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Eighteen strains isolated from human specimens or from food products were characterized as atypical variants of mannitol-negative Streptococcus bovis. They were tested for extended biochemical criteria, ribotyping and DNA-DNA hybridization in order to define their taxonomic status. These strains were demonstrated to constitute a DNA relatedness group that includes strains of DNA group 4 of Farrow et al. (1984). Comparative analysis of 16S rRNA sequences demonstrated that these strains represent a new species which belongs to the Streptococcus bovis/Streptococcus equinus complex and which has been provisionally named S. infantarius by Bouvet et al. (1997). Biotyping and ribotyping allowed differentiation of these strains from the aesculin-positive strains of S. bovis belonging to the previously described biotypes I, II.1 and II.2. The results of the ribotyping and hybridization assays demonstrated the presence of two different DNA subgroups within the 18 strains. On the basis of these data, the names S. infantarius subsp. infantarius (aesculin-negative for five strains out of seven, including the type strain HDP 90056T = NCDO 599T) and S. infantarius subsp. coli (aesculin-positive, reference strain HDP 90248 = NCDO 2620) are proposed as the names for these two subspecies within the S. infantarius species.

A phylogenetic analysis was performed on a red-pigmented, radiation-resistant, Gram-negative, rod-shaped organism originating from irradiated pork. Comparative 16S rRNA gene sequencing showed the bacterium was a member of the Cytophaga-Flavobacterium-Bacteroides line of descent and represents a new subline within the genus Hymenobacter. A new species, Hymenobacter actinosclerus, is described for this novel radiation-resistant bacterium. The type strain of Hymenobacter actinosclerus is CCUG 39621T.

The microbiota of the human large intestine influences health and well-being. Whereas it has long been accepted that gut bacteria play a role in host pathogenesis, current opinion is that certain microflora components can have beneficial effects on gastroenteritis resistance, blood lipids, antitumor properties, lactose tolerance, and gastrointestinal immunity. It is postulated that in the infant gut an elevated bifidobacterial count may be associated with health advantages that breast-fed infants may have over formula-fed infants. Whereas beneficial aspects of the human gut flora still need definitive confirmation and mechanistic explanations, there is now interest in modulating the composition of gut flora such that a potentially more remedial community exists. This may be achieved through the targeted use of dietary supplementation. This article provides an overview of how probiotics, prebiotics, and synbiotics may contribute toward nutritional modulation of the gut microecology, with emphasis on the neonatal intestine where appropriate. The use of modern molecular methods, as an essential step forward for assessing the validity and accuracy of the modulatory approach, is also discussed.

Until recently, all clostridia producing neurotoxins able to cause paralysis symptomatic of botulism were deemed to be Clostridium botulinum. Defining Cl. botulinum on the basis of this single phenotypic trait has resulted in the species encompassing metabolically very diverse organisms, and four distinct phenotypic groups are recognized within this taxon (designated groups I-IV). Nucleic acid hybridization and 16S ribosomal RNA sequencing studies have revealed the presence of four phylogenetically distinct lineages within the species, which correlate with these phenotypic divisions. In addition to marked phenotypic and genotypic heterogeneity between groups, the taxonomy of the species is further complicated by the existence of strains which are closely related, if not genetically identifiable, to members of each Cl. botulinum group, but are non-toxigenic. Furthermore, strains of species other than Cl. botulinum (viz. Cl. baratii, Cl. butyricum) have been found which express botulinum neurotoxin (BoNT). Great advances have been made in recent years in elucidating the nucleotide sequences of genes encoding the various BoNT antigenic types (A through to G). Genealogical trees derived from BoNTs show marked discordance with those depicting 'natural' relationships inferred from 16S rRNA and phenotypic clusters, and strong evidence exists for BoNT gene transfer between some groups of Cl. botulinum (e.g. groups I and II), and with non-botulinum species. Botulinum neurotoxin is produced by Cl. botulinum as a non-covalently bound progenitor toxin complex of two or more protein components. Information on the evolutionary histories of the various non-toxic progenitor proteins is currently limited, although there is evidence of gene recombination. In particular, chimera-like or mosaic non-toxic-non-haemagglutinins (NTNH) genes in group I Cl. botulinum have been described, and it is now apparent that the phylogeny of the NTNHs is not going to 'mirror' that of botulinal neurotoxins, although their genes are physically contiguous. In this article, the current state of knowledge of the phylogenetics of the species Cl. botulinum and its neurotoxins is reviewed, and a view is presented that a nomenclature based rigidly on BoNT production is no longer tenable.

A 5-month-old boy of a Romanian family traveling via Ukraine to Poland developed a respiratory disease that resembled and that was initially diagnosed as pharyngeal diphtheria. The child recovered after treatment with antidiphtheria antitoxin. A coryneform bacterium had been isolated from a nasopharyngeal specimen from the child and was initially identified as an atypical Corynebacterium diphtheriae strain. Seven adults who had contact with either the child or an adult contact person also developed symptoms of pharyngeal diphtheria, were also treated with antitoxin, and recovered uneventfully. Coryneform bacteria similar to that originating from the index patient were also isolated from the throat swabs of three adults. Detailed biochemical and chemotaxonomic investigations revealed that the coryneform bacteria belonged to the genus Corynebacterium and could be differentiated from all other defined species of this genus. Ribotyping and pulsed-field gel electrophoresis demonstrated that all four patients' isolates were of clonal origin. The diphtheria toxin gene and its product were not detected either by PCR assays or by the Elek test, making a possible disease association of the Corynebacterium more unlikely. Comparative 16S rRNA gene sequence analysis revealed that the coryneform bacterium represented a new subline within the genus Corynebacterium, for which the name Corynebacterium imitans sp. nov. is proposed. The type strain is NCTC 13015 (DSM 44264; CCUG 36877).

Four strains of an obligately anaerobic spore-forming bacterium were isolated from soil samples from a donkey pasture in Pakistan. Comparative 16S rRNA sequence analysis demonstrated that the strains are members of phylogenetic cluster I of the genus Clostridium (Collins et al. 1994). The strains are mesophilic, nonsaccharolytic, and nonproteolytic, utilize glutamate and histidine, and produce indole. Acetate, butyrate, ethanol, hydrogen, and carbon dioxide are the products of fermentation. Although the strains phenotypically resemble the classical glutamate-fermenting Clostridia, such as Clostridium cochlearium, Clostridium tetanomor-phum, Clostridium tetani, and especially Clostridium malenominatum, they differ from these organisms in sugar utilization, cellular fatty acid composition, and cellular protein pattern and by a 16S rRNA sequence divergence value of approximately 4 to 8%. Phylogenetically, the strains are more closely related to Clostridium estertheticum (sequence divergence, approximately 5%) and Clostridium subterminale (sequence divergence, approximately 5%) but are phenotypically readily distinguished from these species. On the basis of phenotypic and genotypic criteria, we conclude that the four strains are members of a new species of the genus Clostridium, for which the name Clostridium pascui is proposed. The type strain is strain Cm19 (= DSM 10365).

A comprehensive study was performed on 25 bacterial clinical isolates originally identified as Corynebacterium xerosis. Three reference strains of C. xerosis were also included in the study. On the basis of a variety of phenotypic characteristics tested, all strains could be divided into two separate clusters: reference strains ATCC 373 (the type strain of C. xerosis) and ATCC 7711 showed yellow-pigmented, dry, rough colonies, fermented 5-keto-gluconate, exhibited strong leucine arylamidase and alpha-glucosidase activities, produced lactate as the major end product of glucose metabolism, were susceptible to most of the 19 antimicrobial agents tested, and showed an inhibition zone around disks containing the vibriocidal compound O/129. In contrast, the remaining 26 strains including reference strain NCTC 7243 as well as all clinical isolates formed white-grayish, dry, slightly rough colonies, did not ferment 5-keto-gluconate, exhibited only weak leucine arylamidase and no alpha-glucosidase activity, produced large amounts of propionic acid as the end product of glucose metabolism, and were resistant to most antimicrobial agents tested, including O/129. Chemotaxonomic (cellular fatty acids, mycolic acids, and G+C content) and molecular genetic (16S rRNA gene sequence) investigations revealed that the strains of the second cluster unambiguously belonged to the species C. amycolatum. Our data suggest that most strains reported in the literature as C. xerosis are probably misidentified and correspond to C. amycolatum.

We have studied four organic acids of similar structure to further understand the basis of their developmental toxicity. Valproic acid (2-propyl pentanoic acid), ethylhexanoic acid, and octanoic acid are isomeric C8 organic acids but their teratologic potency varied widely. Valproic acid induced a moderate to severe teratologic outcome after a single oral administration of 6.25 mmoles/kg on day 12 of rat pregnancy. Twice as much ethylhexanoic acid (12.5 mmoles/kg) induced a less severe response. Octanoic acid was nonteratogenic even at the very high dose of 18.75 mmoles/kg. This latter result is undoubtedly due to poor intestinal absorption of octanoic acid, as the maternal plasma levels never reached half of those measured for valproic acid and ethylhexanoic acid. Moreover, only a tiny fraction of that in maternal plasma was actually transferred into the embryo. On the other hand, the peak concentration and duration of exposure to valproic acid and ethylhexanoic acid were very similar despite a more severe teratologic outcome following valproic acid, which indicated higher intrinsic activity of this latter agent. A fourth agent, methylhexanoic acid, was also studied and had no teratogenic effects when given at 14.1 mmoles/kg. Pharmacokinetic studies of this agent revealed higher peak concentrations in maternal plasma and embryo than valproic acid or ethylhexanoic acid, but the duration of exposure was shorter. We conclude that pharmacokinetic parameters can be important determinants of teratologic outcome and thereby help explain differing potencies of structurally similar chemicals.

The phylogenetic interrelationship of species of the genus Zygosaccharomyces was examined by 18S rRNA gene sequencing. Comparative analysis of the sequence data revealed the genus to consist of a number of distinct subdivisions. The most prevalent species associated with food spoilage, Z. bailii, Z. bisporus and Z. rouxii, along with Z. mellis were found to form one subdivision. Zygosaccharomyces cidri and Z. fermentati formed a distinct species pair, as did Z. microellipsoides and Z. mrakii. Zygosaccharomyces florentinus formed a separate line displaying no specific relationship with any of the other Zygosaccharomyces species examined. Comparison with nine published ascosporogenous yeast 18S rRNA gene sequences showed that Z. microellipsoides and Z. mrakii were genealogically very close to Torulaspora delbrueckii (both displaying 99·8% 18S rRNA sequence similarity), raising the possibility that these two Zygosaccharomyces species should be moved to the genus Torulaspora. The topologies of trees derived from complete 18S rRNA gene sequences and from individual domains within the gene were compared and the implications of using partial sequence data for inferring phylogenetic relationships discussed.

A polymerase chain reaction test was developed for identification of the gram-positive urinary tract pathogen Aerococcus urinae. Oligonucleotide primers were based on highly specific sequences within the small-subunit rRNA gene. A confirmatory test based on hybridization of the amplified products to a highly specific internal probe was also developed.

The partial 16S rRNA sequence of an unknown bacterium that was originally isolated from middle ear fluids of children with persistent otitis media was determined by reverse transcription. A comparison of this sequence with sequences from other gram-positive species having low guanine-plus-cytosine contents revealed that this bacterium represents a new line of descent, for which the name Alloiococcus otitis gen. nov., sp. nov., is proposed. The type strain is strain NCFB 2890.

A new species, Propionibacterium innocuum, is proposed to accommodate strains of coryneform bacteria from human skin with phenotypic characters similar to those of the classical propionibacteria but differing in exhibiting primarily aerobic respiration and possessing a unique cell wall composition in which LL-diaminopimelic acid and arabinose occur together. The partial 16S rRNA sequence confirms an affinity with the genus Pro-pionibacterium and indicates that the species represents a distinct line within the genus. The type strain of Propionibacterium innocuum is NCTC 11082.

This paper outlines the results of a recent survey of the UK contract research market, estimated at 900 MECU (1988/89). Most UK contract research organization (CROs) undertake a small but significant amount of overseas contract R & D (both for other Member States and elsewhere), and see this increasing as the Single European Market (SEM) develops. Most UK CROs have participated in EC R & D programmes and viewed involvement as a, generally, positive experience. UK Industrial customers of contract R & D, although more Uk orientated, also believe the SEM will increase the amount of contracting from Member States. Industrial companies involved in EC R & D programmes also noted benefits from involvement. Both UK CROs and the industrial customer organizations saw the SEM and the associated Europeanization process as enhancing commercial contacts with organizations in other Member States.

Maternal diabetes (types 1 and 2) induces a broad array of congenital malformations, including neural tube defects (NTDs), in humans. One of the difficulties associated with studying diabetic embryopathy is the rarity of individual malformations. In an attempt to develop a sensitive animal model for maternal diabetes-induced NTDs, the present study uses chemically induced diabetes in an inbred mouse model with or without the splotch (Sp) mutation, a putatively nonfunctional allele of Pax3. Pax3 deficiency has been associated with an increase in NTDs. Female C57BL/6J mice, either with or without the Sp allele, were injected intravenously with alloxan (100 mg/kg), and plasma glucose was measured 3 days later. A wide range of hyperglycemia was induced, and these diabetic mice were bred to C57BL/6J males, some carrying the Sp allele. Gestational-day-18 fetuses were examined for developmental malformations. Fetuses from matings in which either parent carried the Sp allele were genotyped by polymerase chain reaction. Maternal diabetes significantly decreased fetal weight and increased the number of resorptions and malformations, including NTDs. A significant correlation was found between the level of maternal hyperglycemia and the malformation rate. The sex ratio for live fetuses in diabetic litters was significantly skewed toward male fetuses. Matings involving the Sp allele yielded litters with significantly higher percentages of maternal diabetes-induced spina bifida aperta but not exencephaly, and this increase was shown to be associated with the presence of a single copy of the Sp allele in affected fetuses. Thus, Pax3 haploinsufficiency in this murine model of diabetic embryopathy is associated with caudal but not cranial NTDs.

Two Gram-positive, non-motile and aerobic bacteria were isolated from a water sample of the hypersaline Ekho Lake, Antarctica. The cocci or short rods grew well on oligotrophic PYGV agar of pH 7.5 and at 26 degrees C. Strains EL-17KT and EL-17AT both required thiamine and biotin, strain EL-17AT also required nicotinic acid. Carbon sources utilized by both strains were acetate, pyruvate, alpha-D-glucose, glutamate and (weakly) citrate, but succinate, malate or butyrate were utilized only by EL-17KT. Gelatin, starch and DNA were hydrolyzed, NH, was formed from peptone, and nitrate was reduced aerobically by both strains. The isolates had the same temperature tolerance for growth in the range tested (below 3 to above 33.5 degrees C) and pH range (5.5 to9.5) and were sensitive to chloramphenicol and penicillin G. Their cell walls contained LL-diaminopimelic acid and had a single glycine residue as interpeptide bridge. Strain EL-17AT contained glycine at position 1 of the peptide subunit (peptidoglycan type A 3gamma'). Isolates EL-17KT and EL-17AT differed in their maximum NaCl tolerance, which was 15% or 6-8%, respectively. The major fatty acid of EL-17KT was C18:1 and that of EL-17AT was ai-C15:0. The major respiratory quinones of EL-17KT and EL-17AT were MK-8(H4) and MK-9(H4), respectively. The former isolate had 69 mol% G+C, the latter had 73 mol% G+C. Comparative 16S rRNA gene sequencing revealed phylogenetic relationships of isolate EL-17KT with the genus Nocardioides, with N. pyridinolyticus and N. plantarum as the closest relatives. Phenotypic and genotypic characteristics support the description of a new species, Nocardioides aquaticus sp. nov., with EL-17KT as the type strain (= DSM 11439T). Isolate EL-17AT is related to the genus Friedmanniella, with E antarctica and E spumicola as the closest relatives. The differentiating characteristics support the description of a new species, Friedmanniella lacustris sp. nov., with EL-17AT as the type strain (= DSM 11465T).

Twelve strains isolated from the posterior fornix fluid of the human vagina were identified as Lactobacillus johnsonii, Lactobacillus acidophilus, Lactobacillus gallinarum and Lactobacillus crispatus based on numerical analyses of total soluble cell protein profiles and randomly amplified polymorphic DNA (RAPD)-PCR banding patterns. Five strains grouped with the type strains of Lactobacillus gasseri (DSM 20077T) and Lactobacillus jensenii (DSM 20557T) at r > or = 0.83 in one protein profile cluster, well separated from the other species included in this study. However, numerical analysis of the RAPD-PCR banding patterns of representative strains selected from the L. gasseri-L. jensenii protein cluster clearly indicated that they belong to two different species. Four strains (TV 1010, TG 1013, TV 1018T and TV 1045) grouped into another well separated protein profile cluster at r > or = 0.87. Strains selected from this cluster displayed very similar RAPD-PCR banding patterns and clustered at R2 > or = 0.78, separate from the other strains examined. Sequencing of the 16S rRNA of two representative strains, TV 1018T and TG 1013, of this group indicated that it represents a new member of rRNA group I Lactobacillus, which includes Lactobacillus delbrueckii, the type of the genus, and close relatives Lactobacillus acetotolerans, Lactobacillus kefiranofaciens, Lactobacillus iners, L. jensenii, L. crispatus, L. acidophilus, Lactobacillus helveticus, Lactobacillus amylovorus, Lactobacillus hamsteri, L. johnsonii, L. gasseri and Lactobacillus amylolyticus. The name Lactobacillus fornicalis sp. nov. is proposed for strains TV 1010 (DSM 13172), TG 1013, TV 1018T and TV 1045, with strain TV 1018T (= DSM 13171T = ATCC 700934T) as the type.

When administered to mice during gestation, the heavy metal, cadmium, is known to induce malformations of the neural tube, craniofacial region, limbs, trunk, viscera, and axial skeleton that vary in scope and severity among inbred strains of mice. Two strains, C57BL/6 and SWV, were previously shown to differ in their susceptibility to exencephaly induced by many teratogenic treatments, including sodium 2-ethylhexanoate, hyperthermia, valproic acid, and carbon dioxide, with the SWV strain being consistently more sensitive than the C57BL/6 strain. These findings support the observation of Finnell et al. ([1988] Teratology 38:313-320) of shared hierarchies of relative susceptibility to exencephaly induced by biochemically distinct teratogens, and suggest that the SWV strain would also be more sensitive to exencephaly induced by cadmium. In the present study, pregnant mice from the two strains were exposed to single i.p. injections of cadmium chloride at 4 mg/kg-BW on day 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0 of gestation. Fetuses were removed by cesarean section on day 18 of gestation and scored for malformations. The C57BL/6 strain was observed to be more sensitive than the SWV strain to the induction of exencephaly by cadmium on days 6.5, 7.0, and 8.0, with mean litter percentages of 3.6%, 88.3%, and 62.2%, respectively, compared to 0.0%, 4.1%, and 27.7% for the SWV strain. This finding provides evidence in contrast to the hypothesis of shared hierarchies of sensitivity to teratogen-induced exencephaly. Data on a number of other cadmium-induced malformations are also presented.

A unique coryneform bacterium was isolated from a patient with bacterial vaginosis. Chemotaxonomical investigations demonstrated that the unknown bacterium belonged to the genus Corynebacterium. The yellow-pigmented, slightly lipophilic, oxidative, urea-hydrolyzing bacterium could be phenotypically readily differentiated from the other members of the genus Corynebacterium. Comparative 16S rRNA gene analysis revealed that the bacterium represented a new subline within the genus Corynebacterium for which the name Corynebacterium lipophiloflavum sp. nov. is proposed. The type strain is CCUG 37336 (DSM 44291).

Arthrobacter spp. are very widely distributed in the environment (e.g., soil) but have not been described as causing disease in humans. Over a 6-year period, two reference laboratories isolated or received 11 strains which were eventually identified as belonging to the genus Arthrobacter. These strains had been initially identified as Centers for Disease Control and Prevention coryneform group B-1 and B-3 bacteria (whitishgrayish colonies of 2 mm or greater in diameter after 24 h of incubation, respiratory metabolism, absent or weak acid production from sugars, and hydrolysis of gelatin). However, chemotaxonomic investigations revealed lysine as the diamino acid of the cell wall and the presence of branched cellular fatty acids (with anteiso-pentadecanoic acid predominating) which was compatible with an assignment of the 11 isolates to the genus Arthrobacter only. Peptidoglycan and 16S rRNA gene sequence analyses demonstrated that three of the strains studied were representatives of a new Arthrobacter species for which the name Arthrobacter cumminsii sp. nov. is proposed and that one other strain represented a second new Arthrobacter species for which the name Arthrobacter woluwensis sp. nov. is proposed. This report is the first on the isolation of Arthrobacter spp. from clinical specimens.

A new type of slowly growing, nonphotochromogenic mycobacterium was recovered from two patients with disseminated disease. The growth characteristics, acid fastness, acids were consistent with those for Mycobacterium species. The results of biochemical investigations, lipid analyses, and comparative 16S rRNA sequencing showed that these isolates represent a new slowly growing Mycobacterium species which is named Mycobacterium conspicuum.

CDC coryneform group A-3 and A-4 bacteria were defined by Hollis and Weaver in 1981, but their taxonomic position is still unclear. By using biochemical and chemotaxonomical methods, four clinical strains belonging to CDC coryneform groups A-3 (n = 2) and A-4 (n = 2) were studied and could be assigned to the genus Cellulomonas, resulting in the first description of Cellulomonas strains isolated from clinical specimens. CDC coryneform group A-3 and A-4 strains were compared with the type strains of the seven species constituting the genus Cellulomonas at present as well as with the closely related species Oerskovia turbata, Oerskovia xanthineolytica, and Jonesia denitrificans, but their biochemical patterns were not compatible with the patterns of any of those species. Almost the entire sequences of the 16S rRNA genes of one representative strain of both CDC taxa were determined, and comparative sequence analysis confirmed the placement of the CDC coryneform group A-3 and A-4 strains studied in the Cellulomonas-Oerskovia subbranch of the actinomycetes. Both CDC taxa exhibited > 99% base pair homology within their 16S rDNAs. On the basis of phenotypic and molecular data, we formally propose a new species, Cellulomonas hominis sp. nov., for the CDC coryneform group A-3 bacteria examined. The type strain is DSM 9581. The precise taxonomic status of the CDC coryneform group A-4 strains studied remains to be established by quantitative DNA-DNA hybridizations.

The wave field splitting/invariant imbedding/phase space approach to direct wave propagation in the frequency domain is outlined. The properties and subsequent applications of the reflection and Dirichlet-to-Neumann operator symbols are briefly illustrated by an exact solution case.

Segmented filamentous bacteria (SFB) are nonpathogenic bacteria that are commonly found attached to the intestinal walls of many animals. Until now, these bacteria have not been cultured in vitro. Recently, a 16S rRNA sequence analysis revealed that SFB isolated from mice represent a distinct subline within the Clostridium subphylum of the gram-positive bacteria. Since SFB isolated from mice, rats, and chickens are known to be host specific, we investigated the phylogenetic relationships among SFB obtained from these three hosts. Total DNAs from the intestinal floras of chickens and rats were used as templates for PCR amplification of 16S rRNA genes. PCR products were cloned and screened by a dot blot hybridization procedure to identify homologous sequences that cross-reacted with mouse SFB-specific oligonucleotide probes. A phylogenetic analysis of these 16S ribosomal DNA sequences revealed that SFB isolated from these three hosts form a natural group, which is peripherally related to the genus Clostridium sensu stricto (group I Clostridium). The SFB obtained from chickens, rats, and mice had closely related, albeit different, 16S rRNA gene sequences. The observed levels of 16S rRNA sequence divergence, ca. 1.5 to 3%, together with host specificity, suggest that SFB isolated from mice, rats, and chickens represent different species and that coevolution of the SFB and their hosts occurred. “Candidatus Arthromitus” is proposed as the provisional generic name for this group of organisms.

Over a 1-year period, 11 isolates (including 5 from blood cultures) of the recently described CDC group 3 and group 5 coryneform bacteria were derived from clinical specimens and compared with reference strains. Biochemical characteristics indicated a very close relationship between CDC group 3 and group 5 coryneform bacteria. The ability of CDC group 3 and the inability of CDC group 5 coryneform bacteria to ferment xylose were the only reactions that were different for the two taxa. Chemotaxonomic features of the two groups included the presence of meso-diaminopimelic acid, a lack of mycolic acids, and the presence of predominantly branched cellular fatty acids, a combination found among gram-positive rods only in Brevibacterium spp., Brachybacterium faecium, and Dermabacter hominis. 16S rRNA gene sequence analysis revealed that CDC group 3 and group 5 coryneform bacteria are members of the genus Dermabacter, which to date has been isolated exclusively from human skin.

The 16S rRNA gene sequence of Melissococcus pluton, the causative agent of European foulbrood disease, was determined in order to investigate the phylogenetic relationships between this organism and other low-G + C-content gram-positive bacteria. A comparative sequence analysis revealed that M. pluton is a close phylogenetic relative of the genus Enterococcus.

The 16S rRNA gene sequences of the type strains of Aeromonas enteropelogenes and Aeromonas ichthiosmia were determined by polymerase chain reaction direct sequencing in order to clarify their interrelationships with other aeromonad species. On the basis of 16S rRNA gene sequence analysis, A. enteropelogenes and A. ichthiosmia were found to be identical to Aeromonas trota and Aeromonas veronii, respectively.

23S ribosomal RNa (rRNA) gene sequences of Streptococcus uberis and Strep. parauberis were determined by direct polymerase chain reaction (PCR) sequencing. Oligonucleotide probes specific for Strep. uberis and Strep. parauberis were designed from variable regions of the 23S rRNA gene sequence data. Molecular hybridizations with PCR-amplified rRNA gene targets provided a precise and reliable means of differentiating Strep. uberis and Strep. parauberis from each other and from other streptococcal species.

The large-subunit ribosomal RNA gene sequences of Leuconostoc carnosum, Leuc. mesenteroides, Leuc. oenos, Leuc. paramesenteroides and Lactobacillus confusus were determined by direct sequencing of enzymatically-amplified DNA. Comparative analysis of the large-subunit rDNA sequences revealed that the leuconostocs form three distinct lines of descent: Leuconostoc sensu stricto, Leuc. paramesenteroides group of organisms, and Leuc. oenos. The distance matrix tree constructed from the large-subunit sequence data was in excellent agreement with that derived from small-subunit sequences, and demonstrates the 'synchrony' of the two chronometers. The results of the present and earlier sequence analyses indicate the taxonomy of the genus Leuconostoc is in need of revision, and that the Leuc. paramesenteroides group of organisms and the species Leuc. oenos represent distinct phylogenetic units, separate from Leuconostoc sensu stricto.

The phylogenetic relationships of members of the genera Rhizobium, Agrobacterium, Bradyrhizobium, and Azorhizobium were studied by direct sequencing of their amplified 16S rRNA genes. Comparative analysis of the sequence data confirmed that the genera Bradyrhizobium and Azorhizobium belong to distinct phylogenetic lineages. The genera Rhizobium and Agrobacterium were found to be phylogenetically heterogeneous, and several subgroupings in which Rhizobium and Agrobacterium species were intermixed were evident. The present findings show that the genus and species definitions of these organisms are in need of revision. Different possibilities for this are discussed in the light of sequencing data.

Taxonomic studies were performed on an anaerobic Gram-positive, spore-forming, psychrophilic bacterium originally isolated from spoiled vacuum-packed refrigerated beef. Based on the present finding it is proposed that this unknown psychrophilic bacterium be classified as a new species of the genus Clostridium, as Clostridium estertheticum sp. nov. The type strain is NCIMB 12511.

The primary structures of the small subunit rRNA of Bradyrhizobium japonicum and Blastobacter denitrificans were determined by direct sequencing of enzymatically amplified DNA. Comparative sequence analysis revealed that Bradyrhizobium japonicum and Blastobacter denitrificans are members of the alpha-2 subgroup of the Proteobacteria and show a very close phylogenetic relationship with the genus Afipia.

A 23S rRNA gene of Listeria monocytogenes was cloned into pUC19 on a 6.2-kb Pst I fragment. Hybridisation studies demonstrated the presence of the 5S and partial 16S rRNA genes within the clone. The nucleotide sequence of the region encoding the 23S rRNA was found to be highly homologous with those of other low G + C Gram-positive bacteria. The 16S-23S intergenic spacer region was amplified using PCR technology and revealed two product sizes, the larger of which contained tRNA(Ala) and tRNA(Ileu) genes. Further tRNA genes were found downstream of the 5S rRNA gene.

Primers designed to conserved regions of botulinum and tetanus clostridial toxins were used to amplify DNA fragments from non-proteolytic Clostridium botulinum type F (202F) DNA using polymerase chain reaction technology. The fragments were cloned and the complete nucleotide sequence of the gene encoding type F toxin determined. Analysis of the nucleotide sequence demonstrated the presence of an open frame encoding a protein of 1274 amino acids, similar to other botulinum neurotoxins. Upstream of the toxin gene is the end of an open reading frame which encodes the C-terminus of a protein with homology to non-toxic-non-hemagglutinin component of type C progenitor toxin.

16S rRNA gene sequencing was performed on the species Lactobacillus minutus, Lactobacillus rimae and Streptococcus parvulus in order to clarify their taxonomic position. Based on comparative sequence analyses these organisms represent a hitherto unknown line of descent within the lactic acid group of bacteria for which a new genus, Atopobium gen. nov., is proposed.

A rapid polymerase chain reaction test was developed for specific identification of the human middle ear pathogen Alloiococcus otitis. Primers for the enzymatic amplification reaction were designed from highly specific sequences within the 16S rRNA gene. In addition, a confirmatory test based on hybridization of the polymerase chain reaction products to a specific internal probe was developed.

A polymerase chain reaction/oligonucleotide probe method was developed for the specific identification of the Gram-positive bacterium Aerococcus viridans. Primers for the enzymatic amplification reaction were designed from specific sequences within the 16S rRNA. The method was also highly sensitive and 10 cfu of A. viridans could be detected in 5 h although the reliability of detection was poor in mixed cultures with Escherichia coli.

The primary structures of the 23S ribosomal RNA genes of Bacillus anthracis and an emetic strain of Bacillus cereus were determined by direct sequencing of enzymatically amplified chromosomal DNA. The 23S rRNA gene sequences of B. anthracis and B. cereus were found to be almost identical and showed only two differences (a single nucleotide change, and a single base insertion in B. cereus). The feasibility of using PCR-direct sequencing for the rapid sequence determination of large-subunit rRNA genes is demonstrated.

The phylogenetic interrelationships of members of the genera Aeromonas and Plesiomonas were investigated by using small-subunit ribosomal DNA (rDNA) sequencing. Members of the genus Aeromonas formed a distinct line within the gamma subclass of the Proteobacteria. Plesiomonas shigelloides also clustered within the confines of the gamma subclass of the Proteobacteria but exhibited a closer association with members of the family Enterobacteriaceae than with members of the family Aeromonadaceae. Species of the genus Aeromonas exhibited very high levels of overall sequence similarity (ca. 98 to 100%) with each other. Several of the relationships derived from an analysis of the rDNA sequence data were in marked disagreement with the results of chromosomal DNA-DNA pairing experiments. Diagnostic rDNA signatures that have possible value for differentiating most Aeromonas species were discerned.

A phylogenetic analysis based on 16S rRNA was performed on the genera Planococcus, Marinococcus, Sporosarcina and endospore-forming rods. In agreement with earlier 16S rRNA cataloguing data, Planococcus citreus and Sporosarcina ureae clustered with Bacillus pasteurii and other bacilli containing lysine in their cell walls. Sporosarcina halophila was shown to be genetically distinct from S. ureae and formed a loose association with the main Bacillus subtilis grouping. Marinococcus halophilus (formerly Planococcus halophilus) exhibited low levels of relatedness to all reference species examined and formed a distinct line of descent.

J.L. BROOKS, A.S. MOORE, R.A. PATCHETT, M. D. COLLINS AND R.G. KROLL. 1992. The polymerase chain reaction (PCR) was used selectively to amplify specific rDNA sequences of Carnobacterium divergens, C. mobile, C. piscicola and C. gallinarum in purified DNA extracts, crude cell lysates and food samples. The PCR products were visualized by agarose gel electrophoresis and identified, at species level, by hybridization reactions with three specific oligonucleotide probes for C. divergens, C. mobile and C. piscicola/C. gallinarum designed from 16S rRNA sequence data. The PCR was sufficiently sensitive to amplify DNA from a single bacterium to detectable levels after 30 cycles of amplification. Both radioactive (32P) and non-radioactive alkaline phosphatase labelled probes was able to detect the PCR products. Detection was highly specific and the probes did not hybridize with DNA samples from any other of the bacterial species tested. These methods enabled the rapid and specific detection and identification of carnobacteria from pure cultures and samples of meat.

Phenotypic and genetic studies were performed on some atypical aeromonas strains of uncertain taxonomic position. 16S rRNA gene sequence analysis revealed that these strains represent a hitherto unknown genetic line within the genus Aeromonas, for which the name Aeromonas allosaccharophila sp. nov. is proposed. The type strain is CECT 4199.

We tested 12 reference and 24 clinical strains of lactococci for physiologic characteristics using a conventional test system, the Gen-Probe Enterococcus 2 chemiluminescence assay (Gen-Probe Inc., San Diego, Calif.), the Rapid Strep identification system (Analytab Products, Plainview, N.Y.), and whole-cell protein analysis. The Gen-Probe Enterococcus 2 chemiluminescence assay for Enterococcus identification was negative with all strains. Neither the conventional test nor the Rapid Strep identification system could differentiate between the two Lactococcus spp. most commonly isolated from humans. A simple procedure, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was developed for comparing the whole-cell protein patterns of Lactococcus spp. L. lactis and L. garvieae were differentiated by unique protein patterns.