The zoonotic pathogen Wohlfahrtiimonas chitiniclastica - current findings from a clinical and genomic perspective.

The zoonotic pathogen Wohlfahrtiimonas chitiniclastica can cause several diseases in humans, including sepsis and bacteremia. Although the pathogenesis is not fully understood, the bacterium is thought to enter traumatic skin lesions via fly larvae, resulting in severe myiasis and/or wound contamination. Infections are typically associated with, but not limited to, infestation of an open wound by fly larvae, poor sanitary conditions, cardiovascular disease, substance abuse, and osteomyelitis. W. chitiniclastica is generally sensitive to a broad spectrum of antibiotics with the exception of fosfomycin. However, increasing drug resistance has been observed and its development should be monitored with caution. In this review, we summarize the currently available knowledge and evaluate it from both a clinical and a genomic perspective.

Arcobacter roscoffensis sp. nov., a marine bacterium isolated from coastal seawater.

A novel Gram-negative, aerobic, motile, rod-shaped, beige-pigmented bacterium, strain ARW1-2F2T, was isolated from a seawater sample collected from Roscoff, France. Strain ARW1-2F2T was catalase-negative and oxidase-positive, and grew under mesophilic, neutrophilic and halophilic conditions. The 16S rRNA sequences revealed that strain ARW1-2F2T was closely related to Arcobacter lekithochrous LFT 1.7T and Arcobacter caeni RW17-10T(95.8 and 95.5 % gene sequence similarity, respectively). The genome of strain ARW1-2F2T was sequenced and had a G+C content of 28.7%. Two different measures of genome similarity, average nucleotide identity based on blast and digital DNA-DNA hybridization, indicated that strain ARW1-2F2T represents a new Arcobacter species. The predominant fatty acids were C16 : 1 ω7c/C16 : 1 ω6c and C18 : 1 ω7c/C18 : 1 ω6c. The results of a polyphasic analysis supported the description of strain ARW1-2F2T as representing a novel species of the genus Arcobacter, for which the name Arcobacter roscoffensis sp. nov. is proposed with the type strain ARW1-2F2T (DSM 29169T=KCTC 52423T).

Aminithiophilus ramosus gen. nov., sp. nov., a sulphur-reducing bacterium isolated from a pyrite-forming enrichment culture, and taxonomic revision of the family Synergistaceae.

A novel sulphur-reducing bacterium was isolated from a pyrite-forming enrichment culture inoculated with sewage sludge from a wastewater treatment plant. Based on phylogenetic data, strain J.5.4.2-T.3.5.2T could be affiliated with the phylum Synergistota. Among type strains of species with validly published names, the highest 16S rRNA gene sequence identity value was found with Aminiphilus circumscriptus ILE-2T (89.2 %). Cells of the new isolate were Gram-negative, non-spore-forming, straight to slightly curved rods with tapered ends. Motility was conferred by lateral flagella. True branching of cells was frequently observed. The strain had a strictly anaerobic, asaccharolytic, fermentative metabolism with peptides and amino acids as preferred substrates. Sulphur was required as an external electron acceptor during fermentative growth and was reduced to sulphide, whereas it was dispensable during syntrophic growth with a Methanospirillum species. Major fermentation products were acetate and propionate. The cellular fatty acid composition was dominated by unsaturated and branched fatty acids, especially iso-C15 : 0. Its major polar lipids were phosphatidylglycerol, phosphatidylethanolamine and distinct unidentified polar lipids. Respiratory lipoquinones were not detected. Based on the obtained data we propose the novel species and genus Aminithiophilus ramosus, represented by the type strain J.5.4.2-T.3.5.2T (=DSM 107166T=NBRC 114655T) and the novel family Aminithiophilaceae fam. nov. to accommodate the genus Aminithiophilus. In addition, we suggest reclassifying certain members of the Synergistaceae into new families to comply with current standards for the classification of higher taxa. Based on phylogenomic data, the novel families Acetomicrobiaceae fam. nov., Aminiphilaceae fam. nov., Aminobacteriaceae fam. nov., Dethiosulfovibrionaceae fam. nov. and Thermovirgaceae fam. nov. are proposed.

Nocardia australiensis sp. nov. and Nocardia spumae sp. nov., isolated from sea foam in Queensland, Australia.

Strains USC-21046T and USC-21048T were isolated from foaming coastal marine waters on the Sunshine Coast, Queensland, Australia. Both strains displayed growth and morphological characteristics typical for members belonging to the genus Nocardia. The major polar lipids were diphosphatidylglycerol and phosphatidylethanolamine, and the major fatty acids were C16 : 0, C18 : 1 ω9c, C18 : 0 and C18 : 0 10-methyl. The mycolic acids of strains USC-21046T and USC-21048T consisted of chain lengths between 50-64 and 56-68, respectively. Moreover, both of those strains contained meso-diaminopimelic acid and ribose, arabinose, glucose and galactose as whole cell sugars. Based on the phylogenomic results, both strains belonged to the genus Nocardia with strain USC-21046T showing an 80.4 % genome similarity to N. vinacea NBRC 16497T and N. pseudovaccinii NBRC 100343T, whereas USC-21048T strain showed an 83.6 % genome similarity to N. aobensis NBRC 100429T. Both strains were delineated from their closely related relatives based on physiological (e.g. growth on sole carbon source) and chemotaxonomic (e.g. cellular fatty composition) differences. The digital DNA-DNA hybridization (dDDH) values between USC-21046T and USC-21048T and their closely related relatives were below the dDDH threshold value of ≤70 % used for the taxonomic classification of novel species status. The genome length of strains USC-21046T and USC-21048T were 6 878 863 and 7 066 978 bp, with G+C contents of 65.2 and 67.8 mol%, respectively. For the novel isolates, we propose the names Nocardia australiensis sp. nov. with the type strain USC-21046T (=DSM 111727T=NCCB 100867T) and Nocardia spumae sp. nov. with the type strain USC-21048T (=DSM 111726T=NCCB 100868T).

Vibrio syngnathi sp. nov., a fish pathogen, isolated from the Kiel Fjord.

A new Vibrio strain, K08M4T, was isolated from the broad-nosed pipefish Syngnathus typhle in the Kiel Fjord. Infection experiments revealed that K08M4T was highly virulent for juvenile pipefish. Cells of strain K08M4T were Gram-stain-negative, curved rod-shaped and motile by means of a single polar flagellum. The strain grew aerobically at 9-40° C, at pH 4-10.5 and it tolerated up to 12 % (w/v) NaCl. The most prevalent (>10 %) cellular fatty acids of K08M4T were C16 : 1 ω7c and C16 : 0. Whole-genome comparisons revealed that K08M4T represents a separate evolutionary lineage that is distinct from other Vibrio species and falls within the Splendidus clade. The genome is 4,886,292 bp in size, consists of two circular chromosomes (3,298,328 and 1, 587,964 bp) and comprises 4,178 protein-coding genes and 175 RNA genes. In this study, we describe the phenotypic features of the new isolate and present the annotation and analysis of its complete genome sequence. Based on these data, the new isolate represents a new species for which we propose the name Vibrio syngnathi sp. nov. The type strain is K08M4T (=DSM 109818T=CECT 30086T).

New insights into the energy metabolism and taxonomy of Deferribacteres revealed by the characterization of a new isolate from a hypersaline microbial mat.

Strain L21-Ace-BEST , isolated from a lithifying cyanobacterial mat, could be assigned to a novel species and genus within the class Deferribacteres. It is an important model organism for the study of anaerobic acetate degradation under hypersaline conditions. The metabolism of strain L21-Ace-BEST was characterized by biochemical studies, comparative genome analyses, and the evaluation of gene expression patterns. The central metabolic pathway is the citric acid cycle, which is mainly controlled by the enzyme succinyl-CoA:acetate-CoA transferase. The potential use of a reversed oxidative citric acid cycle to fix CO2 has been revealed through genome analysis. However, no autotrophic growth was detected in this strain, whereas sulfide and H2 can be used mixotrophically. Preferred electron acceptors for the anaerobic oxidation of acetate are nitrate, fumarate and dimethyl sulfoxide, while oxygen can be utilized only under microoxic conditions. Aerotolerant growth by fermentation was observed at higher oxygen concentrations. The redox cycling of sulfur/sulfide enables the generation of reducing power for the assimilation of acetate during growth and could prevent the over-reduction of cells in stationary phase. Extracellular electron transfer appears to be an essential component of the respiratory metabolism in this clade of Deferribacteres and may be involved in the reduction of nitrite to ammonium.

Higher phage virulence accelerates the evolution of host resistance.

Pathogens vary strikingly in their virulence and the selection they impose on their hosts. While the evolution of different virulence levels is well studied, the evolution of host resistance in response to different virulence levels is less understood and, at present, mainly based on observations and theoretical predictions with few experimental tests. Increased virulence can increase selection for host resistance evolution if the benefits of avoiding infection outweigh resistance costs. To test this, we experimentally evolved the bacterium Vibrio alginolyticus in the presence of two variants of a filamentous phage that differ in their virulence. The bacterial host exhibited two alternative defence strategies: (1) super infection exclusion (SIE), whereby phage-infected cells were immune to subsequent infection at the cost of reduced growth, and (2) surface receptor mutations (SRM), providing resistance to infection by preventing phage attachment. While SIE emerged rapidly against both phages, SRM evolved faster against the high- than the low-virulence phage. Using a mathematical model of our system, we show that increasing virulence strengthens selection for SRM owing to the higher costs of infection suffered by SIE immune hosts. Thus, by accelerating the evolution of host resistance, more virulent phages caused shorter epidemics.

Extremophile Metal Resistance: Plasmid-Encoded Functions in Streptomyces mirabilis.

The extreme metal tolerance of up to 130 mM NiSO4 in Streptomyces mirabilis P16B-1 was investigated. Genome sequencing revealed the presence of a large linear plasmid, pI. To identify plasmid-encoded determinants of metal resistance, a newly established transformation system was used to characterize the predicted plasmid-encoded loci nreB, hoxN, and copYZ. Reintroduction into the plasmid-cured S. mirabilis ΔpI confirmed that the predicted metal transporter gene nreB constitutes a nickel resistance factor, which was further supported by its heterologous expression in Escherichia coli. In contrast, the predicted nickel exporter gene hoxN decreased nickel tolerance, while copper tolerance was enhanced. The predicted copper-dependent transcriptional regulator gene copY did not induce tolerance toward either metal. Since genes for transfer were identified on the plasmid, its conjugational transfer to the metal-sensitive Streptomyces lividans TK24 was checked. This resulted in acquired tolerance toward 30 mM nickel and additionally increased the tolerance toward copper and cobalt, while oxidative stress tolerance remained unchanged. Intracellular nickel concentrations decreased in the transconjugant strain. The high extracellular nickel concentrations allowed for biomineralization. Plasmid transfer could also be confirmed into the co-occurring actinomycete Kribbella spp. in soil microcosms. IMPORTANCE Living in extremely metal-rich environments requires specific adaptations, and often, specific metal tolerance genes are encoded on a transferable plasmid. Here, Streptomyces mirabilis P16B-1, isolated from a former mining area and able to grow with up to 130 mM NiSO4, was investigated. The bacterial chromosome, as well as a giant plasmid, was sequenced. The plasmid-borne gene nreB was confirmed to confer metal resistance. A newly established transformation system allowed us to construct a plasmid-cured S. mirabilis as well as an nreB-rescued strain in addition to confirming nreB encoding nickel resistance if heterologously expressed in E. coli. The potential of intra- and interspecific plasmid transfer, together with the presence of metal resistance factors on that plasmid, underlines the importance of plasmids for transfer of resistance factors within a bacterial soil community.

Genomic and proteomic profiles of biofilms on microplastics are decoupled from artificial surface properties.

Microplastics in marine ecosystems are colonized by diverse prokaryotic and eukaryotic communities. How these communities and their functional profiles are shaped by the artificial surfaces remains broadly unknown. In order to close this knowledge gap, we set up an in situ experiment with pellets of the polyolefin polymer polyethylene (PE), the aromatic hydrocarbon polymer polystyrene (PS), and wooden beads along a coastal to estuarine gradient in the Baltic Sea, Germany. We used an integrated metagenomics/metaproteomics approach to evaluate the genomic potential as well as protein expression levels of aquatic plastic biofilms. Our results suggest that material properties had a minor influence on the plastic-associated assemblages, as genomic and proteomic profiles of communities associated with the structurally different polymers PE and PS were highly similar, hence polymer-unspecific. Instead, it seemed that these communities were shaped by biogeographic factors. Wood, on the other hand, induced the formation of substrate-specific biofilms and served as nutrient source itself. Our study indicates that, while PE and PS microplastics may be relevant in the photic zone as opportunistic colonization grounds for phototrophic microorganisms, they appear not to be subject to biodegradation or serve as vectors for pathogenic microorganisms in marine habitats.

Dilution-to-Stimulation/Extinction Method: a Combination Enrichment Strategy To Develop a Minimal and Versatile Lignocellulolytic Bacterial Consortium.

The engineering of complex communities can be a successful path to understand the ecology of microbial systems and improve biotechnological processes. Here, we developed a strategy to assemble a minimal and effective lignocellulolytic microbial consortium (MELMC) using a sequential combination of dilution-to-stimulation and dilution-to-extinction approaches. The consortium was retrieved from Andean forest soil and selected through incubation in liquid medium with a mixture of three types of agricultural plant residues. After the dilution-to-stimulation phase, approximately 50 bacterial sequence types, mostly belonging to the Sphingobacteriaceae, Enterobacteriaceae, Pseudomonadaceae, and Paenibacillaceae, were significantly enriched. The dilution-to-extinction method demonstrated that only eight of the bacterial sequence types were necessary to maintain microbial growth and plant biomass consumption. After subsequent stabilization, only two bacterial species (Pseudomonas sp. and Paenibacillus sp.) became highly abundant (>99%) within the MELMC, indicating that these are the key players in degradation. Differences in the composition of bacterial communities between biological replicates indicated that selection, sampling, and/or priority effects could shape the consortium structure. The MELMC can degrade up to ∼13% of corn stover, consuming mostly its (hemi)cellulosic fraction. Tests with chromogenic substrates showed that the MELMC secretes an array of endoenzymes able to degrade xylan, arabinoxylan, carboxymethyl cellulose, and wheat straw. Additionally, the metagenomic profile inferred from the phylogenetic composition along with an analysis of carbohydrate-active enzymes of 20 bacterial genomes support the potential of the MELMC to deconstruct plant polysaccharides. This capacity was mainly attributed to the presence of Paenibacillus sp.IMPORTANCE The significance of our study mainly lies in the development of a combined top-down enrichment strategy (i.e., dilution to stimulation coupled to dilution to extinction) to build a minimal and versatile lignocellulolytic microbial consortium. We demonstrated that mainly two selectively enriched bacterial species (Pseudomonas sp. and Paenibacillus sp.) are required to drive the effective degradation of plant polymers. Our findings can guide the design of a synthetic bacterial consortium that could improve saccharification (i.e., the release of sugars from agricultural plant residues) processes in biorefineries. In addition, they can help to expand our ecological understanding of plant biomass degradation in enriched bacterial systems.

Natronoglycomyces albus gen. nov., sp. nov, a haloalkaliphilic actinobacterium from a soda solonchak soil.

A haloalkaliphilic hydrolytic actinobacterium, strain ACPA22T, was enriched and isolated in pure culture from saline alkaline soil (soda solonchak) in northeastern Mongolia. The isolate was facultatively alkaliphilic, growing at pH 6.5-10.5 (optimum at 7.3-9.0) and highly salt-tolerant, tolerating up to 3 M total Na+ as carbonates. The hydrolytic nature of ACPA22T was confirmed by two different growth-dependent methods and by the presence of multiple glycosidase-encoding genes in the genome. The 16S rRNA gene-based phylogenetic analysis demonstrated that strain ACPA22T formed a deep-branching lineage within the family Glycomycetaceae, with the highest sequence similarity value to Glycomyces buryatensis 18T (92.1 %) and Salininema proteolyticum Miq-4T (91.8 %). The average amino acid identity values (56.1-61.5 %) between ACPA22T and other Glycomycetaceae members with available genomes did not exceed the threshold reported for different genera. The cell wall of ACPA22T contained meso-diaminopimelic acid, glycine, glutamic acid and alanine in a molar ratio, characteristic of the peptidoglycan type A1γ'. The whole-cell sugars included mannose, galactose, arabinose, ribose and xylose. The major menaquinones were MK-10(Н4) and MK-11(Н4). The identified polar lipids were represented by phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides. In addition, the strain had a few unidentified characteristic polar lipids, including an amine-containing phospholipid with chromatographic mobility similar to that of phosphatidylinositol. The polar lipid fatty acids were dominated by anteiso-C17 : 0 and iso-C16 : 0. The genome included a chromosome of 3.94 Mbp (G+C content 61.5 mol%) encoding 3285 proteins and two plasmids of 59.8 and 14.8 kBp. Based on the data obtained in this study, a new genus and species, Natronoglycomyces albus gen. nov., sp. nov, is proposed with the type strain ACPA22T (=DSM 106290T=VKM Ac-2771T).

Streptomyces bathyalis sp. nov., an actinobacterium isolated from the sponge in a deep sea.

A novel actinobacterium, designated ASO4wetT, was isolated from the unidentified sponge (SO4) in the deep sea collected of the North Atlantic Ocean. Study of 16S rRNA gene sequences indicated that strain ASO4wetT is a member of the genus Streptomyces and showed the closest similarities to Streptomyces karpasiensis K413T (98.87 %), Streptomyces glycovorans YIM M 10366T (98.38 %), and Streptomyces abyssalis YIM M 10400T (97.53 %). Strain ASO4wetT contained MK-9(H8) as the predominant menaquinone and the major fatty acids are iso-C16:0, anteiso-C15:0, and iso-C15:0. Polyphasic taxonomy was carried out between strain ASO4wetT and its phylogenetically closely related Streptomyces strains, which further elucidated their relatedness and revealed that strain ASO4wetT could be distinguished from currently known Streptomyces species. Strain ASO4wetT clearly represents a novel species in genus Streptomyces. We propose the name Streptomyces bathyalis sp. nov., with the type strain ASO4wetT (= DSM 106605T = NCCB 100657T). Analysis of the whole-genome sequence of S. bathyalis revealed that genome size is 7,377,472 bp with 6332 coding sequences.

Synchronous termination of replication of the two chromosomes is an evolutionary selected feature in Vibrionaceae.

Vibrio cholerae, the causative agent of the cholera disease, is commonly used as a model organism for the study of bacteria with multipartite genomes. Its two chromosomes of different sizes initiate their DNA replication at distinct time points in the cell cycle and terminate in synchrony. In this study, the time-delayed start of Chr2 was verified in a synchronized cell population. This replication pattern suggests two possible regulation mechanisms for other Vibrio species with different sized secondary chromosomes: Either all Chr2 start DNA replication with a fixed delay after Chr1 initiation, or the timepoint at which Chr2 initiates varies such that termination of chromosomal replication occurs in synchrony. We investigated these two models and revealed that the two chromosomes of various Vibrionaceae species terminate in synchrony while Chr2-initiation timing relative to Chr1 is variable. Moreover, the sequence and function of the Chr2-triggering crtS site recently discovered in V. cholerae were found to be conserved, explaining the observed timing mechanism. Our results suggest that it is beneficial for bacterial cells with multiple chromosomes to synchronize their replication termination, potentially to optimize chromosome related processes as dimer resolution or segregation.

Discovery of Paenibacillus larvae ERIC V: Phenotypic and genomic comparison to genotypes ERIC I-IV reveal different inventories of virulence factors which correlate with epidemiological prevalences of American Foulbrood.

Paenibacillus larvae is the etiological agent of American Foulbrood (AFB), a highly contagious brood disease of honey bees (Apis mellifera). AFB requires mandatory reporting to the veterinary authority in many countries and until now four genotypes, P. larvae ERIC I-IV, have been identified. We isolated a new genotype, ERIC V, from a Spanish honey sample. After a detailed phenotypic comparison with the reference strains of the ERIC I-IV genotypes, including spore morphology, non-ribosomal peptide (NRP) profiling, and in vivo infections of A. mellifera larvae, we established a genomic DNA Macrorestriction Fragment Pattern Analysis (MRFPA) scheme for future epidemiologic discrimination. Whole genome comparison of the reference strains and the new ERIC V genotype (DSM 106052) revealed that the respective virulence gene inventories of the five genotypes corresponded with the time needed to kill 100 % of the infected bee larvae (LT100) in in vivo infection assays. The rarely isolated P. larvae genotypes ERIC II I-V with a fast-killing phenotype (LT100 3 days) harbor genes with high homology to virulence factors of other insect pathogens. These virulence genes are absent in the epidemiologically prevalent genotypes ERIC I (LT100 12 days) and ERIC II (LT100 7 days), which exhibit slower killing phenotypes. Since killing-retardation is known to reduce the success of hygienic cleaning by nurse bees, the identified absence of virulence factors might explain the epidemiological prevalences of ERIC genotypes. The discovery of the P. larvae ERIC V isolate suggests that more unknown ERIC genotypes exist in bee colonies. Since inactivation or loss of a few genes can transform a fast-killing phenotype into a more dangerous slow-killing phenotype, these rarely isolated genotypes may represent a hidden reservoir for future AFB outbreaks.

The transcription factor WRKY22 is required during cryo-stress acclimation in Arabidopsis shoot tips.

Storage of meristematic tissue at ultra-low temperatures offers a mean to maintain valuable genetic resources from vegetatively reproduced plants. To reveal the biology underlying cryo-stress, shoot tips of the model plant Arabidopsis thaliana were subjected to a standard preservation procedure. A transcriptomic approach was taken to describe the subsequent cellular events which occurred. The cryoprotectant treatment induced the changes in the transcript levels of genes associated with RNA processing and primary metabolism. Explants of a mutant lacking a functional copy of the transcription factor WRKY22 were compromised for recovery. A number of putative downstream targets of WRKY22 were identified, some related to phytohormone-mediated defense, to the osmotic stress response, and to development. There were also alterations in the abundance of transcript produced by genes encoding photosynthesis-related proteins. The wrky22 mutant plants developed an open stomata phenotype in response to their exposure to the cryoprotectant solution. WRKY22 probably regulates a transcriptional network during cryo-stress, linking the explant's defense and osmotic stress responses to changes in its primary metabolism. A model is proposed linking WRKY53 and WRKY70 downstream of the action of WRKY22.

Hypericibacter terrae gen. nov., sp. nov. and Hypericibacter adhaerens sp. nov., two new members of the family Rhodospirillaceae isolated from the rhizosphere of Hypericum perforatum.

Two strains of the family Rhodospirillaceae were isolated from the rhizosphere of the medicinal plant Hypericum perforatum. Cells of both strains were Gram-stain-negative, motile by means of a single polar flagellum, non-spore-forming, non-capsulated, short rods that divided by binary fission. Colonies were small and white. Strains R5913T and R5959T were oxidase-positive, mesophilic, neutrophilic and grew optimally without NaCl. Both grew under aerobic and microaerophilic conditions and on a limited range of substrates with best results on yeast extract. Major fatty acids were C19 : 0 cyclo ω8c and C16 : 0; in addition, C18 : 1ω7c was also found as a predominant fatty acid in strain R5913T. The major respiratory quinone was ubiquinone 10 (Q-10). The DNA G+C contents of strains R5913T and R5959T were 66.0 and 67.4 mol%, respectively. 16S rRNA gene sequence comparison revealed that the closest relatives (<92 % similarity) of the strains are Oceanibaculum pacificum MCCC 1A02656T, Dongia mobilis CGMCC 1.7660T, Dongia soli D78T and Dongia rigui 04SU4-PT. The two novel strains shared 98.6 % sequence similarity and represent different species on the basis of low average nucleotide identity of their genomes (83.8 %). Based on the combined phenotypic, genomic and phylogenetic investigations, the two strains represent two novel species of a new genus in the family Rhodospirillaceae, for which the name Hypericibacter gen. nov. is proposed, comprising the type species Hypericibacter terrae sp. nov. (type strain R5913T=DSM 109816T=CECT 9472T) and Hypericibacter adhaerens sp. nov. (type strain R5959T=DSM 109817T=CECT 9620T).

Genomics-Metabolomics Profiling Disclosed Marine Vibrio spartinae 3.6 as a Producer of a New Branched Side Chain Prodigiosin.

A wide range of prescreening tests for antimicrobial activity of 59 bacterial isolates from sediments of Ria Formosa Lagoon (Algarve, Portugal) disclosed Vibrio spartinae 3.6 as the most active antibacterial producing strain. This bacterial strain, which has not previously been submitted for chemical profiling, was subjected to de novo whole genome sequencing, which aided in the discovery and elucidation of a prodigiosin biosynthetic gene cluster that was predicted by the bioinformatic tool KEGG BlastKoala. Comparative genomics led to the identification of a new membrane di-iron oxygenase-like enzyme, annotated as Vspart_02107, which is likely to be involved in the biosynthesis of cycloprodigiosin and analogues. The combined genomics-metabolomics profiling of the strain led to the isolation and identification of one new branched-chain prodigiosin (5) and to the detection of two new cyclic forms. Furthermore, the evaluation of the minimum inhibitory concentrations disclosed the major prodigiosin as very effective against multi-drug-resistant pathogens including Stenotrophomonas maltophilia, a clinical isolate of Listeria monocytogenes, as well as some human pathogens reported by the World Health Organization as prioritized targets.

Genome and Methylome Variation in Helicobacter pylori With a cag Pathogenicity Island During Early Stages of Human Infection.

BACKGROUND & AIMS: Helicobacter pylori is remarkable for its genetic variation; yet, little is known about its genetic changes during early stages of human infection, as the bacteria adapt to their new environment. We analyzed genome and methylome variations in a fully virulent strain of H pylori during experimental infection. METHODS: We performed a randomized Phase I/II, observer-blind, placebo-controlled study of 12 healthy, H pylori-negative adults in Germany from October 2008 through March 2010. The volunteers were given a prophylactic vaccine candidate (n = 7) or placebo (n = 5) and then challenged with H pylori strain BCM-300. Biopsy samples were collected and H pylori were isolated. Genomes of the challenge strain and 12 reisolates, obtained 12 weeks after (or in 1 case, 62 weeks after) infection were sequenced by single-molecule, real-time technology, which, in parallel, permitted determination of genome-wide methylation patterns for all strains. Functional effects of genetic changes observed in H pylori strains during human infection were assessed by measuring release of interleukin 8 from AGS cells (to detect cag pathogenicity island function), neutral red uptake (to detect vacuolating cytotoxin activity), and adhesion assays. RESULTS: The observed mutation rate was in agreement with rates previously determined from patients with chronic H pylori infections, without evidence of a mutation burst. A loss of cag pathogenicity island function was observed in 3 reisolates. In addition, 3 reisolates from the vaccine group acquired mutations in the vacuolating cytotoxin gene vacA, resulting in loss of vacuolization activity. We observed interstrain variation in methylomes due to phase variation in genes encoding methyltransferases. CONCLUSIONS: We analyzed adaptation of a fully virulent strain of H pylori to 12 different volunteers to obtain a robust estimate of the frequency of genetic and epigenetic changes in the absence of interstrain recombination. Our findings indicate that the large amount of genetic variation in H pylori poses a challenge to vaccine development. ClinicalTrials.gov no: NCT00736476.

Comparison of VITEK 2, MALDI-TOF MS, 16S rRNA gene sequencing, and whole-genome sequencing for identification of Roseomonas mucosa.

The bacterial species Roseomonas mucosa is pathogenic in humans, and although it is rarely detected during routine diagnostics, it is becoming increasingly important clinically. For a long time, R. mucosa was regarded as a classic environmental bacterium. Recent studies, however, revealed that it is part of the physiological human skin flora and mainly affects immunocompromised patients. Furthermore, the use of catheter systems may increase the risk of contracting R. mucosa infections. The bacterium has been linked to severe infections, such as bacteraemia, osteomyelitis and cellulitis. Therefore, it is important to discern the best method of identifying R. mucosa in routine laboratory testing. To facilitate this testing, we compared three suitable methods for routine bacterial identification in the laboratory: VITEK 2, MALDI-TOF MS and 16S rRNA gene sequencing. Additionally, we conducted whole-genome sequencing (WGS) and calculated the average nucleotide identity (ANI). ANI is seen as the gold standard of strain identification; therefore, we decided to use it as a reference method. Both MALDI-TOF MS and 16S rRNA gene sequencing confidently identified the species. However, when using the VITEK 2 technique, isolates were misidentified as Roseomonas gilardii, Rhizobium radiobacter, or Sphingomonas paucimobilis. When conducting WGS and determining the ANI, it became obvious that one isolate belonged to the species R. gilardii rather than R. mucosa. Therefore (although not yet applicable in routine diagnostics), we suggest that WGS is presently the most appropriate technique to reliably identify Roseomonas mucosa. However, after expanding the Biotyper database, MALDI-TOF MS could also be an applicable method.

Leucobacter muris sp. nov., isolated from the nose of a laboratory mouse.

A Gram-stain-positive, rod-shaped, aerobic, non-motile, white, opaque bacterial isolate, designated 924/12T, was isolated from the nose of a laboratory mouse in Düsseldorf, Germany. The 16S rRNA gene sequence analyses indicated the phylogenetic position of the strain within the genus Leucobacter. Similarity levels over 97 % were recorded between the 16S rRNA gene sequence of strain 924/12T and the type strains of the species Leucobacter chironomi DSM 19883T (99.5 %), followed by Leucobacter celersubsp. astrifaciens CBX151T (97.6 %), Leucobacter celersubsp. celer NAL101T (97.5 %), 'Leucobacter kyeonggiensis' F3-P9 (97.5 %), Leucobacter zeae CC-MF41T (97.3 %), Leucobacter chromiiresistens JG31T (97.1 %), Leucobacter triazinivorans JW-1T (97.1 %), Leucobacter corticis 2 C-7T (97.0 %) and Leucobacter aridicolis CIP108388T (97.0 %). DNA-DNA hybridization and whole genomic comparison, mandatory to taxonomically separate strain 924/12T from the type strain of L. chironomi, revealed similarity values of 40.4 and 30.8 %, respectively, thus below the threshold of 70 % recommended differentiating between species. The cell-wall amino acids of the novel isolate were diaminobutyric acid, alanine, glycine, threonine and glutamic acid. The major fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, glycolipid and one unknown lipid, whereas the predominant menaquinones were MK-11 and MK-10. The genomic DNA G+C content of strain 924/12T was 70.6 mol%. Phylogenetic analyses based on the 16S rRNA gene sequences and the phenotypical differences between strain 924/12T and the other closely related type strains of the genus Leucobacter indicated that strain 924/12T represents a novel species within the genus Leucobacter, family Microbacteriaceae, for which the name Leucobacter muris sp. nov. is proposed. The type strain is 924/12T (=DSM 101948T=CCM 8761T).