A Salt-Tolerant Streptomyces Paradoxus D2-8 from Rhizosphere Soil of Phragmites Communis Augments Soybean Tolerance To Soda Saline-Alkali Stress

Soil salinity and alkalization limit plant growth and agricultural productivity worldwide. The application of salt-tolerant plant growth-promoting rhizobacteria (PGPR) effectively improved plant tolerance to saline-alkali stress. To obtain the beneficial actinomyces resources with salt tolerance, thirteen isolates were isolated from rhizosphere saline and alkaline soil of Phragmites communis. Among these isolates, D2-8 was moderately halophilic to NaCl and showed 120 mmol soda saline-alkali solution tolerance. Moreover, the plant growth-promoting test demonstrated that D2-8 produced siderophore, IAA, 1-aminocyclopropane-1-carboxylate deaminase (ACCD), and organic acids. D2-8 showed 99.4% homology with the type strain Streptomyces paradoxus NBRC 14887^T and shared the same branch, and, therefore, it was designated S. paradoxus D2-8. Its genome was sequenced to gain insight into the mechanism of growth-promoting and saline-alkali tolerance of D2-8. IAA and siderophore biosynthesis pathway, genes encoding ACC deaminase, together with six antibiotics biosynthesis gene clusters with antifungal or antibacterial activity, were identified. The compatible solute ectoine biosynthesis gene cluster, production, and uptake of choline and glycine betaine cluster in the D2-8 genome may contribute to the saline-alkali tolerance of the strain. Furthermore, D2-8 significantly promoted the seedling growth even under soda saline-alkali stress, and seed coating with D2-8 isolate increased by 5.88% of the soybean yield in the field. These results imply its significant potential to improve soybean soda saline-alkali tolerance and promote crop health in alkaline soil. Open in a separate window     

Genome sequences and description of novel exopolysaccharides producing species Komagataeibacter pomaceti sp. nov. and reclassification of Komagataeibacter kombuchae (Dutta and Gachhui 2007) Yamada et al., 2013 as a later heterotypic synonym of Komagataeibacter hansenii (Gosselé et al. 1983) Yamada et al., 2013

Strains T5K1 and AV446 isolated from apple cider vinegars during a submerged vinegar production in two separate vinegar facilities showed 94% 16S rRNA gene similarity to its closest neighbors Komagataeibacter maltaceti LMG 1529^T and Gluconacetobacter entanii LTH 4560^T. Further phylogenetic and phenotypic characterizations indicated that the isolates belonged to a novel species of the Komagataeibacter genus. Comparison based on 16S–23S rRNA gene ITS sequences and concatenated partial sequences of the housekeeping genes dnaK, groEL and rpoB, grouped both strains to a single phylogenetic cluster well separated from the other species of the Komagataeibacter genus. Average nucleotide identity of T5K1 and AV446 draft genome sequences compared to other Komagataeibacter type strains was below 94% and at the same time, in-silico DNA–DNA hybridization was below 70%. Both strains on the other hand showed approximately 98% (average nucleotide identity) and 87% (in silico DNA–DNA hybridization) similarity to each other. Strains T5K1 and AV446 can be differentiated from other Komagataeibacter type strains based on their ability to produce 2-keto-d-gluconic acid and at the same time inability to produce 5-keto-d-gluconic acid. Furthermore, strains of the new species do not grow on Asai medium supplemented with d-glucose or d-mannitol. The growth is also absent (T5K1) or weak (AV446) on Hoyer–Frateur medium supplemented with afore mentioned sugars. Both strains produce cellulose. In addition, draft genome analysis revealed that strains T5K1 and AV446 possess genes involved in the synthesis of acetan-like extracellular heteropolysaccharide. We propose the name Komagataeibacter pomaceti sp. nov. for the new species with LMG 30150^T [= CCM 8723^T = ZIM B1029^T] as the type strain. Data collected in this study and in a previous study also revealed that Komagataeibacter kombuchae is a later heterotypic synonym of Komagataeibacter hansenii.     

Influence of D genome of wheat on expression of novel type spike branching in hybrid populations of 171ACS line

A 171ACS line (AABBDD, 2n = 6x = 42) has been crossed with the tetra-(AABB and AAGG, 2n = 4x = 28) and octoploid (AAAABBGG, 2n = 8x = 56) wheat species without the D genome, as well as with hexaploid (AABBDD and AAGGDD, 2n = 6x = 42) wheat species and tetra-(AADD, 2n = 4x = 28) and hexaploid (AADDSS, 2n = 6x = 42) amphidiploids that have the D genome. The inheritance of a novel type of spike branching in these obtained hybrid populations F₁–F₃ was studied. According to the results of a morphogenetic analysis of hybrid populations derived from crossings between 171ACS and wheat species without the D genome, the novel type of branching was found to be controlled by a single recessive gene (although a phenotype of the 171ACS line gives a handle for a doubt about occurrence of the second gene) and the 171ACS line is a source of the novel type branching. However, not a single branched spike plant was observed in hybrid populations that were produced by crosses of the 171ACS line with wheat species, as well as with amphidiploids that have the D genome. This result also experimentally confirmed the inhibitor effect of chromosomes of the D genome on the expression of the spike-branching trait. The appearance of branched-spike forms, together with normal spiked plants in hybrid populations of the 171ACS line and T. araraticum Jakubz. (AAGG) or T. fungicidum Zhuk. (AAAABBGG) confirmed that, as opposed to the D genome, neither genome G nor genome B demonstrated the inhibition of the expression of the spikebranching trait. In conclusion, keeping in mind that branching is exhibited in hybrid progenies obtained from crosses between the 171ACS line and wheat species with AABB and AAGG genomes, it can be said that this gene belongs to the A genome.     

Modestobacter excelsi sp. nov., a novel actinobacterium isolated from a high altitude Atacama Desert soil

A polyphasic study was undertaken to establish the taxonomic status of three Modestobacter strains isolated from a high altitude Atacama Desert soil. The isolates, strains 1G6^T, 1G14 and 1G50, showed chemotaxonomic and morphological properties characteristic of members of the genus Modestobacter. The peptidoglycan contained meso-diaminopimelic acid, the whole cell sugars were glucose and ribose (diagnostic sugars) and arabinose, the predominant menaquinone was MK-9(H₄), polar lipid patterns contained diphosphatidylglycerol, glycophosphatidylinositol, phosphatidylethanolamine (diagnostic component), phosphatidylglycerol and phosphatidylinositol while whole cellular fatty acid profiles consisted of complex mixtures of saturated, unsaturated iso- and anteiso-components. The isolates were shown to have different BOX-PCR fingerprint and physiological profiles. They formed a distinct phyletic line in Modestobacter 16S rRNA gene trees, were most closely related to the type strain of Modestobacter italicus (99.9 % similarity) but were distinguished from this and other closely related Modestobacter type strains using a combination of phenotypic properties. Average nucleotide identity and digital DNA:DNA hybridization similarities between the draft genome sequences of isolate 1G6^T and M. italicus BC 501^T were 90.9 % and 42.3 %, respectively, indicating that they belong to different species. Based on these phenotypic and genotypic data it is proposed that the isolates be assigned to a novel species in the genus Modestobacter, namely as Modestobacter excelsi with isolate 1G6^T (=DSM 107535^T =PCM 3004^T) as the type strain. Analysis of the whole genome sequence of M. excelsi 1G6^T (genome size of 5.26 Mb) showed the presence of genes and gene clusters that encode for properties that are in tune with its adaptation to extreme environmental conditions that prevail in the Atacama Desert biome.     

Extended-Spectrum-β-Lactamase-Producing Enterobacteriaceae Isolated from Vegetables Imported from the Dominican Republic,India, Thailand,and Vietnam

To examine to what extent fresh vegetables imported into Switzerland represent carriers of extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae, 169 samples of different types of fresh vegetables imported into Switzerland from the Dominican Republic, India, Thailand, and Vietnam were analyzed. Overall, 25.4% of the vegetable samples yielded one or more ESBL-producing Enterobacteriaceae, 78.3% of which were multidrug resistant. Sixty isolates were obtained: Escherichia coli, 26; Klebsiella pneumoniae, 26; Enterobacter cloacae, 6; Enterobacter aerogenes, 1; and Cronobacter sakazakii, 1. We found 29 isolates producing CTX-M-15, 8 producing CTX-M-14, 7 producing CTX-M-55, 3 producing CTX-M-65, 1 each producing CTX-M-1, CTX-M-3, CTX-M-27, and CTX-M-63, 5 producing SHV-2, 3 producing SHV-12, and 1 producing SHV-2a. Four of the E. coli isolates belonged to epidemiologically important clones: CTX-M-15-producing B2:ST131 (1 isolate), D:ST405 (1 isolate), and D:ST38 (2 isolates). One of the D:ST38 isolates belonged to the extraintestinal enteroaggregative E. coli (EAEC) D:ST38 lineage. Two of the K. pneumoniae isolates belonged to the epidemic clones sequence type 15 (ST15) and ST147. The occurrence of antibiotic-resistant pathogenic and commensal Enterobacteriaceae in imported agricultural foodstuffs constitutes a source of ESBL genes and a concern for food safety.     

Mating Type Gene (MAT1-2) of Trichophyton verrucosum

Trichophyton verrucosum is a zoophilic dermatophyte species that is the most frequent etiologic agent of bovine dermatophytosis throughout the world. Since no teleomorph of T. verrucosum has been found, polymerase chain reaction (PCR) analysis on the genome of T. verrucosum isolated from the Czech Republic and Japan was performed to confirm the presence of a mating type locus in the genome of the fungus and to clarify its classification and ecological characteristics. The mating type gene (MAT1-2) allele was detected by PCR analysis in all 22 isolates (four isolates from the Czech Republic and 18 isolates from Japan). The nucleotide sequence of the region exhibited 99–100 % identity among all isolates, including the reference strain of T. verrucosum. Phylogenetic analysis revealed that the sequences of the internal transcribed spacer region at the MAT1-2 locus clustered together in the isolates examined, forming a branch distinct from that of the other dermatophyte species. These results suggest that T. verrucosum is a clonal offshoot that has drifted away from Arthroderma benhamiae.     

Sequence analysis of sub-genotype D hepatitis B surface antigens isolated from Jeddah,Saudi Arabia

Little is known about the prevalence of HBV genotypes/sub-genotypes in Jeddah province, although the hepatitis B virus (HBV) was identified as the most predominant type of hepatitis in Saudi Arabia. To characterize HBV genotypes/sub-genotypes, serum samples from 15 patients with chronic HBV were collected and subjected to HBsAg gene amplification and sequence analysis. Phylogenetic analysis of the HBsAg gene sequences revealed that 11 (48%) isolates belonged to HBV/D while 4 (18%) were associated with HBV/C. Notably, a HBV/D sub-genotype phylogenetic tree identified that eight current isolates (72%) belonged to HBV/D1, whereas three isolates (28%) appeared to be more closely related to HBV/D5, although they formed a novel cluster supported by a branch with 99% bootstrap value. Isolates belonging to D1 were grouped in one branch and seemed to be more closely related to various strains isolated from different countries. For further determination of whether the three current isolates belonged to HBV/D5 or represented a novel sub-genotype, HBV/DA, whole HBV genome sequences would be required. In the present study, we verified that HBV/D1 is the most prevalent HBV sub-genotype in Jeddah, and identified novel variant mutations suggesting that an additional sub-genotype designated HBV/DA should be proposed. Overall, the results of the present HBsAg sequence analyses provide us with insights regarding the nucleotide differences between the present HBsAg/D isolates identified in the populace of Jeddah, Saudi Arabia and those previously isolated worldwide. Additional studies with large numbers of subjects in other areas might lead to the discovery of the specific HBV strain genotypes or even additional new sub-genotypes that are circulating in Saudi Arabia.     

The Global Trade in Fresh Produce and the Vagility of Plant Viruses: A Case Study in Garlic

As cuisine becomes globalized, large volumes of fresh produce are traded internationally. The potential exists for pathogens infecting fresh produce to hitchhike to new locations and perhaps to establish there. It is difficult to identify them using traditional methods if pathogens are novel, scarce, and/or unexpected. In an attempt to overcome this limitation, we used high-throughput sequencing technology as a means of detecting all RNA viruses infecting garlic (Allium sativum L.) bulbs imported into Australia from China, the USA, Mexico, Argentina and Spain, and those growing in Australia. Bulbs tested were grown over multiple vegetative generations and all were stably infected with one or more viruses, including two species not previously recorded in Australia. Present in various combinations from 10 garlic bulbs were 41 virus isolates representing potyviruses (Onion yellow dwarf virus, Leek yellow stripe virus), carlaviruses (Shallot latent virus, Garlic common latent virus) and allexiviruses (Garlic virus A, B, C, D, and X), for which 19 complete and 22 partial genome sequences were obtained, including the first complete genome sequences of two isolates of GarVD. The most genetically distinct isolates of GarVA and GarVX described so far were identified from Mexico and Argentina, and possible scenarios explaining this are presented. The complete genome sequence of an isolate of the potexvirus Asparagus virus 3 (AV3) was obtained in Australia from wild garlic (A. vineale L.), a naturalized weed. This is first time AV3 has been identified from wild garlic and the first time it has been identified beyond China and Japan. The need for routine generic diagnosis and appropriate legislation to address the risks to primary production and wild plant communities from pathogens spread through the international trade in fresh produce is discussed.     

Mitochondrial genome sequence diversity of Indian Plasmodium falciparum isolates

We have analysed the whole mitochondrial (mt) genome sequences (each ~6 kilo nucleotide base pairs in length) of four field isolates of the malaria parasite Plasmodium falciparum collected from different locations in India. Comparative genomic analyses of mt genome sequences revealed three novel India-specific single nucleotide polymorphisms. In general, high mt genome diversity was found in Indian P. falciparum, at a level comparable to African isolates. A population phylogenetic tree placed the presently sequenced Indian P. falciparum with the global isolates, while a previously sequenced Indian isolate was an outlier. Although this preliminary study is limited to a few numbers of isolates, the data have provided fundamental evidence of the mt genome diversity and evolutionary relationships of Indian P. falciparum with that of global isolates.     

Comparative Genomics of a Helicobacter pylori Isolate from a Chinese Yunnan Naxi Ethnic Aborigine Suggests High Genetic Divergence and Phage Insertion

Helicobacter pylori is a common pathogen correlated with several severe digestive diseases. It has been reported that isolates associated with different geographic areas, different diseases and different individuals might have variable genomic features. Here, we describe draft genomic sequences of H. pylori strains YN4-84 and YN1-91 isolated from patients with gastritis from the Naxi and Han populations of Yunnan, China, respectively. The draft sequences were compared to 45 other publically available genomes, and a total of 1059 core genes were identified. Genes involved in restriction modification systems, type four secretion system three (TFS3) and type four secretion system four (TFS4), were identified as highly divergent. Both YN4-84 and YN1-91 harbor intact cag pathogenicity island (cagPAI) and have EPIYA-A/B/D type at the carboxyl terminal of cagA. The vacA gene type is s1m2i1. Another major finding was a 32.5-kb prophage integrated in the YN4-84 genome. The prophage shares most of its genes (30/33) with Helicobacter pylori prophage KHP30. Moreover, a 1,886 bp transposable sequence (IS605) was found in the prophage. Our results imply that the Naxi ethnic minority isolate YN4-84 and Han isolate YN1-91 belong to the hspEAsia subgroup and have diverse genome structure. The genome has been extensively modified in several regions involved in horizontal DNA transfer. The important roles played by phages in the ecology and microevolution of H. pylori were further emphasized. The current data will provide valuable information regarding the H. pylori genome based on historic human migrations and population structure.     

Detection,isolation and characterization of Fusobacterium gastrosuis sp. nov. colonizing the stomach of pigs

Nine strains of a novel Fusobacterium sp. were isolated from the stomach of 6–8 months old and adult pigs. The isolates were obligately anaerobic, although they endured 2 h exposure to air. Phylogenetic analysis based on 16S rRNA and gyrase B genes demonstrated that the isolates showed high sequence similarity with Fusobacterium mortiferum, Fusobacterium ulcerans, Fusobacterium varium, Fusobacterium russii and Fusobacterium necrogenes, but formed a distinct lineage in the genus Fusobacterium. Comparative analysis of the genome of the type strain of this novel Fusobacterium sp. confirmed that it is different from other recognized Fusobacterium spp. DNA-DNA hybridization, fingerprinting and genomic %GC determination further supported the conclusion that the isolates belong to a new, distinct species. The isolates were also distinguishable from these and other Fusobacterium spp. by phenotypical characterization. The strains produced indole and exhibited proline arylamidase and glutamic acid decarboxylase activity. They did not hydrolyse esculin, did not exhibit pyroglutamic acid arylamidase, valine arylamidase, α-galactosidase, β-galactosidase, β-galactosidase-6-phosphate or α-glucosidase activity nor produced acid from cellobiose, glucose, lactose, mannitol, mannose, maltose, raffinose, saccharose, salicin or trehalose. The major fatty acids were C16:0 and C18:1ω9c. The name Fusobacterium gastrosuis sp. nov. is proposed for the novel isolates with the type strain CDW1(T) (=DSM 101753(T) = LMG 29236(T)). We also demonstrated that Clostridium rectum and mortiferum Fusobacterium represent the same species, with nomenclatural priority for the latter.     

Characterization of the genomes of a diverse collection of Salmonella enterica serovar Typhimurium definitive phage type 104

Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) has caused significant morbidity and mortality in humans and animals for almost three decades. We completed the full DNA sequence of one DT104 strain, NCTC13348, and showed that significant differences between the genome of this isolate and the genome of the previously sequenced strain Salmonella serovar Typhimurium LT2 are due to integrated prophage elements and Salmonella genomic island 1 encoding antibiotic resistance genes. Thirteen isolates of Salmonella serovar Typhimurium DT104 with different pulsed-field gel electrophoresis (PFGE) profiles were analyzed by using multilocus sequence typing (MLST), plasmid profiling, hybridization to a pan-Salmonella DNA microarray, and prophage-based multiplex PCR. All the isolates belonged to a single MLST type, sequence type ST19. Microarray data demonstrated that the gene contents of the 13 DT104 isolates were remarkably conserved. The PFGE DNA fragment size differences in these isolates could be explained to a great extent by differences in the prophage and plasmid contents. Thus, here the nature of variation in different Salmonella serovar Typhimurium DT104 isolates is further defined at the gene and whole-genome levels, illustrating how this phage type evolves over time.     

Population genetics and microevolution of clinical Candida glabrata reveals recombinant sequence types and hyper-variation within mitochondrial genomes,virulence genes,and drug targets

Candida glabrata is the second most common etiological cause of worldwide systemic candidiasis in adult patients. Genome analysis of 68 isolates from 8 hospitals across Scotland, together with 83 global isolates, revealed insights into the population genetics and evolution of C. glabrata. Clinical isolates of C. glabrata from across Scotland are highly genetically diverse, including at least 19 separate sequence types that have been recovered previously in globally diverse locations, and 1 newly discovered sequence type. Several sequence types had evidence for ancestral recombination, suggesting transmission between distinct geographical regions has coincided with genetic exchange arising in new clades. Three isolates were missing MATα1, potentially representing a second mating type. Signatures of positive selection were identified in every sequence type including enrichment for epithelial adhesins thought to facilitate fungal adhesin to human epithelial cells. In patent microevolution was identified from 7 sets of recurrent cases of candidiasis, revealing an enrichment for nonsynonymous and frameshift indels in cell surface proteins. Microevolution within patients also affected epithelial adhesins genes, and several genes involved in drug resistance including the ergosterol synthesis gene ERG4 and the echinocandin target FKS1/2, the latter coinciding with a marked drop in fluconazole minimum inhibitory concentration. In addition to nuclear genome diversity, the C. glabrata mitochondrial genome was particularly diverse, with reduced conserved sequence and conserved protein-encoding genes in all nonreference ST15 isolates. Together, this study highlights the genetic diversity within the C. glabrata population that may impact virulence and drug resistance, and 2 major mechanisms generating this diversity: microevolution and genetic exchange/recombination.     

Novel species of Frankia,Frankia gtarii sp. nov. and Frankia tisai sp. nov., isolated from a root nodule of Alnus glutinosa

The status of four Frankia strains isolated from a root nodule of Alnus glutinosa was established in a polyphasic study. Taxogenomics and phenotypic features show that the isolates belong to the genus Frankia. All four strains form extensively branched substrate mycelia, multilocular sporangia, vesicles, lack aerial hyphae, but contain meso-diaminopimelic acid as the diamino acid of the peptidoglycan, galactose, glucose, mannose, ribose, xylose and traces of rhamnose as cell wall sugars, iso-C_16:0 as the predominant fatty acid, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol as the major polar lipids, have comparable genome sizes to other cluster 1, Alnus-infective strains with structural and accessory genes associated with nitrogen fixation. The genome sizes of the isolates range from 7.0 to 7.7 Mbp and the digital DNA G + C contents from 71.3 to 71.5 %. The four sequenced genomes are rich in biosynthetic gene clusters predicted to express for novel specialized metabolites, notably antibiotics. 16S rRNA gene and whole genome sequence analyses show that the isolates fall into two lineages that are closely related to the type strains of Frankia alni and Frankia torreyi. All of these taxa are separated by combinations of phenotypic properties and by digital DNA:DNA hybridization scores which indicate that they belong to different genomic species. Based on these results, it is proposed that isolates Agncl-4^T and Agncl-10, and Agncl-8^T and Agncl-18, be recognised as Frankia gtarii sp. nov. and Frankia tisai sp. nov. respectively, with isolates Agncl-4^T (=DSM 107976^T = CECT 9711^T) and Agncl-8^T (=DSM 107980^T = CECT 9715^T) as the respective type strains.     

Genome analysis and CRISPR typing of Salmonella enterica serovar Virchow

Background

Salmonella enterica subsp. enterica serovar Virchow has been recognized as a significant health burden in Asia, Australia and Europe. In addition to its global distribution, S. Virchow is clinically significant due to the frequency at which it causes invasive infections and its association with outbreaks arising from food-borne transmission. Here, we examine the genome of an invasive isolate of S. Virchow SVQ1 (phage type 8) from an outbreak in southeast Queensland, Australia. In addition to identifying new potential genotyping targets that could be used for discriminating between S. Virchow strains in outbreak scenarios, we also aimed to carry out a comprehensive comparative analysis of the S. Virchow genomes.

Results

Genome comparisons between S. Virchow SVQ1 and S. Virchow SL491, a previously published strain, identified a high degree of genomic similarity between the two strains with fewer than 200 single nucleotide differences. Clustered Regularly Interspaced Palindromic Repeats (CRISPR) regions were identified as a highly variable region that could be used to discriminate between S. Virchow isolates. We amplified and sequenced the CRISPR regions of fifteen S. Virchow isolates collected from seven different outbreaks across Australia. We observed three allelic types of the CRISPR region from these isolates based on the presence/absence of the spacers and were able to discriminate S. Virchow phage type 8 isolates originating from different outbreaks. A comparison with 27 published Salmonella genomes found that the S. Virchow SVQ1 genome encodes 11 previously described Salmonella Pathogenicity Islands (SPI), as well as additional genomic islands including a remnant integrative conjugative element that is distinct from SPI-7. In addition, the S. Virchow genome possesses a novel prophage that encodes the Type III secretion system effector protein SopE, a key Salmonella virulence factor. The prophage shares very little similarity to the SopE prophages found in other Salmonella serovars suggesting an independent acquisition of sopE.

Conclusions

The availability of this genome will serve as a genome template and facilitate further studies on understanding the virulence and global distribution of the S. Virchow serovar, as well as the development of genotyping methods for outbreak investigations.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-389) contains supplementary material, which is available to authorized users.     

Draft Genome Sequences of Two Campylobacter jejuni Clinical Isolates,NW and D2600

The Campylobacter jejuni human clinical isolates NW and D2600 colonized C57BL/6 interleukin-10-deficient (IL-10^−/−) mice without inducing a robust inflammatory response (J. A. Bell et al., BMC Microbiol. 9:57, 2009). We announce draft genome sequences of NW and D2600 to facilitate comparisons with strains that induce gastrointestinal inflammation in this mouse model.     

Comprehensive molecular,genomic and phenotypic analysis of a major clone of Enterococcus faecalis MLST ST40

Background

Enterococcus faecalis is a multifaceted microorganism known to act as a beneficial intestinal commensal bacterium. It is also a dreaded nosocomial pathogen causing life-threatening infections in hospitalised patients. Isolates of a distinct MLST type ST40 represent the most frequent strain type of this species, distributed worldwide and originating from various sources (animal, human, environmental) and different conditions (colonisation/infection). Since enterococci are known to be highly recombinogenic we determined to analyse the microevolution and niche adaptation of this highly distributed clonal type.

Results

We compared a set of 42 ST40 isolates by assessing key molecular determinants, performing whole genome sequencing (WGS) and a number of phenotypic assays including resistance profiling, formation of biofilm and utilisation of carbon sources. We generated the first circular closed reference genome of an E. faecalis isolate D32 of animal origin and compared it with the genomes of other reference strains. D32 was used as a template for detailed WGS comparisons of high-quality draft genomes of 14 ST40 isolates. Genomic and phylogenetic analyses suggest a high level of similarity regarding the core genome, also demonstrated by similar carbon utilisation patterns. Distribution of known and putative virulence-associated genes did not differentiate between ST40 strains from a commensal and clinical background or an animal or human source. Further analyses of mobile genetic elements (MGE) revealed genomic diversity owed to: (1) a modularly structured pathogenicity island; (2) a site-specifically integrated and previously unknown genomic island of 138 kb in two strains putatively involved in exopolysaccharide synthesis; and (3) isolate-specific plasmid and phage patterns. Moreover, we used different cell-biological and animal experiments to compare the isolate D32 with a closely related ST40 endocarditis isolate whose draft genome sequence was also generated. D32 generally showed a greater capacity of adherence to human cell lines and an increased pathogenic potential in various animal models in combination with an even faster growth in vivo (not in vitro).

Conclusion

Molecular, genomic and phenotypic analysis of representative isolates of a major clone of E. faecalis MLST ST40 revealed new insights into the microbiology of a commensal bacterium which can turn into a conditional pathogen.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1367-x) contains supplementary material, which is available to authorized users.     

Comparative Genome Analysis Provides Insights into the Pathogenicity of Flavobacterium psychrophilum

Flavobacterium psychrophilum is a fish pathogen in salmonid aquaculture worldwide that causes cold water disease (CWD) and rainbow trout fry syndrome (RTFS). Comparative genome analyses of 11 F. psychrophilum isolates representing temporally and geographically distant populations were used to describe the F. psychrophilum pan-genome and to examine virulence factors, prophages, CRISPR arrays, and genomic islands present in the genomes. Analysis of the genomic DNA sequences were complemented with selected phenotypic characteristics of the strains. The pan genome analysis showed that F. psychrophilum could hold at least 3373 genes, while the core genome contained 1743 genes. On average, 67 new genes were detected for every new genome added to the analysis, indicating that F. psychrophilum possesses an open pan genome. The putative virulence factors were equally distributed among isolates, independent of geographic location, year of isolation and source of isolates. Only one prophage-related sequence was found which corresponded to the previously described prophage 6H, and appeared in 5 out of 11 isolates. CRISPR array analysis revealed two different loci with dissimilar spacer content, which only matched one sequence in the database, the temperate bacteriophage 6H. Genomic Islands (GIs) were identified in F. psychrophilum isolates 950106-1/1 and CSF 259–93, associated with toxins and antibiotic resistance. Finally, phenotypic characterization revealed a high degree of similarity among the strains with respect to biofilm formation and secretion of extracellular enzymes. Global scale dispersion of virulence factors in the genomes and the abilities for biofilm formation, hemolytic activity and secretion of extracellular enzymes among the strains suggested that F. psychrophilum isolates have a similar mode of action on adhesion, colonization and destruction of fish tissues across large spatial and temporal scales of occurrence. Overall, the genomic characterization and phenotypic properties may provide new insights to the mechanisms of pathogenicity in F. psychrophilum.     

Organization of the cpe Locus in CPE-Positive Clostridium perfringens Type C and D Isolates

Clostridium perfringens enterotoxin (encoded by the cpe gene) contributes to several important human, and possibly veterinary, enteric diseases. The current study investigated whether cpe locus organization in type C or D isolates resembles one of the three (one chromosomal and two plasmid-borne) cpe loci commonly found amongst type A isolates. Multiplex PCR assays capable of detecting sequences in those type A cpe loci failed to amplify products from cpe-positive type C and D isolates, indicating these isolates possess different cpe locus arrangements. Therefore, restriction fragments containing the cpe gene were cloned and sequenced from two type C isolates and one type D isolate. The obtained cpe locus sequences were then used to construct an overlapping PCR assay to assess cpe locus diversity amongst other cpe-positive type C and D isolates. All seven surveyed cpe-positive type C isolates had a plasmid-borne cpe locus partially resembling the cpe locus of type A isolates carrying a chromosomal cpe gene. In contrast, all eight type D isolates shared the same plasmid-borne cpe locus, which differed substantially from the cpe locus present in other C. perfringens by containing two copies of an ORF with 67% identity to a transposase gene (COG4644) found in Tn1546, but not previously associated with the cpe gene. These results identify greater diversity amongst cpe locus organization than previously appreciated, providing new insights into cpe locus evolution. Finally, evidence for cpe gene mobilization was found for both type C and D isolates, which could explain their cpe plasmid diversity.