Plasmodiophora bassicae isolates at the 3′-end. After re-sequencing 2069 bp large subunit (LSU) sequences at the 3′-end, the authors confirm that in the main text of our original paper [The authors of Laila et al.  would lial paper : Figuresal paper : Table SP. bassicae isolates and submitted the revised sequences to NCBI. The up-to-date accession numbers of rDNA sequences are given in Here, we have corrected rDNA sequences of 11 Korean P. brassicae of Korean isolates now have over 98% sequence identity with reference sequence KX011115. Korean isolates Yeoncheon, Daejon, Haenam2, Gangneung2, Haenam1 and Geumsan have identical rDNA sequences. These six isolates have less than 1% sequence dissimilarities with another five isolates.The updated rDNA sequences of P. brassicae in the same cluster as shown in Heteromita globosa. The phylogenetic tree grouped rDNA sequences of both Korean and reference isolates, reported by Schwelm et al. , of P. bP. bassicae isolates. There were no complete published rDNA sequences except AB526843 when we started sequencing and obtained rDNA sequences of selected Korean P. bassicae isolates. In a recent article, it was reported that rDNA sequences of NGY isolate were chimeric in nature between P. brassicae and non-parasitic Glissomonadida [We previously used the rDNA sequences of the Japanese NGY isolate, accession number AB526843 [monadida . P. bassicae isolates that mismatched with recently reported sequences (We have now, therefore, designed primers based on the following two sequences, KX011115 and KX011135, published by Schwelm et al.  to correequences . The proP. brassicae isolates Yeoncheon, Daejon, Haenam2, Gangneung2, Haenam1 and Geumsan had identical rDNA sequences. The other five isolates had less than 1% sequence dissimilarities with those six isolates. The rDNA sequences of 11 Korean P. brassicae including LSU had 98% identity with the reference rDNA sequences of European isolates KX011115 and KX011135. In conclusion, Among 11 Korean


Leishmania (L.) parasites depends on polyamine availability, which can be generated by the L-arginine catabolism and the enzymatic activity of arginase (ARG) of the parasites and of the mammalian hosts. In the present study, we characterized and compared the arginase (arg) genes from pathogenic L. major and L. tropica and from non-pathogenic L. tarentolae. We quantified the level of the ARG activity in promastigotes and macrophages infected with pathogenic L. major and L. tropica and non-pathogenic L. tarentolae amastigotes. The ARG's amino acid sequences of the pathogenic and non-pathogenic Leishmania demonstrated virtually 98.6% and 88% identities with the reference L. major Friedlin ARG. Higher ARG activity was observed in all pathogenic promastigotes as compared to non-pathogenic L. tarentolae. In vitro infection of human macrophage cell line (THP1) with pathogenic and non-pathogenic Leishmania spp. resulted in increased ARG activities in the infected macrophages. The ARG activities present in vivo were assessed in susceptible BALB/c and resistant C57BL/6 mice infected with L. major, L. tropica and L. tarentolae. We demonstrated that during the development of the infection, ARG is induced in both strains of mice infected with pathogenic Leishmania. However, in L. major infected BALB/c mice, the induction of ARG and parasite load increased simultaneously according to the time course of infection, whereas in C57BL/6 mice, the enzyme is upregulated solely during the period of footpad swelling. In L. tropica infected mice, the footpads' swellings were slow to develop and demonstrated minimal cutaneous pathology and ARG activity. In contrast, ARG activity was undetectable in mice inoculated with the non-pathogenic L. tarentolae. Our data suggest that infection by Leishmania parasites can increase ARG activity of the host and provides essential polyamines for parasite salvage and its replication. Moreover, the ARG of Leishmania is vital for parasite proliferation and required for infection in mice. ARG activity can be used as one of the main marker of the disease severity.Proliferation of  Leishmania spp. and the pathogenesis of cutaneous leishmaniasis (CL) in the mouse models. Leishmania parasites infect macrophages that can be activated via two major pathways resulting in classical and alternative activated macrophages which metabolize L-arginine differentially. Classically activated macrophages upregulate the enzyme inducible NO synthase (iNOS) and alternatively activated macrophages upregulate the arginase (ARG). ARG hydrolyzes the conversion of its substrate (L-arginine) to L-ornithine and urea. L-ornithine is a key intermediate substrate for the biosynthesis of polyamines, which are crucial nutrients for cellular processes such as growth, differentiation and proliferation of host cells and Leishmania parasites. Leishmania parasites may partly activate ARGs and inactivate the NO production by the host cells and enhance parasite survival via depletion of the iNOS substrate (L-arginine) and reduce NO levels. In this study, we tested the activity of this enzyme in the promastigote forms of the pathogenic L. major and L. tropica, and non-pathogenic L. tarentolae in vitro and assessed the differences between the levels of ARG activity in THP1 cells infected with different Leishmania spp. Moreover, we investigated the relationship between excessive ARG activity and lesion development in susceptible BALB/c and resistant C57BL/6 mice infected with virulent L. major, L. tropica and non-virulent L. tarentolae parasites and its impact on parasite burden during the development of infection. Our results show that ARG is highly upregulated in all pathogenic promastigotes as compared to non-pathogenic L. tarentolae and had a negative correlation between production of ARG and NO. We showed that during the development of the infection in susceptible BALB/c and resistant C57BL/6 mice, ARG is induced in both strains of mice infected with pathogenic Leishmania but not in non-pathogenic counterparts. These results suggest that ARG activity of Leishmania is essential for parasite survival in vitro and in vivo that directly regulates its growth and replication inside the host cells; therefore, it can be used as a significant marker of disease severity in CL and possible tools for drug designing.Over the past decades, there has been a significant improvement in the understanding of immune responses against infection with pathogenic  Leishmania, which are present throughout 98 countries   –42. Absoin vitro and in vivo evaluation, t-test or one-way ANOVA was applied to analyze ARG assay, NO production and footpad swelling in the current study. The p-values less than 0.05  were considered statistically significant. Data were presented as mean ± standard deviation (SD).Statistical analysis was done using Graph-Pad Prism 6.0 for Windows . For Leishmania parasite species and the non-pathogenic L. tarentolae revealed an open reading frame (ORF) consisting of 990 and 987 nucleotides respectively, which encoded 330 amino acids (Leishmania including: L. major Friedlin (XM_003722493.1), L. infantum JPCM5 (XM_001468931.1) and L. mexicana (MNYC/BZ/62/M379) , 98%, 97% and 89% to L. infantum JPCM5 (XM_001468931.1) and 96%, 95% and 88% to L. mexicana ARG, respectively. The L. major and L. tropica ARG proteins are 43% and 38% identical to the two isoforms of human (Homo sapiens) ARG enzymes, ARG 1 and ARG 2, respectively. L. tarentolae ARG is 41% and 38% identical to the ARG 1 and ARG 2, respectively. Furthermore, the L. major and L. tropica ARG proteins are 42% and 39% identical to the two isoforms of mice (Mus Musculus) ARG enzymes, ARG 1 and ARG 2, respectively. L. tarentolae ARG is 40% and 39% identical to the ARG 1 and ARG 2, respectively  in Leishmania instead of histidine (228) in human. It was suggested that these differences between the two ARGs in the vicinity of the active sites or catalytic centers are not conserved and can be exploited in parasites ARG inhibitors. Active sites or catalytic centers of both are vital for protein function and processing [DNA sequence analysis of the two pathogenic no acids . The seq62/M379) . The mulectively . Althougocessing , 43.L. major Friedlin, L. infantum JPCM5 and L. mexicana equivalents, differing only one amino acid in sequenced L. major and L. tropica as well as two amino acids in L. tarentolae proximal to the COOH terminus. A unique and notable feature of Leishmania spp. ARG protein is the existence of a glycosomal  targeting signal SKL and AKL, a tripeptide located in the deduced COOH terminus [arg of Leishmania species [L. major, L. tropica and L. tarentolae arg genes encode this COOH terminus tripeptide that mediate the translocation of the protein to the glycosome. Importantly, the ARG of L. major and L. tropica, two Old World Leishmania species, and also L. tarentolae, encode an AKL  COOH terminus tri-peptide, while the ARG from New World Leishmania spp. like L. amazonensis and L. mexicana, encompass an SKL (serine-lysine-leucin) COOH terminus tripeptide. Both tri-peptides (AKL and SKL) are the most typical topogenic signature for targeting protein to the glycosome in Leishmania spp.  method [Leishmania parasites, two Homo sapiens and two Mus musculus arg genes is presented in Leishmania arg genes, L. major and L. tropica were found to be more similar to the reference L. major Friedlin strain and have similar length in the phylogenetic tree and all nodes supported by high bootstrap values. Our data show that L. tarentolae is less similar to other Leishmania parasites. ARG of distant organisms such as H. sapiens and M. musculus was partly different from Leishmania parasites ARG (30–40%), and according to Leishmania parasites were uncertain because their nodes presented low bootstrap values.Gene-by-gene alignment, comparison and analysis was done for all sequenced ) method . The phyLeishmania spp. expresses their own ARG enzyme [arg gene on both alleles of the parasite was fatal and became auxotrophic for polyamines [Leishmania parasites we investigated in this study. It is crucial to analyze the exact transcript levels of ARG in promastigotes; therefore, we quantified the ARG transcript levels of promastigotes at the logarithmic (Leishmania. As shown in L. tropica expressed the highest transcript levels of ARG in the both logarithmic and stationary growth phases as compared with L. major and L. tarentolae (p<0.05). Although significant ARG transcript levels were detected in the pathogenic Leishmania species, it is low in the non-pathogenic L. tarentolae (p<0.05). We also measured the enzymatic activity of ARG in both pathogenic and non-pathogenic Leishmania promastigotes in the logarithmic and stationary growth phases. Our experiments revealed that significant ARG activity was detected in the both pathogenic and non-pathogenic Leishmania promastigotes (L. tropica and L. major as compared to non-pathogenic L. tarentolae (p<0.05). In the logarithmic phase, the highest and lowest ARG activities were expressed by L. tropica (638.63 mU/mg protein) and L. tarentolae (197.45 mU/mg protein) respectively. In contrast, in the stationary phase L. tropica promastigotes exhibited the highest specific activity of ARG (420.47 mU/mg protein) and L. major the lowest (145.34 mU/mg protein) (p<0.05).It has been previously shown that G enzyme , 23, 48.lyamines , 43, 47.arithmic  and statarithmic  growth pstigotes . As showstigotes  of the pstigotes , and theLeishmania infection outcomes: the induction of ARG results in the catabolism of L-arginine into urea and ornithine; the latter is further catabolized into polyamines that are required for parasite growth while iNOS oxidizes L-arginine in a two steps process into NO, a metabolite responsible for Leishmania parasite clearance. Both enzymes are induced in macrophages and share L-arginine as a substrate and are crucial indicators for the disease outcome [Leishmania infected THP1. Our findings are in agreement with other studies elucidating that ARG is vital for growth and proliferation of Leishmania promastigotes but not intracellular amastigotes [Leishmania infected macrophages [Leishmania produced lower levels of NO as compared to non-infected THP1 cells (p<0.05). As expected, our data show a direct relationship between ARG activity and NO levels, when ARG activity was increased in infected macrophages, the NO production was decreased   ; althougly lower .L. tropica infection in the draining LNs of BALB/c and C57BL/6 mice at distinct time post infection. As shown in L. major infection, L. tropica infection progressed with same pattern as in C57BL/6 mice, with a peak parasite load attain after 10 weeks . Importantly, the L. tropica did not grow uncontrolled in the LNs of BALB/c; the growth of L. tropica in LN of C57BL/6 mice, progressed very slowly and attained a peak number of almost 70 per LN  at the site of pathology at different times post infection to better understand the infection process. We unexpectedly found that, in C57BL/6 mice parasite load was higher than BALB/c whereas there was no significant difference in ARG activities in infected mice .L. tarentolae in draining LNs of BALB/c and C57BL/6 mice. As shown in L. tarentolae infection in both strains of mice. Similar to L. tropica infections, in C57BL/6 mice the ARG activities in both naive control and test groups were considerably higher than in BALB/c mice.Similarly, we checked the ARG activity due to non-pathogenic L. major infected LNs elucidated a strong positive correlation between parasite number and ARG activities in infected BALB/c mice 10 weeks post infection   (2 = 0.8697)  , whereas 0.9974) . There w 0.9974) . Similar 0.9573) . Data sh 0.8697) . Our res in mice , 56. As Leishmania ARG are the C-terminal tri-peptides AKL and SKL, respectively. They have now been genetically confirmed as the peroxisomal targeting signal (PTS-1) that serves as the topogenic signal for targeting ARG to the unique Leishmania glycosome [Leishmania glycosome, a peroxisome like organelle, was found only in kinetoplastids, and the presence of this organelle shows one of the major differences among the Leishmania parasites and the host. da Silva et al have shown that the appropriate localization of Leishmania ARG in this unique organelle (glycosome) is a key factor for ARG activity and proper infectivity. In addition, they showed that dislocated ARG in Leishmania interrupted its infectivity. Therefore, glycosome is vital for enzyme activity in Leishmania parasites [Leishmania PTS-1 showed no equivalent PTS-1 sequence among 31 ARG family members [Leishmania spp. have this unique glycosomal signature and share a high level of homology by reference genes amino acid residues in those areas critical to enzymatic function. The non-pathogenic L. tarentolae ARG homologue shows lower sequence homology to the closest orthologues in L. major. There are some differences that may be due to the fact that L. tarentolae is a Leishmania-like parasite of lizards that belong to the genus Sauroleishmania and partly distinct from Leishmania genus.The most remarkable structural signature of the Old and New World lycosome . Leishmaarasites . Our seq members , 57–59. Leishmania express their own arg gene, and have ARG activity. According to previous studies on ARG deleted mutants, it is a vital gene in Leishmania parasites [Leishmania species, the ARG activity at both enzymatic and transcriptomic levels in the logarithmic growth phase of the parasites is higher than that in the stationary one. Although the exact mechanisms of higher ARG activity in the logarithmic growth phase is unknown, the possible explanation can be the higher replication rate of the parasites and their requirement for particular essential needs at this stage. Also, ARG, is an essential enzyme in Leishmania promastigotes [Leishmania is ready to infect the host cells and may need less metabolite in compare to logarithmic phase. Thus, it is interesting to characterize all key biochemical and immunological pathways such as ARG activity in the pathogenic and non-pathogenic Leishmania species at different stages.Our data demonstrated that all pathogenic and non-pathogenic arasites , 56, 60.stigotes , 47, theLeishmania infected MQs, the activity of ARG were increased over uninfected; therefore, parasite infection indirectly upregulated the ARG activity at in vitro condition. Leishmania parasites may partly activate ARGs, inactivate NO production by host cells, and enhance parasite survival via local depletion of the iNOS substrate L-arginine and reduced NO levels [Leishmania and they may also serve as long-term host cells that facilitate the replication of the parasites [Leishmania proliferation inside macrophages was remarkably diminished in the absence of Leishmania ARG and therefore, ARG deficient parasites were unable to induce host ARG that are in agreement with our study [Leishmania species, no significant differences in the activity of ARG were found between the pathogenic species and non-pathogenic L. tarentolae. This suggests that ARG is essential for all species of Leishmania and they need ARG to survive and multiply inside host cells [This study showed that in O levels . High ARarasites . One preur study . Interesst cells , 48.Leishmania parasites are inside the phagolysosome in infected macrophages, their ARG activity is compartmentalized in their glycosome. Suboptimally activated macrophages due to low concentrations of IFN-γ, iNOS and ARG stay partly inactivated, allowing Leishmania parasites to take up and utilize accessible arginine for their multiplication and proliferation [A direct correlation was seen between ARG activities and NO production. Since feration , 23, 61.L. major leads to non-healing infection and increased parasite load in the LN as well as significantly enhanced ARG activity at the site of infection and in the draining LN. In contrast, infection of genetically resistant C57BL/6 mice with pathogenic L. major resulted in healed lesions with low ARG activity and reduced parasite load in the LN. Studies have shown that the ARG activity of both host and the parasites is important for the replication of L. major in in vivo [Leishmania replication; therefore, it resulted in decreased ARG activity [Leishmania parasites in resistant C57BL/6 and susceptible BALB/c mice infected with L. major have been attributed to polarized Th1/Th2 responses [Leishmania than do resistance mice macrophages. Therefore, in these milieu susceptible mice permits the parasites to express their antigens effectively to reach at the threshold of inhibiting effective cellular immune responses [We report here that infection of susceptible BALB/c mice with pathogenic  in vivo , 62. In activity . Althougesponses , 63, theesponses . Macrophesponses .L. tropica are scarce; therefore, this is the first report showing the ARG activity in pathogenic L. tropica promastigotes that were used to initiate infections in BALB/c and C57BL/6 mice. In both strains of mice, the lesions grew non-progressively and non-ulceratively, parasite replication was slow to develop and remained low. Although the level of ARG activity in both mice were lower than in L. major infected mice, ARG activity in BALB/c mice were higher than C57BL/6 mice. These results are in agreement with those obtained by Anderson et al., where they showed that dermal lesions in the ears of BALB/c and C57BL/6 mice were slow to develop and displayed minimal skin pathology [L. tropica infections develop slower as compared to that of L. major [Published data using mouse models of leishmaniasis caused by athology . A potenL. major .L. tarentolae is a non-pathogenic parasite that does not cause pathology in humans nor in immunodeficient mice and its non-pathogenicity has been recently proven in several studies [L. tarentolae did not induce any swelling in either BALB/c or C57BL/6 mice. Although L. tarentolae can enter into macrophages, they could not induce ARG activity at the infection site and the draining LNs. Interestingly; we found that in LNs of C57BL/6 mice (about 400 parasites/LN) more surviving L. tarentolae than in LNs of BALB/c mice (about 20 parasites/LN). However, there was no significant difference in ARG activities in infected mice, a low number of L. tarentolae parasites replicated in the draining LN of C57BL/6 mice and the parasite numbers reached to background levels at the end of the experiment. Although L. tarentolae can enter macrophages and differentiate into intracellular amastigote forms, there are no clear evidences for their effective replication inside the macrophages [ studies , 64, 65.rophages , 65. TheLeishmania species/strains. Another interesting experiment would be to overexpress ARG in different Leishmania species/strains and test the effects on infectivity of the disease. More studies are required to clarify the contribution of host and parasite ARG activity to the course of cutaneous leishmaniasis [Further research is required to assess whether ARG activity is a pivotal element in the outcome of leishmaniasis in humans, especially in the acute and chronic lesions , and it maniasis . One of Leishmania-derived ARG is a potentially vital enzyme in promastigotes that regulates parasite growth. Furthermore, Leishmania promote their own existence via manipulating the host’s intracellular signaling pathways to repress different enzymatic processes. Therefore, we consider that our work may open new perspectives for the study of ARG in both mammalian host as a favorable environment for parasite growth and in Leishmania parasites as a potential target for novel therapeutic and vaccine research. Thus, determining the main role of ARG in Leishmania provides valuable knowledge that directs to a better understanding of host/parasite interactions and to identification of the efficient targets for vaccination and importantly for their treatments.In summary, S1 FigLeishmania species were aligned. The C-terminal tripeptide (SKL and AKL) signature for glycosomal localization in Leishmania parasites is highlighted with the rectangle box. The alignment was applied by using Clustal W and BioEdit Sequence Alignment Editor Softwares. Stars indicate fully conserved residues; colons indicate highly conserved residues; and periods indicate weakly conserved residues. The sequences analysed are: The retrieved GenBank nucleotide sequences analysed are: Homo sapiens ARG 1 (NP_001231367.1), Homo sapiens ARG 2 (NP_001163.1), Mus musculus ARG 1 (U51805.1), Mus musculus ARG 2 (U90886.1), L. panamensis MHOM/PA/94/PSC-1 (XM_010704259), L. mexicana MNYC/BZ/62/M379 (AY386701.1), L. major Friedlin LMJF_35_1480 (XM_003722493.1), L. infantum JPCM5 (XM_001468931.1), L. donovani LDBPK_351490 (XM_003864686.1), L. braziliensis MHOM/BR/75/M2904 (XM_001568200.1) and L. amazonensis MHOM/BR/1973/M2269 (AF038409.2). Sequenced Leishmania arg genes in the current study are shown in black doted as following: L. tropica MOHM/IR/09/Khamesipour-Mashhad (KU641753), L. major MRHO/IR/75/ER (KU641750) and L. tarentolae Tar II ATCC30267 (KU641752).ARG protein sequences from human, mice and various (TIF)Click here for additional data file.S2 FigHomo sapiens ARG 1 (NP_001231367.1), Homo sapiens ARG 2 (NP_001163.1), Mus musculus ARG 1 (U51805.1), Mus musculus ARG 2 (U90886.1), L. panamensis MHOM/PA/94/PSC-1 (XM_010704259), L. mexicana MNYC/BZ/62/M379 (AY386701.1), L. major Friedlin LMJF_35_1480 (XM_003722493.1), L. infantum JPCM5 (XM_001468931.1), L. donovani LDBPK_351490 (XM_003864686.1), L. braziliensis MHOM/BR/75/M2904 (XM_001568200.1) and L. amazonensis MHOM/BR/1973/M2269 (AF038409.2). Sequenced Leishmania arg genes in the current study are shown in black doted as following: L. tropica MOHM/IR/09/Khamesipour-Mashhad (KU641753), L. major MRHO/IR/75/ER (KU641750) and L. tarentolae Tar II ATCC30267 (KU641752).The written numbers next to the each branch are computed from bootstrap values of 500 replicates. The evolutionary tree was created by user-friendly MEGA 5.05. The retrieved GenBank nucleotide sequences analysed are: (TIF)Click here for additional data file.


Kineosporia sp. strain A_224. Analysis of the predicted genes may improve our knowledge of its role in ochrous formations in natural and technical water systems. This is the first public genome sequence of a Kineosporia aurantiaca strain.We report here the draft genome sequence of the neutrophilic iron-precipitating  Kineosporiaceae was isolated from the floodplain area in the Lower Oder Valley National Park, Germany. Based on 16S rRNA gene sequence analysis, the family Kineosporiaceae , and EzBioCloud (K. mesophila strain YIM 65293 (FJ214362).A medium . Dark, bA medium . Total gA medium . Reads wA medium , followesion 3.0 , and funsion 3.0  revealedBioCloud  searchesMWLN00000000. The version described in this paper is the first version, MWLN01000000.This genome shotgun project has been deposited in GenBank under the accession number


Trypanosoma spp. and Leishmania spp. are a major source of infectious disease in humans and domestic animals worldwide. Fundamental to the host-parasite interactions of these potent pathogens are their cell surfaces, which are highly decorated with glycosylated proteins and other macromolecules. Trypanosomatid genomes contain large multi-copy gene families encoding UDP-dependent glycosyltransferases (UGTs), the primary role of which is cell-surface decoration. Here we report a phylogenetic analysis of UGTs from diverse trypanosomatid genomes, the aim of which was to understand the origin and evolution of their diversity.Trypanosomatid parasites such as Bodo saltans that likely represents the ancestral state of this gene family. The phylogeny of parasite-specific genes shows that UGTs repertoire in Leishmaniinae and salivarian trypanosomes has expanded independently and with distinct evolutionary dynamics. In the former, the ancestral UGT repertoire was organised in a tandem array from which sporadic transpositions to telomeric regions occurred, allowing expansion most likely through telomeric exchange. In the latter, the ancestral UGT repertoire was comprised of seven subtelomeric lineages, two of which have greatly expanded potentially by gene transposition between these dynamic regions of the genome.By combining phylogenetics with analyses of recombination, and selection, we compared UGT repertoire, genomic context and sequence evolution across 19 trypanosomatids. We identified a UGT lineage present in stercorarian trypanosomes and a free-living kinetoplastid Leishmania.The phylogeny of UGTs confirms that they represent a substantial parasite-specific innovation, which has diversified independently in the distinct trypanosomatid lineages. Nonetheless, developmental regulation has been a strong driver of UGTs diversification in both African trypanosomes and The online version of this article (10.1186/s12862-018-1149-6) contains supplementary material, which is available to authorized users. Leishmania spp., which cause various kinds of leishmaniasis; stercorarian trypanosomes such as Trypanosoma cruzi, the cause of Chagas disease in central and south America; and salivarian trypanosomes such as Trypanosoma brucei, the cause of African trypanosomiasis in humans and animals, . Collectively, these vector-borne diseases have a significant impact on human and animal health, and are a profound constraint on the socio-economic development of low and middle-income countries.Trypanosomatid parasites are the causes of several neglected tropical diseases worldwide that put 500 million people and over 60 million cattle at risk of infection . TrypanoGlossina spp.) and extracellular bloodstream-forms in the mammalian host. T. cruzi infects a wide range of mammals and is transmitted by the bite of triatomine bugs. Leishmania spp. alternate between a motile, promastigote form in a sand-fly vector, and an intracellular amastigote form in their mammalian host. Besides these, and many other dixenic parasites, there are multiple genera of monoxenic trypanosomatids that parasitize insects and are transmitted through the faecal-oral route, such as Crithidia, Leptomonas and Lotmaria  that compose the parasite dense glycocalyx, using UDP-galactose as the glycosyl donor [Leishmania papatasi stage-specific adhesion potential is provided by LPG, a glycoconjugate interacting with lectin receptors in the epithelium of the sandfly midgut [In yl donor . Simultayl donor . Whilst y midgut . The galy midgut .T. brucei in both insect and mammal forms [In African trypanosomes, UGTs are involved in the synthesis of complex poly-N-acetyllactosamie-containing type N-linked and GPI-linked glycans. N-linked glycans can have various functions: on VSG, they are predicted to assist the protection of invariant surface antigens by filling the spaces between VSGs ; on the al forms , 31, UGTBodo saltans [The publication of genomes for most trypanosomatid species –4, 32–39 saltans  providesThree main reasons make UGTs sensible study targets: i) Despite being a multi-copy gene family with distinct repertoires across species and important roles in pathogenesis, their diversity across the genus is poorly understood; ii) The understanding of its diversity may elucidate phenotypic differences in disease progression; and iii) Through genomic comparison we can identify shared and species-specific loci, as well as stage-specific isoforms, to expedite the search for suitable drug and transmission targets.Bodo saltans with particular emphasis on African trypanosomes and Leishmania. We aim to identify monophyletic free-living  and parasitic (trypanosomatid) UGTs to understand more about their ancestral form and the origin of family expansion. In this process, we investigate orthology across parasites to know whether UGT expansion was independent in distinct parasites, and understand the role of recombination among paralogs and of selection in gene divergence. Finally, we interpret those results in the context of available gene expression and functional studies, whilst searching for evidence of functional differentiation, since non-redundant paralogs under strong negative selection could offer targets for functional studies and interventions.Here we describe the phylogeny and comparative genomics of UGT genes in trypanosomatids and Trypanosoma cruzi CL Brenner Esmeraldo-like, T. rangeli SC58, T. grayi ANR4, T. brucei TREU927, T. congolense IL3000, T. vivax Y486, Leishmania major Friedlin, L. infantum JPCM5, L. mexicana MHOM/GT/2001/U1103, L. tarantolae Parrot-Tarll, L. enriettii LEM3045, L. braziliensis MHOM/BR/75/M2904, Leptomonas pyrrhocoris H10, and Crithidia fasciculata Cf-Cl hosted by TritrypDB v.28 (http://tritrypdb.org) [Bodo saltans hosted by the GeneDB website (http://genedb.org) [; and Angomonas deanei and Strigomonas culicis hosted by Ensembl Protists v.31 (http://protists.ensembl.org). Additionally, a sequence similarity search with tBLASTn using T. brucei, L. major and B. saltans UGTs as query was performed to identify relevant genes annotated as hypothetical.Annotated UGT sequences were obtained from genome sequences of pdb.org) ; Bodo saedb.org) ; and AngCrithidia acanthocephali and Lotmaria passim unannotated genomes were retrieved from NCBI (http://www.ncbi.nlm.nih.gov/genome). These were inspected for UGTs by sequence similarity search with tBLASTn using its closest relative, C. fasciculata, UGTs as the query. Identified putative UGTs were named L. passim1–4 and C. acanthocephali1–4.To expand the sample repertoire of monoxenic species, the genome sequences from Trypanosoma gambiense DAL972 and Trypanosoma evansi (hosted by TritrypDB v.28 (http://tritrypdb.org) [Trypanosoma equiperdum (hosted by NCBI (http://www.ncbi.nlm.nih.gov/genome)) were also inspected. However, as they present the same repertoire as T. brucei TREU 927, the latter was used as a representative of the Trypanozoon subgenus.UGT Sequences from pdb.org) ), and frThe presence of the conserved UDP catalytic domain previously described (DXD) in the sequences was a requirement for the inclusion in this study .http://www.mbio.ncsu.edu/BioEdit/bioedit.html) and back translated, producing a nucleotide alignment of 1005 nucleotides. Subsequently, aligned nucleotide sequences were translated again, resulting in a protein alignment of 335 amino acids around the catalytic domain, after trimming non-conserved regions. This corresponded to 23–93% of the full glycosyltransferase proteins sequence, due to Leishmania spp. having a large specific insertion. When analyzed separately, the African trypanosome alignment was 305 amino acids long, while the Leishmaniinae alignment was 824 amino acids long.Translated nucleotide sequences were aligned with ClustalW  using BiThe UGT phylogeny was estimated from protein sequence alignments with maximum likelihood (ML) under a WAG+Γ substitution model  using PHLeishmaniinae were estimated from nucleotide sequence alignments with ML using PHYML v3.0 [The UGT phylogenies of African trypanosomes and YML v3.0 , BI usinYML v3.0 , 55, andYML v3.0 , and froYML v3.0 , BI usinYML v3.0 , 55, andYML v3.0 .c). PHYML protein trees were estimated with WAG+Γ (African trypanosomes) [Leishmaniinae) [Leishmaniinae) [Optimal substitution models for PHYML amino acid trees were found with the Smart Model Selection option in PHYML, using the Akaike Information Criterion (AICnosomes)  or LG + aniinae) . Optimalaniinae) . IQTree aniinae)  or VT + aniinae) . Nucleotaniinae)  with 500The BI trees were estimated with gamma rates function in MrBayes and four Markov chain Monte Carlo chains run in parallel over 2,500,000 generations, with a burnin of 5000. The nucleotide BI trees were estimated with default parameters whereas the protein BI trees were estimated with a fixed WAG+Γ model. Posterior probabilities of each node were used to assess accuracy of BI trees.Nucleotide NJ trees were estimated using logdet genetic distances to correct base composition bias  using MEL. major, L. infantum, L. mexicana, T. brucei, and T. congolense. For Leishmania, SCG and SCGR subfamilies were separately analyzed. For African trypanosomes, each lineage was separately analyzed. Four sequences were randomly selected for each species and subject to different tests. In Leishmania, a negative control comprised of four sequences known not to recombine  were included. In African trypanosomes, the negative control was comprised of all genes from lineages 2–5.Evidence for recombination was investigated in c information criterion. The KH test was applied to test for rate heterogeneity to prevent false positives arising from significant topological incongruences rather than recombination. These tests informed on the likelihood of recombination affecting sequence evolution. The breakpoint(s) identified with GARD were used to split the sequences into non-recombinant parts before subsequent analyses of selection to prevent false positives due to recombination.Recombination probability was detected with the pair-wise homoplasy index (PHI)  as part dN/dS ratios [dN/dS ratios at individual sites based on a pre-defined distribution; Partitioning Approach for Robust Inference of Selection (PARRIS) [dN/dS ratio based on Bayesian Inference using a MCMC routine [To evaluate whether positive selection was affecting sequence evolution, full sequences where recombination was unlikely and non-recombinant partial sequences were subject to six site-level selection tests: Single Likelihood Ancestor Counting (SLAC) to perform ancestral reconstruction; Fixed Effects Likelihood (FEL) to directly estimate S ratios ; Random (PARRIS)  to test  routine ; and the routine .p-value < 0.05 and posterior probability > 0.9. For sites to be considered under positive selection, support by 4 out of 5 tests was required. Unless specified, all programs were hosted at the DataMonkey server (http://datamonkey.org).Significance thresholds for recombination were B. saltans, 2 from Angomonas deanei, 1 from Strigomonas culicis, 82 from stercorarian trypanosomes (3 species), 71 from African trypanosomes (3 species), and 70 from Leishmaniinae (10 species). It is immediately clear from this phylogeny that trypanosomatids have greatly expanded their UGT repertoires relative to the free-living B. saltans . Furthermore, UGT repertoires have been elaborated to different extents in trypanosomatid genera, e.g. T. brucei: L. major ratio of 2:1).We estimated a UGT maximum likelihood phylogeny Fig.  from a 3B. saltans sequences and rare orthologs from stercorarian species  (1); a clade of Leishmaniinae sequences (2); two clades of African trypanosomes (3). Most stercorarian trypanosome sequences clustered together  but without adequate node support (4). The lack of species diversity hampers orthology analysis and thus we have not examined stercorarian sequences further in this study.The tree topology broadly reflects the major trypanosomatid lineages and contains four main features, , one T. cruzi (TcCLB.503487.50), and one T. rangeli (TRSC58_00816), all close in length (352 to 495 amino acids). The B. saltans sequences share 31% overall identity between each other and 34–36% identity with T. cruzi, T. rangeli and T. grayi (Tgr.1587.1000). The latter was not included in the phylogeny due to its short length (90 amino acids). The absence of this lineage of UGTs in Leishmaniinae and African trypanosomes suggests post-speciation gene loss. Transcriptomic data from genomic microarrays show TcCLB.503487.50 is constitutively expressed, being the most abundant in amastigotes and the least in epimastigotes [T. rangeli and T. grayi genomes.The ancestral lineage is composed of four genes retaining orthology: two stigotes . The genEuglena gracilis transcriptome [Trypanoplasma borreli [Phytomonas sp. isolates EM1 and Hart1 [Paratrypanosoma confusum [Naegleria fowleri [A search for similar sequences in criptome  and Tryp borreli , Phytomond Hart1 , Paratryconfusum  and Naeg fowleri  genomes Leishmaniinae subfamily so we investigated it further by building a Leishmaniinae-only phylogeny based on a longer multiple sequence alignment ; these are the side chain galactose receptors (SCGR) [L. braziliensis (e.g. Lbr.02.0250). This suggests slow but on-going concerted evolution arising from rapid gene duplication and resulting in the loss of orthology over time. One possible exception to this prevailing pattern is Lbr.02.0260 and its orthologs (denoted by “iv”). Although orthology between L. major and L. braziliensis is mostly absent within the array, this is an example of a lineage present in all Leishmania species (LmjF.02.0230), whose sequences show a unique change in the catalytic domain from DDD to YDD, hinting functional differentiation.All s (SCGR) , 73. TheLeishmania-specific single-copy lineage located in chromosome 14, which has been previously identified in L. major as side-chain galactose ligand (SCGL) [Leishmania ancestor. The absence of a gene at this particular locus in L. braziliensis and L. mexicana indicates loss in these species  . The phyLeishmaniinae sub-family, which comprises the Leishmania-specific side-chain galactose (SCG) genes [Leishmania ancestor and has since expanded to other chromosomes perhaps by telomeric exchange, providing strong evidence for concerted evolution.Unlike the two previous lineages, the last lineage of UGTs in the G) genes , has a dL. infantum [L. major [L. mexicana [L. infantum [L. major showed differential expression at the amastigote stage of LmjF.02.0230 only, but all seven SCG genes and LmjF.02.0190 seem to be more abundant in the amastigote stage L. infantinfantum  reveal tte stage  Leishmania species; SCGR generally constitutively expressed; and SCGL present in very low abundances. This suggests that developmental regulation accounts for some degree of gene differentiation.In summary, SCG genes seem generally more abundant in the amastigote stage of L. major SCG genes to be under recombination, with GARD identifying one significant breakpoint at nucleotide 489. Trees inferred from GARD were fed into six tests for selection. Only PAMLx and FUBAR found evidence for positive selection, but not significant compared to the negative control. Selection tests for sequences where GARD did not predict significant breakpoints were not consistent, but no sites under positive selection were identified in any of the sequence collections by more than 3 out of 6 tests  and a thioestherase-like superfamily protein (Tb927.7.330), but the T. vivax contig containing the former does not span the UGT gene. Similarly, lineage 6 locus seems conserved in all three species, being delimited by a methyltransferase domain containing protein (Tb927.10.12270) and a helicase-like protein (Tb927.10.310), although sequence gaps in T. congolense assembly preclude a final decision.The phylogeny of UGTs in African trypanosomes shows seven lineages present in the common ancestor  and 4 (Tb927.7.300) being differentially expressed in the bloodstream form, and lineages 5 (Tb927.10.12290) and 7 (Tb927.2.3370 and Tb927.4.5240 to Tb927.4.5290) being preferentially expressed in the procyclic form of the life cycle [The analysis of the available expression data at the proteomic level reveals some developmental regulation of fe cycle , 42.At the transcriptomic level, the higher abundance in bloodstream forms of Tb927.3.5660 and Tb927.7.300 is already significant, but not of Tb927.10.12290 in procyclics. Transcriptomic data also shows Tb927.5.2760 as differentially regulated in bloodstream forms. Tb927.2.3370 and Tb927.4.5240 seem to be constitutively transcribed, whilst Tb927.9.800 and Tb927.4.5790 are preferentially transcribed in BSF .Available ribosomal profiling studies agree with proteomic data results and suggest higher abundance at the bloodstream form stage of Tb927.8.8090, Tb927.8.8100, Tb927.4.4290, Tb927.4.4250 and Tb927.4.4270   in bloodstream forms compared to metacyclics (maximum fold change of 1.42), as well as of TvY486_0403900 (lineage 4) when compared to epimastigotes (maximum fold change of 11.02) [Expression data for f 11.02) . Transcrf 11.02) .In summary, the UGTs repertoire of African trypanosomes seems to be under strong developmental regulation, corroborating the hypothesis of functional differentiation within the family.T. congolense genes with three significant breakpoints identified by GARD taking into account rate variation. The six tests for positive selection performed did not show evidence for positive selected sites; only PAML identified one site under positive selection at nucleotide 257 of the alignment.To test the contribution of selection to UGT expansion in African trypanosomes, we first searched for evidence of recombination and subsequently performed six tests of site-level selection. Three tests found evidence for recombination among Selection tests for sequences where GARD did not predict significant breakpoints did not find any evidence for positive selection at the site level, but rather negative selection in lineages 2–6, suggesting UGT family expansion is not driven by positive selection or gene conversion.Angomonas deanei and Strigomonas culicis, have a broad UGT repertoire, which suggests these enzymes play important roles for parasite survival. The lineage present in B. saltans and stercorarian trypanosomes may represent a remnant of the ancestral repertoire, which expanded independently in trypanosomes and Leishmaniinae. The trypanosomatid UGT phylogeny lacks support in the stercorarian trypanosomes and T. grayi nodes, which could potentially be improved through the introduction of sequences from related trypanosomes, such as T. theileri or T. avium. These would strengthen robustness of T. grayi nodes and help deciphering the relative phylogenetic distance between T. grayi UGTs and the remaining trypanosomes.All trypanosomatids sampled, with the exception of B. saltans and stercorarian trypanosomes indicates that the UGT repertoire of the ancestral organism was considerably smaller, with fewer loci, supporting the theory that UGT expansion in trypanosomatids is a parasite-specific innovation. UGT expansion is occurring under different dynamics in Leishmania spp. and trypanosomes and UGTs are evolving to perform specific, essential roles in the life cycles of these parasites. Comparing the UGT ancestral lineage, which we term the ‘protolog’, in the free-living B. saltans with parasite-specific UGTs can be useful to uncover the reasons behind UGT expansion and the benefits gained by these innovations. Comparing ‘the protolog’ with its parasitic homologs can begin to reveal the role of parasite-specific UGTs in the origin of parasitism. At the moment, the phylogeny shows that, in T. cruzi, the gene belonging to the ancestral lineage is constitutively transcribed, but more abundant in amastigotes , which contrasts with the transcriptomic data available for other UGT genes , mostly more abundant in trypomastigotes (the bloodstream stage of the parasite) [The UGT ancestral lineage retained in arasite) .L. major shows functional differentiation between SCG, SCGR and SCGL sub-families [Leishmaniinae subfamily with striking amino acid conservation, particularly in the surroundings of the “DXD motif” catalytic domain. This domain is conserved across most eukaryotic GT-A proteins, but is modified in SCGR1 and 4 (LmjF.02.0230 and Lmj.02.0190) in all Leishmania species (i.e. DDD to YDD), suggesting a parasitic innovation that may result in functional adaptation of these enzymes. When these genes were described in L. major, expression analysis by Western Blot suggested higher abundance in metacyclics [Current knowledge of UGTs in families , 73. Theacyclics , which iLeishmania-specific single-copy SCGL lineage likely arose from a transposition event from the SCGR array in chromosome 2 to chromosome 14. Members of this family are found in a paraphyletic clade with single gene copies in Crithidia and Lotmaria. When first described, LmjF.14.1400 was detected at low levels in all life cycle stages, compared to the high expression of SCGR and SCG members [L. major and L. infantum, where the gene was either not detected [The  members , which wdetected  or constLeishmania-specific whose genes are located at the telomeric regions of several chromosomes. In L. major, these genes have been shown to encode functional proteins, which are expressed in the parasite [L. infantum revealed differential expression at the amastigote level in 3 of the 4 homologs [L. mexicana also suggest preferential expression in amastigotes [L. major comparing metacyclic promastigotes to 4 and 24-h post-infection amastigotes also suggest gradual higher abundance in amastigotes [L. major sequences. Although evidence of positive selection to be acting upon this clade, as previously suggested [Leishmania species.Finally, the SCG lineage is parasite . Most liparasite . Howeverparasite . Similarhomologs , and trastigotes . Existinstigotes . The eviuggested , could nIn summary, the scenario of UGT evolution in Leishmaniinae suggests the UGT repertoire of its ancestor was organised in a tandem array and that transpositions to telomeric regions of various chromosomes might have allowed parasite-specific expansion of the repertoire. This expansion has since become concerted, as a result of the high rate of recombination possibly by telomeric exchange.T. brucei and T. congolense at the subtelomeres  not mucT. brucei and T. congolense and most genes are located in the sub-telomeres  in this clade to be up regulated only 12 h after differentiation induction, continuing until established procyclic stage [T. brucei retains so many procyclic-specific UGTs, and whether members of this lineage are all non-essential or redundant as Tb927.2.3370. If so, it could be hypothesized that the family expansion and its subtelomeric localization may be advantageous for expression. In lineage 1, T. brucei and T. vivax genes seem to be constitutively expressed, but the lack of gene expansion in T. vivax is intriguing. This might be explained by crucial differences in parasite surface coating, such as a lower requirement of GPI-anchored proteins to be secreted.Lineages 1 and 7 have greatly expanded in res Fig. . Subteloic stage . This suTo accommodate the requirements of a parasitic life cycle, both African trypanosomes and Leishmaniinae had to develop survival strategies in the shape of developmental regulation of protein expression. In this paper, we showed that UGTs have greatly expanded and are strongly developmentally regulated in both species. However, the specific characteristics of each life cycle have led to distinct approaches to the same challenge. To succeed in their obligate extracellular life cycle, African trypanosomes have developed mechanisms through which the parasite cell is covered by glycosylated proteins, e.g. procyclin or VSGs, which may account to up to 20% of the total protein in the cell . To syntTrypanosoma and Leishmaniinae have diversified their UGT repertoires, relative to their free-living ancestor, which had considerably fewer UGT genes. The lineage we have discovered in B. saltans and T. cruzi may represent a remnant of this ancestral repertoire, and functional comparison of this lineage with parasite-specific UGT will be important in elucidating the precise benefit conferred on the ancestral parasites by these innovations. At present, gene expression profiles indicate that UGT genes diversified for similar reasons in both Trypanosoma and Leishmaniinae, i.e. to enable developmental regulation of UGTs that, like other functions, is necessary during multi-host life cycles. However, while these expansions may be responses to a common need, we have shown conclusively that they occurred independently. This supports the general hypothesis that dixenic life cycles in Trypanosoma and Leishmania evolved in parallel from different invertebrate-parasitic ancestors [Trypanosoma and Leishmaniinae betray how these two lineages evolved a similar solution to independently meet their superficially common need to decorate their cell surfaces for infection and transmission. In this way, the UGT phylogeny is consistent with the evolutions of the cell-surface proteins that they decorate, which have also evolved independently. This independence only reinforces the importance of cell-surface interactions in determining parasite fitness, and shaping their genomes.The UGT phylogeny shows that ncestors . Among LAdditional file 1:Figure S1. Conserved genomic locus in L. major Friedlin, L. infantum JPCM5, L. braziliensis M2904, and Crithidia fasciculata CfC1. The UDP-glycosyltransferase genes are shaded yellow, flanking genes are shaded orange; sequence homology is illustrated by gray vertical bars. Gene terminology is according to Tritrypdb identifiers. Comparisons were obtained with Artemis Comparison Tool (ACT) [ol (ACT) .  [ol (ACT) , (PNG 35Additional file 3:Table S1. Results of selection tests on Leishmania. PARRIS [. PARRIS  searches. PARRIS  searches. PARRIS , SLAC [6. PARRIS , FEL [64. PARRIS  search f


Enterococcus faecalis, Escherichia coli, Staphylococcus epidermidis, Pseudomonas aeruginosa and Candida albicans biofilms are major causes of catheter-associated urinary tract infections. Antimicrobial-coated or impregnated urinary catheters are seen as a possible way to prevent these infections.E. faecalis isolates from urinary tract infections and to compare several urinary catheters for antimicrobial durability and the inhibitory effects on biofilm formation of different laboratory strains and clinical isolates of E. faecalis.To determine the biofilm-forming ability of 89 In vitro experimental study.E. faecalis isolates was determined by the crystal violet staining and plate counting methods. For comparison of urinary catheters, biofilms of 45 E. faecalis isolates from the catheter samples of hospitalized patients and five laboratory strains of E. coli ATCC25922, S. epidermidis ATCC35984, P. aeruginosa ATCC27853, E. faecalis ATCC29212 and C. albicans ATCC90028 were formed on the catheters in 24-well tissue culture plates. Scanning electron microscopy analysis was performed to observe biofilms.The biofilm forming ability of E. faecalis isolates were found to be biofilm positive. Nitrofurazone-impregnated catheters significantly reduced the cell counts of E. faecalis isolates and completely inhibited the formation of P. aeruginosa and S. epidermidis biofilms compared with the others. Regarding reduction of biofilm cell counts, a hydrophilic-coated catheter was more effective against P. aeruginosa, whereas a silver-coated catheter was found to be more effective against S. epidermidis. The nitrofurazone-impregnated catheter had the best antimicrobial durability.All 89 E. faecalis had considerable ability with respect to biofilm formation. The nitrofurazone-impregnated catheter was the most effective against all tested bacteria; however, the effect of a hydrophilic or silver-coated catheter depends on the species present in it.Urine isolates of  Catheter-associated urinary tract infections (CAUTIs) account for 80% of all hospital-acquired infections and correlate with the presence of microbial biofilms that cause catheter blockage . BacteriIn the CAUTI guidelines, the utilization of antimicrobial-coated or impregnated urinary catheters is suggested as a possible preventive strategy to fight these infections ,5. SilveA variety of species such as Escherichia coli, Staphylococcus epidermidis, Pseudomonas aeruginosa and Candida albicans are considered significant causative agents of nosocomial urinary tract infections (UTIs). Enterococcus species have also been associated with various nosocomial infections including UTIs and account for 15%-30% of CAUTIs . EnterocE. faecalis clinical isolates from urine infections. In the second part, we aimed to evaluate the antimicrobial durability and the inhibition of biofilm formation in different types of commercial urinary catheters.In this context, this study was divided in two parts. In the first part, we aimed to evaluate the ability of biofilm formation in This study was approved by the ethics committee.E. faecalis clinical isolates were included in this study. These isolates were taken from urinary catheter (n=45) and urine samples (n=44) of hospitalized intensive care unit patients  who were admitted to a University Hospital from 2000 to 2011.A total number of 89 E. faecalis, isolates of 45 hospitalized patients with catheters and laboratory strains of E. coli ATCC25922, E. faecalis ATCC29212, P. aeruginosa ATCC27853, S. epidermidis ATCC35984 and C. albicans ATCC90028 were used in catheter assays.In addition to Biofilm formation and quantification: Final inoculum suspensions of all clinical E. faecalis strains were adjusted to approximately 106 colony forming units (CFU) mL-1. Each experiment included the biofilm-forming E. faecalis ATCC29212 strain as a positive control. Sterile tryptic soy broth (TSB)  with 0.25% glucose was used as the blank.For each test condition, 12 wells of a flat-bottomed polystyrene 96-well microtiter plate were inoculated with 100 μL of the final inoculum suspension. After 4 h of incubation at 37 °C without shaking, non-adhered cells were removed and rinsed with 100 μL of 0.9% physiological saline (PS), then 100 μL of fresh TSB with 0.25% glucose was added, and the plates were incubated for an additional 20 h for biofilm maturation. After 24 h, supernatants were removed, and each well was rinsed with PS before quantifying the sessile cells.Crystal violet staining: The biomass quantification of E. faecalis biofilms was performed according to an optimized assay (ed assay . After rThe number of cells in mature biofilms was quantified via plate counting using tryptic soy agar (TSA) medium. Biofilms were detached by vortexing (5 min) followed by sonication (5 min). Serial dilutions of sonication fluids were made and plated on TSA to determine the number of (CFU) per millilitre of the isolates. Each isolate was tested in twelve wells in each assay, and each assay was carried out in duplicate (n=24).We tested five types of commercially 18 Fr Foley catheters including:1. A nitrofurazone-impregnated silicone catheter (RelaseNF®),2. A silver-coated silicone catheter (Dover®),3. A hydrophilic-coated silicone catheter without an antimicrobial agent,4. A silico-latex catheter (Rüsch®) without antimicrobial agent, and5. A silicone catheter without an antimicrobial agent (Rüsch®).Catheters were cut into 1-cm long segments followed by cutting in half lengthwise to expose the interior surface.E. faecalis ATCC29212, S. epidermidis ATCC35984, P. aeruginosa ATCC27853 and C. albicans ATCC90028. Each catheter was tested in duplicate in each assay.The antimicrobial durability of the nitrofurazone-impregnated and the silver-coated silicone catheter was assessed over time. We used a previously described method, with some modifications . BrieflyE. faecalis isolates obtained from the inpatients with catheters, E. coli ATCC25922, E. faecalis ATCC29212, P. aeruginosa ATCC27853 and S. epidermidis ATCC35984 and approximately 105 CFU mL-1 of C. albicans ATCC90028 were added separately to the wells for each catheter. The plates were then placed individually in the incubator at 37 °C for 24 hours for biofilm formation. After the incubation time, the contents of the wells were removed and segments were washed three times in 0.9% PS and aseptically transferred to tubes containing 1 mL phosphate-buffered saline. Then, the tubes were sonicated (15 min) and vortexed (30 seconds) three times. Serial dilutions were made in sterile PS and plated onto TSA. Each catheter was tested three times in each assay.Sterile catheter segments were placed in sterile 24-well tissue culture plates  and 1 mL of TSB containing approximately 106 CFU mL-1 of the 45 The catheter segments were washed with sterile distilled water following 24 hours of incubation. Then, the segments were fixed in a buffer containing 2% glutaraldehyde and 0.1 M cacodylate for 30 min followed by rinsing three times for 10 min in 0.2 M cacodylate buffer. After passage through serial ethanol solutions, samples were dried and then coated with gold-palladium .Statistical analysis was performed using the software program Statistical Package Version 21 . The normal or non-normal distributions of the data were verified using the Shapiro-Wilk test. For the comparison of urinary catheters, we used the one-way ANOVA test.E. faecalis isolates were tested to assess biofilm formation ability by two different methods. All isolates produced biofilms as determined by the plate counting assay, and in terms of biofilm forming ability, no statistically significant difference was determined between the isolates from the inpatients with and without catheters. Although all isolates from the inpatients with catheters were detected as biofilm positive by the CV staining assay, only 2 of 44 isolates from the urine samples of the inpatients were found to be biofilm negative. The results obtained by the CV staining assay and plate counting method for the isolates from the inpatients with and without catheters are presented in In this study, 89 C. albicans, E. faecalis and S. epidermidis, and E. coli and P. aeruginosa, respectively. This catheter was found to be more effective against gram-negative than gram-positive bacteria and to have no effect on C. albicans, as no inhibition zone was detected. However, for the second tested silver-coated silicone catheter, an inhibition zone was not detected just after the incubation period (data not shown).The antimicrobial effects of the nitrofurazone-impregnated silicone catheter were observed immediately after the start of incubation and at the end of the first and fifth days against E. faecalis isolates obtained from the hospitalized patients with catheters and laboratory strains per centimetre of catheter segment after 24 hours of incubation. The number of viable biofilm cells of the clinical isolates on the nitrofurazone-impregnated silicone catheter was lower than that on the silver- or hydrophilic- coated catheters, and the difference between catheters was found to be statistically significant (p<0.001)  .E. faecalis, P. aeruginosa, S. epidermidis and C. albicans, the nitrofurazone-impregnated catheter completely inhibited the growth of viable cells of P. aeruginosa and S. epidermidis after 24 hrs of incubation. The mean log10 counts of viable sessile cells on the nitrofurazone-impregnated silicone catheter, except for those of C. albicans, was significantly lower than on the silver- and hydrophilic-coated silicone catheters (p<0.05)  .E. faecalis ATCC 29212 were consistent with the results of the plate counting method. As in E. faecalis (The scanning electron microscope (SEM) results for faecalis .E. faecalis isolates in microtiter plates was analysed both by CV staining and plate counting assay. When we compared the isolates from inpatients with and without urinary catheters, the results indicate that all isolates were biofilm positive by the plate counting method. For all samples, although the starting bacteria concentration was normalized to 6 log10 (106 CFU/mL) after the incubation period, the lowest bacterial number in the wells was found to be 7.8 log10, indicating that all the isolates attached and grew on the walls of the wells in microtiter plates . Researchers reported that the novel antimicrobial Foley catheter coatings had a potential in the prophylaxis of catheter-related UTIs and reduced biofilm formation . Mean sessile cell numbers of E. faecalis isolates on the silver-coated catheter were significantly lower than on the silicolatex and silicone catheters ; however, there was no significant difference between the silver- and hydrophilic-coated catheters, p=0.286). Our results reveal that the nitrofurazone-impregnated silicone catheter is the most effective at reducing the number of sessile cells of E. faecalis isolates. These findings agree with previous in vitro assessments of antimicrobial catheters (22). It was concluded that the adherence of E. faecalis to the nitrofurazone-impregnated and hydrogel-coated silicone catheters was significantly decreased compared with the silicone-only catheters. Furthermore, the silver coating had little effect on bacterial adherence, whereas nitrofurazone impregnation had a significant effect that lasted up to 5 days . The number of viable biofilm cells of E. coli and E. faecalis on the nitrofurazone-impregnated silicone catheter was significantly lower than that on the silver-coated silicone, hydrophilic-coated silicone, silicone-only and silico-latex catheters (p<0.001)  . For C. atheters . Anotheratheters .E. faecalis obtained from urinary catheters. The nitrofurazone-impregnated catheter was found to be the catheter with the lowest number of E. faecalis cells, followed by the silver-coated urinary catheter.SEM analysis revealed an intense network of cellular layers consistent with the results of sessile cell counts of


Plasmodium species that infect humans has been hampered by a lack of genetic information for three human-infective species: P. malariae and two P. ovale species P. malariae and a manually curated draft P. o. curtisi genome, we are now able to accurately place these species within the Plasmodium phylogeny. Sequencing of a P. malariae relative that infects chimpanzees reveals similar signatures of selection in the P. malariae lineage to another Plasmodium lineage shown to be capable of colonization of both human and chimpanzee hosts. Molecular dating suggests that these host adaptations occurred over similar evolutionary timescales. In addition to the core genome that is conserved between species, differences in gene content can be linked to their specific biology. The genome suggests that P. malariae expresses a family of heterodimeric proteins on its surface that have structural similarities to a protein crucial for invasion of red blood cells. The data presented here provide insight into the evolution of the Plasmodium genus as a whole.Elucidation of the evolutionary history and interrelatedness of  P. malariae was produced from clinically isolated parasites and sequenced using long-read sequencing technology . Two genes expressed in blood stages are under significant balancing selection in both comparisons (ama1) and an uncharacterized conserved protein. Genes under significant selection in both comparisons are enriched for ‘pathogenesis’ and ‘entry into/exit from host cell’ Gene Ontology terms. The increased signatures of selection in the P. malariae lineage reflect a genome-wide increase in fixed nonsynonymous mutations in this species , prompting us to hypothesize that human genes encoding transmembrane proteins that act as potential RBP1a receptors would have to be conserved in red blood cells between humans and New World primates but not with chimpanzees  is only pseudogenized in P. o. wallikeri and may be linked to the difference in relapse times between the two P. ovale speciesManual curation of about 5,000 gene models of both  content , with so content . Genes pentified . The man species  to be an species , and a hin PF3D7_27500 is P. malariae and P. ovale, approximately 40% of the total genome is subtelomeric. However, the gene content of the subtelomeres differs substantially between the two species , suggesting an even smaller functional repertoire.Large multigene families are a defining feature that distinguish the genomes of malaria species but are refractory to detailed analysis in non-curated draft genome data. In  species ; Table 1d subset . The ancuishable . Despiteifferent . MoreoveP. malariae is the presence of two large gene families that were not apparent in earlier partial genome datafam-l and fam-m . Most fam-l and fam-m genes face the telomeres and occur as doublets -score > 0.5)P. falciparum RH5 protein (TM-score > 0.8)P. falciparum protein that is essential for erythrocyte invasion, through binding to basigin on the erythrocyte surfaceP. vivaxRh and RBP genes. This suggests that fam-l and fam-m genes also have an adhesion role, possibly binding host receptors.The most notable difference in the subtelomeres of nd fam-m . Proteindoublets , and we doublets . ProteinPlasmodium genus, invasion-related genes are consistently found to be rapidly evolving. The RBP family is highly expanded  and P. ovale (AB354571) mitochondrial genome sequences against those of P. falciparumP. vivaxP. knowlesiCox1 gene that contained two or more species-specific single-nucleotide polymorphisms (SNPs). We searched for these 15-bp species-specific barcodes within the sequencing reads of all 2,512 samples from the Pf3K global collection that mapped to the P. falciparum mitochondrial genome . Samples that contained at least two sequencing reads matching one or more of the 15 bp barcodes for a specific species were considered to be positive for that species  had high numbers of sequencing reads for all three P. ovale barcodes and were used for reference genome assembly and SNP calling respectively.We aligned the  species . We founP. ovale samples were obtained from symptomatic patients diagnosed with a P. falciparum infection. The two P. o. curtisi samples  identified through co-infection mining (see above), were from two patients that tested positive on a CareStart (HRP2-based) rapid malaria diagnostic test kit at the Navrongo War Memorial hospital, Ghana. One P. o. wallikeri sample (PowCR01) and one P. o. curtisi sample (PocCR01) were from patients with uncomplicated malaria that tested positive by light microscopy at the Mile 16 Bolifamba Health Centre, Buea, Cameroon. The other P. o. wallikeri sample (PowCR02) was obtained from an individual with asymptomatic parasitaemia enrolled through a community survey in Mutengene, Cameroon. For all samples, following consent obtainment, about 2–5 ml of venous blood was obtained and then diluted with one volume of PBS. This was passed through CF11 cellulose powder columns to remove leukocytes before parasite DNA extraction. Ethical approval was obtained for all P. ovale samples used in this study.All P. malariae-like samples, PmlGA01 and PmlGA02, were extracted from Chimpanzee blood  obtained during routine sanitary controls of animals living in a Gabonese sanctuary . Blood collection was performed following international rules for animal health. Within six hours after collection, host white blood cell depletion was performed on fresh blood samples using the CF11 methodP. malariae infections by Cytb PCR and sequencingThe two P. malariae sample, PmMA01, was collected from a patient with uncomplicated malaria in Faladje, Mali. Venous blood (2–5 ml) was depleted of leukocytes within 6 h of collection through CF11 cellulose powder columns as previously describedOne P. malariae were obtained from travellers returning to Australia with malaria. PmUG01 and PmID01 were sourced from patients returning from Uganda and Papua Indonesia, respectively, who presented at the Royal Darwin Hospital, Darwin, with microscopy-positive P. malariae infection. PmMY01 was sourced from a patient presenting at the Queen Elizabeth Hospital, Sabah, Malaysia, with microscopy-positive P. malariae infection. Patient sample PmGN01 was collected from a patient who presented to Royal Brisbane and Women’s Hospital in 2013 on return from Guinea. Venous blood samples were subject to leukodepletion within 6 h of collection. PmUG01 was leukodepleted using a commercial Plasmodipur filter ; custom-made cellulose-based filters were used for PmID01 and PmMY01, whereas PmGN01 was leukodepleted using an inline leukodepletion filter present in the venesection pack . DNA extraction was performed on filtered blood using commercial kits . For samples PmUG01, PmID01 and PmMY01, ethical approval for the sample collection was obtained from the Human Research Ethics Committee of NT Department of Health and Families and Menzies School of Health Research (HREC-2010-1396 and HREC-2010-1431) and the Medical Research Ethics Committee, Ministry of Health Malaysia (NMRR-10-754-6684). For sample PmGN02, ethical approval was obtained from the Royal Brisbane and Women’s Hospital Human Research Ethics Committee (HREC/10/QRBW/379) and the Human Research Ethics Committee of the Queensland Institute of Medical Research (p1478).Four samples of P. malariae sample, PmUG01, was selected for long-read sequencing, using Pacific Biosciences (PacBio), owing to its low host contamination and abundant DNA. Passing through a 25-mm blunt-ended needle, 6 (μg of DNA was sheared to 20–25 kb. SMRT bell template libraries were generated using the PacBio issued protocol (20 kb Template Preparation using the BluePippin Size-Selection System). After a greater than 7 kb size-selection using the BluePippin Size-Selection System (Sage Science), the library was sequenced using P6 polymerase and chemistry version 4 (P6/C4) in 20 SMRT cells . Before applying MaSURCAP. falciparum 3D7 reference genomeP. vivax PVP01 (ref. http://www.genedb.org/Homepage/PvivaxP01) assembly  or against the P. malariae PacBio assembly . The assemblies were manually filtered on GC, coverage, and BLASTN hits to the non-redundant nucleotide databaseThe Illumina-based genome assemblies for P01 ref.   and for P. ovale curtisi . The genome sequences have been deposited into the European Nucleotide Archive (http://www.ebi.ac.uk/ena). Accession numbers for all reads generated for this study can be found in The genome sequences and annotations are currently available on GeneDB for Plasmodium species . In addition, genes were predicted ab initio using AUGUSTUSP. malariae and P. ovale genes respectively. Non-coding RNAs and tRNAs were identified using Rfam 12.0 .Following orthologue assignment using BLASTPivax P01 , P. cynoP. malariae  and all P. malariae-like samples  was generated using PhyMLP. malariae genes. For each sample, the raw SNPs, as called using the SNP calling pipeline (see below), were mapped onto all genes to morph them into sample-specific gene copies using BCFtoolsA phylogenetic tree of four P. malariae, PocGH02 for P. o. curtisi, PowCR02 for P. o. wallikeri, and PmlGA02 for P. malariae-like) was used to morph the respective assembly using iCORNP. o. wallikeri and the P. o. curtisi chromosomes were aligned against each other, as were the P. malariae and P. malariae-like chromosomes. The alignments were split into 1 kb loci, removing those that contained gaps, masked regions, and coding regions to conform with the neutral loci prediction of G-PhoCShttp://beast.bio.ed.ac.uk/Tracer) confirmed that this was sufficient for convergence of the MCMC chain in all cases. In the model, we assumed a variable mutation rate across loci and allowed for on-going gene flow between the populations. The tau values obtained from this were 0.0049 for P. malariae and 0.0434 for P. ovale.Species divergence times were estimated using the Bayesian inference tool G-PhoCSG) / m, where G is the generation time in years and m is the mutation rate. Testing a number of different generation time and mutation rate estimates in order to optimize the P. falciparum and P. reichenowi split to 4 million years ago as estimated previously−10 SNPs per site per lifecycleP. malariae, a generation time of 100 days was used owing to the longer intra-erythrocytic cycle.The tau values were used to calculate the date of the split, using the formula (tau × http://www.zhanglab.ccmb.med.umich.edu/I-TASSER) was used for 3D protein structure prediction. Predicted structures with a TM-score of over 0.5 were considered reliable as suggested in the I-TASSER user guidelinesThe I-TASSERPlasmodium species used for the phylogenetic analysis (see above), we examined clusters containing only genes of hypnozoite-forming species: P. vivax P01 genes, P. cynomolgiP. ovale species.Using the OrthoMCLP. o. curtisi orthologues of previously published hypnozoite gene candidatesAdditionally, we examined P. malariae, P. ovale, and P. vivax P01 genes were compared pairwise using BLASTPAll P. malariae, P. o. curtisi and P. o. wallikeri protein sequences for Plasmodium interspersed repeat (pir) genes, excluding pseudogenes, were combined with those from P. vivax P01 (P. knowlesiP. chabaudi AS v3 (http://www.genedb.org/Homepage/Pchabaudi), P. yoelii yoelii 17X v2  were compared pairwise using BLASTPThe fam-m and fam-l doublets that were confidently predicted as being paired-up were manually selected on the basis of their dispersal throughout the subtelomeres of different chromosomes. The Mirrortreehttp://csbg.cnb.csic.es/mtserver/) was used to construct mirror trees for these 79 doublets. 35 doublets with recent branching from another doublet were manually selected to enrich for genes under recent selection .Full-length RBP genes were manually inspected using ACTP. malariae  and P. o. curtisi  samples were mapped back against the reference genomes using SMALT   and P. o. wallikeri  were also mapped against the P. malariae and P. o. curtisi genomes respectively. The resulting ‘bam’ formatted files were merged for either of the two genomes, and GATKP. vivax samples (SRR3400910 and SRR332566) and two P. falciparum Pf3K field samples  for comparative purposes.Additional  -i 300) . As outgP. malariae, P. o. curtisi, P. vivax, P. falciparum) and one comparison for species with two samples . These estimates were then averaged by species .The filtered SNPs were used to morph the reference genomes using BCFtoolsP. malariae and within P. o. curtisi) and inter-specifically . The intraspecific comparisons were averaged to obtain the nucleotide diversity π of the genes and these were divided by the average inter-specific comparisons, the nucleotide divergence, to get the HKA ratio (HKAr) for each gene.For the HKAP value, as previously described2(((Npoly + 1) / (Spoly + 1)) / ((Nfix + 1) / (Sfix + 1))), where Npoly and Nfix are polymorphic and fixed non-synonymous substitutions respectively, and Spoly and Sfix refer to the synonymous substitutions.The MK testKa/Ks ratioP. malariae and P. malariae-like . The ‘Bio::Align::DNAStatistics’ module was used to calculate the Ka/Ks values for each pairTo calculate the average P. falciparumP. falciparum 3D7 reference genomeP. malariae and P. ovale were classified by the maximum expression stage of their P. falciparum orthologue if the difference between the maximum expression stage and the second highest stage was larger than the difference between the second and third highest stage, otherwise the gene was classified as having no peak expression.Using existing RNA sequencing data from seven different life-cycle stages in P. falciparum 3D7 genes orthologous to the P. malariae and P. o. curtisi genes included in the analysis were used. Collated tables for all molecular evolution measures for all genes can be found in Source Data File 1 for P. malariae and P. o. curtisi.The Gene Ontology term enrichment analysis was performed in R, using TopGOP. brasilianum  results in chronic infection in the common marmoset−10.To find the putative RBP1a receptor, we performed an OrthoMCLhttp://www.ebi.ac.uk/ena/data/view/PRJEB14392). The individual accession numbers are as follows for PmUG01 , PocGH01 , PowCR01  and PmlGA01 .All raw data has been deposited as described in Supplementary Information is available in the online version of the paper.Supplementary Data