Fritillaria belongs to the widely distributed Liliaceae. The bulbs of Fritillaria, F. ussuriensis and F. cirrhosa are valuable herbaceous medicinal ingredients. However, they are still used indiscriminately in herbal medicine. Identification and molecular phylogenic analysis of Fritillaria species are therefore required. Here, we report the complete chloroplast (CP) genome sequences of F. ussuriensis and F. cirrhosa. The two Fritillaria CP genomes were 151,524 and 151,083 bp in length, respectively, and each included a pair of inverted repeated regions  that was separated by a large single copy region , and a small single copy region . A total of 111 genes in F. ussuriensis and 112 in F. cirrhosa comprised 77 protein-coding regions in F. ussuriensis and 78 in F. cirrhosa, 30 transfer RNA (tRNA) genes, and four ribosomal RNA (rRNA) genes. The gene order, content, and orientation of the two Fritillaria CP genomes exhibited the general structure of flowering plants, and were similar to those of other Fritillaria species. Comparison of the six Fritillaria species’ CP genomes indicated seven highly divergent regions in intergenic spacers and in the matK, rpoC1, rpoC2, ycf1, ycf2, ndhD, and ndhF coding regions. We established the position of the six species through phylogenic analysis. The complete chloroplast genome sequences of the two Fritillaria species and a comparison study are useful genomic information for identifying and for studying the phylogenetic relationship among Fritillaria species within the Liliaceae.The genus  Fritillaria belongs to the Liliaceae, which consists of 140 known species. The bulbs of several Fritillaria species  are important ingredients in herbal drugs that are used in oriental medicine, and have great economic value in Asian countries. Fritillaria species are distributed in temperate regions of the Northern hemisphere [Fritillaria (F.) cirrhosa is mainly distributed in the alpine regions of Northwestern China  at altitudes of 3200–4600 m, whereas F. ussuriensis is distributed in the lowland regions  of the northeastern and the far-eastern areas of China, Russia, and Korea [Fritillaria ussuriensis and F. cirrhosa display morphological differences in their floral bud, bract, stem, and capsule [F. ussuriensis is 50–60 cm (maximum: 100 cm); the stem length of F. cirrhosa is less than 60 cm. Fritillaria cirrhosa has three bracts per flower, whereas F. ussuriensis has two. The tapals are yellow or yellowish green in F. cirrhosa; the petals are purple in F. ussuriensis. The capsules of F. cirrhosa are narrowly winged; the capsules of F. ussuriensis are wingless. While it is difficult to classify the plants according to the morphological characteristics of their bulbs, the flowering plants can be easily distinguished. The bulbs are divided into two and five types in the Korean Pharmacopoeia and the Chinese Pharmacopoeia, respectively [Fritillaria ussuriensis Maxim. and Fritillaria cirrhosa D. Don are used in different herbal medicines—namely, Fritillariae Ussuriensis Bulbus (Ping-bei-mu in Chinese) and Fritillariae Cirrhosae Bulbus (Chuan-bei-mu in Chinese), respectively. Fritillariae Cirrhosae Bulbus and Fritillariae Ussuriensis Bulbus have separately been used for clinical purposes in traditional Korean medicine. In detail, the former has been used to treat cough due to deficiency of the lung, asthenia of the viscera, and tidal fever, and the latter has mainly been used to treat cough due to exogenous dryness and deficiency of Um . Furthermore, Fritillariae Cirrhosae Bulbus and Fritillariae Ussuriensis Bulbus have been prescribed to treat pulmonary carbuncle and bloody sputum, respectively [Fritillaria have value in herbal medicine, different Fritillaria species are still used indiscriminately because of their morphological similarity and similar names [Fritillaria species  is required to identify medicinal plants and the drugs derived from them [The genus rom them .Marchantia polymorpha [matK and rbcL are used as universal plant DNA barcodes [Chloroplasts play an important role in photosynthesis and carbon fixation as well as in the biosynthesis of starch, fatty acids, and amino acids ,9. The clymorpha  was repolymorpha . With thlymorpha . Throughbarcodes .F. ussuriensis and F. cirrhosa CP genomes using the Illumina platform. This is the first comparative analysis of Fritillaria CP genomes in conjunction with previously reported CP genomes. This study aims to investigate global structural patterns of six Fritillaria CP genomes and also to discover highly divergent regions among the species. We also analyzed phylogenic relationships among the six Fritillaria species. The results provide basic knowledge on characteristics of Fritillaria species and enhance our understanding of Fritillaria species evolution within the Liliaceae.Here, we report the de novo assembly of F. ussuriensis (KY646166) and F. cirrhosa (KY646167) were collected from medicinal plant plantations, and the samples were used for CP genome sequencing. Fritillaria ussuriensis and F. cirrhosa were given identification numbers, and specimens were registered in the Korean Herbarium of Standard Herbal Resources (Index Herbariorum code KIOM) at the Korea Institute of Oriental Medicine (KIOM). The extraction of DNA was conducted with the DNeasy Plant Maxi kit , according to the manufacturer’s instructions. Illumina short-insert paired-end sequencing libraries were constructed and generated using the NextSeq platform  by LabGenomics, Korea. The CP genomes were obtained by the de novo assembly method from low-coverage whole-genome sequence derived from the Phyzen pipeline [Fritillaria hupehensis (NC024736) [Fresh leaves of pipeline . Trimmedpipeline . The pripipeline , and aliC024736) .F. ussuriensis and F. cirrhosa CP genomes was performed using DOGMA annotation [Fritillaria CP genomes were obtained using OGDRAW [Fritillaria CP genomes using the F. ussuriensis CP genome as reference [Fritillaria CP genomes were downloaded from GenBank .Gene annotation of notation , and mannotation . The cirg OGDRAW . GC contg OGDRAW . The mVIeference . Five FrF. ussuriensis and F. cirrhosa CP genomes were detected using MISA [SSRs in ing MISA  with theing MISA . IRs wering MISA . Fritillaria unibracteata (KF769142), Fritillaria taipaiensis (NC023247), Fritillaria hupehensis (NC024736), Fritillaria thunbergii (KY646165), Lilium superbum (NC026787), Lilium longiflorum (KC968977), Smilax china (HM536959), Acorus gramineus (NC026299), and Cocos nucifera (KX028884). A total of 71 protein-coding genes were aligned with MAFFT [Fritillaria species CP genomes were aligned using MAFFT, and the sequences were manually adjusted with Bioedit [A molecular phylogenetic tree was constructed using 74 protein-coding genes from 11 species. Among these 11 taxa, nine completed CP genomes were downloaded from NCBI: eus NC0269, and Cosis NC0237, Fritil Bioedit . To calc Bioedit . Window Fritillaria ussuriensis and Fritillaria cirrhosa, respectively. From the de novo assembly sequence that uses low-coverage whole-genome sequencing (WGS), we obtained ten and eight contigs covering the whole chloroplast genome sequences of F. ussuriensis and F. cirrhosa, respectively . In addition, both F. ussuriensis and F. cirrhosa chloroplast genomes were also separated by large single copy  and small single copy  regions (Fritillaria chloroplast genomes were AT-rich (63% in both species), but the LSC  and SSC  regions were more AT-rich than the IR regions , making the LSC and SSC regions more similar to other chloroplast genomes [Illumina sequencing generated 5.0 and 4.5 Gb of trimmed paired-end reads from ectively . Single ectively . Paired- program . Both F. regions , Table 1 genomes ,34,35.Fritillaria CP genomes. There were 111 and 112 predicted genes in F. ussuriensis and F. cirrhosa, respectively. Of these, 94 in F. ussuriensis and 95 in F. cirrhosa were unique to the LSC and SSC regions, and 18 were duplicated in the IR regions . The gene cemA encodes a heme-binding protein in the chloroplast envelope membrane, which was lost in land plants due to the introduction of an internal stop codon. [Fritillaria CP genomes had 30 tRNAs, with 17 duplicated genes including seven tRNAs , four rRNAs , and six protein-coding genes . They also had 18 intron-containing genes, among which 15 (nine protein-coding genes and six tRNA genes) had a single intron and two genes (ycf3 and clpP) had two introns each  that were duplicated in the IR regions. The rps12 gene was trans-spliced because the 5′ end was located in the LSC region and the 3′ end in the IR region. The trnK-UUU gene had the largest intron region (2613 bp in F. ussuriensis and 2562 bp in F. cirrhosa), including matK. The genes psbT, rpl2, and ndhD had the alternative start codon ACG, and rps19 started with GTG. Use of ACG and GTG as start codons are common features of a variety of genes in the chloroplast genomes of land plants [The gene content, order, and orientation were similar in  regions . The 111p codon. ,36. The ons each . Thirteed plants ,38,39,40Fritillaria chloroplast genomes consisted of protein-coding genes , 1.9% of tRNAs (2876 bp in both species), and 6.0% of rRNAs (9048 bp in both species). The remaining 40.1% consisted of intergenic regions, non-coding introns, and pseudogenes. The ycf1 gene located between the inverted repeat b (IRb) and the small single copy (SSC) region had premature stop codons in the coding sequence, and has been annotated as a pseudogene in other angiosperm chloroplast genomes [F. ussuriensis and 26,611 in F. cirrhosa. Among these codons, those for leucine and isoleucine were the most common in both Fritillaria genomes, as observed previously in other land plant CP genomes  were more abundant in non-coding regions than in coding regions, as evidenced by the presence of 63% of all SSRs in the non-coding regions of both genomes. Furthermore, most SSRs were located in the LSC region. In addition, 48 polymorphic SSRs were detected between the two Fritillaria CP genomes  or microsatellites are tandem repeat sequences consisting of 1–6 nt sequence motifs in prokaryotic and eukaryotic genomes ,49. SSRs genomes . Almost  genomes . Most SSF. ussuriensis and 13 tandem repeats in F. cirrhosa of more than 20 bp (F. ussuriensis (located in the trnT-UGU/trnL-UAA IGS) and 94 bp in F. cirrhosa (located in the trnG-UCC/trnR-UCU IGS). Four  tandem repeats represented the same region in both CP genomes. Four and six palindromic repeats were also detected in F. ussuriensis and F. cirrhosa, respectively  were similar in both CP genomes  ranged from 0 to 15.8% with a mean of 0.9%. Ten highly divergent loci included matK, atpI, trnY-GUA, trnE-UUC, trnT, ycf3, rps4, ycf4, petA, rpl16, rps3, rps19, ccsA, ndhD, and ycf1 (ycf4-petA (IGS) region showed 15.8% sequence variability. In the coding region, rpl16 showed the highest degree of nucleotide variability (4.3%). The ten loci had a much higher sequence divergence value than the other regions (Pi > 2.7%). Among the six Fritillaria CP genomes, Pi values varied from 0 to 2.1% with a mean of 0.4% .We performed multiple sequence alignments between six g mVISTA . The non regions . These r regions ,52. The and ycf1 . The mos of 0.4% . The locFritillaria species within the Liliaceae, 74 protein-coding sequences shared by 11 CP genomes were aligned  and maximum parsimony (MP) analyses revealed that six out of eight nodes had 100% bootstrap values. Both the ML and MP phylogenetic results strongly indicated that the genera—Fritillaria and Lilium—were clearly separated, and the six Fritillaria species were closely related within Liliaceae. Fritillaria cirrhosa and F. unibracteata formed a cluster. Subsequently, a monophyletic clade formed a cluster with F. taipaiensis, F. hupehensis, and F. thunbergii, which were related to F. ussuriensis as a monophyletic branch.Chloroplast genome sequences have been successfully used in numerous phylogenetic studies of angiosperms ,54. To i aligned . Two speFritillaria chloroplast genomes and applied comparative analysis to six Fritillaria species CP genomes. The two Fritillaria CP genomes contained a pair of IRs, and LSC and SSC regions. The two Fritillaria genomes have similar genome structures, gene orders, and gene contents, including introns and base compositions. They show characteristics that are typical of land plant CP genomes [Advances in NGS technologies make it possible to complete the entire chloroplast genome with the discovery of molecular markers ,56. We u genomes ,32.F. ussuriensis and F. cirrhosa CP genomes, respectively (Fritillaria species, F. cirrhosa and F. ussuriensis (Fritillaria species, including species identification, assessment of genetic diversity, ecological genetic studies, and evolution studies [SSRs are suitable molecular markers because they are distributed throughout the whole genome and display high polymorphism between species, locus-specific co-dominance, and high transferability . SSRs plectively . In addiuriensis . These a studies .Fritillaria genomes. The SC/IR boundaries showed only slight differences, for example, at ycf1 and ѱycf1 (ndhF, rps19, and trnH, had almost identical locations and sizes among Fritillaria. Multiple sequences of alignment between the six Fritillaria genomes indicated that the IR regions were more conserved than the LSC and SSC regions due to copy corrections by gene conversion in the IR regions [matK, rpoC1, rpoC2, ycf1, ycf2, ndhD, and ndhF. Previous studies reported similar divergent regions [rpoC2, rpoC1, and ycf1 genes are known to contain hotspots of sequence variation [Fritillaria CP genomes contain general hotspot regions for genetic variation.IR regions are the most conserved regions in the chloroplast genome . The connd ѱycf1 . This ph regions . The mos regions ,69. For  regions ,52. The ariation ,51,52,70Fritillaria species and found that the six species were well clustered according to the type of herbal medicine (F. ussuriensis could be clearly separated from other Fritillaria species, as previously reported [Fritillaria at the species level with 100% bootstrap values. Fritillaria hupehensis and F. thunbergii formed monophyly with 100% bootstrap values. Another monophyletic group clustered F. cirrhosa and F. unibracteata with F. taipaiensis. Fritillaria ussuriensis showed a distant genetic relationship to the other five Fritillaria species. In a previous study, a phylogenetic tree was constructed to verify the relationship between ninety-two Fritillaria species using a combined chloroplast region consisting of matK, rbcL, and rpl16. Ninety-two Fritillaria species were classified into eight Fritillaria subgenera while considering their geographic distributions [Fritillaria thunbergii, F. cirrhosa, and F. ussuriensis were well classified into the Fritillaria subgenus—consistent with our results. Although the phylogenetic tree comprised only a few Fritillaria species, our results are well supported by the phylogenetic relationships of Fritillaria species reported previously [Fritillaria species—in particular, details of their positions at the species level with 100% bootstrap values. However, the results suggested that additional large-scale genomic analyses using numerous accurately identified Fritillaria species are required to clarify the taxonomy and phylogenetic relationships of Fritillaria species at low taxonomic levels.Chloroplast genomes provide an abundant genomic resource for phylogenetic analysis, and many studies have used protein-coding sequences or whole chloroplast genome sequences in these analyses ,69,71. Imedicine . Furtherreported ,72. In tFritillaria CP genome sequences determined in this study could provide useful basic genomic information along with the previously reported Fritillaria species for studying phylogeny. The sequence variability from comparative analyses could be used as potential resources for further development of DNA molecular markers such as SSR or microsatellite marker. These additional developments could be used to study evolutionary properties and/or population genetics of Fritillaria species as well as DNA barcodes or SCAR markers.Molecular genetic tools using complete CP genome sequences provide an efficient and accurate way to authenticate herbal medicines. Universal DNA barcoding is widely used as a reliable genetic tool for identifying plant species; however, these methods are still limited in their ability to identify and discriminate medicinal plants at the species level for several taxa. The complete CP genome sequences could be useful for identifying plant species solely as a super DNA barcode, which provide high resolution at lower taxonomic levels . In addi


Mycoplasma species to daptomycin. Mycoplasma orale and M. arginini showed low-level resistance to daptomycin , whereas M. hyorhinis was high-level resistant (MIC = 32 mg/L). However, some Mycoplasma isolates developed higher resistance to daptomycin after failed treatments with inadequate doses or durations. An aminoglycoside (gentamicin) was still active against M. hyorhinis and could be used in Orientia cultures. For complete eradication of mycoplasmas in Rickettsia cultures, we recommend a 3-week treatment with daptomycin at 256 mg/L. In contaminated Orientia cultures, daptomycin at 32 mg/L was effective in eradicating M. orale, whereas either gentamicin or amikacin (100 mg/L) was effective in eradicating M. hyorhinis. Unlike each drug alone, the combinations of daptomycin plus clindamycin and/or quinupristin/dalfopristin proved effective in eradicating M. hyorhinis. In summary, our study demonstrated the in vitro anti-mycoplasma activity of daptomycin and its application as a new mycoplasma decontamination method for Rickettsia and Orientia cultures.Mycoplasma contamination detrimentally affects cellular functions and the growth of intracellular pathogens in cell cultures. Although several mycoplasmacidal agents are commercially available for sterile cell cultures, they are not applicable to rickettsia-infected cells. In our attempt to find an anti-mycoplasma drug for contaminated rickettsial cultures, we determined the susceptibilities of three common  M. orale, M. arginini, M. hyorhinis, M. hominis, M. fermentans, and Acholeplasma laidlawii [Rickettsia and Orientia [Mycoplasma organisms are a serious threat during cell culture because they grow well extracellularly in vitro, contaminate cell lines for a long time without being recognized, and easily spread to other cell lines. It has been reported that approximately 15–35% of continuous cell lines are contaminated by mycoplasmas . The comOrientia .Mycoplasma species have been identified, some of which are pathogenic in humans or animals [M. pneumoniae and M. genitalium. The former is a common cause of community-acquired pneumonia, but it can cause extra-pulmonary diseases as well [M. genitalium is an emerging causative agent of sexually transmitted infections [Mycoplasma species [Mycoplasma species are associated with diseases in humans or animals. M. hominis usually causes genitourinary tract infections; it can be an opportunistic pathogen causing disseminated diseases in immunocompromised hosts [M. hyorhinis colonizes the nasal cavity and can cause diseases in pigs. M. orale, M. fermentans (in humans), and M. arginini  are commensal bacteria in the oral cavity [Mycoplasma organisms are the smallest bacteria with the smallest genomes . Current animals . The bes as well . On the fections . The two species ,9. Both  species . However species ,11. On t species . In addied hosts . M. hyorl cavity ,14. All l cavity , which aOrientia cultures, only two mycoplasma species  were tested in that study [Orientia culture due to the intrinsic resistance of Orientia to these classes of antibiotics [Orientia spp., Rickettsia spp. are susceptible in vitro to fluoroquinolones and to high gentamicin concentrations [Several antibiotics can get rid of mycoplasma organisms in cell culture, but these drugs may simultaneously kill rickettsial organisms. Lincosamides are a drug of choice for common mycoplasma species in cell culture ,15 and hat study . Fluoroqibiotics . Howevertrations .Rickettsia and Orientia are Gram-negative bacteria, we hypothesized that they are high-level resistant to daptomycin. Quinupristin/dalfopristin is a combination of streptogramin antibiotics targeting the 23S rRNA of most Gram-positive bacteria as well as those of certain Gram-negative bacteria. The combination of two streptogramins renders this compound synergistic and bactericidal against susceptible bacteria [Mycoplasma spp. [In the search for new antibiotic regimens for mycoplasma decontamination of rickettsial cultures, daptomycin (a lipopeptide antibiotic) is interesting because it is rapidly bactericidal against Gram-positive bacteria. Since daptomycin directly targets the cell membrane , it may bacteria ,23. As qsma spp. , it may Rickettsia and Orientia cultures.Our study aimed to find an antimicrobial agent for mycoplasma decontamination in rickettsial cultures. We demonstrated the in vitro anti-mycoplasma activity of daptomycin against some common mycoplasma species in cell cultures. We also summarized the antimicrobial susceptibilities of common mycoplasma species to other antibiotics that may be used for mycoplasma decontamination. In addition, we demonstrated the acquisition of high-level resistance by some mycoplasma isolates after failed treatment with inadequate daptomycin concentrations or durations. Accordingly, our study provides a new option together with data for antibiotic selection in mycoplasma-contaminated Mycoplasma—four M. orale isolates, two M. arginini isolates, and one M. hyorhinis isolate—in our rickettsial cultures according to the DNA sequences of internal transcribed spacer (ITS) regions. The DNA sequences showed 100% similarities among these four M. orale isolates and two M. arginini isolates. Therefore, we could not conclude whether these Mycoplasma isolates were derived from the same or different sources.We found three species of M. orale, M. arginini, and M. hyorhinis isolates. M. orale and M. arginini were both 2 mg/L. In contrast, M. hyorhinis exhibited higher resistance to daptomycin (MIC = 32 mg/L). Similar to other Gram-negative bacteria, Rickettsia spp. exhibited a very high-level intrinsic resistance to daptomycin (MIC > 256 mg/L). The MIC of daptomycin for O. tsutsugamushi (128 mg/L) was lower than that for Rickettsia spp., possibly due to the absence of lipopolysaccharides in the Orientia cell wall [M. orale and M. arginini, but it may be problematic for M. hyorhinis eradication, especially in Orientia cultures. We determined the antimicrobial susceptibilities of all of the in MIC > 6 mg/L. Tell wall . From thLegionella and Moraxella [R. typhi (MIC = 8 mg/L) was more resistant to quinupristin/dalfopristin than O. tsutsugamushi (MIC = 1 mg/L). We determined the MIC of quinupristin/dalfopristin for M. hyorhinis, which is resistant to daptomycin. Unlike other species, including M. pneumoniae, M. hominis, and M. fermentans [M. hyorhinis in our study was resistant to quinupristin/dalfopristin  are susceptible to lincosamides, including clindamycin [Rickettsia and Orientia are high-level resistant to clindamycin  as well as in other studies [Mycoplasma spp. showed variable susceptibilities to aminoglycosides [Rickettsia species are low-level resistant in vitro (MIC = 4–16 mg/L) [Orientia is high-level resistant to aminoglycosides [M. hyorhinis in Orientia cultures.Aminoglycosides may be effective for our  studies ,32. In cycosides ,34. Rick16 mg/L) , whereasycosides   in patients at a therapeutic dose of 6 mg/kg [M. orale isolates and one M. arginini isolate. However, another M. arginini isolate developed high-level resistance, and its MIC increased from 2 to 64 mg/L. The isolate of M. hyorhinis was inhibited but not killed by a daptomycin concentration of 32 mg/L. After treatment, its MIC increased from 32 to 256 mg/L. At the beginning of our trial, we determined an MIC of only one isolate of  6 mg/kg ) for onlRickettsia cultures, we recommended using a high concentration (256 mg/L) of daptomycin for 3 weeks to prevent mycoplasma relapse, although lower concentrations of 32 mg/L were effective for all M. orale isolates. Even though one M. arginini isolate in a R. japonica culture had acquired resistance to daptomycin (64 mg/L) after a failed treatment, we still completely eradicated this isolate by applying a high concentration (256 mg/L) of daptomycin  must be used instead of high doses because the MIC of daptomycin for Orientia is 128 mg/L. We successfully used daptomycin (32 mg/L) for 3 weeks to eradicate two isolates of M. orale in cultures of Karp and Kato strains. At 64 mg/L of daptomycin, O. tsutsugamushi still grew well for 6 days during our antimicrobial testing experiment, but we did not continue the experiment beyond that period. Aminoglycosides (gentamicin or amikacin at 100 mg/L) can be used alone or in combination with daptomycin. Although M. hyorhinis (in O. tsutsugamushi strain Gilliam culture) had intrinsic and acquired high-level resistances to daptomycin, we completely eradicated this mycoplasma isolate by treatment with either gentamicin or amikacin at 100 mg/L for 3 weeks  for 3 weeks, but failed to eliminate M. hyorhinis (data not shown).Studies have reported that lincosamides can be used for mycoplasma decontaminations of cell cultures ,29. We cM. hyorhinis in L929 cultures without rickettsiae. We inoculated M. hyorhinis (MIC daptomycin = 32 mg/L) into sterile L929 cultures and added daptomycin 256 mg/L into these contaminated cultures. We found that daptomycin alone failed to completely eradicate M. hyorhinis. Since certain mycoplasma species, including M. hyorhinis and M. hominis, have been reported to survive inside eukaryotic cells [M. hyorhinis. A remarkable finding was that the quinupristin/dalfopristin concentration of 2 mg/L was the sub-MIC level that, when used alone, did not inhibit the growth of extracellular M. hyorhinis and intracellular R. typhi . After 3 weeks of treatment, mycoplasmas became undetectable (<103 organisms/mL). The treated samples remained mycoplasma-free for 2 consecutive weeks after treatment was discontinued.All of the effective treatment regimens in M. orale and M. arginini, but not M. hyorhinis. However, the combination of daptomycin and protein synthesis inhibitors  did completely eradicate M. hyorhinis. Besides, gentamicin or amikacin alone can be used for M. hyorhinis contamination in Orientia cultures. Our new results from using antibiotics for mycoplasma elimination in rickettsial cultures, as well as data on antimicrobial susceptibilities, will be useful in work on cell cultures of intracellular organisms.Mycoplasma contamination is a common and serious problem in cell cultures. Although several mycoplasma removal agents are suitable for use in sterile cell cultures, these drugs may not be applicable to cell cultures with intracellular bacteria such as rickettsiae. In this study, we demonstrated the in vitro anti-mycoplasma activities of some antibiotics against three common mycoplasma species in cell cultures. We paid attention to daptomycin because we hypothesized that it might exert rapid bactericidal action against extracellular mycoplasmas, which are Gram-positive bacteria. In addition, we demonstrated that some mycoplasma isolates developed high-level resistance to daptomycin if it was used in inadequate concentrations or for inadequate durations. We demonstrated that daptomycin or clindamycin alone completely eliminated M. hyorhinis isolate in this study displayed higher resistance to daptomycin than M. orale and M. arginini did. We suspect that the resistance of M. hyorhinis to daptomycin and quinupristin/dalfopristin is intrinsic, because daptomycin is not widely used and quinupristin/dalfopristin is still unavailable in Thailand. The susceptibilities of the three Mycoplasma species to daptomycin and quinupristin/dalfopristin may have differed because of cell membrane structural differences and variations in 23S rRNA sequences; therefore, further exploratory studies are necessary. Despite the resistance of M. hyorhinis to quinupristin/dalfopristin, we hypothesized that this drug combination may, nonetheless, be active against intracellular mycoplasmas because the intracellular concentrations of quinupristin and dalfopristin are 50 and 30 times higher, respectively, than the extracellular concentrations [M. hyorhinis eradication if used in combination with daptomycin. The trations . TherefoM. orale and M. arginini. Even for M. arginini with acquired resistance (MIC = 64 mg/L), a daptomycin concentration (256 mg/L) of only four times above the MIC was enough to eradicate this M. arginini isolate. In contrast, M. hyorhinis (MIC = 32 mg/L) contamination was intractable with high doses of daptomycin alone. Previous studies have shown that certain Mycoplasma species, including M. hominis (a human pathogen) and M. hyorhinis (a swine pathogen), are able to invade and persist inside mammalian cells [M. hyorhinis organisms. The quinupristin/dalfopristin concentration of 2 mg/L in this study did not inhibit the growth of extracellular M. hyorhinis. Clindamycin, on the other hand, could temporarily inhibit M. hyorhinis growth, but the organisms rapidly regrew after discontinuation of clindamycin, suggesting that clindamycin is bacteriostatic against extracellular M. hyorhinis. However, the combination of daptomycin plus quinupristin/dalfopristin and/or clindamycin did, as mentioned, permanently eradicate M. hyorhinis. We postulated that the intracellular concentrations of clindamycin and quinupristin/dalfopristin may be high enough to exceed the minimum bactericidal concentrations against M. hyorhinis. Collectively, these protein synthesis inhibitors are efficacious against intracellular mycoplasmas and thereby enhance the effectiveness of daptomycin, which primarily kills extracellular mycoplasmas.As mentioned previously, our results demonstrated that daptomycin alone permanently eradicated an cells ,38,39. TRickettsia and Orientia are intrinsically resistant to daptomycin, the MIC of daptomycin for O. tsutsugamushi (128 mg/L) was lower than that for Rickettsia spp. Accordingly, daptomycin cannot be used at very high concentrations in Orientia culture. Nevertheless, gentamicin or amikacin can be used alone or in combination with low daptomycin concentrations because of the intrinsic high-level resistance of Orientia to aminoglycosides. Evidence demonstrates that aminoglycosides accumulate in phagosomes, function in non-acidic conditions, and are active against some intracellular bacteria [M. hyorhinis.Although both bacteria . TherefoMycoplasma isolates can develop high-level resistance to daptomycin if the treatment is maintained for only 2 weeks. Therefore, we recommend extended treatment to prevent relapse; in fact, we did not observe any relapse after an effective treatment for 3 weeks. The acquired resistance to daptomycin after treatment has also been observed in other bacteria, such as in staphylococci [Rickettsia cultures) or aminoglycosides (for Orientia cultures) show promise for use in combination with daptomycin because of their high intracellular concentrations. Since the MICs of quinupristin/dalfopristin may vary among different Rickettsia species, the optimal concentration should be determined before use in decontamination.Despite their strong in vitro susceptibility to daptomycin, some ylococci ,43. FurtAntibiotic powders of daptomycin  and quinupristin/dalfopristin  were dissolved in sterile water for injection and stored as stocks at −20 °C. Sterile liquid formulations of clindamycin , moxifloxacin , gentamicin , and amikacin  were stored at 4 °C. Final antibiotic solutions were freshly prepared before use by diluting stock solutions with cell culture medium.2. O. tsutsugamushi , R. typhi and spotted fever rickettsiae - infected cells were maintained at 37, 35, and 32 °C, respectively. These rickettsial organisms were obtained from Prof. Didier Raoult (Aix-Marseille Université) and Dr. Wuttikorn Rodkvamtook . Heavily infected cells were detached by cell scrapers, cryo-preserved in RPMI 1640 with 25% fetal bovine serum and 7% DMSO, and stored as rickettsia-infected cell stocks at −80 °C. For some experiments, infected cells were disrupted by repeated passage through a 25-G needle using a syringe [g for 5 min to discard cell pellets. The supernatants containing extracellular rickettsiae were frozen in a cryopreservative medium consisting of RPMI 1640 with 25% fetal bovine serum and 7% DMSO at −80 °C until use.The L929 mouse fibroblast cell line was obtained from Prof. Stuart Blacksell  and cultivated in RPMI 1640 supplemented with 5% fetal bovine serum  in a humidified atmosphere with 5% CO syringe . The celMycoplasma. We identified the Mycoplasma species by PCR with DNA sequencing of nuclear ribosomal internal transcribed spacer (ITS) region. Mycoplasma organisms were detected by real-time PCR with hydrolysis (Taqman) probes targeting the 16S rRNA gene of the genus 5 mycoplasma organisms per ml were inoculated onto L929 monolayers at a multiplicity of infection of 2:1. The contaminated cells were then grown in RPMI 1640 medium with 5% fetal bovine serum with different antibiotic concentrations. We quantified extracellular mycoplasma DNA copy numbers in cell culture supernatants on days 0, 1 and 2 by real-time PCR and compared them to that of time 0. The mycoplasma count at the MIC was confirmed again on day 3. We chose real-time PCR because it yields rapid results and represents the real culture conditions of rickettsiae with 5% serum.Mycoplasma organisms were cultivated in cell culture media containing L929 monolayers in order to obtain a large number of organisms in the log phase. We extracted DNA from supernatants containing mycoplasmas using the QIAamp DNA mini kit . We used real-time PCR to quantify the copy number of mycoplasmas using the standard curve method. To determine the MICs, approximately 5 × 10O. tsutsugamushi and Rickettsia spp. was performed as previously described [Antimicrobial testing of escribed ,35. BrieWe pre-incubated mycoplasma-contaminated rickettsiae with the indicated antibiotics and used them to infect mycoplasma-free L929 cells. We manipulated only one mycoplasma-contaminated culture at a time to prevent cross-contamination from another mycoplasma isolate. We trypsinized the infected cells and transferred them to new flasks every 3–4 days. Rickettsiae from infected cells with cytopathic effect were passaged into new cells, as appropriate. We grew the infected cells in media with antibiotics for 3 weeks and subsequently in media without antibiotics for 2 weeks. Mycoplasma testing was performed every week after the discontinuation of antibiotics.M. orale and M. arginini in Rickettsia and Orientia cultures. For daptomycin-resistant M. hyorhinis in Orientia cultures, an aminoglycoside can be used instead of daptomycin. Moreover, the combinations of daptomycin plus protein synthesis inhibitors  were demonstrated to be effective in eradicating M. hyorhinis in sterile cell cultures. A treatment duration of at least 3 weeks is recommended to prevent relapse. Our mycoplasma decontamination methods with antibiotics are more convenient and practical than other methods involving in vivo passage in mice, which requires a longer duration [Mycoplasma are needed to determine the effectiveness and consistency of our protocols.In conclusion, our results demonstrated that daptomycin is suitable for complete eradication of duration  and animduration . Further


Tenacibaculum mesophilum DSM 13764T is presented. T. mesophilum is a close relative of the fish pathogen T. maritimum, which causes significant fish disease outbreaks in aquaculture facilities. The T. mesophilum genome sequence will serve as an important resource for comparative genomics approaches.Here, the complete genome sequence of sponge-associated  Tenacibaculum mesophilum DSM 13764T is presented. T. mesophilum is a close relative of the fish pathogen T. maritimum, which causes significant fish disease outbreaks in aquaculture facilities. The T. mesophilum genome sequence will serve as an important resource for comparative genomics approaches.Here, the complete genome sequence of sponge-associated  Tenacibaculum species have often been found in association with marine hosts (Tenacibaculum soleae and Tenacibaculum ovolyticum) (Tenacibaculum mesophilum) (Tenacibaculum amylolyticum) . The central metabolism of T. mesophilum DSM 13764T is similar to that of T. maritimum, whose metabolic potential has been previously reported  , 2, sponophilum) , and alglyticum) . It has aculosis , 4, a cou, Japan ). A totaCanu 1.8  was usedia HGAP4 ) with devailable , resultereported . A compaCP045192 (Canu assembly) and CP032544 (HGAP4 assembly). The version of the described Canu assembly in this paper is the first version, CP045192.1. PacBio RS II raw data are available under SRA accession number SRR9020974, and Illumina MiSeq raw data are available under SRA accession number SRR9020973.This whole-genome shotgun project has been deposited in GenBank under the accession numbers


Oxynaia Haas, 1911 is thought to be comprised of two geographically disjunct and morphologically variable species groups but the monophyly of this taxon has yet to be tested in any modern cladistic sense. This generic hypothesis has important systematic and biogeographic implications as Oxynaia is the type genus of the currently recognized tribe Oxynaiini (Parreysiinae) and is one of the few genera thought to cross several biogeographically important barriers in Southeast Asia. Morphological and molecular data clearly demonstrate that Oxynaia is not monophyletic, and the type species and its allies (O. jourdyi group) belong to the Unioninae, and more specifically as members of the genus Nodularia Conrad, 1853. Therefore, neither Oxynaiasyn. nov. nor Oxynaiini Starobogatov, 1970 are applicable to the Parreysiinae and in the absence of an available name, Indochinellagen. nov. and Indochinellini trib. nov. are described. Several combinations are proposed as follows: Indochinella pugio  gen. et comb. nov., Nodularia jourdyi  comb. res., N. gladiator  comb. res., N. diespiter  comb. res. and N. micheloti  comb. res. Finally, we provide an updated freshwater biogeographic division of Southeast Asia.The freshwater mussel genus  However, the vast majority of recent systematic research has focused on the North American and European fauna, while the comparatively diverse tropical lineages have received disproportionately less attention2. Although, several recent systematic efforts focused on Asian lineages have dramatically improved our understanding of the classification, morphological evolution, and biogeography of many tropical freshwater mussel clades9, many biographically interesting and systematically important taxa remain poorly understood from a phylogenetic perspective. This is particularly true of the genus Oxynaia, which has an unusual disjunct geographic distribution in Myanmar and northern Vietnam and is the type genus of the tribe Oxynaiini Starobogatov, 1970.Integrative taxonomic studies are of substantial practical importance to conservation stakeholders as accurate information on the systematics and distributions of biodiversity forms the foundation of taxon- and habitat-based conservation efforts. The application of basic systematic and phylogenetic research has played a critical role in the conservation of freshwater mussels , which are among the most threatened groups of animals worldwideet al.10 recently transferred the tribe Oxynaiini from the subfamily Ambleminae to the Parreysiinae on the basis of recovering Oxynaia pugio  among the latter subfamily in a molecular phylogeny. Whereas, traditional morphological classifications consistently consider Oxynaia a member of the subfamily Unioninae (in its various usages14). However, these seemingly irreconcilable subfamily-level classifications of Oxynaia (Unioninae vs Parreysiinae) have relied primarily on only one of the two geographically disjunct species groups, suggesting that Oxynaia may not be monophyletic.Whelan Oxynaia each share the presence of a strongly pointed posterior end (Greek: oxy – ‘sharp’), but can be divided into two distinct geographic species groups, the O. jourdyi group and the O. pugio group15  (generic type), O. gladiator , O. diespiter , and O. micheloti . The Oxynaia pugio group contains a single described species (O. pugio) from Myanmar14.Species in the genus Oxynaia, evaluate the morphological traits of the resultant suprageneric clades containing Oxynaia species, and to make the appropriate taxonomic changes to more accurately reflect our hypotheses of evolutionary history.Our objective herein is to test the monophyly of the genus COI + 16S rRNA + 28S rRNA) reveals that Oxynaia is not monophyletic . The Oxynaia pugio is recovered in a well-supported clade with the genus Radiatula Simpson, 1900. The genus Indonaia Prashad, 1918 appears to be the closest relative of the Oxynaia pugio + Radiatula clade, but with only moderate support values .Our family-level phylogenetic analyses based on mitochondrial and nuclear markers  is united with all other members of the subfamily Parreysiinae on the basis of possessing a tetragenous brooding condition , unhooked glochidia, as well as having the ascending lamella of the inner demibranchs attached to the visceral mass across their entire length (vs. attached only anteriorly in the Unioninae). This combination of traits is a novel method of recognizing the Parreysiinae (sans Lamellidentini). Several shell characters also unite the Oxynaia jourdyi group with representatives of the Unioninae, especially Nodularia, including the position, elevation, and sculpturing of the umbo, as well as several features of the dentition (Table Nodularia differs from Indochinellagen. nov. by having a very pronounced and elevated umbo (vs. not pronounced and not elevated), umbo location in the first half of the shell (vs. in the first third), nodulose wrinkles umbo sculpture (vs. v-shaped), and a rectangular and sharp anterior pseudocardinal tooth and a thick and pyramidal posterior pseudocardinal tooth in the left valve .Comparisons of the soft anatomy and larval morphology of representatives of the major freshwater mussel clades in the Oriental Region clearly demonstrate the polyphyly of to Table . The Oxyon Table . NodularOxynaia s. lato. The type species and its allies (i.e. the Oxynaia jourdyi group) are unambiguously placed in the subfamily Unioninae, rendering both Oxynaia and Oxynaiini inapplicable to the Parreysiinae. The tribe Oxynaiini Starobogatov, 1970 is herein recognized as an available family-group level name of the Unioninae Rafinesque, 1820. In the absence of an available name for the Oxynaia pugio species group and the larger Parreysiinae clade including the O. pugio group, Radiatula, and Indonaia (i.e. the former Oxynaiini), the genus Indochinellagen. nov and tribe Indochinellini trib. nov. are described here.The combination of the molecular phylogeny, soft anatomy, larval characters, and shell morphology clearly demonstrate the polyphyly of Oxynaia have clearly distinct ranges . Zieritz et al.17 listed two species, Oxynaia gladiator and O. micheloti, from the Mekong River but those reports refer to misidentified specimens (MNHN IM-2014-6880) or suspect localities (FMNH 20402 and NCSM 84425).The two species groups of ges Fig. . All theTaxonomic AccountFamily Unionidae Rafinesque, 1820Subfamily Unioninae Rafinesque, 1820Unio Philipsson in Retzius, 1788Type genus: GenusNodulariaConrad, 1853Unio douglasiae Griffith & Pidgeon, 1833 Type species: Type locality: Unknown.Oxynaia Haas, 1911 syn. nov. = Comments: We recognize Oxynaia as a junior synonym of Nodularia, and transfer the four Vietnamese species previously treated as Oxynaia to Nodularia as well. Additionally, we considered Nodularia dorri as the fifth member of this group. The close geographic proximity of the type localities and the conchological similarity of the named taxa raises questions about their validity and deserves further research.Nodularia jourdyi  comb. res.Unio jourdyi Morlet (1886): p. 7618.Nodularia jourdyi Simpson (1900): p. 81619.Oxynaia jourdyi Haas (1911): Pl. 1620; Haas (1913): p. 15215.Type locality: Tonkin. Environs de Dang-son [Đặng Sơn] (Jourdy) [p. 77]18; Bac-Hat [Bắc Hà], étangs du bord de la rivière Claire (Jourdy) [p. 290]18.Distribution: Red and Cả River drainage basins, northern Vietnam.Nodularia gladiator  comb. res.Unio gladiator Ancey (1881): p. 46821.Oxynaia micheloti Haas (1913): p. 15615.Nodularia gladiator Simpson (1914): p. 99122.Type locality: Yon-Bag, Tonkin21.Distribution: Red River drainage basin, northern Vietnam. Record from the Mekong Basin17 is erroneous.Nodularia diespiter  comb. res.Unio diespiter Mabille (1887): p. 16223; Simpson (1900): p. 86119.Nodularia diespiter Simpson (1914): p. 99322.Oxynaia diespiter Haas (1911): Pl. 1520; Haas (1913): p. 15415.Type locality: Tonkin23.Distribution: Red River drainage basin, northern Vietnam.Nodularia micheloti  comb. res.Unio micheloti Morlet (1886): p. 7718.Nodularia micheloti Simpson (1900): p. 81419.Oxynaia micheloti Haas (1911): Pl. 1420; Haas (1913): p. 15615.Type locality: Tonkin, environs de Chu18.Distribution: Cầu River drainage basin, northern Vietnam. Record from the Mekong Basin17 is erroneous.Nodularia dorri Unio dorri Wattebled (1886): p. 7124.Nodularia dorri Simpson (1900): p. 80919.Type locality: Les arroyos des environs de Hué24.Distribution: Perfume River, central Vietnam. Zieritz et al.17 erroneously listed N. dorri as an endemic species of the Mekong Basin.Subfamily Parreysiinae Henderson, 1935Type genus:Parreysia Conrad, 1853Comments: This subfamily includes five tribes: Parreysiini Henderson, 1935, Coelaturini Modell, 1942, Lamellidentini Modell, 1942, Leoparreysiini Vikhrev, Bolotov & Kondakov, 20177 and Indochinellini trib. nov., a new tribe described here.Tribe Indochinellini Bolotov, Pfeiffer, Vikhrev & Konopleva trib. nov.Indochinella Bolotov, Pfeiffer, Vikhrev & Konopleva gen. nov.Type genus: Figures 4Diagnosis: The Parreysiini and Leoparreysiini are the most morphologically similar family-group level taxa to the Indochinellini. However, the Indochinellini can be distinguished from the Parreysiini and Leoparreysiini by having a much more elongate shell outline (vs. circular) and a nearly straight to convex ventral margin (vs. strongly convex).Description: Adults small (22 mm) to medium (54 mm) sized for family. Shell outline narrow, elongate, strongly inequilateral, always with a straight or convex ventral margin. Moderately inflated, posterior ridge rounded with moderately to very steep posterior slope. Umbo only slightly elevated above hinge line usually with v-shaped umbo sculpture. Green zigzag sculpturing on shell disc common, but absent in some individuals and taxa, e.g., Indonaia caerulea , Radiatula humilis , R. pilata , and Indochinella pugiogen. et comb. nov. Shells moderately thick. Pseudocardinals erect and stumpy to long and bladelike; two in left valve (may become one in blade-like teeth) and one in right valve occasionally with a second rudimentary anterior tooth. Laterals are moderately short and diverging; two in left, one in right. Unhooked glochidia brooded in all four gills. Mantle margin ventral to incurrent aperture with many prominent simple papillae. Incurrent aperture papilose, excurrent and supra-anal apertures smooth. Ascending lamella of inner demibranch attached to visceral mass for its entire length.Distribution: Southeast and South Asia7.Comments: This tribe includes at least three valid genera: Indochinellagen. nov., Radiatula Simpson, 1900, and Indonaia Prashad, 1918.GenusIndochinellaBolotov, Pfeiffer, Vikhrev & Konopleva gen. nov.Unio pugio Benson, 1862.Type species: 25.Type locality: Regione Ava [Mandalay]Figure 2Etymology: The name of this genus is derived from the greater Indochinese Peninsula.Diagnosis: The genus is distinguished from Nodularia by the presence of tetragenous brooding of unhooked glochidia (vs. ectobranchous brooding of hooked glochidia), as well as complete fusion of the ascending lamella of the inner demibranchs to the visceral mass (vs. only anterior fusion) , fine v-shaped beak sculpture (vs. wrinkled and nodular), triangular posterior pseudocardinal , and moderately short lateral teeth (vs. elongate) (Table Indochinella (with exception of a lineage from the Tavoy River) can be distinguished from all other representatives of the tribe by the presence of a sharp posterior ridge (vs. rounded) and very steep posterior slope . Pseudocardinal teeth in Indochinella also tend to be more strongly developed than in other Indochinellini.n) Table . Indochie) Table . Adult IDescription: Shell moderately thick; elongate with rounded anterior end and strongly pointed posterior end. Posterior ridge sharp. Posterior slope steep. Umbo hardly elevated above hinge line, fine v-shaped umbo sculpture, shell disc smooth to strongly sculptured. Moderately inflated. Pseudocardinal teeth strong, two in the left valve, one in the right. Laterals moderately short, two in left valve, one in right. Tetragenous brooding condition , unhooked glochidia.Distribution: The genus is primarily known from Myanmar in the Irrawaddy, Sittaung, and Tavoy River drainages. The genus may also inhabit several other river basins in Myanmar, e.g., the Great Tenasserim, Salween, and some coastal rivers of the Bay of Bengal28. A few records from India 14 are in need of future studies because these specimens could have been collected within the Irrawaddy Basin.Comments: We assigned a single described species to the genus, although the divergent molecular lineages from the Sittaung and Tavoy River drainage basins may be worthy of formal taxonomic recognition.Indochinella pugio  gen. et comb. nov.Unio pugio Benson (1862): p. 19325.Nodularia pugio Simpson (1900): p. 81419.Oxynaia pugio Haas (1911): Pl. 1420; Haas (1913): p. 15815.Figures 2Material examined: Myanmar: Irrawaddy River basin, Nant Phar Lake, 24.2972°N, 97.2610°E, 29.xi.2016, 4 specimens , Vikhrev leg. Myanmar: Irrawaddy River basin, Myaung Lake, 24.2387°N, 97.1658°E, 01.xii.2016, 4 specimens , Vikhrev leg. Myanmar: Irrawaddy River basin, Pauk In Lake, 21.81347°N, 95.19746°E, 13.x.2009, 5 specimens (CAS 180788), Pitotrowski et al. leg. Myanmar: Irrawaddy River basin, Irrawaddy River near Myingyan, 21.48187°N, 95.30501°E, 15.x.2009, 5 specimens (CAS 189963), Pitotrowski et al. leg., Myanmar: Irrawaddy River basin, Chindwin River near confluence with Irrawaddy, 21.498445°N, 95.26631°E, 09.x.2009, 5 specimens (CAS 180796), Pitotrowski et al. leg. Myanmar: Sittaung River basin, Myit Kyi Pauk Stream, 18.9613°N, 96.4455°E, 26.xi.2016, 3 specimens , Vikhrev leg. Myanmar: Tavoy River, 14.5012°N, 98.1557°E, 26.iv.2015, 26.iv.2015, 38 specimens , Bolotov leg.Redescription: Shell shape cuneiform, elongated, inequilateral, not inflated, rather thick. Posterior ridge sharp, posterior slope steep. Maximum shell length 53.4 mm, height to 24.3 mm, width to 18.7 mm. Fine v-shaped sculpture on umbo, umbo only slightly elevated above hinge line. Periostracum smooth, grey-brown to yellow-green, with dark parts along the radial lines; nacre whitish. Left valve with two parallel rather short lateral teeth and two ribbed parallel pseudocardinal teeth. Right valve with a single slightly curved lateral tooth and two pseudocardinal teeth, anterior tooth reduced, posterior tooth high, ribbed and strong. Umbo cavity not very deep, nacre in umbo cavity commonly tinted peach to golden-brown. Anterior adductor scar well pronounced, funneled; posterior adductor scar rounded. The Sittaung lineage differs from the Irrawaddy lineage in having a shorter and higher shell, more pronounced and curved lateral teeth, and a moderately strong sculpture on shell disc. The Tavoy lineage differs from the two other lineages in having an oval-shaped shell, more rounded posterior ridge, more gradual posterior slope, and distinct zigzag ridges across shell disc.Distribution: Irrawaddy, Sittaung and Tavoy River basins. In the Irrawaddy River, it is known as far north as Mya Taung and as far south as Hinthada. A few records from India 14 are in need of future studies because these specimens could have been collected within the Irrawaddy Basin. The lineages from the Sittaung and Tavoy River catchment areas may represent separate species- or subspecies-level taxa but requires further systematic research.Habitat and ecology: The species seems to be rather a habitat generalist, and it is known from the mainstream of large, medium-sized and small rivers, as well as from their floodplain lakes.Comments:Unio digitiformis Sowerby, 1868 from India is not a synonym of Indochinella pugiogen. et comb. nov29. but a separate species, the generic placement of which is unclear. Haas13 noted that the location of this form is certainly not India and that it most likely belong to the Lanceolaria Conrad, 1853.Oxynaia is polyphyletic. This result has clear implications regarding the higher-level classification of the Unionidae, the morphological characteristics of the Parreysiinae, and corroborates broader biogeographic patterns in Southeast Asia. Molecular and morphological data reject the monophyly of Oxynaia with its former constituents being recovered in phylogenetically divergent and morphologically diagnosable clades. The Oxynaia jourdyi group is unambiguously placed in the Unioninae on the basis of our molecular phylogeny and several morphological synapomorphies and is herein considered a junior synonym of Nodularia, rendering the tribe name Oxynaiini inapplicable to the Parreysiinae. This taxonomic rearrangement required the description of a new genus for the “Oxynaia” pugio group (=Indochinellagen. nov.) and a new tribe (=Indochinellini trib. nov.) to recognize these morphologically cohesive clades of Parreysiinae.Our integrative molecular and morphological approach has determined that the genus Oxynaia was thought to be one of the few genera distributed across much of Southeast Asia from central Myanmar to northeastern Vietnam17. This distribution was thought to be unusual in that it crossed two important biogeographic barriers in Southeast Asia: (1) the Salween/Mekong river drainage divide separating the Western Indochinese freshwater mussel assemblage from the Sundaland assemblage, and (2) the Mekong/northern Vietnamese drainage divides separating the Sundaland assemblage from the East Asian fauna. However, the seemingly large distribution of Oxynaia s. lato is discovered here to be spurious and based on incorrect interpretations of common ancestry. The geographic distributions of Nodularia and Indochinella closely follow these two influential biogeographic barriers. These two biogeographic barriers divide eastern Asia into three faunistically distinct subregions , five tribes and eight genera8. The fauna of the Greater Sunda Islands  appears similar to that of mainland Southeast Asia and may also belong to this subregion, as suggested by the molecular data for northern Borneo9 and by the putative connections of paleo-drainages during the Pleistocene30, but further systematic research is necessary to delineate these boundaries. The Sundaland Subregion is comprised at least five subfamilies, i.e. Parreysiinae, Rectidentinae, Pseudodontinae, Gonideinae, and the monotypic Modellnaiinae. A single member of the Unioninae 32 occurs in the region, although, it has likely been introduced. Two large endemic monophyletic radiations of freshwater mussels, i.e., the tribes Pilsbryoconchini and Rectidentini, were recorded in this region7. The highest levels of diversity of these clades occur within the Mekong River basin, with a few representatives inhabiting the Chao Phraya River, the Malay Peninsula, and the Greater Sunda Islands9. The Indochinellini is the only tribe of the Parreysiinae distributed in the Sundaland Subregion, whose constituents belong to a subclade Radiatula that appears to have significant levels of cryptic diversity  Yangtze-Huang, from the Pei south to the Qiantang and Taiwan; (2) Indochina, including southern China and the Mekong west to the Salween; (3) India–Burma, from the Indus to the Irrawaddy; and (4) Sunda Islands–Philippines. However, our new scheme  of the subfamily-level diversity and endemism of the Unionidae in Asia, i.e. (1) Southeast Asian Hotspot harboring the highest diversity of the Rectidentinae, Gonideinae (+Pseudodontinae), Parreysiinae, and Modellnaiinae, and (2) Chinese Hotspot dominated by the Unioninae (+Anodontini) lineages. Our biogeographic division is largely congruent with this diversity-based model, i.e., the Western Indochinese and Sundaland subregions correspond to the Southeast Asian diversity hotspot and the East Asian Subregion correlates with the Chinese diversity hotspot.Zieritz http://zoobank.org), the online registration system for the ICZN. The LSID for this publication is: urn:lsid:zoobank.org:pub:C585DACD-6AB1-4692-B68A-50ABA47940A4. The electronic edition of this paper was published in a journal with an ISSN, and has been archived and is available from PubMed Central.The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature (ICZN), and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank , British Museum of Natural History, London, UK (NHMUK), Muséum National d’Histoire Naturelle, Paris, France (MNHN), Museo Civico di Storia Naturale di Genova, Genoa, Italy (MSNG), California Academy of Natural Sciences (CAS), North Carolina State Museum (NCSM), and Florida Museum of Natural History (UF). Additionally, we accessed the images of the types of several nominal taxa at the MUSSELp DatabaseOxynaia species groups (Table 16. Character states were observed using a Leica M27s dissecting scope and a Leica DM LB2 compound microscope. Representative taxa relevant to previous classifications of Oxynaia were also included, as were other major lineages present in the region (Table Parreysia corrugata was scored using literature sources36, as no soft anatomy for this species was available. A more detailed comparison of the shell characters that distinguish the three most commonly confused genera are provided in Table et al.37.Soft anatomy and larval characters were scored for representatives of both ps Table . These tOxynaia jourdyi sequences in the recent phylogenetic data set of Bolotov et al.7 to test the monophyly of Oxynaia. This data set was simplified to include only one haplotype of each species, with exception of the Oxynaia pugio sequences (Supplementary Table Parreysia spp. and Indonaia spp. from India were left as the members of the Parreysiinae. The sequence alignment of COI, 16S rRNA and 28S rRNA gene fragments was performed separately using the Muscle algorithm implemented in MEGA638. The alignment data sets were joined in a multi-locus alignment. Lacking sites were treated as missing data. We performed maximum likelihood and Bayesian inference phylogenetic analyses using RAxML v. 8.2.6 HPC Black Box39 and MrBayes v. 3.2.640, respectively. The settings of the analyses were as described in Bolotov et al.7. The phylogenetic models were calculated at the San Diego Supercomputer Center through the CIPRES Science Gateway41.We included novel The sequences used in this study are available from GenBank. Accession numbers for each specimen are presented in Supplementary Table Supplementary Info


Background: Whether patients with subclinical cardiomyopathy (CMP) are more prone to experience Takotsubo syndrome (TTS) than patients without CMP, is unknown. We present a patient with TTS as the initial manifestation of a hitherto unrecognized genetic CMP. Method: case report. Results: At age 55 after the unexpected death of her father, a now 61-year-old female had developed precordial pressure. Work-up revealed moderately reduced systolic function, dyskinesia of the interventricular septum, and indications for a TTS. Coronary angiography was normal but ventriculography showed TTS. Cardiac MRI confirmed reduced systolic function and TTS. TTS resolved without treatment and sequelae. At age 57 atrial fibrillation was recorded. After deterioration of systolic function at age 59 dilated CMP was diagnosed. Despite application of levosimendan, sacubitril, valsartan, and ivabradine, complete remission could not be achieved. Upon genetic work-up by means of a gene panel, the heterozygous mutation c.1489G > T (p. E497X) in exon 9 of the titin gene was detected and made responsible for the phenotype. Neurological work-up precluded involvement of the skeletal muscles. The further course was complicated by ventricular arrhythmias, requiring implantation of an implantable cardioverter defibrillator (ICD). Conclusions: previously subclinical CMP may initially manifest as TTS. Since patients with titin CMP are at risk of developing ventricular arrhythmias and thus to experience sudden cardiac death, appropriate anti-arrhythmic therapy needs to be established. WhetherThe patient is a 61-year-old Caucasian female, height 168 cm, weight 58 kg, with an uneventful previous history until age 55, when she developed precordial pressure after exposure to psychosocial stress after the unexpected death of her father. ECG showed left anterior hemiblock, missing R-progression until V4, and flat T-waves in III, aVL, and V1. Echocardiography revealed moderately reduced systolic function, dyskinesia of the interventricular septum, and regional wall motion abnormalities, indicative of TTS. Coronary angiography was normal but ventriculography was indicative of TTS . Cardiacn, 0–247 ng/L). The EF on cMRI had slightly improved (48%) compared to the previous cMRI. After initiation of a neurohumoral therapy with carvedilol, angiotensin-converting enzyme inhibitors (ACEI), and a statin, systolic function improved, stress test became normal, and proBNP declined to 152 ng/L. At age 59 the EF deteriorated again to 48% and the LVEDD to 59 mm. Despite re-establishing β-blockers, the EF further decreased to 40%, the LVEDD increased to 68 mm, and the proBNP to 1058 ng/L. ECG showed stable sinus rhythm but there was easy fatigability upon psychosocial stress.At age 57 tachycardious atrial fibrillation (AF) and isolated ventricular ectopic beats were recorded, which resolved spontaneously. ProBNP was 866 ng/L .After pneumonia at age 60, severe heart failure developed with an EF of 18%. Echocardiography showed mitral insufficiency and pulmonary hypertension. Coronary angiography was normal again. Myocarditis was excluded upon cMRI . Levosimtitin gene. The neurological history was noteworthy for intense myalgias during gripal infections since years and sore muscles during one month with pneumonia. Since clinical neurologic exam and creatine-kinase (CK) were normal, no further invasive work-up for myopathy was conducted. At discharge she was on a therapy with sacubitril (97 mg/d), valsartan (103 mg/d), nebivolol , amiodarone (50 mg/d), spironolactone (25 mg/d), furosemide (40 mg/d), and duloxetine (30 mg/d).Genetic testing by means of a gene panel covering 40 genes associated with dilated CMP (dCMP) revealed the heterozygous mutation c.1489G > T (p. E497X) in exon 9 of the titin mutation c.1489C > T, for TTS as the initial manifestation of titin CMP, and for the absence of skeletal muscle involvement.The presented case is interesting for dCMP due to the Heart failure is a major health burden and affects approximately 40 million people worldwide . One of titin gene, located on chromosome 2q31. The titin gene encodes titin, the largest known protein in biology, spanning half the cardiac and muscle sarcomere and, as such a basic structural and functional unit of striated muscles [titin gene can cause dCMP, hypertrophic CMP, myopathy, or overlap forms [titin mutation may present phenotypically with different manifestations in the same family or different families [titin gene are detectable in 20–25% of the hereditary forms of dCMP and in 18% of the patients with the sporadic type of dCMP [titin mutations manifest with dCMP, hCMP, myopathy or overlap phenotypes is unknown. dCMP due to the titin mutation c.1489G > T has not been reported so far. Nonetheless, the new variant was regarded as pathogenic since pathogenic mutations in the near surrounding of this variant had been reported. However, in case of additional stress, systolic dysfunction or heart failure may ensue. There are also indications that truncating titin variants represent a modifier of hypertrophic CMP and an increased risk of cardiovascular death [titin variants may be also associated with chemotherapy-induced CMP [One of the dCMP genes is the  muscles . Mutatioap forms . There afamilies . Mutatio of dCMP ,10. How ar death . Subclinuced CMP . Importauced CMP . In animtitin mutations most frequently manifest in the skeletal muscle as limb-girdle muscular dystrophy type 2J [titin variant, 32% had arrhythmias  and 18% had CMP [Concerning titin-related myopathy,  type 2J , recessi type 2J . In some type 2J . Cardiac type 2J . In a st had CMP . Titin m had CMP .titin variant may initially manifest as TTS. Patients with titin CMP seem to be at risk of developing ventricular arrhythmias and thus to experience sudden cardiac death, why appropriate anti-arrhythmic therapy needs to be established. The LifeVest® is the most appropriate tool to effectively bridge the interval between indication and implantation of an ICD but in-hospital telemetry or anti-arrhythmic drugs could be an alternative.In conclusion, this case shows that a previously subclinical dCMP due to a


Helicobacter pylori and that of commensal bacteria in the stomach. The abundances of Variovorax paradoxus and Porphyromonas gingivalis were correlated with histological gastritis, but not with endoscopic or symptomatic gastritis. The total PAGI-SYM score showed a stronger correlation with the duodenal microbiota (Prevotella nanceiensis and Alloprevotella rava) than with the gastric microbiota . Different correlations of the gastric and duodenal microbiota with histological, endoscopic, and symptomatic gastritis were observed for the first time at the species level. H. pylori-negative gastritis is not associated with endoscopic or symptomatic gastritis. Only H. pylori-induced endoscopic gastritis requires gastric cancer surveillance. Owing to the weak correlation with H. pylori, symptomatic gastritis should be assessed separately from histological and endoscopic gastritis.Mucosal inflammation is characterized by neutrophil and mononuclear cell infiltration. This study aimed to determine the gastric and duodenal microbiota associated with histological, endoscopic, and symptomatic gastritis. Dyspeptic adults who presented for evaluation were included. Subjects with either comorbidities or recent drug intake were excluded. Three endoscopic biopsies were obtained from the antrum, body, and duodenum. Next-generation sequencing for 16S ribosomal RNA V1–V2 hypervariable regions was performed. The correlation between the composition of microbiota and the degree of inflammatory cell infiltration, endoscopic findings, and Patient Assessment of Gastrointestinal Disorders Symptom Severity Index (PAGI-SYM) score was analyzed. In 98 included subjects, microbial communities in the antrum and body showed Bray–Curtis similarity; however, those in the duodenum showed dissimilarity. Histological and endoscopic gastritis was associated with the abundance of  Helicobacter pylori-infected subjects among the patients with functional dyspepsia [H. pylori-induced organic dyspepsia and functional dyspepsia, histopathological and endoscopic findings are more reliable for diagnosing H. pylori infection and estimating the risk of gastric cancer [H. pylori-negative autoimmune gastritis (type A gastritis) and H. pylori-induced antral-dominant type B gastritis [H. pylori, other species may induce gastritis; however, the contribution of other microbiota to symptom generation, mucosal inflammation, and endoscopic findings is still uncertain.Gastritis remains challenging for clinicians, because symptoms may appear even in the absence of histological or endoscopic gastritis. As gastritis-related symptoms are nonspecific, it is thus difficult to detect yspepsia . Owing tc cancer ,3. Furthastritis . Aside fH. pylori infection [H. pylori [H. pylori affects adjacent organs, and is more abundant in the duodenal lumen than in the duodenal mucosa [H. pylori that contribute to mucosal inflammation, carcinogenesis, or symptom generation remain unknown.Next-generation sequencing (NGS) analysis of the 16S ribosomal RNA (rRNA) hypervariable regions has enabled the understanding of changes in the composition of the gastric microbiota induced by nfection ,6. Priornfection ,7. Acute. pylori ,11,12,13l mucosa . Becausel mucosa , the comA pathogenic species may induce mucosal inflammation, specific endoscopic findings, and gastrointestinal symptoms. Conversely, the microbiota that plays a protective role against inflammation might be underrepresented in the inflamed mucosa. In this study, we attempted to determine the composition of gastric and duodenal microbiota according to the degree of inflammatory cell infiltration and the presence of endoscopic gastritis. To understand the differences among histological, endoscopic, and symptomatic gastritis, we also analyzed the correlation between the composition of microbiota and gastrointestinal symptoms.Korean adults who visited our Digestive Disease Center for the evaluation of dyspeptic symptom were enrolled in a prospective setting. Subjects with either comorbidities or recent drug intake within the last 3 months  were excluded. Ninety-eight subjects  provided signed informed consents prior to the procedure, and their details were omitted to ensure anonymity. None of the included subjects were vegetarians.https://cris.nih.go.kr).Ethics approval: This study was approved by the institutional review board of Konkuk University Medical Center which confirmed that the study followed the Declaration of Helsinki. This study is registered as KCT0000718 at the Clinical Research Information Service  to 5 (very severe) on the 20-item Patient Assessment of Gastrointestinal Disorders Symptom Severity Index (PAGI-SYM) questionnaire, as described previously . EndoscoH. pylori-related endoscopic findings [Endoscopic findings were described according to the updated Sydney classification system in addition to findings ,4,17,18.Gastric and duodenal biopsy specimens were evaluated by one pathologist (Dr. H. S. Han) blinded to the subjects’ clinical information and status. All the biopsied specimens were fixed with 10% neutral-buffered formalin and were embedded in paraffin blocks. After hematoxylin-eosin (H&E) and Giemsa staining, microscopic findings were described. The specimens were observed with the aid of microscope . Degrees of neutrophil and mononuclear cell infiltration, atrophy, and intestinal metaplasia were scored from 0 to 3  according to the updated Sydney classification system .The 16S rRNA V1–V2 hypervariable regions were analyzed based on recent study findings on the gastric and/or duodenal mucosal microbiota using endoscopic biopsied specimens ,19,20. TBacterial 16S rRNA V1–V2 hypervariable regions were amplified using primers  . PCR ampEnd-repair, ligation, nick-repair, and final amplification processes for sequencing libraries were performed using Ion Plus Fragment Library Kit and Ion Xpress Barcode Adapters  according to the manufacturer’s protocol. The 16S rRNA libraries were purified using Agencourt AMPure XP reagent . The respective size of each library and the absence of contaminants were verified using high-sensitivity DNA Kit on Agilent 2100 Bioanalyzer .Template preparation and sequencing were performed with the aid of Ion Chef System and Ion S5 XL System with Ion 530 Chip Kit . The FASTQ data were generated using the Torrent Suite™ software version 5.8  by sequencing the V1–V2 hypervariable regions of 16S rRNA gene. The FASTQ sequence files are available in the National Center for Biotechnology Information (NCBI) Bioproject under the accession number of PRJNA486978.The FASTQ data were further analyzed for quality control. After obtaining the sequences from each sample, trimmed reads were mapped. The final files were generated by demultiplexing the data and removing the primer. Reliable data were obtained by excluding Phred quality scores below 20, polyclonal reads with low quality, and 3′-end adaptors of reads.Ion Reporter™ software version 5.6  was used to process the FASTQ files. Read sequences were checked for a minimum read length of 150 bp. Data filtering was performed by removing low copy numbers (threshold <10) and reads with an alignment coverage of <90%. Based on Ion Reporter Metagenomics 16S algorithms, automatic analyses were conducted using the metagenomics workflow in the Ion Reporter software. The linked Quantitative Insights into Microbial Ecology (QIIME) workflow on the Ion Reporter Server processed the FASTQ files as input data, and reported operational taxonomic unit (OTU) tables as output data.The sequence reads were aligned against the reference sequences exhibited in Greengenes version 13.5 (Greengenes Database Consortium) and MicroSEQ 16S rRNA Reference Library version 2013.1 . With respect to taxonomy assignment, OTUs with ≥10 copy numbers were taxonomically classified using two databases. The reads were matched with clean OTUs showing a sequence similarity of >97% for the entire length, as described previously .To minimize the effects of uneven sampling, diversity indices were calculated after normalizing the read numbers in each sample. Alpha diversity was exhibited as Chao1, Shannon, and Simpson indices using the linked QIIME workflow. To determine beta diversity, transformed OTU counts were used for principal coordinate analysis (PCoA). A PCoA plot based on the Bray–Curtis dissimilarity was constructed using the linked QIIME workflow.t-test. For continuous variables with asymmetrical distribution, median values and ranges were provided using the Kruskal–Wallis test. Categorical variables were described as frequencies using chi-square test or Fisher’s exact test. Analysis was performed using PASW version 17.0 . A p-value of <0.05 was considered statistically significant.For the comparison of PCoA plots, permutational multivariate analysis of variance test was performed using “vegan” R package version 2.2-1. For continuous variables, means and standard deviations were provided using the H. pylori-infected and non-infected subjects, >1% H. pylori relative abundance was defined as H. pylori infection [r) with a p-value. Furthermore, correlation analysis was performed to evaluate the association between the relative abundance of microbiota and PAGI-SYM scores. To exclude correlations found by chance, statistical significance was set at p < 0.0083  after multiple testing correction.For the comparison between the nfection . For mulPrevotella pallens (p = 0.009) and Streptococcus sp. (p = 0.007) in the stomach. The median relative abundance of P. pallens was higher in women  than in men . The median relative abundance of Streptococcus sp. was higher in men  than in women .A total of 573 different species  were detected in the stomach in 98 subjects. In the duodenum, 500 different species  were found. Beta diversity indicated a significant similarity between the antrum and the body . Common H. pylori in the antrum and H. pylori in the body . The correlation coefficient values for the relative abundance of H. pylori in the duodenum and H. pylori in the antrum and body were 0.064 (p = 0.528) and 0.260 (p = 0.010), respectively. Regardless of the presence of H. pylori infection, Brevundimonas aurantiaca was dominant in the duodenum in 89 (90.8%) subjects. Only 14 subjects showed H. pylori in the duodenum among the 31 H. pylori-infected subjects. H. pylori-infected subjects showed lower diversity indices in the stomach, and higher diversity indices in the duodenum than non-infected subjects  and two species in the duodenum (H. pylori and Leptotrichia genomosp).The composition of the gastric microbiota differed according to the combined inflammation score A. The coPropionibacterium acnes (r = −0.475), Pseudomonas veronii (r = −0.349), Pseudomonas sp. (r = −0.345), Dechloromonas sp. (r = −0.291), Staphylococcus epidermidis (r = −0.272), Cloacibacterium rupense (r = −0.267), Escherichia coli (r = −0.265), Hydrogenophilus hirschi, (r = −0.227), and Bacillus sp. (r = −0.225) was negatively associated with the combined inflammation score in the stomach. In the duodenum, the relative abundance of Moraxella osloensis (r = −0.297), S. epidermidis (r = −0.245), and Actinomyces odontolyticus (r = −0.217) was negatively correlated with inflammation. Gastric and duodenal microbiota that showed significant correlation with inflammatory cell infiltration at the genus or species level are summarized in The relative abundance of H. pylori was more abundant in subjects with hemorrhagic spots (n = 6), hypertrophic rugae (n = 7), advanced atrophy (n = 9), and mucosal nodularity (n = 13) than in their counterparts (n = 3), hematin deposits (n = 3), linear hyperemic streaks (n = 2), and gastric xanthoma (n = 1)). P. acnes, P. veronii, Pseudomonas sp., S. epidermidis, and C. rupense were abundant in the presence of regular arrangement of collecting venules, which indicates intact gastric mucosa without endoscopic gastritis  were found among the species associated with histological gastritis.terparts . No corrastritis . All spePrevotella nanceiensis (r = 0.273) and Alloprevotella rava (r = 0.209) in the duodenum. Stronger correlations were found with the duodenal microbiota than the gastric microbiota . Correlation with the duodenal microbiota was mostly observed with bloating, nausea, vomiting, and lower abdominal pain , bloating (p = 0.001), and lower abdominal pain (p = 0.001) were significantly higher than those without hemorrhagic spots  and P. gingivalis (r = 0.259) in the stomach (S. epidermidis (r = −0.346) and M. osloensis (r = −0.305). No duodenal microbiota showed a positive correlation with the combined inflammation score.In 61  stomach B. None oOnly eight subjects had specific endoscopic findings . No significant correlation was found between the presence of these endoscopic findings and the composition of the microbiota.of N. elongata (r = 0.207), C. segmentosum (r = 0.235), P. pallens (r = −0.237) in the stomach and that of A. odontolyticus (r = 0.243), Pseudomonas grimontii (r = 0.238), and Paracoccus sp. (r = −0.207) in the duodenum. Stronger positive correlations were found with the duodenal microbiota than with the gastric microbiota , M. osloensis (p = 0.018), P. acnes (p = 0.011), and Bacillus sp. (p = 0.004) differed between the 23 antrum- and 16 corpus-dominant cases of atrophic gastritis. The median relative abundance of H. pylori (0.62% vs. 0%) was significantly higher in the antrum- than in the corpus-dominant gastritis cases. Conversely, M. osloensis (0.08% vs. 0.06%), P. acnes (9.20% vs. 3.20%), and Bacillus sp. (1.13% vs. 0.08%) were more abundant in the corpus- than in the antrum-dominant gastritis cases. Nevertheless, none of these species showed significant differences in their relative abundance between the 79 specimens with atrophy (43 antrum and 36 body) and the 117 specimens without atrophy (55 antrum and 62 body).A mild degree of atrophy was found in the antrum in 43 subjects and in the body in 36 subjects. Among these subjects, 20 showed a mild degree of atrophy in both the antrum and the body. Neither a moderate nor marked degree of atrophy was found in this study. The relative abundance of H. pylori in the stomach was associated with the most cases of histological and endoscopic gastritis, but only some cases of symptomatic gastritis. Only a weak correlation was observed between symptom scores and H. pylori abundance. Symptom scores showed stronger correlation with the duodenal microbiota than with the gastric microbiota. Therefore, avoiding an overgrowth of H. pylori may prevent most histological and endoscopic gastritis, but only some symptomatic gastritis. Furthermore, cases of H. pylori-negative gastritis involved abundant V. paradoxus and P. gingivalis. Such abundances were correlated with histological gastritis, but not with endoscopic or symptomatic gastritis.In the present study, microbial communities in the duodenum showed dissimilarity with those in the antrum and body. The relative abundance of H. pylori. V. paradoxus and P. gingivalis were also linked to inflammation; however, their correlation coefficient values and relative abundance were not comparable to those of H. pylori. Only H. pylori-dominant dysbiosis showed an extremely high relative abundance of up to 98.4% with significant changes on endoscopic and histological findings. These findings are additive to those of previous studies that reported microbial changes during H. pylori-induced chronic inflammation [H. pylori-dominant condition and was lower in the body than in the antrum. This supports our study findings that correlations between the combined inflammation score and species abundance of V. paradoxus and P. gingivalis are more prominent in the body than in the antrum. The impact of certain species seems to be low in a diverse environment such as that in the antrum.Among the 573 gastric species detected in this study, the strongest correlation was observed between the degree of inflammatory cell infiltration and the relative abundance of ammation ,11,12. IH. pylori and those of P. acnes, P. veronii, Pseudomonas sp., C. rupense, and S. epidermidis. Discrepancy between the species related to histological and endoscopic gastritis was noted for V. paradoxus and P. gingivalis. The abundances of V. paradoxus, and P. gingivalis were correlated with histological gastritis, but not with endoscopic or symptomatic gastritis. The abnormal endoscopic findings observed in this study are consistent H. pylori-related endoscopic findings with an increased risk of gastric cancer. Intestinal metaplasia and gastric corpus atrophy increase the risk of intestinal-type gastric cancer, whereas hypertrophic rugae, diffuse redness, and nodularity increase the risk of diffuse-type gastric cancer [Endoscopic and histological gastritis were associated with the relative abundance of c cancer ,25,26,27P. gingivalis is a well-known periodontal pathogen associated with esophageal cancer [V. paradoxus nor P. gingivalis was associated with atrophy. Only the abundance of H. pylori was associated with antrum-dominant atrophic gastritis. These findings support that H pylori-negative gastritis does not progress to precancerous lesions [l cancer ; howeverl cancer ,17,18. T lesions .H. pylori explains why other factors  are more correlated with symptoms than H. pylori [P. nanceiensis and A. rava) and gastric microbiota  may negatively affect ENS modulation via neurogenic inflammatory process. This increases understanding of the symptoms of H. pylori-negative subjects.In this study, the species associated with symptom scores were mostly inconsistent with those related to histological and endoscopic gastritis. A stronger correlation with the duodenal microbiota than with the gastric microbiota was observed only in symptomatic gastritis. The weak correlation between the total PAGI-SYM score and the abundance of . pylori ,30,31,32. pylori ,34, our S. epidermidis, P. acnes, P. veronii, Pseudomonas sp., and C. rupense play a defensive role in reducing histological and endoscopic gastritis. Even in H. pylori-negative subjects, the degree of inflammatory cell infiltration increased with the relative abundance of pathogens , and none of the commensals demonstrated statistically significant differences. The beneficial role of commensal bacteria requires further investigation.The present study has limitations. First, PAGI-SYM questionnaires were used instead of Rome criteria, because the validity of the Korean version of the Rome III questionnaires was shown to be low . NeverthH. pylori, V. paradoxus, and P. gingivalis. H. pylori-negative gastritis was not associated with endoscopic or symptomatic gastritis. H. pylori was the only pathogen associated with endoscopic gastritis, which requires gastric cancer surveillance.Different correlations of the gastric and duodenal microbiota with histological, endoscopic, and symptomatic gastritis were observed for the first time at the species level. Histological gastritis was associated with the relative abundances of P. nanceiensis and A. rava) than the gastric microbiota . Thus, factors other than H. pylori infection status should be assessed in cases of symptomatic gastritis.Symptomatic gastritis should be evaluated and managed differently from histological and endoscopic gastritis, because it is more strongly correlated with the duodenal microbiota (


Histophilus somni is a Gram-negative opportunistic pathogen associated with respiratory disease in cattle. Here, we report the draft genome sequences of 12 Histophilus somni strains isolated from feedlot cattle in Alberta, Canada, which were diagnosed with respiratory disease. Histophilus somni is a Gram-negative opportunistic pathogen associated with respiratory disease in cattle. Here, we report the draft genome sequences of 12 Histophilus somni strains isolated from feedlot cattle in Alberta, Canada, which were diagnosed with respiratory disease. Histophilus somni, formerly Haemophilus somnus, is a member of the family Pasteurellaceae. Histophilus somni, along with two other members of Pasteurellaceae (Pasteurella multocida and Mannheimia haemolytica), is associated with bovine respiratory disease (BRD) complex , Saskatoon, Canada. These short-read whole genomes were previously aligned against a reference genome for the detection of single nucleotide polymorphisms and insertion and deletion sites  and QUAST v5.0.2 (http://quast.sourceforge.net/quast) were used for quality assessment and trimming of sequenced files before and after assembly, respectively , as well as reads of less than 50 nucleotide base pairs, that were >10% Ns, and those with a >15-bp overlapping sequence with the Illumina adaptors. FASTQ files with a Q score of at least 98% were used for further in silico analysis. The genomes were assembled de novo using SPAdes v3.13.0 (http://cab.spbu.ru/software/spades/) (https://www.ncbi.nlm.nih.gov/genome/annotation_prok/) with default thresholds , and the DNA Data Bank of Japan (DDBJ), under the accession numbers provided in The genome sequences and associated data for 12