Streptomyces marokkonensis sp. nov., isolated from rhizosphere soil of Argania spinosa L.

The novel actinomycete strain Ap1(T) was isolated from rhizosphere soil of the argan tree (Argania spinosa L.) in the south of Morocco. Strain Ap1(T) has been reported as a novel producer of the pentaene polyene macrolide isochainin, which strongly inhibits the growth of pathogenic yeasts and phytopathogenic fungi. Strain Ap1(T) shows a greyish-white aerial mycelium with chains of smooth-surfaced spores of the Spiralis type and a cell wall containing ll-diaminopimelic acid. Based on chemotaxonomy and morphological features, strain Ap1(T) was identified as a member of the genus Streptomyces. 16S rRNA gene sequence similarities based on almost-complete 16S rRNA gene sequences showed that strain Ap1(T) is closely associated with members of the Streptomyces violaceoruber species group (S. violaceoruber, S. coelescens, S. violaceorubidus, 'S. caesius', 'S. lividans', S. violaceolatus and S. humiferus) and others (Streptomyces aurantiogriseus, S. lienomycini, S. chattanoogensis, S. rubrogriseus and S. tendae). However, protein profiling, DNA-DNA hybridization and BOX-PCR fingerprinting proved a relationship above the species level. In addition, the phenotype also allowed for the differentiation of strain Ap1(T) from its closest neighbours. As a result of this polyphasic approach, we conclude that strain Ap1(T) represents a novel species of the genus Streptomyces, for which the name Streptomyces marokkonensis sp. nov. is proposed. The type strain is Ap1(T) (=R-22003(T) =LMG 23016(T) =DSM 41918(T)).

Identification of lactic acid bacteria in Moroccan raw milk and traditionally fermented skimmed milk 'lben'.

AIMS: To identify lactic acid bacteria (LAB) present in Moroccan dairy products to establish and preserve their microbial species diversity. METHODS AND RESULTS: Thirty-seven samples were collected from different farms. A total of 146 LAB were isolated and subjected to (GTG)(5)-PCR analysis. Comparison of the profiles with data available at the Moroccan Coordinated Collections of Micro-organisms allowed identification of 85 isolates. The remaining 61 were subjected to SDS-PAGE analysis of whole cell proteins. Comparison of the profiles with data available at the Belgian Coordinated Collections of Micro-organisms allowed identification of 43 isolates. Several of the remaining 18 isolates exhibited identical protein electrophoretic fingerprints. Therefore, eight representatives of them were subjected to partial pheS gene sequencing which allowed identification of all remaining isolates. In raw milk, six genera were found while in 'lben', three were found. This is the first report of Leuconostoc kimchii in dairy products. CONCLUSIONS: LAB diversity was established using a stepwise polyphasic identification approach. It used the expertise of both research bodies involved in this study and proved to be cost-effective for the identification of all isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: To establish LAB diversity in Moroccan dairy products which could be a source of strains with specific properties.

Identification of lactobacilli isolated from the cloaca and vagina of laying hens and characterization for potential use as probiotics to control Salmonella Enteritidis.

AIMS: To select Lactobacillus strains from laying hens for potential use as probiotic to control Salmonella Enteritidis infection. METHODS AND RESULTS: One hundred and eighty-six lactobacilli were isolated from the cloaca and vagina of laying hens, and identified at the species level by a polyphasic taxonomic approach. All isolates belonged to the Lactobacillus acidophilus, Lactobacillus reuteri or Lactobacillus salivarius phylogenetic groups, with the L. reuteri group being the most predominant group. Based on genetic diversity, about 50 representative strains were selected and tested for in vitro properties that could be predictive for probiotic activity in laying hens. Salmonella inhibition was shown to be species dependent, and correlated to some extent with the production of lactic acid. A selection of strains was evaluated in a S. Enteritidis challenge experiment. Two strains, L. reuteri R-17485 and Lactobacillus johnsonii R-17504 significantly decreased the colonization of chicks by S. Enteritidis in caeca, liver and spleen. CONCLUSIONS: Lactobacilli isolated from laying hens were observed to inhibit Salmonella growth in vitro, most probably through production of lactic acid, and to decrease in vivo the S. Enteritidis colonization of chicks. SIGNIFICANCE AND IMPACT OF THE STUDY: The data demonstrate that Lactobacillus isolates from laying hens may have probiotic potential in reducing S. Enteritidis infection.

Antimicrobial susceptibility of Bifidobacterium strains from humans, animals and probiotic products.

OBJECTIVES: The aim of this study was to assess the antimicrobial susceptibility of a taxonomically diverse set of Bifidobacterium strains to different classes of antimicrobial agents using a recently described medium. METHODS: The susceptibility of 100 strains encompassing 11 bifidobacterial species originating from humans, animals and probiotic products to 12 antimicrobial agents was tested by agar overlay disc diffusion. Based on these results, one or two strains per species were selected for susceptibility testing to nine antibiotics by broth microdilution using the Lactic acid bacteria Susceptibility test Medium (LSM) supplemented with cysteine. The genotypic basis of atypical tetracycline resistance was further characterized using PCR, Southern blotting and partial sequencing. RESULTS: Based on the distribution of inhibition zone diameters and MIC values, all strains tested were susceptible to amoxicillin, chloramphenicol, erythromycin, quinupristin/dalfopristin, rifampicin and vancomycin. Our data also reinforce earlier observations indicating that bifidobacteria are intrinsically resistant to gentamicin, sulfamethoxazole and polymyxin B. Susceptibility to trimethoprim, trimethoprim/sulfamethoxazole, ciprofloxacin, clindamycin, tetracycline and minocycline was variable. The tet(W) gene was responsible for tetracycline resistance in 15 strains including 7 probiotic isolates belonging to the taxa Bifidobacterium animalis subsp. lactis and Bifidobacterium bifidum. This gene was present in a single copy on the chromosome and did not appear to be associated with the conjugative transposon TnB1230 previously found in tet(W)-containing Butyrivibrio fibrisolvens. CONCLUSIONS: The use of the LSM + cysteine medium allowed us to discriminate between intrinsic and atypical resistance properties of bifidobacteria and sets the scene for future definition of epidemiological cut-off values for all important Bifidobacterium species. The presence of an acquired tet(W) gene in several probiotic product isolates stresses the need for a minimal safety evaluation during the selection of Bifidobacterium strains for probiotic use.

Vibrio gigantis sp. nov., isolated from the haemolymph of cultured oysters (Crassostrea gigas).

Polyphasic analysis of four new Vibrio isolates originating from the haemolymph of diseased cultured oysters is described. The new isolates were closely related to Vibrio splendidus, having 98 % 16S rRNA gene sequence similarity. Phylogenetic analysis based on DNA gyrase subunit B (gyrB), RNA polymerase sigma70 factor (rpoD), replication origin-binding protein (rctB) and transmembrane regulatory protein (toxR) genes, fluorescent amplified fragment length polymorphism and DNA-DNA hybridization experiments clearly showed that the new isolates form a tight genomic group that is different from the currently known Vibrio species. It is proposed that these new isolates should be accommodated in a novel species, Vibrio gigantis sp. nov. Phenotypic features that differentiate V. gigantis from other known Vibrio species include arginine dihydrolase, gelatinase and beta-galactosidase activities, NO(2) production, growth at 35 degrees C, and utilization of sucrose, melibiose, amygdalin, glycerol, galactose, starch and glycogen. The type strain is LGP 13T (=LMG 22741T=CIP 108656T).

Phylogeny and molecular identification of vibrios on the basis of multilocus sequence analysis.

We analyzed the usefulness of rpoA, recA, and pyrH gene sequences for the identification of vibrios. We sequenced fragments of these loci from a collection of 208 representative strains, including 192 well-documented Vibrionaceae strains and 16 presumptive Vibrio isolates associated with coral bleaching. In order to determine the intraspecies variation among the three loci, we included several representative strains per species. The phylogenetic trees constructed with the different genetic loci were roughly in agreement with former polyphasic taxonomic studies, including the 16S rRNA-based phylogeny of vibrios. The families Vibrionaceae, Photobacteriaceae, Enterovibrionaceae, and Salinivibrionaceae were all differentiated on the basis of each genetic locus. Each species clearly formed separated clusters with at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively. The genus Vibrio was heterogeneous and polyphyletic, with Vibrio fischeri, V. logei, and V. wodanis grouping closer to the Photobacterium genus. V. halioticoli-, V. harveyi-, V. splendidus-, and V. tubiashii-related species formed groups within the genus Vibrio. Overall, the three genetic loci were more discriminatory among species than were 16S rRNA sequences. In some cases, e.g., within the V. splendidus and V. tubiashii group, rpoA gene sequences were slightly less discriminatory than recA and pyrH sequences. In these cases, the combination of several loci will yield the most robust identification. We can conclude that strains of the same species will have at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively.

Polyphasic study of Chryseobacterium strains isolated from diseased aquatic animals.

Members of most Chryseobacterium species occur in aquatic environments or food products, while strains of some other species are pathogenic to humans and animals. A collection of 52 Chryseobacterium sp. strains isolated from diseased fish, one frog isolate and 22 reference strains were included in a polyphasic taxonomy study. Fourteen clusters of strains were delineated following the comparison of whole-cell protein profiles. Most of these clusters were confirmed when the phenotypic and RAPD profiles and the 16S rRNA gene sequences were compared. Fatty acid composition helped differentiate the Chryseobacterium strains from members of related genera. None of the fish isolates could be allocated to the two species previously reported from fish but two isolates belonged to C. joostei, while the frog isolate was identified as Elizabethkingia meningoseptica, a human pathogen previously included in the genus Chryseobacterium. Three clusters grouping from 3 to 13 isolates will probably constitute the core of new Chryseobacterium species but all other isolates occupied separate or uncertain positions in the genus. This study further demonstrated the overall high similarity displayed by most Chryseobacterium strains whatever the technique used and the resulting difficulty in delineating new species in the genus. Members of this bacterial group should be considered potential emergent pathogens in various fish and frog species, farming conditions and geographical areas.

Leuconostoc durionis sp. nov., a heterofermenter with no detectable gas production from glucose.

Three lactic acid bacterial (LAB) strains obtained from a Malaysian acid-fermented condiment, tempoyak (made from pulp of the durian fruit), showed analogous but distinct patterns after screening by SDS-PAGE of whole-cell proteins and comparison with profiles of all recognized LAB species. 16S rRNA gene sequencing of one representative strain showed that the taxon belongs phylogenetically to the genus Leuconostoc, with its nearest neighbour being Leuconostoc fructosum (98 % sequence similarity). Biochemical characteristics and DNA-DNA hybridization experiments demonstrated that the strains differ from Leuconostoc fructosum and represent a single, novel Leuconostoc species for which the name Leuconostoc durionis sp. nov. is proposed. The type strain is LMG 22556(T) (= LAB 1679(T) = D-24(T) = CCUG 49949(T)).

Photobacterium rosenbergii sp. nov. and Enterovibrio coralii sp. nov., vibrios associated with coral bleaching.

Six new Vibrio-like isolates originating from different species of bleached and healthy corals around Magnetic Island (Australia) were investigated using a polyphasic approach. Phylogenetic analyses based on 16S rRNA, recA and rpoA gene sequences split the isolates in two new groups. Strains LMG 22223(T), LMG 22224, LMG 22225, LMG 22226 and LMG 22227 were phylogenetic neighbours of Photobacterium leiognathi LMG 4228(T) (95.6 % 16S rRNA gene sequence similarity), whereas strain LMG 22228(T) was related to Enterovibrio norvegicus LMG 19839(T) (95.5 % 16S rRNA gene sequence similarity). The two new groups can be distinguished from closely related species on the basis of several phenotypic features, including fermentation of d-mannitol, melibiose and sucrose, and utilization of different compounds as carbon sources, arginine dihydrolase activity, nitrate reduction, resistance to the vibriostatic agent O/129 and the presence of fatty acids 15 : 0 iso and 17 : 0 iso. The names Photobacterium rosenbergii sp. nov. (type strain LMG 22223(T)=CBMAI 622(T)=CC1(T)) and Enterovibrio coralii sp. nov. (type strain LMG 22228(T)=CBMAI 623(T)=CC17(T)) are proposed to accommodate these new isolates. The G+C contents of the DNA of the two type strains are respectively 47.6 and 48.2 mol%.

Vibrio crassostreae sp. nov., isolated from the haemolymph of oysters (Crassostrea gigas).

Polyphasic analysis of five new Vibrio isolates originating from the haemolymph of diseased cultured oysters is described. The new isolates were closely related to Vibrio splendidus, having 98 % 16S rRNA gene sequence similarity. gyrB phylogenetic analysis, fluorescent amplified-fragment length polymorphism (FAFLP) fingerprinting and DNA-DNA hybridization experiments clearly showed that the new isolates form a tight genomic group that is different from the currently known Vibrio species. It is proposed to accommodate these isolates in a novel species, Vibrio crassostreae sp. nov. (type strain LGP 7(T)=LMG 22240(T)=CIP 108327(T)). Phenotypic and chemotaxonomic features that differentiate V. crassostreae from other known Vibrio species include arginine dihydrolase, utilization and fermentation of various carbon sources, beta-galactosidase activity, NO(2) production and the presence of the fatty acids 14 : 0 iso and 16 : 0 iso.

Use of recA as an alternative phylogenetic marker in the family Vibrionaceae.

This study analysed the usefulness of recA gene sequences as an alternative phylogenetic and/or identification marker for vibrios. The recA sequences suggest that the genus Vibrio is polyphyletic. The high heterogeneity observed within vibrios was congruent with former polyphasic taxonomic studies on this group. Photobacterium species clustered together and apparently nested within vibrios, while Grimontia hollisae was apart from other vibrios. Within the vibrios, Vibrio cholerae and Vibrio mimicus clustered apart from the other genus members. Vibrio harveyi- and Vibrio splendidus-related species formed compact separated groups. On the other hand, species related to Vibrio tubiashii appeared scattered in the phylogenetic tree. The pairs Vibrio coralliilyticus and Vibrio neptunius, Vibrio nereis and Vibrio xuii and V. tubiashii and Vibrio brasiliensis clustered completely apart from each other. There was a correlation of 0.58 between recA and 16S rDNA pairwise similarities. Strains of the same species have at least 94 % recA sequence similarity. recA gene sequences are much more discriminatory than 16S rDNA. For 16S rDNA similarity values above 98 % there was a wide range of recA similarities, from 83 to 99 %.

Vibrio kanaloae sp. nov., Vibrio pomeroyi sp. nov. and Vibrio chagasii sp. nov., from sea water and marine animals.

The taxonomic position of the fluorescent amplified fragment length polymorphism fingerprinting groups A46 (five isolates), A51 (six isolates), A52 (five isolates) and A53 (seven isolates) obtained in a previous study were further analysed through a polyphasic approach. The 23 isolates were phylogenetically related to Vibrio splendidus, but DNA-DNA hybridization experiments proved that they belong to three novel species. Chemotaxonomic and phenotypic analyses further disclosed several features that differentiate between the 23 isolates and known Vibrio species. The names Vibrio kanaloae sp. nov. (type strain LMG 20539(T) = CAIM 485(T); EMBL accession no. AJ316193; G + C content 44.7 mol%), Vibrio pomeroyi sp. nov. (type strain LMG 20537(T) = CAIM 578(T); EMBL accession no. AJ491290; G +C content 44.1 mol%) and Vibrio chagasii sp. nov. (type strain LMG 21353(T) = CAIM 431(T); EMBL accession no. AJ316199; G + C content 44.6 mol%) are respectively proposed to encompass the five isolates of A46, the six isolates of A51 and the 12 isolates of A52/A53. The three novel species can be distinguished from known Vibrio species by several phenotypic features, including utilization and fermentation of various carbon sources, beta-galactosidase activity and fatty acid content (particularly of 12 : 0, 14: 0, 14 : 0 iso and 16 : 0 iso).

Vibrio tasmaniensis sp. nov., isolated from Atlantic salmon (Salmo salar L.).

We describe the polyphasic characterization of four Vibrio isolates which formed a tight AFLP group in a former study. The group was closely related to V. cyclitrophicus, V. lentus and V. splendidus (98.2-98.9% similarity) on the basis of the 16S rDNA sequence analysis, but by DNA-DNA hybridisation experiments it had at maximum 61% DNA similarity towards V. splendidus. Thus, we propose that the isolates represent a new Vibrio species i.e. V. tasmaniensis (LMG 20012T; EMBL under the accession numbers AJ316192; mol% G+C of DNA of the type strain is 44.7). Useful phenotypical features for discrimination of V. tasmaniensis from other Vibrio species include gelatinase and beta-galactosidase activity, fatty acid composition (particularly 14:0), utilisation and fermentation of different compounds (e.g. sucrose, melibiose and D-galactose) as sole carbon source.

Re-examination of the genus Acetobacter, with descriptions of Acetobacter cerevisiae sp. nov. and Acetobacter malorum sp. nov.

Thirty-four Acetobacter strains, representing Acetobacter aceti, Acetobacter pasteurianus, Acetobacter pomorum, Acetobacter peroxydans, Acetobacter lovaniensis, Acetobacter estunensis, Acetobacter orleanensis, Acetobacter indonesiensis and Acetobacter tropicalis, were subjected to a polyphasic study that included DNA-DNA hybridizations, DNA base ratio determinations, 16S rDNA sequence analysis and phenotypic characterization. Two novel species are proposed, Acetobacter cerevisiae sp. nov. and Acetobacter malorum sp. nov. The type strains of these species are respectively LMG 1625T (= DSM 14362T = NCIB 8894T = ATCC 23765T) and LMG 1746T (= DSM 14337T).

Enterovibrio norvegicus gen. nov., sp. nov., isolated from the gut of turbot (Scophthalmus maximus) larvae: a new member of the family Vibrionaceae.

Twenty-two isolates originating from the gut of healthy cultured turbot larvae in Norway were investigated using a polyphasic approach. Amplified fragment length polymorphism fingerprinting analysis showed that the isolates have typical patterns and form two main groups. Phylogenetic analysis revealed that the isolates belong to the gamma-Proteobacteria, with Vibrio hollisae as their closest neighbour. DNA-DNA hybridization, chemotaxonomic and phenotypic analyses further proved that these isolates represent a tight novel taxon that differs from currently described species in the family Vibrionaceae. It is proposed that these novel isolates be accommodated in a new genus, Enterovibrio gen. nov., with Enterovibrio norvegicus sp. nov. as the type species. Isolates were motile by a polar flagellum, positive for oxidase, catalase, arginine dihydrolase and beta-galactosidase, but negative for the Voges-Proskauer reaction. They produced indole, did not reduce nitrate and were resistant to the vibriostatic agent O/129. The DNA G+C content of E. norvegicus was 47.1-47.9 mol%. The type strain is E. norvegicus LMG 19839(T) (= CAIM 430(T)).

Enterococcus haemoperoxidus sp. nov. and Enterococcus moraviensis sp. nov., isolated from water.

A polyphasic taxonomic approach was used to study atypical enterococci isolated from surface waters. All strains were characterized by physiological and biochemical tests as well as by genotyping. The results of biochemical tests and tRNA intergenic length polymorphism analysis (tDNA-PCR) divided all studied strains uniformly into two groups. Because these groups were clearly separated from all enterococcal species described to date, 16S rDNA sequence analysis, DNA base composition analysis and DNA-DNA hybridization of representative strains were done to elucidate the taxonomic position of the analysed groups. On the basis of the results obtained, the names Enterococcus haemoperoxidus (type strain CCM 4851T = LMG 19487T) and Enterococcus moraviensis (type strain CCM 4856T = LMG 19486T) are proposed for the two hitherto undescribed species. The type strains and reference cultures have been deposited in the Czech Collection of Microorganisms (CCM), Masaryk University, Brno, Czech Republic, and in the BCCM/LMG Culture Collection, Ghent University, Belgium.

Enterococcus villorum sp. nov., an enteroadherent bacterium associated with diarrhoea in piglets.

The taxonomic positions of five enteroadherent bacterial pig isolates, showing phenotypic characteristics most similar to those of Enterococcus durans and Enterococcus hirae, were investigated in a polyphasic study that included 16S rDNA sequence analysis, DNA-DNA hybridizations, DNA base-ratio determinations, whole-cell protein fingerprinting, D11344-primed PCR typing and an extensive examination of phenotypic properties. The results demonstrated that the organisms represent a new species in the Enterococcus faecium species group, for which the name Enterococcus villorum sp. nov. is proposed. The type strain is LMG 12287T (= CCM 4887T).

Sphingomonas alaskensis sp. nov., a dominant bacterium from a marine oligotrophic environment.

Seven Gram-negative strains, isolated in 1990 from a 10(6)-fold dilution series of seawater from Resurrection Bay, a deep fjord of the Gulf of Alaska, were identified in a polyphasic taxonomic study. Analysis of 16S rDNA sequences and DNA-homology studies confirmed the phylogenetic position of all strains in the genus Sphingomonas and further indicated that all of the strains constitute a single homogeneous genomic species, distinct from all validly described Sphingomonas species. The ability to differentiate the species, both phenotypically and chemotaxonomically, from its nearest neighbours justifies the proposal of a new species name, Sphingomonas alaskensis sp. nov., for this taxon. Strain LMG 18877T (= RB2256T = DSM 13593T) was selected as the type strain.

Genomic diversity amongst Vibrio isolates from different sources determined by fluorescent amplified fragment length polymorphism.

The genomic diversity among 506 strains of the family Vibrionaceae was analysed using Fluorescent Amplified Fragments Length Polymorphisms (FAFLP). Isolates were from different sources (e.g. fish, mollusc, shrimp, rotifers, artemia, and their culture water) in different countries, mainly from the aquacultural environment. Clustering of the FAFLP band patterns resulted in 69 clusters. A majority of the actually known species of the family Vibrionaceae formed separate clusters. Certain species e.g. V. alginolyticus, V. cholerae, V. cincinnatiensis, V. diabolicus, V. diazotrophicus, V. harveyi, V. logei, V. natriegens, V. nereis, V. splendidus and V. tubiashii were found to be ubiquitous, whereas V. halioticoli, V. ichthyoenteri, V. pectenicida and V. wodanis appear to be exclusively associated with a particular host or geographical region. Three main categories of isolates could be distinguished: (1) isolates with genomes related (i.e. with > or =45% FAFLP pattern similarity) to one of the known type strains; (2) isolates clustering (> or =45% pattern similarity) with more than one type strain; (3) isolates with genomes unrelated (<45% pattern similarity) to any of the type strains. The latter group consisted of 236 isolates distributed in 31 clusters indicating that many culturable taxa of the Vibrionaceae remain as yet to be described.

Polyphasic characterization of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (p(HB-co-HV)) metabolizing and denitrifying Acidovorax sp. strains.

For the purpose of denitrification in small drinking water plants, a bacterial mixed population was isolated from a packed bed column bioreactor with poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P(HB-co-HV)) as a substrate for the denitrification of ground water (10 degrees C). Isolates 2nIII from the mixed culture, with the ability to denitrify and metabolize P(HB-co-HV), were used as starter cultures for the elimination of nitrate in ground water. The strains were characterized by diverse techniques. Classical phenotypic studies lead to rRNA group III of the genus Pseudomonas. Results obtained by molecular techniques demonstrated that the 2nIII strains are members of the Comamonadaceae and shows similarities to the genus Acidovorax. However, an integration of the 2nIII isolates within one of the known Acidovorax species is not possible for the moment. The 2nIII starter cultures clustered close to Av. temperans according to their whole cell proteins and fatty acids, whereas in DNA/DNA hybridization no significant DNA binding (< 25%) was found. In contrast a significant but low degree of DNA/DNA hybridization was found between the 2nIII strains and Av. facilis and Av. delafieldii. Our polyphasic results lead to the conclusion that the 2nIII strains may constitute a separate Acicdovorax species.