillus sphaericus isolates from Argentinean soils

Eighty soil samples of different origin (from urban, agricultural, forested and horticultural areas) which had not previously been treated with bioinsecticides, were collected and examined to investigate the presence of Bacillus thuringiensis and B. sphaericus. From a total of 1473 bacterial isolates examined by differential staining techniques and growth on nutrient agar with the addition of penicillin and streptomycin, 31 (2.1%) strains of Bacillus sphaericus and 25 (1.6%) strains of Bacillus thuringiensis were isolated. These strains were tested for their pathogenicity against Diptera (Culex quinquefasciatus) and Lepidoptera (Anticarsia gemmatalis and Spodoptera frugiperda). Seven strains of Bacillus thuringiensis subspecies kurstaki were found to be pathogenic to Spodoptera frugiperda and twenty-two strains showed a pathological effect against Anticarsia gemmatalis. None of the strains of Bacillus thuringiensis nor the Bacillus sphaericus investigated, showed pathogenic activity against Culex quinquefasciatus. The strains of Bacillus thuringiensis were characterized serologically as belonging to six serotypes (darmstadiensis, entomocidus, kurstaki, muju, sotto and xianguangiensis). One strain seemed to be a new serotype. The electrophoretic profiles of the strains of Bacillus thruringiensis showed bands of 130 kDa similar to those found in strains pathogenic against Lepidoptera. Some physicochemical characteristics were also studied in the soil samples, in order to relate them to the presence or absence of these Bacillus species.     

Psychrotolerant species from the Bacillus cereus group are not necessarily Bacillus weihenstephanensis

Twenty-six strains of Bacillus cereus from different sources were determined to be either mesophilic or psychrotrophic by growth at 6 and 42 degrees C. The strains were also screened by two polymerase chain reaction (PCR) methods designed to discriminate between mesophilic and psychrotrophic types. Seventeen of the 26 strains were able to grow at 6 degrees C, but only four conformed to the new psychrotolerant species Bacillus weihenstephanensis. Among the 26 strains were two which caused outbreaks of food poisoning in Norway, and three others that were isolated from food suspected of causing illness. The presence of the gene components encoding production of enterotoxins Nhe, Hbl, EntT and a recently described cytotoxin K was determined by PCR. All the strains possessed genes for at least one of these toxins, and 19 of the 26 strains were cytotoxic in a Vero cell assay. We conclude that there are psychrotrophic B. cereus strains which cannot be classified as B. weihenstephanensis, and that intermediate forms between the two species exist. No correlation between cytotoxicity and the growth temperature of the strains was found.     

Distribution and characteristics of Bacillus bacteria associated with hydrobionts and the waters of the Peter the Great Bay, Sea of Japan

Bacilli of the species Bacillus subtilis, B. pumilus, B. mycoides, B. marinus and B. licheniformis (a total of 53 strains) were isolated from 15 invertebrate species and the water of the Vostok Bay, Peter the Great Bay, Sea of Japan. Bacilli were most often isolated from bivalves (22.7%) and sea cucumbers (18.9%); they occurred less frequently in sea urchins and starfish (13.2 and 7.5%, respectively). Most of bacilli strains were isolated from invertebrates inhabiting silted sediments. No Bacillus spp. strains were isolated from invertebrates inhabiting stony and sandy environments. The species diversity of bacilli isolated from marine objects under study was low. Almost all bacterial isolates were resistant to lincomycin. Unlike B. pumilus, B. subtilis isolates were mostly resistant to benzylpenicillin and ampicillin. Antibiotic sensitivity of B. licheniformis strains was variable (two strains were resistant to benzylpenicillin and oxacillin, while one was sensitive). A significant fraction of isolated bacilli contained pigments. Pigmented strains were more often isolated from seawater samples, while colorless ones predominated within hydrobionts. B. subtilis colonies had the broadest range of colors. In the Bacillus strains obtained, DNase, RNase, phosphatase, elastolytic, chitinase, and agarolytic activity was detected. Bacilli strains with hydrolytic activity occurred in invertebrates more often than in seawater.     

The phylogenetic diversity of thermophilic members of the genus Bacillus as revealed by 16S rDNA analysis

Abstract Sixteen thermophilic strains of the genus Bacillus , representing eight validly described and six invalidly described species, as well as one unassigned strain, were investigated by comparative 16S rDNA analyses and the sequences compared to the existing database for the genera Bacillus and Alicyclobacillus . The majority of strains were found to cluster in two groups represented by B. stearothermophilus and B. pallidus. Bacillus smithii, B. thermocloacae , and B. thermoruber are phylogenetically well separated and cluster within the radiation of mesophilic bacilli. The as yet undescribed taxon 'B. flavothermus' warrants species status. B. schlegelii and B. tusciae group peripherally with members of Alicyclobacillus and may be reclassified when more phenotypic data support their phylogenetic position.