Salmonella enterica serovar Kentucky (S. Kentucky) sequence type 198 has emerged as a global zoonotic pathogen. We explored Salmonella enterica serovar Kentucky ST198 samples from the broiler chicken supply chain and patients between 2010 and 2016. Here, we collected 180 S. Kentucky isolates from clinical cases and the poultry supply chain. We performed XbaI pulsed-field gel electrophoresis and multilocus sequence typing. We assessed mutations in the quinolone resistance-determining regions and screened for the presence of the Salmonella genomic island 1 (SGI1). We determined that 63 (35.0%) of the 180 isolates were S. Kentucky ST198. Chinese strains of S. Kentucky ST198 have a high transmission of ciprofloxacin resistance  and a high risk of multidrug resistance. The quinolone resistance of the S. Kentucky ST198 strain found in China may be due to mutations in its quinolone resistance-determining region. Our study firstly revealed that ciprofloxacin-resistant S. Kentucky ST198 strains can undergo cross-host transmission, thereby causing a serious foodborne public health problem in China. Salmonella enterica is a major global foodborne pathogen [Salmonella serovars have been identified worldwide [Salmonella infections, and the annual medical cost for salmonellosis in the USA is estimated at $3.7 billion. In China, approximately 70% to 80% of foodborne disease outbreaks are caused by Salmonella, and most of these disease outbreaks are associated with the ingestion of contaminated livestock and poultry products [Salmonella [pathogen . To dateorldwide . There aorldwide . In the orldwide , accountproducts . Food oflmonella .Salmonella enterica serotype Kentucky (S. Kentucky) is one of the serovars most frequently isolated from poultry carcasses in the USA [Salmonella enterica serotype Kentucky has rarely been associated with human illness. However, the ciprofloxacin-resistant Salmonella enterica subsp. enterica serovar Kentucky sequence type 198 (ST198) has emerged as a global human pathogen [S. Kentucky ST198 is also established in poultry in France [S. Kentucky. Here, we have systematically studied the epidemiology and molecular characteristics of drug resistance in S. Kentucky ST198 isolates in China, which can support basic research, clinical practice, and public health risk forecasts for Salmonella. the USA . Historipathogen . CiproflSalmonella strains were collected from 23 provinces, including Shanghai, Guangxi, Hubei, Chongqing, Fujian, Xinjiang, and Guangdong. Clinical Salmonella strains were provided by the provincial Centers for Disease Control and regional hospitals. Food and environmental samples were collected from a retail market. Salmonella isolation and identification was performed according to the US FDA Bacteriological Analytical Manual, as previously described, with some modifications [Between 2010 and 2016, ications ,9,11,12.ications .www.mlst.warwick.ac.uk/mlst/dbs/Senterica). The PCR cycling conditions were as indicated in instructions posted on the website. PCR products were purified with Sephadex G-50 fine resin . Nucleotide cycle-sequencing was performed directly on purified PCR templates using automated Sanger dideoxychain termination methods and the primers described on the MLST website. Sequences of seven housekeeping genes  were compared with the available MLST database (http://mlst.warwick.ac.uk/mlst/dbs/Senterica) to get the allele number and sequence typing (ST) number for each isolate. Sequence information for newly assigned alleles and STs was deposited in the MLST database [Genomic DNA was isolated using the InstaGene Matrix  according to the manufacturer’s protocol. All primer sequences for amplification and sequencing were obtained from the MLST Databases at the University of Warwick .The antimicrobial susceptibility of all strains was determined using standard agar dilution method on Mueller–Hinton agar (MH) . The folgyrA, gyrB, parC, and parE) were analyzed using PCR and sequencing. Plasmid-mediated resistance genes, including oqxAB, qepA, aac(6′)-Ib, qnrS, qnrD, qnrC, qnrB, and qnrA (fluoroquinolone resistance), were analyzed via PCR [Salmonella was used as a template for PCR amplification, and the amplified products were detected by agarose gel electrophoresis [Four fluoroquinolone regulatory genes ( via PCR . Accordiphoresis .S. Kentucky isolates were subtyped via pulsed-field gel electrophoresis (PFGE) [XbaI. Salmonella H9812 was used as the standard control strain. PFGE results were analyzed by BioNumerics software .s (PFGE)  to deterS. Kentucky were identified: 105 (0.9%) of 12,011 clinical Salmonella strains isolated from 15 provinces , and 75 (1.8%) of 4236 chicken and environmental samples from eight provinces . Of the 180 isolates of S. Kentucky obtained in China, 63 (35.0%) were S. Kentucky ST198, consisting of 40 strains from patient fecal samples, 18 strains from chicken, and 5 strains from environmental sources .A total of 180 strains of S. Kentucky ST198 isolates, the analysis of antimicrobial susceptibility testing , 28 (44.4%) of the 63 isolates were resistant to ampicillin, and 9 (14.3%) of the 63 isolates were resistant to cephalosporin. We defined strains resistant to three or more antibiotics as multidrug-resistant strains. For S. Kentucky ST198 strains and for all strains of S. Kentucky, the rate of multidrug resistance increased from 2014 to 2015 and again in 2016  and parC  genes , and the parC(Ser80Ile) mutation could give rise to resistance to ciprofloxacin (p < 0.05) and ofloxacin (p < 0.05). Here, S. Kentucky ST198 strains carried four plasmid-mediated quinolone resistance genes: aac(6′)-Ib-cr (30.2%), oqxAB (4.8%), qnrS (7.9%), and qnrB (6.3%).e) genes . StatistS. Kentucky strains, mainly to study SGI1-Ks and its derivatives of the S. Kentucky ST198 strain in China. We found that SGI1-Ks and its derivatives were detected in 28.6% of S. Kentucky ST198 strains in China  was the main ST type from different hosts in Shanghai, Guangdong, Guangxi, Henan, and Xinjiang . This means that ST198 has caused a public health threat in China that needs to be taken seriously. Thus, for food safety and preventive public health measures, it is important that we have presented the first report on the prevalence and resistance characteristics of ST198 in China.h safety ,18. S. Klsewhere . HoweverS. Kentucky has spread widely around the world, which has conferred multidrug resistance under the stress of antibiotics [S. Kentucky ST198 that were isolated from different provinces and regions in China from 2010 to 2016. Global spread of mobile antimicrobial drug resistance determinants in human and animal Escherichia coli and Salmonella strains causing community-acquired infections [S. Kentucky has been increasing its multidrug resistance rate since 2014. Fluoroquinolones are the preferred method of clinical treatment for nontyphoid salmonellosis [gyrA and gyrB) and topoisomerase IV (parC and parE); the mutations in the gyrA and parC genes are the most common mechanisms of bacterial resistance to quinolone antibiotics generally [S. Kentucky ST198 isolates that were resistant to fluoroquinolone was high, with resistance rates greater than 60.0%. Statistical analysis revealed that the mutations of gyrA  and parC (Ser80Ile) have a major influence on the high level of drug resistance of quinolones . As can be seen in this study, quinolone resistance caused by the quinolone resistance-determination region mutation was the same as the internationally common ST198 resistance mechanism [Recently, ibiotics ,22. In tfections ,24,25. Inellosis ; at the nellosis . The mosenerally . It is nechanism ,27. It iS. Kentucky ST198 strain can spread among broiler supply chain and patients, which has been involved in cross-host transmission in China. The study found a higher proportion of multidrug resistance among S. Kentucky ST198 isolates than the average over all S. Kentucky strains in China (S. Kentucky ST198 strains in China.Our study displayed that the ciprofloxacin-resistant in China . The mulS. Kentucky ST198 through the broiler supply chain and patients in China. High risks for development of multidrug resistance make infections with ciprofloxacin-resistant S. Kentucky ST198 a serious public health concern in China.In conclusion, this is the first report about the prevalence of ciprofloxacin-resistant


Mycobacterium xenopi is a slow-growing mycobacterial organism for which pathogenic features are unclear. Here, we report the complete genome sequence of an M. xenopi type strain. This sequence will provide essential information for future taxonomic and comparative genome studies of these mycobacteria. Mycobacterium xenopi is a slow-growing mycobacterial organism for which pathogenic features are unclear. Here, we report the complete genome sequence of an M. xenopi type strain. This sequence will provide essential information for future taxonomic and comparative genome studies of these mycobacteria. Mycobacterium xenopi is a slow-growing, scotochromogenic, thermophilic nontuberculous mycobacterial (NTM) species (Xenopus laevis), and its infection of humans was confirmed in 1959  was purchased from the Japan Collection of Microorganisms. The strain was inoculated on 2% Ogawa medium and incubated at 37°C for 2 weeks. Genomic DNA was extracted by a standard phenol-chloroform method (https://github.com/rrwick/Filtlong). The remining reads  were de novo assembled into one contig  with the suggestCircular flag, using Canu v.1.8 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc), these reads were mapped to the assembly with Burrows-Wheeler Aligner v.0.7.17 . The ANI values with respect to two reported draft genomes of the strain (named DSM43995 and NCTC10042 in the NCBI database) were 99.89% (versus strain DSM43995) and 99.87% (versus strain NCTC10042). Also, the ANI values with respect to draft genomes of Mycobacterium heckeshornense (strain RLE) and Mycobacterium noviomagense (strain DSM45145), which are the mycobacterial species phylogenetically closest to M. xenopi  and DRR201557 (Illumina).The genome sequence and annotations of


Vigna mungo is cultivated in approximately 5 million hectares worldwide. The chloroplast genome of this species has not been previously reported. In this study, we sequenced the genome and transcriptome of the V. mungo chloroplast. We identified many positively selected genes in the photosynthetic pathway  and RNA polymerase genes  from the comparison of the chloroplast genome of V. mungo, temperate legume species, and tropical legume species. Our transcriptome data from PacBio isoform sequencing showed that the 51-kb DNA inversion could affect the transcriptional regulation of accD polycistronic. Using Illumina deep RNA sequencing, we found RNA editing of clpP in the leaf, shoot, flower, fruit, and root tissues of V. mungo. We also found three G-to-A RNA editing events that change guanine to adenine in the transcripts transcribed from the adenine-rich regions of the ycf4 gene. The edited guanine bases were found particularly in the chloroplast genome of the Vigna species. These G-to-A RNA editing events were likely to provide a mechanism for correcting DNA base mutations. The V. mungo chloroplast genome sequence and the analysis results obtained in this study can apply to phylogenetic studies and chloroplast genome engineering. Vigna mungo (L.) Hepper or black gram is a diploid plant with 2n = 2x = 22 chromosomes. It belongs to the family Leguminosae, subfamily Papilionoideae, clade Millettioid [Vigna species, which provides high-protein food [lettioid , and is ein food .Phaseolus [Glycine [Vigna [Cajanus [Vigna radiata has been sequenced [Chloroplast is an essential organelle that harbors about 120–130 genes in its own genome . The genhaseolus , Glycine[Glycine , Vigna [e [Vigna , and Caj[Cajanus  are exam[Cajanus . The chlequenced ,10, wherequenced ,12,13. Tequenced ,15. The equenced ,17. Receequenced . Moreoveequenced ,20,21. Fequenced  and saltequenced  and to cequenced . Pharmacequenced , interfeequenced , and proequenced . TherefoV. mungo. We identified positively selected genes in the V. mungo chloroplast genome by performing comparative analysis with chloroplast genomes from the related legume species that preferred different climates to grow. We additionally used PacBio isoform sequencing (Iso-seq) reads to show polycistronic transcription units and used Illumina short reads to identify RNA edited sites in different V. mungo tissues.In this study, we sequenced the chloroplast genome of V. mungo was 151,294 base pairs (bp) long (V. mungo chloroplast genome to the GenBank Nucleotide Database with the accession number MT418597. The average depth coverage across the genome was 61.98× (V. radiata (mungbean) [V. mungo chloroplast genome has 108 genes including 75 protein-coding genes, 29 tRNA genes, and four rRNA genes  boundary. The ψrpl33 was shown be specific to Phaseolinae chloroplast genomes, while ψrps16 was found to be absent in the Ceratonia siliqua and Glycine max chloroplast genomes [We obtained 4.4 Gb from 15 million Illumina paired-end (PE) reads for chloroplast genome assembly. The raw reads were deposited to the National Center for Biotechnology Information (NCBI) database under the BioProject accession number of PRJNA623719. The assembled chloroplast genome of bp) long A. We havs 61.98× . The genNA genes . The LSC genomes .V. mungo chloroplast genome sequenced in this study with 11 other Leguminosae chloroplast genomes that were downloaded from the GenBank database. Five of these species were V. radiata, Vigna angularis, Vigna unguiculata, and Phaseolus vulgaris, and G. max was from the tribe Phaseoleae. The first four species are member of the subtribe Phaseolinae, while G. max is from the subtribe Glycininae. The other four species were Glycyrrhiza glabra, Cicer arietinum, Pisum sativum, and Medicago sativa from the IR-lacking clade. Two other species were Arachis hypogaea from the tribe Dalbergieae and Ceratonia siliqua from the subfamily Caesalpinioideae.We compared the Arachis and Ceratonia, respectively. V. mungo formed a monophyletic group with V. radiata at the deepest branch of the Phaseolinae clade, indicating a close evolutionary relationship between these two species. We also calculated the phylogenetic tree of these species with 57 other species to confirm the placement of these Fabales species in relative to other diverse species. This calculation used 38 orthologous protein sequences from the chloroplast genomes. In resulted phylogenetic tree , followed by ndhF (7 sites), atpF and rps2 (5 sites), ndhH (4 sites), matK, psbT and rpoC1 (3 sites), and other genes (<2 sites)  to synonymous (Ks) substitution ratio (Ka/Ks) for each of the 60 protein-coding genes shared by the analyzed legumes. The Ka/Ks ratio showed the strength and mode of natural selection on the protein-coding genes . The Ka/a/Ks > 1 ,35) incl2 sites) .P. sativum (dry pea) and C. arietinum (chickpea) are members of the cool-season food legumes, while V. radiata (mungbean), V. mungo (black gram), and P. vulgaris (common bean) grow well under hot and humid conditions [rbcL, matK, ndhF, atpF, and rpoC2, in the V. mungo chloroplast genome were found to be involved in ecological adaptations in other plant species. For example, an adaptive evolution in the rbcL gene is linked with photosynthetic performance under temperature, drought and carbon dioxide concentration variations [2 affinity, which limits the rate of carbon assimilation and net photosynthesis [rbcL positive selection that was widespread in many land plants [matK was also identified in many plants suggesting that this gene experienced different ecological selective pressures [Citrus, positive selection of matK and ndhF was considered to play roles in the adaptation of Australian species to hot and dry climate [ndhF was believed to be able to delay drought-induced senescence in Haberlea rhodopensis [Quercus species and evergreen Quercus species revealed that the positive selection of atpF and rpoC2 played a role in the adaptation of the deciduous Quercus species to the winter or the dry season [Amino acid changes due to the selection pressure can drive evolution within a specific taxonomic lineage . Positivnditions . We usedriations . Rubiscoynthesis . The impd plants . The posressures ,42. In t climate . In addidopensis . The geny season . The posOryza that were grouped into shade-tolerant and sun-loving rice species showed a correlation between the positive selection of photosynthetic genes, e.g., rbcL, ndh, and psb, and the adaptation of rice species to different sunlight levels [In addition to climatic conditions, some legumes prefer different light spectrums as shown by a study indicating that the red light significantly suppressed pod growth in soybean, but promoted the growth in cowpea . A studyt levels . The posV. mungo chloroplast genome.The transcriptional regulation of chloroplast genes has characteristics found in both prokaryotes and eukaryotes . PlastidV. mungo chloroplast genome. We retrieved 10 monocistronic units, seven dicistronic units, and 11 polycistronic units . The degradation of a long polycistronic unit into smaller oligocistronic units could result from intercistronic processing activities [Arabidopsis thaliana, the binding of HCF152 to the intergenic region between psbH and petB was involved in the digestion of the psbB-psbT-psbH-petB-petD polycistronic units by exonucleases to psbB-psbT-psbH tricistronic units and petB-petD dicistronic units [psbB-psbT-psbN-psbH-petB and psbN-psbH-petB-petD polycistronic units, suggesting that there may be binding sites in the intergenic region between psbT and psbN for a protein with a similar function to HCF152 in the V. mungo chloroplast genome.This study used PacBio Isoform Sequencing (Iso-Seq) to identify polycistronic transcription units of the ic units B. We foutivities . For exaic units . In thisaccD polycistronic transcripts in V. mungo were transcribed from the rps16 promoter, locating further upstream of accD, due to a 51-kb inversion between accD/rps16 and rbcL/trnK-UUU. We observed that the promoter sequence of accD in V. mungo is truncated and lacks the GAA-box compared to that of C. siliqua, a legume without the 51-kb inversion. These results suggested that the 51-kb inversion affect transcriptional regulation of the accD polycistronic transcripts.The Iso-Seq data also provided evidence that the ycf3 and clpP) with two introns in the V. mungo chloroplast genome. We have mapped our iso-seq reads to the genome. However, the results showed no evidence of an alternative splicing event in either of these genes.The iso-seq reads also allow for the detection of alternative splicing events. There are two genes  that was commonly found in all five tissues  are absent from core asterids and Poaceae but are retained in most of rosids [Vicia faba and C. arietinum are two legumes that lost CLB19 and clpP-559 editing [clpP can be used as a good marker for studying phylogenetics.In this study, we identified 34 RNA editing events in 18 plastid genes from leaf, shoot, flower, fruit, and root tissues of seq data . Ninety-thaliana . The num tissues . Our Ka/t genome . clpP hasurvival . The disity rate . clb19 alpP clpP-6 that walpP clpP-6 that waycf4 from leaves , although they had low editing efficiency. G-to-A editing has also been reported in ndhF, rpoC2, and ycf1 genes of the A. thaliana [Betula platyphylla [V. mungo chloroplast genome, we found the edited G bases in two A-rich regions . Frozen tissues (CN80 accession) were homogenized, and DNA was extracted using QIAGEN Genomic-tip 100/G based on the manufacturer’s protocol . We assessed DNA integrity with the Pippin Pulse Electrophoresis System . Total RNA was isolated from leaf, root, stem, flower, and three-week-old pod using the CTAB buffer . RNA was extracted from the aqueous phase three times using 25:24:1 phenol:chloroform:isoamylalcohol. Poly(A) enrichment with Dynabeads mRNA Purification Kit  was used to get mRNAs.For DNA sequencing, we prepared sequencing library from a total of 1.25 ng of high molecular weight DNA using the Chromium Genome Library Kit & Gel Bead Kit v2, the Chromium Genome Chip Kit v2, and the Chromium i7 Multiplex Kit following the manufacturer’s instructions . We used a single lane of Illumina HiSeq X Ten (2 × 150 bp paired-end reads) to sequenced the library.For RNA sequencing, RNA integrity was assessed with a Fragment Analyzer System . To obtain short-read RNA sequences, six RNA libraries (one for each tissue type) were prepared according to the protocol reported in Pootakham et al., 2018. Briefly, 200 ng of poly(A) mRNA was used to construct a library using the Ion Total RNA Sequencing Kit . The libraries were sequenced on the Ion S5 XL using the Ion 540 TM chip . For RNA isoform sequencing, two PacBio Iso-seq libraries were prepared following protocols described in  using thThe raw reads were trimmed and filtered for high quality reads by fastp program with default parameters . We usedV. mungo chloroplast genome with chloroplast genome of V. radiata (NC_013843.1), V. angularis (NC_021091.1), V. unguiculata (NC_018051.1), P. vulgaris (NC_009259.1), G. max (NC_007942.1), G. glabra (NC_024038.1), C. arietinum (NC_011163.1), P. sativum (NC_014057.1), M. sativa (NC_042841.1), A. hypogaea (NC_037358.1), and C. siliqua (NC_026678.1). The sequence of the C. siliqua chloroplast genome was used as a reference for visualizing the LCB relative locations. To analyze IR expansion/contraction, IRscope [We used Mauve aligner software  with def IRscope  was usedp-value cutoff of 0.05 was used to compared between the fitness of the alignment data to the M8 model (with the Ka/Ks > 1 parameter) and the M7 model (without the Ka/Ks > 1 parameter). Thereby, the identified positively selected genes had a high probability to experience positive selection than neutral selection.The codon-based alignment of the protein-coding gene sequences were conducted with the integrated MUSCLE alignment program of MEGA-CC software ,70. The V. mungo chloroplast genome with Bowtie 2 software [We mapped RNA short reads from the Illumina deep RNA sequencing to the software . RNA edisoftware . We seleV. mungo chloroplast genome using BLASTN [−3 E-value cutoff. We selected the Iso-seq reads that had a 100% coverage and >95% identity alignment with the chloroplast genome sequence.To find genes on a polycistronic transcription unit, we aligned PacBio Iso-seq reads to the g BLASTN  with a 1V. mungo chloroplast. The sequence and structure of the assembled chloroplast genome was consistent with the chloroplast genome of the closely related species of V. mungo. Our comparisons between the chloroplast genome of V. mungo and other legume species that grew in different habitats revealed many positively selected genes. Our RNA sequencing results showed that the 51-kb DNA inversion conserved among Papilionoideae legume species could affect the transcriptional regulation of the accD polycistronic transcription. Finally, we found RNA editing events that change guanine to adenine in the RNA molecules that were transcribed from the adenine-rich regions of the ycf4 gene in the V. mungo chloroplast genome. The chloroplast genome sequence and the knowledge gained from this study can be used for plant phylogenetic studies and for the engineering of the chloroplast genome of V. mungo and other related legume species.In this study, we sequenced the genome and transcriptome of the


Carduus, commonly known as plumeless thistles, is a genus in the Asteraceae family that exhibits both medicinal value and invasive tendencies. However, the genomic data of Carduus  have not been sequenced.Carduus species using the Illumina Miseq sequencing system and Geneious Prime. Phylogenetic relationships between Carduus and related taxa were reconstructed using Maximum Likelihood and Bayesian Inference analyses. In addition, we used a single nucleotide polymorphism (SNP) in the protein coding region of the matK gene to develop molecular markers to distinguish C. crispus from C. acanthoides and C. tenuiflorus.We sequenced and assembled the chloroplast genome (cpDNA) sequences of three C. crispus, C. acanthoides, and C. tenuiflorus ranged from 152,342 bp to 152,617 bp in length. Comparative genomic analysis revealed high conservation in terms of gene content  and gene order within the three focal species and members of subfamily Carduoideae. Despite their high similarity, the three species differed with respect to the number and content of repeats in the chloroplast genome. Additionally, eight hotspot regions, including psbI-trnS_GCU, trnE_UUC-rpoB, trnR_UCU-trnG_UCC, psbC-trnS_UGA, trnT_UGU-trnL_UAA, psbT-psbN, petD-rpoA, and rpl16-rps3, were identified in the study species. Phylogenetic analyses inferred from 78 protein-coding and non-coding regions indicated that Carduus is polyphyletic, suggesting the need for additional studies to reconstruct relationships between thistles and related taxa. Based on a SNP in matK, we successfully developed a molecular marker and protocol for distinguishing C. crispus from the other two focal species. Our study provides preliminary chloroplast genome data for further studies on plastid genome evolution, phylogeny, and development of species-level markers in Carduus.The cpDNA sequences of  Carduus L. , commonly known as plumeless thistles, comprises 90 species native to Eurasia and Africa , C. tenuiflorus Curtis (sheep thistle), C. pycnocephalus L. , and C. crispus Guirão ex Nyman (welted thistle), all of which originate in Eurasia and Africa, are considered invasive in North America , its chloroplast genome has not been sequenced. It is therefore worthwhile to study the genome of Carduus, and particularly that of C. crispus, which has potential medicinal benefits.d Africa . Severalntinents . Four sp America . Carduusin Korea . This spl bracts . Among Cdiseases . Specifid cancer . While CmatK region (Taraxacum kok-saghyz LE Rodin), horseweed (Conyza sp.), Indian Chrysanthemum (Chrysanthemum indicum L.), the endemic herb Aster savatieri Makino, and the invasive plant Tithonia diversifolia (Hemsl.) A Gray .In most angiosperms, the chloroplast genome (cpDNA) contains genes essential to photosynthesis . GenomicK region . Among t) A Gray . In addi) A Gray . cpDNA s) A Gray . Previouear loci . Howeverted taxa . In partTyrimmus . As suchCarduus crispus, C. acanthoides, and C. tenuiflorus, which exhibit both invasive tendencies and potential medical utility (particularly C. crispus). We then conducted comparative genomic analyses to explore genomic diversity among the three species with respect to highly variable regions, and the types and numbers of repeats. In addition, we reconstructed the formerly ambiguous relationship between Carduus and related taxa based on 78 protein-coding regions and non-coding sequences. Finally, we developed a specific molecular marker for C. crispus based on a SNP in the matK gene. This molecular marker provides useful information for managing C. crispus invasions, particularly with respect to the identification of immature (vegetative) individuals, which tend to be morphologically similar to other Carduus species . This molecular marker may also support positive identification of C. crispus for medical usage.We used next-generation sequencing (NGS) to sequence and characterise the chloroplast genomes of C. crispus, C. acanthoides, and C. tenuiflorus were collected and dried in silica gel powder for NGS analysis. In addition, to test the efficiency of molecular markers (PRJNA645567). Using Geneious Prime, filtered NGS data for each species were mapped to the reference chloroplast genome sequences of Arctium lappa (NCBI accession number MH375874), Saussurea polylepis (MF695711), Carthamus tinctorius (KP404628), Centaurea diffusa (KJ690264), Silybum marianum (KT267161), Cirsium arvense (KY562583), Cynara humilis (KP299292), and Atractylodes chinensis (MG874805) to isolate cpDNA reads of which the similarity to reference was over 95%. Isolated cpDNA reads were then assembled using the de novo function in Geneious to create various contigs of chloroplast genome sequences. The newly created contigs were de novo re-assembled to construct complete cpDNA sequences for each focal species. We confirmed the results from Geneious Prime by reconstructing the complete cpDNA of Carduus using NOVOplasty and following Dierckxsens et al. (2017). Using Geneious Prime, gene content and order of sequenced cpDNA were annotated based on existing complete cpDNA sequences of other Asteraceae taxa. Annotations that had over 95% similarity in comparison with references were retained, and the start and stop codons in the protein coding regions were verified  was used as an outgroup;  markers , leaves  markers . High-qu markers . Raw datverified . tRNA severified . Completg OGDraw . Complethttp://iqtree.cibiv.univie.ac.at), using 1,000 bootstrap replications to calculate branch support values (http://tree.bio.ed.ac.uk/software/figtree/) was used to visualise phylogenetic trees. Other datasets, including whole chloroplast genomes (excluding one IR region), non-coding regions of cpDNA, and hotspot regions derived from the cpDNA of Carduus species, were used in phylogenetic analysis in addition to protein-coding regions  and inner primer pairs  were 0.75 pM and 0.5 pM, respectively. The PCR procedure consisted of 1 min at 94 °C, followed by denaturing for 1 min at 94 °C, annealing for 40 s at 55 °C, an extension stage of 50 s at 72 °C, and an additional extension of 7 min at 72 °C.The complete ify SNPs ; Fig. S1 species . Based o species . Primer  samples . By cont species . Reactiorps19 and ycf1 coding regions, respectively, but were longer in C. acanthoides (rps19 = 61 bp and ycf1 = 568 bp) than in the other two species (rps19 = 60 bp and ycf1 = 565 bp). In addition, pairwise identity indicated that C. crispus is more similar to C. tenuiflorus (99.6%) than C. acanthoides (99.2%). Observations of nucleotide diversity indicated that 119 of 131 surveyed regions differ among the three focal species (psbC-trnS (0.0171) and psbH-petB (0.0161) regions. The highest value in coding regions was 0.00696 for ycf1  were identified in C. tenuiflorus and C. acanthoides; by contrast, only forward and palindrome repeats were found in C. crispus. Forward repeats were more abundant than reverse and palindrome repeats . Similarly, trnT_GGU was completely deleted or pseudogenised in the tribe Gnaphalieae  were used to reconstruct phylogenetic relationships, the position of Carduus remained unresolved  between the chloroplast genomes of Carduus and those of related taxa, the newly acquired cpDNA sequences have value as a resource in future studies of the evolution of the chloroplast genome in Carduoideae and Asteraceae. Additionally, the 78 protein-coding regions of the chloroplast genome revealed uncertainty regarding the position of Carduus within the subfamily Carduoideae, and suggested the need for additional studies to reconstruct relationships not only among thistles, but among other members of the Asteraceae as well. The methods and protocols used in developing molecular markers for C. crispus are easy to apply and may be useful as a standard method in other studies of Asteraceae species.In this study, we provided the first complete cpDNA sequences for 10.7717/peerj.10687/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.10687/supp-2Data S1Click here for additional data file.10.7717/peerj.10687/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.10687/supp-4Supplemental Information 4Carduus chloroplast genomes. Table S4: Feature of SSR in three Carduus species. Table S5: Feature of repeats in three Carduus species. Figure S1: The alignment of matK among three Carduus. The asterisk indicates the positions of single nucleotide polymorphism (SNP). The red square indicates SNP site for design primer pairs. Figure S2: The design of the primer pairs based on SNP site that is specific for Carduus crispus. Figure S3: The Bayesian Inference tree of Carduus and related taxa inferred from whole cpDNA sequences (A), non-coding regions of cpDNA (B), and eight hotspot regions (C).Figure S4: The PCR results of specific primer pairs for Carduus crispus. The combination of matK_463F, matK_1162R, CD_SNP_F1, and CD_SNP_R1. The number from 1 to 4: Carduus crispus; from 5 to 8: Carduus acanthoides; from 9 to 12: Carduus tenuiflorus. Figure S5: The PCR results of specific primer pairs for Carduus crispus. The combination of matK_463F, matK_1162R, CD_SNP_F2, and CD_SNP_R2. The number from 1 to 4: Carduus crispus; from 5 to 8: Carduus acanthoides; from 9 to 12: Carduus tenuiflorus. Figure S6: The MAUVE alignment of chloroplast genomes among Carduus and related species.Table S1: List of species for phylogenomic analysis and whole cpDNA alignment. Table S2: List of regions for calculating Pi values. Table S3. Genes composition of the Click here for additional data file.10.7717/peerj.10687/supp-5Supplemental Information 5Click here for additional data file.


Scedosporium boydii infection initially misdiagnosed as aspergillosis. The patient presented to the hospital with shortness of breath and chest and abdominal pain. Laboratory tests revealed leukocytosis and elevated serum liver enzymes, myoglobin and lipase. He died of hypotensive shock and brain abscesses despite antibiotic treatment. Autopsy revealed invasive fungal infection in the heart, thyroid, and brain with presence of 45-degree angled, branching hyphae. The initial diagnosis of aspergillosis was made; however, further molecular studies identified the organism as S. boydii. This report reveals the potential pitfalls of morphologic diagnosis alone; and the necessity of other testing modalities to render an accurate diagnosis which is crucial for appropriate.Aspergillosis is a commonly diagnosed fungal infection. Histopathologic examination alone can have diagnostic pitfalls due to the overlapping of fungal morphology. We report a case of  Aspergillus species. In recent years, other filamentous fungal pathogens have emerged, such as species of Fusarium, Penicillium, Rhizopus, Rhizomucor, Scedosporium boydii , and agents of phaeohyphomycosis [Aspergillus remains the most common filamentous mold, the differentiation from other emerging filamentous fungal agents is important due to different treatment regimens. For example, innate resistance or erratic susceptibility to amphotericin B is a characteristic of S. boydii [Aspergillus and Mucorales. However, morphology is not always specific. Other diagnostic modalities including fungal culture and fungal ribosomal rRNA gene sequencing are necessary for a definitive diagnosis of the causative fungal agent. Here, we report an autopsy case of systemic invasive fungal infection in a patient with chronic renal failure. Blood and lung cultures submitted at the time of autopsy were negative. Microscopic examination of tissue sections showed the presence of 45-degree angle branching fungal elements. Initial autopsy diagnosis was attributed to systemic aspergillosis. Subsequent broad-range fungal PCR and sequence analysis of DNA extracted from paraffin tissue sections revealed S. boydii (P. boydii) as the causative agent of the infection. Differentiation of S. boydii from Aspergillus species is very important for patient management so that the right antifungal agents can be administered.Over the last two decades, there have been an increased number of life-threatening systemic fungal infections due to both pathogenic and opportunistic fungi. High-risk populations include immunocompromised patients . One of the most frequently encountered filamentous fungal agents is omycosis –3. Altho. boydii , 5. In t3 to 61,000/mm3. The patient's blood pressure dropped precipitously, and he was treated with vasopressive drugs. The patient also had diarrhea; a subsequent colonoscopy was negative. Other laboratory tests were all normal including blood and sputum cultures, serology for CMV, Brucella species, Rickettsia rickettsii, serum alpha-fetal protein, smooth muscle antibody, mitochondrial antibody, antinuclear antibody, and direct Coombs test. With persistent hypotension, the patient developed atrial fibrillation and had decreased responsiveness. A brain CT scan was performed and showed multiple areas of decreased attenuation with focal mass effect and subacute intraparenchymal hemorrhage in the left frontal lobe  fistula, and a recent surgery for a new AV fistula in the left upper extremity one month prior to admission. Following admission, the patient was intubated, and initial laboratory tests were remarkable for leukocytosis, elevated liver enzymes with a negative hepatitis panel, and increased myoglobin, lipase, and creatinine. In addition, the patient exhibited azotemia, metabolic disturbance, and mild coagulopathy . Chest Xtal lobe . The difμm thickness, stained with hematoxylin eosin (HE), Gomori-Grocott's methenamine silver (GMS), and Gram stains.Numerous specimens from different organ systems were taken at the time of autopsy, fixed in 10% formalin and paraffin embedded. Paraffin sections were cut at 5 Paraffin-embedded tissues from the brain, heart, and thyroid lesions with the microscopic presence of fungal elements were studied. Deparaffinized sections were processed using the QIAamp DNA FFPE Tissue Kit  for DNA extraction. DNA sequencing for identification was performed by analysis of internal transcribed spacer region 2 (ITS2) and the 28S rRNA gene. Polymerase chain reactions (PCR) were performed using the primer pairs 5.8S-F (GTG AAT CAT CGA RTC TTT GAA C) and 28S-1 R (TAT GCT TAA GTT CAG CGG GTA) and D1/2-R (GGT CCG TGT TTC AAG ACG G) as previously described using 30–160 ng of extracted DNA [S. boydii.At autopsy, pertinent gross findings included multiple small hemorrhagic skin papules on the chest, severe atherosclerotic coronary artery disease, multiple necrotic nodular lesions in the heart, thyroid, liver, and lung, and multiple hemorrhagic lesions in the brain . MicroscS. boydii (previous naming-P. boydii) is a pathogenic species of the ascomycete genus Scedosporium. Members of the genus along with those of Pseudallescheria have been reclassified [S. auranticum, S. S. dehoogii, S. minutisporum, and S. desertorum. The previously named pathogenic species S. prolificans has been renamed Lomentospora prolificans along with L. inflatum. These species are ubiquitous filamentous fungi present in soil, sewage, and polluted water. S. boydii can colonize airways of patients with existing disease such as poorly draining bronchi or paranasal sinuses, and “fungus ball” formation in preformed cavities is similar to that seen in Aspergillus [S. boydii in immunocompetent patients are usually caused by traumatic events such as following implantation and penetrating injuries [µm (average 4.5 µm) [µm) and right-angle branching nonseptate hyphae [S. boydii fungal hyphae in tissue sections closely resembles that of Aspergillus species (µm (average 3.3 µm). Aspergillus seems to have 45-degree alternating side branching, whereas S. boydii shows 45-degree fork-shaped branching hyphae, in this case  . The maje hyphae . In this species  with 45-his case . However species . Culturel agents . Althougactivity . Echinoc


Nature Communications 10.1038/s41467-018-07858-8, published online 2 January 2019.Correction to: The original version of this article omitted from the author list the 19th author Andrew Fisher, who is from the Newcastle Fibrosis Research Group, Newcastle University Translational and Clinical Research Institute, Newcastle upon Tyne, UK. Consequently, the following was added to the Acknowledgements: ‘A.J.F. acknowledges funding support via a collaborative framework agreement between GSK and Newcastle University and from the NIHR Newcastle Biomedical Research Centre (Newcastle BRC).’ Additionally, the following was added to the Author Contributions: ‘A.J.F. obtained ethical approvals, consented patients and provided patient tissue and reviewed and approved the final manuscript.’ Finally, the following was added to the Competing Interests: ‘A.J.F. has no declarations directly related to this manuscript. His institution has received industry-academic research funding from GlaxoSmithKline R&D, Genentech, Pfizer and Nuformix. His institution has received consultancy fees from Mallinckrodt Pharmaceuticals.’ This has been corrected in both the PDF and HTML versions of the article.


Helicobacter pylori (H. pylori) is a common and universally distributed bacterial infection. However, in children, active gastritis and ulcer are rarely seen. H. pylori infection and to compare the clinical, endoscopic, and histopathological findings between infected and noninfected pediatric patients at Makassed General Hospital.  The aims of this study were to establish the prevalence of H. pylori was confirmed by a CLO test and/or its presence on biopsy specimens. Demographic data, clinical characteristics, endoscopic and histopathological findings, and gastritis score were recorded retrospectively.  Patients aged between 1 month and 17 years who underwent upper gastrointestinal endoscopy from January 2011 to January 2017 were included. The diagnosis of H. pylori infection was 16.5%. The infection was most commonly seen among children aged between 6 and 10 years (43%). A large number of family members were associated with increased risk of infection . Epigastric pain was more associated with H. pylori . Nodular gastritis was commonly seen in infected patients . Mild and moderate gastritis was seen more in infected versus noninfected patients .  During the study period, 651 children underwent upper gastrointestinal endoscopy. The main indication was abdominal pain (61%). The prevalence of H. pylori, other diagnoses should be considered since the infection are rarely symptomatic in children. Antral nodularity was associated with H. pylori infection; however, its absence does not preclude the diagnosis. Although epigastric pain was associated with  Helicobacter pylori (H. pylori) is one of the most common and universally distributed bacterial infection. It was identified in gastric biopsies of gastritis patients by Warren and Marshall in 1983 [H. pylori infection is intimately correlated to the age, race, family members, and country's socioeconomic level. A higher incidence is found in patients aged 5 to 10 years, high family members, and developing countries [H. pylori may lead to gastroduodenal ulcer or gastric cancer. However, these findings are rarely seen in children [H. pylori rarely give rise to symptoms in children in the absence of peptic ulcer disease (PUD) [H. pylori infection [H. pylori infection [ in 1983 . The ratchildren . Recent nfection . Howevernfection H. pylori infection and histologic gastritis, its specificity remains questionable [H. pylori infection according to ESPGHAN and NASPGHAN guidelines is either histopathological identification of the bacterium with at least one other positive biopsy-based test  or positive culture [Although nodular gastritis predicts tionable . In addi culture .H. pylori-infected and noninfected pediatric patients.The aim of this study was to investigate the clinical, endoscopic, and histopathological findings in This was a retrospective cross-sectional study, held at the Pediatrics Department of Makassed General Hospital over a 6-year period between January 2011 and January 2017.Patients aged from one month to 17 years who underwent upper gastrointestinal endoscopy for gastrointestinal symptoms, failed to thrive, and/or have short stature were included in the study. Patients who received H2 blockers, antimicrobials, or proton pump inhibitors (PPI) within 2-30 days of the procedure were excluded from data analysis.Data regarding the age, sex, anthropometric measures, place of residence, number of family members, indications of gastroscopy, and family history of PUD or gastric cancer were recorded. The endoscopic and histopathological findings were collected from the medical records.The materiel used for endoscopy was (Pentax®) video gastroscope (EG-2490K) (outer diameter: 8 mm). The examination was performed in the operating room under deep sedation or anesthesia. The possible endoscopic diagnosis included normal, superficial gastritis, nodular gastritis, and peptic ulcer disease. The endoscopic aspect of nodular gastritis was based on the irregular appearance of the mucosa as a cobblestone pavement.H. pylori was found on histopathologic examination in biopsy specimens, the diagnosis of H pylori infection was made.Five gastric biopsy specimens were collected from each patient: two from the gastric antrum , two from the corpus (from the greater curvature), and one from the area next to the incisura angularis for use in the CLO™ test (campylobacter-like organism test) on which the specimens were sent for histopathological analysis. No specimen was taken for culture since it is not available in our country. Biopsy specimens were fixed in 10% formalin embedded in paraffin and were stained with hematoxylin-eosin and modified Giemsa coloration technique. The same pathologist interpreted the biopsy results during the study period. When the CLO™ test showed red-violet color within 24 hours at room temperature and/or H. pylori density and the type of inflammation. The inflammation was divided into monocyte infiltration, neutrophil infiltration, glandular atrophy, or intestinal metaplasia and scored as mild, moderate, or severe using the visual analogue scale applied to microscopic examination results according to updated Sydney system [The histologic findings were used to classify the y system . The sumt-test was used to compare continuous variables. A multivariate analysis was conducted to control for confounding variables. A p value ≤ 0.05 was considered significant.The study was approved by the IRB. The Statistical Package for Social Sciences  program was used for data analysis. Bivariate analysis was carried out by using the chi-square for comparing categorical variables. Student's H. pylori positive and negative patients concerning the indications of upper gastrointestinal endoscopy.During the study period, 651 patients underwent upper gastrointestinal endoscopy. Ten of them were excluded for prior use of H2 blockers, PPI, and antibiotics. Abdominal pain was the most common indication for upper gastrointestinal endoscopy during the study period (67%), followed by failure to thrive (37.7%), suspected celiac disease (30.2%), short stature (25.5%), recurrent vomiting (16%), chronic constipation (7.6%), and hematemesis (2.8%). There was no statistical significance between H pylori infection was 16.5% (106/641). The CLO test was positive in 99/106 of H. pylori-positive patients (93.4%). p value (p < 0.0001). There was no significant difference between H. pylori-negative (Hp(-)) and H. pylori-positive (Hp(+)) groups concerning gender (p = 0.35). Higher weight (28 ± 14 vs. 21.6 ± 13.5 kg) and height (122.6 ± 20.6 vs. 108.5 ± 27.6 cm) were seen in H. pylori-positive versus H. pylori-negative patients, respectively, with a statistical significance (p < 0.0001). A higher number of family members were noted in H. pylori-positive patients versus H. pylori-negative patients  with a statistical significance (p = 0.02). There was no significant difference between the two groups regarding the place of residence, the educational level of the guardian, the family history, and the socioeconomic level.The overall prevalence of H. pylori-negative patients versus H. pylori-positive patients . However, epigastric pain was seen most frequently in H. pylori-infected patients . But the frequency and duration of epigastric pain did not differ between the two groups.According to the characteristics of abdominal pain, diffuse abdominal pain was seen most commonly in H. pylori-positive patients . In comparison, the gastric mucosa was normal in 44.8% of H. pylori-negative patients with superficial gastritis in 46.3% of them. No peptic ulcer disease was seen in infected patients. H. pylori-infected and noninfected patients. Chronic and active gastritis were the most common findings in H. pylori-infected patients . However, glandular atrophy and intestinal metaplasia were rarely seen in both groups. As for the gastritis score, mild and moderate scores were seen in H. pylori-infected patients . However, mild gastritis was seen in (14%) of H. pylori-negative patients. Severe degree of gastritis was seen only in 4.7% of infected patients. Superficial and nodular gastritis were the most common endoscopic finding in H. pylori infection in a pediatric age group based on invasive testing. Naous et al. in 2007 reported a fecoprevalence of H. pylori infection in asymptomatic Lebanese pediatric patients of 21% [H. pylori is used for the confirmation of eradication of the pathogen after the treatment and not as a diagnostic tool [To the best of our knowledge, our study is the first one done in Lebanon reporting the prevalence of s of 21% . But acctic tool .H. pylori infection varies worldwide ranging between 5 and 21% and reaching 79.4% in Bahrain [H. pylori infection was comparable to those reported in the literature. The highest prevalence was seen among children aged between 6 and 10 years and decreased thereafter. This finding was supported by Rowland et al. and Sykora et al. who reported an increased risk of H. pylori in the same age group [H. pylori infection in children more than 15 years [The prevalence of  Bahrain . In our ge group , 13. Sim15 years . This co15 years .H. pylori infection was comparable in boys and girls. Similarly, Martel and Parsonnet in their meta-analysis on 10 studies showed that there is no significant relationship between sex and incidence of H. pylori infection [H. pylori-infected children. This is supported by the lack of evidence that H. pylori infection could alter the growth in children [In our study, the prevalence of nfection . Surprischildren –19.H. pylori infection include poor socioeconomic conditions, family overcrowding, child care attendance, poor hygiene, and living with an infected family member and are higher in developing countries than in developed countries [H. pylori infection was associated with a higher number of family members with poor socioeconomic status.The risk factors of ountries . In our H. pylori infection, which was also reported by Yang et al. and Ng et al. [H. pylori infection with persistent upper gastrointestinal symptoms [Abdominal pain was the main indication of upper gastrointestinal endoscopy in our study. In addition, there was a statistical significant association between epigastric pain and g et al. , 21. Thisymptoms . HoweverH. pylori infection [Antral nodularity may be a pathognomonic macroscopic finding of childhood nfection . The posnfection , 23.H. pylori-infected children . Similarly, Luzza et al. found a high prevalence of antral nodularity in H. pylori-infected children followed by superficial gastritis . Erosions and ulcers were present in 1% of H. pylori-positive patients [H. pylori which necessitates the identification of the bacterium histopathologically. In our study, none of the H. pylori-infected patients developed an ulcer disease.In our study, superficial and nodular gastritis were most commonly seen among patients . Thus, tH. pylori was significantly associated with chronic and active gastritis  [H. pylori-infected patients . At the same time, H. pylori infection may be associated with normal appearing mucosa as reported by Cárdenas-Mondragón et al. [Histologically, Mazigh Mrad et al. found that infection with ctively) . Similarn et al. . In our H. pylori-infected children than severe gastritis . Our results were comparable to those reported by Cárdenas-Mondragón et al. who found a mild to moderate degree of gastritis in H. pylori-infected patients compared to severe one  [H. pylori immune reaction in children is completely different from adults due to the presence of T-regulatory cells which downregulates the inflammation and ulceration induced by H. pylori in children [According to gastritis score, mild and moderate gastritis were seen more commonly in ctively) . This cochildren .H. pylori infection. This highlights the importance of invasive testing of this pathogen in patients with persistent upper abdominal symptoms. Although antral nodularity was related to the histopathological presence of H. pylori, its absence does not exclude the diagnosis. As a result, the combination of the clinical, endoscopic, and histopathological findings remains crucial for a definitive diagnosis of H. pylori infection.In our study, we found a positive correlation between epigastric pain and


Pseudomonas is one of the most diverse bacterial genera identified in the environment. Genome sequence analysis has indicated that this genus can be clustered into three lineages and ten groups. Each group can adopt different mechanisms to thrive under zinc-depleted or high-zinc conditions, two environments that are frequently encountered during their environmental propagation. The response of three prominent Pseudomonas strains  to minimal inhibitory concentrations of zinc were compared using RNA-seq and ultra-performance liquid chromatography–tandem mass spectrometry analysis. Results demonstrated that the three strains shared only minimal similarity at the transcriptional level. Only four genes responsible for zinc efflux were commonly upregulated. P. aeruginosa PAO1 specifically downregulated the operons involved in siderophore synthesis and the genes that encode ribosomal protein, while upregulated the genes associated with antibiotic efflux and cell envelope biosynthesis. The membrane transporters in P. putida KT2440 were globally downregulated, indicating changes in cell permeability. Compared with P. aeruginosa PAO1 and P. putida KT2440, the most remarkable transcriptional variation in P. fluorescens ATCC 13525T is the significant downregulation of the type VI secretion system. Metabolite quantitative analysis showed that low concentrations of the metabolites involved in central carbon metabolism and amino acid synthesis were detected in the three strains. In summary, the cellular responses of the three strains under high-zinc condition is quite divergent. Although similar metal efflux systems were upregulated, the three strains employed different pathways to reduce zinc intrusion. In addition, zinc treatment can increase the difficulties of scavenging P. aeruginosa from its colonization area, and reduce the competitiveness of P. fluorescens in microbiota. Fertilizing use of these sludges may cause severe zinc pollution in soils.Zne et al.  analyzed. Klimek  reportedu et al.  used a fPseudomonas is a ubiquitous bacterial genus with high adaptability to various environments , and glucose-6-phosphate dehydrogenase (G6PDH), was markedly increased in P. fluorescens. Meanwhile, the most upregulated NADPH-generating enzyme in P. putida KT2440 was ferredoxin-NADP+ reductase and the transcription of ME and G6PDH remained unchanged. In addition to P. fluorescens and P. putida, P. aeruginosa represents another important phylogenetic group in the Pseudomonas genus  were challenged with external zinc at minimal inhibitory concentrations (MICs). Their responses were compared to provide a comprehensive understanding of the genes, the cellular processes and the cellular molecules that play crucial roles in zinc adaptation.In the current study, three prominent P. putida KT2440 (ATCC 47054), P. aeruginosa PAO1 (ATCC 47085), and P. fluorescens ATCC 13525T were obtained from American Type Culture Collection . These strains were routinely grown in a semi-synthetic cation-defined medium (CDM) . The MIC values of zinc ions were determined as described by Mergeay et al. . The MIC values were defined as the lowest concentrations of zinc sulfate at which no colonies were observed after 48 h of incubation at optimum temperature. For zinc treatment, the exponential cells grown in the CDM culture were harvested via centrifugation and inoculated into new CDM containing zinc sulfate at MIC to achieve a cell density of 107 CFU mL−1. After zinc treatment for 1 h, the cells were stabilized using RNA protect bacterial reagent  at a ratio of 2:1 (2 mL of reagent per 1 mL of culture). The control groups were grown in CDM without zinc. Three biological replicates were performed for each treatment.y et al. . The expThe total RNAs was extracted separately from each replicate using Trizol reagent . Residual genomic DNA contamination was removed from the total RNA samples via DNAase I digestion. The quality and quantity of total RNA was measured using an Agilent 2100 Bioanalyser. RNA-seq libraries were prepared and sequenced at the Beijing Genomics Institute . 150 bp pair-end reads were sequenced using an Illumina HiSeq 2500 platform .Pseudomonas genomes  controlling for the false discovery rate. Genes with average absolute fold changes higher than 2.0 and Padj values <0.01 were classified as differentially expressed genes (DEGs). The functional clusters of orthologous groups classification of all DEGs was performed automatically using the Integrated Microbial Genomes portal , the cell pellets were frozen with liquid nitrogen. To extract the metabolites, 50 mg of the frozen samples were dispersed in 1 mL of liquid containing methanol/acetonitrile/water (2:2:1), sonicated for 10 min at 0°C, and then centrifuged at 14,000 × g for 20 min at 4°C. The resulting supernatants were lyophilised and reconstituted in 100 μL of acetonitrile/water (1:1) before UPLC-MS/MS analysis.The bacterial samples used for the metabolome analysis were prepared as described by Chavez-Dozal et al. . Zinc-tr−1 CH3COONH4 and 25 mmol L−12O, Solvent A), and acetonitrile (Solvent B). The dilution programme was operated for 0.5 min 95:5 (B:A) then switched to 40:60 for 8.5 min before returning to 95:5 for 9 min further. The mass spectra were recorded by an Agilent 6540 Q-TOF mass spectrometer operating in the positive and negative ion modes using an Agilent electrospray ionization source. After each spectrum was normalized to the total spectral intensity, the profile data were imported into SIMCA-P+ software  and pareto-scaled to perform orthogonal partial least squares-discriminate analysis (OPLS-DA). The significance of the metabolites was ranked using the variable importance in projection score from the OPLS-DA model. For the univariate analysis of a specific metabolite, statistical significance was determined using Student's two-sample t-test. The metabolites with average absolute fold changes higher than 2.0 and Padj values <0.05 were defined as statistically different.An Agilent 1290 HPLC system  equipped with an HILIC analytical column  was used to analyse the extract of each sample. The mobil phase was water  plate that contained more than 0.7 mmol L−1 zinc completely inhibited its colony formation. Therefore, in the following RNA-seq and UPLC-MS/MS analysis, 0.7, 1.1, and 0.4 mmol L−1 zinc sulfate were used to compare the zinc-induced cellular responses in P. aeruginosa PAO1, P. putida KT2440, and P. fluorescens ATCC 13525T.Given that metal ions exert does-dependent effects on bacterial cells, comparing the cellular responses of different strains at the same zinc stress level is important. In this study, the MIC of ZnSOP. aeruginosa PAO1, P. putida KT2440, and P. fluorescens ATCC 13525T are provided in P. aeruginosa PAO1 with their control samples identified 529 genes that were differently transcribed. Amongst which, 406 genes, 7.1% of the total coding DNA sequence (CDS) in the genome, were upregulated, whereas, 123 (2.2%) genes were downregulated. The comparison of the zinc-treated P. putida KT2440 with their control bioreplicates showed that, the expression of 607 genes was significantly altered, with the majority  displaying significant induction in transcription compared with those  demonstrating decreased transcription. The lowest number of DEGs was observed in P. fluorescens ATCC 13525T, with only 48 induced genes and 19 repressed genes  during the zinc treatment of ed genes . In genesm class , particucadA, cadR, czcR, and czcS) commonly upregulated by the three strains were involved in metal efflux. cadA has been recognized as an efficient P-type ATPase that moves several divalent heavy metal ions out of the cellular membrane , were strongly upregulated (>5 fold), whereas oprD and kguT were mutually downregulated. In the other two groups, genes associated with central metabolism and those responsible for protein folding and degradation were generally upregulated. For P. fluorescens, the most prominent response mutually identified in P. fluorescens and P. aeruginosa was the upregulation of acyl-CoA dehydrogenase  and protein-tyrosine-phosphatase (RS10450). A ton-dependent receptor (RS23050) and a non-ribosomal peptide synthatase were markedly down-regulated (RS27315) (P. fluorescens and P. putida shared three more transcripts (All the four DEGs (RS27315) . In addinscripts . Two of nscripts .P. aeruginosa PAO1 are shown in P. aeruginosa to alleviate zinc-induced toxicity. The proton-translocating NADH:ubiquinone oxidoreductase complex (nuoGHIJLMN) and the pyruvate dehydrogenase complex (PA3416-3417), which transforms pyruvate into acetyl-CoA were upregulated by ~2.3-fold, whilst the synthesis of ribosomal protein was significantly downregulated . A distinct group of operons involved in lipid A synthesis/modification was highly induced (arnBCADEF), suggesting that the P. aeruginosa cells were in a cell-envelope-stressed state. In support of this, there was also significant upregulation of other genes previously linked to bacterial cell envelope homeostasis, such as glmM (phosphoglucosamine mutase) and pagL (lipid A 3-O-deacylase). The direct replacement of iron from their binding sites has been recognized as an important mechanism for zinc to exhibit cytotoxicity. Therefore, it is reasonable that the transcription of pvdQAPNOFEDJ that responsible for pyoverdine biosynthesis was downregulated. Using UV-visible spectrophotometry analysis  reduced about 66.8% compared with control samples (121.4 μmol L−1)  .P. aeruginosa is an opportunistic pathogen, monitoring the transcriptional variation of pathogenicity related genes is important. The MIC of zinc strongly enhanced the transcription of mexRAB-oprM  was significantly activated. The overproduction of exopolysaccharide alginate caused mucoid conversion in P. aeruginosa   the transcription of genes from four functional groups, including metal transporting genes, genes associated with membrane homeostasis, antioxidant-encoding genes and genes involved in basic cellular metabolism  and arsenic (arsBR) efflux systems in P. putida KT2440. Such phenomenon was not observed in the other two strains.In our previous study, the transcriptional response of sporters , which iP. aeruginosa PAO1 and P. putida KT2440, the downregulation of type VI secretion system (T6SS) was the most remarkable feature of P. fluorescens ATCC 13525T under zinc stresses   . TherefoPseudomonas strains . The metabolite profiles of P. aeruginosa and P. putida shared more similarity, 18 metabolites were commonly regulated  and idh (isocitrate dehydrogenase) in P. putida were only about 70% of that calculated in the cells without zinc treatment, and the transcription of eda  were downregulated about 1.7 and 1.5-fold in P. putida and P. fluorescens, respectively. The metabolites involved in amino acids metabolism were also reduced. For example, a wide range of amino acids  or intermediates involved in their biosynthesis were decreased in P. putida. And lower concentrations of metabolites involved in Arg, Glu, Lys, Cys, and Met metabolisms were observed in P. aeruginosa.Considering that large quantities of DEGs identified in the transcriptome analysis were genes associated with metabolism, we detected the concentrations of metabolites in these bacterial cells. Metabolic profiles of whole-cell extracts were obtained from zinc-treated and control samples exponentially cultured in CDM. Six replicates were performed for each sample. A total of 83 annotated metabolites were significantly changed, and approximately 76% of which were down-regulated. Only two metabolites were commonly regulated by the three egulated . Howevermilarity . SeveralPseudomonas is one of the most diverse bacterial genera, comprising ~200 species isolated from sources ranging from vegetation to water and contaminated soil to animal clinical samples  and P. putida . High percentages of branched amino acids were observed, indicating that these dipeptides were not generated by protein degradation. The changes of Ile/Leu/Val containing short peptides were also observed by Jousse et al.  and proline. Proline has been reported to play important roles in protecting protein denaturation and stabilizing protein synthesis, the upregulation of which commonly occurs in cells under heavy metal stresses  and P. putida KT2440  was observed (<2 fold), the presence of high concentrations of nucleotide and base may be produced by the recycling of denatured nucleic acid.As transcriptome analysis showed that large quantities of DEGs identified in e et al. . Their mPseudomonas strains from different phylogenetic groups. The analysis of the obtained transcriptomic profiles showed that 1,120 genes transcriptionally changed by more than twofold. The exclusion of cytoplasmic ions and reducing the unexpected intrusion of excess zinc are important for P. aeruginosa, P. putida, and P. fluorescens to withstand external zinc stress. Metabolic analysis showed that low concentrations of the metabolic intermediates involved in central carbon metabolism and amino acid biosynthesis were identified. Dipeptides that contained branched-chain amino acids may play important roles during their zinc adaptation.In this study, we compared the zinc-induced cellular responses of three prominent PRJNA606809.The RNA-seq datasets for this study can be found in the NCBI short read archive database under the bioproject accession number LL and PL conceived the experiment. LL, WL, and JC performed the experiment. LL and PL wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Seed dispersal is crucial to gene flow among plant populations. Although the effects of geographic distance and barriers to gene flow are well studied in many systems, it is unclear how seed dispersal mediates gene flow in conjunction with interacting effects of geographic distance and barriers. To test whether distinct seed dispersal modes  have a consistent effect on the level of genetic connectivity  among populations of riverine plant species, we used unlinked single-nucleotide polymorphisms (SNPs) for eight co-distributed plant species sampled across the Rio Branco, a putative biogeographic barrier in the Amazon basin. We found that animal-dispersed plant species exhibited higher levels of genetic diversity and lack of inbreeding as a result of the stronger genetic connectivity than plant species whose seeds are dispersed by water or wind. Interestingly, our results also indicated that the Rio Branco facilitates gene dispersal for all plant species analyzed, irrespective of their mode of dispersal. Even at a small spatial scale, our findings suggest that ecology rather than geography play a key role in shaping the evolutionary history of plants in the Amazon basin. These results may help improve conservation and management policies in Amazonian riparian forests, where degradation and deforestation rates are high. FST; The proportion of total genetic variability that resides among populations  have a consistent effect on the level of historical and contemporary  genetic connectivity among populations of riverine plant species . We alsoAn adequate understanding of gene flow patterns and genetic diversity in the Amazon can help guide effective conservation and management policies  of riverThe Rio Branco is a medium-sized river that has been used as a model system in various studies due to its size. This is due to the fact that narrow and wide rivers tend to affect gene flow in various taxa in the same way, regardless of their dispersal modes. The Rio Branco in particular has been shown to represent an important barrier to gene flow for birds e.g.,  and primAdenocalymma schomburgkii (DC.) L.G. Lohmann (Bignoniaceae), Amphirrhox longifolia (A. St.-Hil.) Spreng. (Violaceae), Anemopaegma paraense Bureau & K. Schum (Bignoniaceae), Bignonia aequinoctialis L. (Bignoniaceae), Pachyptera kerere (Aubl.) Sandwith (Bignoniaceae), Passiflora spinosa (Poepp. & Endl.) Mast. (Passifloraceae), Psychotria lupulina Benth. (Rubiaceae), and Tanaecium pyramidatum (Rich.) L.G. Lohmann (Bignoniaceae). These species were chosen due to their varied dispersal modes  and combined this information with data available from the same location for A. longifolia, P. spinosa, and P. lupulina  on population structure, while considering distinct dispersal modes. Toward this end, we collected new genomic data for five plant species  is the largest tributary of the Rio Negro; it is located on the Guiana Shield, within a sediment-rich “white-water” river ecoregion, and flows southward into the Negro-Amazon Rivers , A. longifolia (n = 78), A. paraense (n = 84), B. aequinoctialis (n = 78), P. kerere (n = 84), P. spinosa (n = 78), P. lupulina (n = 78), and T. pyramidatum (n = 78). Those individuals were distributed among 14 sampling locations situated on both banks (left and right) of the Rio Branco during the wet seasons of 2015 and 2016  while reducing the number of samples randomly, from six to two using a custom script in R (described by A. schomburgkii an FST (n = 6) = 0.014, 95% CI  compared with an FST (n = 2) = 0.013, 95% CI , for A. paraense an FST (n = 6) = 0.015, 95% CI  compared with an FST (n = 2) = 0.022, 95% CI , for B. aequinoctialis an FST (n = 6) = 0.052, 95% CI  compared with an FST (n = 2) = 0.051, 95% CI , for P. kerere an FST (n = 6) = 0.022, 95% CI  compared with an FST (n = 2) = 0.010, 95% CI , and for T. pyramidatum an FST (n = 6) = 0.032, 95% CI  compared with an FST (n = 2) = 0.033, 95% CI .For each sampling location, sample sizes ranged from two to six individuals . The comngifolia , P. spinlupulina . To ensuA. schomburgkii, A. paraense, B. aequinoctialis, P. kerere, and T. pyramidatum. The genomic libraries were created using a double digest RADseq (ddRAD) protocol . The genomic libraries were sequenced on Illumina HiSeq 2500 flow cell  to generate single-end 100 bp reads at The Centre for Applied Genomics, Toronto, Canada. More details of the protocol can be found in A. longifolia, P. spinosa, and P. lupulina  and reads missing the expected EcoRI cut site (options –barcode_dist 2 -q -e ecoRI). All sequences were processed in ustacks to produce consensus sequences of RAD tags. The program ustacks takes a set of short-read sequences from a single sample as input and aligns them into exactly matching stacks. For each species, a maximum-likelihood framework (m) to create a stack was set at three sequences, the maximum distance allowed between stacks was two nucleotides, and the maximum number of stacks allowed per de novo locus was three. The stacks assembly enabled the Deleveraging algorithm (-d), which resolves overmerged tags, and the Removal algorithm (-r), which drops highly repetitive stacks and nearby errors from the algorithm. The alpha value for the SNP model was set at 0.05. As reported by Files containing the raw sequence reads were analyzed separately for each plant species in Stacks 1.35  using deramework  was applk tests using the sequential Bonferroni procedure  and the inbreeding coefficient (Wright’s Fixation Index F) for each population. Population genetic statistics were averaged across loci using the R package diveRsity , we performed Pearson product-moment correlation test of uHE and HO with the geographic distance along the river course using the package ggpubr implemented in R 3.3.1 , we analyzed the five abiotically dispersed plant species for which new data was obtained using the methods described in In order to investigate how the river affects the population structure of plant species with different seed dispersal modes , we assessed the historical and contemporary genetic structure and connectivity of 1.We assessed the genetic structure and the connectivity patterns between sampling locations along and across the river using complementary genetic analyses. For all plant species, we investigated the historical relationships among sampling locations using RAxML 8.2  in post-processing. As the most likely number of k populations was unknown, it was treated as a simulated variable along with the MCMC simulations (1 ≤ k ≤ N), where N represents the number of sampling locations for each plant species, which ranged from 13  to 14 . The modal number of genetic groups of the best run  was considered as the number of genetic clusters (K).To more precisely characterize the geographic distribution of genetic variation, we applied a Bayesian model that uses a Markov Chain Monte Carlo (MCMC), as implemented in the R package GENELAND 4.0.6 . BayesiaFST; To investigate whether species with different dispersal modes displayed congruent patterns of genetic structure, we estimated pairwise genetic differentiation FST;  using thFST/(1 – FST); 4 permutation test of significance for the coefficient of correlation. A Mantel test was also applied to pairs of sampling locations situated (1) across river banks and (2) along each bank of the Rio Branco.In order to investigate the similarity between genetic and geographic distance, we conducted a test for isolation by distance (IBD)  between 4 random permutations.Finally, to examine the effect of the Rio Branco on the partitioning of genetic variation among sampling locations, we performed a Molecular Analysis of Variance AMOVA; . We defim) using a Bayesian inference framework implemented in BayesAss3-SNPs 1.1 (n) of six individuals per sampling location  of six, four, and two individuals in two populations. The results indicated that there are no differences in the migration rates (m) even when a small number of individuals  are used in the analysis .We obtained estimates of contemporary bidirectional migration rates  did not vary much among populations of A. schomburgkii, A. longifolia, A. paraense, B. aequinoctialis, P. kerere, P. spinosa, P. lupulina, and T. pyramidatum  to 0.376 (P. lupulina), while the mean observed heterozygosity ranged from 0.113  to 0.410  to 0.025 ± 0.007 95% CI (A. schomburgkii). Due to the excess of observed heterozygotes, the mean fixation index was negative for all animal-dispersed plant species  for A. schomburgkii, 0.015  for A. paraense, 0.052  for B. aequinoctialis, 0.025  for P. kerere, and 0.033  T. pyramidatum. For the three animal-dispersed plant species the average pairwise estimate of FST was 0.021  for A. longifolia, 0.031  for P. spinosa, and 0.031  for P. lupulina . Results were also not significant when applied between pairs of sampling locations across river banks . Likewise, results of simple matrix correlation between genetic and geographic distance were insignificant when applied separately along each bank of the Rio Branco , but significant congruence between water- and wind-dispersed plant species . These results indicated that the biotically dispersed plant species are more connected in the Rio Branco. All these findings were consistent when different datasets  were considered  and 3L , while the expected proportion of migrants from 4L into 2L , and 3L  were much smaller. Asymmetrical migration rates were also detected among some pairs of sampling locations for the animal-dispersed plant species A. longifolia, P. spinosa, and P. lupulina . For P. kerere, exclusively, we found a significant and positive association between contemporary migration rates and geographic distance, although this association was restricted to the right river bank of the Rio Branco  were observed for all but one plant species . Howevero Branco . This reLife-history traits, such as dispersal mode/ability can have an effect on the species’ response to geographical and evolutionary processes e.g., , with coA. schomburgkii, A. paraense, B. aequinoctialis, P. kerere, and T. pyramidatum) and those reported for three animal-dispersed plant species  shape the genetic structure of shrub plant species with different seed dispersal modes.The population structure observed for the five abiotically dispersed plant species studied here  in this large-sized river  and P. lupulina  showed higher levels of genetic variation  than A. longifolia. The effectiveness of seed dispersion in A. longifolia – a plant species with a mixed seed dispersal mode  – can be influenced by flood dynamics throughout the year, which may be more restricted in periods of non-flooding, especially if the plant is located far from the river banks. Thus, the contribution of ballistic dispersal may have a negative effect on the levels of intrapopulation genetic diversity in A. longifolia. Indeed, animal-dispersed plant species tend to have higher levels of genetic diversity than ballistic-dispersed plant species , life-history traits such as pollination mode and growth form  these results can help guide conservation and restoration policies in Amazonian riparian forests areas, where degradation and deforestation rates are rising . Yet, siAmphirrhox longifolia, Passiflora spinosa, and Psychotria lupulina populations are available for download from the Dryad Digital Repository (https://doi.org/10.5061/dryad.f53j0dk). For Adenocalymma schomburgkii, Anemopaegma paraense, Bignonia aequinoctialis, Pachyptera kerere, and Tanaecium pyramidatum populations, the SNP datasets are available for download at https://doi.org/10.5061/dryad.573n5tb6c.SNP datasets for the AGN and LGL designed the study and coordinated sample collection. AGN conducted molecular work, performed analyses, and led the writing of the manuscript with input from all co-authors. LLK provided analytical support and troubleshooting. CWD provided laboratory assistance. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.