Reclassification of Pseudomonas sp. PB-6250T as Lysobacter firmicutimachus sp. nov.

Strain PB-6250T, isolated from soil in Japan, was first identified in 1992. In contrast to its original taxonomic classification, its 16S rRNA gene sequence showed the highest similarity (99.2 %) to the sequence of Lysobacter enzymogenes DSM 2043T, with Lysobacter antibioticus DSM 2044T being the next most closely related species (98.7 %) with a validly published name. Chemotaxonomic data (fatty acid profile, quinone and polar lipid composition) and the G+C content of strain PB-6250T were compared with those of the closely related type strains L. enzymogenes LMG 8762T, L. antibioticus LMG 8760T, L. capsici DSM 19286T and L. gummosus LMG 8763T; this supported the affiliation of strain PB-6250T to the genus Lysobacter. Phylogenetic analyses, DNA-DNA-hybridization data, biochemical and physiological characteristics strongly supported the genotypic and phenotypic differentiation of strain PB-6250T from species of Lysobacter with validly published names. Strain PB-6250T, therefore represents a novel species, for which the name Lysobacter firmicutimachus sp. nov. is proposed. The type strain is PB-6250T (=LMG 28994T=DSM 102073T).

Lutibacter holmesii sp. nov., a marine bacterium of the family Flavobacteriaceae isolated from the sea urchin Strongylocentrotus intermedius, and emended description of the genus Lutibacter.

Seven Gram-staining-negative, strictly aerobic, pale-yellow-pigmented, rod-shaped and non-motile strains were isolated from the sea urchin Strongylocentrotus intermedius collected from Troitsa Bay, Sea of Japan. Phylogenetic analyses based on 16S rRNA gene sequences showed that these isolates were affiliated with the family Flavobacteriaceae. The novel isolates showed 99.9-100 % 16S rRNA gene sequence similarity to each other and were closely related to the type strains of the recognized members of the genus Lutibacter with sequence similarities of 95.8-98.4 %. The G+C content of the genomic DNA was 35-36 mol%. DNA-DNA relatedness among the sea urchin isolates was 95-99 % and between strain KMM 6277T and its most closely related type strains, Lutibacter agarilyticus KCTC 23842T and Lutibacter litoralis JCM 13034T, was 38 and 27 %, respectively. The prevalent fatty acids were iso-C15 : 0, anteiso-C15 : 0, summed feature 3 (comprising iso-C15 : 0 2-OH and/or C16 : 1 ω7c fatty acids), iso-C15 : 1 and C15 : 0. The polar lipid profile was composed of the phosphatidylethanolamine, one unknown aminolipid and one unknown lipid. The main respiratory isoprenoid quinone was MK-6.The results of phylogenetic, phenotypic and genotypic analyses indicated that the novel strains represent a novel species within the genus Lutibacter, for which the name Lutibacter holmesii sp. nov. is proposed. The type strain is KMM 6277T ( = CCUG 62221T = LMG 26737T).

Thiouracil-Forming Bacteria Identified and Characterized upon Porcine In Vitro Digestion of Brassicaceae Feed.

In recent years, the frequent detection of the banned thyreostat thiouracil (TU) in livestock urine has been related to endogenous TU formation following digestion of glucosinolate-rich Brassicaceae crops. Recently, it was demonstrated that, upon in vitro digestion of Brassicaceae, fecal bacteria induce TU detection in livestock (porcine livestock > bovines). Therefore, the present study was intended to isolate and identify bacteria involved in this intestinal TU formation upon Brassicaceae digestion and to gain more insight into the underlying mechanism in porcine livestock. Twenty porcine fecal inocula (gilts and multiparous sows) were assessed through static in vitro colonic-digestion simulations with rapeseed. After derivatization and extraction of the fecal suspensions, TU was analyzed using liquid chromatography-tandem mass spectrometry (LC-MS(2)). On average, lower TU concentrations were observed in fecal colonic simulations in gilts (8.35 ng g(-1) rapeseed ± 3.42 [mean ± standard deviation]) than in multiparous sows (52.63 ng g(-1) ± 16.17), which correlates with maturation of the gut microbial population with age. Further exploration of the mechanism showed cell-dependent activity of the microbial conversion and sustained TU-forming activity after subjection of the fecal inoculum to moderate heat over a time span of up to 30 min. Finally, nine TU-producing bacterial species were successfully isolated and identified by a combination of biochemical and molecular techniques as Escherichia coli (n = 5), Lactobacillus reuteri (n = 2), Enterococcus faecium (n = 1), and Salmonella enterica subsp. arizonae (n = 1). This report demonstrates that endogenous formation of TU is Brassicaceae induced and occurs under colonic conditions most likely through myrosinase-like enzyme activity expressed by different common intestinal bacterial species.

Echinimonas agarilytica gen. nov., sp. nov., a new gammaproteobacterium isolated from the sea urchin Strongylocentrotus intermedius.

A novel Gram-negative, facultatively anaerobic and motile bacterial strain, designated KMM 6351(T), was isolated from the sea urchin Strongylocentrotus intermedius and examined using a polyphasic taxonomic approach. A phylogenetic analysis based on 16S rRNA gene sequencing revealed that the strain formed a distinct phyletic line in the class Gammaproteobacteria and was most closely related to the genera Aliivibrio, Photobacterium and Vibrio. Strain KMM 6351(T) grows at 4-40 °C and with 0.5-12 % NaCl and decomposes aesculin, agar, gelatin, starch, chitin and DNA. The DNA G+C content of the strain was determined to be 46.1 mol%. The prevalent fatty acids were found to be C(16:0), C(18:1) ω7c, C(12:0) 3-OH and summed feature 3 (comprising C(16:1) ω7c and/or iso-C(15:0) 2-OH fatty acids). The major polar lipids were determined to be diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified aminolipid. The predominant ubiquinone was found to be Q-8. The results of the phenotypic, chemotaxonomic and genotypic analyses clearly indicated that the novel strain should be assigned to a new genus and species within the class γ-Proteobacteria for which the name Echinimonas agarilytica gen. nov., sp. nov. is proposed. The type strain is KMM 6351(T) (=KCTC 22996(T) = LMG 25420(T)).

Bacillus berkeleyi sp. nov., isolated from the sea urchin Strongylocentrotus intermedius.

A bacterial strain, designated KMM 6244(T), was isolated from the sea urchin Strongylocentrotus intermedius and subjected to a polyphasic taxonomic investigation. The bacterium was found to be heterotrophic, aerobic, non-motile and spore-forming. Comparative phylogenetic analysis based on 16S rRNA gene sequencing placed the marine isolate in the genus Bacillus. The nearest neighbor of strain KMM 6244(T) was Bacillus decolorationis LMG 19507(T) with a 16S rRNA gene sequence similarity of 98.0%. Sequence similarities with the other recognized Bacillus species were less than 96.0%. The results of the DNA-DNA hybridization experiments revealed a low relatedness (37%) of the novel isolate with the type strain of B. decolorationis LMG 19507(T). Strain KMM 6244(T) grew at 4-45°C and with 0-12% NaCl. It produced catalase and oxidase and hydrolyzed aesculin, casein, gelatin and DNA. The predominant fatty acids were anteiso-C(15:0), iso-C(15:0), anteiso-C(17:0), C(15:0), iso-C(16:0) and iso-C(14:0). The DNA G + C content was 39.4 mol%. A combination of phylogenetic, genotypic and phenotypic data clearly indicated that strain KMM 6244(T) represents a novel species in the genus Bacillus, for which the name Bacillus berkeleyi sp. nov. is proposed. The type strain is KMM 6244(T) (KCTC 12718(T) = LMG 26357(T)).

An MLSA-based online scheme for the rapid identification of Stenotrophomonas isolates.

An online scheme to assign Stenotrophomonas isolates to genomic groups was developed using the multilocus sequence analysis (MLSA), which is based on the DNA sequencing of selected fragments of the housekeeping genes ATP synthase alpha subunit (atpA), the recombination repair protein (recA), the RNA polymerase alpha subunit (rpoA) and the excision repair beta subunit (uvrB). This MLSA-based scheme was validated using eight of the 10 Stenotrophomonas species that have been previously described. The environmental and nosocomial Stenotrophomonas strains were characterised using MLSA, 16S rRNA sequencing and DNA-DNA hybridisation (DDH) analyses. Strains of the same species were found to have greater than 95% concatenated sequence similarity and specific strains formed cohesive readily recognisable phylogenetic groups. Therefore, MLSA appeared to be an effective alternative methodology to amplified fragment length polymorphism fingerprint and DDH techniques. Strains of Stenotrophomonas can be readily assigned through the open database resource that was developed in the current study (www.steno.lncc.br/).

An MLSA-based online scheme for the rapid identification of Stenotrophomonas isolates

An online scheme to assign Stenotrophomonas isolates to genomic groups was developed using the multilocus sequence analysis (MLSA), which is based on the DNA sequencing of selected fragments of the housekeeping genes ATP synthase alpha subunit (atpA), the recombination repair protein (recA), the RNA polymerase alpha subunit (rpoA) and the excision repair beta subunit (uvrB). This MLSA-based scheme was validated using eight of the 10 Stenotrophomonas species that have been previously described. The environmental and nosocomial Stenotrophomonas strains were characterised using MLSA, 16S rRNA sequencing and DNA-DNA hybridisation (DDH) analyses. Strains of the same species were found to have greater than 95 percent concatenated sequence similarity and specific strains formed cohesive readily recognisable phylogenetic groups. Therefore, MLSA appeared to be an effective alternative methodology to amplified fragment length polymorphism fingerprint and DDH techniques. Strains of Stenotrophomonas can be readily assigned through the open database resource that was developed in the current study (www.steno.lncc.br/).

Actinobaculum schaalii causing Fournier's gangrene.

Actinobaculum schaalii, which belongs to the group of Gram-positive rods, is difficult to culture. Using molecular genetics, Actinobaculum schaalii could be identified as a causing microorganism in a case of Fournier's gangrene.

Screening for endophytic nitrogen-fixing bacteria in Brazilian sugar cane varieties used in organic farming and description of Stenotrophomonas pavanii sp. nov.

A Gram-negative, rod-shaped, non-spore-forming and nitrogen-fixing bacterium, designated ICB 89(T), was isolated from stems of a Brazilian sugar cane variety widely used in organic farming. 16S rRNA gene sequence analysis revealed that strain ICB 89(T) belonged to the genus Stenotrophomonas and was most closely related to Stenotrophomonas maltophilia LMG 958(T), Stenotrophomonas rhizophila LMG 22075(T), Stenotrophomonas nitritireducens L2(T), [Pseudomonas] geniculata ATCC 19374(T), [Pseudomonas] hibiscicola ATCC 19867(T) and [Pseudomonas] beteli ATCC 19861(T). DNA-DNA hybridization together with chemotaxonomic data and biochemical characteristics allowed the differentiation of strain ICB 89(T) from its nearest phylogenetic neighbours. Therefore, strain ICB 89(T) represents a novel species, for which the name Stenotrophomonas pavanii sp. nov. is proposed. The type strain is ICB 89(T) ( = CBMAI 564(T)  = LMG 25348(T)).

Phylogenetic variation of Aggregatibacter actinomycetemcomitans serotype e reveals an aberrant distinct evolutionary stable lineage.

The periodontal pathogen Aggregatibacter actinomycetemcomitans that comprises six serotypes (a-f), is often identified by PCR-based techniques targeting the 16S rRNA gene. In this study, 16S rRNA gene sequence analysis revealed an aberrant cluster of 19 strains within serotype e, denoted as serotype e'. The 16S rRNA gene sequence similarities found between serotype e' strains ranged from 99.7% to 100.0%, whereas 96.8-97.5% sequence similarity was obtained with members of the other serotypes, indicating that the serotype e' strains might not be true members of A. actinomycetemcomitans. However, DNA-DNA hybridizations between a representative serotype e' strain and representative strains of serotypes b, d and e of A. actinomycetemcomitans revealed 68-75% DNA-DNA relatedness, demonstrating that the serotype e' strains do belong to the species A. actinomycetemcomitans. AFLP analysis of 33 A. actinomycetemcomitans strains, representing all serotypes (a-f), but mainly serotype e' strains, showed that the latter form a distinct cluster, demonstrating that these strains are also closely related on the whole genome level. Moreover, the serotype e' strains were unable to ferment starch and glycogen in contrast to almost all other A. actinomycetemcomitans strains tested. Overall, the data obtained in this study suggest that the serotype e' strains form an evolutionary relatively stable distinct subgroup within A. actinomycetemcomitans.

Staphylococcus devriesei sp. nov., isolated from teat apices and milk of dairy cows.

Ten non-motile, Gram-stain-positive, coagulase-negative staphylococci were isolated from bovine milk and teat apices. All isolates were catalase-positive, with anteiso-C(15 : 0), iso-C(15 : 0), anteiso-C(17 : 0), iso-C(17 : 0) and C(18 : 0) as predominant fatty acids and diphosphatidylglycerol and phosphatidylglycerol as major polar lipids. The results of sequence analysis of the 16S rRNA gene and four housekeeping genes (rpoB, hsp60, tuf and dnaJ) in combination with tRNA-intergenic spacer length analysis showed that the isolates form a separate branch within the genus Staphylococcus. Based on 16S rRNA gene sequencing, the phylogenetically most closely related species are Staphylococcus haemolyticus, S. hominis and S. lugdunensis, with >98.7 % sequence similarity. The DNA G+C content varies from 33.3 to 33.7 mol%, and DNA-DNA hybridization with the nearest neighbours, based on 16S rRNA gene sequences, confirmed that the isolates represent a novel Staphylococcus species. All isolates induced a small zone of complete haemolysis on Columbia agar with 5 % sheep blood and exhibited a homogeneous biochemical fingerprint that is discriminative from the phylogenetically most closely related species. Based on these results, it is proposed to classify the ten isolates as Staphylococcus devriesei sp. nov., with strain KS-SP 60(T) (=LMG 25332(T) =CCUG 58238(T)) as the type strain.

Arcobacter mytili sp. nov., an indoxyl acetate-hydrolysis-negative bacterium isolated from mussels.

Three Arcobacter isolates, recovered from mussels (genus Mytilus), and one isolate from brackish water in Catalonia (north-east Spain) showed a novel pattern using a recently described identification method for members of the genus Arcobacter, 16S rRNA gene RFLP. Enterobacterial repetitive intergenic consensus PCR fingerprinting demonstrated that the three isolates from mussels belonged to two genotypes and that the fourth isolate from water belonged to a third genotype. Analysis of the 16S rRNA and rpoB gene sequences showed that the new isolates formed a separate lineage within the genus Arcobacter. This was also confirmed by the low DNA-DNA relatedness values (16-30 %) of the isolates with the type strains of recognized Arcobacter species. Hydrolysis of indoxyl acetate, a characteristic trait for all species of the genus Arcobacter, was negative for the novel isolates. The susceptibility of the novel isolates to cefoperazone, together with the lack of urease production and nitrate reduction, further enabled them to be differentiated from recognized Arcobacter species based on physiological characteristics. Genotypic and phenotypic characteristics indicated that the new isolates represent a novel species of the genus Arcobacter, for which the name Arcobacter mytili sp. nov. is proposed, with the type strain F2075(T) (=CECT 7386(T) =LMG 24559(T)). The DNA G+C content of strain F2075(T) was 26.9 mol%.

Bacterial diversity from benthic mats of Antarctic lakes as a source of new bioactive metabolites.

During the MICROMAT project, the bacterial diversity of microbial mats growing in the benthic environment of Antarctic lakes was accessed for the discovery of novel antibiotics. In all, 723 Antarctic heterotrophic bacteria belonging to novel and/or endemic taxa in the α-, ß- and γ-subclasses of the Proteobacteria, the Bacteroidetes branch, and of the high and low percentage G+C Gram-positives, were isolated, cultivated in different media and at different temperatures, and then screened for the production of antimicrobial activities. A total of 6348 extracts were prepared by solid phase extraction of the culture broths or by biomass solvent extraction. 122 bacteria showed antibacterial activity against the Gram-positives Staphylococcus aureus and to a lower extent Enterococcus faecium, and versus the Gram-negative Escherichia coli. Few of these strains showed also some antifungal activity against Cryptococcus neoformans, Aspergillus fumigatus and to a lower extent Candida albicans. LC-MS fractionation of extracts from a subset of strains (hits) that exhibited relatively potent antibacterial activities evidenced a chemical novelty that was further investigated. Two strains of Arthrobacter agilis produced potent antibacterial compounds with activity against Gram-positives and possibly related to novel cyclic thiazolyl peptides. To our knowledge, this is the first report of new antibiotics produced by bacteria from benthic microbial mats from Antarctic lakes. With no doubts these microbial assemblages represent an extremely rich source for the isolation of new strains producing novel bioactive metabolites with the potential to be developed as antibiotic compounds.

Enterobacter pulveris sp. nov., isolated from fruit powder, infant formula and an infant formula production environment.

Six Gram-negative, facultatively anaerobic, non-spore-forming, coccoid rod-shaped isolates were obtained from fruit powder (n=3), infant formula (n=2) and an infant formula production environment (n=1) and investigated in a polyphasic taxonomic study. Comparative 16S rRNA gene sequence analysis combined with rpoB gene sequence analysis allocated the isolates to the family Enterobacteriaceae. The highest rpoB gene sequence similarities (91.2-95.8%) were obtained with Enterobacter helveticus, Enterobacter radicincitans, Enterobacter turicensis and Enterobacter sakazakii and the phylogenetic branch formed by these species was supported by a high bootstrap value. Biochemical data revealed that the isolates could be differentiated from their nearest neighbours by their ability to utilize melibiose, sucrose, D-arabitol, mucate and 1-O-methyl-alpha-galactopyranoside and their negative reactions for D-sorbitol utilization and the Voges-Proskauer test. On the basis of the phylogenetic analyses, DNA-DNA hybridization data, and unique physiological and biochemical characteristics, it is proposed that the isolates represent a novel species of the genus Enterobacter, Enterobacter pulveris sp. nov. The type strain is 601/05(T) (=LMG 24057(T)=DSM 19144(T)).

Acetobacter senegalensis sp. nov., a thermotolerant acetic acid bacterium isolated in Senegal (sub-Saharan Africa) from mango fruit (Mangifera indica L.).

A thermotolerant acetic acid bacterium, designated strain CWBI-B418(T), isolated in Senegal from mango fruit (Mangifera indica), was characterized in detail by means of genotypic and phenotypic methods. The novel strain was strictly aerobic and exhibited optimal growth on YGM medium at 35 degrees C. Cells were Gram-negative, motile and coccoid. The strain was assigned to the genus Acetobacter on the basis of 16S rRNA gene sequence analysis. DNA-DNA hybridization experiments with its phylogenetically closest relatives showed that strain CWBI-B418(T) represented a novel Acetobacter genospecies. The DNA G+C content of strain CWBI-B418(T) was 56.0 mol%. Phenotypic characteristics enabling the differentiation of strain CWBI-B418(T) from phylogenetically related Acetobacter species were: production of 2-keto-D-gluconic acid from D-glucose, but not 5-keto-D-gluconic acid, production of catalase but not oxidase, growth on yeast extract with 30 % d-glucose, growth with ammonium as sole nitrogen source with ethanol as carbon source, utilization of glycerol and ethanol but not maltose or methanol as carbon sources, and growth in the presence of 10 % ethanol. Based on the genotypic and phenotypic data presented, strain CWBI-B418(T) clearly represents a novel Acetobacter species, for which the name Acetobacter senegalensis sp. nov. is proposed. The type strain is CWBI-B418(T) (=LMG 23690(T)=DSM 18889(T)).

Enterobacter turicensis sp. nov. and Enterobacter helveticus sp. nov., isolated from fruit powder.

Four Gram-negative, facultatively anaerobic, non-spore-forming isolates of coccoid rods were obtained from fruit powder and investigated in a polyphasic taxonomic study. Comparative 16S rRNA gene sequence analysis allocated the isolates to the family Enterobacteriaceae. Their phylogenetic position within the family Enterobacteriaceae was confirmed by rpoB sequence analysis and as the highest rpoB sequence similarities were obtained with Enterobacter radicincitans, Enterobacter cowanii and Enterobacter sakazakii, the isolates clearly belong to the genus Enterobacter. Biochemical data revealed that the isolates can be separated into two distinct groups that represent two novel species, as confirmed by DNA-DNA hybridizations. The two novel species can be differentiated from their nearest neighbours by the following characteristics: the utilization of sucrose, D-sorbitol, putrescine and mucate, the hydrolysis of aesculin and a negative result in the Voges-Proskauer reaction. It is therefore proposed that these novel isolates are classified as Enterobacter turicensis sp. nov. (type strain 508/05(T)=LMG 23730(T)=DSM 18397(T)) and Enterobacter helveticus sp. nov. (type strain 513/05(T)=LMG 23732(T)=DSM 18396(T)).

Rhodanobacter spathiphylli sp. nov., a gammaproteobacterium isolated from the roots of Spathiphyllum plants grown in a compost-amended potting mix.

Three Gram-negative, yellow-pigmented strains were isolated from the rhizospheres of Spathiphyllum plants grown in a compost-amended potting mix. The strains showed biological control activity towards the root-rot plant pathogen Cylindrocladium spathiphylli, and were characterized to determine their taxonomic position. Cells of the strains were non-motile rods, and the strains were oxidase- and catalase-positive and unable to ferment most sugars tested. The three strains showed differences in growth temperature range, optimal growth temperature and some biochemical reactions. The majority of the fatty acids were branched, and large amounts of 15 : 0 iso and 17 : 1 iso omega9c were present. The 16S rRNA gene sequence (1,497 bp) of strain B39(T) showed the highest level of similarity (98.5 %) to that of Rhodanobacter fulvus IAM 15025(T), followed by Rhodanobacter lindaniclasticus LMG 18385(T) (96.0 %; strain no longer extant), Dyella koreensis CCUG 50883(T) (96.4 %), Dyella japonica DSM 16301(T) (96.3 %), Frateuria aurantia LMG 1558(T) (96.2 %) and Fulvimonas soli LMG 19981(T) (95.9 %). Less than 90 % 16S rRNA gene sequence similarity was observed for other members of the Gammaproteobacteria. The mean DNA-DNA reassociation value for the three strains was 100 % and was 25 % when the strains were compared with DNA from R. fulvus LMG 23003(T). The strains had a mean DNA G+C content of 67.6 mol%. On the basis of their phylogenetic, genomic and phenotypic properties, the three strains represent a novel species within the genus Rhodanobacter, for which the name Rhodanobacter spathiphylli sp. nov. is proposed. The type strain is strain B39(T) (=LMG 23181(T)=DSM 17631(T)).

Tetrathiobacter mimigardefordensis sp. nov., isolated from compost, a betaproteobacterium capable of utilizing the organic disulfide 3,3'-dithiodipropionic acid.

In this study, a novel betaproteobacterium, strain DPN7(T), was isolated under mesophilic conditions from compost because of its capacity to utilize the organic disulfide 3,3'-dithiodipropionic acid. Analysis of the 16S rRNA gene sequence of strain DPN7(T) revealed 98.5 % similarity to that of Tetrathiobacter kashmirensis LMG 22695(T). Values for sequence similarity to members of the genera Alcaligenes, Castellaniella and Taylorella, the nearest neighbours of the genus Tetrathiobacter, were about 95 % or less. The DNA G + C content of strain DPN7(T) was 55.1 mol%. The level of DNA-DNA hybridization between strain DPN7(T) and T. kashmirensis LMG 22695(T) was 41 %, whereas it was much lower between strain DPN7(T) and Alcaligenes faecalis LMG 1229(T) (7 %) or Castellaniella defragrans LMG 18538(T) (5 %). This genotypic divergence was supported by differences in biochemical and chemotaxonomic characteristics. For this reason, and because of the differences in the protein and fatty acid profiles, strain DPN7(T) should be classified within a novel species of Tetrathiobacter, for which the name Tetrathiobacter mimigardefordensis sp. nov. is proposed. The type strain is strain DPN7(T) (=DSM 17166(T) = LMG 22922(T)).

Identification of Paenibacillus larvae to the subspecies level: an obstacle for AFB diagnosis.

This study was initially aimed at developing a PCR-test to differentiate between the pathogenic agent of American foulbrood (Paenibacillus larvae subsp. larvae) and powdery-scale disease (P. larvae subsp. pulvifaciens) of the honeybee. The test was based on the "insert of clone 9" (iC9), referring to a cloned 1.9 kB HaeIII fragment that occurs only in the P. larvae subsp. larvae reference strains and possibly correlates with American foulbrood virulence. It was shown that an iC9-based PCR-test discriminates between the BCCM/LMG reference strains of both subspecies. However, the screening of 179 Belgian field strains revealed five isolates that gave no iC9-based amplicon, thus rather resembling to P. larvae subsp. pulvifaciens. In addition, they all produced acid from mannitol, a characteristic previously assigned to the pulvifaciens subspecies. Because the reference strains gave conflicting data, this carbohydrate acidification was not conclusive. Therefore, the exact taxonomic position of the five retained strains was determined by a polyphasic approach using SDS-PAGE, AFLP, and ERIC-based PCR. Four iC9-negative field strains could be identified as P. larvae subsp. larvae; the taxonomic position of the fifth field strain remained ambiguous. The latter was provisionally classified as a subspecies pulvifaciens strain on the basis of SDS-PAGE. The present paper demonstrates the existence of field strains that do not fit well in the subdivision of the species P. larvae into two subspecies. Knowing that only one of both subspecies represents the pathogenic agent of AFB, this is a serious obstacle for the diagnosis of this honeybee disease.

Alcaligenes aquatilis sp. nov., a novel bacterium from sediments of the Weser Estuary, Germany, and a salt marsh on Shem Creek in Charleston Harbor, USA.

Four nitrite-dissimilating strains, isolated from Weser Estuary sediments, were investigated using a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA gene sequences indicated that these strains belong to the 'Betaproteobacteria' and are related to the genus Alcaligenes. The highest level of sequence similarity (100 %) was found with strain M3A (=ATCC 700596), a dimethyl sulfide-producing marine isolate that was included in this study. DNA-DNA hybridizations between the five strains and related Alcaligenes faecalis strains confirmed that the former belong to a single and novel species within the genus Alcaligenes. The isolates are Gram-negative, motile, rod-shaped cells with a DNA G+C content of about 56 mol%. The whole-cell fatty acid profiles of the isolates were very similar and included C(16 : 0), C(17 : 0) cyclo, C(18 : 1)omega7c, summed feature 2 (comprising any combination of C(12 : 0) aldehyde, an unknown fatty acid of equivalent chain length 10.928, C(16 : 1) iso I and C(14 : 0) 3-OH) and summed feature 3 (C(15 : 0) iso 2-OH and/or C(16 : 1)omega7c) as the major fatty acid components. On the basis of their phylogenetic, genomic and phenotypic properties, the five novel strains can be assigned to the genus Alcaligenes as a novel species, for which the name Alcaligenes aquatilis sp. nov. is proposed. The type strain is LMG 22996T (=CCUG 50924T).