Stefanie Van Trappen

Diversity of 746 heterotrophic bacteria isolated from microbial mats from Ten Antarctic lakes

Microbial mats, growing in Antarctic lakes constitute unique and very diverse habitats. In these mats microorganisms are confronted with extreme life conditions. We isolated 746 bacterial strains from mats collected from ten lakes in the Dry Valleys (lakes Hoare and Fryxell), the Vestfold Hills (lakes Ace, Druzhby, Grace, Highway, Pendant, Organic and Watts) and the Larsemann Hills (lake Reid), using heterotrophic growth conditions. These strains were investigated by fatty acid analysis, and by numerical analysis, 41 clusters, containing 2 to 77 strains, could be delineated, whereas 31 strains formed single branches. Several fatty acid groups consisted of strains from different lakes from the same region, or from different regions. The 16S rRNA genes from 40 strains, representing 35 different fatty acid groups were sequenced. The strains belonged to the alpha, beta and gamma subclasses of the Proteobacteria, the high and low percent G+C Gram-positives, and to the Cytophaga-Flavobacterium-Bacteroides branch. For strains representing 16 fatty acid clusters, validly named nearest phylogenetic neighbours showed pairwise sequence similarities of less than 97%. This indicates that the clusters they represent, belong to taxa that have not been sequenced yet or as yet unnamed new taxa, related to Alteromonas, Bacillus, Clavibacter, Cyclobacterium, Flavobacterium, Marinobacter, Mesorhizobium, Microbacterium, Pseudomonas, Saligentibacter, Sphingomonas and Sulfitobacter.

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%.

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)).

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)).

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-, β- 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.

Actinobaculum schaalii Causing Fournier's Gangrene

ABSTRACT 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 Fourniers gangrene.

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.

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/).

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.

ABSTRACT 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-MS2). 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.