Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, and S. Paratyphi A and B, causative agents of paratyphoid fever, are major public health threats throughout the world. Although two licensed typhoid vaccines are currently available, they are only moderately protective and immunogenic necessitating the development of novel vaccines. A major obstacle in the development of improved typhoid, as well as paratyphoid vaccines is the lack of known immunological correlates of protection in humans. Considerable progress has been made in recent years in understanding the complex adaptive host responses against S. Typhi. Although the induction of S. Typhi-specific antibodies  and memory B cells, as well as their cross-reactivity with S. Paratyphi A and S. Paratyphi B has been shown, the role of humoral immunity in protection remains undefined. Cell mediated immunity (CMI) is likely to play a dominant role in protection against enteric fever pathogens. Detailed measurements of CMI performed in volunteers immunized with attenuated strains of S. Typhi have shown, among others, the induction of lymphoproliferation, multifunctional type 1 cytokine production, and CD8+ cytotoxic T-cell responses. In addition to systemic responses, the local microenvironment of the gut is likely to be of paramount importance in protection from these infections. In this review, we will critically assess current knowledge regarding the role of CMI and humoral immunity following natural S. Typhi and S. Paratyphi infections, experimental challenge, and immunization in humans. We will also address recent advances regarding cross-talk between the host’s gut microbiota and immunization with attenuated S. Typhi, mechanisms of systemic immune responses, and the homing potential of S. Typhi-specific B- and T-cells to the gut and other tissues. Salmonella enterica serovar Typhi (S. Typhi) and paratyphoid fever caused largely by S. enterica serovars Paratyphi A and B (S. Paratyphi) ] to S. Typhi and S. Paratyphi acquired through natural infection, experimental challenge, and vaccination. For discussions of the “mouse model of Salmonella infection,” the reader is referred to excellent reviews included in this “Frontiers in Immunology Research Topic” compilation.Enteric fevers encompass typhoid fever caused by the Gram-negative intracellular bacterium ratyphi) , 2. MostS. Typhi . However in Asia –7. Typho in Asia . Humans  in Asia . In indung world –13. It ing world –13. Baseng world . Furtheratyphi A , 16. Thuatyphi A , 10, 17.Salmonella Typhi is a facultative intracellular bacterium that causes an acute generalized infection of the reticuloendothelial system (RES), intestinal lymphoid tissue, and gallbladder in humans , passage through enterocytes in endocytic vacuoles, and/or disruption of tight junctions (paracellular route)  , 21. OncS. Typhi natural infection originates from studies involving individuals living in typhoid endemic areas , H (flagellin), Vi , porins, and heat-shock proteins , among others, have been well documented in the sera of acute and convalescent typhoid fever patients (S. Typhi secretory IgA (SIgA) was also described in intestinal fluids of typhoid patients , which increase with age has also been reported in healthy residents of typhoid endemic areas -γ, interleukin (IL)-12, IL-23, and STAT1 receptors  , which elicited antibody responses in these patients during the acute and convalescent phases. These results confirmed and extended previous studies by the same group using a different technique  in Bangladeshi patients who were bacteremic with S. Paratyphi A and S. Typhi  cytokines but decreased levels of IL-10 were reported in patients with early non-typhoidal gastroenteric Salmonella bacterial clearance in stools as compared to the non-clearance group (eff) and T regulatory (Treg) responses following wild-type infection.Regarding CMI, as reported in typhoid fever, elevated serum levels of IFN-γ, IL-6, TNF-R p55, and TNF-R p75 were reported in patients . Moreovece group . It is rSalmonella is that although increases in circulating cytokines/ chemokines are widely considered to be associated with protective responses, this might not necessarily be an accurate interpretation. In fact, it is likely that the levels of cytokines/chemokines in the microenvironments of the gut and the “RES”  are not necessarily reflected in circulation. These are the sites in which most immune responses are likely to be generated, and where Salmonella find their niche(s) for long-term persistence, representing important sites for localized immune responses. With the information currently available, it is not possible to rule out the notion that serum/plasma levels might be a representation of a generalized pro-inflammatory response  in response to a systemic bacterial infection  rather than an effective targeted host response leading to protection.Another issue to consider regarding the cytokine/chemokine data in natural infections with typhoidal and non-typhoidal S. Typhi is a human-restricted pathogen, i.e., there are no good animal models that faithfully recapitulate S. Typhi infection  in 1918  has re-established this model. This CHI model followed in the steps of previous studies by challenging healthy adult subjects with wild-type S. Typhi Quailes strain (3 or 104 colony-forming units) resulted in attack rates of 55 or 65%, respectively. Interestingly, participants who developed typhoid infection demonstrated serological responses to flagellin and LPS antigens by day 14, while no changes were observed in the titers of these antibodies in participants not succumbing to infection after challenge. It is reasonable to speculate that the increased anti-LPS responses in subjects who developed typhoid was largely the result of clinical disease involving local and systemic infection rather than representing a protective mechanism at play. Moreover, anti-S. Typhi antibody baseline titers did not correlate with subsequent infection risk  but only modest rises in anti-Vi antibody titers  polysaccharide parenteral vaccine and the oral live-attenuated S. Typhi strain Ty21a vaccine. Both vaccines are moderately protective and have been shown to induce herd immunity  has shown an efficacy of ~90% in 2–5-year-old children (S. Typhi strain Ty2 conjugated to tetanus toxoid (TT) as a carrier protein, which can be given to infants older than 6 months  to extend the generation of immunity to other relevant specific antigens   England . At that England –68. Currimmunity , 70. Theimmunity , 71–75. immunity , 69, 76.immunity , 70, 77.on Table , 78–92. on Table , 93–96. children , 96–99. 6 months , 101. Otths  and heat-shock proteins (htrA). The latter vaccine candidates, designated CVD 906 , have engineered new attenuated typhoid vaccine strains that aim to be as safe as Ty21a but immunogenic and protective following the ingestion of only a single dose. These vaccine candidates include Ty800 , M01ZH09 CVD 906 , 106, CV CVD 906 –109, CVD06   . Althougvaccine) . Althougnjugates –126, no S. Typhi strains as live-vector vaccines. S. Typhi presents multiple advantages as a live-vector, including (a) oral delivery, (b) targeting of M cells overlying gut-associated lymphoid tissue (inductive sites for immune responses), (c) internalization by DC and macrophages, and (d) stimulation of broad immune responses , as well as the interaction between T, B, and antigen-presenting cells  are all likely to contribute to an effective acquired immune response against typhoid fever  IgM and both IgG1 and IgG2 seroconversions were detected  based on structural, antigenic, and functional differences . The subge fluid . IgG cange fluid . Interesbclasses . In contbclasses . Moreovedetected . UnfortuS. Typhi, there have been few investigations of the functional properties of these antibodies. Early studies indicated that S. Typhi-specific IgA was responsible for antibody dependent cellular cytotoxicity (ADCC) following Ty21a immunization -transformed lymphoblastoid B cell lines (B-LCL) are able to effectively stimulate CD4+ cells as well as classical and non-classical CD8+ cells  . In Salmor sites , 154–157al blood . In factal blood . Followi IgG ASC , 158. Of IgG ASC . Ingestiific ASC , 158. Noific ASC , 144. Otific ASC . ConsequM) are long-lived antigen primed cells that upon antigenic stimulation during a secondary response undergo rapid terminal differentiation into plasmablasts and plasma cells  (M responses against both T-cell-dependent (flagella) and T-cell-independent (LPS and Vi) antigens were identified in volunteers primed with CVD 909 (a Vi expressing live-attenuated S. Typhi vaccine candidate). These results suggest that immunization with CVD 909 was capable of mucosally priming the immune system to deliver robust and sustained Vi-specific BM responses to a subsequent parenteral exposure. LPS-specific BM responses were also observed in volunteers primed with CVD 909, but these responses were of lower magnitude than those against Vi. Similar to findings for ASC, LPS-specific IgA BM cells predominated over LPS-specific IgG BM responses. In the same study, we observed that volunteers immunized with Ty21a also developed IgA BM responses to LPS, but only a single volunteer developed IgG BM responses against LPS. Moreover, both CVD 909 and Ty21a were capable of inducing anti-S. Typhi flagella IgG and IgA BM responses. Finally, we observed a strong association between the frequency of antigen-specific BM cells and antibody levels, supporting an important role of this cell population in the generation of humoral responses. Recent studies have shown that S. Typhi porins can induce short- and long-lasting IgG and IgM responses in humans, a response likely to be mediated by BM   and desclla, Vi) . Notablyed by BM . Interesi porins . This storins  B cells as detected by flow cytometry. Although naïve B cells that interacted with S. Typhi were observed, phosphorylation of Stk, Akt, and p38MAPK were not identified in this subset. In contrast, Um B cells showed multi-phosphorylation of all three proteins assayed, as well as cells that phosphorylated only p38MAPK or Akt and p38MAPK. Interestingly, different antigenic structures appeared to induce different patterns of phosphorylation. For example, the phosphorylation patterns induced by S. Typhi were dramatically different from the phosphorylation patterns induced by the Gram-positive bacterium Streptococcus pneumoniae. These novel studies provide the first glimpse of the activation pathways of S. Typhi-specific B cell responses in humans. Further characterization of these mechanisms can provide key information to help advance the generation of novel vaccine strategies.Early signaling events that occur following encounter of B cells with  B cells . We repoSalmonella in humans is derived from studies using peripheral blood, effector immunity in the local microenvironment of the gut is likely to be of paramount importance in the understanding of protection against S. Typhi infection. Mucosal derived circulating IgA ASC detected after administration of live oral typhoid vaccines have been used to estimate the degree of priming of the local intestinal immune system (4β7 and chemokine (C–C motif) receptor (CCR)9 (S. Typhi-specific IgM and IgA ASC toward chemokine (C–C motif) ligand (CCL)25 and CCL28, the ligands for CCR9 and CCR10, respectively, were noted  (S. Typhi-LPS are predominantly CD19+ CD27+ (a phenotype associated with BM and plasmablasts) with selective gut homing potential  , as well as to other lymphoid tissues where S. Typhi resides following systemic dissemination.Although most of our knowledge of immune responses against e system . These ce system . Selectir (CCR)9 , while Cr (CCR)9 . The prir (CCR)9 . Followire noted . In contse cells , 158. Moression) . We have CD62L−) . Of noteS. Typhi infection rely largely on two types of cells: CD4+ and CD8+ T-cells -stimulated PBMC, as well as B-LCL  cells. Induction of strong and persistent memory T-cell responses is one of the hallmarks of successful vaccination , and effector memory T-cells (TEM) , as well as the chemokine receptor CCR7 and CD62L (L-selectin) molecules, which allow efficient homing to peripheral lymph nodes , which could lead to improved control against re-infection. Recently, these results were confirmed and extended using multichromatic flow cytometry to measure six cytokines simultaneously   , 179. TCph nodes , 179. TEy” cells , 179. ReS. Typhi . In thesn nature . We alsoesponses . We spec MIP-1β) . In thisaccinees . These fcination  or exposcination , 182 andrculosis , 184.reg and pro-inflammatory Teff responses influence the clearance or persistence of S. Typhimurium (reg are characterized by the expression of high levels of the IL-2 receptor (CD25) and transcription factor Forkhead box P3 (FoxP3). Activated Treg may traffic to the sites of specific immune responses and exert their regulatory functions via cell–cell interactions , consumption of IL-2, and production of anti-inflammatory factors  -β] . Observa antigen , 123 andS. Typhi, is the presence of background S. Typhi-specific responses among individuals prior to immunization, even in the absence of travel to endemic areas , but not by uninfected cells , and contributing to clear the infection without causing overt disease once the specific adaptive immune responses are fully developed.A common finding when measuring T-cell immune responses in humans vaccinated against enteric bacteria, such as ic areas , 152. Thic areas , 151 or ic areas –190. Altantigens . It is aantigens –194. Prenfection , 196. Thnfection , 196. OfS. Typhi regulation of the development of specific T-cell responses in humans remains unclear. Studies in mice have shown that DC can either directly  or indirectly (by bystander mechanisms) elicit Salmonella-specific CD8+ T-cells  underlying  T-cells . DC are  T-cells . In addi T-cells . This al T-cells . Althoug from volunteers immunized with S. Typhi vaccine strain Ty21a when stimulated in vitro produced around 10-fold more IFN-γ than the remaining populations (CD45RA− β7− or CD45RA− β7intermediate)  and effector  memory T populations  subsets. Interestingly, while CD8+ TEM included both integrin α4β7+ and integrin α4β7− cells, CD8+ TEMRA were predominantly integrin α4β7+   . Also, uV. cholerae O1 vaccine CVD 103-HgR, Lagos and colleagues demonstrated that excessive bacterial growth  in the small intestine of children in less developed countries might contribute to the low-antibody response to the vaccine . Although the number of volunteers analyzed was small, this study provides additional information supporting the potential influence of the gut microbiota on the immune response elicited by oral immunization, and perhaps, in protection. Additional studies involving larger numbers of volunteers and a multiplicity of vaccines administered via the oral route are necessary to extend our understanding of the complex role of the gut microbiota in modulating host immunity and vaccination in humans, and its possible role in vaccine efficacy.We recently initiated studies to directly investigate the interactions between the microbiome and vaccination with attenuated oral vaccines. We observed that, although Ty21a is a live-attenuated  vaccine . HoweverS. Paratyphi A and S. Paratyphi B in humans. In fact, most of the immune responses believed to be elicited by S. Paratyphi A have been inferred from S. Typhi studies. Interestingly, field trials of Ty21a have shown modest cross-protection against S. Paratyphi B  as S. Typhi-specific ASC. We also reported the induction of antibodies and BM to S. Typhi LPS and OMP antigens, which cross-react with S. Paratyphi A and S. Paratyphi B. However, IgA BM reactive to S. Typhi was of higher magnitude than those against S. Paratyphi A and S. Paratyphi B. In contrast, BM to outer membrane proteins (OMP) from S. Paratyphi B were similar to those observed for S. Typhi-OMP, but higher than those for S. Paratyphi A OMP. In a subsequent study, we reported in Ty21a and CVD 909 vaccines the presence of cross-reactive serum antibodies able to mediate opsonophagocytosis of S. Paratyphi A and S. Paratyphi B, albeit at lower levels than those against S. Typhi , capable of resolving more than 35 measurements per cells using rare metal-conjugated monoclonal antibodies with minimal signal overlap (reg cells) and the possible mechanisms leading to protection against infection. In fact, it is likely that it is the balance  between effector and regulatory responses that holds the key to understanding protective immunity. Mass cytometry, in conjunction with traditional immunological assays and state-of-the-art genomics, transcriptomics, proteomics, and metabolomics approaches and the availability of human challenge models provide, for the first time, the necessary tools to uncover the mechanisms underlying protective immunity, both systemically and in the gut microenvironment. This information will be invaluable in accelerating the development of novel vaccine strategies to prevent enteric fevers. In addition, the expected explosion of knowledge regarding the gut microbiome and its role in modulating immunity to oral vaccines is also likely to provide significant insights in coming years in understanding the observed differences in immunogenicity between vaccine responses in developed and developing countries.Despite decades of effort, the mechanisms of protective immunity in natural infection and vaccination remain largely undefined and many questions remain Box . The vas overlap –232; a pBox 1What are the relative contributions of humoral and cellular responses to protection?What are the precise roles of effector and memory B and T-cells, as well as innate immune cells in protection?How can an appropriate balance between pro-inflammatory and regulatory responses be achieved, resulting in protection without causing excessive inflammation?S. Typhi, S. Paratyphi A, and S. Paratyphi B and how can long-lasting responses be preferentially induced?What are the mechanisms of enduring protection against What are the characteristics of protective local gut immune responses?What are the differences and similarities between local and systemic immune responses?What is the role of the gut microbiota in modulating immune responses against enteric fevers?S. Typhi, S. Paratyphi A, and S. Paratyphi B be exploited to develop broad-spectrum vaccines against enteric fevers?Can cross-reactive immune responses between 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.


Zygogonium ericetorum, the type species of the genus, was studied from a natural population collected in Mt. Schönwieskopf, Tyrol, Austria. Generic concepts of Zygogonium and Zygnema were tested with atpB, psbC, and rbcL gene sequence analysis, which showed a sister relationship between Z. ericetorum and Mesotaenium, in an early branching clade sister to a grouping of Zygnema and several other filamentous and unicellular zygnematalean taxa. A variety of light, confocal, transmission electron microscopy, and cytochemical techniques provided new data on the variable chloroplast shape of Z. ericetorum, and its aplanospore structure and development, which has been previously considered taxonomically important but has been ambiguously interpreted. Zygogonium can be distinguished from other zygnematophytes (particularly Zygnema), based on the combination of two characters: (i) irregular, compressed plate-like chloroplasts and (ii) residual cytoplasmic content left in sporangia outside of the fully developed aplanospores or zygospores. The presence of a sporangial wall that separates the spores from the parent cell should be excluded from the definition of Zygogonium, because it is also observed in Zygnema. Similarly, the ecological characterization of Zygogonium as acidophilic is not unique to the genus. The names of 18 species currently belonging to Zygogonium are here changed to Zygnema, because of incompatibility with this new proposed Zygogonium concept. In the species transferred to Zygnema, chloroplasts are typically stellate in three-dimensions, and the entire content of fertile cells is transformed into the spore, so there is no cytoplasmic residue. Zygogonium Kützing and its type species, Zygogonium ericetorum, have confounded phycologists for decades with their peculiar vegetative and reproductive morphology. Zygogonium and Zygnema C. Agardh synonymous because of vegetative similarities. Zygogonium based on three characteristics: (i) vegetative cells with a pair of rounded, plate-like or irregular pillow-shaped chloroplasts, each with a central pyrenoid; (ii) formation of a sporangium wall which divides the gametangium before the formation of the spore wall, and (iii) presence of cytoplasmic residue in the mature gametangia. Despite the clear delineation of chloroplast shape by Zygogonium species in a questionable fashion. For instance, in Zygogonium tunetanum Gauthier-Lièvre the chloroplasts have been reported as irregularly spherical or stellate  could be confidently identified and thoroughly studied.rs e.g., , such asrs e.g., . Molecul Zygnema . StancheZygnema and Zygogonium, we will limit further discussion of systematics and ecology of Zygogonium to those few well-documented records that are entirely consistent with that of the type species. Z. ericetorum is a cosmopolitan species and based on the few confirmed sites, occasionally forms large single-species communities in acid pools, sphagnum peat bogs and on wet acid soils  evaluate its phylogenetic placement within Zygnematophyceae and (ii) determine vegetative and reproductive characters relevant to generic concepts of Zygogonium and Zygnema.Despite the interesting ecophysiological and ultrastructural features described, no phylogenetic characterization of is genus . TherefoZ. ericetorum collected from Mt. Schönwieskopf , at 2,350 m a.s.l. near Obergurgl, Tyrol, Austria as previously reported  and fixation for transmission electron microscopy . Zygogonium ericetorum filaments were incubated in water from the habitat and placed in plastic petri dishes with Bold’s Basal Medium . The DNA was extracted using the Nucleon Phytopure DNA extraction kit , as previously described  was performed following the methods described above and presented in Filaments of escribed . Portionrotocols . Attemptrotocols . Brieflyrotocols  with 10 rotocols , 2008 virotocols . Analyserotocols  were alsrotocols  using twrotocols ; each loAn Olympus BX41 light microscope and Olympus IX50 inverted fluorescence microscope with an attached Olympus MicroFire S99809 digital camera  were used for specimen observation and microphotography. The size data given in the descriptions are based on a minimum of 20 specimens measured and analyzed using Rincon image analysis software . The chemical composition of the cell wall was examined using cytochemical techniques for the localization of pectic substances , equipped with a 63×1.4 NA objective lens. An argon laser was used at 488 nm in single track mode , for excitation of the chloroplast autofluorescence and the emitted signal was collected with a 560-nm long pass filter and false colored red. In parallel, a bright field image was collected at channel D with a transmission photomultiplier tube. The two images were either merged or only the chloroplast autofluorescence is shown here.4 at 4°C, dehydrated in increasing ethanol concentrations and transferred to modified Spurr’s resin . Data sets for these three chloroplast loci containing a large number of conjugating green algae (Zygnematophyceae) and Coleochaete (as an outgroup) were assembled. The atpB data set included 62 taxa and 1,207 nt characters of which 544 characters were variable and 504 characters were parsimony informative. The psbC data set included 38 taxa and 1,202 nt of which 518 were variable and 468 were parsimony informative. The rbcL data set included 62 taxa and 1,335 nt of which 567 were variable and 512 were parsimony informative. The combined, 3-locus data set contained 3,744 characters of which 1,629 characters were variable and 1,482 characters were parsimony informative.Zygogonium ericetorum n JH1396 . We obtaZ. ericetorum was sister to Mesotaenium sp. JH0031 .Each locus was aligned and analyzed independently. In all analyses,  of rbcL  with varrbcL, the clade of Z. ericetorum, Mesotaenium sp. JH0031, Mesotaenium sp. AG2009-1, and F. pyrenoidosa was embedded in a clade containing Mesotaenium cf. chlamydosporum . Because there was no statistically strong disagreement among loci in bootstrap analyses of single locus data sets, these loci were combined into a 3-locus data set that was analyzed as one large partition as well as with three partitions corresponding to each gene. In the single-partition 3-locus data set, Z. ericetorum was sister to Mesotaenium sp. JH0031 with high statistical support  Itzigsohn and Rothe was strongly supported in likelihood analyses , but poorly supported (74) in parsimony analyses , Mesotaenium kramstai Lemmermann, and most Cylindrocystis spp. In the BI analyses of the combined data set, the Z. ericetorum + Mesotaenium sp. JH0031 clade was found in a polytomy with Cylindrocystis brebissonii  DeBary UTEX1259 and a clade containing most Zygnematales (except Spirogyra and N. digitus) (not shown). ML analysis of the 3-locus data set in RAxML found the Z. ericetorum + Mesotaenium sp. JH0031 clade sister to all other Zygnematales , but the placement of the Z. ericetorum + Mesotaenium sp. JH0031 clade with respect to other lineages in the Zygnematales remained poorly supported (Sister relationships between the upported .Z. ericetorum and Mesotaenium sp. JH0031 (1.0), and strong support for their inclusion in the main clade of Zygnematales (1.0), but weak support for relationships among deeper clades of the Zygnematales (data not shown). In all analyses of all data sets, Z. ericetorum was sister with Mesotaenium sp. JH0031 and outside of the main clade of Zygnema spp. (including Z. tunetanum).The topology of the 3-locus data set partitioned by locus found using BI was the same as the topology from the RAxML analysis of the combined data set  filaments, numerous vegetative cells formed aplanospores. Their morphology and developmental stages are illustrated in Zygnema, and their chloroplasts remained intact during the resting period, a minimum of 30 d. Longitudinal and transverse cell walls of filaments thickened with the aging of aplanospores, and cytoplasmic residue became more condensed and purple-brownish . We were unable to confirm the identity of this strain because of a lack of reproductive characteristics, and thus the taxonomic status of Zygogonium remained in question . What is clear is that Z. ericetorum is phylogenetically distantly related to other zygnematophytes in terms of sequence divergence of the three genes sampled, including the core group of Zygnematales  is phylogenetically distinct from Zygnema.This study on the type species of ematales . Zygogonma clade . We concZ. ericetorum previously considered taxonomically important. Many of these characteristics are ambiguously and inaccurately described in the literature (see discussion below), and thus poorly understood. We applied a variety of LM, confocal, cytochemical and TEM techniques, which allowed us to provide a more thorough description of the extremely variable chloroplast shape as well as to provide new structural data on the aplanospores of Z. ericetorum.In this study, we assessed the value of morphological features of Z. ericetorum are in agreement with the existing drawings of its vegetative and reproductive properties in the literature . In the exsiccatae, Kützing lists the author of C. ericetorum as Roth with no reference to Bory de Saint-Vincent . C. erictorum, but when C. ericetorum to Leda, he stated that “our C. ericetorum adopted by Roth is probably one of the species.” Because C. ericetorum Roth is a later homonym of C. ericetorum Bory de Saint-Vincent, Guiry (2013) concluded that C. ericetorum Roth was illegitimate and that Kützing should be listed as the author of Z. ericetorum and we follow his suggestion. Regardless of the nomenclatural status of Z. ericetorum, this is likely the same species as C. ericetorum Bory and C. ericetorum Roth (illegitimate)  .Z. ericetorum of thick, multilayered cell walls with H-shaped structures in response to desiccation , and therefore the latter genus needs careful taxonomic revision.This finding is in contrast to the radial symmetry of the chloroplasts in roplasts . Because species , many ofZ. erictorum, but according to Zygogonium. On the other hand, the aplanospores and zygospores in Zygnema aplanosporum Stancheva, J. D. Hall et Sheath, illustrated by Zygogonium, as a similar structure has been observed in some species of Zygnema , outside the aplanospore. These observations of aplanospore structure and associated purple residue agreed well with detailed descriptions and illustrations by Z. ericetorum for the first time by TEM. While the aplanospores contain all organelles also found in the vegetative cell, the cytoplasmic residue consists of a large vacuole with granular content of varying electron density. The granular material is tightly packed against the newly formed aplanospore wall. Moreover, the area outside the aplanospores included small granules and rarely chloroplast fragments, but no intact organelles.The presence of cytoplasmic residue left in the gametangia after zygospore (and aplanospore) formation is the third diagnostic characteristic of Mougeotia C. Agardh and Zygogonium are similar, because the zygospore is surrounded by a sporangial wall separating it from the gametangial cells, and cytoplasmic residue is left in gametangia. However, the structure and composition of cytoplasmic residue is also a character that differs among these genera. For instance in Mougeotia, Mougeotiella Yamagishi and Neozygnema Yamagishi described a granular appearance or irregularly shrunken membranous structure (Zygnemopsis (Skuja) Transeau and Debarya Wittr. (syn. Transeauina Guiry) the lumen of the gametangia outside the spore is filled completely with a bluish-white, refractive pectic or cellulosic layer as the protoplast contracts to the middle of the cell  Kützing (Zygnema spp. (Mesotaenium berggrenii (Wittrock) Lagerheim or Ancylonema nordenskiöldii Berggern The Zygnematophyceae are known to synthesize phenolic compounds e.g., , which m Kützing . More reema spp. . Purple Z. ericetorum, the purple pigmentation may have UV shading and antioxidative capacities due to high amounts of soluble phenolic compounds identified , although confident phylogenetic placement was not possible with our data. Structurally, our specimen of Z. ericetorum was largely consistent with its original description and the observations made by subsequent authors  only when both vegetative and reproductive material is present, based on the combination of two characters: (i) irregular, compressed plate-shaped chloroplasts and (ii) residual cytoplasmic content left in sporangia outside the aplanospore and zygospore. Z. ericetorum seems to be extraordinarily well adapted to the terrestrial environment as indicated by its unusual physiology.Our results require a careful reconsideration of the taxonomy and ecology of rs e.g.,  for that species . TherefoZ. ericetorum, only a few species are consistent with its peculiar developmental characteristics. Other taxa currently classified as Zygogonium, having radial, nearly stellate chloroplasts and lacking cytoplasmic residue after spore formation should be transferred to other genera, most likely Zygnema. Many of these species have zygospores with multilayered colored and ornamented mesospores, and are surrounded by a sporangial wall, similar to other Zygnema species . When the chloroplast shape was described it was stellate, except for the irregular sub-globular to globular chloroplasts in Zygogonium sinense C.-C. Jao  Stancheva, J. D. Hall, McCourt et Sheath comb. nov.Basionym: Zygogonium aquaticumZygnema cyanosphaeroidicum (O. Bock and W. Bock)Synonym: Zygogonium cyanosphaeroidicum (O. Bock and W. Bock) Kadłubowska 1972Zygnema exuvielliforme (C.-C. Jao) Krieger in Kolkwitz and Krieger 1944Zygogonium exuvielliforme C.-C. Jao in Taylor 1935Basionym: Zygnema guineense (Gauthier-Lièvre) Stancheva, J. D. Hall, McCourt et Sheath comb. nov.Basionym: Zygogonium guineenseZygnema heydrichiiSchmidle 1897Synonym: Zygogonium heydrichii (Schmidle) Transeau 1933Zygnema heydrichii var. indicumRandhawa 1938Synonym: Zygogonium indicum (Randhawa) Transeau 1951Zygnema jaoi Krieger in Kolkwitz and Krieger 1944Synonyms: Zygogonium orientale Y. X. Wei 1979, and Zygogonium orientale var. reniforme H. J. Hu 1979 (according to Zygogonium reniforme (H. J. Hu) Zygogonium sinenseC.-C. Jao 1935Zygnema laetevirensKlebs 1886Synonym: Zygogonium laetevirens (Klebs) Migula 1907Zygnema maghrebianum (Gauthier-Lièvre) Stancheva, J. D. Hall, McCourt et Sheath comb. nov.Basionym: Zygogonium maghrebianumZygnema marocanum (Gauthier-Lièvre) Stancheva, J. D. Hall, McCourt et Sheath comb. nov.Basionym: Zygogonium marocanumZygnema mayyanadense (Kothari) Stancheva, J. D. Hall, McCourt et Sheath comb. nov.Basionym: Zygogonium mayyanadenseZygnema norvegicum (Kadłubowska et Langangen) Stancheva, J. D. Hall, McCourt et Sheath comb. nov.Zygogonium norvegicumBasionym: Zygnema plakountiosporum (C.-C. Jao) Krieger in Kolkwitz and Krieger 1944Basionym: Zygogonium plakountiosporum C.-C. Jao in Taylor 1935Zygnema seuratiiGauthier-Lièvre 1941Synonym: Zygogonium seuratii (Gauthier-Lièvre) Gauthier-Lièvre 1965Zygnema sphagnophilum (Gauthier-Lièvre) Stancheva, J. D. Hall, McCourt et Sheath comb. nov.Basionym: Zygogonium sphagnophilumZygnema stephensiae Transeau in Transeau et al. 1934Synonym: Zygogonium stephensiae (Transeau) Transeau 1951Zygnema sudanense (Gauthier-Lièvre) Stancheva, J. D. Hall, McCourt et Sheath comb. nov.Basionym: Zygogonium sudanenseZygnema tunetanum (Gauthier-Lièvre) Stancheva, J. D. Hall, McCourt et Sheath comb. nov.Basionym: Zygogonium tunetanumS1rbcL showing the relationship between Zygogonium ericetorum and a broader sampling of Mesotaenium strains. Support values follow Figure S1. Phylogeny of Zygnematophyceae based on a RAxML analysis of S2Zygogonium ericetorum, showing their variable morphology: (A–D) sterile vegetative filaments, (E–J) filaments with aplanospores. Scale bars: 10 μm.Figure S2. Confocal laser scanning microscopic images of chloroplasts in S3Zygogonium ericetorum chloroplast ultrastructure in vegetative cells (A–D) and aplanospore (E). (A) transverse section through chloroplast, pillow-shaped central part with starch grains clearly visible, flat parts emerging to both sides, numerous plastoglobues (arrow) adjacent to the starch grains, (B) Pyrenoid surrounded by starch grains, plastoglobules (arrow) in the close to starch grains, (C) fine structure of thylakoids in the edge of a chloroplast wing, (D) surface section through central part of chloroplast showing starch grains and numerous plastoglobues, (E) irregular arrangement of thylakoids and plastoglobules in aplanospore. PG, plastoglobules; Py, pyrenoid; S, starch; V, vacuole. Scale bars: (A) 2 μm;  1 μm; (C) 200 nm.Figure S3. Transmission electron micrographs of S4Zygogonium tunetanum UTCC136. Scale bar: 10 μm.Figure S4. Light and fluorescence microscopic images of chloroplasts in Table S1Table S1. List of strains used in this study as well as the GenBank number for the sequenced loci. Newly determined sequences are presented in bold typeface.


Colonic lymphoma is an uncommon presentation of extranodal lymphoma. Colonic mucosa-associated lymphoid tissue lymphoma is a different entity from gastric mucosa-associated lymphoid tissue lymphoma, and very rare. The presentation and management of colonic mucosa-associated lymphoid tissue are highly variable in the literature.We report the case of a 59-year-old Taiwanese man who underwent a colonoscopy after a positive test for fecal occult blood. His past history included hypertension and hyperthyroidism. The colonoscopy revealed an adenomatous polyp and mucosa-associated lymphoid tissue lymphoma. We successfully performed a polypectomy and endoscopic mucosal resection. The lymphoma was staged according to the Ann Arbor system modified by Musshoff as E-I. Our patient showed no lymphoma recurrence over a 3-year follow-up.Endoscopic mucosal resection for colonic mucosa-associated lymphoid tissue lymphoma without disseminated disease may be feasible. We successfully used colonoscopic treatment without adjuvant therapy to treat early-stage pathogen-free colonic mucosa-associated lymphoid tissue lymphoma. The gastrointestinal tract is the most common site for extranodal lymphomas, with the colon being the least involved area , 2. ColoOur patient was a 59-year-old Taiwanese, well-nourished man with a history of hypertension for 4 years and hyperthyroidism for one more year that were well controlled. He was a heavy smoker (1–2 packs per day) for 30 years, and had only quit for a few months. He had received a lumbar laminectomy to treat spinal stenosis 6 years previously. He had no relevant family history. He had developed thyrotoxic exophthalmos 2–3 months prior to presentation and received steroid pulse therapy for compressive optic neuropathy. The symptoms were relieved for 2–3 months after the steroid therapy.He visited our clinic after a positive fecal occult blood test 132 ng/mL) but reported no change to his bowel habits. A physical examination revealed bilateral eyes proptosis and mild obesity. Complete colonoscopy revealed one flat polyp, 0.3 cm in size, located 35 cm from his anal verge, and another polypoid polyp with a wide base, 2.0 cm in size with a slightly irregular border, located 25 cm from his anal verge. The smaller polyp was removed by polypectomy. We biopsied the larger polyp because of its wide base. A pathological examination of the specimen revealed it was a hyperplastic polyp. We did not perform an endoscopic ultrasound because we intended to directly remove the polyp. The polyp was then removed by endoscopic mucosal resection followed by clipping . During the last 2 years, he has had no epigastralgia, no acid reflux, and no tarry stool.According to his history and throughout his clinical course, our patient had no symptoms or signs of a peptic ulcer. We did not perform a panendoscopy nor test for H. pylori infection [MALT-type lymphoma was first defined by Isaacson and Wright in 1983 . Colon lnfection , 9. MALTnfection . It compnfection . Colon Mnfection –11.H. pylori infection. The prevalence of H. pylori infection is as high as 80 % in patients with gastric MALT lymphoma [H. pylori eradication [H. pylori infection in only 0–57 % of patients [et al. reviewed H. pylori-negative gastric MALT lymphomas and suggested that possible mechanisms include antibiotic eradication for non-H. pylori bacteria and the immunomodulatory effect of clarithromycin [The most well-known gastrointestinal lymphoma is gastric MALT lymphoma. Gastric MALT lymphoma has a strong predisposing factor, lymphoma , 16. Howdication , 18. Furpatients , 19. Asahromycin .et al. reported the resemblance between tonsil MALT lymphoma and EBV-related tonsillar hyperplasia [Other associated diseases, such as EBV infection and autoimmune diseases, have also been reported in the literature . Kaneko erplasia . These terplasia , tonsil erplasia , and ocuerplasia . Most oferplasia , 23.H. pylori eradication [et al. reported a MALT lymphoma of the cecum presenting as an acute bowel obstruction; however, no H. pylori nor other etiologies could be identified [Unlike gastric MALT lymphoma, colon MALT lymphoma does not have any obvious predisposing factors. Although chronic antigen stimulation is a hypothetical etiology , it was dication , 10. Ourentified . Terada entified . His diaentified .H. pylori eradication, chemotherapy, and radiotherapy [The presentation and management of colonic MALT lymphoma are still highly variable. The symptoms include a positive test for fecal occult blood, constipation, abdominal pain, gastro-intestinal bleeding, and tenesmus, or it may be asymptomatic –11. Manaotherapy , 10, 11.otherapy . HoweverAlthough more and more MALT lymphomas have been reported, colon involvement is still relatively rare. We report the case of a patient with a colonic MALT lymphoma with previous hyperthyroidism. Our patient was asymptomatic and underwent endoscopic mucosal resection without antibiotic eradication for a stage E-I tumor. He attended regular follow-up appointments without lymphoma recurrence for 3 years. A “wait and see” policy for resectable stage E-I colonic MALT lymphoma without pathogens seems to be a reasonable course of action. However, more studies about standard management for the disease are needed.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


Clostridium difficile infection (CDI) remains a substantial burden on healthcare systems and is likely to remain so given our reliance on antimicrobial therapies to treat bacterial infections, especially in an aging population in whom multiple co-morbidities are common. Antimicrobial agents are a key component in the aetiology of CDI, both in the establishment of the infection and also in its treatment. The purpose of this review is to summarise the role of antimicrobial agents in primary and recurrent CDI; assessing why certain antimicrobial classes may predispose to the induction of CDI according to a balance between antimicrobial activity against the gut microflora and C. difficile. Considering these aspects of CDI is important in both the prevention of the infection and in the development of new antimicrobial treatments. Clostridium difficile infection (CDI) remains a substantial worldwide burden on healthcare systems, despite significant research and investment over the past four decades. CDI normally, but not exclusively, emerges following one or more course(s) of antimicrobial therapy for an unrelated condition, which perturbs microbial colonisation resistance within the host. Other risk factors that have been linked to the development of CDI include proton pump inhibitors and the use of anticancer agents. Antimicrobial therapy facilitates germination of C. difficile spores within the colon and production of toxin A and/or toxin B. C. difficile is the aetiological agent of pseudomembranous colitis (PMC) and is implicated in approximately 30% of cases of antibiotic-associated diarrhoea [C. difficile and its toxins, antimicrobial agents, their effect of the indigenous gut microflora and C. difficile, and the virulence of the infecting C. difficile strain  may be influenced by antimicrobial susceptibility. We will also explore how the susceptibility of important indigenous gut microflora groups may influence the risk of CDI.This review will concentrate on antimicrobial susceptibility and resistance in C. difficile and its cytotoxic activity were demonstrated in faeces from PMC patients [C. difficile has identified multiple possible resistance mechanisms. Indeed, before highly discriminatory molecular typing methods were available, resistance to clindamycin was one characteristic that suggested that C. difficile subtypes existed within the same endemic ribotype (RT) 001 strains [C. difficile to clindamycin varies substantially between RTs and also according to geographical location. Freeman and colleagues recently published the results of a prospective susceptibility testing trial (ClosER) which encompassed 22 European countries and 953 C. difficile isolates [C. difficile have also been observed in other studies in Europe, Asia, North America , and the Middle East [C. difficile have been reported for RT027 [Clindamycin was the first antimicrobial agent linked to pseudomembranous colitis in the mid-1970s by Tedesco and colleagues in a prospective study of 200 patients [ strains . Antimicdle East ,22,23. Ior RT027 ,25 and oor RT027 , with clC. difficile with MICs of 16 to >256 mg/L often possess the erythromycin ribosomal methylase B, encoded by erm(B), which is often located on mobile genetic elements [erm(B) negative, and clindamycin-susceptible erm(B) positive C. difficile have been reported [erm(B)-independent 23S rRNA methylation [C. difficile.A true understanding of MICs is governed by the susceptibility testing method employed. For simplicity, many testing studies use antimicrobial-gradient based methods such as the E-test  or the M.I.C Evaluator strip . A limitation of such methods is that definitive MICs often cannot be determined due to the pre-defined concentration range of the test strip; with the highest concentration for clindamycin being 256 mg/L. Indeed, even for gold-standard methods such as agar incorporation MIC testing, antimicrobial concentrations are doubling dilutions, often with 256 mg/L as the highest concentration; drug solubility often limits the testing of higher concentrations for many agents. Clindamycin resistant elements ,28 and felements . Erm(B) reported ; thus, ahylation , efflux,C. difficile may be resistant to erythromycin via expression of erm(B) [erm(FS) [erm(B)-independent) [erm(B) negative C. difficile) [cme efflux pump [C. difficile isolates collected over 10 years, resistance (MICs 32 to >128) was seen in 3/4 of the most prevalent RT , and in 80% of all C. difficile isolates [erm(B) (E1-E17) reported in C. difficile [C. difficile (erm(B) positive) appear to demonstrate reduced fitness in vitro in competition assays with susceptible strains, and in growth rate experiments [in vivo remains to be determined.Erythromycin is a macrolide antimicrobial agent that inhibits protein synthesis in bacteria by binding the 50S ribosomal subunit and impairing the elongation cycle by preventing movement of the ribosome along the mRNA. Macrolides are not considered antimicrobial agents that have a strong association with CDI, although true risk data are difficult to obtain given that macrolides are often co-administered with other antibiotics; for example, with penicillins in the treatment of community-acquired pneumonia. f erm(B) ,30 or er[erm(FS) , methylapendent) , mutatiofficile) , and a plux pump . Similarisolates . Erythroisolates ,21 and 0isolates , and othisolates , and 017isolates . There aeriments ; whetherC. difficile was documented over 30 years ago and was demonstrated as transferable between C. difficile strains [C. difficile in isolates from the UK and the Netherlands, 14.3% resistance in isolates from Poland, 21.4% resistance in isolates from Hungary, and 38.9% resistance in isolates from Greece; compared to 9.2% across the entire study [C. difficile tetracycline resistance most often manifests as a consequence of TetM production but may also be due to TetW [tet genes in C. difficile are carried on transposons related to Tn916 [i.e., Tn5397 [tetM and tetW in three isolates of RT048 and one RT012 [tet elements elicits any further reduction in tetracycline MIC. Using a MIC breakpoint of ≥8 mg/L to examine human and pig isolates from the Netherlands and UK, 75/102 human strains and 15/56 porcine strains were resistant to tetracycline [916-like transposon harbouring the tet(M) gene. DNA fingerprinting of strains demonstrated relatedness of porcine and human strains, which is notable given the widespread use of tetracyclines in animal husbandry [Tetracyclines inhibit protein synthesis by preventing the attachment of aminoacyl-tRNA to the ribosomal acceptor (A) site . This fa strains ,38 and a strains . Resistare study . In genere study ,40, resire study , and higre study  and in are study . Resistare study . In C. d to TetW ,43. Both to TetW . The tetto Tn916 ,45, i.e., Tn5397 ,46, Tn61, Tn5397 , Tn6235 , Tn5397 . Spigaglacycline . All tetusbandry .C. difficile including strains with reduced susceptibility to metronidazole [in vitro models of CDI [C. difficile strains are inhibited by linezolid at concentrations far below the CLSI breakpoint for resistance (>4 mg/L), sporadic isolates with MICs of 8–16 mg/L have been reported [C. difficile (RT023 and RT067) with MICs of 8 mg/L in a study of 118 C. difficile isolates; further testing of 27 RT023 isolates yielded no further resistant isolates [C. difficile yielded 9 isolates with elevated MICs , RT017 (6/9), and RT078 (1/9)) and PCR and sequencing identified the presence of cfr, which encodes a rRNA methyltransferase, in 7/9 C. difficile isolates on Tn6218, but not in the two RT001 isolates [Linezolid is an oxazolidinone that is active against Gram-positive bacteria by inhibition of protein synthesis via targeting of bacterial 23S rRNA . Linezolnidazole ,54,55, as of CDI . Althougreported ,56,57. Bisolates . FurthergyrA and gyrB in C. difficile, which encode the A and B subunits of the enzyme responsible for supercoiling bacterial DNA. Resistance to fluoroquinolones is well documented in C. difficile usually due to alterations in target structure via nucleotide substitutions (gyrA and/or gyrB) [C. difficile is unclear [C. difficile fluoroquinolone susceptibility within RT and between countries, with certain RT which are currently circulating being uniformly resistant to fluoroquinolones. The breakpoints for ciprofloxacin and moxifloxacin resistance are ≥32 mg/L (EUCAST ECOFF) and ≥8 mg/L respectively. A European prospective susceptibility testing surveillance programme (ClosER) recently reported 2011–2012 results; 40% of 953 C. difficile were resistant to moxifloxacin, in multiple RTs, especially RT027 and RT356 [gyrB was associated with high-level resistance to moxifloxacin [C. difficile strains may possess two concurrent substitutions in either gyrase gene, or a concurrent mutation in both gyrA and gyrB [gyrA substitution but Asp71-Glu, Pro116-Ala, Ala118-Ser, and Thr82-Ala also have been reported [gyrB may involve Asp426-Val, Asp426-Asn, Glu466-Val, Ser366-Ala, Leu444-Phe [gyrA substitution, in a clinical environment where fluoroquinolone use was substantial in the 1990–2000s [C. difficile that were initially postulated as responsible for RT027 hypervirulence and transmission, were in fact present in pre- and post-epidemic RT027; yet the acquisition of fluoroquinolone resistance was the defining moment of the evolution and spread of the two lineages of this RT [Fluoroquinolones are synthetic derivatives of the naphthyridone molecule nalidixic acid, and were developed to increase inhibition of target molecules and broaden the antimicrobial spectrum . Fluoroqor gyrB) ,60,61,62 unclear ,63. Consfloxacin , whereasfloxacin ,65; suchreported ,67,68. Mu444-Phe ,59,64,6590–2000s , was a pC. difficile; rifaximin (but not rifampicin) is poorly absorbed after oral administration [C. difficile has been reported in 0–17.5% of isolates [C. difficile isolates, O’Connor et al observed seven rpo(B) substitutions (14 C. difficile strains): Arg505-Lys, His502-Asn, -Tyr, -Arg, Ser488-Thr, Asp492-Asn, and Iso548-Met; therefore, several distinct substitutions, some at the same location, are associated with MICs >256 mg/L [C. difficile, typically with results ≤0.016 mg/L and >256 mg/L [et al. observed mutations in the β-subunit of RNA polymerase, encoded by rpo(B) (1 or 2 substitutions); specifically His502-Asn and Arg505-Lys (92% of resistant strains), Arg505-Lys alone (6% of resistant isolates), or His502-Asn alone (2% of isolates) [rpo(B) between positions 488–548 may either disrupt the direct interaction between rifamycins and RpoB, or modify the rifamycin-binding pocket and therefore reduce the affinity of target for the antimicrobial [The rifamycins achieve selective toxicity in bacteria by targeting bacterial DNA-dependent RNA polymerase. Rifamycins (rifampicin and rifaximin) have been used to treat CDI due to very low MICs in (susceptible) stration . Rifampiisolates ,17,71,72isolates . High prisolates . In a st256 mg/L . Spigaglsolates) . Rifampisolates) ,22,72, iicrobial . Resistaicrobial ,75,76. AC. difficile, with MICs generally 0.02–0.25 mg/L [et al. conducted laboratory studies exposing C. difficile to sub-inhibitory concentrations of fidaxomicin over 10 serial passages, with whole genome sequencing to characterise resistance mechanisms, and demonstrated a Glu-Arg nucleotide substitution at position 1073 in rpo(B) or mutation within CD22120 (a marR homolog); resulting in a fidaxomicin MIC of 4 mg/L [Fidaxomicin was the first new therapy to be licensed for the treatment of CDI in over 25 years; it is now recommended for use in initial or recurrent episodes of CDI . Fidaxom.25 mg/L . Rare re.25 mg/L ,81 or re.25 mg/L . Howeveret al. [Metronidazole achieves its antibacterial action by directly damaging bacterial DNA following reduction of its nitro group once inside a bacterium. Several reduction systems have been highlighted as important for the activation of metronidazole, but perhaps most important is the pyruvate-ferredoxin/ flavodoxin oxidoreductase (PFOR) system; pyruvate is decarboxylated to acetyly-CoA and concurrently electrons are transferred to the nitro group of metronidazole by ferredoxin or flavodoxin . Metroniet al.  in a proet al. .C. difficile were resistant to metronidazole (MIC ≥ 8 mg/L), with maximal MICs of 8 mg/L observed in a single RT106 isolate from the UK [C. difficile RT001 (but not RT106 or RT027) in 2008 [C. difficile have been reported in other studies [C. difficile within a population observed after extended incubation periods using E-test [Freeman and colleagues recently demonstrated that only 0.11% of 953  in 2008 , but the studies ,88,89,90g E-test . It is ig E-test ,92.C. difficile [C. difficile have remained elusive; resistant strains have not been described with nimA-J [C. difficile [et al. used comparative whole genome sequencing to analyse a stable metronidazole-resistant RT027 strain and a clone that reverted to metronidazole-susceptible after freeze-thawing [spo0A) and germination (cspC) loci, the authors demonstrated mutations in the ferric uptake regulator (fur), a mutation in a putative nitroreductase gene (in both RT027 strains), and a mutation in the corporphyrinogrn III oxidase gene (hemN) , CDI treatment failure has not been linked to antimicrobial resistance in ifficile . Until rh nimA-J ,95, which nimA-J . Three rifficile ,98,99. Le (hemN) . In a suC. difficile, including against strains with reduced metronidazole susceptibility and also epidemic strains that tend to have higher geometric mean metronidazole MICs. Vancomycin binds to the C-terminal dipeptide, D-alanyl-D-alanine, of the NAM-pentapeptide of peptidoglycan (PG) precursors during PG synthesis, so preventing transpeptidation (cross-linking) reactions between adjacent peptide side chains of adjacent PG strains. Acquired resistance to vancomycin is well studied in Staphylococcus aureus and Enterococcus spp. and results from expression of one of 9 van gene clusters, which encode ligase enzymes that modify the terminal D-ala-D-ala of PG precursors [C. difficile has been shown to possess a vanG homolog [CdvanG), which was recently assessed for its ability to confer vancomycin resistance. CdvanG is inducible by vancomycin, but does not promote vancomycin resistance in C. difficile. When CdvanG was cloned into a vancomycin-susceptible Escherichia coli strain, vancomycin resistance was conferred; therefore, it was postulated that there is some mechanism in C. difficile that prevents expression of vancomycin resistance [C. difficile isolates (MIC ≤ 2 mg/L). Reduced susceptibility to vancomycin has begun to emerge over the past 5 years , but remains relatively sporadic. Recently, single isolates with intermediate-resistance (MIC 4 mg/L) have been seen from the Czech Republic, Ireland, Latvia, and Poland, and multiple isolates from Italy and Spain, resistant to vancomycin (MIC ≥ 8 mg/L), from multiple ribotypes  have been detected. No underlying mechanisms for such reduced susceptibility have been reported [S. aureus septicaemia [Much like metronidazole, vancomycin has been a first-line therapy for CDI for the past 3 decades; vancomycin concentrations typically exceed 1000 mg/L in faeces after oral administration. Vancomycin retains good activity against  homolog ,102 withticaemia .C. difficile susceptibility to β-lactams varies depending on the antimicrobial, with general susceptibility to β-lactam-β-lactamase inhibitor combinations, resistance to cephalosporins , and intermediate resistance or susceptibility to aminopenicillins. Noren et al. reported penicillin V and piperacillin MIC90 of 8 mg/L and 32 mg/L, respectively, in C. difficile isolates from Sweden over a 15 year period, with some isolates demonstrating high-level resistance (>256 mg/L) [Broad spectrum β-lactam antimicrobial agents are well recognised for their propensity to induce CDI, e.g., aminopenicillins and cephalosporins. Conversely, some β-lactams when given as combination products with β-lactamase inhibitors, e.g., piperacillin-tazobactam, are not noted for their propensity to induce CDI, and indeed have been used in preference to cephalosporins to reduce CDI rates ,104. C. 56 mg/L) .in vitro experiments exploring colonisation resistance [C. difficile specific; and (3) have a favourable pharmacokinetic/pharmacodynamic profile. Researchers have assessed in vitro and in vivo the effects of antimicrobial agents commonly linked to CDI; measured endpoints include which populations of the indigenous gut microflora are adversely affected or are promoted, whether C. difficile can be isolated and what is the duration of toxin production following spore germination [C. difficile, further research is needed to delineate which are the critical microbial factors in disease pathogenesis. Recurrent CDI occurs in approximately 20% of CDI cases following treatment with metronidazole or vancomycin, and this remains a key goal for improving therapeutic outcomes.From early sistance , and itssistance , it was mination ,123,124.mination ,126,127.mination  and feacmination ) has demin vitro and in vivo studies it is clear that antimicrobial agents that are potent inducers of CDI elicit widespread reductions in the anaerobic microflora, particularly Bacteroides spp. and Bifidobacterium spp., with a concurrent increase in certain populations of facultative anaerobes either during or after antimicrobial dosing, e.g., Enterococcus spp. and lactose-fermenting Enterobacteriaceae.Antimicrobial agents most commonly associated with CDI cause a shift from an anaerobe-dominated gut microflora in favour of facultative-anaerobes. Within the indigenous gut microflora, alterations in the community structure may occur over time in response to diet, stresses, antimicrobial agent exposure, and age, but the total concentrations of microbes remain relatively constant. The changes in gut microflora community structure associated with the administration of selected antimicrobial agents are presented in et al., used 16S rRNA microarray to show that the Bacteroidetes and Firmicutes phyla were associated with development of CDI [Lactobacillus and Clostridium are two important members of the Firmicutes phylum. Additionally, Antharam and colleagues recently reported the results of a study of 39 CDI patients , assessing the gut microflora composition, using culture-independent 454 16S rRNA Pyrosequencing [Firmicutes phylum sequence reads. Furthermore, associations between the depletion of Ruminococcaceae, Lachnospiraceae, and butyrogenic bacteria in the gut microbiota and nosocomial diarrhoea, including CDI were noted [Culture-independent techniques have supplemented the data generated from culture-based studies. In a study of the intestinal microbiota of 25 CDI patients compared with those for 50 matched controls, Manges t of CDI . Lactobaquencing . The sture noted . Bifidobacterium spp. are in general susceptible to penicillins, macrolides, glycopeptides, and fluoroquinolones, but variations between species are observed [Bifidobacterium spp. possess resistance determinants such as tetW [erm(X) [Bacteroides spp. vary in susceptibility to co-amoxiclav, with MIC90s ranging from 4 to >128 mg/L [et al. raised an interesting hypothesis that antimicrobial-resistant gut microflora may be protective in the gut when a patient is exposed to antimicrobial agents [Bacteroides thetaiotamicron was dosed orally into mice for 3 days, prior to administration of subcutaneous ceftriaxone and oral C. difficile spores, and showed that prior colonisation with the B. thetaiotamicron strain inactivated intra-intestinal cephalosporin and also prevented overgrowth by C. difficile [Clostridium scnidens may be a key protective species against CDI [Such changes in gut microflora community structure, based on viable count data, correlate well with susceptibility studies and the concentrations of antimicrobial agent that are observed in faeces following dosing . Bifidobobserved . Some Bi as tetW , and erm [erm(X)  to confe128 mg/L  and also128 mg/L ,141. Stil agents . A cephaifficile . If key inst CDI .C. difficile [C. difficile and its toxins [Bifidobacterium spp. and Bacteroides spp.) and this, combined with rapid decline of antimicrobial concentration following cessation of dosing , may allow C. difficile to compete for key nutrients [Treatments for CDI also may adversely affect the indigenous gut microflora and therefore prolong the perturbation of the microflora; thus, putting an individual at risk of recurrent CDI either because persistent spores may germinate (CDI relapse) or if a new strain is acquired (CDI re-infection). The reduced efficacy of metronidazole may be to be due to a perturbed microflora ,145,146 ifficile ,91, and/s toxins . Mean mes toxins ,149,150 s toxins ; therefoutrients  that areEnterococcus spp. and Bacteroides spp., and increased numbers of facultatively-anaerobic Gram-negative bacilli  [in vitro gut model reported similar alterations in bacterial community structure [Bacteroides spp. was demonstrated following vancomycin administration, in accordance with the observations of Edlund and colleagues [et al. demonstrated susceptibility to vancomycin in seventeen isolates of B. fragilis with MICs ranging from 16–128 mg/L; thus, very high concentrations of this anti-Gram-positive antibiotic are sufficient to adversely affect Gram-negative populations such as Bacteroides spp. [Very high vancomycin levels occur in faeces following oral administration (1345–1406 mg/L) ,150. In er spp.) . Previouer spp.) ,153. Instructure . Vancomytructure . Intereslleagues . Citron des spp. .et al. analysed 175 faecal specimens from CDI patients who underwent a 200 mg twice-daily dose of fidaxomicin for 10 days and reported a mean (±SE) faecal concentration of 1396 mg/kg (±77.0) [C. difficile. With fidaxomicin achieving such high faecal concentrations there is the potential that it may disrupt the indigenous gut microflora, even species with raised MICs. Susceptibility testing data indicated that fidaxomicin has good activity against C. difficile, C. perfringens, C. innocuum (MICs < 0.125 mg/L), but also has activity against other Gram-positive bacteria such as Bacillus spp. (MIC 1 mg/L), Enterococcus spp. (MIC 4 mg/L), Lactobacillus casei (but not L. acidophilus) (MIC 1 mg/L), Micrococcus luteus (MICs ≤ 0.06 mg/L), Bifidobacterium spp. (MICs ≤ 0.25 mg/L), and anaerobic cocci (MICs ≤1 mg/L). Additionally, some anti-Gram-negative activity was demonstrated against Moraxella cattharalis (MIC ≤ 2 mg/L) and Acinetobacter calccoaceticus (MIC 1 mg/L) [Sears  (±77.0) . An acti 1 mg/L) ,156. Conin vivo studies offer insights into the effects of CDI therapeutics on gut microflora [in vitro gut model fidaxomicin is effective in treating simulated primary and recurrent CDI [Bifidobacterium spp. was seen in three of four gut model experiments and the microflora became dominated by lactose-fermenting Enterobacteriaceae. Despite these microflora changes, fidaxomicin instillation was not associated with recurrent CDI, possibly due to persistence of supra-MICs of fidaxomicin for 2–3 weeks after dosing cessation, so preventing C. difficile spore germination. No inhibitory activity against Bacteroides spp. was observed, as would be predicted from in vitro susceptibility testing [C. difficile spore outgrowth [Gut model and croflora ,157. In rent CDI ; inhibit testing ,156. Anoutgrowth .C. difficile to antimicrobial agents is not as simple as it might first appear, since CDI may result from the administration of antimicrobial agents to which the infecting strain is either susceptible or resistant. It is the timing of CDI that may differ; earlier infection may occur if a C. difficile strain is resistant to an antimicrobial agent. In vitro experiments with a human chemostat model of the colon clearly demonstrate C. difficile spore germination, outgrowth, and toxin production occur once bioactive antimicrobial concentrations decline to sub-MIC levels [Trying to assess the importance of the susceptibility of C levels ,111. The−1) is complicated by the variability between subjects. For the present predictions, the gut transit time was based upon the rate of washout of vancomycin observed in the study of Abujamel et al. (vancomycin concentrations kindly supplied by Dr Curtis Donskey) [−1), washout to <1 mg/L vancomycin would be achieved by day 11 after dosing ceased, which reflects the data published in [C. difficile strain with a vancomycin MIC of 4 mg/L, spores would be able potentially to germinate and outgrow 2 days before a strain that had an MIC of 1 mg/L; i.e., day 9 vs. 11 post cessation of vancomycin dosing, assuming a faecal concentration of 1350 mg/L at the end of dosing. This may therefore provide a competitive advantage for strains with reduced vancomycin susceptibility; strains with an MIC of 8 mg/L could potentially germinate/outgrow a day earlier still.By using a mass-balance analysis of faecal concentrations of antimicrobial agents, it is possible to predict how quickly antimicrobials will be washed out of the gut and therefore analyse the potential importance of elevated MICs . It shouDonskey) . By seleished in   and/or the therapeutic agent. If we consider a “fidaxomicin-resistant” C. difficile strain with an MIC of 16 mg/L, spores could germinate/outgrow 7 days after cessation of fidaxomicin dosing, whereas susceptible strains (MIC 0.06 mg/L) would be inhibited from outgrowing until 15 days after cessation of fidaxomicin dosing if we assume concentrations in faeces were 1400 mg/L and no additional persistence due to spore binding or sequestration within gut biofilms. For metronidazole reduced susceptibility strains (MIC 4–8 mg/L) there is the possibility that luminal metronidazole concentrations during CDI therapy would be sub-MIC and therefore not inhibit C. difficile. If we assume that 15 mg/L of metronidazole was present at the end of a dosing regimen, then spores of C. difficile with MICs of 4 or 8 mg/L would be able to germinate/outgrow after 1 to 2 days following cessation of metronidazole dosing, compared to 5 days for a strain with an MIC of 0.5 mg/L. Abujamel et al. commented that there was a 3 week vulnerable period for re-establishment of C. difficile colonization after CDI treatment with vancomycin or metronidazole [C. difficile that are susceptible to vancomycin/metronidazole.Importantly, germination, outgrowth, and proliferation of nidazole . This obC. difficile with reduced drug susceptibility or resistance, when proliferation may occur before gut microflora colonisation resistance has normalised.These same principles can be applied to the other antimicrobial agents which commonly induce CDI , with anC. difficile ribotypes, low potential for mutational resistance, narrow spectrum activity that results in minimal gut microflora disruption, a good pharmacokinetic profile that yields high enough luminal concentrations sufficient to inhibit/kill C. difficile that leads to washout of residual toxins and spores , and finally no recurrence of CDI after excretion of antibiotic. The efficacy of the antimicrobial against C. difficile and the indigenous gut microflora, and likelihood of recurrence can be assessed using in vitro models and to a lesser extent in animal models of CDI, but human trials are where the most pivotal data are generated.So what does all of this mean for existing and new CDI treatments? The preferred characteristics of the ideal CDI therapeutic are easy to state: Potent bactericidal activity against all C. difficile and the normal gut microflora at the levels of gene transcription and translation. Antimicrobial agents have been studied for their effects against C. difficile at sub-MIC in several studies at the transcriptional and also the whole organism levels [C. difficile. These studies demonstrate interesting observations but based on the faecal concentrations presented in C. difficile being exposed to sub-MIC which may not be the case in all CDI patients. Novel antimicrobial agents, both CDI therapeutics and agents for other infections, would ideally not stimulate C. difficile to express virulence determinants or elevate their levels of expression. However, such considerations are not necessarily criteria for discounting the development of an agent and these types of studies are not often performed in drug development programmes. In terms of preventing CDI, it is imperative that resistance development in C. difficile is limited as this provides C. difficile with a competitive advantage as it may facilitate CDI earlier in the course of a therapeutic antimicrobial. Reduced susceptibility to metronidazole may potentially result in therapeutic failure due to low bioactive drug concentrations in the colon and could predispose to greater likelihood of recurrent CDI, although clinical data supporting this hypothesis are lacking. Conversely, reduced susceptibility to fidaxomicin and vancomycin is unlikely to affect primary treatment efficacy for CDI due to very high luminal concentrations in the gut. Antimicrobial agents are integral in the aetiology of CDI and although resistance is not a prerequisite for CDI to occur, there is now substantial evidence that mutation to a resistant phenotype may confer a significant selective advantage within the gut ecosystem. Limiting the spread of antimicrobial resistance in C. difficile should be a primary goal for clinicians and scientists, and prospective monitoring of resistance phenotypes and genotypes are essential in achieving this.One aspect not evaluated extensively in this review is the effect of the antimicrobial agent on the physiology of m levels ,171,172


Neurodegenerative diseases (NDD), such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS), are one of leading causes of death all over the world. Traditional medicine (TM), especially traditional Chinese medicine (TCM), usually having good clinical tolerability, is applied to treat NDD in China based on TCM or modern pharmacological theories. This issue on TM for treatment of NDD compiles 12 exciting manuscripts, most of which reported the efficacy and possible mechanisms of action of TM or active constituents on AD, PD, and ALS both in clinical and experimental levels.The effects of TCM on AD both in clinical and experimental levels are addressed in ten manuscripts. Dr. P. Wang et al. performed a survey of TCM treatment for AD, including Chinese herbal compound prescriptions  and Chinese materia medica ingredients, and confirmed the certain complementary cognitive benefits of TCM therapy. This paper is accompanied by other 8 manuscripts associated with TM or TCM.For clinical study, Dr. W. Pan et al. evaluated the effects of Shen-Zhi-Ling oral liquid on the behavioral and psychological symptoms of dementia in 98 patients with AD. Moreover, Dr. L. An et al. reported an updated meta-analysis of placebo-controlled RCTs of Huperzine A on patients with AD and vascular dementia (VD). Ginkgo biloba extract, GBE50, on activated microglia by inhibiting signal transduction through the NF-κB p65 and p38 MAPK pathways, which suggest the potential role of GBE on neuroinflammation related AD. Dr. L.-B. Zou et al. studied that xanthoceraside, a triterpene extracted from the husks of Xanthoceras sorbifolia Bunge, ameliorates mitochondrial dysfunction contributing to the improvement of learning and memory impairment in mice with intracerebroventricular injection of Aβ1-42. Dr. L. Zhang and L. Li found that cornel iridoid glycoside (CIG), an ingredient extracted from a traditional Chinese herb Cornus officinalis, attenuated tau hyperphosphorylation at multiple AD-related sites by increasing the activity of protein phosphatase 2A (PP2A) in SK-N-SH cells. Dr. R. Pan et al. reported that the crude extract of Polygala tenuifolia improved memory function of the aged mice probably via its antioxidant properties and decreased the activities of MAO and AChE. Dr. S.-J. Song et al. developed a bioactivity-oriented screening platform based on a modified Ellman's method and HPLC-QTOF MS technique and rapidly screened and confirmed some active compounds of Anemarrhena asphodeloides Bge. Dr. W.-X. Zhou and Y.-X. Zhang reported that Danggui-Shaoyao-San, could alleviate cognitive dysfunction in female senescence-accelerated mouse prone 8 (SAMP8) via modulation of estradiol.For experimental study, Dr. Z. Zhang et al. reported the potential suppressing effects of a new type of Panax quinquefolius (American ginseng), markedly improved the locomotor, motor balance, coordination, and apomorphine-induced rotations in 6-OHDA lesioned rats, and suggested its potent anti-Parkinson's property through inhibiting free radical formation and stimulating endogenous antioxidant release. Dr. W. Pan et al. investigated the use of integrative therapies  in patients with ALS in Shanghai, China.In addition to AD, two manuscripts reported the effects of TCM on PD and ALS, respectively. Dr. C.-F. Wu et al. reported that pseudoginsenoside-F11 (PF11), a component of Drosophila models, to evaluate the effects of SuHeXiang Wan, Gardenia jasminoides Ellis components, and Gastrodia elata Blume extract, celastrol as well as curcumin.As for the experimental model to evaluate the effect of TCM on NDD, Dr. KS Cho reviewed the successful application ofBy compiling these papers, we hope to enrich our readers and researchers with respect to the recent advancement of traditional medicine therapy on NDD.Chun Fu WuChun Fu WuWenxia ZhouWenxia ZhouChuthamanee SuthisisangChuthamanee SuthisisangJing Yu YangJing Yu Yang


Australopithecus anamensis, Australopithecus africanus, Paranthropus robustus and South African early Homo juveniles. Long-period line periodicities range from at least 6–12 days (possibly 5–13 days), and do not support the hypothesis that australopiths have lower mean values than extant or fossil Homo. Crown formation times of australopith and early Homo postcanine teeth fall below or at the low end of extant human values; Paranthropus robustus dentitions have the shortest formation times. Pliocene and early Pleistocene hominins show remarkable variation, and previous reports of age at death that employ a narrow range of estimated long-period line periodicities, cuspal enamel thicknesses, or initiation ages are likely to be in error. New chronological ages for SK 62 and StW 151 are several months younger than previous histological estimates, while Sts 24 is more than one year older. Extant human standards overestimate age at death in hominins predating Homo sapiens, and should not be applied to other fossil taxa. We urge caution when inferring life history as aspects of dental development in Pliocene and early Pleistocene fossils are distinct from modern humans and African apes, and recent work has challenged the predictive power of primate-wide associations between hominoid first molar emergence and certain life history variables.Until recently, our understanding of the evolution of human growth and development derived from studies of fossil juveniles that employed extant populations for both age determination and comparison. This circular approach has led to considerable debate about the human-like and ape-like affinities of fossil hominins. Teeth are invaluable for understanding maturation as age at death can be directly assessed from dental microstructure, and dental development has been shown to correlate with life history across primates broadly. We employ non-destructive synchrotron imaging to characterize incremental development, molar emergence, and age at death in more than 20  Evolutionary biologists have debated the evolution of human ontogeny for nearly a century –7, whichAustralopithecus africanus Taung child, which was often aged at ~3 or ~6 years according to ape and human developmental standards, respectively [Exploring the evolution of life history is of particular significance for understanding unique attributes of extant humans, such as early weaning ages, long childhoods, and extended post-reproductive periods. Furthermore, the fossil record provides scant evidence to assess the rate and duration of neurological or skeletal developmental beyond the information recorded in teeth. There are no alternative methods available to age juveniles without employing extant populations, which leads to circular reasoning when comparing fossil to modern taxa. This problem of circularity is particularly apparent in the history of debates about the ectively , 30–33. Enamel and dentine formation are characterized by the production of long- and short-period increments that represent the position of the forming front and the daily products of the secretory cells, respectively. Long-period features in enamel are known as Retzius lines, and are equivalent to Andresen’s lines in dentine. External manifestations of long-period lines on tooth crowns and roots are known as perikymata (enamel) and periradicular bands (dentine), respectively –36. ShorA. africanus, their age at death for Sts 24 was subsequently used to infer that Taung was also approximately 3.3 years of age, as it died at a similar developmental stage, although this has subsequently been revised to 3.7–3.9 years based on information from StW 402 [Bromage and Dean’s innovative study  of incre StW 402 . Assessm StW 402 , these s StW 402 , 47. Sev StW 402 –50 tende StW 402 .Although daily secretion rates (cross-striation spacing), enamel long-period line (Retzius line or perikyma) numbers, and long-period line periodicities have been documented in numerous Pliocene and early Pleistocene hominin teeth , 53, theAustralopithecus anamensis, A. africanus, Paranthropus robustus, South African early Homo, and two individuals of uncertain taxonomic attribution  .Long-period incremental lines, neonatal (birth) lines, and developmental defect patterns were imaged on complete teeth with voxel sizes around 5 μm , 55. In In order to characterize long-period line periodicity, selected areas of one or more teeth of each individual were scanned using sub-micron resolution local phase contrast X-ray synchrotron microtomography, and virtual histological slices  were produced following 3D optimized orientation . TemporaU test was employed for comparisons of periodicity values between taxa represented by four or more individuals. The fidelity of virtual histology has been demonstrated through comparisons of virtual and histological sections [Cuspal enamel thickness was measured on 2D slices with Adobe Photoshop CS5 from the dentine horn tip to the approximate position of the first-formed long-period line (perikyma) at the crown surface. Labio-lingual sections were cut for anterior teeth, buccal-lingual sections were cut for premolars, and buccal-lingual sections were cut for the mesial and distal cusps of molars following a standardized virtual orientation technique . Long-pesections , and lonsections .Homo samples. For Stw 151, which has been attributed to either early Homo or A. africanus [A. africanus [Crown formation time was calculated as the sum of cuspal and lateral formation times. Cuspal formation time was typically calculated by dividing the linear enamel thickness of each tooth cusp by the species-specific average cuspal daily secretion rates in ref. . Daily sfricanus , estimatfricanus , resultiAge at death was typically calculated by identification of the neonatal (birth) line in permanent first molars (M1s) or deciduous premolars, and summation of subsequent crown and root formation determined from incremental features. In three individuals  it was not possible to identify a neonatal line, and thus the M1 cusp employed was assumed to have initiated at birth, as species-specific initiation ages were not available for these particular cusps. For older juveniles, developmental defects (hypoplasias or accentuated lines) were registered across the dentition to link developmentally-overlapping teeth , 36, 54.Tooth calcification was determined from virtual 2D sections of each tooth for those individuals whose age could be determined. The section planes employed to assess calcification stages often differed from those employed to assess cuspal enamel thickness. Sections were cut to optimize developing root profiles for calcification stage determination, and a numerical score was assigned to each tooth following the commonly employed classification system of Moorrees and colleagues . CalcifiP. robustus juveniles that possessed the appropriate teeth and for which it was possible to estimate age at death histologically.While additional aging systems from extant human tooth calcification and/or emergence studies are available , 74, theParanthropus robustus individuals range from 6–12 days, and do not show a significant difference when compared with Australopithecus africanus, Homo neanderthalensis, fossil Homo sapiens, extant humans, gorillas, or previously reported values for this species  do not show smaller ranges or lower average periodicity values than extant or fossil Homo . Previo  formation times in A. africanus and P. robustus are shorter than those of extant humans and chimpanzees, while A. africanus mandibular premolar values exceed mean values for extant humans from South Africa  and chimpanzees . Comparisons of molar crown formation times also show a complex pattern, which is difficult to generalize as each molar tooth and cusp must be considered independently [A. anamensis, Australopithecus afarensis, A. africanus, P. robustus, and Homo erectus values [Homo individual DNH 67/70/71 are most similar to times for both gorillas and extant humans from South Africa (Hominin incisor formation times are shorter than chimpanzees (contra ), and arendently . Our news values , 65, 82,s values , 83. In h Africa , underscA. afarensis specimen LH 2 was estimated to have died at 3.25 years of age [A. afarensis possessed a similar range of long-period line values to A. anamensis and A. africanus  and one A. africanus individual (MLD 11/30), which show particularly rapid dental development that exceeds similarly-aged humans and chimpanzees. Age at death was also predicted for two P. robustus individuals with captive chimpanzee standards [Since our age at death estimates do not rely on extant human or great ape developmental information, we compared ages derived from the calcification stage of each tooth of each individual  and a 3.4 year-old A. africanus individual (MLD 11/30). More subtle minor wear facets were also observed on the maxillary and mandibular M1s of a 4.4 year-old A. africanus individual (Sts 24), implying variation in A. africanus M1 emergence that is comparable to chimpanzees, which may erupt their first molars between ~2.1–4.5 years of age [P. robustus individuals sampled here are also of interest for consideration of M1 emergence ages: 3.1 year-old SK 62 had not yet erupted its M1s ; the mandibular M1 of DNH 107 appeared to be nearing full occlusion (with slight facets) at 4.8 years of age  The maxillary M1 emergence status and wear in StW 151 appears to be more similar to the 5.4 or 5.5 year-old P. robustus individual DNH 108. Although it is difficult to precisely compare emergence among these individuals, our results suggest that M1 emergence in A. anamensis and/or A. africanus may have been somewhat earlier than in P. robustus.None of the individuals in this study appear to have died during the initial skeletal phase of M1 emergence . Marked wear facets are apparent on the mandibular M1 of a 3.6 or 4.2 year-old KNM-KP 345 and a 3s of age , 86–88. us; ref. ) appearsSubstantial intraspecific variation exists in aspects of dental development quantified in this study , which has also been demonstrated for extant human populations and African apes , 89, 90.P. robustus teeth  may resemble that of African apes [We present histologically-derived ages at death for 16 Pliocene and early Pleistocene hominins, illustrating an approach that may eventually lead to the development of australopith calcification standards in order to estimate the age of other juveniles. Importantly, these ages were determined from developmental information quantified directly from the specimens themselves and supplemented with species-specific information on daily secretion rates , 57 and ld: ref. ). Moreovcan apes , 99, whiStudies of dental development have also been of great interest for reconstructing the evolution of human life history, although scholars have recently begun to question the association between molar emergence and life history variables among extant apes and humans , 86, 97.Homo relative to species of Australopithecus or Paranthropus due to limited samples and uncertainty over the taxonomic identification of particular elements [Homo cuspal enamel thickness and crown formation time involve analyses of naturally fractured teeth, which may be of limited comparative value due to section obliquity [Homo erectus [While it is tempting to compare aspects of dental development in order to speculate on the taxonomic identity of StW 151 or KB 5223, additional data are required from securely identified Pliocene and early Pleistocene hominins. Little is known about dental development and overall tooth morphology in early elements , 57, 100bliquity , or the  erectus , and gre erectus .S1 FileFig. A. Linear cuspal enamel thickness values in four hominin taxa. Fig. B. Long-period line periodicity in two A. anamensis individuals. Fig. C. Developmental plate used to assess tooth calcification in A. anamensis (KNM-KP 31712). Fig. D. Developmental plate used to assess tooth calcification in A. anamensis (KNM-KP 34725). Fig. E. Developmental plate used to assess tooth calcification in A. africanus (Sts 2). Fig. F. Developmental plate used to assess tooth calcification in A. africanus (Sts 24). Fig. G. Developmental plate used to assess tooth calcification in A. africanus (MLD 11/30). Fig. H. Developmental plate used to assess tooth calcification in P. robustus (SK 62). Fig. I. Developmental plate used to assess tooth calcification in P. robustus (TM 1536). Fig. J. Developmental plate used to assess tooth calcification in P. robustus (DNH 44). Fig. K. Developmental plate used to assess tooth calcification in P. robustus (DNH 47). Fig. L. Developmental plate used to assess tooth calcification in P. robustus (DNH 84). Fig. M. Developmental plate used to assess tooth calcification in P. robustus (DNH 107). Fig. N. Developmental plate used to assess tooth calcification in P. robustus (DNH 108). Fig. O. Developmental plate used to assess tooth calcification in early Homo (DNH 35). Fig. P. Developmental plate used to assess tooth calcification in early Homo (DNH 83). Fig. Q. Developmental plate used to assess tooth calcification in StW 151. Fig. R. Developmental plate used to assess tooth calcification in KB 5223. Fig. S. Ages at death predicted from modern human calcification standards compared to known- or histologically-derived ages. Fig. T. Recently erupted lower right first molar of DNH 107, a 4.8 year-old P. robustus individual from Drimolen. Table A. Long-period line periodicity (in days) for individuals in this study. Table B. Long-period line periodicity (in days) for fossil hominins, extant humans, and chimpanzees. Table C. Results of Mann-Whitney U test for comparisons of long-period line periodicity in P. robustus and A. africanus with fossil hominin taxa, extant humans, African apes, and previously published values. Table D. Cuspal enamel thickness values (in microns) for fossil hominins in this study. Table E. Age at death calculations for individual hominin specimens. Table F. Comparison of age at death estimates for two complete P. robustus mandibular dentitions. Table G. Histologically-determined initiation ages in Pliocene and early Pleistocene hominins.(DOC)Click here for additional data file.


Enterococcus faecium contribute beneficially to human health and food fermentation. However, other E. faecium strains are opportunistic pathogens due to the acquisition of virulence factors and antibiotic resistance determinants. To characterize E. faecium from soybean fermentation, we sequenced the genomes of 10 E. faecium strains from Korean soybean-fermented foods and analyzed their genomes by comparing them with 51 clinical and 52 non-clinical strains of different origins. Hierarchical clustering based on 13,820 orthologous genes from all E. faecium genomes showed that the 10 strains are distinguished from most of the clinical strains. Like non-clinical strains, their genomes are significantly smaller than clinical strains due to fewer accessory genes associated with antibiotic resistance, virulence, and mobile genetic elements. Moreover, we identified niche-associated gene gain and loss from the soybean strains. Thus, we conclude that soybean E. faecium strains might have evolved to have distinctive genomic features that may contribute to its ability to thrive during soybean fermentation.Certain strains of  Enterococcus faecium is a Gram-positive bacterium found in the gastro-intestinal tracts (GIT) of animals [E. faecium is a lactic acid bacterium, which produces lactic acid as a final product of carbohydrate fermentation [E. faecium have beneficial roles in GIT health as commensal or probiotic bacteria [E. faecium strains have emerged as critical hospital pathogens due to their resistance to vancomycin treatments [ animals , ferment animals ,3, and a animals . E. faecentation , and it entation . Some stbacteria ; others bacteria . Recentleatments .E. faecium has not been yet fully defined, many antibiotic resistance (AR) genes and virulence factors (VF) have been identified [E. faecium strains than community-associated non-clinical strains [E. faecium strains revealed different lineages that represent clinical and non-clinical strains [Although the pathogenicity of entified . Mobile entified . AR, VF, strains . Moreove strains –10.Enterococcus spp were found at considerable levels in fermented soybeans at the early stage of fermentation [E. faecium strains were frequently isolated from the fermented products [E. faecium in soybean fermentation, genomic features and contents of E. faecium have never been evaluated by using genomic information. Here, we sequenced genomes of 10 E. faecium strains isolated from fermented soybeans to characterize their genomic features.Many Korean foods have long been prepared based on fermentation of soybeans. Fermented soybean blocks (Meju), fermented soybean paste (Doenjang), and soy sauce (Ganjang) are representative fermented soybean foods in Korea. A recent study showed that entation , and E. products . DespiteE. faecium strains used in this study were obtained from a Microorganism Collection, Korean Agricultural Culture Collection (KACC). They were all isolated from fermented soybean products as listed in The 10 Bacterial cells were harvested from overnight BHI (Brain-Heart Infusion) broth culture. Harvested cells were washed two times with 1X PBS buffer. The cells were further processed to extract genomic DNA using G-spin Genomic DNA Extraction Kit . Genomic DNA was fragmented using NEBNext dsDNA Fragmentase . The fragmented DNA was further processed to construct a genomic DNA library using NEBNext Ultra DNA Library Prep Kit for Illumina . Genomic DNA libraries were constructed with ~350-bp inserts and sequenced by Illumina HiSeq2500 for 100 bp paired-end reads.Sequenced reads were quality-filtered using in-house Perl scripts . In brieE. faecium nucleotide sequences were downloaded on 07/03/2014 from the NCBI GenBank database. We inspected protein coding sequences (CDS) from GenBank information using in-house Perl scripts. In brief, we excluded premature stop codons, codon shifts by deletions and insertions, errors in CDS length, etc. as shown previously [E. faecium nucleotides and our 10 annotated E. faecium genomes. The CDS were clustered based on the sequence similarity as suggested previously [E. faecium strains  from the 113 strains. We used 990 core genes that are commonly present in all genomes. Due to incomplete genome assemblies, 100 core genes are excluded from SNP selection. To collect core gene sequences from each genome, ortholog sequences were aligned to each genome using GASSST under the 75–100% sequence similarity option. The aligned regions in each genome were extracted and collected for SNP selection. The collected core gene sequences from each genome were aligned among 113 strains using the alignment tool MUSCLE [To analyze clonal relatedness among soybean strains and assign their lineage among 113 l MUSCLE . On the l MUSCLE . The evol MUSCLE  with bool MUSCLE  and the l MUSCLE , respecthttp://bagel.molgenrug.nl/) [As previously defined , 34 VF grug.nl/) . Each geE. faecium from all three different origins; soybean origin, non-soybean non-clinical origin, and clinical origin. Genes with higher frequency in the soybean origin than other origins were regarded as soybean-specific. Significance of different frequencies was evaluated by Fisher's Exact Test at P < 0.05.The frequency of each orthologous CDS was counted for Before we describe the differences between 10 soybean strains and others from different origins, we need to demonstrate whether any specific strains are clonally related, which may affect subsequent comparisons. In total, 8,850 SNPs from 990 core genes, which are commonly found in 113 strains, were identified among the 10 soybean strains. We constructed a phylogenetic tree  that wasE. faecium strains from fermented soybean were sequenced and assembled to obtain draft genomes (P = 0.006), but not compared to that of non-clinical strains . From our pan-genome analysis, we identified 1,090 core CDS that are found in all 113 E. faecium genomes . The number of accessory CDS that were found only in some strains was compared among the three groups (P<0.001). Non-clinical strains  also have the higher numbers of accessory CDS compared to clinical strains (P<0.001). The average GC content of soybean-isolated strains .Ten  genomes . The num genomes  was signe groups . SoybeanE. faecium genomes were positioned as shown in According to previous studies ,11, non-E. faecium species [E. faecium strains, we analyzed the prevalence of these genes  were found in all soybean strains; however, the 3 major VF genes  that are used for safety assessment in Europe [E. faecium is more similar to non-clinical strains than to the clinical strains.AR, VF, ME, and BA genes contribute not only to the pathogenicity of  species  but also species ,11,23,24se genes . The soyr debate , and no r debate ,11. Somen Europe  were notE. faecium strains are similar to non-clinical strains as described above, we expected that they would have their own niche-enriched genes that are not frequently found in isolates of other origins. We identified not only such niche-enriched genes, but also missing genes in soybean E. faecium strains (P<0.005) and non-clinical strains (P<0.005). Among the 81 genes, 6 gene clusters were identified , and another cluster (Cluster 4) was found not only in many E. faecium contigs but also in Enterococcus faecalis (GenBank Accession No. KJ756557) and Enterococcus durans (GenBank Accession No. CP012367). Clusters 1, 4, 5, and 6 are related to sugar metabolism, cell wall-associated function, the enterocin AS-48 system, and mannose/fructose transportation, respectively. We identified 17 genes that were frequently found in E. faecium from both clinical and other origins but were frequently missing from the soybean E. faecium strains. Among the 17 genes, we identified two major clusters . The soybean strains (1927.5±74.4 genes) have a lower number of accessory genes than CL and NC strains (2178.5±292.0 genes), which differentially influenced the GC contents of the entire genome. That is, lower-GC accessory genes are more abundant in NC and CL strains than in soybean strains. More specifically, ME genes (37.05%) were lower in GC content than other genes , and there were significantly fewer such ME genes in the soybean strains (75.2±15.8 genes) than in both CL and NC strains . However, soybean-enriched genes may have a limited effect on the higher GC contents of the soybean strains, although they are significantly lower in GC content (34.20%) than other genes . For now, the reason for the slightly higher GC contents in soybean strains is not fully understood. Other types of accessory genes or codon usage bias may play a role in such a high GC content, which must be clarified in future studies.Genomic GC contents of the soybean strains were slightly higher than those of CL and NC strains. The higher GC contents of the soybean strains were influenced by accessory genes, which are lower in GC content than core genes Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.