Helicobacter pylori infection, but there are controversies on nodule regression in adults. The aim of this study was to analyze the factors that are related to the nodule regression in adults diagnosed as NG.Nodular gastritis (NG) is associated with the presence of H. pylori infection during esophagogastroduodenoscopy (EGD) at our center were included. Changes in the size and location of the nodules, status of H. pylori infection, upper gastrointestinal (UGI) symptom, EGD and pathology findings were analyzed between the initial and follow-up tests.Adult population who were diagnosed as NG with H. pylori eradication, whereas 9.9% (9/99) of the non-eradicated NG patients showed spontaneous nodule regression without H. pylori eradication (P < 0.001). Nodule regression was more frequent in NG patients with antral nodule location (P = 0.010), small-sized nodules (P = 0.029), H. pylori eradication (P < 0.001), UGI symptom (P = 0.007), and a long-term follow-up period (P = 0.030). On the logistic regression analysis, nodule regression was inversely correlated with the persistent H. pylori infection on the follow-up test (odds ratio (OR): 0.020, 95% confidence interval (CI): 0.003 - 0.137, P < 0.001) and short-term follow-up period < 30.5 months .Of the 117 NG patients, 66.7% (12/18) of the eradicated NG patients showed nodule regression after H. pylori eradication is the most significant factor associated with nodule regression. Long-term follow-up period is also correlated with nodule regression, but is less significant than H. pylori eradication. Our findings suggest that H. pylori eradication should be considered to promote nodule regression in NG patients with H. pylori infection.In adults with NG,  Helicobacter pylori infection plays a role in the pathogenesis of NG [H. pylori test-and-treat strategy as an initial management approach to Korean adults presenting as functional dyspepsia [Nodular gastritis (NG) is characterized by an unusual goose-flesh appearance on esophagogastroduodenoscopy (EGD). There is accumulating evidence that is of NG . Most cais of NG , but somis of NG , 6. Howeyspepsia , 4, 7. WThere are two endoscopic patterns of nodules in NG, and the clinicopathological findings do not appear to differ significantly between the two patterns . A granuH. pylori eradication [H. pylori infection.The prognosis differs in adults with NG; some might progress to a diffuse-type gastric cancer and a few may progress to a lymphofollicular malignancy , 10. Sevdication , 11, 12.H. pylori infection at our center between August 2005 and April 2013. NG patients were included when EGD was followed up at our center after the initial diagnosis. NG patients were excluded when the age was below 20 years old, had a history gastric surgery, or had other significant disease at the time of diagnosis. Furthermore, those without preserved gastric biopsied specimens were also excluded from the study.This cross-sectional study included Korean adults who were diagnosed as NG with All of the included NG patients gave their written informed consent before EGD and answered to the questionnaires on their recent symptoms and medical history. This study was approved by the Institutional Review Board (IRB) of Konkuk University School of Medicine which confirmed that the study was in accordance with the ethical guidelines of the Helsinki Declaration. After the IRB approval at our center (KUH 1010574), it was registered as ClinicalTrials.gov ID: KCT0001148 (https://cris.nih.go.kr).EGD was performed with either EG-2990i endoscope  or GIF-H260 endoscope  at our center. Endoscopic findings of the background mucosa was classified as normal finding, chronic superficial gastritis, hemorrhagic gastritis, erosive gastritis, verrucous gastritis, chronic atrophic gastritis, metaplastic gastrtiis, and hypertrophic gastririts. Other notable findings such as nodular changes or hemorrhagic spots were recorded additionally.NG was defined as diffuse chicken-skin-like mucosal changes or unusual goose-flesh apprearance on the endoscopic finding. When the nodules exhibit regular protrusion with a size < 5 mm, NG was subclassified as a small granular-type. When the nodules exhibit larger size with uniform protrusion, it was subclassified as a large nodular-type.H. pylori infection. Based on the updated Sydney system, the grades were scored as either none, mild, moderate, or marked for chronic changes including atrophy and intestinal metaplasia . In addition, the grades were scored for acute changes including activity (the intensity of acute polymorphonuclear cell infiltrates) and inflammation (the intensity of chronic mononuclear cell infiltrates).Gastric biopsied specimens were fixed in formalin. They were stained with hematoxylin and eosin (H&E) and with modified Giemsa staining. Positive Giemsa staining was regarded as H. pylori infection was determined when more than one of the invasive tests showed positive finding. For the invasive tests, histology and Giemsa stain were performed during EGD with or without the rapid urease test .The presence of H. pylori eradication was performed using a triple therapy with a proton pump inhibitor , amoxicillin (1 g twice) and clarithromycin (500 mg twice) daily for 1 week according to the guideline [NG patients were sent to the Digestive Disease Center if the patient agreed on further management. For the treatment, uideline . If the H. pylori test at our center as the initial tests. Changes in the size and location of the nodules, status of H. pylori infection, UGI symptom, EGD and pathology findings were compared with the previous findings by one gastroenterologist (Dr. J. W. Kim) to exclude any interobserver variability. Improvement of NG was defined as nodule regression on EGD findings.After the initial diagnosis, NG patients underwent EGD and t-test and were expressed as the mean ± standard deviation (SD) values. Variables exhibiting skewed distributions were expressed as median values with ranges , and differences were tested using the Kruskal-Wallis test. Categorical variables were analyzed using the Chi-square test and were presented as percentage. For the cut-off value of follow-up periods, a receiver operating characteristic (ROC) curve was constructed by plotting sensitivity (true-positive rate) against 1-specificity  over all possible threshold levels of follow-up periods that were found to be related to the nodule regression. Based on the area under the ROC curve (AUC) analysis with 95% confidence interval (CI) and standard error (SE) values, the accuracy of the follow-up period for detecting nodule regression was caluculated. With the cut-off value provided by the ROC curve analysis, logistic regression analysis was performed to identify variables that were associated with nodule regression. Odds ratio (OR) and 95% CI were analyzed for each significant variable. All analyses were performed using SPSS software, Windows version 19.0. A P-value of less than 0.05 was considered to be statistically significant.Continuous variables were analyzed using the Student’s A total of 117 NG patients fulfilled the study criteria and underwent follow-up EGD at our center after the initial diagnosis of NG. Most of the NG patients (85.5%) did not have UGI symptom at the time of endoscopic examination .H. pylori eradication after the diagnosis. At the follow-up tests, none of the eradicated patients showed H. pylori infection, whereas all of the non-eradicated patients showed persistent H. pylori infection. Nodule regression was noticed at the follow-up tests in 66.7% of the 18 eradicated NG patients after H. pylori eradication, whereas it was noticed in 9.9% of the 99 non-eradicated NG patients (P < 0.001). None of these patients progressed to gastric cancer during the follow-up period.Of the included NG patients, 18 (15.4%) underwent H. pylori eradication  were related to nodule regression (H. pylori eradication (P < 0.001), and small size (P = 0.029) of the nodules , UGI symptom , nodule size  and gression . When th nodules .Furthermore, NG patients with nodule regression showed a longer follow-up period (38.2 ± 12.1 months) than those without nodule regression . Using the ROC analysis, the cut-off value of follow-up period on nodule regression was 30.5 months  with a sensitivity of 71.4% and specificity of 53.1%.H. pylori eradication and follow-up period were significant on the logistic regression analysis  and short-term follow-up period < 30.5 months . Conversely, persistent nodules in NG patients were significantly higher in the non-eradicated group  and short-term follow-up period < 30.5 months .Of all significant variables, only analysis . Nodule H. pylori infiltration (P = 0.880) scores, but median intestinal metaplasia score was significantly higher in those with spontaneous nodule regression (0.25 ranging from 0 to 3) than those without regression .Of 99 NG patients without eradication, nine (9.1%) showed spontaneous nodule regression. Between these nine NG patients with nodule regression and other 90 NG patients with persistent nodules, there were no significant differences on age (P = 0.605), gender (P = 0.224), nodule size (P = 0.165), location (P = 0.279), UGI symptom (P = 0.502), hemorrhagic spots (P = 0.351), and follow-up period (P = 0.131). With regard to the scores based on the updated Sydney system, there were no significant differences on inflammation (P = 0.351), activity (P = 0.824), atrophy (P = 0.094), and H. pylori at the follow-up tests, and 12 (66.7%) showed nodule regression. Between these 12 NG patients with nodule regression and other six NG patients with persistent nodules, there were no significant differences on age (P = 0.944), gender (P = 0.344), nodule size (P = 0.294), location (P = 0.073), UGI symptom (P = 0.563) and hemorrhagic spots (P = 0.569). Mean follow-up period of the 12 improved NG patients (37.6 ± 12.6 months) was significantly longer than that of the six persistent NG patients . On the pathology findings, there were no significant differences on activity (P = 0.258), atrophy (P = 0.201), intestinal metaplasia (P = 0.621), and H. pylori infiltration (P = 0.285), but mean inflammation score was significantly higher in NG patients with persistent nodules (2.3 ± 0.5) than in those with nodule regression .All the eradicated patients exhibited no H. pylori eradication is the most important factor related to nodule regression in NG. In addition, longer follow-up period was a significant independent factor for nodule regression in NG patients with H. pylori eradication. Neither the gender nor the age of the patient was related to the prognosis of NG, despite NG being more common in young women.This study found that H. pylori eradication in a Chinese study [H. pylori eradication should be performed to the adults with NG for nodule regression [In this study, two-thirds of the eradicated NG patients exhibited complete improvement of NG at the follow-up tests. Similarly, endoscopic evidence of NG disappeared in 63.2% of the NG patients at 2 months after se study . The obsgression , 11, 12.gression . This stH. pylori eradication in the present study exhibited spontaneous regression of the nodules, even with persistent H. pylori infection. Few studies have found a tendency for antral nodules to regress more frequently than those on the body [H. pylori-induced inflammation on the antrum and body, and because antral nodules tend to regress in association with atrophic changes [H. pylori eradication. The presence of UGI symptom was not an idependent signficant factor for nodule regression in this study.Interestingly, 9.1% of the NG patients without the body . This fi changes , 14. Alt changes , nodule With regard to nodule size, small ones (granular-type NG) tended to regress more often than the large ones (nodular-type NG) in our NG patients. This can be explained by the histological findings, whereby smaller lymphoid follicles resolve more easily than large lymphoid follicles with other inflammatory cells. Although we were unable to determine why the smaller antral nodules regressed more readily than the large body nodules, we suggest that this is due to the different proportions of histopathological features, including the presence of gastric atrophy, intestinal metaplasia, lymphoid aggregates, or eosinophil infiltrates in the mucosal layer, as shown previously . The hisThis study found that pathology findings could be related to nodule regression. In case of eradicated NG patients, those with persistent nodules even after the eradication exhibited higher inflammation score. Our findings suggest that higher intensity of chronic mononuclear cell infiltrates in these NG patients might interfere nodule regression by continous aggregation of the lymphoid follicles. On the other hand, in case of non-eradicated NG patients, those with spontaneous nodule regression exhibited higher intestinal metaplasia score. This is consistent with previous studies that NG improves upon senile changes of the gastric mucosa such as atrophic gastriits and intestinal metaplasia , 14.H. pylori eradication was not performed in all of the NG patients, because it is not covered by insurance in Korea. It was performed only in NG patients who agreed on additional payment for eradication. Secondly, there was no malignant change in all NG patients at the follow-up tests. Therefore, we could not find a correlation between NG and gastric malignancy in this study. Despite all these limitations, there was a significant difference on nodule regression between the NG patients with H. pylori eradication and those without eradication.There are several limitations in this study. Firstly, H. pylori eradication is the most significant factor associated with the prognosis of NG in adults. Although there is a tendency for NG patients with longer follow-up to regress nodules, the significance as a prognostic factor for nodule regression is low when compared to that of H. pylori eradication. Together, the findings of this study suggest that adults with NG should undergo H. pylori eradication to promote nodule regression regardless of the size and location of the nodules.


Lactobacillus plantarum strains, was identified in Lactobacillus plantarum NCFB 965 and Weissella viridescens NCFB 1655. The receptors of the two species had 66% identical amino acid sequences and belong to the amino acid-polyamine-organocation (APC) transporter protein family. The resistant mutants contained point mutations in the protein-encoding gene resulting in either premature stop codons, leading to truncated versions of the protein, or single amino acid substitutions. The secondary structure of the W. viridescens protein was predicted to contain 12 transmembrane (TM) helices, a core structure shared by most members of the APC protein family. The single amino acid substitutions that resulted in resistant strains were located in a confined region of the protein that consists of TM helix 10, which is predicted to be part of an inner membrane pore, and an extracellular loop between TM helix 11 and 12. By use of template-based modeling a 3D structure model of the protein was obtained, which visualizes this mutational hotspot region and further strengthen the hypothesis that it represents a docking site for plantaricin JK.By whole-genome sequencing of resistant mutants, a putative receptor for plantaricin JK, a two-peptide bacteriocin produced by some  Bacteriocins produced by lactic acid bacteria (LAB) are antimicrobial peptides that often kill closely related bacteria by inducing membrane leakage . Many ofLactobacillus plantarum is present in a great variety of foods and beverages such as dairy products, vegetables, meat, fish and wine [L. plantarum are also widely used as probiotics [L. plantarum-strains harbor genes encoding bacteriocins, including so-called two-peptide bacteriocins such as plantaricin JK, plantaricin EF and plantaricin S [Lactobacillus plantarum NCFB 965 and the opportunistic human pathogen Weissella viridescens NCFB 1655 [and wine . Selecteobiotics , 15. Sevaricin S . These taricin S , 17. Plaaricin S  kill senaricin S . As for CFB 1655 .W. viridescens NCFB 1655, Oppegård et al. recently identified a member of the amino acid-polyamine-organocation (APC) family of transport proteins as a putative plantaricin JK-receptor [By whole-genome sequencing of plantaricin JK-resistant mutants of receptor . The 610receptor , predictreceptor . The locL. plantarum NCFB 965. In addition, we generated two new resistant mutants of W. viridescens NCFB 1655, as a control and to confirm previous results. An APC protein family member was identified in both L. plantarum and W. viridescens as the putative receptor for plantaricin JK, and the structure of a possible bacteriocin docking site in this transporter was investigated using bioinformatics tools for prediction of TM helices and their orientation in the membrane and by template-based modeling of the 3D structure.In the present study, the whole-genome sequencing approach was used to identify the plantaricin JK-receptor in W. viridescens NCFB 1655 (LMGT 2314) and L. plantarum NCFB 965 (LMGT 2003) and were kindly provided by professors I. F. Nes and D. B. Diep at the Laboratory of Microbial Gene Technology (LMG), Norwegian University of Life Sciences. Stationary phase cultures of wild type or mutant variants of either W. viridescens NCFB 1655 or L. plantarum NCFB 965 were diluted 1:50 and added to the microtiter plate to obtain a final volume of 200 μl in each well. The microtiter plates were incubated for 6–8 hours at 30°C, after which growth of indicator cells was measured spectrophotometrically at 600 nm. The minimum inhibitory concentration (MIC50) value is defined as the concentration of bacteriocin needed to obtain 50% growth inhibition of the sensitive bacteria in the microtiter plate assay. A detailed description of this protocol is publicly available at protocols.io (https://dx.doi.org/10.17504/protocols.io.i66chhe).The two peptides of plantaricin JK, PlnJ and PlnK, were produced by solid phase synthesis as described by Hauge et al. . The purSpontaneous mutants with increased plantaricin JK-resistance were isolated as previously described by Oppegård et al. . In shorL. plantarum NCFB 965 and W. viridescens NCFB 1655 was isolated from 6 ml of overnight culture according to the manufacturer’s protocol using the Qiagen DNeasy Blood & Tissue Kit . The genomes were sequenced using an Illumina MiSeq instrument at the Norwegian Sequencing Center and evaluated using the polymorphism detection tool VAAL [W. viridescens consisting of 243 contigs [L. plantarum genome consisting of 188 contigs was obtained from wild type sequence reads using the A5-miseq assembly pipeline [http://www.geneious.com). The genome assemblies are available at the European Nucleotide Archive (ENA) with the following accession numbers: PRJEB22014 for the strain L. plantarum NCFB 965 and PRJEB10916 for the strain W. viridescens NCFB 1655.Genomic DNA of mutants derived from ool VAAL  as previool VAAL . In the  contigs . A crudeW. viridescens NCFB 1655 was built using I-TASSER (https://zhanglab.ccmb.med.umich.edu/I-TASSER/), an automated web-service for protein structure and function predictions based on amino acid sequences as input. In addition to the structure evaluation report performed by I-TASSER, an evaluation of the backbone RMSD values was done using MOLMOL (V1.0.7) [https://bioinformatics.cineca.it/PMDB/main.php) with the following PM-id: PM0081188.A 3D structure model of the APC family protein from (V1.0.7) . The higL. plantarum NCFB 965 strain is about 400 times more sensitive to plantaricin JK than the wild type W. viridescens NCFB 1655 strain , whereas only partial resistance (about 50 fold) was obtained with the W. viridescens mutants  to plantaricin JK, carried mutations in an APC family protein 66% identical to the one identified in W. viridescens, further emphasizing that this protein is a probable plantaricin JK-receptor. In one of these mutants, Lp.JK-c, a stop codon had been introduced at codon 83. In the second mutant, Lp.JK-b, 14 nucleotides had been inserted in codon 98 leading to sequence alteration and truncation of protein 14 amino acids downstream of the insertion point. The two remaining mutants, Lp.JK-d and Lp.JK-a, contained a 1047 nucleotide transposon-like insert at codon 182 and 270, respectively, also leading to sequence alteration and truncation of protein after 5 and 19 amino acids post insertion, respectively. In addition, Lp.JK-a had a codon change from ACA to ATA in a cytosine permease gene leading to a Thr to Ile alteration in position 91 of the protein  and it is also present in a number of complete Lactobacillus genomes in GenBank (results not shown).To further investigate the involvement of the APC family protein in resistance against plantaricin JK, we also analyzed plantaricin JK-resistance in  protein . Three fThe mutants Wv.JK-a, Wv.JK-b and Lp.JK-a had additional mutations outside the APC transporter gene . The posW. viridescens and Lp.JKa-d from L. plantarum, all contained mutations giving rise to truncated versions of the protein, either due to premature stop codons or to insertions. Interestingly, the L. plantarum NCFB 965 truncating mutations led to full resistance against the bacteriocin  and the previous study [W. viridescens NCFB 1655, but not in L. plantarum NCFB 965.In the previous study by Oppegård et al. , all mutus study , that leW. viridescens NCFB 1655 and L. plantarum NCFB 965 regarding the amino acids comprising TM helix 10 and the extracellular loop between TM helix 11 and 12 reveals that the sequences are, respectively, approximately 88 and 60 percent identical in these two regions . Conserved amino acids comprising TM helix 10 includes an Ala residue at position 377 where a mutation has been observed twice, once in this study and once in our previous study [W. viridescens and F425 in L. plantarum), although the M425K-mutation in W. viridescens (JK11) resulted in 400-fold increase in resistance . Conserus study . Interesd et al. ). This md et al. .W. viridescens and L. plantarum also are conserved among other related strains, a search using Basic Local Alignment Search Tool (BLAST) with the relevant part of the APC family protein from W. viridescens as query was performed. The 200 closest related GenBank protein sequences were aligned using Clustal Omega [weblogo.berkeley.edu). As the logo in To further investigate whether the identical amino acids in these two regions of al Omega , and a sApart from a His or Arg residue at position 428 and a Phe residue at position 429, none of the residues in the loop between TM helix 11 and 12 seem to be highly conserved. In contrast, out of the 24 residues comprising TM helix 10, all but three of them are identical . This inW. viridescens NCFB 1655 as input. The highest ranked 3D model (Model 1) by I-TASSER was constructed using another APC superfamily protein, ApcT from Methanocaldococcus jannaschi [In an attempt to visualize the regions of interest, a protein structure model was constructed using I-TASSER –30 with annaschi  (PDB-ID:W. viridescens protein using Phyre2 [We also modeled the g Phyre2  and obtag Phyre2 . Using MW. viridescens protein is in concordance with the predicted secondary structure of the protein (Figs In the predicted structure, the mutations at position 425 and 428 are located at or near the extracellular loop between TM helix 11 and 12. Furthermore, the mutations located at 377 and 387 are positioned in the middle or near the upper part of TM helix 10. The identification and characterization of bacteriocin receptors and the bacteriocin docking site on these receptors is important for gaining detailed insight into the mode of action of bacteriocins and their development into therapeutic drugs and/or natural additives in food. The method used in this study, possibly in combination with molecular dynamic simulations, should be an applicable and general approach for identification of bacteriocin receptors and docking sites.


Clostridium luticellarii, which has straight or slightly curved rod-shaped cells, polar endospores, and peritrichous flagella, is used for the production of strong aromatic Chinese liquors. C. luticellarii is able to produce butanoic acid. The draft genome sequence consists of 3.757 Mbp, including 3,632 predicted protein-encoding genes.The strictly anaerobic, Gram-positive bacterium  Clostridium luticellarii strain DSM 29923, which was first characterized in 2015 . Illumina paired-end sequencing libraries were generated from the isolated DNA, as recommended by the manufacturer . Sequencing was performed by employing the MiSeq reagent kit version 3 and the MiSeq sequencing platform, as described by the manufacturer (Illumina). Quality improvement of the recovered reads was obtained with Trimmomatic version 0.36 -transferase subunits A and B in the genome indicated production of butanoic acid in C. luticellarii DSM 29923 via this enzyme.Chromosomal DNA of ion 0.36 . Assemblion 0.36  was emplPVXP00000000. The version described here is version PVXP01000000.The whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number


Pleopeltis minima (Polypodiaceae), and compared it to plant miRNA databases, allowing for the identification of miRNA families that are shared by all land plants, shared by all vascular plants (tracheophytes) or shared by euphyllophytes (ferns and seed plants) only. Using the recently described transcriptome of another fern, Lygodium japonicum, we also estimated the degree of conservation of fern miRNA targets in relation to other plant groups. Our results pinpoint the origin of several miRNA families in the land plant evolutionary tree with more precision and are a resource for future genomic and functional studies of fern miRNAs.MicroRNAs (miRNAs) are short, single stranded RNA molecules that regulate the stability and translation of messenger RNAs in diverse eukaryotic groups. Several miRNA genes are of ancient origin and have been maintained in the genomes of animal and plant taxa for hundreds of millions of years, playing key roles in development and physiology. In the last decade, genome and small RNA (sRNA) sequencing of several plant species have helped unveil the evolutionary history of land plants. Among these, the fern group (monilophytes) occupies a key phylogenetic position, as it represents the closest extant cousin taxon of seed plants, i.e. gymno- and angiosperms. However, in spite of their evolutionary, economic and ecological importance, no fern genome has been sequenced yet and few genomic resources are available for this group. Here, we sequenced the small RNA fraction of an epiphytic South American fern,  Th. ThP. miP. minima, by comparing its miRNA repertoire to those of other land plant groups. P. minima, showing that the fern miRNA repertoire is very conserved in this regard  family. In angiosperms, C3HDZ genes are expressed in the adaxial side of developing leaves and are necessary for the proper dorsoventral patterning of these structures . In the HDZ familAGO1, DCL1 and AGO2 are targets of miR168, miR162 and miR403, respectively [AGO1 is also a miR168 target in the Chinese fir, a gymnosperm [AGO1 and DCL1 with a different set of miRNAs. Thus, miR902 and miR1047 target AGO1 and DCL1 in the moss P. patens [AGO1 in the liverwort, M. polymorpha [L. japonicum AGO1 in a site that, given its position in the mRNA, seems to be homologous to the AGO1 site targeted by miR168 in angiosperms , which originated in the early stages of the colonisation of land by plants and has roles in pathogen defence [L. japonicum and the liverwort, P. endiviifolia, PPO is targeted at the 3'-UTR by miR408  J. Prado & R.Y. Hirai  de la Sota.) [Specimens of a Sota.)  were colP. minima fertile fronds from two different specimens each were subjected to a protocol for total RNA extraction enriched for small RNAs using the mirVana miRNA Isolation Kit (Thermo Fisher Scientific). The quantity and quality of the total RNAs were analised with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) and an Agilent 2100 Bioanalyzer . Small RNA libraries were generated from both P. minima independent RNA samples using the Illumina Truseq SmallRNA Preparation kit. Total sRNAs were ligated to 3p and 5p adapters (ADTs), and the corresponding cDNA was obtained by reverse-transcription PCR. The purified cDNA library was used for cluster generation on Illumina’s Cluster Station and then sequenced on Illumina GAIIx . Raw sequencing reads were obtained using Illumina’s Sequencing Control Studio software version 2.8 (SCS v2.8) following real-time sequencing image analysis and base-calling by Illumina's Real-Time Analysis version 1.8.70 (RTA v1.8.70).Two independent samples of http://rfam.janelia.org) were removed, as well as reads mapping to RepeatMasker (http://www.repeatmasker.org/). The non-redundant sRNA reads were mapped to plant precursor and mature miRNA sequences stored in miRBase 21.0 (http://www.mirbase.org/) [http://www.mirbase.org/search.shtml) using BLASTN and SSEARCH to confirm the identity of the miRNAs. The final set of conserved P. minima miRNAs . Raw reads were filtered to remove adaptor sequences and reads which were too short (<15 nt) or too long (>40 nt). Sequences corresponding to mRNA fragments, rRNA, tRNA, snoRNA and snRNA mapping to Rfam (se.org/)  using Boa miRNAs  are eith entries  to discaP. minima sequences were also checked against a large plant miRNA database generated recently [M. quadrifolia. The miRNAs that were reported for M. quadrifolia by Chávez-Montes et al [http://www.mirbase.org/) and miRNA sequences that exhibited low similarily to miRBase entries  were discarded. Next, the abundance of each miRNA sequence was checked against the Comparative Sequencing of Plant miRNA database  [M. quadrifolia and other plants [bona fide conserved miRNAs were incorporated into the descriptions of miRNA distribution and evolution shown in Figs To better characterize the phylogenetic distribution of plant miRNAs, recently  which ines et al  were reaer.org/)  and sequr plants  were disP. minima conserved miRNA sequences identified  [http://bioinf.mind.meiji.ac.jp/kanikusa/) that consists of a transcriptome assembled from sequenced RNA samples from prothalli, trophophylls, rhizomes and sporophylls obtained using Roche 454 GSFLX and Illumina HiSeq sequencers [The aponicum , for potTarget/) . Searchequencers . Search quencers . For somP. minima small RNA transcriptome was submitted to the NCBI Sequence Read Archive under the accession number SRR4294182.The raw sequencing data of S1 Fig(A) Sequence of a transcript (Locus_14997) encoding a Squamosa-binding protein-like (SPL) homologue from the fern L. japonicum. The region predicted to be targeted by pmi-miR156 or pmi-miR529 is indicated in yellow. The starting ATG and stop codon are highlighted in blue. (B) Alignment of part of the SPL transcripts from L. japonicum (Lja), the liverwort M. polymorpha (Mpo), the gymnosperm Pinus tabuliformis (Pta), the dicot A. thaliana (Ath) and the monocot O. sativa (Osa). Residues displaying over 75% identity are highlighted. The region targeted by miR156/529 is indicated in red, and the conserved SBP-DNA binding domain in green. Note that the miRNA-targeted region is conserved in all mRNAs and species. (C) Predicted pairing between pmi-miR156 and miR529 and L. japonicum Locus_14997. The E-complementarity score between miRNA and target RNA as estimated by the psRNATarget program is shown.(DOCX)Click here for additional data file.S2 Fig(A) Sequence of a transcript (Locus_7173) encoding a MYB transcription factor protein from the fern L. japonicum. The region predicted to be targeted by pmi-miR159 or pmi-miR319 is indicated in yellow. The starting ATG and stop codon are highlighted in blue. (B) Alignment of part of the MYB transcripts from L. japonicum (Lja), the liverwort M. polymorpha (Mpo), the moss P. patens (Ppa), the gymnosperm Pinus tabuliformis (Pta), the basal angiosperm Amborella trichopoda (Atr), the dicot A. thaliana (Ath) and the monocot O. sativa (Osa). Residues displaying over 75% identity are highlighted in blue. The region targeted by miR156/529 is indicated in red, and the conserved MYB DNA binding domain in green. Note that the miRNA-targeted region is conserved in all mRNAs and species. (C) Predicted pairing between pmi-miR159 and miR319 and L. japonicum Locus_7173. The E-complementarity score between miRNA and target RNA as estimated by the psRNATarget program is shown.(DOCX)Click here for additional data file.S3 Fig(A) Sequence of a transcript (Locus_3676) encoding a RKD transcription factor protein from the fern L. japonicum. The region predicted to be targeted by pmi-miR159 or pmi-miR319 is indicated in yellow. The starting ATG and stop codon are highlighted in blue. (B) Alignment of part of the RKD transcripts from L. japonicum (Lja), the liverwort M. polymorpha (Mpo) and the moss P. patens (Ppa). Residues displaying over 75% identity are highlighted. The region targeted by miR159 (or miR319) is indicated in red, and the conserved RWP-RK DNA binding domain in green. Stop codons are encircled in yellow. Note that the miRNA-targeted region is conserved in all mRNAs and species. (C) Predicted pairing between pmi-miR159 and miR319 and L. japonicum Locus_3676. The E-complementarity score between miRNA and target RNA as estimated by the psRNATarget program is shown.(DOCX)Click here for additional data file.S4 Fig(A) Sequence of a transcript (Locus_3364) encoding an ARF transcription factor protein from the fern L. japonicum. The region predicted to be targeted by pmi-miR160 is indicated in yellow. The starting ATG and stop codon are highlighted in blue. (B) Alignment of part of the ARF transcripts from L. japonicum (Lja), the liverwort M. polymorpha (Mpo), the lycopod S. moellendorffii (Smo), the moss P. patens (Ppa), the gymnosperms Picea abies (Pab) and Cycas rumphii (Cru), the dicot A. thaliana (Ath) and the monocots O. sativa (Osa) and Brachypodium distachyon (Bdi). Residues displaying over 75% identity are highlighted. The region targeted by miR160 is indicated in red, and the conserved Auxin response domain (ARF) in green. Note that the miRNA-targeted region is conserved in all mRNAs and species. (C) Predicted pairing between pmi-miR160 and L. japonicum Locus_3364. The E-complementarity score between miRNA and target RNA as estimated by the psRNATarget program is shown.(DOCX)Click here for additional data file.S5 Fig(A) Sequence of a transcript (Locus_1230) encoding an C3HD-ZIP transcription factor protein from the fern L. japonicum. The region predicted to be targeted by pmi-miR166 is indicated in yellow. The starting ATG and stop codon are highlighted in blue. (B) Predicted pairing between pmi-miR166 and L. japonicum Locus_1230. The E-complementarity score between miRNA and target RNA as estimated by the psRNATarget program is shown. (C) Alignment of part of the C3HD-ZIP transcripts from ferns L. japonicum (Lja) and Psilotum nudum (Pnu), the liverwort M. polymorpha (Mpo), the lycopod S. moellendorffii (Smo), the moss P. patens (Ppa), the gymnosperms Cunninghamia lanceolata (Cla), Taxus globosa (Tgl) and Gingko biloba (Gbi); the basal angiosperm Amborella trichopoda (Atr), the dicots A. thaliana (Ath), Medicago truncatula (Mtu), Solanum lycopersicum (Sly) and Populus trichocarpa (Ptr), and the monocots O. sativa (Osa) and Brachypodium distachyon (Bdi). Residues displaying 100% identity are highlighted in blue. The region targeted by miR166 is indicated in red, the START domain in green, and HOX Homeodomain in light blue. Note that the miRNA-targeted region is conserved in all mRNAs and species.(DOCX)Click here for additional data file.S6 Fig(A) Sequence of a transcript (Isotig_21625) encoding AGO1 protein from the fern L. japonicum. The region predicted to be targeted by pmi-miR168 is indicated in yellow. The starting ATG and stop codon are highlighted in blue. (B) Alignment of part of the AGO1 transcripts from fern L. japonicum (Lja), the gymnosperm Cunninghamia lanceolata (Cla), the dicot A. thaliana, and the monocots O. sativa (Osa) and Brachypodium distachyon (Bdi). Residues displaying 100% identity are highlighted in blue. The region targeted by miR168 is indicated in red, and the Argonaute N-terminal domain domain (ArgoN) in green. Note that the miRNA-targeted region is conserved in all mRNAs and species. (C) Predicted pairing between pmi-miR168 and L. japonicum Isotig21625. The E-complementarity score between miRNA and target RNA as estimated by the psRNATarget program is shown.(DOCX)Click here for additional data file.S7 Fig(A) Sequence of a transcript (Locus_11016) encoding a Scarecrow-like(SCL)/GRAS domain transcription factor protein from the fern L. japonicum. The region predicted to be targeted by pmi-miR168 is indicated in yellow. The transcript is incomplete and lacks the starting ATG, but the stop codon is highlighted in blue. (B) Alignment of part of the SCL transcripts from fern L. japonicum (Lja), the liverwort M. polymorpha (Mpo), the lycopod S. moellendorffii (Smo), the moss P. patens (Ppa), the gymnosperms Pinus tabulliformis (Pta) and Pinus radiata (Pra); the basal angiosperm Amborella trichopoda (Atr), the dicots A. thaliana (Ath), Solanum lycopersicum (Sly) and Populus trichocarpa (Ptr), and the monocots O. sativa (Osa) and Brachypodium distachyon (Bdi). Residues displaying over 75% identity are highlighted. The region targeted by miR171 is indicated in red, and the GRAS domain in green. Note that the miRNA-targeted region is conserved in all mRNAs and species. (C) Predicted pairing between pmi-miR171 and L. japonicum Locus_11016. The E-complementarity score between miRNA and target RNA as estimated by the psRNATarget program is shown.(DOCX)Click here for additional data file.S8 Fig(A) Sequence of a transcript (Locus_9880) encoding a Apetala-2 transcription factor protein from the fern L. japonicum. The region predicted to be targeted by pmi-miR172 is indicated in yellow. The starting ATG and stop codon are highlighted in blue. (B) Alignment of part of the AP2 transcripts from ferns L. japonicum (Lja) and Ceratopteris thalictroides (Cta), the gymnosperms Pinus tabulliformis (Pta) and Ginkgo biloba (Gbi); the basal angiosperm Amborella trichopoda (Atr), the dicot A. thaliana  and Brachypodium distachyon (Bdi). Residues displaying over 75% identity are highlighted. The region targeted by miR172 is indicated in red, and the AP2 DNA-binding domain in green. Note that the miRNA-targeted region is conserved in all mRNAs and species. (C) Predicted pairing between pmi-miR172 and L. japonicum Locus_9880. The E-complementarity score between miRNA and target RNA as estimated by the psRNATarget program is shown.(DOCX)Click here for additional data file.S9 Fig(A)TAS3 RNAs (Locus 20755 and Locus 39179) from L. japonicum predicted to be targeted by pmi-miR390. The regions predicted to pair with miR390 are indicated in blue, and the trans-acting small-interfering RNAs (tasi-RNAs) derived from TAS3 processing are shown in yellow. The tasi-RNAs are predicted to target mRNAs encoding ARF3/4 transcription factors. (B)TAS3 RNAs were aligned with the CLUSTAL Omega program. Regions predicted to be targeted by pmi-miR390 are indicated in blue, with the base pairing with miR390 indicated. Note that the pairing between pmi-miR390 is more extensive at the 5' site than at the 3' site, indicating that RISC-mediated cleavage of the RNAs probably occurs only at the 5' site. The tasiRNAs derived from TAS3 processing are shown in yellow. (C) Alignment of part of the TAS3 transcripts from L. japonicum (Lja) and a TAS3 from the dicot A. thaliana (Ath). Residues displaying 100% identity are highlighted. (D) Sequence of a transcript (Isotig23398) encoding a ARF transcription factor protein from the fern L. japonicum. The region predicted to be targeted by pmi-tasiR-ARF is indicated in yellow. The starting ATG and stop codon are highlighted in blue. (E) Alignment of part of ARF transcripts from fern L. japonicum (Lja), the gymnosperm Pinus pinaster (Ppi); the basal angiosperm Amborella trichopoda (Atr), the dicot A. thaliana (Ath) and the monocot Brachypodium distachyon (Bdi). Residues displaying 100% identity are highlighted. The region targeted by pmi-tasiR-ARF is indicated in red, and the B3 DNA binding and the Auxin responsive factor domains are also indicated. Note that the tasiR-ARF-targeted region is conserved in all mRNAs and species.(DOCX)Click here for additional data file.S10 Fig(A) Sequence of a mRNA encoding Polyphenol oxydase  from L. japonicum predicted to be targeted by pmi-miR408. The targeted region is located in the 3'-UTR of the mRNA (yellow). The starting ATG and stop codon are highlighted in blue. (B) Predicted pairing between pmi-miR408 and L. japonicum Locus_9880. The E-complementarity score between miRNA and target RNA as estimated by the psRNATarget program is shown.(DOCX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Myxobolus carnaticus, infecting the gill lamellae of mrigal carp, Cirrhinus mrigala, was characterized by the 18S rRNA gene sequence. The DNA sequence of M. carnaticus clustered phylogenetically with other gill infecting Myxobolus spp. of freshwater clades, forming a dichotomy with closely related M. pavlovskii (HM991164) that infects the gill lamellae epithelium of silver carp, Hypophthalmichthys molitrix with 95% similarity. Evolutionary pair-wise distances among M. carnaticus and other species of myxosporeans indicated high genetic diversity among myxosporeans. The present study demonstrated that tissue tropism, host specificity and habitat play important roles in phylogenetic relationships among myxozoan species.Myxozoans are an economically important group of microscopic parasites best known for the diseases they cause in commercially important fish hosts. The classification of myxosporeans is generally based on the morphology of their myxospores. Without molecular data, it is very difficult to identify new or existing species. DNA sequence information is therefore, a prerequisite to taxonomic and phylogenic studies of myxosporeans. In the present study, a myxozoan parasite,  Myxobolus, Thelohanellus, Henneguya, Kudoa, Myxidium, Myxosoma and several others have been found to be the causative agents of the disease [Myxobolus carnaticus infecting the gill lamellae of Cirrhinus mrigala are presented.Myxoboliasis, caused by myxosporean parasites, is one of the most widely distributed fish diseases. Myxosporean species of the genera  disease . These a disease . Heavy c disease . The cur disease  were theMyxobolus species infecting the inner base of gill lamellae of Cirrhinus mrigala , collected from Garia , South 24 Parganas District, West Bengal, India, during the routine survey of carp parasitic diseases in 2013, was characterized by morphometric and molecular techniques. Myxosporean identification was performed according to Lom and Arthur [Myxobolus species, viz., DNA extraction, PCR amplification, purification of amplicon, sequencing, electrophoresis and data analysis was done as described in Mondal et al. [For the purpose of the present study, the d Arthur . The unil et al.  and Abra-Expectation (MUSCLE) program [Phylogenetic analysis was performed on a selection of 18S rRNA gene sequences comprising the new sequence KF796620) and 25 additional sequences from closely related species of freshwater and marine origin available in the NCBI GenBank database using the basic local alignment search tool (BLAST). Sequence alignment was performed by Multiple Sequence Comparison by Log6620 and  program . Bayesia program  in TOPAL program . A total program  in MEGA6 program . IncludeC. mrigala. Plasmodium was very small, white to pale coloured and elongated. Mature spores (n=20) measured 9.49±0.98 (8.10-12.90) μm in length and 8.27±0.62 (7.20-10.00) μm in breadth , the large polar capsule length to breadth ratio (1:0.67) and the small polar capsule length to breadth ratio (1:0.69) differed slightly from original descriptions  of M. carnaticus [Myxosporean species was isolated from the inner base of the gill lamellae of sub- adult  breadth . Small a Manohar  from thernaticus , but didM. carnaticus were successfully amplified (M. carnaticus showed 82-95% homogeneity with other myxosporeans from both freshwater and marine clades (M. carnaticus and other gill-infecting Myxobolus spp. was87-95%. Our previously characterized carp gill-infecting M. orissae (KF448527) [M. catmrigalae (KC933944) exhibited 89% and 87% similarity with M. carnaticus (KF796620), respectively, while the carp fin-infecting Thelohanellus caudatus (KC865607) [M. carnaticus. Maximum similarity (95%) was observed with M. pavlovskii (HM991164), which infects the epithelium of gill lamellae of Hypophthalmichthys molitrix in Hungary.About 1984 bp fragments of the 18S rRNA gene of mplified  by the ue clades . The 18Sicus KF79620, respM. carnaticus clustered phylogenetically with other gill infecting Myxobolus spp. of freshwater clades, and formed a dichotomy with carp gill infecting M. pavlovskii (HM991164) with a high node support. All marine species comprising of Ceratomyxa sp. (DQ333431), Kudoa funduli (AF195510), Myxidium gadi (GQ890675), and Myxidium maxi (KF179055) were phylogenetically clustered as a separate lineage  to 0.30 (Kudoa funduli AF195510 and Ceratomyxa sp. DQ333431) is a possible indicator of a high level of genetic diversity among myxosporeans.The phylogenetic tree established by Bayesian Estimation for the new sequence  was simi lineage . Thelohalgorithm  from 0.0M. carnaticus (KF796620) infecting the gill lamellae of C. mrigala. Earlier, we characterized M. cuttacki KF465682 [M. orissae KF448527 [T. caudatus KC865607 [This report is the first of its kind to desribe the molecular phylogeny of KF465682  and M. oKC865607 . AccordiKC865607 , site spOur study also demonstrated that tissue tropism, host specificity and habitat playimportant roles in phylogenetic relationships among myxozoan species. As the list of myxosporean parasites described from India is growing , molecul


Bartonella infections were investigated in seven species of bats from four regions of the Republic of Georgia. Of the 236 bats that were captured, 212 (90%) specimens were tested for Bartonella infection. Colonies identified as Bartonella were isolated from 105 (49.5%) of 212 bats Phylogenetic analysis based on sequence variation of the gltA gene differentiated 22 unique Bartonella genogroups. Genetic distances between these diverse genogroups were at the level of those observed between different Bartonella species described previously. Twenty-one reference strains from 19 representative genogroups were characterized using four additional genetic markers. Host specificity to bat genera or families was reported for several Bartonella genogroups. Some Bartonella genotypes found in bats clustered with those identified in dogs from Thailand and humans from Poland. Bartonella parasitize erythrocytes and endothelial cells of a wide range of mammals and recently were reported in bats from Africa, Asia, America, and northern Europe. A human disease case in the USA was associated with a novel Bartonella species, which later was identified in bats in Finland. This human case has demonstrated the zoonotic potential of bat-borne Bartonella and underscores the need for extended surveillance and studies of these pathogens. The present work assesses prevalence and diversity of Bartonella in bats in the country of Georgia (southern Caucasus), characterizes reference strains representing diverse genogroups by variation of genetic loci, and evaluates the links between identified Bartonella genogroups and bat hosts. Importantly, some Bartonella genotypes found in bats were close or identical to those identified in dogs and humans. The data indicate that the public health impact of Bartonella carried by bats should be investigated.Bacteria of the genus  Bartonella are small and slow-growing Gram-negative aerobic bacilli. These bacteria parasitize erythrocytes and endothelial cells of a wide range of mammals. During the last six years, diverse Bartonella strains were identified in bats from Europe [Bartonella, demonstrating that strains of Bartonella in bats tend to cluster according to bat families, superfamilies, and suborders [Bartonella strains [Leptospira strains [Bartonella spp. can spillover into phylogenetically distant hosts, including humans [Bartonella species  are hosts of a wide range of zoonotic pathogens. Their significance as reservoirs of emerging infectious diseases, predominantly of viral origin, has been increasinglyecognized during recent decades ,2. In com Europe –6, Africm Europe –12, Asiam Europe ,14, and m Europe –19. Receuborders ,21. Host strains ,22 and b strains . Despiteg humans ,25. A reonensis; ), which Bartonella in bats in theRepublic of Georgia (southern Caucasus) with the following objectives: 1) to compare prevalence of Bartonella infection in diverse bat species from different geographic locations within Georgia; 2) to determine the genotypes of obtained strains by variation in gltA sequences, a gene commonly used for discrimination of Bartonella species; 3) to characterize reference strains representing diverse genogroups by variation of multiple genetic loci; and 4) to evaluate the links between identified Bartonella genogroups and bat hosts.The goal of the present work was to identify prevalence and diversity of All animal work has been conducted according to relevant NCDC, national, and international guidelines.Bats were collected from two distinct parts of Georgia in June 2012. Four locations are situated in Eastern Georgia: three sites in the Kakheti region near Davit Gareja, one site in the Kvemo Kartli region in Gardabani district. The other four locations are in Western Georgia: two sites in the Samegrelo-Zemo Svaneti region (Martvili district and Chkhrotsku district) and two sites in the Imereti region . The number of captured bats from each site is shown in Bats were captured manually or using nets from different roosts in caves and buildings . The list of bat species and the number of animals per roost or colony availablefor sampling was approved by the Ministry of Environmental and Natural Resources Protection ofGeorgia. Species of captured bats were identified based on external morphological characteristics. Captured bats (n = 236) were delivered to the processing site in individual cotton bags where they were processed. Bats were anesthetized with the use of ketamine (0.05–0.1 mg/g body mass) and exsanguinated by cardiac puncture. All bats were sexed and measured. The procedures of handling animals were performed in compliance with the protocol approved by the CDC Institutional Animal Care and Use Committee (protocol 2096FRAMULX-A3). Blood specimens were transported on dry ice to the NCDC Laboratory, Tbilisi where they were stored at -80°C until they were shipped on dry ice to the CDC’s laboratory, Fort Collins, Colorado. Upon arrival at CDC, the samples were stored at -80°C until they were analyzed.Bartonellawere passaged onto a new plate to obtain pure cultures. In an attempt to capture possible Bartonella coinfections, all morphologically unique colonies growing from a single sample were sub-passaged and sequenced. All resulting isolates were collected in a 10% glycerol solution. Crude DNA extracts were obtained from isolates by heating a heavy suspension of themicroorganisms for 10 minutes at 95°C. Polymerase chain reactions (PCR) with the gltA primersBhCS781.p (5’-GGGGACCAGCTCATGGTGG-3’) and BhCS1137.n (5’-AATGCAAAAAGAACAGTAAACA-3’) [B. doshiae) and negative (nuclease free water) control samples were included in each PCR assay to evaluate the presence of appropriately sized amplicons and to rule out contamination of reagents, respectively. Positive PCR products were purified using QIAquick PCR purification Kit  and sequenced with an ABI 3130 Genetic Analyzer . Forward and reverse reads were assembled into consensus sequences with the SeqMan Pro program in Lasergene v. 11 .Bat blood was diluted 1:4 in Brain Heart Infusion (BHI) with 5% Fungizone (amphotericin B), and 100μl of the sample was placed on a chocolate agar plate following the protocol published previously . InoculaAACA-3’)  were perhttp://blast.ncbi.nlm.nih.gov/Blast.cgi) search of the GenBank database was performed with all assembled gltA sequences to verify their Bartonella origin. Positive sequences were aligned with Bartonella reference sequences available in GenBank which included sequences obtained from various bats in previous studies. Brucella abortus sequence was used as outgroup. Alignment was performed with MAFFT v7.187 using the local, accurate L-INS-i method [Bartonella sequences from Georgian bats were all isolated at the same date and thus could not be used for calibration of another clock model; furthermore, our analysis did not seek to accurately deduce branch times, and the strict clock was adequate. No codon partitioning was used due to the fact that gltA sequences represent only a 367 base pair fragment of the entire gene; codon partitioning with limited genetic information can substantially reduce the effective sample size of estimated parameters forseparate codon positions [Bartonella species [A BLAST  that have been previously used for bartonellacharacterization [gltA gene. Genogroups Vesp-7, Vesp-13, and Rhin-3 were not analyzed by MLST, while three isolates of Vesp-6 were selected for analysis to examine within-genogroup variation. The primers and cycle conditions used to generate sequences for each loci have been previously published [Bartonella species and other Bartonella sequences obtained from bats with MAFFT v7.187 using the L-INS-i method [gltA tree as described above. Separate maximum likelihood gene trees were generated using the GTRCAT model in RAxML [Five genetic loci , were collapsed into a single principlecomponent that explained 95% of variation in size. However, bat size was strongly predicted by batspecies  and sex , so size was not included as acovariate in the global model. Model selection was based on AICc [Bartonella prevalence among bat species, capture locations, and bat sexes wereapproximated with the Agresti-Coull method [A logistic model was used to examine important predictors of  on AICc . Additio on AICc ,49. Binol method . All stal method  andvalueEptesicus serotinus ; Schreibers's long-fingered bat, Miniopterus schreibersii sensu lato  [Myotis blythii ; Geoffroy's myotis, Myotis emarginatus ; whiskered myotis, Myotis mystacinus ; soprano pipistrelle, Pipistrellus pygmaeus ; Mediterranean horseshoe bat, Rhinolophus euryale ; and greater horseshoe bat, Rhinolophus ferrumequinum . The number of species and specimens obtained varied per site  ; lesser per site .Bartonella testing. The amount of blood from thesingle captured My. mystacinus was not sufficient for culturing. Except for this, bartonellae weresuccessfully cultured from all bat species tested . All the harvested colonies were confirmed as Bartonella by PCR and sequencing of gltA fragments. The overall prevalence of Bartonella in bats by culturing was 49.5% (105/212). Bartonella isolates were obtained from each of the eight sites. The prevalence of culture-positive bats varied from 16.7% at the Lavra site in Davit Gareja to 64.6% at Gliana Cave in Tskaltubo.A total of 212 bats of seven species were available for s tested . BartoneP. pygmaeus to 88.9% in Mn. schreibersii. The best model based on AICc included bat species only with a good amount of predictive power(AUC = 0.71) [Bartonella prevalence. Prevalence of Bartonella in My. blythii , Mn. schreibersii , and R. euryale  was significantly higher.The range of prevalence varied from 16.7% in  = 0.71) . Based oBartonella-like colonies were observed with morphologies that visually varied by size  and/or texture . The number of visually different colonies per plate varied from one unique colony among TNTC similar colonies to an equal number of two unique colony morphologies. We did not attempt to estimate colony forming units (CFU) for individual bats suspected of coinfection. Sequencing analysis confirmed a coinfection with two different Bartonella sequences from these 16 samples , but from distant locations. Other sequences, notably from genogroups Mini-1.1, Mini-3, and Vesp-6, clustered with bartonella sequences identified in dogs from Thailand [In some cases, Georgian bat sequences matched very closely with other Thailand  and in hThailand .ftsZ, gltA, nuoG, rpoB, and groEL) confirmed that most Bartonella genogroups from Georgian bats formed well-supported clades (posterior probability > 90%) with other Bartonella genogroups identified in bats. (The phylogeny based on concatenated sequences from five genetic loci (in bats. Bartonella genogroups found in African pteropodid (Eidolon helvum andRousettus aegyptiacus) [Hipposideros sp. and Triaenops persicus) [Coleura afra) [Bartonella genogroup found in Miniopterus schreibersii from Taiwan [Candidatus Bartonella naantaliensis found in Myotis daubentonii from Finland [Genogroups Mini-1, Mini-1.1, Mini-2, Mini-3, Rhin-2, Rhin-4, Rhin-5, and Vesp-10 formed a well-supported clade with other ptiacus) ,9, hippoersicus) , andembara afra)  bats. Gem Taiwan . GenogroEw from Eidolon helvum [Bartonella mayotimonensis isolated from a human endocarditis patient [Eptesicus nilssonii and Myotis daubentonii) [Bartonella vinsonii subspecies, with low posterior probability support (50%). Finally, genogroup Vesp-12 clustered with genogroup E4 from Eidolon helvum [Bartonella clarridgeiae and Bartonella rochalimae. The network phylogeny  where high prevalence and diversity of Bartonella strains have been reported [Mn. schreibersii and below 17% in P. pygmaeus) as well as between study sites. The difference inprevalence between locations can be likely explained by bat community composition  Mn. schreibersii tested were coinfected with two different Bartonella genotypes  from Mn.schreibersii aligned with genogroups Mini-1, Mini-1.1., Mini-2, or Mini-3  from Madagascar [Rhinolophus spp.  belonged to genogroupsRhin-1, Rhin-2, Rhin-3, or Rhin-4. Genogroups Rhin-2 and Rhin-3 cluster with Bartonella sequences identified in R. acuminatus and R. sinicus from Vietnam [Eptesicus, Myotis, and Pipistrellus spp.) were members of genogroups Vesp-1 to Vesp-12 with closely matching sequences found in other vespertilionid bats [We observed several coinfections among sampled bats. The phenomenon of coinfections with two or three different  rodents . Intereshe world ,16,20. Fr Mini-3 . Based odagascar . Thirty- Vietnam . Most isnid bats ,56.Bartonella into atypical hosts. For example, isolates of Bartonella from genogroup Mini-1 were found in E. serotinus, My. blythii, and P. pygmaeus, and isolates of Bartonella from genogroups Rhin-1 and Rhin-3 were found in My. emarginatus and My. blythii, respectively  has been collected from E. serotinus, My. blythii, P. pygmaeus, and R. ferrumequinum [Cimex pipistrelli (Hemiptera: Cimicidae) has been reported parasitizing E. serotinus, My. blythii, My. emarginatus, P. pygmaeus, and R. ferrumequinum [Spinturnix myoti (Mesostigmata: Spinturnicidae) has been recorded on E. serotinus, Mn. schreibersii, My. blythii, R. euryale, and R. ferrumequinum [Bartonella genogroups in some bat hosts can potentially be explained by sharing of ectoparasites. Future analyses exploring the influence of ectoparasite distributions on sharing of Bartonella genogroups among bats are in progress.Despite these general host associations, specificity of genogroups at the genus or family levelwas not strict, with some instances of apparent spillover of ectively . Though ectively , whereinhsteinii , Basiliaattereri , Nycterieibersii , and Phthus spp. . Neverthrginatus , Basiliaerotinus , Nycterimequinum ,63, Peni blythii , and Phtrginatus ,64. Othemequinum ,65,66. Cmequinum ,68. Addimequinum –71. ThisftsZ, gltA, nuoG, rpoB, and groEL) along with the network phylogenetic analysis strongly indicated that many genogroups characterized in our study can be segregated into new Bartonella species according to established demarcationcriteria considering loci separately [Bartonella genogroup. The host associations observed for most of identified genetic clusters also supports the biological basis for discrimination of the species. As was reasoned previously [Bartonella species compared to sequence analysis alone. For example, genogroups Vesp-1, Vesp-2, and Vesp-3 share 92%, 93%, and 92% nucleotide identity, respectively, with Bartonella mayotimonensis, the bacterial species discovered in a human patient in the United States [B. mayotimonensis is closest (95%) at the gltA locus to a sequence identified in a bat fly Anatrichobius scorzai taken from a bat Myotis keaysi in Costa Rica [B. mayotimonensis species, but using the gltA locus alone creates an artifactual split among the genogroups. When all five concatenated loci were considered, genogroups Vesp-1, Vesp-2, and Vesp-3 shared pairwise sequence identities between 96.9–98.11%. Considering their relatedness and apparent specificity to vespertilionid bats  [B. mayotimonensis ranged 95.1–95.5%, which is near the previously established minimum threshold for distinguishing between Bartonella species (95.4% for rpoB sequences [Eptesicus and Myotis) [Bartonella species. This is also true for genogroups Vesp-4 and Vesp-5 found in one bat species, My. blythii (96.3% sequence identity) and genogroups Mini-1 and Mini-1.1 found in Mn. schreibersii (96.6% sequence identity).The sequence characterization of five house-keeping genes  and we closest 9% at the Bartonella, which are similar to Bartonella strains previously reported in humans and in dogs. Thepublic health relevance of bat-borne Bartonella infection has been discussed since the identification ofsuch bacteria in bats from Kenya [Bartonella surveillance inbats, as it can help to identify potential wildlife reservoirs of human cases. Although some sequences of Bartonella found in Georgian bats clustered with B. mayotimonensis, the genetic distances were relatively long, as noted above. We might speculate that Bartonella more closely related to thishuman case are circulating in vespertilionid bats in the North and South America rather than in Europe. Even more unexpected was the discovery of Bartonella strains in Georgian bats which wereidentical or very similar to ones reported in forest workers from Poland. The study in Poland wasconducted to evaluate the level of exposure of 129 forest workers to diverse tick-borne pathogens [Bartonella antibodies were reported in about 30% of tested individuals, but more importantly, threeserologically-positive samples were also positive for Bartonella nucleic acids by PCR and sequencing. The gltA sequences identified in that study were distinct from all previously reported. They were closest (90% similarity) to B. koehlerae, B. clarridgeiae and a genotype from an arthropod from Peru. They were deposited in GenBank  as uncultured Bartonella spp. [gltA sequences to the HM116785 sequence. Vesp-6 is the largest genogroup found in bats from Georgia containing 18 sequences from My. blythii (n = 15), My. emarginatus (n = 2), and E. serotinus (n = 1). All of these bat species are listed as occurring in southern Poland where the investigation of forest workers was conducted [The most intriguing and important results from this study is the identification of bat-borneom Kenya . Our resathogens .Bartonellla spp. . All stronducted –75.Bartonella strains observed in this study were closely related to those identified in stray dogs from Thailand., Bai et al. [Bartonella infections and diverse Bartonella species in the blood of stray dogs from Bangkok and Khon Kaen (northeastern province of Thailand). Besides two Bartonella species (B. elizabethae and B. taylorii) detected in stray dogs from Khon Kaen, the authors also reported two genotypes (KK20 and KK61) that could potentially represent a new species [Bartonella strains found in bats from Georgia, we found that sequences of the strains from genogroup Mini-1.1 obtained from Mn. schreibersii (n = 7) and R. euryale (n = 1) were 99% similar to those dog sequences from Thailand . Likewise, seven sequences from Mn. schreibersii (genogroup Mini-3) were 99% similar to the sequences of the strain KK20 from stray dogs from Khon Kaen, Thailand (GenBank accession FJ946854). Bat species belonging to the genus Miniopterus  are present in Thailand [Another surprising discovery was that i et al.  provided species . PerformThailand .Bartonella strains in Georgian bats, which are identicalor similar to the strains previously described in humans and in companion animals in other geographic areas grants special attention in future studies to evaluate their role as potential zoonotic agents. Aparticular question remains regarding the route of transmission of bat-associated Bartonella to people. Itis easier to speculate how stray dogs, which may scavenge for grounded bats, can become infected withbat-associated Bartonella, but the question concerning transmission of bat-borne strains to humans ismore challenging [Bartonellaspecies [Argas vespertilionis and Cimex pipistrelli [Bartonella surveillance should include not only mammals, but also their vectors whenever possible to better understand the risks of disease transmission.The identification of diverse llenging . Howeveraspecies ,26 suggeistrelli –80. ThusS1 Appendix(DOCX)Click here for additional data file.


Anodontoides and Strophitus, primarily generic placement and species boundaries. We sequenced 3 loci to create our molecular matrix: cytochrome c oxidase subunit I, NADH dehydrogenase subunit I, and the nuclear-encoded ribosomal internal transcribed spacer I. Aligned sequences were used in phylogenetic analyses and to identify diagnostic nucleotides for Strophitus pascagoulaensis, Strophitus radiatus, Strophitus sp. cf. pascagoulaensis, and Strophitus williamsi. Linear morphometrics  were also implemented to further evaluate species boundaries within Strophitus. For further details and experimental findings, please refer to the article published in Molecular Phylogenetics and Evolution  This data article presents the multi-locus DNA alignments, morphometric data, and details on specimens examined to resolve the evolutionary history of  Specifications TableValue of the data•Provided sequence data and alignments can be directly utilized in future phylogenetic studies on freshwater mussels .•All data are tied to museum vouchers, ensuring repeatability of results and facilitating further investigation of additional DNA loci and morphological characters on the same specimens used in our study.•Sequence data are available to confirm species-level identification of existing museum specimens or samples collected during future field surveys.•Strophitus radiatus, including diagnostic nucleotides that distinguish species within this complex.Molecular and morphological data provides insight on cryptic diversity within 1Strophitus pascagoulaensis, Strophitus radiatus, Strophitus sp. cf. pascagoulaensis, and Strophitus williamsi. Our molecular matrix can be used as a tool for confirming identification of vouchered or field collected specimens. Additionally, by depositing vouchered specimens and providing full access to our DNA alignment files, morphometric measurements, and specimen collection information, our data provide a valuable resource for future research on freshwater mussel systematics, distribution, and conservation.We present multi-locus sequence data for 97 individuals representing 18 species of North American freshwater mussels  belonging to the subfamily Anodontinae. We also provide shell measurements  taken from 142 specimens used to evaluate morphological differences between 22.1cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit I (NDI), and the nuclear-encoded ribosomal internal transcribed spacer I (ITSI). Tissue samples were preserved in 95% ethanol and DNA was isolated using a modified plate extraction protocol Our taxon sampling focused on North American freshwater mussels in the subfamily Anodontinae . We sequ2.2S. pascagoulaensis, S. radiatus, S. sp. cf. pascagoulaensis, and S. williamsi were identified using MEGA6 S. pascagoulaensis, S. radiatus, S. sp. cf. pascagoulaensis, and S. williamsi. We used the mitochondrial genome sequence of Cristaria plicata (GenBank Accession: KM233451) to determine nucleotide positions for COI and NDI. Base pair positions at ITSI were determined by using an alignment consisting of only A. radiatus . We used the sequence of our only A. radiatus from the Pearl River as a reference (Genbank accession number MG199854). Diagnostic nucleotides are provided in Chromatograms were assembled and edited in Geneious v 6.1.2 2.3S. pascagoulaensis, S. radiatus, S. sp. cf. pascagoulaensis, and S. williamsi specimens used in genetic analyses along with additional museum specimens from focal drainages. Three measurements were taken for morphological analyses: maximum length (anterior to posterior), height , and width  to the nearest 0.01 mm using digital calipers. One person conducted all measurements, and height and width were measured at the posterior end of the hinge ligament for consistency. Raw measurements are provided in We conducted linear morphometrics of external shell dimensions for all


Bacillus thuringiensis (Berliner) (Bt) to express insecticidal Cry proteins. Increasing temperatures profoundly affect insect life histories and agricultural pest management. However, the implications of climate change on Bt crop–pest interactions and insect resistance to Bt crops remains unexamined. We analysed the relationship of temperature anomaly and Bt adoption with field-evolved resistance to Cry1Ab Bt sweet corn in a major pest, Helicoverpa zea (Boddie). Increased Bt adoption during 1996–2016 suppressed H. zea populations, but increased temperature anomaly buffers population reduction. Temperature anomaly and its interaction with elevated selection pressure from high Bt acreage probably accelerated the Bt-resistance development. Helicoverpa zea damage to corn ears, kernel area consumed, mean instars and proportion of late instars in Bt varieties increased with Bt adoption and temperature anomaly, through additive or interactive effects. Risk of Bt-resistant H. zea spreading is high given extensive Bt adoption, and the expected increase in overwintering and migration. Our study highlights the challenges posed by climate change for Bt biotechnology-based agricultural pest management, and the need to incorporate evolutionary processes affected by climate change into Bt-resistance management programmes.Increased temperature anomaly during the twenty-first century coincides with the proliferation of transgenic crops containing the bacterium  Sixteen of the 17 warmest years recorded since 1880 occurred in the twenty-first century, and land surface temperature anomaly (difference as compared to twentieth century) during 2016 reached 1.43°C [Insects are major economic pests, and increasing temperature anomalies and warming have profound influences on insect biology and agricultural pest management. Rising temperatures accelerate insect phenology , and couBacillus thuringiensis (Berliner) (Bt) to express insecticidal toxins. Bt crops are safe for human health and provide many benefits through reduced pest populations and pesticide usage, and increased profits [Currently, a key insect pest management strategy is the genetic engineering of crops through agricultural biotechnology. The period of increased temperature anomaly in the twenty-first century coincides with the proliferation of crops engineered with genes from the bacterium  profits –17. Bt c profits . HoweverZea mays L.) and Bt cotton (Gossypium sp. L.) provides economic benefits by suppressing pest populations and damage from lepidopteran pests [sensu Tabashnik et al. [Agricultural biotechnology, through molecular breeding and genetic engineering, could significantly help to mitigate the effects of climate change on agricultural production ,20. To uan pests ,17,21–23an pests , is not an pests . Resistak et al. ) to Bt ck et al. ,27.While the role of biotechnology regulatory policies and insect resistance management strategies for the sustainability of Bt technology is well documented , Bt cropHelicoverpa zea (Boddie), a key pest of many agricultural crops, including sweet corn, field corn, cotton, tomato, Solanum lycopersicum L., and grain sorghums, Sorghum bicolor (L.). In North America, H. zea is a long-distant migrant [One of the targets of the Bt crops is the polyphagous corn earworm,  migrant  distribu migrant . However migrant , leading migrant .H. zea which is more tolerant to the Bt toxins. Owing to this ‘moderate dose’ expression, the risk of evolution of resistance increases in areas where Bt field corn is widely adopted, and H. zea overwinters successfully [et al. [H. zea to multiple Cry toxins, including pyramided traits expressed in transgenic sweet corn. During 1996–2016, the rate of increase for the indicators of resistance was significantly different between Bt and non-Bt hybrids with steeper increases for Bt than non-Bt hybrids , clearly et al. , H. zea H. zea resistance evolution. Contrastingly, models also predict that resistance could evolve rapidly in other Helicoverpa sp. when Bt crops are rare in the landscape, rather than common [H. zea resistance to Cry proteins and Bt crop adoption in the landscape are not available. Such information is necessary to predict vulnerability of crops to increased damage due to the spread of Bt resistant H. zea under climate change scenarios. In this study, we use two decades of data on indicators of resistance to Bt sweet corn and examine the role of temperature anomaly and Bt adoption on field-evolved resistance in H. zea to Cry toxins.Simulations predict that the regional deployment of Bt crops affects average gene frequencies, and therefore incomplete technology adoption can slow resistance evolution ,43. Convn common . However2.2.1.et al. [H. zea to Bt Cry proteins (measures of control efficacy and changes in susceptibility). Dively et al. [H. zea infestations and abundances during 1996–2016 in Maryland, USA. We used percentage of ears damaged by H. zea, mean kernel area consumed, mean instars and proportion of late instars (4th–6th instars) in Bt hybrids  as indicators of field-evolved resistance to Cry1Ab in sweet corn. See Dively et al. [H. zea moths in black light traps during 1996–2014 (henceforth counts) as measure of population abundance.We used the data provided by Dively et al. ,45 that y et al.  establisy et al.  for deta2.2.https://www.nass.usda.gov/Charts_and_Maps/Crops_County/boundary_maps/md.gif) as a measure of Bt adoption. The Bt events and Cry proteins expressed in field corn are also expressed in sweet corn (except for Cry1F). For e.g. Attribute  and Attribute II  hybrids from Syngenta Seeds, and Performance Series (PS) hybrids  from Seminis Seeds. Therefore, Bt field corn acreage, which represented 81% of all corn acreage in the USA in 2015, is a good measure of the evolutionary selection pressure for resistance development in H. zea against Bt Cry proteins. Area under corn production differed among the study sites, representing a range of selection pressure for resistance development. Crops (particularly corn) are planted extensively around the sites on the Eastern Shore, while western Maryland sites include urban areas. We estimated the Bt corn acreage in the agricultural districts as the product of total area of corn planted each year [As predictors of field-evolved resistance, we used Bt corn (including field corn) acreage in the agricultural crop districts of the study sites in Maryland (ach year  and the ach year .https://www.ncdc.noaa.gov/monitoring-references/maps/us-climate-divisions.php). Temperature anomalies, particularly warmer temperatures, during the growing season (spring to summer) are related to increased number of generations [http://www.ncdc.noaa.gov/cag/time-series).We compiled the temperature anomaly data for the respective climatic divisions  as response variables, site as a random effect accounting for repeated measurement, and the interaction, additive, or individual effect of (log) Bt acreage and temperature anomaly as fixed effects. We tested the interactive influence of Bt acreage and temperature anomaly prior to analysing additive or individual influences. Where applicable, the response variables were square root transformed to conform to linear model assumptions.Linear mixed effects models (LMMs) were used to analyse the influence of temperature anomaly and Bt acreage on F-tests with Kenward–Roger approximation. Also, a likelihood-ratio based pseudo-R2 value [R2 values were calculated using package ‘MuMIn’ [We determined the statistical significance of the fixed effects through Wald R2 value  was calcR2 value ; pp. 174R2 value  was used ‘MuMIn’ . Estimat ‘MuMIn’  and ‘vis ‘MuMIn’ , all in  ‘MuMIn’ .3.H. zea over the past two decades .The interactive influences of temperature anomaly and log Bt acreage significantly influenced the abundance of F1,89 = 20.3, p < 0.001; Bt acreage: F1,87 = 15.9, p < 0.001, pseudo-R2 = 0.49). Damage to corn ears increased with temperature anomaly (a) and log Bt acreage . The effect of temperature anomaly on kernel consumption increased with Bt acreage. Mean instar was significantly positively associated with Bt acreage .For indicators of field-evolved resistance, damage was significantly positively associated with additive effects of temperature anomaly and log Bt acreage  and levels of biological organization (molecular to biosphere) [Musca domestica L. resistance to pyrethroids; [H. zea [H. zea [The mechanisms through which the interactive influences of increased Bt acreage and temperature anomaly influence evolution of osphere) ,59. Reduosphere) –62. Alsothroids; ). Regard [H. zea –65. The  [H. zea ,67, and  [H. zea –68 probaH. zea, probably exacerbated the selection pressure for resistance development.At the local spatial scale, increased feeding at high temperatures ,8 resultHelicoverpa zea sampling for monitoring and testing for resistance is currently concentrated in the southern US regions where both Bt corn and Bt cotton are grown. Expanded resistance monitoring for all Cry proteins registered against corn earworm in all high corn production regions would help detect resistance development as the overwintering range of corn earworm expands due to climate change.Our results have important implications for understanding the risk of resistance increasing and spreading, sustainability of Bt technology and regional insect pest management strategies for climate change preparedness. The expansion of overwintering range north of 40° N, increased migration due to climate change ,25, and H. zea can benefit from warmer temperatures [Our study highlights risks and limitations of Bt technology for agricultural pest management in the context of climate change. Polyphagous, migratory species such as eratures ,8, and ieratures .H. zea resistance to Cry1Ab is driven by environmental conditions, and a complex genetic basis underlies insect resistance to Cry1A Bt toxins in Lepidoptera species [The evolutionary basis of  species . We ende


Enterococcus faecium is of special concern, as it can easily acquire new antibiotic resistances and is an excellent colonizer of the human intestinal tract. Several clinical studies have explored the potential use of beneficial bacteria to weed out opportunistic pathogens. Specifically, the widely studied Lactobacillus rhamnosus strain GG has been applied successfully in the context of VRE infections. Here, we provide new insight into the molecular mechanism underlying the effects of this model probiotic on VRE decolonization. Both clinical VRE isolates and L. rhamnosus GG express pili on their cell walls, which are the key modulators of their highly efficient colonization of the intestinal mucosa. We found that one of the VRE pilus clusters shares considerable sequence similarity with the SpaCBA-SrtC1 pilus cluster of L. rhamnosus GG. Remarkable immunological and functional similarities were discovered between the mucus-binding pili of L. rhamnosus GG and those of the clinical E. faecium strain E1165, which was characterized at the genome level. Moreover, E. faecium strain E1165 bound efficiently to mucus, which may be prevented by the presence of the mucus-binding SpaC protein or antibodies against L. rhamnosus GG or SpaC. These results present experimental support for a novel probiotic mechanism, in which the mucus-binding pili of L. rhamnosus GG prevent the binding of a potential pathogen to the host. Hence, we provide a molecular basis for the further exploitation of L. rhamnosus GG and its pilins for prophylaxis and treatment of VRE infections.Vancomycin-resistant enterococci (VRE) have become a major nosocomial threat. IMPORTANCE Concern about vancomycin-resistant Enterococcus faecium causing nosocomial infections is rising globally. The arsenal of antibiotic strategies to treat these infections is nearly exhausted, and hence, new treatment strategies are urgently needed. Here, we provide molecular evidence to underpin reports of the successful clinical application of Lactobacillus rhamnosus GG in VRE decolonization strategies. Our results provide support for a new molecular mechanism, in which probiotics can perform competitive exclusion and possibly immune interaction. Moreover, we spur further exploration of the potential of intact L. rhamnosus GG and purified SpaC pilin as prophylactic and curative agents of the VRE carrier state. For years, enterococci were viewed as harmless endogenous members of the human microbiota , with so3–Enterococcus species are successful nosocomial agents since they are excellent colonizers, show striking genome plasticity, and persist for long periods of time in the intestinal tract, on medical devices, or on household appliances (van genes) is particularly worrisome  of E. faecium E1165 and the SpaCBA pili of L. rhamnosus GG revealed a high level of sequence similarity between the two pilus gene clusters. Hence, we tested the capacity of the L. rhamnosus GG pili, and in particular the adhesive SpaC subunit, as well as antibodies raised against these components to interfere with E. faecium E1165 and its mucus interaction. Our findings not only demonstrate the existence of a novel immunogenic probiotic mechanism in the widely exploited intestinal isolate L. rhamnosus GG, but they also hold great promise for the development of prophylactic treatments.In this work, we provide new molecular insight into the mechanism underlying the role of pacities –20. L. rtributed –24. Earlisolates , 26. We Lactobacillus rhamnosus GG and its pilus-deficient derivative strain PB12 were grown at 37°C in MRS broth (Difco) in nonshaking conditions. Enterococcus faecium was grown stationary in brain heart infusion (BHI) broth (Difco) at 37°C. The three strains were grown in semianaerobic conditions, i.e., stationary growth and minimal oxygen. Escherichia coli was cultured aerobically at 37°C in LB medium under shaking conditions.E. faecium isolate E1165 was extracted using the Wizard genomic DNA purification kit (Promega) per the manufacturer's recommendations. Genomic DNA sequencing of E. faecium isolate E1165 was performed using the Pacific Biosciences sequencing technology of the DNA Sequencing and Genomics Laboratory of the Institute of Biotechnology, University of Helsinki. In short, the PacBio library kit was used to construct a library with size targeted to 10 kb. Two single-molecule real-time (SMRT) cells were run on a PacBio RS II system using P6/C4 chemistry and 240 min of video time. A total of 78,536 reads  were assembled using the HGAP.3 implemented in the SMRT analysis system . Apart from the complete chromosome , a megaplasmid of 192,533 bp and one smaller plasmid of 32,402 bp were obtained with mean coverages of 190×, 210×, and 260×, respectively. Additionally, a circular phage genome  was found. Using the RAST server , excluding the C-terminal cell wall sorting signal and the N-terminal signal peptide, was PCR amplified from a codon-optimized spaCBA gene cluster (primers BG3764 ttcTCATGActgataacattcgcccaacc and BG3765 ttcGCGGCCGCcggcaaaattgcaagtgg). The purified PCR product was digested with BspHI and NotI  and cloned into the pWUR533 vector, resulting in pWUR533_SpaC. This vector is a derivative of the pET52-1b vector in which the C-terminal thrombin protease site was replaced by an HRV 3C protease site and a Strep-tag II instead of a His tag. One percent of an overnight culture of E. coli BL21(DE3)/pSJS1244, in which the pWUR533_SpaC vector was introduced, was grown in LB medium with ampicillin (100 μg/ml) and spectinomycin (50 μg/ml). After 3 h of incubation at 37°C, protein production was induced by isopropyl-β-d-thiogalactopyranoside (IPTG) (1 mM). Three hours later, cells were harvested by centrifugation. Purification of the recombinant SpaC protein was performed in accordance with the Strep-Tactin resin manufacturer's recommendations (IBA). The Strep-tag was removed using the HRV 3C protease (EMD Millipore) following the protocol of the manufacturer. The tag and protease were subsequently removed using a Vivaspin 20 centrifugal concentrator (Sartorius).The coding sequence of 3H]thymidine (Perkin-Elmer Life Sciences). Bacteria  were applied to a Nunc MaxiSorp plate coated with 0.5 mg/ml type II porcine mucus . Optionally, 1/100 dilutions of antisera against total L. rhamnosus GG cells  under project ID L. rhamnosus GG on VRE shown in clinical trials   , and hos in PGCs , 26.L. rhamnosus GG and E. faecium resulted in a striking similarity (>50%) between the SpaCBA-SrtC1 pilus cluster of L. rhamnosus GG and the PGC-3 cluster of E. faecium. Scanning the organization of the PGC-3 gene cluster, harboring the PilB-type pili . This similarity rose up to 49% when taking the amount of positive matches into account. Overall, mapping of the SpaC protein sequence to the E. faecium taxonomy identification number (taxid) 1352 revealed that in some strains, the sequence similarity even surpasses 50% (54% and up to 65% when considering positive matches) (data not shown).A homology search exploring potential similarity between the PGCs of ype pili , revealenosus GG , 24, 36 nosus GG . Closer E. faecium strain E1165, originally isolated from the wound/soft tissue of a hospitalized patient in 1997 . Expression of the PGC-3 cluster-encoded PilB-type pili in E. faecium E1165 was already confirmed previously  located on a megaplasmid of 192.533 kb   . These rby PGC-3 . This crL. rhamnosus GG administration on VRE colonization   for strain E1165 than those for L. rhamnosus GG, which may be indicative of a stronger binding of E1165 to mucus or the presence of more pili per cell. In view of the similarity between the pili of E. faecium and those of L. rhamnosus GG, we hypothesized that the E. faecium E1165 pili are the principal actors of its strong mucus adhesion. Hence, we sought to study the capacity of antibodies raised against L. rhamnosus GG and its SpaC pilin to affect E. faecium E1165 mucosal adhesion. Antibodies that were raised against the SpaC adhesive pilin effectively interacted with the binding of E1165 to mucus (L. rhamnosus GG cells could significantly interfere with E. faecium E1165 mucus binding (P < 0.0001)  the mucus binding of E. faecium E1165 (and L. rhamnosus GG) (data not shown)  . E. faeche mucus . Interacto mucus . In addi 0.0001) . The efft shown) . In concE. faecium or VRE have become a major nosocomial threat  carrier state of VRE. Our results shed light on a unique mechanism to specifically target VRE without harming commensal microbiota.In this work, we have successfully elucidated a molecular mechanism underlying the successful application of reatment . L. rham