Fructobacillus spp. in fructose-rich niches belong to the family Leuconostocaceae. They were originally classified as Leuconostoc spp., but were later grouped into a novel genus, Fructobacillus, based on their phylogenetic position, morphology and specific biochemical characteristics. The unique characters, so called fructophilic characteristics, had not been reported in the group of lactic acid bacteria, suggesting unique evolution at the genome level. Here we studied four draft genome sequences of Fructobacillus spp. and compared their metabolic properties against those of Leuconostoc spp.Fructobacillus species possess significantly less protein coding sequences in their small genomes. The number of genes was significantly smaller in carbohydrate transport and metabolism. Several other metabolic pathways, including TCA cycle, ubiquinone and other terpenoid-quinone biosynthesis and phosphotransferase systems, were characterized as discriminative pathways between the two genera. The adhE gene for bifunctional acetaldehyde/alcohol dehydrogenase, and genes for subunits of the pyruvate dehydrogenase complex were absent in Fructobacillus spp. The two genera also show different levels of GC contents, which are mainly due to the different GC contents at the third codon position.Fructobacillus spp. suggest reductive evolution that took place to adapt to specific niches.The present genome characteristics in The online version of this article (doi:10.1186/s12864-015-2339-x) contains supplementary material, which is available to authorized users. Fructobacillus spp. in the family Leuconostocaceae are found in fructose-rich environments such as flowers, (fermented) fruits, or bee guts, and are characterized as fructophilic lactic acid bacteria (FLAB)  was characterized as Fructobacillus-specific whereas adhE gene coding bifunctional acetaldehyde/alcohol dehydrogenase [EC1.2.1.10 1.1.1.1] was characterized as Leuconostoc-specific. There was no alternative acetaldehyde dehydrogenase gene in Fructobacillus. These results are consistent with our previous study reporting the lack of adhE gene and acetaldehyde dehydrogenase activity in Fructobacillus spp. [Interestingly the lus spp.  and theilus spp. , 8. No plus spp. , 6, 9.Fructobacillus-specific H under the presence of oxygen. This helps to keep the NAD(P)/NAD(P)H balance, since their sugar metabolism produces imbalance in NAD(P)/NAD(P)H cycling as described above. Indeed, Fructobacillus spp. can be easily differentiated from Leuconostoc spp. based on the reaction to oxygen [Fructobacillus spp. grew well under aerobic conditions but poorly so under anaerobic conditions on GYP medium H dehydrogenase gene was found as o oxygen . In our ium Fig. . PresencFructobacillus, but were found in Leuconostoc. Fructobacillus also lack TCA cycle genes. This suggests that, in Fructobacillus, pyruvate produced from the phosphoketolase pathway is not dispatched to the TCA cycle but metabolized to lactate by lactate dehydrogenase. The lack of pyruvate dehydrogenase complex was also reported in Lactobacillus kunkeei [Genes for subunits of the pyruvate dehydrogenase complex were undetected in the genomes of  kunkeei , which i kunkeei , 36.Fructobacillus-specific  in this study. A number of genes coding peptidases and amino acids transport/synthesis/metabolism were also found as Leuconostoc-specific genes (Additional file Leuconostoc spp. can survive various environments with different amino acid compositions. Several PTS related genes and genes for teichoic acid transport were also characterized as Leuconostoc-specific. LAB cells usually contain two distinct types of teichoic acid, which are wall teichoic acid and lipoteichoic acid. The identified genes are involved in biosynthesis of wall teichoic acid in Bacillus subtilis [Leuconostoc spp. and none in Fructobacillus spp.The levansucrase gene was also characterized as reported , 8, and ependent , and dexsubtilis . Few stuFructobacillus spp. and Leuconostoc spp., a phylogenetic tree was produced based on concatenated sequences of 233 orthologous genes which were conserved as a single copy within the tested strains. The tree showed a clear separation of the two genera (Fig. Fructobacillus spp. have distinct phylogenetic position from Leuconostoc spp. This agrees well with the previous reports using 16S rRNA gene or house-keeping genes [To confirm the phylogenetic relationship between era Fig. , indicatng genes , 8.Fig. Fructobacillus spp. and Leuconostoc spp. Fructobacillus spp. possess smaller numbers of CDS in smaller genomes compared to Leuconostoc spp. This is mainly due to the absence of carbohydrate metabolic systems. Similar genomic characteristics have been reported for L. kunkeei [Genome-based analysis on conserved genes and metabolic characteristics clearly indicated the distinction between  kunkeei , a membe kunkeei –12, and


Pinus elliottii. The phospholipid fatty acids (PLFA) combined with community level physiological profiles (CLPP) method was used to assess these characteristics in the rhizospheric soils of P. elliottii. We found that the soil microbial communities were significantly different among different stand ages of P. elliottii plantations. The PLFA analysis indicated that the bacterial biomass was higher than the actinomycic and fungal biomass in all stand ages. However, the bacterial biomass decreased with the increasing stand ages, while the fungal biomass increased. The four maximum biomarker concentrations in rhizospheric soils of P. elliottii for all stand ages were 18:1ω9c, 16:1ω7c, 18:3ω6c  and cy19:0, representing measures of fungal and gram negative bacterial biomass. In addition, CLPP analysis revealed that the utilization rate of amino acids, polymers, phenolic acids, and carbohydrates of soil microbial community gradually decreased with increasing stand ages, though this pattern was not observed for carboxylic acids and amines. Microbial community diversity, as determined by the Simpson index, Shannon-Wiener index, Richness index and McIntosh index, significantly decreased as stand age increased. Overall, both the PLFA and CLPP illustrated that the long-term pure plantation pattern exacerbated the microecological imbalance previously described in the rhizospheric soils of P. elliottii, and markedly decreased the soil microbial community diversity and metabolic activity. Based on the correlation analysis, we concluded that the soil nutrient and C/N ratio most significantly contributed to the variation of soil microbial community structure and metabolic activity in different stand ages of P. elliottii plantations.Soil microbes play an essential role in the forest ecosystem as an active component. This study examined the hypothesis that soil microbial community structure and metabolic activity would vary with the increasing stand ages in long-term pure plantations of  Pinus elliottii is a tree species native to the southeastern United States, and has been widely planted in China since 1980s due to its forage characteristics of high yield and fast growth [P. elliottii have attracted an increasing level of interest from many ecologists and foresters [Cunninghamia lanceolata, Larix gmelinii, Pinus massoniana, Eucalyptus spp., and Populus spp. [P. elliottii continuous pure plantations, including their growth regulation, biomass, and allelopathy [P. elliottii long-term pure plantation patterns on the below-ground soil microbial community has not yet been reported.t growth . Howeveroresters . Previouoresters . This phlus spp. –9. It halus spp. . In addilus spp. . Due to elopathy –13. Howeet al. discovered that without any soil-health management practices, long-term safflower (Carthamus tinctorius) plantations resulted in a shift of soil fertility from high (bacteria dominant) to low (fungi dominant) over time [et al. found that the amount of fungal biomass increased markedly in Chinese fir plantations over successive rotations [Pinus elliottii plantations.Soil microbes play an active and essential role in the forest ecosystem . The comver time . Li et aotations . Furtherotations . The vegotations , 19. HowP. elliottii. The goal of this study was to answer two questions: (1) how different stand ages of P. elliottii plantations affect soil microbial community structure and metabolic activity? and (2) What are the possible environmental factors leading to these differences? For these objectives, PLFA combined with CLPP methods were used to detect the variation of soil microbial community structure and metabolic activity in P. elliottii plantations. Our study will help to further understand ecological linkages between plant diversity aboveground and microbial diversity underground, and will therefore facilitate the establishment of scientific-based, effective management to achieve better ecosystem balance in man-made forests.To describe the composition of the microbial communities in forest soils, culture-independent methods have been widely applied. Compared to other methods, phospholipids fatty acid analysis (PLFA) and community level physiological profiles (CLPP) methods are quantitative, have a relatively high throughput, and allow rapid analyses for the high number of samples needed for field-based microbial ecology investigations. In this study, we addressed the hypotheses that soil microbial community structure and metabolic activity would vary with increasing stand ages in pure plantations of This study has been approved by the Fujian Agriculture and Forestry University, which takes care of the planning and protecting of Xiqin Forest Farm. The study did not involve any endangered or protected species. All the data in this study can be published and shared.2 forested area in the subtropical monsoon region. This is one of the earliest P. elliottii plantations in China. The annual mean temperature is 19.4°C with an extreme high and low temperature of 39.0°C and -0.6°C respectively. Annual mean relative humidity is 80.2%, and the region has an average of 84 fog days per year. Annual precipitation reaches 1817.0 mm, most of which (57.56%) falls in summer (May through August). The soil is dominated by yellow-red soil.The experiment was conducted at the Xiqin Forest Farm , Fujian Province, South China, an 11.6 kmP. elliottii plantations aged 3, 10, 18 and 25 years and established in 2011, 2004, 1996 and 1989, respectively, were selected for our study. We established three 20 m×20 m soil sampling plots for each stand age in September 2014. Sampling plots were selected to have similar environmental characteristics such as altitude, slope position, and slope aspect. Soil samples were randomly collected from 0–20 cm depths in each plot using a soil core sampler (diameter of 2.0 cm). Twenty cores were mixed into one soil sample, which was then sieved (2 mm) to remove soil impurities, hand-mixed and stored in plastic bags. Half of each soil sample was stored at 4°C for microbe characteristics analysis, and the other half was air-dried and sieved to determine soil pH, total organic carbon (TOC), total nitrogen (TN), available nitrogen (AN), total phosphorus (TP), available phosphorus (AP), total potassium (TK), and available potassium (AK) as described by Wu et al. [Four et al. [et al. [et al., Huygens et al. and Brockett et al. [PLFAs were extracted and derivatized as described by Zelles et al. . Phospho [et al. . We usedt et al. –25. The et al. [Community level physiological profiling (CLPP) owes its beginnings to the development of the BIOLOG system in the late 1980s. In this study, the microbial catabolic diversity was assessed using BIOLOG Eco Microplate system, which contains 31 different carbon substrates, allowing triplicate samples arranged on a single 96-well plate . The optet al. . The detC was the optical density within each well and R was the absorbance value of the control well. To test significant differences among soil samples, we performed one way analysis of variance (ANOVA) followed by Least Significant Difference (LSD) tests (P < 0.05) using DPS software version 7.05. Principal component analysis (PCA) and correlation analysis were performed using SPSS11.5 software.Microbial activity in each microplate, expressed as average well-color development (AWCD) was determined by the equation P<0.05, Most soil physicochemical properties selected for analysis in this study were significantly different among the soil samples P<0.05, . The pH Our results demonstrated a significant effect of stand age on soil microbial community composition. The Gram (+) bacteria, Gram (-) bacteria, Fungi, and actinomycete components are listed in The content of all individual PLFAs was subjected to the principal component analysis (PCA) . Four soThe AWCD values, as a measure of the total microbial activity, increased and showed a typical sigmoid course curve across the 168 h incubation for all samples . HoweverWe divided 31 single carbon substrates into 6 categories: amino acids, polymers, phenolic acids, amines, carboxylic acids and carbohydrates as described by Chowdhury and Dick . OverallP. elliottii plantations. The bacterial biomass decreased with the increasing stand ages, while the fungal biomass increased. The carbon source utilization rate and community diversity of soil microbes gradually decreased with increasing stand ages. Overall, both the PLFA and CLPP illustrated that the long-term pure plantation pattern exacerbated the microecological imbalance previously described in the rhizospheric soils of P. elliottii, and markedly decreased the soil microbial community diversity and metabolic activity. Similar trends were also reported in other tree species [P. elliottii stands is the decline of soil nutrient content. P. elliottii is a fast-growing tree species, and has a high requirement and utilization rate of soil nutrients to maintain this growth. Although litterfall can return nutrients to the soil and improve fertility to some extent, this rate of natural nutrient cycling is too slow [p<0.05) and 0.851 (p<0.05), respectively. Consequently, the decline of soil nutrient content inevitably leads to a decrease in soil microbial diversity. Furthermore, the effects of spatial change on soil nutrient content are also very important [Our study demonstrated that soil microbial community diversity and metabolic activity decreased with increasing stand ages in  species . The imp species . Therefotoo slow . In thismportant –32. We wet al. found that soil C/N ratio and soil pH are important factors influencing soil microbial community composition [et al. concluded that the variation of C/N ratio caused by tree species changed the soil microbial community structure profoundly [P. elliottii litterfall contains many slowly-degrading components such as wax, cellulose, and lignin, and the C/N ratio in these substances are often relatively high [et al. concluded the higher microbial C/N ratio indicated soil microorganisms activated by C-rich rhizodeposits with a high N demand and a greater potential for immobilization of N in the microbial biomass [P. elliottii plantations, adequate soil nutrition was beneficial to the reproduction of bacteria, which are favored in high-nutrient soil. Soil nutrient content decrease in increasing stand ages, however, resulted in an increase in fungal abundance, as fungal growth is favored in soil with lower fertility.In addition to nutrient content, soil C/N ratio is another important factor affecting soil microbial community structure. This study showed that the soil microbial community diversity was significantly negatively correlated with C/N ratio, indicating that high C/N ratio is not conducive to microbial growth. This result is also consistent with previous studies. For example, Högberg position . Ushio eofoundly . P. elliely high . Therefo biomass . High C/ biomass . In thisP. elliottii result in a change in soil properties over time, and therefore cause a corresponding change in soil microbial community composition and metabolic activity. However, the CLPP and PLFA methods used here have limitations in their detection of soil microbial community characteristics. The experimental result of CLPP and PLFA are influenced by environmental change and physiological status of microbes under some environmental stresses. These results can be strengthened with future work combing with other soil microbial research methods such as terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE). Our studies help to more deeply understand the ecological linkages between aboveground and belowground biota, and thereby facilitate to establishment of an effective forest management practice for the improvement of forest ecosystem balance. Based on the results as mentioned above, improved management practices, such as microbial fertilizer application, organic matter amendment and enhanced plant diversity, could be used to relieve the consecutive monoculture problems of P. elliottii in future.Our conclusions indicate that long-term monoculture plantations of S1 Appendixhttp://dx.doi.org/10.6084/m9.figshare.1423487.(DOC)Click here for additional data file.S2 Appendixhttp://dx.doi.org/10.6084/m9.figshare.1423488.(DOC)Click here for additional data file.S1 Tablehttp://dx.doi.org/10.6084/m9.figshare.1423489.(XLS)Click here for additional data file.S2 Tablehttp://dx.doi.org/10.6084/m9.figshare.1423490.(XLS)Click here for additional data file.


Eruca sativa (Cruciferae family) is an ancient crop of great economic and agronomic importance. Here, the complete mitochondrial genome of Eruca sativa was sequenced and annotated. The circular molecule is 247 696 bp long, with a G+C content of 45.07%, containing 33 protein-coding genes, three rRNA genes, and 18 tRNA genes. The Eruca sativa mitochondrial genome may be divided into six master circles and four subgenomic molecules via three pairwise large repeats, resulting in a more dynamic structure of the Eruca sativa mtDNA compared with other cruciferous mitotypes. Comparison with the Brassica napus MtDNA revealed that most of the genes with known function are conserved between these two mitotypes except for the ccmFN2 and rrn18 genes, and 27 point mutations were scattered in the 14 protein-coding genes. Evolutionary relationships analysis suggested that Eruca sativa is more closely related to the Brassica species and to Raphanus sativus than to Arabidopsis thaliana. Brassica hirtaSilene conicaMitochondria supply energy in the form of ATP through oxidative phosphorylation in almost all eukaryotic cells Mitochondria play an important role in plant growth and development. Genomic rearrangements involving substoichiometric shifting (SSS), a consequence of intermediate repeat DNA exchange Eruca sativa Mill.or Eruca vesicaria subsp. sativa (Miller) (Garden rocket), a member of the Cruciferae family, has several desirable agronomic traits, such as resistance to salt, drought, white rust and aphids E. sativa into economically important cultivated species will promote crop improvement E. sativa with other species of the family Cruciferae, including B. rapa, B. juncea, and B. oleracea, have been reported Cruciferae family have been sequenced, including Arabidopsis thaliana (tha) Raphanus sativus (sat) Brassica genus, i.e., B. napus , B. rapa (cam), B. oleracea (ole), B. juncea (jun), and B. carinata (car) E. sativa and provide a comparison with other sequenced cruciferous mt genomes. This research will help to characterize the E. sativa crop and further our understanding of the evolution of mitochondrial genomes within the Cruciferae family.To date, several mt genomes from the E. sativa was used in this study. Mitochondrial DNA was isolated from 7-day-old etiolated seedlings according to Chen's methods , and stored at −80°C until use. Genome sequencing was performed using the GS-FLX platform . The reads were assembled into contigs using Newbler v.2.6. Sanger sequencing of PCR products was used to join the contigs to form the complete genome.A commercial cultivar of http://www.ncbi.nlm.nih.gov/) was searched for mitochondrial sequences annotation, using previously annotated mitochondrial genes from angiosperms as query sequences. The tRNAs were identified using the tRNA scan-SE software (http://lowelab.ucsc.edu/tRNAscan-SE/). Putative open reading frames (ORFs) with a minimum size of 100 codons were predicted and annotated using ORF-Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The circular map was drawn using OGDraw v1.2 (http://ogdraw.mpimp-golm.mpg.de/). Repeats analysis was performed as previously described The NCBI database , B. oleracea (GenBank: NC_016118), B. juncea (GenBank: NC_016123), B. carinata (GenBank: NC_016120), B. napus (GenBank: NC_008285), B. napus cultivar Polima (EMBL: FR715249), R. sativus (GenBank: JQ083668) and A. thaliana (GenBank: NC_001284), using NCBI-blastn. For comparison, the exons of 32 protein coding genes , which were shared by these nine species, were extracted and sequentially joined together. A neighbor-joining tree The E. sativa was assembled as a single circular molecule of 247 696 bp (Cruciferae. The largest part of the E. sativa mtDNA comprises the non-coding sequences (85.14%), which is slightly smaller than the average non-coding sequences content (89.4±3.1%) in other reported angiosperm mitochondrial genomes The mitochondrial genome of atp9) to 7 979 bp (nad4), including 18 genes for components of the electron transport chain and ATP synthase: nine subunits of complex I , one subunit of complex III (cob), three subunits of complex IV (cox1-3) and five subunits of complex V . In addition, there are five genes for cytochrome c biogenesis , eight genes for ribosomal proteins , two genes for maturase (matR) and one gene for other functions (tatC). The total length of the 33 PCGs of E. sativa mtDNA is 58 569 bp, accounting for 23.64% of its total mtDNA genome length, which is lower than that of the Brassica and R. sativus mitotypes. Nine genes had an exon–intron structure. All exons of ccmFC , nad2 (a-e), cox2 , nad4 (a-d), nad7 (a-e), rps3  and rpl2  were cis-spliced, whereas some exons of nad1 and nad5 were trans-spliced as follows: nad1a/nad1b-e; nad5a, b, d, e/nad5c (the slash indicating trans-spliced exons). ATG is the most commonly used initiation codon for mitochondrial PCGs in E. sativa, except for nad1 (start with ACG), matR (start with AGA) and tatC (start with ATT), as predicted by previous studies . Ten genes  are predicted to terminate with TGA and six  with TAG; other PCGs use TAA as their termination codon.Using BLAST and tRNA scan-SE, 54 genes were identified, including 33 protein coding genes, three rRNA genes  and 18 transfer RNA genes , Table 1E. sativa mtDNA (18 tRNA sequences (1 383 bp) were found in va mtDNA , in the E. sativa mitochondrial genome. Among the 50 ORFs, only the orf112, orf121, orf122, and orf275 have two copies. All others are single-copy ORFs. Most of the ORFs are between 300 and 500 bp in length, except for 10 ORFs that are longer than 500 bp, including the 1 200 bp orf399 and the 1 911 bp orf636.Using ORF-Finder and BLAST searching, 50 ORFs longer than 100 codons were identified in the Brassica hirta) . The repeats in the E. sativa mitochondrial genome were analyzed. Three pairs of large repeats were identified, accounting for 13.48% of the genome. The large repeats were designated as R1, R2 and R3  have been identified in most of the seed plants analyzed, except for white mustard  were detected in 14 genes when compared with B. napus  switch in atp1, a V to I (18aa) and an H to F (51aa) switch in atp6, a P to L (107aa) switch in ccmB, an R to K (113aa) switch in ccmFC, an H to Y (285aa) switch in cob, a P to L (112aa) switch in cox1, an S to L (126aa) and an S to N (438aa) switch in matR, a C to R (72aa) switch in nad2, an S to L (29aa) switch in rpl2, an L to P (172aa) switch in rpl5, and an M to I (50aa) switch in rps7. Of these 27 SNPs, most were transitions and only three were transversion . All tRNAs in the B. napus mitochondrial genome were detected in E. sativa mtDNA. However, the ORFs were quite different between these two mitotypes.We compared the sequences of the mtDNAs from ifferent  and a 25va mtDNA  comparedB. napus . ThirteeE. sativa mtDNA and other reported Cruciferous mtDNAs were compared using BLASTN E. sativa mtDNA was used as the reference sequence and similar regions in two or more mtDNA sequences were aligned. The alignable E. sativa sequence (93%) was 81% identical to that of R. sativus mtDNA. The sequence identity shared by the mtDNA of E. sativa and Brassica was more than 83%, with a coverage in the range of 83–85%. Only 63% of the E. sativa mtDNA matched those of Arabidopsis thaliana, with an identity of more than 68%, and the longest fragment was only 8.0 kb. This result suggested that the evolutionary relationship of mitochondrial genomes among E. sativa, the Brassicas and R. sativus is closer than that between E. sativa and A. thaliana.To further illustrate the evolution of mitochondrial genomes within the Cruciferae family, the E. sativa and A. thaliana are shorter than those between E. sativa and Brassica or R. sativus. Additionally, the distribution of syntenic regions between the mtDNAs of E. sativa and A. thaliana is more dispersed, and the identity is lower, than that between E. sativa and the Brassica mitotypes  compared with other seed plants. The E. sativa mt genome (248 kb) is larger than most Brassica mitotypes, but smaller than that of B. oleracea (360 kb) and A. thaliana (367 kb). Comparison of the E. sativa mtDNA with the B. napus mtDNA revealed that the cox2-2 gene was absent from the E. sativa mt genome. This gene was also absent from the genomes of B. oleracea, B. carinata, and Ogura-cms-cybrid (oguC) rapeseed mitotypes ccmFN genes are divided into two reading frames (ccmFN1 and ccmFN2) ccmFN2 has been confirmed in A. thaliana mitochondria, which demonstrated that ccmFN2 was not a pseudo gene, although it lacks a classical ATG initiation codon ccmFN2 from reported Cruciferae mtDNAs showed that the first 45 bp of the putative ccmFN2 gene in E. sativa mt genome is quite different from the ccmFN2 gene in Brassica and A. thaliana mitotypes  to their chloroplast counterparts. The chloroplast-derived trnH-GTG, trnM-CAT, trnS-GGA, trnW-CCA, trnD-GUC, and trnN-GTT genes, which are frequently found in mitochondrial genomes of angiosperms E. sativa mtDNA. An additional chloroplast-originating tRNA gene (trnL-CAA), which is found in the R. sativus and Brassica mitotypes E. sativa mitochondrial genome. This result indicated that mt tRNA genes are frequently transferred from chloroplast genomes during the evolution of angiosperms. However, another two gene (trnP-GGG and trnQ-UUG) transfer events reported in dicots E. sativa.18 tRNA genes were identified in Cruciferae mitotypes, especially for the protein coding genes. However, the mitochondrial genomes structural differences are remarkable among the Cruciferae family. Multipartite structures of mtDNA mediated by large repeats have been commonly observed in plant species Brassica (except the oguC rapeseed) mitotypes, was not found in the E. sativa mtDNA. Instead, three pairwise large repeats were identified. Large repeat R1 in E. sativa mtDNA showed significantly high sequence similarity to the 6 580-bp large repeat R in B. carinata mitochondria  with the large repeats in A. thaliana and R. sativus mtDNA, respectively. The tripartite structure of the mitochondrial genome, including one master circle and two smaller subgenomic circles, has been reported in Brassica species (except the ole mitotype) and R. sativusE. sativa is more complex than other Cruciferae species because of the three pairwise large repeats, including six master circles and four smaller subgenomic circles.Genes with known functions are relatively conserved among the chondria . The 1 5E. sativa, a member of the Cruciferae family. The E. sativa mtDNA is 247 696 bp and harbors 33 known protein coding genes, three rRNAs  and 18 tRNAs. In addition, the cox2-2 gene is absent, the ccmFN2 and rrn18 genes have different lengths and 27 SNPs are involved in 14 protein coding genes in comparison with B. napus mtDNA. Reorganization of the genome may have occurred via three pairs of large repeats, resulting in a more dynamic structure of the E. sativa mtDNA compared with other cruciferous mitotypes. This may produce six master circles and four smaller subgenomic circles. The evolutionary relationships analysis among reported Cruciferous mitotypes revealed that the mitochondrial genome of E. sativa is divergent from A. thaliana, but closely related to those of Brassica and R. sativus. This study will improve our understanding of the E. sativa crop and the evolution of mitochondrial genomes within the Cruciferae family.In this study, we reported the complete mitochondrial genome sequence of Figure S1Sequence alignments of ccmFN2 from reported Cruciferae mtDNAs. The highly and partly conserved amino acids are shaded black or grey respectively. The black block diagram indicates the un-conserved region of ccmFN2 in E. sativa mtDNAs compared to other reported Cruciferae mtDNAs.(TIF)Click here for additional data file.Figure S2Sequence alignments of rrn18 from reported Cruciferae mtDNAs. The highly and partly conserved amino acids are shaded black or grey respectively. The black block diagram indicates the deletion region of rrn18 in E. sativa mtDNAs compared to other reported Cruciferae mtDNAs.(TIF)Click here for additional data file.Figure S3Alignment of the large repeats in Eruca sativa mtDNA with the large 6.6 kb repeats in car. The alignment was made using Mauve. Blocks of the same color denote homologous regions; the B. carinata blocks above or below the middle line represent direct or inverted, respectively, compared with E. sativa. The extent to which a block is filled indicates the similarity of the syntenic region.(TIF)Click here for additional data file.


The study of candidemia in cancer patients has been limited. This retrospective study aims to investigate the epidemiologic characteristics and prognostic factors of candidemia among cancer patients.Candida species were retrospectively reviewed.From 2009 to 2012, cancer patients with candidemia were identified at a hospital in Taiwan. The medical records of all patients with bloodstream infections due to C. albicans  was the most common pathogen, followed by C. tropicalis , C. parapsilosis , and C. glabrata . Seventeen patients had polycandidal candidemia, and 77 patients had concomitant bacteremia. Approximately one-third of the patients required admission to the intensive care unit (ICU) or mechanical ventilation, and the overall in-hospital mortality was 50.8%. Multivariable analysis showed that the in-hospital mortality was significantly associated with only the non-use of antifungal agents and acute respiratory failure (P<.001).During the four-year period, a total of 242 episodes of candidemia were identified among cancer patients. Half of these patients were classified as elderly (≥65 years old), and more than 95% of the candidemia episodes were classified as healthcare-associated infections. Among the 242 cancer patients with candidemia, head and neck cancer was the most common, followed by gastrointestinal tract and lung cancer. Additionally, most of the patients had variable underlying conditions, such as the presence of CVC (99%) or prior exposure to broad-spectrum antibiotics (93%) and were receiving an immunosuppressant (86%). Overall, Candidemia can develop in patients with both solid cancer and hematological malignancy, especially for patients with underlying conditions. Overall, the associated morbidity and mortality due to Candidemia remain high. It was also determined that the non-use of antifungal agents and acute respiratory failure conditions were associated with in-hospital mortality. Candida species have become one of the major pathogens that cause nosocomial infections through bloodstream infection (BSI) C. albicans remains the main cause of invasive candidiasis and accounts for more than 50% of all cases, the number and proportion of candidemia cases caused by non-albicans Candida species, including C. tropicalis, C. glabrata, and C. parapsilosis, are increasing in all populations in nosocomial infection settings Most cases of candidemia develop inn the patients at risk of receiving invasive procedures/devices or intravascular catheters, broad-spectrum antimicrobial agents, mechanical ventilation, immunosuppressive agents or parenteral nutrition Candida species were retrospectively reviewed, and the following information was collected: age; gender; underlying conditions ; risk factors , recent abdominal surgery, and prior use of broad spectrum antibiotics); laboratory data; microbiological findings; and outcomes. The records and information of patients were anonymized and de-identified prior to analysis. Therefore, informed consent was not required and was specifically waived by the Institutional Review Board. An eEthics approval was obtained from the Institution Review Board of Chi Mei Medical Center.This study was retrospectively conducted at the Chi Mei Medical Center, which is a 900-bed hospital in southern Taiwan. The hospital had a total of 136831 admissions and 41440 cancer patient admissions between 2009 and 2012. Cancer patients with candidemia were identified from the Microbiology Laboratory database. The medical records of all patients with BSI due to Candida species was confirmed using the API 20C and Vitek YBC systems .Fungal blood cultures obtained at the hospital from January 2009 to December 2012 were analyzed. BACTEC Myco/F Lytic bottles  containing 5–10 mL of blood were incubated in the BACTEC 9240 culture system at 35°C. Each patient was included only once at the time of detection of the first bloodstream infection. The identification of Candida species in patients with compatible clinical signs/symptoms of infections. Healthcare-associated infection was defined according to the National Nosocomial Infection Surveillance guideline 5 CFU/ml and pyuria. If no primary focus could be identified, the candidemia was classified as primary. In-hospital mortality was defined as death due to any cause during hospitalization. Polycandidal candidemia was the assigned classification if at least two Candida pathogens grew from the blood samples of any one patient at the same time.The presence of candidemia was defined by at least one set of positive blood cultures for P value<0.05 was considered statistically significant.The continuous variables are expressed as means ± standard deviations. Comparisons between each variable/category were conducted using the chi-square test. A multivariable forward logistic regression model was used to identify risk factors for mortality. All statistical analyses were conducted using the statistical package SPSS for Windows , and a During the four-year period, a total of 242 episodes of candidemia among cancer patients were identified, and the overall incidence of candidemia was 1.77 episode per 1000 admissions (or 5.84 episodes per 1000 cancer patient admissions). The clinical characteristics of the cancer patients with candidemia are summarized in C. albicans and C. glabrata, four had C. albicans and C. tropicalis, three had C. albicans and C. parapsilosis, two had C. tropicalis and C. parapsilosis, and one had C. tropicalis and C. glabrata. Overall, C. albicans  was the most common pathogen, followed by C. tropicalis , C. parapsilosis , and C. glabrata . The trends of species distribution and the ratio of non-albicans Candida by year are shown in Candida species among four major cancer categories; however, no significant differences were found among the types of cancer. In addition, 77 patients had concomitant bacteremia. Among these 77 cases, coagulase-negative Staphylococci  was the most common bacteria, followed by enterococcus , Staphylococcus aureus , and E. coli . Moreover, several antibiotic-resistant pathogens were noted, including methicillin-resistant Staphylococcus aureus (n = 8), vancomycin-resistant enterococci (n = 6), extend-spectrum β-lactamase (ESBL) E. coli (n = 2), and multidrug-resistant Acinetobacter baumannii (n = 1).Seventeen patients had polycandidal candidemia. Among them, seven had concomitant ns n = 13, 54.5% wC. albicans, hypoalbuminemia, elevated bilirubin, and no use of antifungal agents than surviving patients. Furthermore, the rates of complications including ICU admission and acute respiratory failure were significantly higher among patients with mortality than survival. Overall, a multivariable analysis showed that the in-hospital mortality was only significantly associated with no use of antifungal agents  and acute respiratory failure .Although more than 90% of the patients received a variable antifungal agent, approximately one-third required intensive care unit (ICU) admission or mechanical ventilation, and the overall in-hospital mortality was 50.8%. This large study investigated the clinical features of candidemia among cancer patients in a single institution and had several significant findings. Although several studies have reported on this issue, most of them focus on hematological malignancy, especially in relation to leukemia In this study, more than 80% of the cancer patients with candidemia had CVC or a port-A catheter, received an immunosuppressant or had prior exposure to broad-spectrum antibiotics. This finding is consistent with previous studies that found cancer patients with candidemia often have variable underlying conditions, in addition to underlying malignancy Staphylococcus aureus, vancomycin-resistant enterococci, extend-spectrum β-lactamase (ESBL) E. coli, and multidrug-resistant Acinetobacter baumannii were found to be co-pathogens in cancer patients. This finding reminds us that while treating patients with candidemia, the presence of possible concomitant bacteremia cannot be neglected. In this critical condition, broad-spectrum antibiotics should be administered to cover antibiotic-resistant bacteria.Consistent with a previous study In this study, the in-hospital mortality due to candidemia remained as high as 50% in cancer patients. This finding is similar to a previous study C. albicans was the most common causative agent of fungemia, accounting for more than half of all episodes of candidemia among cancer patients in our hospital. In addition, C. tropicalis (21.5%), C. parapsilosis (15.7%), and C. glabrata (12.0%) were the most frequently isolated non-albicans Candida species. This result differs from a surveillance study of candidemia in cancer patients in China C. albicans was the most common species (48.8%), followed by C. parapsilosis (24.4%), C. glabrata (9.8%), C. tropicalis (9.8%), C. lusitaniae (4.9%), and C. famata (2.3%). Another study C. albicans was the most common pathogen, followed in frequency by C. parapsilosis , C. krusei  and C. glabrata , while in 150 oncology patients, the second most common species was C. glabrata , followed by C. parapsilosis . In Portugal C. albicans (48.7%), followed by C. parapsilosis (20.2%), C. tropicalis (8.4%), C. krusei (6.7%) and C. glabrata (5.0%). However, several studies examining candidemia in Taiwan showed similar findings to those of the present work; C. tropicalis was the second most common Candida species that caused invasive infections We found that in vitro antifungal susceptibility profiles of the Candida species. Therefore, we cannot evaluate the association between inappropriate antifungal agent and outcome. Second, approximately one-third of the patients treated at our institution had underlying malignancies; therefore, our findings may not be generalized to other hospitals. Third, this study is a retrospective investigation that may suffer from sources of bias, such missing data. However, the impact on this study due to missing data was minimal. Fourth, we used all-cause mortality for outcome analysis and did not evaluate the mortality attributable to candidemia. However, this study attempts to focus on candidemia in oncology patients and still provides useful information regarding this issue.There were several limitations to this study. First, we did not determine the C. albicans is the most common Candida species, followed by C. tropicalis.In conclusion, candidemia can develop in both patients with solid cancer and hematological malignancy, especially among patients with risk factors. Both polycandidal candidemia and concomitant bacteremia are not uncommon in this clinical entity. Overall, candidemia-associated morbidity and mortality remain high. No use of antifungal agents and acute respiratory failure conditions are independently associated with in-hospital mortality. Among cancer patients in Taiwan,


Xanthomonas pathogenic to various plants.Comparative genomics provides insights into the diversification of bacterial species. Bacterial speciation usually takes place with lasting homologous recombination, which not only acts as a cohering force between diverging lineages but brings advantageous alleles favored by natural selection, and results in ecologically distinct species, e.g., frequent host shift in Xanthomonas campestris that infected Brassicaceae, and X. citri, pathogenic to a wider host range. Genetic differentiation between two incipient races of X. citri pv. mangiferaeindicae was attributable to a DNA fragment introduced by phages. In contrast to most portions of the genome that had nearly equivalent levels of genetic divergence between subspecies as a result of the accumulation of point mutations, 10% of the core genome involving with homologous recombination contributed to the diversification in Xanthomonas, as revealed by the correlation between homologous recombination and genomic divergence. Interestingly, 179 genes were under positive selection; 98 (54.7%) of these genes were involved in homologous recombination, indicating that foreign genetic fragments may have caused the adaptive diversification, especially in lineages with nutritional transitions. Homologous recombination may have provided genetic materials for the natural selection, and host shifts likely triggered ecological adaptation in Xanthomonas. To a certain extent, we observed positive selection nevertheless contributed to ecological divergence beyond host shifting.Using whole-genome sequences, we examined the genetic divergence in Xanthomonas was likely governed by natural selection that played a key role in helping the deviating populations to explore novel niches (hosts) or respond to environmental cues, subsequently triggering species diversification.Altogether, mediated with lasting gene flow, species formation in The online version of this article (doi:10.1186/s12864-015-1369-8) contains supplementary material, which is available to authorized users. Diversification of prokaryotes attracts much attention from ecologists because it plays a critical role in ecosystem equilibrium and dynamics. Speciation of bacteria is distinct from eukaryotes, especially given the predominantly asexual reproduction  and the Xanthomonas, of the γ-subdivision of Proteobacteria, cause plant diseases, e.g., bacterial spots and blights in leaves and fruits [X. citri pv. citri exclusively infecting citrus, with various genetic mechanisms associated with the host specificity [mangiferaeindicae that causes bacterial black spot in mango, and in pv. vesicatoria that attacks pepper and tomato, displaying a wide phylogenetic range of hosts in X. citri [X. campestris pathovars predominantly infect crucifers [Xanthomonas [Xanthomonas, and the contributions of host shifts and homologous recombination to the adaptive diversification between species.Many of the bacteria in the genus d fruits . These pd fruits , e.g., Xcificity . Host shX. citri ,16. In crucifers . It has rucifers . During rucifers . Lu et athomonas . In thisXanthomonas taxa, including X. campestris pv. campestris (3 strains) [X. campestris pv. raphani [X. citri pv. vesicatoria [X. citri pv. citrumelo [X. citri pv. citri, X. albilineans [X. oryzae pv. oryzae (3 strains) [X. oryzicola [X. citri pv. mangiferaeindicae, X. citri pv. citri, and X. citri pv. citrumelo, three taxa that are closely related to X. citri. pv. vesicatoria [X. citri pv. mangiferaeindicae strain LMG 941 from India with 195 contigs was obtained by pyrosequencing [mangiferaeindicae strain from Taiwan. As the two strains LMG 941 and BCRC 13182 of X. citri pv. mangiferaeindicae diverged only recently, how they became genetically differentiated is quite fascinating. In this study, for exploring the nature of adaptive diversification in Xanthomonas, comparative genomic analyses were conducted on two nonsister species complexes of Xanthomonas, i.e., X. citri and X. campestris. The patterns of genomic divergence, homologous recombination, and genes under positive selection were examined to elucidate the ecological interactions among Xanthomonas taxa.To date, whole genomes of 8 strains) -23, X. c raphani , X. citricatoria , X. citritrumelo , X. citrilineans , X. oryzstrains) -29, and ryzicola , have beryzicola . Phylogeicatoria ,31. ReceX. citri pv. mangiferaeindicae BCRC 13182 (XCM-B) was sequenced, and 4,292,719,080 bases of paired-end data  and 1,185,355,594 bases of mate-paired data  were retrieved. These sequences were de novo assembled to 221 contigs, and scaffolded into 43 scaffolds, which comprised 5,355,324 bp with a GC content of 64.75% and indicated the sequence coverage is 1022.92. The largest scaffold was 1,286,619 bp long, and the N50 statistic was 549,958 bp, with an average length of 124,542 bp  and 4 X. campestris taxa   were detected with genetic recombination, which included 83 genes with cross-overs occurring between X. citri and X. campestris, 172 genes only detected within species, while 28 have likely derived from other distant species , a near-neutral indicator of genetic divergence, to assess the divergence along the genome. In the comparison between 2 XCM strains based on the orthologous genes between XCM and XCC, only one major peak was observed in the putative prophage region, while the remaining regions displayed more or less uniform levels of genomic variation . In addition, comparisons between closely related strains, i.e., XCM-B vs. XCM-L and XCCA vs. XCC8, were also performed. Similar to the comparisons of host-shifting pairs, significant positive correlations were also observed in the strain pairs without host-shifting   were detected under positive selection. Of these positively selected genes, only 3 were shared between X. citri and X. campestris, while 125 were exclusively in X. citri, and 51 were only in X. campestris . We further tested the association between homologous recombination and positive selection. In total, 98 (57%) out of 184 positively selected genes were identified with recombination, with 73 in the citri-group, and 26 in the campestris-group, all significantly deviating from a random distribution  (Table citri-group and 96.3% (52/54) in the campestris-group agreeing with the species tree   and India (XCM-L) and two strains of X. campestris (XCCA and XCC8) displayed slight genetic differentiation, providing a window for investigating incipient genetic divergence. As expected, given a shorter time for isolation, strains had lower levels of genetic divergence than that of species/pathovars. It is noticeable that prophage-introduced DNA fragments were detected in X. citri pv. mangiferaeindicae and pv. citri [X. citri. Several salient features were observed in these inserted DNA fragments. First, long DNA fragments of 28,787 bp were lost in genomes of the two pathovars  became eliminated along with the divergence time. Third, higher nucleotide substitution rates were detected in these inserted DNAs than in the host bacterial genome. These facts indicate that the foreign genes introduced by the phage tended to lose functions and were likely to be removed from the genome eventually , although gene residues may still remain [Bacterial diversity, which often results from species diversification through ecological interactions, greatly influences the ecosystem health. v. citri , an evenl remain .Xanthomonas strains at the incipient stage, as shown by the positive and significant correlation with genomic divergence  with short DNA fragments. Apparently, those genes displaying trees inconsistent with the species tree represented footprints of gene flow between species. This result suggests that the divergence of Xanthomonas tends to follow parapatric speciation, in which gene flow between species can be long lasting and nonstop [Xanthomonas reflects a scenario of deep divergence between species, mediated by recurrent gene flow at the same time [In this study, we found frequent genetic recombination in  nonstop . This is nonstop . It is i nonstop  in bacteame time .RpfB (XCM0815) and RpfC (XCM0813) of the Rpf gene cluster, which regulates the virulence of Xanthomonas [X. citri pv. citri lineage  has been reported to be associated with copper tolerance [Xanthomonas species, including X. citri [DsbD knockout mutant was highly sensitive to environmental copper [DsbD allele was capable of reducing the impacts of the copper-containing bactericides, thus enabling the bacterium to escape from the agricultural control.In this study, 98 out of 283 genes with recombination were under positive selection , that encodes ATPase involved in putrescine transport, was shaped by positive selection in the X. citri pv. vesicatoria lineage. Putrescine is a ubiquitous polyamine that enhances the interaction between plants and arbuscular mycorrhizal fungi (AMF) [Xanthomonas [PotG gene that decreases AMF colonization of host plant will be favored by natural selection, subsequently helping X. citri pv. vesicatoria to explore a new niche (host). In addition, BtuB gene (XCM3148) was positively selected in X. citri pv. mangiferaeindicae lineage. BtuB is responsible for the iron uptake, while iron dependent superoxide dismutases are vital in inhibiting the reactive oxygen species (ROS) responses in host cells, thus increasing the infection rates [-1 dry weight) [We found that some positively selected genes were associated with the host shifting events. It is notable that the gi (AMF) , which ithomonas . Alternaon rates . Interes weight) ,59. The Hrp regulon gene XCM1612 that encodes components for the type III secretion system (T3SS) [citri. The pathogenicity of Xanthomonas mainly depends on the T3SS, which is highly conserved among plant and animal pathogenic bacteria [Hrp pili are hypervariable in different subspecies of bacterial pathogens [Hrp pili provide evidence for strong positive selection in Xanthomonas spp. and diversifying selection in Pseudomonas syringae [Hrp gene cluster has been found to be associated with the adaptation and plant pathogenicity in Xanthomonas [HrpF gene may be responsible for the invasion routes and the host specificity of Xanthomonas. Furthermore, two genes involved in type IV secretion system were detected with positive selections exclusively in X. campestris, seemingly agreeing that type IV secretion system may not be involved in the infection of X. citri on citrus [EcpD (XCM0369) that encodes an adhesion protein to help pili assembly was positively selected in XCM-L. EcpD has been shown to facilitate the polymerization of EcpA to form pilus in Escherichia coli and involve in host cell recognition or biofilm formation [Xanthomonas species only possessed EcpD while lacks other members of Ecp operon. We therefore hypothesized that EcpD might participate the assembly of the other pili in association with the virulence in Xanthomonas. On the other hand, a xylanase gene XynA (XCM1323) was positively selected at the branch leading to X. campestris pv. campestris . Xylan is a major component in the cell walls of land plants and exists in all plant tissues [X. citri pv. vesicatoria and X. oryzae pv. oryzae [xcs and xps of the Type II secretion system (T2SS) have been shown to control the secretion of xylanases in XCV and are associated with the virulence [In addition to the genes related to the host shifting, an m T3SS)  was detebacteria . Curiousathogens ,64. The syringae ,66. The thomonas . Thus, wn citrus . Moreoveormation . XanthomcpD XCM03 that enc. oryzae ,71. Two irulence . NeverthSS  was dX. citri pv. mangiferaeindicae and conducted genomic analyses of 9 taxa of X. citri and campestris. Between the 2 strains of XCM, only the prophage region displayed sharp differentiation, while gradually losing the constituting genes. In addition, we found homologous recombination frequently occurring in the Xanthomonas genomes, which likely represented footprints of gene flow between species, thus most likely suggesting parapatric speciation. It is noticeable that facilitated accumulations of positively selected genes occurred along the lineages with host shifts. Interestingly, most of the favored genes were acquired from homologous recombination. Taken together, the genes with recombination enabled Xanthomonas species to explore novel niches and respond to environmental stresses, subsequently resulting in adaptive diversification in this pathogenic genus.In this study, we sequenced a genome of X. citri pv. mangiferaeindicae was purchased from the Food Industry Research and Development Institute and sequenced in this study. The whole genome was sequenced using a high-throughput sequencing technique with Illumina GA IIx and HiSeq sequencers [de novo assembler ABySS version 1.2.7 [Bacterial strain BCRC 13182 (NCHUPP Xma1) from Taiwan of quencers . Paired-on 1.2.7 . After con 1.2.7 , and 43 http://ncbi.nlm.nih.gov) and the Clusters of Orthologous Groups (COG) database [-5. All protein-coding regions were manually curated with the EMBOSS analysis package according to the BLAST results [The draft genome was analyzed using an integrated annotation pipeline glued by the perl programming language. Glimmer version 3.02  was useddatabase  with a c results . We used results ,79.http://www.ncbi.nlm.nih.gov/genbank/) in addition to the newly assembled genomes of X. citri pv. mangiferaeindicae BCRC 13182, including X. citri pv. citri 306, X. citri pv. citrumelo F1, X. citri pv. vesicatoria 85–10, X. campestris pv. campestris ATCC 33913, X. campestris pv. campestris 8004, X. campestris pv. campestris B100, X. campestris pv. raphani 756C, and a draft genome of X. citri pv. mangiferaeindicae LMG 941  for identifying genetic recombination, to confirm the results of geneconv. Using the PhiPack program, recombination was identified for p-values lower than 0.05. Taken together, a gene with recombination was recognized when recombination was detected with geneconv and at least two additional methods implemented in PhiPack.Putative genetic recombination events occurring in geneconv  and PhiP[PhiPack . First, Ks) and per nonsynonymous site (Ka) were calculated using codeml program implemented in the PAML package with the codon-based model [Ks values and the number of genes with recombination were made with a window size of 50 genes and an overlap of 10 genes. Spearman’s rho coefficient was used to examine the correlations between Ks values and the number of the genes with recombination. In addition, the significant of difference of Ks distributions between genes with and without recombination were evaluated with Mann–Whitney U test. Both tests were performed by using SPSS Statistics 17.0.Numbers of substitutions per synonymous site  was adopted [Ka/Ks) values of 0.5, 1, and 1.5, respectively, while the null model was fixed with the omega value at 1. Likelihood ratio tests were used to assess the significance between the test and null models, and the P values were adjusted with a false discovery rate (FDR) of 0.01 for multiple testing corrections. We performed codeml analyses on each branch of individual gene trees. For above three tests, branches detected with significance were deemed to be affected by positive selection. Assuming the number of positively selected genes was proportional to the branch length, we used a chi-square test to examine the homogeneity in the distribution of positively selected genes between branches with and without host shifts. Fisher’s exact tests were also used to assess the differences of the number of positively selected genes between outlier and background genes, as well as between the genes with and without homologous recombination.For detecting genes under positive selection, we applied the  adopted . Three i(http://treebase.org/treebase-web/home.html).Alignments and phylogenetic trees of the core orthologous genes were deposited in TreeBASE


Treponema are characteristic of traditional gut microbiomes. Moreover, through genome reconstruction  and functional potential characterization, we discover these Treponema are diverse, fall outside of pathogenic clades and are similar to Treponema succinifaciens, a known carbohydrate metabolizer in swine. Gut Treponema are found in non-human primates and all traditional peoples studied to date, suggesting they are symbionts lost in urban-industrialized societies.Recent studies suggest that gut microbiomes of urban-industrialized societies are different from those of traditional peoples. Here we examine the relationship between lifeways and gut microbiota through taxonomic and functional potential characterization of faecal samples from hunter-gatherer and traditional agriculturalist communities in Peru and an urban-industrialized community from the US. We find that in addition to taxonomic and metabolic differences between urban and traditional lifestyles, hunter-gatherers form a distinct sub-group among traditional peoples. As observed in previous studies, we find that  The gut microbiomes of urban-industrialized societies differ from those of traditional rural societies and hunter-gatherers. Here the authors perform a comparative analysis of available and new gut microbiome data to provide fresh insight into these differences. Yet, despite large-scale sequencing efforts, the full extent of human gut microbial diversity remains underexplored. Extant people living traditional lifestyles are especially under-studied, limited to one population of hunter-gatherers from TanzaniaBecause of their unique cultural, behavioural and ecological environment, we hypothesize that remote hunter-gatherer communities harbour novel microbiome profiles that depart from those previously described in urban and semi-urban settings, and that may be tailored to the specific dietary sources within each population. To test this hypothesis, here we use a combination of high throughput 16S ribosomal RNA (rRNA) gene amplicon sequencing and shotgun metagenomic sequencing to characterize the gut microbiota of peoples from three different lifeways: traditional hunter-gatherers, traditional agriculturalists and urban-industrialized peoples. In addition to previously published data, we provide novel data from: (1) the Matses, a remote hunter-gatherer population from the Peruvian Amazon; (2) Tunapuco, a traditional agricultural community from the Andean highlands; and (3) residents of Norman, Oklahoma, a typical US university community that serves as a comparative population following an urban-industrialized lifestyle.Manihot spp.) and invasive plantains (Musa spp.) (Solanum tuberosum spp.) and root tubers like oca  and mashua (Tropaeolum tuberosum), which they eat at every meal. Tocosh, a typical dish of the central Andes made out of potatoes that have been fermented in wet soil, is eaten at least once a week by families in Tunapuco  . Fish isTunapuco . ResidenThis study was conducted under the supervision of the University of Oklahoma and the Ethics Committee of the Peruvian National Institute of Health, in collaboration with the Matses and Tunapuco communities . Our modPrevious reports have indicated that Western populations have lower microbial richness than non-Western populationsP<0.001 and P<0.001 respectively). Further, the Tunapuco population is characterized by high interpersonal variation, evident in both PC axes 1 and 2. Supervised learning using Random ForestsNext, we compared microbial community structure (beta diversity) among the three populations using Principal Coordinates Analysis (PCoA) transformation of weighted UniFracP<0.0006)  . Three o<0.0006) . In addi<0.0006) . Finally<0.0006) .P<0.05; Bifidobacterium, Succinivibrio and Treponema, respectively . The Norman population consistently had a 100% classification accuracy at all taxonomic levels. In contrast, the Matses and Tunapuco populations had a 93% and 100% classification accuracy, respectively, at the genus level, reducing to 77% and 91% at the phylum level . MisclasFinally, we compared genus-level taxa abundance profiles between our populations, and those from two previous studies of remote agrarian and hunter-gatherer human gut microbiomes1de novo using Ray-MetaWe performed shotgun metagenome sequencing  to investigate whether the Matses, Tunapuco and Norman gut microbiomes harbour differences in functional capacity. To improve annotation quality, the short reads obtained from metagenome sequencing were assembled Supervised clustering using KEGG Orthology (KO)13To identify KOs showing differential abundance between the three populations, we performed Kruskal–Wallis tests on KO tables. Overall, we identified 112 KOs showing a significant difference in abundance in at least 1 population . Of thesTo further characterize some of these functional differences, we performed statistical analyses on orthologue tables annotated using Enzyme Commission (EC) codesBifidobacterium, a genus thought to be associated with dietary dairy consumption. As the number of children of age <3 years in our study is limited to four individuals from the Matses, we instead performed correlation analyses between age and PC axes generated from a weighted UniFrac distance matrix. A negative correlation was observed between the first PC axis and age for the Matses population . While the relative abundance of Bifidobacterium in children shows no direct correlation with age, 10 out of 13 individuals  showing presence of the genus were <7 years old. In contrast, all individuals sampled from our Norman population showed presence of Bifidobacterium, with no correlation between age and levels of Bifidobacterium. This is consistent with regular dairy consumption self-reported by the Norman individuals.A previous study on US, Malawi and Venezuelan populations3Treponema. Phylogenetic analysis of these Spirochaetes indicates the presence of at least five Treponema OTUs  from four Matses samples. These samples were selected based on high frequencies of Treponema observed in their taxonomic profiles. Samples from Tunapuco were not included in this analysis as they had lower sequencing coverage and often contained multiple Treponema strains leading to poor assemblies. Phylogenetic analysis using 16S rRNA gene sequences retrieved from these contigs confirmed the presence of two distinct strains of Treponema within these samples, one with ~99% sequence similarity to T. succinifaciens  and the other with ~90% sequence similarity to T. succinifaciens  . We annotated these partial assemblies using the ‘prokka’ pipelineTreponema strains cluster most closely with T. succinifaciens and are nested with other gut-associated treponemes reported from termites (T. azotonutricium and T. primitia)Treponema reported from cattle (T. brennaborense)Brachyspira clade of Spirochaetes, along with several gut-associated bacteria from other phyla, including Ruminococcus, Eubacterium and Butyrivibrio. In contrast several pathogenic Spirochaetes including T. pallidum (syphilis), Borrellia burgdorferi (Lyme disease) and T. denticola , form a functionally separate clade outside of the gut-associated bacteria. Overall, these results give further support for a potential metabolic role for the Treponema strains observed in the gut microbiomes of traditional human populations.To further characterize the phylogenetic and functional relationships of the Matses gut train 2) . A secon samples . OverallCharacterizing microbial communities and their functions in populations living relatively ancestral lifestyles is essential for understanding the coevolution of humans as a species with their microbiomes. Our results strongly support the need for human microbiome research on a larger sampling of human lifeways and traditions. Such work with vulnerable populations is challenging, especially with respect to building trust and establishing reasonable informed consent, but remains possible, even with very remote and traditional peoples. Without these insights, the benefit of research may be more applicable to the Westernized, affluent, urban populations, further exacerbating health disparities for the underrepresented. Here we present a microbiome profile that may be more consistent with the ancestral state of human biology. Such information provides a potential foundation for understanding microbiome-associated ‘diseases of civilization’.Collaborative research with remote human communities requires careful planning and extensive outreach. As with many other indigenous populations, the Matses and Tunapuco have experienced and resent the idea of safari or helicopter research, a common model of research on indigenous populations. In addition, foreign companies’ recent attempts to extract oil from the Matses’ natural reservation have fuelled the Matses’ distrust towards the outside world. To maximize protection of the communities, we consulted with colleagues at the Center for Intercultural Health of the Peruvian Institute of Health from the early stages of the study design.Recognizing communities’ vulnerabilities and concerns, in addition to the official efforts aiming to protect them, we initiated our work by engaging political and traditional authorities in the review of our protocol. Political authorities included regional and national authorities. The traditional authority we first approached was the Peruvian leader of the ethnic group. All concerns from these authorities were addressed in the protocol before submission to the ethics committee of the Peruvian National Institute of Health, which approved the protocol.The protocol for the Matses community includes oversight by additional local authorities. On protocol approval, and with the authorization of the leader of the Matses ethnic group, we presented our project to the local authority of the District Yaquerana, who oversees all activities in the Matses reservation, and later to the leader of the Comunidad Nativa Matses Anexo San Mateo, who introduced us to the community members. Such structures are unavailable for Tunapuco. For both communities, we implemented a public meeting for community consultation and obtained community consent. In addition to community consent, all volunteer participants were individually consented when they arrived to deposit their samples.In an effort to maximize benefits and prevent potentially coercive incentives for the community, we avoided individual presents or compensation. Instead, we offered on-site parasite screening, making a microscope available for the community to observe the analysis we performed. This experience also served to anchor the discussion about microorganisms, emphasizing the informed part of the consent process. A Matses interpreter, who was fluent in Spanish, mediated communication with the Matses community.For both the Matses and Tunapuco communities, once preliminary results became available we returned to the community to disseminate our findings. We obtained authorization from the community for data publication and to use the community’s name in association with our findings.n=25) and Tunapuco populations (n=31) were stored in ice for up to 4 days until arriving at Lima, and they were kept frozen until DNA was extracted at the laboratory in Oklahoma. In addition, faecal samples were collected from 23 individuals from Norman, Oklahoma to serve as a comparative population with an urban-industrial lifestyle. These samples were kept on ice during transport to the laboratory and frozen within 24 h of collection.Faecal samples from participants were collected in polypropylene containers. Samples from the Matses  following manufacturer’s instructions, with the addition of two heating steps: 10 min at 60 °C before vortexing the samples with the PowerBeads and later 10 min at 90 °C. For the Norman faecal samples, DNA extraction was performed using the PowerMicrobiome RNA Isolation Kit (MoBio) with the exclusion of the DNase I step. Both extraction methods included an initial bead-beating step.To characterize the taxonomic profile of the gut microbiome, we amplified the V4 hypervariable region of the bacterial 16S rRNA gene using the universal bacterial/archaeal primers F515 (5′-CACGGTCGKCGGCGCCATT-3′) and R806 (5′-GGACTACHVGGGTWTCTAAT-3′)https://github.com/najoshi/sickle) to remove bases with a quality score <30, followed by discarding sequences with ambiguous bases (‘N’) and a length <90 bp. These trimmed reads were demultiplexed, chimera filtered (‘usearch’)de novo using a 97% sequence similarity threshold, and the resulting OTU table merged with the one generated using the closed-reference approach. Comparative 16S rRNA data sets were obtained from previously published studies1n=27), rural agriculturalists  and urban populations . The data set composed of Venezuela, Malawi and USA individualsde novo clustering strategy using a 97% sequence similarity threshold to maximize read assignment to OTUs. All comparisons between sequences generated in this study and the two previously published data sets are limited to genus-level taxa tables.The 16S rRNA sequencing data from the Illumina runs were filtered and trimmed using the programme ‘sickle’  and a length <25 bp. P values for differences among categories . Machine learning analyses utilized Random Forest classifiers with 10-fold cross-validation and 1,000 trees.Alpha diversity analyses were performed using observed species and PD indices, as implemented in QIIME7Treponema genus were filtered. Two strains of Treponema were identified in our samples. Strain 1, found in all four samples, had a >99% sequence identity to T. succinifaciens at the 16S rRNA locus (nucleotide) and 35 single copy marker loci . The second strain (Strain 2), found in samples SM23 and SM42 had ~90–91% sequence identity (nucleotide) at the 16S rRNA locus and ~88% sequence identity  at the single copy marker loci to T. succinifaciens. Several of the single copy marker loci co-assembled on contigs. Depth of coverage was consistent for marker loci on different contigs. Further, in samples with both strains (SM23 and SM42) the strains were observed to have different depths of coverage, consistently observed across their respective contigs. Additional contigs were assigned to the two strains using a combination of NCBI-BLAST, depth of coverage and GC%. Assembly evaluation was performed using the ‘reapr’ pipelineTreponema strains were compared using hierarchical clustering with a collection of reference genomes, including other Spirochaetes and several gut-associated bacteria across other phyla.Contigs assembled from shotgun metagenomic reads obtained from four Matses individuals  were screened for 16S rRNA gene sequences and 35 single copy marker loci sequences38C.M.L. led the project and C.M.L., A.J.O.-T. and R.Y.T., conceived the initial project design, with inputs from J.M., K.S. and R.K. during the later phases. C.M.L., A.J.O.-T., R.Y.T., L.M.-R., O.T.-V., A.T.O., E.G.-P., L.T.-C. designed the field study, human subjects protocols and consent, and collected the samples. C.M.L., A.J.O.-T., R.Y.T., J.M., K.S., J.C.C., L.K.U., Z.Z.X., W.V.T., R.K., P.M.G., C.W. and A.T.O. performed the experiments and analyzed the data. C.M.L. provided financial support for the initial project design, with additional materials and bioinformatic support provided by R.K., P.M.G., M.F., P.S. and P.L. at later phases. C.M.L., A.J.O.-T. and R.Y.T. wrote the initial manuscript with significant contributions from J.M., K.S. and R.K., and critical input from all other authors. The funders had no role in this study design, data collection and analysis, decision to publish or preparation of the manuscript.Accession codes: 16S rRNA gene sequences from the study have been deposited in the QIIME database under study id 1448 (Illumina HiSeq V4). Shotgun sequence data sets have been deposited in the NCBI SRA database under the BioProject id PRJNA268964.How to cite this article: Obregon-Tito, A. J. et al. Subsistence strategies in traditional societies distinguish gut microbiomes. Nat. Commun. 6:6505 doi: 10.1038/ncomms7505 (2015).Supplementary Figures 1-4, Supplementary Tables 1-13 and Supplementary References


Yersinia ruckeri, a Gram-negative rod-shaped enterobacterium. It has a wide host range, broad geographical distribution, and causes significant economic losses in the fish aquaculture industry. The disease gets its name from the subcutaneous hemorrhages, it can cause at the corners of the mouth and in gums and tongue. Other clinical signs include exophthalmia, darkening of the skin, splenomegaly and inflammation of the lower intestine with accumulation of thick yellow fluid. The bacterium enters the fish via the secondary gill lamellae and from there it spreads to the blood and internal organs. Y. ruckeri can be detected by conventional biochemical, serological and molecular methods. Its genome is 3.7 Mb with 3406–3530 coding sequences. Several important virulence factors of Y. ruckeri have been discovered, including haemolyin YhlA and metalloprotease Yrp1. Both non-specific and specific immune responses of fish during the course of Y. ruckeri infection have been well characterized. Several methods of vaccination have been developed for controlling both biotype 1 and biotype 2 Y. ruckeri strains in fish. This review summarizes the current state of knowledge regarding enteric redmouth disease and Y. ruckeri: diagnosis, genome, virulence factors, interaction with the host immune responses, and the development of vaccines against this pathogen.Enteric redmouth disease (ERM) is a serious septicemic bacterial disease of salmonid fish species. It is caused by  Introduction2. Identification and classification3. Clinical signs and pathology of the diseaseYersinia ruckeri4. Distribution of 5. Transmission and epidemiology6. Route of entry and spread7. Genome8. Diagnosis9. Virulence factors10. Host immune response11. Control/treatment11.1 Antibiotherapy11.2 Probiotics11.3 Vaccination12. Conclusions13. Abbreviations14. Competing interests15. Authors’ contributions16. Acknowledgements17. ReferencesYersinia ruckeri, a Gram-negative rod-shaped enterobacterium, which was first isolated from rainbow trout (Oncorhynchus mykiss) in the Hagerman Valley of Idaho, USA [Y. ruckeri, including the present distribution and diagnosis of ERM, the route of entry of Y. ruckeri into the fish, its genome, virulence factors, interactions with the host and immune responses, and methods to control yersiniosis.Fish constitute a major source of protein, fatty acids, vitamins, minerals and essential micronutrients for an expanding segment of the world population. Consequently, aquaculture is the fastest growing food production sector and accounts for approximately 50% of the fish consumed worldwide . Diseaseaho, USA  and is caho, USA ,5. Althoaho, USA -4. Rainbaho, USA . Herein,Yersinia is a genus of Gram-negative, rod-shaped, facultative anaerobes within the family Enterobacteriaceae. Yersinia comprises several pathogenic species, which cause diseases in humans and other animals, including fish. Yersinia ruckeri is the causative agent of enteric redmouth disease in various species of salmonids worldwide. It was described from rainbow trout in the Hagerman Valley of Idaho, USA in the 1950s [Y. ruckeri bacillus is approximately 0.75 μm in diameter and 1–3 μm in length. Y. ruckeri has a 3.7Mb genome, with a ~47% G + C ratio [Yersinia species [Yersinia species has confirmed that Y. ruckeri shares the same core set of genes with the other members of the genus [he 1950s . The Y.  C ratio ,7, the s species . High-thY. ruckeri have been reported and categorized on the basis of serotypes, biotypes and outer-membrane protein types. In 1993, the typing scheme was updated and species were further subdivided into four serotypes with different subgroups: Serotype O1 is subdivided into two subgroups O1a (serovar I) and O1b (serovar III) and serotype O2 (serovar II) into three subgroups O2a, O2b and O2c. The remaining serotypes are designated as serotype O3 (serovar V) and serotype O4 (serovar VI) [Different strains of ovar VI) . The vasovar VI) .Y. ruckeri have also been subdivided into two biotypes. Strains of biotype 1 are positive for motility and lipase secretion, whereas strains of biotype 2 are negative for both tests [Y. ruckeri during the natural infection [Y. ruckeri is characterized biochemically as glucose-fermentative, catalase-positive, nitrate-reductive, oxidase-negative, with the ability to secrete b-galactosidase, lysine and ornithine decarboxylases but neither hydrogen sulfide nor indole [Strains of th tests . Howevernfection . Y. ruckr indole .Y. ruckeri have been investigated using molecular tools including multilocus enzyme electrophoresis, pulsed-field gel electrophoresis (PFGE), fatty acid methyl ester profiles, ribotypes and interspersed repetitive sequences-PCR. These have shown that O1a strains of Y. ruckeri have high levels of genetic homogeneity [Y. ruckeri strains using a multilocus sequence typing scheme. These authors suggested the existence of two major clonal complexes (CC1 and CC2) within the Y. ruckeri population structure. They support the ‘epidemic’ model of clonal expansion, in which populations of well-adapted clones explode to be widely distributed. Genetic and antigenic differences have been found between biotype 1 and biotype 2 strains, using 16S rRNA sequence analysis, genotyping (including ERIC-PCR and (GTG)5-PCR), and Western blot analysis [Y. ruckeri using fliR gene primers, restriction enzyme digestion and sequencing of the resulting fragments. This assay identified four mutant alleles in biotype 2 strains of Y. ruckeri that are presently circulating in Europe and the United States, and which can cause outbreaks in vaccinated fish.The genetic structure and variations within ogeneity ,13. Bastogeneity  investiganalysis ,16. Furtanalysis  developeERM can affect fish from all age classes but it is most acute in young fish (fry and fingerlings). The disease appears as a more chronic condition in older/larger fish. Disease outbreaks start with low level mortalities that are sustained over time, resulting in high cumulative stock losses ,4. ChangHistopathological examination shows general septicaemia with inflammation in most organs, and particularly kidney, spleen, liver, heart, gills and in areas with petechial haemorrhage. Pathological changes in the gills, including hyperemia, oedema and desquamation of the epithelial cells in the secondary lamellae have been described ,4,18. FoY. ruckeri infection in rainbow trout in the USA [Y. ruckeri has been isolated from animals other than fish, including muskrat (Ondatra zibethicus), kestrel , sea gulls (Laridae), turtles (Cheloniidae) and humans [Y. ruckeri has a wide host range and geographical distribution, and can cause both epizootics and zoonosis.Since the first report of  the USA , the pat the USA ,5,19. Add humans ,20,21. TY. ruckeri infections can be transmitted by direct contact between infected and non-infected fish. A carrier state for Y. ruckeri was demonstrated, where infectious fish can survive 2 months after both experimental [Y. ruckeri in the lower intestine. The bacteria can then be released when the carrier fish become stressed. For example, it was observed that carriers transmitted Y. ruckeri to clinically healthy fish when the temperature was raised to 25 °C, but no transmission occurred from unstressed carrier fish [Y. ruckeri can survive at least 4 months outside the host [Y. ruckeri strain that was able to form biofilms on the solid supports frequently found in fish farm tanks. These biofilms are reported to be a source of recurrent infection in rainbow trout facilities [Y. ruckeri has also been linked to putative vectors, which include aquatic invertebrates and birds [Y. ruckeri has been recovered from disinfected non-fertilized eggs of Chinook salmon (Oncorhynchus tshawytscha), whose offspring experienced low mortalities from fertilization to 12 weeks on feed [Y. ruckeri DNA in unfertilized eggs and ovarian fluid of Chinook salmon suggests that the pathogen could be transmitted vertically. However, this study was unable to verify the occurrence of bacterial cells within the chorion of the egg [rimental  and naturimental ,22. Buscrimental  verifiedier fish . Sheddinthe host . The bacthe host . The ovethe host ,27. Coqund birds . While v on feed . Further the egg .Y. ruckeri indicated that gills are an important portal of entry for Y. ruckeri. Thereafter, Y. ruckeri spreads to the other organs [Y. ruckeri initially infects the secondary gill lamellae, then spreads to the blood system via the gill pavement cells, as rapidly as 1 min post infection (mpi). It could be detected in the lumen of the intestine at 30 mpi, in the kidney at 3 days post infection (dpi), and in the liver, spleen, brain and heart at 7 dpi. Y. ruckeri was no longer detectable in the liver, spleen, brain and heart at 21 dpi [Histological examination of rainbow trout experimentally infected with r organs . This spr organs . The autt 21 dpi .Y. ruckeri are currently available: the motile CSF007-82 strain, isolated from diseased rainbow trout and the motile O1b 37551 strain, isolated from vaccinated Atlantic salmon   [Y. ruckeri strains from different geographical locations are still needed to gain insight into the differences between motile and non-motile strains.Annotated whole genome sequences of two strains of ctively) . The genctively) . The avaY. ruckeri, including culturing, serological tests and molecular biological techniques [Y. ruckeri has been isolated using Tryptic soy agar, Columbia blood agar and MacConkey agar [Y. ruckeri can grow at a wide range of temperatures, its thermal optimum is 20–28 °C [Y. ruckeri can be detected using ELISA, agglutination and immunofluorescence antibodies [Y. ruckeri in tissues of infected fish [Y. ruckeri quorum sensing system. This ERM-LAMP assay is sensitive, rapid and the amplification products can be detected by visual inspection or gel electrophoresis  have been shown to reproduce the clinical signs associated with the hemorrhagic form of the disease when injected into their host [Serratia-like haemolysin YhlA, which has cytolytic and haemolytic activity [yrpA and yrpB have been demonstrated to be induced in the gut of rainbow trout [50 of the bacterium [Y. ruckeri virulence [Y. ruckeri, raising questions about the necessity of the cdsAB operon [Several virulence mechanisms of eir host . Multiplactivity , an azocactivity , and theactivity ,47. Receow trout . In addiacterium . Heat seacterium . Howeverirulence . Similarirulence  and has irulence . HoweverB operon .Y. ruckeri [Y. ruckeri under iron-limited conditions [Y. ruckeri during the infection process in the fish.Finally, as is often the case in microbial pathogens, the expression of these virulence factors appears tightly regulated and linked with both the availability of iron  and the  ruckeri  and has nditions . More reY. ruckeri strains have been studied extensively. For example, both O-antigen and formalin-inactivated Y. ruckeri cells induced an immune response in rainbow trout [Y. ruckeri bacterin induced a significant immune response in rainbow trout at 146 days post injection [Y. ruckeri has been observed in the body cavity of rainbow trout [Y. ruckeri biotype 1 strains [Y. ruckeri biotype 2 strains using quantitative real-time PCR [Y. ruckeri may play a role in protection against disease [Y. ruckeri infection [Y. ruckeri biotypes 1 and 2 at the proteomics level still need to be investigated, both to understand the proteomic background for observed proteomic changes, and to elucidate the mechanism of action of the proteins whose expression differs between the biotypes.Non-specific and specific immune responses of fish against ow trout ,58, prodow trout . Similarow trout . Gene ex strains -64 and Ytime PCR ,66. Rece disease . Serum anfection . All thenfection . MechaniY. ruckeri isolates has shown that only 1 out of 50 was resistant to florfenicol [Y. ruckeri [Y. ruckeri readily develops resistance against oxolinic acid, oxytetracycline and potentiated sulphonamide [Y. ruckeri is both a natural producer of and naturally resistant to the antibiotic holomycin [As is often the case with fish bacterial pathogens, post-infection treatment relies mostly on the use of antibiotics. Unfortunately, only a limited spectrum of compounds is routinely used : amoxicirfenicol . Similarrfenicol . While a ruckeri , there i ruckeri . In vitrhonamide . Finallyolomycin .Y. ruckeri: oral administration of Bacillus subtilis and Bacillus licheniformis protects rainbow trout against subsequent infections [B. subtilis and outer membrane components including LPS of Aeromonas sobria, were also protective [Concern about the development of antibiotic resistance has spurred research into alternative methods for controlling bacteria, in particular, beneficial probiotic bacteria and yeast . Some sufections . The autotective .Bacillus sp., and Aeromonas sobria, was confirmed as protective by Brunt et al. [Bacillus sp. treatment) and 6% (A. sobria treatment). Similarly, Enterobacter cloacae fed alongside Bacillus mojavensis was shown to reduce mortalities from Y. ruckeri challenge from 65to 0.8% [Dietary supplementation with t et al. : it redu5to 0.8% .Carnobacterium maltaromaticum B26 and Carnobacterium divergens isolated from the normal intestinal microbiota of rainbow trout were found to be protective against further Y. ruckeri infections [Carnobacterium sp. but found no antagonistic activity in vivo, suggesting that, as seems to be the case for B. subtilis, protection is achieved by stimulation of host defense rather than direct anti-microbial effect.Feed supplementation with fections . Interesfections  confirmeLactobacillus lactis was shown to be antagonistic to Y. ruckeri and, as with Lactobacillus fermentum, significantly reduces adhesion of the pathogen to fish mucus [Y. ruckeri.In addition, sh mucus . Comparash mucus  who screY. ruckeri [Recently, it was reported that feeding fish with a plant-based diet modified the composition of their gut microflora, and affected their immune response to  ruckeri , suggestY. ruckeri.Jaafar et al.  investigY. ruckeri serotype O1 biotype 1, which can be administered to fish by several routes, e.g. immersion, injection and oral. It provides good levels of protection against Y. ruckeri biotype 1 strains [Y. ruckeri Yrp1 protease, aroA gene, extracellular product and lipopolysaccharide and these provide good protection against Y. ruckeri biotype 1 strains [Y. ruckeri biotype 2 strains are harder to combat and have been responsible for disease outbreaks in fish that had been vaccinated against biotype 1 strains; thus monovalent vaccines fail to induce protection against biotype 2 infection [Y. ruckeri strains, and provides good protection against the biotype 2 strains [Salmo salar) challenged with Y. ruckeri O1b biotype 1, have led to detection of genes and biosignatures which may be useful as predictive indicators of vaccine success. These targets include: non-protective/pathological response genes , vaccine-induced protective genes  and transcriptional biosignature of predominantly immune-relevant genes including hepcidin, immunoglobulin mu heavy chain, mylelin and lymphocyte protein. Detection of these bioindicators demonstrates that there is a range of potential targets for future vaccine development [The significant economic losses in salmonid fish aquaculture can be controlled to some extent by use of vaccinations. ERM was one of the first fish diseases for which an effective commercial vaccine was developed ,88. The  strains –93, as s strains -96. Howenfection ,31. A bi strains ,97. cDNAelopment .Table 1Y. ruckeri causes significant economic losses, particularly in salmonid aquaculture. Two whole genome sequences of motile strains of Y. ruckeri have been annotated and can now be used for comparative genomic analysis of Y. ruckeri strains, investigation of gene-level pathogenicity, development of potential drug targets and vaccines. Quantitative proteomic analysis of multiple geographic isolates of biotypes 1 and 2 Y. ruckeri strains are not yet completed, and are required to create a proteomic map and understand proteomic changes and differences between the biotypes and strains. Some potential virulence factors of Y. ruckeri have been identified but more research on the bacterium’s virulence mechanisms is needed to understand the full pathogenicity of Y. ruckeri during the course of infection. Investigations have revealed aspects of the fish immune response to Y. ruckeri infections, however there is still an urgent need to improve our understanding of the biochemical changes that occur in host tissues and organs during infection. Biotype 2 strains of Y. ruckeri have been responsible for outbreaks in rainbow trout that had been vaccinated against biotype 1, thereby confirming the failure of monovalent vaccines to protect the fish against infection. Formalin inactivated bivalent vaccines can significantly reduce mortalities due to infections with biotype 2 strains but the development of more efficient vaccines against both biotypes of Y. ruckeri is still needed.