Isoprenoid quinones in the classification of coryneform and related bacteria.

Menaquinones were the only isoprenoid quinones found in 85 of the 95 coryneform bacteria examined. Dihydromenaquinones having nine isoprene units were the main components isolated from Corynebacterium bovis, from other glutamic acid-producing strains, and from Arthrobacter globiformis and related species. Dihydromenaquinones with eight isoprene units were found in Brevibacterium linens, the remaining Corynebacterium species and strains probably belonging to the genus Rhodococcus. Tetrahydromenaquinones with eight isoprene units were found in Arthrobacter simplex and Arthrobacter tumescens, and with nine isoprene units in Cellulomonas and Oerskovia. Kurthia and Curtobacterium were characterized by menaquinones with seven and nine isoprene units, respectively, and Microbacterium lacticum and Corynebacterium aquaticum had comparable amounts of menaquinones with 10 and 11 isoprene units. Strains received as Brevibacterium leucinophagum, Corynebacterium autotrophicum, Corynebacterium nephridii, Mycobacterium flavum, Mycoplana rubra and Protaminobacter ruber contained uniquinones as their sole isoprenoid quinones. The isoprenoid quinone data correlate well with major trends in coryneform taxonomy and are of value in the classification of coryneform and related bacteria.     

Chemotaxonomic differentiation of coryneform bacteria isolated from biofilters.

Coryneform bacteria that were isolated from biofilters which are used for waste gas treatment of animal-rendering plant emissions were differentiated and partially identified by using chemotaxonomic methods. On the basis of the results of a numerical analysis of whole-cell fatty acid profiles, 79 isolates were divided into two major groups; the members of the first group contained saturated and monounsaturated fatty acids, whereas the members of the second group were characterized by iso- and anteiso-branched fatty acids. Division into subclusters was based mainly on quantitative differences in fatty acid composition and was confirmed by the results obtained for additional chemical markers (e.g., respiratory quinones, mycolic acids, polar lipids, cell wall amino acids, and whole-cell sugar patterns). By combining the results obtained for chemotaxonomic analyses that were performed for strains containing saturated and monounsaturated fatty acids, we were able to identify the genus Corynebacterium (two Corynebacterium species were differentiated on the basis of the occurrence of tuberculostearic acid), the genus Gordona, and the genus Mycobacterium. Among the strains that produced iso-anteiso fatty acid patterns, one subgroup was affiliated with the "nicotianae" group of the genus Arthrobacter; however, some strains contained a new combination of chemical markers. Peptidoglycan type A4 alpha, L-Lys-Gly-L-Glu was combined with menaquinones MK-7 and MK-8, whereas peptidoglycan type A4 alpha, L-Lys-L-Glu occurred together with MK-8 and MK-9. The second subgroup was characterized by a new type B peptidoglycan and MK-11, as well as small amounts of MK-12. Differentiation that was based first on chemotaxonomy and second on physiology gave reliable results. Thus, coryneform strains with new characteristics were isolated from biofilters.     

Lipids in the Classification and Identification of Coryneform Bacteria Containing Peptidoglycans Based on 2, 4-diaminobutyric Acid

Strains of 2, 4-diaminobutyric acid-containing coryneform bacteria were degraded by acid methanolysis and the non-hydroxylated fatty acid esters released examined by thin-layer and gas chromatography. The major fatty acid structural types were straight-chain, anteiso - and iso -methyl branched-chain acids. Polar lipids of the test strains were examined by two-dimensional thin-layer chromatography. All strains possessed very characteristic polar lipid patterns consisting of diphosphatidylglycerol, phosphatidylglycerol and a number of uncharacterized glycolipids. Menaquinones (vitamin K) were the sole isoprenoid quinones detected in the test strains. Corynebacterium insidiosum, Cor. michiganense, Cor. nebraskense and Cor. sepedonicum contained unsaturated menaquinones with nine isoprene units, whereas unsaturated menaquinones with 10 isoprene units predominated in strains of Cor. iranicum and Cor. tritici and a strain labelled Arthrobacter sp. The single strain of Cor. aquaticum examined contained comparable amounts of menaquinones with 10 and 11 isoprene units whereas strains of Cor. mediolanum and Flavobacterium dehydrogenans contained major amounts of menaquinones with 11 and 12 isoprene units. The results of the present study indicate that lipid markers may be of considerable value in the classification and identification of 2, 4-diaminobutyric acid-containing phytopathogenic and saprophytic coryneform bacteria.     

Polar lipid and isoprenoid quinone composition in the classification of Staphylococcus

Representatives of 13 species of Staphylococcus were examined using a small-scale procedure for the sequential extraction of isoprenoid quinones and polar lipids. Menaquinones were the only isoprenoid quinones found in the 77 test strains which were divided into three groups based upon the predominant isoprenologue detected: (i) S. hyicus subsp. hyicus, S. sciuri subsp. lentus and S. sciuri subsp. sciuri contained unsaturated menaquinones with six isoprene units; (ii) S. capitis, S. cohnii, S. epidermidis, S. haemolyticus, S. hominis, S. hyicus subsp. chromogenes, S. intermedius, S. saprophyticus, S. simulans, S. warneri and S. xylosus contained unsaturated menaquinones with seven isoprene units and (iii) S. aureus contained unsaturated menaquinones with eight isoprene units and varying amounts of the corresponding lower isoprenologue. All of the organisms contained very similar polar lipid patterns consisting of diphosphatidylglycerol, phosphatidylglycerol, beta-gentiobiosyl diacylglycerol and a number of glycolipids and phospholipids. One of the glycolipids was chromatographically indistinguishable from beta-gentiotriosyl diacylglycerol. Lysylphosphatidylglycerol was a major component in S. aureus and S. intermedius but was usually present in minor amounts in the coagulase-negative strains. The polar lipid data underline the homogeneity of the genus Staphylococcus and distinguish staphylococci from aerobic, Gram-positive cocci and from the phylogenetically related aerobic, endospore-forming bacteria. Menaquinone composition can also be used to separate staphylococci from other aerobic, Gram-positive cocci.     

Flexibacter elegans and Myxococcus fulvus: Aerobic gram-negative bacteria containing menaquinones as the only isoprenoid quinones

The aerobic Gram-negative gliding bacteria, Flexibacter elegans (Cytophagales) and Myxococcus fulvus (Myxobacterales) contain exclusively menaquinones as isoprenoid quinones. Each strain produces 3 different menaquinones. The menaqinone content of Flexibacter elegans was 1.5 μmol/g dry cells, with MK-7 as the main compound (>99%). MK-7 was identified by mass and n.m.r. spectroscopy. Myxococcus fulvus contained 2.1 μmol menaquinones per g dry mass, with more than 90% being MK-8.     

Characterization of the lipids of psychrophilic bacteria <Emphasis Type="Italic">Shewanella frigidimarina</Emphasis> isolated from sea ice of the Sea of Japan

Lipids of ten Shewanella frigidimarina strains isolated from sea ice samples of coastal areas of the Sea of Japan and of the type strains of psychrophilic bacteria S. frigidimarina ACAM 591^T and S. hanedai JCM 20706^T were analyzed. Most of the new isolates contained isoprenoid quinones typical of the genus Shewanella (Q-7, Q-8, MK-7, and MMK-7), a high level of branched acids (i-13:0 and i-15:0), and polyunsaturated fatty acid (20:5 ω3). Phospholipid fractions of marine isolates and the type strain S. frigidimarina ACAM 591^T contained not only the main phospholipids (phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol), but also an unknown phosphoaminolipid, which is probably typical of this bacterial species. The isolates exhibited a high level of phylogenetic similarity but were phenotypically heterogeneous. Two strains distinguished by their phenotypic characteristics differed also in the composition of fatty acids, isoprenoid quinones, and phospholipids. The use of chemotaxonomic markers for primary species identification of psychrophilic bacteria of the genus Shewanella is discussed.     

Lactococcus lactis produces short-chain quinones that cross-feed Group B Streptococcus to activate respiration growth

Quinones are essential components of the respiration chain that shuttle electrons between oxidoreductases. We characterized the quinones synthesized by Lactococcus lactis , a fermenting bacterium that activates aerobic respiration when a haem source is provided. Two distinct subgroups were characterized: Menaquinones (MK) MK-8 to MK-10, considered as hallmarks of L. lactis , are produced throughout growth. MK-3 and demethylMK-3 [(D)MK-3] are newly identified and are present only late in growth. Production of (D)MK-3 was conditional on the carbon sugar and on the presence of carbon catabolite regulator gene ccpA . Electron flux driven by both (D)MK fractions was shared between the quinol oxidase and extracellular acceptors O₂, iron and, with remarkable efficiency, copper. Purified (D)MK-3, but not MK-8–10, complemented a menB defect in L. lactis . We previously showed that a respiratory metabolism is activated in Group B Streptococcus (GBS) by exogenous haem and MK, and that this activity is implicated in virulence. Here we show that growing lactococci donate (D)MK to GBS to activate respiration and stimulate growth of this opportunist pathogen. We propose that conditions favouring (D)MK production in dense microbial ecosystems, as present in the intestinal tract, could favour implantation of (D)MK-scavengers like GBS within the complex.     

Lipoquinones of some bacteria and mycoplasmas,with considerations on their functional significance

In a comparative study the lipoquinones of some chemoorganotrophic, facultatively aerobic bacteria, and representative Acholeplasma, Mycoplasma, Spiroplasma, and Thermoplasma strains were investigated. The quinones were partly purified by preparative thin layer chromatography of lipid extracts, and characterized by their difference spectra (reduced minus oxidized) and R_f values. Respiring bacteria expectedly contained benzoquinones and/or naphthoquinones in micromolar concentrations whereas some aerotolerant, cytochrome-less, gram-positive bacteria were found to contain menaquinones in nanomolar concentrations, or even no quinones; only Streptococcus faecalis, an organism supposed to use a rudimentary, flavin-terminated respiratory chain system produced desmethyl menaquinone in amounts ranging between high and low quinone contents. Among the mycoplasmas investigated, only Thermoplasma acidophilum was found to be capable of synthesizing quinones (MK-7) in the micromolar order of magnitude indicating a respiratory electron transport system. The presence of energetically useful respiratory chain systems in Acholeplasma, Mycoplasma, and Spiroplasma is questioned since these organisms contain quinones (MK-4) in nanomolar concentrations, or no quinones, depending on the presence of exogeneous MK-6 in the growth medium. The possible metabolic role of menaquinones present in low amounts, as well as the role of NADH oxidase systems more or less tightly bound to the cytoplasmic membrane with the mycoplasmas deserves further investigation.     

Lipid composition of novel Shewanella species isolated from far Eastern seas

A comparative study of the lipid composition of 26 strains (including type strains) of marine Gammaproteobacteria belonging to the genera Shewanella, Alteromonas, Pseudoalteromonas, Marinobacterium, Microbulbifer, and Marinobacter was carried out. The bacteria exhibited genus-specific profiles of ubiquinones, phospholipids, and fatty acids, which can serve as reliable chemotaxonomic markers for tentative identification of new isolates. The studied species of the genus Shewanella were distinguished by the presence of two types of isoprenoid quinones, namely, ubiquinones Q-7 and Q-8 and menaquinones MK-7 and MMK-7; five phospholipids typical of this genus, namely, phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), lyso-PE, and acyl-PG; and the fatty acids 15:0, 16:0, 16:1 (n-7), 17:1 (n-8), i-13:0, and i-15:0. The high level of branched fatty acids (38-45%) and the presence of eicosapentaenoic acid (4%) may serve as criteria for the identification of this genus. Unlike Shewanella spp., bacteria of the other genera contained a single type of isoprenoid quinone: Q-8 (Alteromonas, Pseudoalteromonas, Marinobacterium, and Microbulbifer) or Q-9 (Marinobacter). The phospholipid compositions of these bacteria were restricted to three components: two major phospholipids (PE and PG) and a minor phospholipid, bisphosphatidic acid (Alteromonas and Pseudoalteromonas) or DPG (Marinobacterium, Microbulbifer, and Marinobacter). The bacteria exhibited genus-specific profiles of fatty acids.     

Isoprenoid Quinone Composition as a Guide to the Classification of Sporolactobacillus and Possibly Related Bacteria

Menaquinones with seven isoprene units were the major isoprenoid quinones detected in the chloroform-methanol extracts of representative strains of the genera Bacillus and Sporolactobacillus. Neither menaquinones nor ubiquinones were detected in similar extracts of strains of the genus Lactobacillus.     

Isolation of bacteria of the genus <Emphasis Type="Italic">Variovorax</Emphasis> from the <Emphasis Type="Italic">Thioploca</Emphasis> mats of Lake Baikal

Three strains of gram-negative bacteria were isolated from the mats of colorless sulfur bacteria Thioploca (Lake Baikal). The cells of new strains are motile with peritrichous flagella. Bacteria are aerobic, obligate chemoorganoheterotrophs growing within the pH range of 3.0–8.8 with the optimum at 8.3 and within the temperature range of 5–42°C with the optimum at 28°C. The cells contained menaquinones MK-8 H2 as the major component, as well as MK-7 H2 (less than 15%), while the content of ubiquinone Q8 was at least an order of magnitude lower. The G+C content of DNA in the new strains varied from 67.4 to 69.9 mol %. The level of DNA-DNA hybridization between the strains ranged from 80 to 94%, indicating that all the isolates belonged to one species. Analysis of the 16S rRNA gene nucleotide sequences of the type strain (Gen-Bank HQ400611) revealed close homologues among the known species of the genus Variovorax: 98% resemblance with the type strains of the species V. paradoxus, V. soli, V. ginsengisoli, and V. boronicumulans and 96% similarity with the type strain of V. dokdonensis. However, since the isolates differed significantly in the composition of fatty acids and isoprenoid quinones from the nearest neighbors in the phylogenetic tree, they cannot be related implicitly to the known species.     

Phylogeny of 54 representative strains of species in the family Pasteurellaceae as determined by comparison of 16S rRNA sequences.

Virtually complete 16S rRNA sequences were determined for 54 representative strains of species in the family Pasteurellaceae. Of these strains, 15 were Pasteurella, 16 were Actinobacillus, and 23 were Haemophilus. A phylogenetic tree was constructed based on sequence similarity, using the Neighbor-Joining method. Fifty-three of the strains fell within four large clusters. The first cluster included the type strains of Haemophilus influenzae, H. aegyptius, H. aphrophilus, H. haemolyticus, H. paraphrophilus, H. segnis, and Actinobacillus actinomycetemcomitans. This cluster also contained A. actinomycetemcomitans FDC Y4, ATCC 29522, ATCC 29523, and ATCC 29524 and H. aphrophilus NCTC 7901. The second cluster included the type strains of A. seminis and Pasteurella aerogenes and H. somnus OVCG 43826. The third cluster was composed of the type strains of Pasteurella multocida, P. anatis, P. avium, P. canis, P. dagmatis, P. gallinarum, P. langaa, P. stomatis, P. volantium, H. haemoglobinophilus, H. parasuis, H. paracuniculus, H. paragallinarum, and A. capsulatus. This cluster also contained Pasteurella species A CCUG 18782, Pasteurella species B CCUG 19974, Haemophilus taxon C CAPM 5111, H. parasuis type 5 Nagasaki, P. volantium (H. parainfluenzae) NCTC 4101, and P. trehalosi NCTC 10624. The fourth cluster included the type strains of Actinobacillus lignieresii, A. equuli, A. pleuropneumoniae, A. suis, A. ureae, H. parahaemolyticus, H. parainfluenzae, H. paraphrohaemolyticus, H. ducreyi, and P. haemolytica. This cluster also contained Actinobacillus species strain CCUG 19799 (Bisgaard taxon 11), A. suis ATCC 15557, H. ducreyi ATCC 27722 and HD 35000, Haemophilus minor group strain 202, and H. parainfluenzae ATCC 29242. The type strain of P. pneumotropica branched alone to form a fifth group. The branching of the Pasteurellaceae family tree was quite complex. The four major clusters contained multiple subclusters. The clusters contained both rapidly and slowly evolving strains (indicated by differing numbers of base changes incorporated into the 16S rRNA sequence relative to outgroup organisms). While the results presented a clear picture of the phylogenetic relationships, the complexity of the branching will make division of the family into genera a difficult and somewhat subjective task. We do not suggest any taxonomic changes at this time.     

反向间接胶乳凝集试验法检测粪便A型产气荚膜梭菌肠毒素的结果及评价

本文以１０种５２株供试菌分别与７个不同年龄组的健康人粪便混合,共配成３６４份模拟标本,采用反向间接胶乳凝集（ＲＰＬＡ）试验法与生物学试验法（小鼠致死试验、豚鼠皮肤血管透性因子试验,Ｖｅｒｏ细胞毒性试验）检测各标本中的Ａ型产气荚膜梭菌肠毒素（简称Ｃｐ－Ｅｎｔ）。除产气荚膜梭菌之外的其他菌种培养液２３８份标本（３４株）,ＲＰＬＡ与生物学试验结果完全一致,均为阴性。产气荚膜梭菌１２６份标本（１８株）中有７０份标本的ＲＰＬＡ同生物学诸法完全一致地检出了Ｃｐ－Ｅｎｔ．有１株７份标本（ＣｐＮＣＴＣ８７９７）的ＲＰＬＡ为阳性,而各生物学试验却均为阴性,该菌株经ＰＣＲ检查证明确为肠毒素原性产气荚膜梭菌,表明ＲＰＬＡ比生物学试验法更灵敏。有５株（ＣｐＮＣＴｃ８２３８,ＣｐＮＣＴＣ１０６１１,ＣｐＮＣＴＣ１０６１４,ＣｐＮＣＴＣ１０６１２,ＣｐＬ－５２）３５份标本ＲＰＬＡ与各生物学试验结果均为阴性,但经ＰＣＲ检吉证明该５株菌均为肠毒素原性产气荚膜梭菌,后经超声波破碎菌体提取物对其中部分菌株进行试验的结果仍然显示了ＲＰＬＡ与生物学法的一致性。有２株（ＣｐＮＣＴＣ８６８６,ＣｐＮＣＴＣ８４４９）１４份标本的所有结果均为阴性,ＰＣＲ     

Immunoadjuvant activities of cell walls and their water-soluble fractions prepared from various gram-positive bacteria.

The cell walls from all 21 species of gram-positive bacteria examined, except lysozyme-susceptible Micrococcus lysodeikticus (NCTC 2665) and lysozyme-resistant Staphylococcus epidermidis (ATCC 155), were found to be definitely adjuvant-active in both stimulation of increased serum antibody levels and induction of delayed-type hypersensitivity to ovalbumin when administered to guinea-pigs as water-in-oil emulsions. Using various cell wall lytic enzymes, the immunoadjuvant principles were solubilized with full retention of the adjuvant activities from walls of Staphylococcus aureus (Copenhagen), Streptococcus pyogens (group A, type 6; S43/100), Streptococcus salivarius (IFO 3350), Streptococcus faecalis (IFO 12580), Streptococcus mutans (BHT), Lactobacillus plantarum (ATCC 8014), Bacillus megaterium (IFO 12068), Corynebacterium diphtheriae (Park-Williams No. 8), Mycobacterium smegmatis, and Actinomyces viscous (ATCC 15987). Evidence was obtained that the non-peptidoglycan portion of the cell walls is not essential for manifestation of immunoadjuvancy.     

Capsular polysaccharides and lipopolysaccharides from two Bacteroides fragilis reference strains: chemical and immunochemical characterization.

Fermentor growth of Bacteroides fragilis under controlled conditions in a complex medium containing 1% glucose and 10% fetal calf serum resulted in high yields of bacteria. After hot phenol-water extraction of the organisms, capsular polysaccharide was isolated from the aqueous phase and purified by Sephacryl S-300 chromatography in a buffer with 3% sodium deoxycholate. Lipopolysaccharide was isolated by phenol-chloroform-light petroleum ether extraction. The capsular polysaccharide from B. fragilis strain NCTC 9343 contained six sugars: L-fucose, D-galactose, D- and L-quinovosamine, D-glucosamine, and galacturonic acid. The capsule of strain ATCC 23745 also contained D-glucose, L-fucosamine, L-rhamnosamine, and a 3-amino-3,6-dideoxyhexose but lacked D-quinovosamine. The latter capsule also contained alanine (4%). The capsular polysaccharides were different immunochemically by ELISA inhibition. The lipopolysaccharide of both strains contained the same sugars (L-rhamnose, D-glucose, D-galactose, and D-glucosamine) and fatty acids (13-methyl-tetradecanoic and 3-hydroxy-hexadecanoic and 3-hydroxy-15 methyl-hexadecanoic as major constituents) and were identical by ELISA inhibition.     

Flavonols and an oxychromonol from Piliostigma reticulatum

The leaf extract from the plant Piliostigma reticulatum was found to exhibit antimicrobial activity against some bacteria and fungi such as Staphylococcus aureus (NCTC 6571), Escherichia coli (NCTC 10418), Bacillus subtilis (NCTC 8236), Proteus vulgaris (NCTC 4175), Aspergillus niger (ATCC 10578) and Candida albicans (ATCC 10231). Upon investigation of the chemical constituents present in the leaf extract, a total of seven compounds were isolated and their structures were unambiguously established by spectroscopic methods including HR-MS and NMR spectrometry. Four of the isolated compounds were novel, namely 6-C-methyl-2-p-hydroxyphenyloxychromonol (piliostigmol), 1, 6,8-di-C-methylquercetin-3,3',7-trimethyl ether, 2, 6,8-di-C-methylquercetin-3,3'-dimethyl ether, 3 and 3',6,8,-tri-C-methylquercetin-3,7-dimethyl ether, 4. The other three were known C-methylated flavonols and they were isolated from P. reticulatum for the first time. These were 6-C-methylquercetin-3-methyl ether, 5, 6,8-di-C-methylkaempferol-3-methyl ether, 6 and 6-C-methylquercetin-3,3',7-trimethyl ether 7. All the isolated compounds were tested for cytotoxicity using the brine shrimp toxicity assay and all of them were active albeit at different levels. With respect to antibacterial activity piliostigmol, 1 showed the highest activity against E. coli (MIC=2.57 microg/ml, 0.006 micromol), which is three times more that of Amoxicillin, where as 4 and 7 showed the least activity.     

Burkholderia thailandensis E125 harbors a temperate bacteriophage specific for Burkholderia mallei

Burkholderia thailandensis is a nonpathogenic gram-negative bacillus that is closely related to Burkholderia mallei and Burkholderia pseudomallei. We found that B. thailandensis E125 spontaneously produced a bacteriophage, termed phiE125, which formed turbid plaques in top agar containing B. mallei ATCC 23344. We examined the host range of phiE125 and found that it formed plaques on B. mallei but not on any other bacterial species tested, including B. thailandensis and B. pseudomallei. Examination of the bacteriophage by transmission electron microscopy revealed an isometric head and a long noncontractile tail. B. mallei NCTC 120 and B. mallei DB110795 were resistant to infection with phiE125 and did not produce lipopolysaccharide (LPS) O antigen due to IS407A insertions in wbiE and wbiG, respectively. wbiE was provided in trans on a broad-host-range plasmid to B. mallei NCTC 120, and it restored LPS O-antigen production and susceptibility to phiE125. The 53,373-bp phiE125 genome contained 70 genes, an IS3 family insertion sequence (ISBt3), and an attachment site (attP) encompassing the 3' end of a proline tRNA (UGG) gene. While the overall genetic organization of the phiE125 genome was similar to lambda-like bacteriophages and prophages, it also possessed a novel cluster of putative replication and lysogeny genes. The phiE125 genome encoded an adenine and a cytosine methyltransferase, and purified bacteriophage DNA contained both N6-methyladenine and N4-methylcytosine. The results presented here demonstrate that phiE125 is a new member of the lambda supergroup of Siphoviridae that may be useful as a diagnostic tool for B. mallei.     

Bacteria decomposing technical oils

Fifty bacterial cultures were isolated from technical oils stored for different periods of time. Twenty out of the fifty strains decomposed technical oils. The bacteria were identified and classified as Bacillus strains. Bacillus subtilis, strains 7 and 10, and B. pumilus, strain 13, isolated from oils MK-8 + 10% Akor-1 and MT-16p as well as from AMG-10 were found to be most active and can be recommended as test cultures to study the resistance of technical oils against bacterial degradation.     

Comparison of the cellular fatty acid composition of a bacterium isolated from a human and alleged to be Bacillus sphaericus with that of Bacillus sphaericus isolated from a mosquito larvicide.

The cellular fatty acid (CFA) composition of the cytoplasmic membrane of a bacillus isolated from a human lung and deposited in the National Collection of Type Cultures as Bacillus sphaericus NCTC 11025 was determined by gas-liquid chromatography. The CFA composition of B. sphaericus 2362, isolated from a microbial larvicide, and those of B. sphaericus reference strains obtained from public collections were also determined. Samples were grouped by hierarchical cluster analysis based on the unpaired-group method using arithmetic averages. Samples that linked at a Euclidean distance of < or = 2.0 U were considered to belong to the same strain. NCTC 11025 and the type strain of B. sphaericus, ATCC 14577, were mixed; all other isolates were monotypic. The predominant fatty acid in NCTC 11025 was 12-methyltetradecanoic acid, while the predominant fatty acid in the remaining isolates was 13-methyltetradecanoic acid. NCTC 11025 linked to the other isolates at a Euclidean distance of 83.8 U, and we concluded that it belongs to a different species that we could not identify. We could distinguish among six DNA homology groups of B. sphaericus by using fatty acids. Within DNA homology group IIA, strain 2362 could be distinguished from other strains belonging to serotype H5a, 5b. We concluded that CFA analysis is a useful technique to determine if future human isolates identified as B. sphaericus in fact belong to other species of bacteria or whether the isolates originated from commercial products.