S. mansoni adults worm antigen (SWA) and S. mansoni egg antigen (SEA) using ELISA. Infection by helminths  was diagnosed using real-time PCR, urine circulating cathodic antigen (CCA) and stool microscopy (Kato-Katz method) while malaria infection was diagnosed using microscopy. We analysed the relationship between helminth and malaria infections and KSHV seropositivity using regression modelling, allowing for survey design. At baseline, 56% of the participants were male while 48% of the participants were male in the final survey. The most prevalent helminth infection was S. mansoni . KSHV seropositivity was 66% (baseline) and 56%  among those 1–12 years and >80% in those 13+ years in both surveys; malaria parasitaemia prevalence was 7% (baseline) and 4% . At baseline, individuals infected with S. mansoni (detected by microscopy) were more likely to be KSHV seropositive  p = 0.012) and had higher anti-K8.1 antibody levels  p = 0.02). In the final survey, S. mansoni , p = 0.028) and malaria parasitaemia (aOR = 3.49 (1.08–11.28), p = 0.038) were positively associated with KSHV seropositivity. Additionally, KSHV seropositive participants had higher S. mansoni-specific IgE and IgG antibody concentrations in plasma. Furthermore, HIV infected individuals on cART were less likely to be KSHV seropositive compared to HIV negative individuals  p = 0.002). Schistosoma species skew the immune response towards Th2 and regulatory responses, which could impact on KSHV reactivation if co-infected with both organisms.We investigated the impact of helminths and malaria infection on Kaposi’s sarcoma associated herpesvirus (KSHV) seropositivity, using samples and data collected from a cluster-randomised trial of intensive versus standard anthelminthic treatment. The trial was carried out in 2012 to 2016 among fishing communities on Lake Victoria islands in Uganda. Plasma samples from 2881 participants from two household surveys, the baseline (1310 participants) and the final (1571 participants) surveys were tested for KSHV IgG antibody responses to K8.1 and ORF73 recombinant proteins using ELISA. The baseline survey was carried out before the trial intervention while the final survey was carried out after three years of the trial intervention. Additionally, a subset sample of 372 participants from the final survey was tested for IgE, IgG and IgG4 antibody concentrations to  Schistosoma mansoni and malaria parasites is associated with being infected or exposed to KSHV. These parasite infections interfere with the proper functioning of the immune system to control viral infections. Although not shown in the current study, these parasite infections might lead to reactivation of KSHV in infected people increasing the likelihood of having detectable KSHV antibodies. Consequently, this viral reactivation may increase the spread of KSHV in sub-Saharan Africa.Kaposi’s sarcoma associated herpesvirus (KSHV), the causative agent of Kaposi’s sarcoma cancer, varies geographically. KSHV infections are highest in sub-Saharan Africa, with Uganda having the highest prevalence reported to date. Infection with KSHV is lifelong with an intermittent revival of the virus, leading to viral spread. In this study, we show that infection with  The prevalence of Kaposi’s sarcoma associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV8), varies geographically, unlike that of other herpesviruses which are ubiquitous –3. UgandSchistosoma is characterised by the production of IL4, IL5 and IL13 cytokines, typical of a T helper (Th) type 2 response and IL10, a regulatory cytokine [Schistosoma co-infection on herpesviruses and other viruses has been demonstrated in animal models, where Schistosoma mansoni infection led to IL4-mediated reactivation of murine herpesvirus 68 and M2 macrophage polarization [Co-infection with helminths has been shown to modulate immune responses to other infections and vaccines –14. Chrocytokine , 16. Thecytokine –19. The rization , 18.P. falciparum and helminths (hookworm and Mansonella perstans) in rural [Our group has documented associations between KSHV antibodies and parasite infections including in rural  and periin rural –22 populin rural –26. No sThis cross-sectional study used samples from an open cluster-randomised trial of intensive versus standard anthelminthic treatment, the Lake Victoria Island Intervention Study on Worms and Allergy-related diseases (LaVIISWA) (ISRCTN47196031) . The stuSchistosoma mansoni Egg Antigen (SEA) and Schistosoma mansoni adult Worm Antigen (SWA) were measured using ELISA. Antigen concentrations of 8 μg/mL (SWA) and 2.4 μg/mL (SEA) plus sample dilutions of 1/20 (IgE), 1/200 (IgG4) and 1/3000 (IgG) were used, as previously reported [IgG antibodies to K8.1 and ORF73 recombinat proteins were measured using ELISA to determine KSHV seropositivity, as previously described . Each plreported , 30, 31.S. mansoni infection intensities: 1–99 eggs per gram of stool as light intensity, 100–399 eggs per gram of stool as moderate intensity and greater than 400 eggs per gram of stool as heavy intensity. An aliquot of the stool was stored in 70% ethanol and later used to detect helminths by real-time PCR. The multiplex real-time PCR assay was used to detect Necator americanus, Strongyloides stercoralis and Schistosoma mansoni as previously described [S. mansoni antigens were also tested in urine using the Circulating Cathodic Antigen (CCA) kits  [Mansonella perstans infection was detected using the modified Knott’s method [Duplicate slides were made from each stool sample and analysed independently by two technicians for helminth infection and intensity using the Kato-Katz (KK) method . The folescribed , 34. S.  Africa) . Mansones method  and thic10 transformed prior to linear regression modelling. For assessing the effect of intensive versus standard treatment, the analysis was done at the cluster level. The proportion of KSHV seropositive participants was calculated for each village, and the mean of these taken for the two trial arms. The risk ratio (RR) was then calculated by dividing the mean KSHV prevalence in the intensive arm by that in the standard arm, and a Taylor approximation was used to calculate a 95% confidence interval for this RR. The p-value was generated from a t-test comparing the village-level prevalences between the two arms. A similar approach was used to assess the effect of intensive versus standard treatment on KSHV antibody levels. A p-value of less than 5% was considered statistically significant. Multivariable models included age (grouped), sex, HIV status, S. mansoni, hookworm and malaria parasite infection. Participants not tested for HIV were also included in the analyses.Statistical analysis was carried out using STATA version 13 . The survey study design of the main trial was non self-weighting . To allow for this non self-weighting design and to ensure that our analyses are representative of the study area, we therefore took into account clustering within villages and applied village-level weights to allow for the different village sizes for all observational analyses . LogistiSchistosoma mansoni was the most prevalent in both surveys, as expected, due to the close proximity of the study sites to the waters of Lake Victoria. The prevalence was 86% by CCA, 50% by PCR and 34% by microscopy in the final survey , 11% for Trichuris trichiura (using KK), 0.1% for Ascaris lumbricoides (using KK) and 3% for Mansonella perstans , 9% for Trichuris trichiura (using KK), 0.04% for Ascaris lumbricoides (using KK) and 0.9% for Mansonella perstans  of 3 to 33 years. In the final survey, the median age was 24 years with an interquartile range (IQR) of 9 to 33 years. The overall proportion of males was 56% at baseline and 48% in the final survey. Details of the socio-demographic characteristics of the study population are shown in l survey . At baseperstans . While iperstans . At basecroscopy .Overall KSHV seropositivity was 84% at baseline and 75% in the final survey. At baseline, the prevalence increased from 66% at 1–12 years to 93% in 31–44 years and 90% in 45-74-year-olds . In the We investigated associations of KSHV seropositivity with parasite infections and other factors at baseline and in the final survey. Overall, KSHV prevalence was higher in males compared to females (adjusted Odds Ratio (aOR) = 1.72 , p = 0.001) in the final survey . HIV serS. mansoni infection status and malaria infection status , p = 0.038) were more likely to be KSHV seropositive in the final survey . Althougn status . Helmintl survey . Other hS. mansoni at baseline  p = 0.012 (S. mansoni infection intensity at baseline (p for trend = 0.013)   . In the = 0.068) . We did sitivity . There wsitivity .S. mansoni infection and infection intensity detected by microscopy and KSHV IgG antibody levels to K8. 1 and ORF73 separately. At baseline, microscopy positive individuals for S. mansoni had higher antibodies to K8.1 compared to microscopy negative individuals  p = 0.02). Furthermore, this association increased with increasing infection intensity . Antibody levels to both K8.1 and ORF73 were neither associated with S. mansoni infection nor with S. mansoni infection intensity in the final survey , IgG (n = 372) and IgG4 (n = 370) antibody concentrations against mparable . After amparable .KSHV prevalence can vary, even between geographically proximate areas . We haveThe HIV prevalence in the studied communities was high (17%). We observed a lower risk of being KSHV seropositive among individuals treated for HIV compared to HIV negative individuals. Since our study was cross-sectional and given the fact that there was missing HIV data, this finding should be treated with caution. However, ART has been shown to lead to tumour regression among AIDS-KS patients –41. AddiS. mansoni in these island communities is very high. We showed that being infected with S. mansoni, detected by microscopy, was associated with an increased risk of being KSHV seropositive. Others have reported no association between S. mansoni and KSHV infections, possibly due to the low prevalence of KSHV or low infection intensity of S. mansoni in the study areas [In vitro reactivation of the model gammaherpesvirus MHV68 by S. mansoni was demonstrated by Reese et al., mediated through IL4 production [S. mansoni and KSHV seropositivity. Furthermore, at baseline (before antihelminthic treatment), anti-K8.1 antibody levels were higher in S. mansoni infected individuals compared to uninfected individuals, and S. mansoni infection intensity was associated with higher antibody levels to K8.1 and ORF73. These associations were not observed in the final survey after antihelminthic treatment. Nonetheless, higher antibody levels to KSHV antigens, and particularly the lytic antigen K8.1, has been associated with viral reactivation and KS disease progression [S. mansoni infection, although a major limitation in interpretation is that we are unable to assign causation. An additional limitation is that we did not measure KSHV viral load in blood due to the unavailability of appropriate samples, and this would reaffirm the role of S. mansoni infection in KSHV reactivation.The high untreated HIV prevalence, coupled with other factors including parasite infections, may contribute to the high prevalence of KSHV in this area. The burden of dy areas , 46. In oduction . Our humgression –50. Our S. mansoni are associated with an increased risk of being KSHV seropositive in older individuals. The (Th) 2 immune response to S. mansoni, characterised by IgE, IL4 and IL5 production has been linked to protection from S mansoni reinfection, while IgG4 production has been linked to susceptibility to reinfection [S. mansoni, through (Th) 2 immune response upregulation, may reactivate KSHV latently infected cells.We also showed that increasing IgE and IgG, but not IgG4 antibodies to nfection . This prS. mansoni infection could be caused by increased inflammation due to new S. mansoni infections, leading to an increase in KSHV antibody levels in individuals already infected with KSHV. However, the association with anti-K8.1 (a lytic antigen) but not anti-ORF73 (a latent antigen) antibodies with S. mansoni infection at baseline may imply specific effects of S. mansoni on KSHV reactivation as opposed to non-specific inflammatory effects. Furthermore, higher anti-Ag85A (a Mycobacteria tuberculosis antigen unrelated to S. mansoni) IgG4, but not IgG, has been reported in S. mansoni infected individuals compared to S. mansoni uninfected individuals [S. mansoni on immunity to other unrelated chronic infections.Alternatively, the association between KSHV seropositivity and ividuals . Taken tS. mansoni and KSHV seropositivity was only observed if S. mansoni was detected using microscopy (KK) but not PCR or CCA, in this study. Microscopic examination of faecal samples is the WHO recommended diagnostic test for S. mansoni [S. mansoni by CCA may be influenced by cross-reactivity, leading to false positives, and may explain the larger numbers diagnosed by this method in our study [S. mansoni [S. mansoni infection, as well as the reduced infection intensity in the final survey due to the three years antihelminthic treatment, may have obscured the effect that S. mansoni has on KSHV antibody levels. Heavy S. mansoni infections might have a larger effect which could easily be observed with the current sample size. Furthermore, a light infection intensity could have effects on KSHV incident cases, as opposed to prevalent cases. Because of the observational study design, incident cases were not specifically detected, and our study likely picked up prevalent cases.The association between  mansoni , 53, and mansoni . Detectiur study . The PCR mansoni –54. The We did not see any effect of intensive versus standard anthelminthic treatment on KSHV seropositivity. This was not surprisingly as such an intervention would likely affect incident KSHV infections, rather than prevalent KSHV infections and antibody titres against KSHV would be unlikely to change over the short follow-up period. Migration rates in these Island communities were very high. Therefore very few people were tested both at baseline and in the final survey. Consequently, the majority of the participants analysed in the final survey might have been new immigrants. The original trial intended to obtain community/population-wide effects as opposed to individual participant effects.Plasmodium falciparum, the main cause of malaria disease in Africa, induces inflammation which is normally regulated through induction of regulatory T cells and production of IL10 and TGF-β. Increased IL10 levels, a cytokine mainly produced by regulatory cells, has been reported in disseminated KS [P. falciparum infection has also been shown to lead to the reactivation of some herpesviruses such as EBV, HSV-1 and VZV [We also found malaria parasitemia to be associated with KSHV seropositivity, consistent with our previous findings , 21, andnated KS . Plasmodnated KS . The imm and VZV –61. We t and VZV , 62, 63,S1 TextaOR: odds ratios. bCI: confidence intervals. cCCA (circulating cathodic antigen). Statistical analysis was performed using logistic regression, allowing for the survey design.KSHV seropositivity defined reactivity to either ORF73 or K8.1 protein. KSHV antibodies detected using ELISA. (DOCX)Click here for additional data file.S2 TextaOR: odds ratios. bCI: confidence intervals. cCCA (circulating cathodic antigen). dPCR: polymerase chain reaction. Statistical analysis was performed using logistic regression, allowing for the survey design.KSHV seropositivity defined reactivity to either ORF73 or K8.1 protein. KSHV antibodies detected using ELISA. (DOCX)Click here for additional data file.S3 TextSchistosoma mansoni was determined from a single stool sample using Kato-Katz method. aCoef.: linear regression coeffient. bCI: Confidence Intervals. c adjusted for age, sex, HIV status, S. mansoni, N. americanus and malaria parasiteamia.KSHV antibodies were detected using ELISA. Statistical analysis was performed using linear regression, allowing for the survey design. (DOCX)Click here for additional data file.S4 Text2 test, allowing for the survey design.P value obtained from a Chi(DOCX)Click here for additional data file.


Entomophthora muscae species complex  commonly kill their insect hosts and manipulate host behaviors in the process. In this study, we made use of public transcriptome data to identify and characterize eight new species of mitoviruses associated with several different E. muscae isolates. Mitoviruses are simple RNA viruses that replicate in host mitochondria and are frequently found in more phylogenetically apical fungi  as well as in plants. E. muscae is the first fungus from phylum Zoopagomycota, and thereby the most phylogenetically basal fungus, found to harbor mitoviruses to date. Multiple UGA (Trp) codons are found not only in each of the new mitovirus sequences from E. muscae but also in mitochondrial core-gene coding sequences newly assembled from E. muscae transcriptome data, suggesting that UGA (Trp) is not a rarely used codon in the mitochondria of this fungus. The presence of mitoviruses in these basal fungi has possible implications for the evolution of these viruses.Fungi constituting the  Fungi currently applied by the National Center for Biotechnology Information  includes eight major phyla: Ascomycota, Basidiomycota, Mucoromycota , Zoopagomycota , Blastocladiomycota, Chytridiomycota, Cryptomycota, and Microsporidia, in approximate order of increasing time since they emerged as divergent taxa [Ascomycota and Basidiomycota, as well as from subphylum Glomeromycotina in phylum Mucoromycota [The classification scheme for kingdom ent taxa ,2. Fungaromycota ,4,5. We Mitovirus, family Narnaviridae. They have small plus-strand RNA genomes and replicate persistently in host cell mitochondria [Mitoviruses are currently classified in genus chondria ,6,7,8. Tchondria ,4,5, thechondria ,10. Sevechondria ,12 mightchondria . Mitovirchondria ,10,14. Mchondria ,16,17,18chondria .Entomophthoromycotina is one of three subphyla that currently constitute fungal phylum Zoopagomycota [Conidiobolus coronatus  and Basidiobolus ranarum  [Entomophthora muscae species complex  [E. muscae is also known to manipulate fly behaviors, in particular inducing the behavior known as “summiting”, in which the fly climbs to a high surface where it becomes affixed via fungal outgrowths and then strikes a characteristic pose considered to aid in dispersal of spores from the subsequent carcass [gomycota ,2. A fewbolales) ,21. Entobolales) . The Enthorales)  is one s carcass .E. muscae isolates, in a broad effort to understand their host specificity and pathogenicity. In that report, the authors mention the presence of several apparent viral transcripts in their transcriptome shotgun assemblies but provide no further descriptions of those viruses. As described in detail below, we subsequently discovered a number of apparent mitovirus sequences from that transcriptome study by searching for mitovirus-like sequences within the Transcriptome Shotgun Assembly (TSA) database at NCBI. Recognizing several interesting features of these apparent new mitoviruses from E. muscae, we have gone on to characterize their sequences in detail, as we report here. As a part of this work, we have also identified additional strains of these mitoviruses by assembling sequence reads from two other transcriptome studies of E. muscae, as obtained from the Sequence Read Archive (SRA) database at NCBI. Included among our findings is identification of numerous UGA (Trp) codons in the mitovirus sequences from E. muscae, as well as in mitochondrial core-gene coding sequences from E. muscae, leading us to conclude that UGA (Trp) is not a rarely used codon in E. muscae, unlike the case in many other basal fungi [E. muscae is hereby the first fungus from phylum Zoopagomycota, and also the most phylogenetically basal fungus [De Fine Licht et al.  have repal fungi . E. muscl fungus ,2, that http://biosrv.cab.unina.it/webcap3/ or galaxy.pasteur.fr/. Terminal residues in each assembled contig were trimmed back to those that agree between at least two mitovirus strains of the same species. Following contig assembly, mapped reads were re-identified using MegaBLAST, and coverage depths were determined using the “Map Reads to Reference” tool in CLC Genomics Workbench v7. RPKM and coverage depth values are shown in The main steps in this process are detailed in Results. All searches of the TSA, SRA, and NR databases were performed using the BLAST web server at NCBI. The 15 TSA hits from the initial TBLASTN searches were: GEMZ01003603.1, GEMZ01006022.1, GEMZ01006112.1, GEMZ01008256.1, GEMZ01008924.1, GEMZ01011847.1, GEMZ01008924.1, and GEMZ01021189.1; GENA01003603.1, GENA01006022.1, GENA01006112.1, GENA01008256.1, GENA01008924.1, GENA01011847.1, GENA01008924.1, and GENA01021189.1; and GEND01018947.1 ,27,28. Thttps://usegalaxy.org/ and rnaSPAdes v3.13.0 [For generating transcriptome shotgun assemblies from SRA libraries, we used Trinity v2.2.0  with def v3.13.0  with def v3.13.0  in BLASThttp://www.bioinformatics.org/sms2/. Pairwise and multiple sequence alignments were performed using MAFFT v7 [https://mafft.cbrc.jp/alignment/server/. For determining % sequence identity, pairwise alignments were performed using EMBOSS Needleall as implemented at http://www.bioinformatics.nl/cgi-bin/emboss/needleall. Maximum-likelihood phylogenetic analyses were performed using the program IQ-Tree [https://www.hiv.lanl.gov/content/sequence/HIV/HIVTools.html.Codon usage was analyzed using the Codon Usage tool in Sequence Manipulation Suite at MAFFT v7  as imple IQ-Tree , includi IQ-Tree  and the  IQ-Tree , as implE. muscae and were then used as queries of the SRA transcriptome libraries from E. muscae isolate Berkeley: atp6, GENA01019820.1; atp9, GENA01019993.1; cob, GEND01033240.1; cox1, GEND01036136.1 and GEND01034538.1; cox2, GEND01003800.1; cox3, GEND01033140.1; nad1, GENA01026764.1 and GENA01028096.1; nad2, GENA01003576.1; nad3, GENA01020377.1; nad4, GEND01006792.1 and GEND01006791.1; nad5, GENA01001061.1; and nad6, GEND01032932.1 and GENA01021432.1 [Programs were used as described above for mitovirus sequences. The following TSA hits arose from the initial TBLASTN searches for mitochondrial gene coding sequences from 021432.1 . RPKM anE. muscae isolate are listed in E. muscae isolate KVL-14-117, the SRA accessions only from media-grown fungus (not fly-grown fungus) were used for assembling mitovirus contigs, in an effort to ensure that the identified viruses were derived from E. muscae, and not the fly hosts or a different fly symbiont. For E. muscae isolate Berkeley, the SRA accessions from only (i) flies infected with E. muscae (not uninfected flies) and (ii) later times post-infection  were used for assembling mitovirus or mitochondrial transcript contigs, in an effort to increase the proportion of reads that were derived from E. muscae, not the fly hosts.SRA accessions that we used in this study for assembling mitovirus or mitochondrial transcript contigs from each E. muscae isolates KVL-14-117, KVL-14-118, HHdFL130914-1, HHdFL050913-1, and Berkeley have been deposited in GenBank as accessions MK682513.1–MK682534.1 and BK010729.1–BK010736.1. Mitochondrial core-gene coding sequences from E. muscae isolate Berkeley have been deposited in GenBank as accessions BK010748–BK010759. In addition, the mitovirus and mitochondrial core-gene sequences from all five of these E. muscae isolates have been included in the Coding-complete mitovirus sequences from E. muscae isolate HHdFL130914-1 (accession SRX2782457) have not been reported in a peer-reviewed article to date, and the methods are therefore described here. A stranded cDNA library was generated using an Illumina TruSeq Stranded RNA LT kit . mRNA was purified from 1 µg of total RNA using magnetic beads containing poly-T oligos. mRNA was fragmented and reverse-transcribed using random hexamers and Superscript II  followed by second strand synthesis. The fragmented cDNA was treated with end repair, A-tailing, adapter ligation, and eight cycles of PCR. The prepared library was quantified using KAPA Biosystem’s next-generation sequencing library qPCR kit  and run on a Roche LightCycler 480 real-time PCR instrument . The quantified libraries were then prepared for sequencing on the Illumina HiSeq platform using an Illumina TruSeq Rapid paired-end cluster kit. Sequencing of the flow cell was performed on the Illumina HiSeq2500 sequencer using Illumina HiSeq TruSeq SBS sequencing kits, following a 2 × 150-nt indexed run recipe.The sequence reads from Ascomycota, phylum Basidiomycota, or subphylum Glomeromycotina) in the TSA database at NCBI, using RdRp sequences of previously reported fungal mitoviruses as queries. The searches yielded 15 strong hits , all derived from the same transcriptomics study of muscoid-fly pathogens from the E. muscae species complex (BioProject PRJEB10825) [E. muscae sensu stricto isolates from house flies (Musca domestica) and E. muscae sensu lato isolates from cabbage flies (Delia radicum). The lengths of these 15 hits range from 2321 to 2813 nt, within the expected interval for mitovirus genomes [We used TBLASTN to search entries from basal fungi , and one mitovirus-like sequence from fly-grown E. muscae cabbage fly isolate HHdFL050913-1. A small number of reads  matching the mitovirus from isolate HHdFL050913-1 were also identified from fly-grown E. muscae cabbage fly isolate HHdFL040913-2 [Before analyzing these apparent new mitovirus sequences, we examined the TSA metadata to learn about the samples and to identify the SRA accessions from which the TSA hits had been assembled. After reviewing the metadata and the sequences themselves, we chose to reassemble the mitovirus-like contigs for each 040913-2  but wereE. muscae or other Entomophthoromycotina members, we found such accessions from five other BioProjects: PRJNA372837 for E. muscae house fly isolate HHdFL130914-1  [Conidiobolus thromboides isolate FSU 785, PRJNA67455 for Conidiobolus coronatus isolate NRRL 28638, and PRJNA501640 for Zoophthora radicans isolate ATCC 208865/ARSEF 4784. We therefore used the newly assembled mitovirus-like sequences described above as queries to search the SRA transcriptome libraries from these five other BioProjects and obtained numerous strong hits from two of them, those for E. muscae house fly isolate HHdFL130914-1 and E. muscae fruit fly isolate Berkeley. Assembling these SRA reads from isolates HHdFL130914-1 and Berkeley then gave rise to seven distinct mitovirus-like sequences from each of these additional isolates, for a total of 29 mitovirus-like sequences assembled from five different E. muscae isolates (By reviewing the SRA database for other transcriptome-associated accessions that are annotated as relating to a hydei) , PRJNA25isolates .E. muscae, we reasoned that there might be sequences of yet other, divergent mitoviruses remaining to be identified in the SRA transcriptome libraries. To address this possibility, we generated our own shotgun assemblies from the libraries analyzed in the preceding paragraph and then searched these new assemblies for other mitoviruses. In this manner, we were able to identify one additional mitovirus-like sequence from E. muscae isolate Berkeley  (E. muscae isolates.Given the large number of mitovirus-like sequences associated with 2479 nt) , bringinE. muscae isolate HHdFL130914-1 (BioProject PRJNA346904), an isolate that we showed above to harbor sequences from seven mitovirus strains in its RNA transcriptome data. These six SRA libraries from E. muscae DNA were found to register no MegaBLAST hits to the seven mitovirus sequences, providing evidence against these virus sequences being derived from endogenized elements. A small number of mitovirus-matching reads  found in three SRA libraries arising from Illumina runs on genomic DNA from the same BioProject as the PacBio SMRT runs seem unlikely to be significant, though they may be reminiscent of evidence for non-genomic DNA fragments detected for Gigaspora margarita mitoviruses [E. muscae mitoviruses found as hits from the Whole Genome Shotgun (WGS) database at NCBI are in fact derived from the transcriptome study BioProject PRJEB10825 [Especially in plants, fragments of mitovirus genome sequences have been commonly endogenized in host DNA ,38,39,40ng reads  total foZoopagomycota [E. muscae isolate Berkeley in The lengths of our 30 newly assembled mitovirus-like contigs range from 2300 to 2806 nt , within gomycota ,42,43), Basidiomycota or subphylum Glomeromycotina in which UGA(Trp) is a rarely used mitochondrial codon [E. muscae described above fit the more typical pattern for fungal mitoviruses, in that a substantial fraction of their Trp codons are UGA (vs. UGG): 38–77%, numbering between 5 and 13 UGA(Trp) codons in the different apparent mitovirus sequences  harbors strains of all eight of the new mitovirus species, three of the E. muscae isolates  harbor strains of even of the new mitovirus species, and the remaining one isolate  harbors a strain of only one of the new mitovirus species , i.e., from another cabbage fly isolate of E. muscae.Features of the mitovirus-like contigs described above led us to suspect that there are multiple new mitovirus species represented by these sequences. To address this possibility, we performed pairwise comparisons of the nt sequences using EMBOSS Needleall. These comparisons grouped the sequences into eight distinct clusters, with pairwise identities >86% within each cluster and <50% between any two clusters . The degd clades . In addi species . We suggE. muscae mitoviruses relative to others in current genus Mitovirus. A representative set of 83 previously reported, full-length mitovirus RdRp sequences were obtained from the Protein database at NCBI and then used in multiple sequence alignments along with the eight mitovirus RdRp sequences from E. muscae isolate Berkeley, plus two RdRp sequences from members of current genus Narnavirus as an outgroup. Each multiple sequence alignment was analyzed using the ModelFinder module within IQ-TREE to identify the best-fit substitution model, which was then directly applied for maximum-likelihood phylogenetic analyses within IQ-TREE. As shown by the representative phylogram in Mitovirus, within a portion of the major clade previously designated Ia. This clustering suggests that all eight of these mitoviruses from E. muscae shared a most recent common ancestor, from which they all diverged, near the root of this subclade  culisetae from subphylum Kickxellomycotina, phylum Zoopagomycota, the only member of this basal phylum for which a complete mitochondrial genome sequence had been annotated as such at NCBI at that time (accession NC_006837.1) [Entomophthoromycotina, phylum Zoopagomycota. The evidence presented above for numerous UGA(Trp) codons in the mitovirus sequences from E. muscae, however, run counter to this expectation. To address this possible discrepancy, we sought to determine whether UGA(Trp) codons are found, too, in mitochondrial core genes of E. muscae. We first performed TBLASTN searches for transcript contigs representing mitochondrial core genes in TSA database entries for subphylum Entomophthoromycotina, using the deduced sequences of 14 mitochondrial core proteins from Z. culisetae as queries. Strong hits, all from the E. muscae transcriptome study BioProject PRJEB10825 [atp6, atp9, cob, cox1, cox2, cox3, nad1, nad2, nad3, nad4, nad5, and nad6.A previous report has revealed that UGA(Trp) is a rarely used codon in mitochondrial core genes of many basal fungi , includi06837.1) . SimplisE. muscae were assembled as described above, beginning with the SRA transcriptome libraries from E. muscae isolate Berkeley (BioProject PRJNA435715) [E. muscae isolate Berkeley  for each protein consistently derived from Conidiobolus species C. coronatus or C. heterosporus, the latter for which a complete mitochondrial genome sequence has only recently been deposited and annotated at NCBI (accession MK049352.1) [E. muscae, along with those from other representative fungi, placed the E. muscae proteins adjacent to those of C. heterosporus within the Zoopagomycota clade  as queries to search the SRA libraries from which the mitovirus sequences from A435715) . Using tBerkeley . Additiota clade .E. muscae isolate Berkeley represent the respective mitochondrial genes of E. muscae, we used the contigs to search the SRA transcriptome libraries from the four other E. muscae isolates KVL-14-117, KVL-14-118, HHdFL130914-1, and HHdFL050913-1. The results summarized in E. muscae isolates. Complete coding sequences were assembled for eight to ten of these genes from each isolate and partial coding sequences for the other genes  codons. At least one and as many as five UGA (Trp) codons each are found in nine of these sequences, namely, in all but those for atp6, atp9, and nad3, which also contain no or only one UGG (Trp) codon each  might be expected to be a rarely used codon in the mitochondria of all basal fungi thus appears to have been incorrect.We lastly examined the 12 mitochondrial coding sequences from don each . Notablyisolates . OverallZoopagomycota (subphylum Entomophthoromycotina). Before this, the most phylogenetically basal fungi [Mucoromycota (subphylum Glomeromycotina) [Entomophthora muscae might have been acquired by horizontal transmission, perhaps from some more apically diverging fungi given their juxtaposition with ascomycete and basidiomycete mitoviruses in the E. muscae mitoviruses first entered the fungal lineage in an ancestral species that predated the divergence of phylum Zoopagomycota from the three more apically diverging phyla Mucoromycota, Basidiomycota, and Ascomycota, and was then transmitted largely by vertical means over the subsequent course of fungal evolution, including to E. muscae. See Conidiobolus heterosporus. The reciprocal possibility, i.e., that E. muscae mitoviruses have been horizontally transmitted to some more apically diverging fungi, must also be considered given their juxtapositions in the The eight mitovirus species newly identified in this report are the first to be discovered in a fungal host from phylum al fungi ,2 that hycotina) ,26,27,28E. muscae isolates whose mitoviruses were characterized in this study were each found to contain strains of either seven or eight different mitovirus species. Although this large number might seem odd, similar findings have been made in other fungi. For example, strains of at least seven different mitovirus species have been found in the same isolate, Ld, of the Dutch elm disease fungus Ophiostoma novo-ulmi (phylum Ascomycota) [Rhizoctonia solani (phylum Basidiomycota) [Cronartium ribicola (phylum Basidiomycota) [E. muscae is multinucleate throughout all stages of its life cycle, and both protoplast cells and asexual spores may contain up to 20 nuclei each [E. muscae isolates whose sequence reads were analyzed for this report originated not from individual spores, but from conidial showers from infected flies, meaning that there remains a possibility that these isolates are mixtures of E. muscae strains, with differences in mitovirus content between strains.Four of the omycota) ,44,45; somycota) ; and stromycota) . Such mulei each . Althouglei each , the conE. muscae can translate codon UGA (Trp) more efficiently than can that in many other basal fungi [E. muscae mitochondria are more like those of most members of the more apically diverging phyla Ascomycota and Basidiomycota. As noted in Results, UGA (Trp) has been shown to be a rarely used mitochondrial codon in Zancudomyces culistae  codons in the 12 mitochondrial core-gene coding sequences examined here), such that there appears to be some variation in mitochondrial UGA (Trp) codon usage across members of phylum Zoopagomycota. In the recently reported mitochondrial genome sequence of Conidiobolus heterosporus  [C. heterosporus encodes a mitochondrial tRNA sequence with anticodon UCA, which should allow for more efficient translation of UGA (Trp). A complete mitochondrial genome sequence for E. muscae, and for other members of subphylum Entomophthoromycotina, may be helpful for understanding why UGA (Trp) codons are more commonly used in the mitochondria of E. muscae than in those of related species C. heterosporus .The findings in this report, including both mitovirus and mitochondrial coding sequences, suggest that the mitochondrial protein synthesis machinery in al fungi . In thisomycota) , we findE. muscae isolates KVL-14-118, Berkeley, and HHdFL050913-1 in the original transcriptome studies (BioProjects PRJEB10825 and PRJNA435715) [E. muscae itself. Importantly, however, the samples from E. muscae isolate KVL-14-117 analyzed in the original transcriptome studies (BioProject PRJNA372837) [E. muscae isolate KVL-14-117 [E. muscae isolate Berkeley, the original transcriptome study (BioProject PRJNA435715) [E. muscae, and the SRA libraries from those control samples contain no or very few sequence reads matching the E. muscae mitoviruses  ,25 were A435715)  includedoviruses . Moreoveculation . Both of article  and alsocox1 ORF of E. muscae contains a conserved in-frame GUG codon well-upstream of the first in-frame AUG  (E. muscae detailed above). Thus, although Hubei narna-like virus 25 has been previously reported as an insect mitovirus [E. muscae or perhaps a related fungus. These findings provide further support for the conclusion that these and perhaps other yet-to-be-discovered mitoviruses are widespread in naturally occurring populations of E. muscae. Coyle et al.  recentlyNA318834 ). Upon pEnmuMV7) . Only thitovirus , it is nE. muscae on host flies are fascinating and are indeed the subjects of ongoing genetic and neuroscientific investigations in Drosophila [E. muscae, an iflavirus , has been recently reported by Coyle et al. [E. muscae. The E. muscae mitoviruses that we describe here might also be considered as possible contributors in that regard. On a related note, two novel mitoviruses were recently identified in the entomopathogenic fungus Ophiocordyceps sinensis (phylum Ascomycota) [E. muscae in flies, and it is fascinating to speculate that again in this case the fungal mitoviruses might in some way contribute to the manipulation of insect host behaviors by the entomopathogenic fungus.The behavioral effects of osophila . Interese et al.  and specomycota) , which comycota) . These e


Vicia faba L.) from Panxi, China, studied their plant growth promoting ability with nitrogen free hydroponics, genetic diversity with clustered analysis of combined ARDRA and IGS-RFLP, and phylogeny by sequence analyses of 16S rRNA gene, three housekeeping genes and symbiosis related genes. Eleven strains improved the plant shoot dry mass significantly comparing to that of not inoculated plants. According to the clustered analysis of combined ARDRA and IGS-RFLP the isolates were genetically diverse. Forty-one of 65 isolates represented Rhizobium anhuiense, and the others belonged to R. fabae, Rhizobium vallis, Rhizobium sophorae, Agrobacterium radiobacter, and four species related to Rhizobium and Agrobacterium. The isolates carried four and five genotypes of nifH and nodC, respectively, in six different nifH-nodC combinations. When looking at the species-nifH-nodC combinations it is noteworthy that all but two of the six R. anhuiense isolates were different. Our results suggested that faba bean rhizobia in Panxi are diverse at species, plant growth promoting ability and symbiosis related gene levels.We isolated 65 rhizobial strains from faba bean ( Vicia faba L.) and rhizobial bacteria can form a symbiotic relationship in which the legume host provides the rhizobia with nutrients and niche while rhizobia provide the host with fixed atmospheric dinitrogen in the form of ammonia. Owing to symbiosis, legumes can act as pioneer plants in nitrogen deficient areas and improve soil fertility . Mountains occupy 80% of the total area of Panxi and the altitude differences in this area reach 5,600 m. Panxi receives plenty of rainfall and strong solar radiation, and the climate ranges from southern Asian semitropical climate to northern temperate climate with xerothermic climate as the main characteristic of the arid-hot river valley area. Faba bean is one of the main crops in Panxi. Cultivation relies on seeds produced by farmers themselves. N fertilizers would be unnecessary if the soils hosted compatible, plant growth promoting rhizobia.Panxi region in Sichuan, southwestern China, is on the western margin of Yangtze Block, between Tibet Plateau, Yunnan-Guizhou Plateau and Sichuan basin. Panxi is within the South-West China mountains biodiversity hotspot  medium  cultivar Hanyuan dabaidou. Seeds of faba bean were immersed in 95% ethanol for 5 min, rinsed for 5 min with 0.2% mercury bichloride (HgCl2) and 8 times (10 min per time) with sterilized water. After surface sterilization, the seeds were soaked in sterilized water overnight to soften the thick and hard seed coat. The seeds were transferred on 0.5% water-agar for germination. The seedlings were transplanted in sterile 250 ml infusion bottles containing Jensen's solution  analysis (P = 0.05) was done using Excel 2010  and SPSS 17.0 .The nodulation ability and symbiotic efficiency of the isolates was tested on the local faba bean . The fragments were separated by gel electrophoreses in 2% agarose with 0.5 μg ml−1 ethidium bromide at 80 V for 3 h and photographed. Amplified ribosomal DNA restriction analysis (ARDRA) and IGS-RFLP were done by combining the results from the four restrictions. Clustered analysis of combined ARDRA and IGS-RFLP (CACAI) was conducted by UPGM clustering algorithm in the NTSYS program  method  using BLASTN. Phylogenetic analyses of sequences from our isolates and reference sequences from databases were done using a Neighbor-Joining method in MEGA 6.0  were considered as potential inoculant strains  were observed: type a included 54 strains, types b, f, and h included two strains each, and types c, d, e, g, and i included one strain each Table .For the majority of strains, IGS PCR resulted in a single band ranging from 1,900 to 2,200 bp, whereas for strains SCAUf90 and SCAUf99 IGS PCR resulted in two and three bands, respectively (Table Rhizobium (R group) and two to Agrobacterium (A group). Four strains clustered with Agrobacterium radiobacter type strain with 98.3–99.8% similarities. SCAUf144 clustered with R. fabae with 100% similarity, SCAUf100 clustered with R. vallis with 98.6% similarity, and SCAUf86, SCAUf90, SCAUf94, SCAUf99, and SCAUf133 clustered with Rhizobium sophorae into clade R2 with 97.9–99.9% similarities. The other eight strains clustered into a distinct clade with R. gallicum, Rhizobium anhuiense, R. laguerreae, and Rhizobium leguminosarum with similarities ranging from 99.8 to 100%.Based on CACAI groups as well as considering the sites of isolation of the strains, 19 representative strains were selected for sequencing. In the 16S rDNA phylogenetic tree Figure , the stratpD, glnII and recA, the 19 representative strains clustered into nine distinct clades related to Rhizobium and Agrobacterium species  based on housekeeping genes s Figure . SCAUf86nifH and nodC amplification was not successful with one primer pair only, possibly due to differences in primer binding sites. Approximately 700 bp fragments were obtained using primer pair nifHctg/nifHI (13 representative strains), and 400 bp products using primer pair nifH1F/ nifH1R . Amplification of nifH from SCAUf149 and SCAUf150 was not successful. Seventeen strains clustered into four clades in the nifH phylogenetic tree (Figure nifH of Agrobacterium sp. II SCAUf93 and R. anhuiense SCAUf104 were 99.7 and 99.6%, respectively, similar to that of R. anhuiense CCBAU 23252T. The nifH of R. anhuiense SCAUf127 and R. fabae SCAUf144 were 100% similar to that of R. fabae type strain. The nifH of R. sophorae SCAUf86, R. anhuiense SCAUf105 and SCAUf131 clustered with that of R. leguminosarum USDA 2370T with 98.4% similarity. R. vallis SCAUf100 clustered with R. leguminosarum CCBAU 43200 with 100% similarity. The strains R. anhuiense SCAUf91, Rhizobium sp. I SCAUf90, Rhizobium sp. II SCAUf94, SCAUf106 and SCAUf109 carried nifH 100% similar to that of R. leguminosarum CCBAU 71124.For both nodC fragments were amplified from thirteen representative strains using primer pair nodC540/nodC1160. Amplification from strains SCAUf105 and SCAUf140 was successful only while using R. leguminosarum sv. viciae nodC specific primer pair nodCf/nodCr. Amplification of nodC from strains assigned as Agrobacterium was not successful. Fifteen strains clustered into five clades in the nodC phylogenetic tree (Figure nodC of R. anhuiense SCAUf131, SCAUf127, and SCAUf104, R. fabae SCAUf144 and R. sophorae SCAUf86 were 100% similar to that of R. fabae type strain (Figure nifH analysis, the nodC of R. vallis SCAUf100 clustered with nodC from non-type strains. Seven strains carried nodC 100% similar to that of R. leguminosarum non-type strain. The nodC of R. anhuiense SCAUf140 and SCAUf105 (Figure R. laguerreae.Nearly 600 bp e Figure . The nodn Figure . Similar5 Figure  were 100Leucaena leucocephala isolates from Panxi (Xu et al., Due to overcutting and mining Panxi in Southwestern China has suffered serious soil degradation and heavy metal contamination (Xu et al., R. fabae, R. leguminosarum, R. anhuiense, R. laguerraeae and A. radiobacter strains, and the dominant species is different in different regions (Tian et al., Rhizobium and Agrobacterium. The Rhizobium strains were assigned as representing R. anhuiense, R. fabae, R. sophorae, and R. vallis, and two putative new species in the genus Rhizobium. Similar to subtropical provinces in East China (Xiong et al., R. anhuiense was the dominant species among the faba bean nodulating rhizobia in Panxi. To our knowledge, R. sophorae, a symbiont of medicinal legume Sophora flavescens (Jiao et al., R. vallis, a symbiont of Phaseolus vulgaris (Wang et al., The diversity and identity of the strains were assessed using molecular methods. Faba bean is nodulated by nifH gene that is the structural gene encoding the nitrogenase Fe protein (Masson-Boivin et al., nodC that is the gene encoding enzymes involved in the synthesis of the core structure of the Nod-factor (Geremia et al., R. leguminosarum strains that carried four different types of nodulation gene nodD had all similar nodC (Table nodC detected in this study suggest higher diversity at symbiosis related gene level. However, considering the six different nifH-nodC combinations in our study, the faba bean isolates from Yunnan (Tian et al., The rhizobia-legume symbiosis benefits sustainable agriculture due to the symbiotic nitrogen fixation capacity that needs two key points: nodule infection and nitrogen fixation, both of which need the regulation of symbiosis related genes (Masson-Boivin et al., dC Table  (Tian etdC Table . The fivDesmodium nodulating rhizobium strains in Panxi region were quite different from those in other places such as temperate and subtropical region of China and Central and North America, possibly due to the special environmental conditions (Xu et al., nifH-nodC combinations it is noteworthy that all but two of the six R. anhuiense isolates were different. The symbiosis and nitrogen fixation related genes of rhizobia can be transferred laterally (Sullivan et al., The R. anhuiense, the dominant faba bean nodulating species in this area, was diverse both at plant growth promoting ability and symbiosis related gene levels.In conclusion, eleven out of 65 faba bean strains in Panxi area could significantly promote plant growth, and were thus considered as potential inoculants. The nodule isolates in this area were diverse belonging to nine species. KX and YC conceived and designed the experiments. KX supervised the experiments. YC, LZ, PP, and KX contributed to discussion of the results, and writing and revising the manuscript. LZ performed most of the experiments and analyzed data. QC and CW participated in collecting faba bean nodules and relevant soil information, and relevant meteorological information from the Sichuan meteorological bureau. QL created the map in Figure The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Olmesartan, an angiotensin II receptor blocker (ARB), is associated with gastrointestinal symptoms resembling sprue-like enteropathy. Some have proposed that enteropathy may be a class effect rather than olmesartan-specific. We performed a systematic review to identify literature of sprue-like enteropathy for all ARBs.Case reports, case series and comparative studies of ARBs were searched on PubMed and Embase databases through 21 November 2018 and then assessed.A total of 82 case reports and case series as well as 5 comparative studies, including 248 cases, were selected and analysed. The ARBs listed in the case reports were olmesartan , telmisartan , irbesartan , valsartan , losartan  and eprosartan . The periods between ARB initiation and onset of symptoms ranged from 2 weeks to 13 years. Histologic results were reported in 218 cases, in which 201 cases (92.2%) were villous atrophy and 131 cases (60.1%) were intraepithelial lymphocytosis. Human leucocyte antigen (HLA) testing was performed in 147 patients, among whom 105 (71.4%) had HLA-DQ2 or HLA-DQ8 haplotypes. Celiac-associated antibodies were tested in 169 patients, among whom 167 (98.8%) showed negative results. Gluten exclusion from the diet failed to relieve symptoms of enteropathy in 127 (97.7%) of 130 patients with information. Complete remission of symptoms after discontinuation of ARB was reported in 233 (97.4%) of the 239 patients with information. Seven cases (2.8%) reported recurrence of symptoms after restarting olmesartan; rechallenge was not reported for the non-olmesartan ARBs. The retrospective studies conducted worldwide had inconsistent study designs (e.g. differences in periods of study and case definition) and findings.Although enteropathy is rare, clinicians should remain vigilant of this potential adverse event even years after medication initiation. Angiotensin II receptor blockers (ARBs) are among the most commonly used blood-pressure-lowering drugs in the world , 2. Theyet al. [In 2012, Rubio-Tapia et al.  reportedet al. . The patet al. . This imet al. . Similaret al. , followeet al. . These cet al. .However, limited epidemiological studies compared enteropathy outcomes between olmesartan and other anti-hypertensive medications, including other ARBs and angiotensin-converting enzyme (ACE) inhibitors, and results have been mixed. However, limited epidemiological studies compared enteropathy outcomes between olmesartan and other anti-hypertensive medications, including other ARBs and ACE inhibitors, and results have been mixed. We performed a systematic review of the published literature to identify case reports, case series and comparative studies of ARB-related enteropathy.We performed a literature search through 21 November 2018 in PubMed and Embase databases. We searched the articles using international ARB brand and generic drug names according to Micromedex/Martindale to have the broadest search possible . MicromeAny article that mentioned an ARB including case reports, case series and comparative studies (prospective and retrospective) was included at the abstract level of review. Titles, abstracts and full texts were assessed independently by two reviewers (A.K. and A.P.) to determine the eligibility of the studies according to inclusion and exclusion criteria. If a case report did not mention diarrhea, it was excluded. All studies that did not include humans  were excluded. Comparative studies were included if they mentioned conditions associated with enteropathy, including celiac disease. During the full-text review, the reviews, editorials and guidelines were excluded. Studies that reported on conditions or symptoms consistent with enteropathy were included. Online systematic review management software, Covidence, was used for title/abstract screening, full-text screening, data abstraction and quality assessment ; it was Two reviewers (A.K. and A.P.) extracted demographic, exposure and outcome information from the included studies into Microsoft Excel 2013. The data were independently collected for each study and were cross-checked. Discrepancies were resolved through consensus. If multiple articles reported on the same patients, only the later report of the patient was included. We extracted the following information for case reports and case series: information on demographics; type of ARB used; the period between the ARB initiation and symptom onset; HLA-DQ haplotypes; antibodies associated with celiac disease; response to gluten exclusion from the diet; and symptomatic, endoscopic and histologic response to ARB discontinuation and response to resuming ARB use . For the comparative studies, we extracted information on demographics, outcome definition and rate of intestinal malabsorption among olmesartan, other ARBs and angiotensin-converting-enzyme-inhibitor (ACEI) users. We collected information on the factors adjusted for the analysis and required that the extracted rate adjusted for a minimum of age and sex.The final search yielded 2510 articles in PubMed and 9308 in Embase before de-duplication. The total number of articles screened at the title/abstract level was 7502 articles; 82 articles reporting cases and 5 comparative studies were included. These studies were published between 2012 and 2018 .There were 248 cases resembling sprue-like enteropathy  . The ARBAmong 147 patients who underwent HLA testing, 105 (71.4%) presented HLA-DQ2 or HLA-DQ8 haplotypes . Among 169 patients in whom celiac-associated antibodies were tested, 167 (98.8%) showed negative results ; 2 (1.2%) olmesartan users showed positive results.Histologic results were reported in 218 patients, among whom 201 (92.2%) had villous atrophy  and 131 (60.1%) developed intraepithelial lymphocytosis .Gluten exclusion from the diet failed to resolve symptoms in 127 (97.7%) of 130 patients with information. Three olmesartan users responded to dietary exclusion; however, the dietary exclusion occurred while the olmesartan treatment was finished, and therefore exclusive response to gluten exclusion could not be established. Complete remission of symptoms after discontinuation of ARB was reported in 233 (97.4%) of the 239 patients with information . Seven cases (2.8%) reported recurrence of symptoms after receiving olmesartan. Rechallenge was not reported for the other ARBs.Five comparative studies included in the presented study were retrospective analyses of large databases.et al. [A case–control study by Marild et al.  linked het al. [A retrospective French study by Basson et al.  identifiet al. [You et al.  conducteet al. [A German and Italian retrospective study conducted by Malfertheiner et al.  includedet al. [Five USA commercial claims and federal databases were pooled to compare olmesartan vs. other ARBs in this retrospective study conducted by Dong et al. . This stThe present literature review included all case reports, case series and comparative studies reporting ARB-related enteropathy that were published before our search. We reported all cases reports and comparative studies published in the literature reporting ARB-related enteropathy. Our search identified 87 studies, of which 82 were case reports or series and 5 were retrospective studies. Overall, 248 cases of enteropathy associated with ARBs were reported with a fairly even male-to-female ratio and ages at diagnosis ranging from 45 to 89 years. Celiac disease-specific antibodies were negative in 99% of the cases. HLA DQ2 or DQ8 haplotype was present in 71% of patients . DQ2/DQ8The mechanism driving this adverse event is unclear. The most common causes of villous atrophy include celiac disease, medication, collagenous sprue and common variable immunodeficiency, human immunodeficiency virus, tropical sprue, giardiasis, whipple disease and viral disease . ARBs maThe comparative studies found that the absolute rate of enteropathy was low after the use of olmesartan, valsartan, irbesartan, losartan, telmisartan, candesartan, eprosartan and ACEI. Pooling of the comparative studies was difficult because of differences in the design. Studies had different periods of observation, different definitions of cases and different comparison groups. Studies that included cases diagnosed after May 2013 , 35 may Our study has several strengths. We included cases using common criteria to define enteropathy across cases. The previous systematic reviews did not search for all ARBs , 10. HowARB-related enteropathy reported in the majority of case reports was associated with the treatment of olmesartan. In the 12 patients receiving other ARBs, discontinuation of the medication resulted in symptom remission. Olmesartan appears to be associated with a higher risk of enteropathy than other ARBs. ARB-related enteropathy should be considered a distinct clinical entity and should be included in differential diagnosis of diarrhea in hypertensive patients. Although enteropathy is rare, clinicians should remain vigilant when treating enteropathy, even after years following medication initiation.A.K. acquired, analysed, interpreted data and drafted and critically revised the manuscript for important intellectual content. A.P. acquired and critically revised the manuscript for important intellectual content. C.F., E.G.V., D.L. and P.W. critically revised the manuscript for important intellectual content. S.H. conceived and designed this study, analysed and interpreted data, drafted and critically revised this manuscript, and supervised this study. All authors approved the final version of the manuscript.None.


Burkholderia pseudomallei is the etiological-agent of the neglected and life-threatening emerging infection melioidosis. The distribution of B. pseudomallei in West Africa is unknown. In the present study we aimed to determine whether B. pseudomallei and B. thailandensis are present in the environment of central Sierra Leone.The soil-dwelling bacillus B. pseudomallei was not identified in the soil, but we identified seven novel B. thailandensis sequence types with multi-locus sequence typing (MLST) and 16S rRNA gene sequence analyses.In June-July 2017, we conducted an environmental surveillance study–designed in accordance with existing consensus guidelines—in central Sierra Leone. A total of 1,000 soil samples (100 per site) were collected and cultured. B. pseudomallei was not demonstrated, however, multiple novel B. thailandensis sequence types were identified. More environmental and sequencing studies are needed to further understand the genetic diversity, evolution and virulence of these emerging organisms.The presence of  Burkholderia pseudomallei is the cause of melioidosis, an often-fatal but neglected infection prevalent across tropical areas. B. thailandensis is a member of the B. pseudomallei complex, rarely causes disease in humans and is considered a-virulent. Modelling studies have estimated a high prevalence of B. pseudomallei in western Africa. In this study, we performed an environmental surveillance study in the West African country of Sierra Leone. Remarkably, we could not demonstrate the presence of B. pseudomallei in the soil of Sierra Leone. However, by both culture and sequencing methods, we identified multiple B. thailandensis strains and novel genotypes. Patients with debilitating B. thailandensis infection have been occasionally reported in among others Southeast Asia and the US. Environmental and sequencing studies on both B. pseudomallei and B. thailandensis are essential to further understand the genetic diversity and evolution of these neglected but emerging organisms.The environmental bacterium  Burkholderia pseudomallei is the etiological agent of melioidosis, an emerging but neglected infectious disease. Disease presentations vary from abscess formation to fulminant sepsis [B. pseudomallei primarily occurs in people who are in regular contact with soil and water [B. thailandensis is a member of the B. pseudomallei complex, is considered a-virulent [B. pseudomallei and B. thailandensis, however, is limited.The Gram-negative environmental bacterium t sepsis . Melioidt sepsis . Infectind water ,2. B. thvirulent ,4 and ravirulent –10. KnowB. pseudomallei and B. thailandensis in its soils [B. pseudomallei and B. thailandensis are present in the soil of central Sierra Leone.Patients from sub-Saharan Africa reported with melioidosis are few and isolated , which most probably is the result of under-recognition and under-reporting. These cases may represent the ‘Tip of the Iceberg’ ,11. Fromts soils . In the B. pseudomallei and B. thailandensis presence  [Burkholderia isolates from soil samples was done as described previously [Burkholderia colonies every 24 hours for a period of 7 days. Initial identification of suspected Burkholderia growth was done by colony morphology, positive oxidase test result, antimicrobial drug susceptibility pattern (susceptible to amoxicillin-clavulanic and resistant to gentamicin and colistin) and latex-agglutination tests [During the rainy season (June-July 2017), an environmental surveillance study–designed in accordance with existing consensus guidelines —was condfarming) ,14. Oralfarming)  was takeeviously . First, on tests   [All isolates were identified as yses see . B. pseu3 (ST73) , but theBurkholderia isolates using the MiSeq platform  as described previously [B. pseudomallei database . Cluster analysis was subsequently performed in the MEGA 6.06 using the UPGMA algorithm and the Jukes-Cantor model. Bootstrap test was performed for 500 repetitions.Multi-locus sequence typing (MLST) and 16S rRNA gene sequence analyses were used to determine the species of the isolated Oral informed permission was obtained from landowners and written informed permission from the paramount chief of Yele, Sierra Leone, prior to soil sampling.Burkholderia strains from 25 soil samples. Four Burkholderia strains showed a negative latex-agglutination test; the rest showed (possible) positive latex-agglutination test results.A total of 1,000 samples (100 per site) were collected at ten sampling sites in the Tonkolili and Bombali District, central Sierra Leone see . InitialB. thailandensis STs available in the PubMLST database  [B. thailandensis showed cross-reactivity with the B. pseudomallei latex-agglutination test. MLST data and microarray based comparative genomic hybridization revealed earlier that there is a separate subgroup of B. thailandensis isolates  containing BTCV strains, which are genetically different from the other B. thailandensis isolates [Two main clusters are presented in a phylogenetic tree based on the concatenated sequences of the seven household genes of all database . Clustere (BTCV)  possiblyisolates .B. thailandensis clusters (I and II) were observed. Various studies have reported human infections by B. thailandensis belonging to both cluster I  and cluster II (ST73 and ST101)  [B. thailandensis isolates within cluster II are more virulent than those in cluster I [B. pseudomallei infection and include soft tissue infection, abscess formation, pneumonia and sepsis [B. thailandensis case occurred in a 29-year old diabetic woman with an infected wound and swelling of her forearm after a car incident in Arkansas, US [In this study, two clearly separated luster I , but eviluster I ,17. Clinnsas, US .B. pseudomallei and B. thailandensis reside could help to determine which sites to study in future environmental surveillance studies. Furthermore, bacteria could have been present in a viable, but not cultivable state. It remains difficult to differentiate B. thailandensis and other members of the B. pseudomallei complex from B. pseudomallei by methods commonly available in clinical labs, even in developed countries, which may result in diagnostic confusion. Therefore, improving the detection and differentiation of members of the B. pseudomallei complex to improve patient care and appropriate public health responses is desired. Taken together, B. pseudomallei was not cultured from the soil of central Sierra Leone, but B. thailandensis with novel genotypes were found. B. thailandensis infection in humans have been sporadically reported in the literature in both the US and Asia [B. thailandensis in general is considered a-virulent. As a result, clinical disease attributed to B. thailandensis is important. This also holds true for to the melioidosis research community, because no strict biocontainment conditions for B. thailandensis are required. The true clinical relevance of this soil-dwelling bacillus, however, remains to be elucidated. We encourage further environmental and sequencing studies on both B. pseudomallei and B. thailandensis to further understand the genetic diversity, virulence and evolution of these emerging organisms.Our study has several limitations, including the lack of standard blood culture services for febrile patients across Sierra Leone. This limits targeted soil sampling studies centred around an index case. In addition, we cannot dismiss the possibility of sampling-error, although consensus guidelines were followed . More knand Asia –10. B. tS1 Table(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S1 Supporting Methods(DOCX)Click here for additional data file.


Fusarium oxysporum f.sp. lycopersici, several other Fusarium species are known to cause extensive worldwide crop losses in tomatoes. Prevalence and identities of Fusarium species infecting tomatoes in Northwest Pakistan is currently not known. In this study, we surveyed and characterized Fusarium species associated with symptomatic tomatoes in Northwest Pakistan using morphological and molecular analyses. Pathogenicity tests revealed varying degrees of virulence with some Fusarium sp. causing severe disease symptoms whereas others displaying mild symptoms. Molecular identification based on Internal Transcribed Spacer (ITS) region and TEF-1α gene sequencing classified all isolates into four major species with a majority (68.9%) belonging to Fusarium incarnatum-equiseti species complex (FIESC), followed by F. graminearum (20.7%), F. acuminatum (6.8%), and F. solani (6.8%). ISSR analyses revealed substantial genetic variability among all the Fusarium population infecting tomatoes. Genetic distance between populations from the central region and the type strain F.o. f.sp. lycopersici from Florida was the highest (0.3662), whereas between the south and central region was the lowest (0.0298), which showed that genetic exchange is negatively effected by distance. High genetic variability suggests that these Fusarium species have the potential to become a major production constraint for tomato growers. Findings in this report would greatly facilitate identification of Fusarium species in developing countries and would provide groundwork for devising and implementing disease management measures for minimizing losses caused by Fusarium species in tomatoes.In addition to the well-known  Solanum lycopersicum), a member of family Solanaceae, is considered one of the world’s most important and popular vegetables ..FusariumFusarium symptoms such as wilting, vascular discoloration, and yellowing of leaves at different growth stages ranging from young seedlings to mature flower- and fruit-bearing plants throughout the crop season. In general, incidence and severity of the disease were higher in Northern (Swat and Mansehra) and Southern (Peshawar and Bannu) districts, and comparatively lower in central districts   .Fusarium species isolates showed differential responses , Fusarium equiseti , Fusarium graminearumm  and unknown Fusarium species .Macroscopic and microscopic characteristics of all the isolates, including colony characteristics, mean spore length of macro and microconidia, presence and absence of chlamydospores are summarized in s groups . The ideITS and TEF-1α. Targeted regions of both ITS and TEF-1a regions were successfully amplified from all isolates. BLASTn analyses conducted using nucleotide databases at the FUSARIUM-ID [http://www.ncbi.nlm.nih.gov/) revealed a 98–100% identity for ITS and 99–100% identity for TEF-1a with the reported Fusarium species isolates. Based on ITS sequences, 19 isolates were identified as F. equiseti, four as F. graminearum, three as F. acuminatum, two as F. solani, and one as F. verticillioides  (F. verticillioides (Pak-13) and F. acuminatum (Pak-26) based on ITS sequences were identified as F. graminearum. Differences between the results of a few isolates based on morphology, and ITS and TEF-1a sequencing are most likely due to close resemblance in morphological and genetic characteristics among many closely-related species in the genus Fusarium. Similarly, ITS sequences are not always accurate in accurately classifying Fusarium species [TEF-1a is recommended [TEF-1a sequencing, of the total 29 isolates tested, 20 isolates (68.9%) belonged to FIESC, 6 isolates (20.7%) belonged to F. graminearum, 2 isolates (6.8%) belonged to F. acuminatum and 2 isolates (6.8%) belonged to F. solani.All 30 isolates were identified using DNA sequences of llioides . BLASTn d on ITS . A major (FIESC) . Two iso species . Since, ommended . As is sF. equiseti. Isolates in clade III represented different geographical areas and different levels of virulence including the most virulent isolates, Pak-1 and Pak-6. One pathogenic isolate, Pak-2, with low virulence from district Swat formed a separate branch within clade III. The F. graminearum isolates  formed a separate clade (Clade II) supported by 100% bootstrap values. Isolates in this group were non-pathogenic on tomatoes. Interestingly all isolates in this group were from the Southern part of the province. Clade IV and V consisted of non-pathogenic isolates, which were identified as F. acuminatum and F. verticillioides. Clade VI consists of only F. solani isolates.Phylogenetic analysis of the ITS sequences classified all isolates into six major groups with high bootstrap values . Sixty fTEF-1α gene separated Fusarium population into five major clades, which were all supported by high bootstrap values 9T and (GA)9C showed robust results and they were selected based on their high retention power and reproducibility in producing polymorphic band patterns for all the 30 isolates representing five different species of Fusarium. These primers produced a total of 303 bands ranging in size from 400–4500 bp. However, the minimum and the maximum number of polymorphic ISSR fragments varied from three to seven per isolate , while the lowest distance was between the South and central regions (0.0298).Genetic diversity parameters of different populations based on geographic origin are shown in . ResultsFusarium species infecting tomatoes in Khyber-Pakhtunkhwa province. Our study shows that Fusarium wilt is present in a majority of the tomato growing areas at all growth stages, most likely because of lack of sanitation, using infected seeds, and mono-culturing, which could lead to build-up of soil-borne inoculum. In addition, inappropriate use of fungicides could also lead to the development of fungicide-resistant strains thus increasing the prevalence and incidence of the disease [To the best of our knowledge, this is the first study reporting epidemiological and phylogenetic analyses of  disease .Fusarium infection and survival [Fusarium attack as reported earlier [Fusarium is a soil- and seed-borne pathogen, seedlings are readily infected right after emergence when grown from infected seeds. It is well known that crop rotation increases disease suppressive conditions, which help reduce soil-borne inoculum [Fusarium diseases in tomatoes. Use of healthy disease-free seeds would also reduce disease incidence and fungicide sprays. Reducing fungicide sprays in turn would help reduce emergence of fungicide-resistant virulent strains.Incidence of the disease was higher in the northern and southern regions of the province and lower in the central region. Soil in the northern area is sandy clay loam with an acidic pH 5–6 and a temperature range of 25–28°C during tomato growth season, which is suitable for survival –38. In d earlier . Due to inoculum , 41. ThuFusarium [Fusarium wilts. Furthermore, including leguminous crops in rotation allows nitrogen-fixing bacteria such as Rhizobia to fix atmospheric nitrogen to nitrates and nitrites, which raise soil pH that could limit the growth of Fusarium species and reduce disease incidence in tomatoes [In contrast, in the central region of the province, disease incidence and severity were lower. In these areas, growers use Nitrate fertilizers, which, by raising soil pH, appear to limit the growth of Fusarium . Growerstomatoes , 43.Fusarium isolates, Pak-1 and Pak-6, were highly virulent on tomato leaves whereas the remaining 27 isolates were either moderately and weakly pathogenic or non-pathogenic on tomatoes. The difference in virulence may be due to differences in genes responsible for pathogenicity and virulence and/or genetic makeup of host plants that could differentiate pathogenic isolates from non-pathogenic isolates [Pathogenicity assay showed that two isolates .F. equiseti was found to be the most prevalent Fusarium species followed by F. graminearum, F. solani, and F. acuminatum associated with tomatoes in Khyber-Pakhtunkhwa. Our results are supported by finding in other studies, which reported different Fusarium species including F. oxysporum, F. verticillioides [F. redolens, F. proliferatum, F. equiseti, and F. solani associated with wilt of tomatoes [Fusarium species use ß-tubulin, translation elongation factor (TEF-1α), RPB 1 and 2, intergenic spacer (IGS), mitochondrial small subunit (mtSSU) rRNA, and internal transcribed spacer (ITS) region of the ribosomal RNA genes [TEF-1α genes, which identified all isolates accurately to the species levels. Our results showed that TEF-1α sequences outperformed ITS in identifying Fusarium species associated with the disease, particularly for members of the FIESC by providing superior resolution. It is also reported that for some fungi, such as Trichoderma and Fusarium, TEF-1α are recommended for species elucidation [Fusarium lineages in phylogenetic trees [TEF-1α, and RPB 1 and 2 are more reliable for inferring deep phylogenies, whereas gene sequences undergoing faster evolutionary rates such as TUB2 are more susitable for recent evolutionary and speciation events. The TEF-1α gene satisfy both these requirements consisting of both conserved exonic and variable intronic sequences [Fusarium species associated with a disease.Based on morphological identification using micro and macroconidia, llioides , F. redotomatoes –50. MostNA genes , 51–54. cidation . ITS seqic trees . In geneequences . AccordiFusarium equiseti, which belongs to F.incarnatum-equiesti species complex (FIESC), was the main species responsible for wilt of tomato in Khyber-Pakhtunkhwa. Within the FIESC clade, isolates from different areas were scattered with no evidence of phylogenetic structure with respect to their pathogenicity and geographic origin, which are consistent with previous findings [Fusarium species pathogenic on tomato plants in Khyber-Pakhtunkhwa was F. solani, which displayed moderate virulence. F. solani is a common pathogen of tomato causing wilt, and pre- and post-emergence damping off in different parts of the world such as India and Turkey [F. solani is the principal cause of wilt of tomato in F. oxysporum resistant tomato varieties [Based on molecular identification, findings . The secd Turkey , 58. In arieties .Fusarium is a complex genus with many similar, different, and not well-researched species. To sustain durable resistant or tolerant varieties, which is the most efficient mean of control for Fusarium wilt of tomato, knowing changes in the genetic diversity of different Fusarium species pathogenic on tomato is imperative [Fusarium isolates, identified in our study as F.equiesti, F. graminearum, F. solani, F. acuminatum, and the type species F. o. f.sp. lycopersici, revealed high genetic diversity among and within populations of Fusarium species isolated from tomato in Khyber-Pakhtunkhwa. Cluster analysis showed that pathogenic and non-pathogenic isolates of different Fusarium species tend to cluster separately irrespective of their geographic distribution, indicating that there is some correlation between the pathogenic and non-pathogenic isolates. These results are consistent with a previous study showing that genetic diffferentiation and genetic distances are not associated with geographic distribution [F. equiesti and F. solani isolates reflected polyphyletic nature of Fusarium species usually attributed to high evolutionary rate within the microsatellite regions [F.equiesti were closely related and showed low genetic diversity. This narrow variation could be due to agricultural practices in which growers mainly rely on local varieties thus restricting the introduction of new genotypes of the pathogen [Fusarium species of several major crops showing that ISSR markers are a reliable and authentic source for inferring genetic relationship within and between different Fusarium species [perative . Employiperative . ISSR anribution . Further regions . Howeverpathogen . Similar species , 60.Fusarium isolates based on parameters such as percentage of polymorphism (PP), observed number of alleles (NA), effective number of alleles (NE), Nei’s gene diversity (H), Shannon’s information index (Is), and Nm gene flow [Fusarium population from the Norther region of the province. Given the fact that a majority (68.9%) of these isolates belonged to F. equiseti indicated high intraspecific variation within the F. equiseti isolates. The high genetic variation among different Fusarium species and the genetic partitioning within F. equiseti are in agreement with the estimates of expected heterozygosity reported for other Fusarium species [m) value of 1.0 and a genetic differetiation (GST) value of 0.25 are considered as treshholds, beyond which significant differentiation occurs [Fusarium populations reported in our studies were just above the treshold level . We reported the highest genetic identity (0.9706) and lowest genetic distance (0.0298) among the poulations of southern and central regions, which could be due to local tranfer of planting material among growers of these areas [In this studies, we report the genetic structure of ene flow . We repo species , 64. In n occurs . Considese areas .


Bacillus thuringiensis (Bt) is widely used in producing biological insecticides. Phage contaminations during Bt fermentation can cause severe losses of yields. Lots of strategies have been engaged to control extrinsic phage contamination during Bt fermentation, but their effectiveness is low. In this study, the candidate endogenous prophages (prophages) in 61 Bt chromosomes that had been deposited in GenBank database were analyzed. The results revealed that all chromosomes contained prophage regions, and 398 candidate prophage regions were predicted, including 135 putative complete prophages and 263 incomplete prophage regions. These putative complete prophages showed highly diverse genetic backgrounds. The inducibility of the prophages of ten Bt strains  was tested, and the results showed that seven of the ten strains’ prophages were inducible. These induced phages belonged to the Siphoviridae family and exhibited a broad host spectrum against the non-original strains. The culture supernatants of the two strains  could lyse Bt cells, but no virions were observed, which was speculated to be caused by lysin. The functional analysis of the putative complete prophage proteins indicated that some proteins, such as antibiotic resistance-associated proteins and restriction endonucleases, might increase the fitness of the Bt strains to different environments. The findings of this study provided understanding on the high prevalence and diversity of Bt prophages, as well as pointed out the role of prophages in the life cycle of Bt.  Bacillus thuringiensis (Bt) is a ubiquitous Gram-positive, spore-forming bacterium that produces parasporal crystals at the end of its life cycle. Due to the insecticidal ability of the parasporal crystal proteins, Bt is widely used in producing bio-pesticides [sticides ,2. Howevsticides . immB in telomere phages [Though many strategies—such as screening phage-resistant Bt strains, treating the seed broth with a high temperature ≥85 °C), and strengthening the air filtration system—have been employed to control phage contamination outside bacterial cells, it is still a problem that interferes with Bt fermentation. For the purpose of understanding Bt phage diversity and providing instructions for constructing phage-resistant genetically engineered Bt strains, many Bt phages have been isolated and their interactions with the host bacteria have been characterized ,5,6,7. T °C, and e phages , and thee phages . The Bt e phages ,18. Aftee phages ,20,21,22e phages . The resDesulfovibrio, 128 prophage-like elements were screened in 46 of the 47 analyzed genomes, and 53 were identified as complete prophages [Escherichia coli O157 possesses 18 prophages, but most of them contain genetic defects and many of the defective prophages could be induced and released from the host cell [Temperate phages are common, and bacterial genomes are full of integrated prophages . The invrophages . Escheriost cell . Though https://www.ncbi.nlm.nih.gov/genome/genomes/486) before 30 December 2018. These Bt chromosome sequences were collected (https://www.ebi.ac.uk/seqdb/confluence/display/THD/PSI-BLAST) and VirFam (http://biodev.cea.fr/virfam/). The prophages containing all structural genes were regarded as putative complete prophages, and their sequences were extracted from the strain genomes and used for further bioinformatic analysis.The complete genome sequences or chromosomes of 61 Bt strains had been deposited in the GenBank database  [Bacillus strains was conducted using Gepard [https://www.lynnon.com/). The proteins encoded by the prophage regions were predicted by using Genmarks [http://weizhong-lab.ucsd.edu/metagenomic-analysis/server/cog/) [In this study, we mainly focused on the putative complete prophages in Bt strains. The distance between the sequences of 135 putative complete prophage genomes was calculated by using MegAlign , and thembl.de/) . The dotg Gepard . The comg Gepard . The aliGenmarks , and theB. thuringiensis serovar. jinghongiensis YGd22-03, guiyangiensis KK31-01, rongseni Scg04-02, pingluonsis NXP15-04, zhaodongensis HZ39-04, sinensis YK30-04 and wuhaniesis 140) were identified and stored by our laboratory [g for 30 min, and the supernatants were collected and filtered through a filter with a pore size of 0.22 μm. The filtrate was incubated with 10% polyethylene glycol (PEG) 8000 and 0.5 M NaCl overnight at 4 °C [600) at intervals of 1 h. The strains without induction were used as controls.The Bt strains  [http://topaz.gatech.edu/GeneMark/gmhmmp.cgi) [http://pfam.xfam.org/search/sequence) [https://toolkit.tuebingen.mpg.de/#/tools/hhpred) [https://www.ncbi.nlm.nih.gov/sutils/pasc/viridty.cgi?cmdresult=main&id=428) [The Bt strains, in plates which contain the most large and clear lysis zones, were used for phages purification. The HD-29 strain was used for the purification of induced phage from HD73 strain. The exponential growth strains (200 μL) were mixed with gradient diluted supernatants (200 µL), and then poured onto a solid medium plate as an overlay overnight. The plaques formed on the plates were used for phage purification using the method previously reported . Simply,ntrifuge . After cntrifuge . The genast.cgi)  and Genemmp.cgi) , and theequence)  and HHpr/hhpred) . The sim&id=428) . The phy&id=428) .2, 1 mM CaCl2, pH 6.5) overnight at 37 °C, as previously described [The bacterial cultures induced by using mitomycin C were collected by centrifugation and subsequently filtered through a 0.22 μm filter. The filtrates were then concentrated by incubation with PEG 8000 and NaCl, followed by being centrifuged and re-filtered. The concentrated and re-filtered supernatants were further separated using SDS-PAGE containing 0.4% of the exponential growth and autoclaved host cells. The strains without mitomycin were used as controls. After separation, the gel was rinsed four times with 2.5% Triton X-100 and incubated in a renaturation buffer  and major capsid protein (95% coverage and 92.22% identity with Bacillus phage phiCM3), respectively. The similarities of the prophage regions to existing phage genomes were analyzed . The resd phi4B1 . Four pud phi4B1 B, and thThe proteins encoded by 135 putative complete prophages were predicted and used for functional classification. Totally, 9477 proteins were predicted, and 5163 of them were homologous with proteins in the COG database. Functional analysis of the prophage proteins revealed that the majority of them were associated with the life cycle of phages, such as structural proteins, DNA replication associated proteins, lytic and lysogenic regulation proteins, and phage host lytic system proteins A,B. The Escherichia coli. These two beta-lactamases possess identical catalytic site residues , including two phages with different morphologies induced from the HD-521 strain. The morphological analysis showed that all the induced phages belonged to the e family , which ce family H. The cue family K,L, whil10 viron/mL. The phage, which was named phiHD73, was similar with Bacillus phage phi4J1, with a similarity of 32.27% in PASC. This phage was classified into the TP21-L-like group using phylogenetic analysis . The titration of the recovered phage was 10analysis .Pneumococcus and Desulfovibrio genii. Overall, 689 prophage-like fragments were identified from 482 pneumococcal strains, and the average number in each strain was 1.45 [E.coli K-12, prophage genes were proven to increase host resistance to 11 β-lactam antibiotics, including the most common penicillins and cephalosporins [Bacillus, that can survive in harsh environments must also be prepared to confront adverse growth conditions. Any resistance factor or metabolic superiority offered by prophages is of great significance and can heighten the rivalrousness of host cells [The diversity of prophage regions in Bt strains seems higher than in other bacteria, such as the was 1.45 , and 128was 1.45 . Additiowas 1.45 ,53. The was 1.45 . In E.coosporins . The bacosporins . Restricosporins . In addiosporins ,58,59. Bst cells .The contamination of phages during Bt fermentation causes severe losses in yields, and can sometimes cause a failure of fermentation. The findings of this study reveal that putative complete prophages could be induced by mitomycin C. A previous study on Bt temperate phages revealed that the prophages in Bt strains could spontaneously enter the lytic cycle at a high frequency without induction . CombineThe construction of Bt engineering strains is one of the most important ways to improve fermentation properties . Nowaday


C. albicans readily forms biofilms in conjunction with other oral microbiota including both bacteria and yeast. Adhesion between species is important to the establishment of these mixed biofilms, but interactions between C. albicans and many NACS are not well-characterized. We adapted a real-time flow biofilm model to study adhesion interactions and biofilm establishment in C. albicans and NACS in mono- and co-culture. Out of five NACS studied, only the filamenting species C. tropicalis and C. dubliniensis were capable of adhesion with C. albicans, while C. parapsilosis, C. lusitaniae, and C. krusei were not. Over the early phase (0–4 h) of biofilm development, both mono- and co-culture followed similar kinetics of attachment and detachment events, indicating that initial biofilm formation is not influenced by inter-species interactions. However, the NACS showed a preference for inter-species cell-cell interactions with C. albicans, and at later time points (5–11 h) we found that dual-species interactions impacted biofilm surface coverage. Dual-species biofilms of C. tropicalis and C. albicans grew more slowly than C. albicans alone, but achieved higher surface coverage than C. tropicalis alone. Biofilms of C. dubliniensis with C. albicans increased surface coverage more rapidly than either species alone. We conclude that dual culture biofilm of C. albicans with C. tropicalis or C. dubliniensis offers a growth advantage for both NACS. Furthermore, the growth and maintenance, but not initial establishment, of dual-species biofilms is likely facilitated by interspecies cell-cell adherence.Non- Candida albicans is by far the most predominant organism isolated from oral samples in OPC, however non-albicans Candida species (NACS) are frequent co-isolates  is prevalent in populations of immunocompromised individuals, but treatment is hampered by limited drug options  genes] and architectural composition of biofilms (extracellular matrix and subpopulations of persister cells)   were used as WT control. NACS strains used were C. dubliniensis Wü284 ; C. krusei AR0397 and C. lusitaniae AR0398 were obtained from the antimicrobial resistance bank of the Centers for Disease Control and Prevention (CDC). All strains were routinely grown in YPD broth medium  at 30°C with shaking at 220 rpm. For biofilm experiments, liquid Spider medium (pH 7.2) prepared as described previously  and 50 mg/ml of uridine (Sigma-Aldrich), were used.Fluorescently labeled C. albicans CAF2-dTomato strain  supplemented with 1.25% N-Acetyl glucosamine (GlcNAc), and incubated for 4 h at 37°C to induce filamentation in mono-culture. For NACS strains, stationary phase cells were incubated in YNB supplemented with 1.25% glucose instead of GlcNAc. Cells were harvested by centrifugation, washed once in 1X PBS, and then resuspended in 1X PBS. Each NACS were then co-incubated with CAF2-dTomato in 1:1 ratio in PBS buffer for 60 min with gentle shaking and observed through Zeiss AxioObserver Z1 inverted fluorescence microscope  using chambered slides  showed distinct cell to cell adhesion . By 4 h of dual species growth, both C. dubliniensis and C. tropicalis had higher biofilm surface coverage area than C. albicans—a reflection of the higher density of cell clusters of adherent NACS to C. albicans hyphae  was directly proportional to the biofilm biomass. Therefore, we employed Spider media to achieve similar levels of filamentation and biofilm growth for dual species quantitation. Furthermore, C. albicans biofilms grown in Spider media, which contains beef extract as the component that induces filamentation, are more susceptible to fluconazole treatment and leukocyte infiltration than biofilms formed in RPMI medium  of these dual-species biofilms. However, over the first 4 h of biofilm growth, the percentage  of C. dubliniensis and C. tropicalis cells adhering to C. albicans increased, indicating a preference for interspecies cell-cell interactions. Additionally, up to 4 h, C. dubliniensis and C. tropicalis dominated over C. albicans based upon quantitation of biofilm surface coverage, which may be due to higher initial attachment. At later time points, the growth of each species was visibly influenced by interspecies interactions. Co-species biofilms of Ca+Cd increased surface coverage faster, while Ca+Ct was slower, than for biofilms of mono-culture C. albicans. C. albicans predominated over the growth of C. dubliniensis and C. tropicalis when the biofilms reached maturation, though neither species was eliminated. Therefore, growth and maintenance, but not initial establishment, of dual-species biofilms is likely facilitated by interspecies cell-cell adherence. Significantly, we found that hyphal production of C. tropicalis was suppressed when grown together with C. albicans despite being cultured in Spider media. This repression is likely due to production of quorum sensing molecule farnesol by C. albicans; and C. tropicalis is particularly sensitive to farnesol (Weber et al., Candida species in vivo.We found that the early phases of dual-species biofilm development followed similar kinetics of attachment and/or detachment events as for mono-culture biofilms. Even though both Candida albicans is the most frequently isolated Candida species found in studies of carriage in healthy individuals and multiple disease states, while C. dubliniensis and C. tropicalis are isolated less often (Patel et al., C. albicans usually remains the predominant species (Patel et al., C. tropicalis was the most common Candida isolate (Jain et al., C. tropicalis has also been identified as the most common cause of candidemia in cancer patients (Wu et al., C. albicans cells tend to predominate over NACS in mature biofilms in healthy individuals. However, disease states may modulate this balance—for example by the presence of blood products that promote filamentation in NACS that do not typically produce hyphae.in vivo. We observed a lag in biofilm growth in Ca+Ct co-culture compared to Ca alone; however, surface coverage of the biofilm past 8.5 h ultimately exceeded what C. tropicalis was capable of producing alone, indicating a clear benefit for C. tropicalis. The potential advantages of rapidly establishing and participating in a mature biofilm are numerous, including protection from environmental stresses (Pemmaraju et al., C. tropicalis have also been shown to be more resistant to antifungal diffusion than C. albicans biofilms (Al-Fattani and Douglas, C. albicans in Ca+Ct biofilms. Nonetheless, the outcome of interactions between C. albicans and NACS is highly dependent upon growth environment. A study by Kirkpatrick et al. (C. dubliniensis is able to tolerate rigorous competitive pressure from C. albicans in a catheter biofilm model, while C. albicans dominates in planktonic co-culture (Kirkpatrick et al., C. tropicalis and C. albicans was reported in another study in which reduction of C. albicans filamentation and reduced expression of C. albicans virulence genes was found in the presence of C. tropicalis (de Barros et al., C. tropicalis filamentation was suppressed by C. albicans. However, in spite of this competition, C. tropicalis still garners an advantage in biofilm growth from association with C. albicans.Our results suggest that there may be a benefit for both species in a co-colonized environment; the rapid establishment of a mature biofilm that occurs in Ca+Cd co-culture indicates a potential competitive advantage against other microbiota k et al.  discoverCandida modify behavior and fitness in biofilms. We conclude that dual culture biofilm of C. albicans and C. tropicalis or C. dubliniensis offers a growth advantage for both NACS. We have identified the normalized rate of substrate detachment as the primary determinant for both mono- and dual-species biofilm formation. In addition, we found that both C. tropicalis and C. dubliniensis, in dual biofilm growth with C. albicans, show a preference for inter-species cell-cell associations so that populations of NACS are additionally maintained within mature dual species biofilms. Our results indicate that growth and maintenance, but not initial establishment, of dual-species biofilms are supported by interspecies adherence. Therefore, among filamenting NACS, dual species growth benefits both species in biofilm development.This study offers insight into the way that inter-species interactions of The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.RP, AM, and ME conceived and designed the experiments. RP, AM, HN, and ME wrote the manuscript. RP, AM, and HN conducted the experiments and analyzed the flow biofilm data. RP conducted the other experiments.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.